Synthesis, antimycobacterial and antifungal evaluation of 3-arylaminopyrazine-2,5-dicarbonitriles

Article abstract of DOI:10.1002/ardp.200700119

This paper describes preparation and biological evaluation of pyrazinamide analogues. Pyrazinamide with its simple structure gives a good opportunity for further modification regarding an increase of its antimycobacterial activity. We prepared a series of compounds derived from pyrazine-2,5-dicarbonitrile with arylamino substitution in position 3. All compounds were assayed in vitro against major Mycobacterium and various Fungi species. The best activity was found in 3-{[3-(trifluoromethyl)phenyl]amino}pyrazine-2,5-dicarbonitrile 11 with the value of 6.25 μmol^-1 against M. tuberculosis H₃₇Rv and moderate activity against minor Mycobacterium pathogens.

Full text of DOI:10.1002/ardp.200700119

Arch. Pharm. Chem. Life Sci. 2008, 341, 61–65
L. Palek et al.
61
Full Paper
Synthesis, Antimycobacterial and Antifungal Evaluation of
3-Arylaminopyrazine-2,5-dicarbonitriles
1
1
2
2
3
ˇ
LukꢀÐ Palek , Jaroslav Dvorꢀk , Michaela Svobodovꢀ , Vladimꢁr Buchta , Josef Jampꢁlek ,
1
ˇ
Martin Dolezal
1 Department of Medicinal Chemistry and Drug Control, Charles University Prague, Faculty of Pharmacy in
Hradec Krꢀlovꢁ, Hradec Krꢀlovꢁ, Czech Republic
2 Department of Clinical Microbiology, Charles University Prague, Faculty of Medicine and University Hospital
in Hradec Krꢀlovꢁ, Hradec Krꢀlovꢁ, Czech Republic
3 Zentiva a. s., Prague, Czech Republic
This paper describes preparation and biological evaluation of pyrazinamide analogues. Pyrazina-
mide with its simple structure gives a good opportunity for further modification regarding an
increase of its antimycobacterial activity. We prepared a series of compounds derived from pyra-
zine-2,5-dicarbonitrile with arylamino substitution in position 3. All compounds were assayed in
vitro against major Mycobacterium and various Fungi species. The best activity was found in 3-{[3-
(trifluoromethyl)phenyl]amino}pyrazine-2,5-dicarbonitrile 11 with the value of 6.25 lmol– 1
against M. tuberculosis H37Rv and moderate activity against minor Mycobacterium pathogens.
Keywords: Antibacterial agents / Antimycotic activity / Antitubercular activity / Pyrazinamide analogues /
Received: June 5, 2007; accepted: October 17, 2007
DOI 10.1002/ardp.200700119
made defeating tuberculosis worldwide very difficult.
Therefore, here is an urgent need for new drugs with
novel modes of action and higher selectivity and biologi-
cal activity.
Introduction
In recent days, the health of people, who live in so-called
third-world countries, is becoming a problem of the
whole world, because of globalization and traveling-busi-
ness boom. Most of the health complications are in rela-
tionship with infectious diseases, such as AIDS, tubercu-
losis, sleeping sickness, or yellow fever. These are reasons
having influenced the main priorities of the World
Health Organization (WHO) global healthcare program
[1] or European Union Sixth Framework Research Pro-
gram [2].
Current treatment of tuberculosis consists in the com-
bination of two to four drugs with different modes of
action for three to nine months of continuous therapy
[3]. In fact, conditions of medical care in poor countries
and the increased resistance of Mycobacterium genus has
Our research follows the pathway of known drug-ana-
logues preparation [4, 5]. Various pyrazine derivatives
exhibit a wide range of biological actions including anti-
infective activity [6]. This study deals with pyrazinamide
analogues and shows structures derived from newly pre-
pared 3-chloropyrazine-2,5-dicarbonitrile that are
formed by nucleophilic substitution of chlorine in posi-
tion 3. Reports that 5-chloropyrazine-2-carboxamide has
a different mode of action compared to pyrazinamide
itself makes this direction in research more than relevant
[7]. We chose 5-chloropyrazine-2,5-dicarbonitrile – that
shows good activity itself – as our model compound [8].
Results and discussion
ˇ
Correspondence: Martin Dolezal, Department of Medicinal Chemistry
Twelve compounds were prepared of which nine were
new and not described so far in the literature. We fully
characterized all of them and they were screened for
anti-tuberculosis and/or antifungal activity.
and Drug Control, Charles University Prague, Faculty of Pharmacy in
Hradec Krꢀlovꢁ, Heyrovskꢁho 1203, 500 05 Hradec Krꢀlovꢁ, Czech Re-
public.
E-mail: Martin.Dolezal@faf.cuni.cz
Fax: +420 49 551-2423
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

62
L. Palek et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 61–65
Scheme 3. Formation of the final compounds.
Scheme 1. Oxidization of pyrazinamide and further reductive
chlorination.
Figure 1. Dependence of experimentally determined p distribu-
tive parameters on p values described in the literature.
Scheme 2. Amidation of 6-chloropyrazin-2-carbonitrile and its
further dehydration to 3-chloropyrazine-2,5-dicarbonitrile.
The chlorine of 3-chloropyrazine-2,5-dicarbonitrile
then undertakes nucleophilic substitution with some
ring-substituted anilines in dry toluene or dimethylfor-
mamide (24 h, reflux) with pyridine as a base (Scheme 3).
We used pyrazinamide as a starting material. After oxi-
dization with hydrogen peroxide (6 h, 908C) which is nec-
essary to activate the pyrazine ring for further homolytic
substitution and yields three intermediates (three iso-
meric pyrazine-N-oxides), comes chlorination and dehy-
dration (POCl3, 1 h, 908C) using phosphorus oxychloride
to give 6-chloropyrazine-2-carbonitrile or 5-chloro or 3-
chloro, respectively (Scheme 1). Since there is no good
possibility to purify the three isomeric N-oxides from the
first reaction, the chlorination gives three products as
well. The isomeric carbonitriles can be separated using
column flash chromatography [9].
The yielded pure isomer is then allowed to react with
formamide using ammonium persulfate (2 h, 908C) to
form 5-cyan-3-chloropyrazine-2-carboxamide followed by
crystallization from water. This compound is then dehy-
drated to 6-chloropyrazine-2,5-dicarbonitrile (Scheme 2)
using phosphorus oxychloride (1 h, 908C).
1
The newly prepared structures were confirmed by H-
NMR, 13C-NMR, IR, melting point, LowRes MS (LRMS),
microanalysis, and HPLC determination. Reproduced
structures were determined by 1H-NMR and melting
point. Purification of the final products has been made
using chromatography followed by recrystallization
from water and ethanol.
The HPLC determination (Table 1) showed that the
measured p parameters correlate to the p parameters
obtained from literature relatively well, except for com-
pound 6 (Fig. 1). This compound exhibits low lipophilic-
ity compared to anilide with the same ring substitution,
maybe because of intra-molecular interactions. See refer-
ence [10] for detailed explanation of structure-lipophilic-
ity relationships.
Table 1. HPLC lipophilicity determination and the distributive parameters p as determined by HPLC and obtained from literature [15].
Comp.
R
UV
kmax/log e
Purity
(%)
Log K
Log P
ACD/Log P
Log P/ClogP
ChemOffice
p
p
Determined Described
5
6
7
8
9
10
11
12
3-Cl
3,5-Br-4-OH
4-F
2-OH-4-NO2
2-SH
2-CF3
283.2 / 3.60
259.5 / 3.91
279.6 / 3.09
222.9 / 3.76
247.6 / 3.33
284.3 / 3.20
284.3 / 3.36
301.0 / 3.68
83.66
99.46
98.53
99.24
98.23
99.93
98.20
95.99
0.7578
0.6603
0.6789
0.5632
0.7829
0.7831
0.7836
0.5860
3.33 l 0.52
4.36 l 0.65
2.75 l 0.57
3.11 l 0.55
2.39 l 0.57
3.50 l 0.56
3.51 l 0.55
2.32 l 0.57
3.01 / 2.41376
3.72 / 2.32156
2.60 / 1.84376
0.34 / 0.972751
2.64 / 2.08923
3.38 / 2.58717
3.38 / 2.58717
0.23 / 0.886165
0.0946
–0.0029
0.0157
–0.1000
0.1197
0.1199
0.1204
–0.0772
0.77
1.31
0.15
–0.19
a)
1.04
1.1
–0.11
3-CF3
2-CN-4-NO2
a)
Data not found in literature.
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2008, 341, 61–65
New Pyrazinamide Analogues as Potential Anti-Infectives
63
Table 2. Antifungal and antituberculosis activities of the presented compounds.
Mycobacterium
Strain
CA
CT
CK
CG
TB
AF
AC
TM
kansasii avium
avium
tuberculosis
Compound
Nr.
80/72
80/72
152/74
H37Rv
(lmol/L)
(lmol/L)a) (%)b)
c)
c)
c)
c)
c)
4
5
6
7
8
9
10
11
3,90
15,63
62,5 >125
15,62
15,63
31,25 7,81
15,63 31,25
15,62
31,25
>125
>500
A500
A125
A125
125
7,81
15,63
>125
125
A500
A125
A125
125
15,62
31,25
>125
500
A500
A125
A125
125
15,62
15,63
125
16
32
>128
64
>128
>128
>128
>128
16
28
0
58
>125
>125
500
A500
A125
A125
125
>128
16
125
A500
A125
250
A500
A125
500
A500
A125
A125
125
62,5 >128
500 64
128
>128
16
53
c)
c)
c)
c)
c)
A125
125
125
31,25
1,95
62,5 A125
128
128
16
A128
>128
128
A128
>128
>128
16
8
32
33
62,5
3,91
0,06
125
15,63
0,12
94d)
16
12
7,82 15,63
3,91 0,98
15,63
0,24 >125
7,82
31,25
>125
Fluconasole
Pyrazinamide
A128
A128
A128
6 to 60[10]
a)
MIC determination by the University Hospital in Hradec Krꢀlovꢁ [12].
TAACF screening; data given in % of inhibition at 6.25 lmol/L.
no data available at the time of publication.
b)
c)
d)
structure released into level 2 of TAACF screening [10].
See Table 3 for further data. Fungi strains are given in abbreviations: CA – Candida albicans; CT – Candida tropicalis; CK – Candida
krusei; CG – Candida glabrata; TB – Trichosporon beigelii; AF – Aspergillus fumigatus; AC – Absidia corymbifera; TM – Trichophyton
mentagrophytes.
Table 3. TAACF data report – MIC determination, IC50 determi-
nation and calculated SI (selectivity index).
vents were dried or redistilled. Reaction kinetics was checked
using Merck UV 254 TLC plates (Merck, Darmstadt, Germany).
Purification of compounds was made using Flash Master Per-
sonal chromatography system from Argonaut Chromatography
(Argonaut Technologies, Redwood City, CA, USA). As sorbent,
Merck silica gel 60 with 40–63 lm granity was used (Merck). 1H-
and 13C-NMR spectra were taken on Varian Mercury VX-BB 300 at
300 and 75 MHz, respectively (Varian, Palo Alto CA, USA).
(CH3)4Si was used as an inner standard. Microanalyses were
recorded on Fisons Instruments EA1110CE CHN analyst (Fisons
Instruments, Mainz, Germany). Mass spectra were taken using
Voyager-DE STR (Applied Biosystems, Framingham, MA, USA).
Infrared spectra were taken at Nicolet Impact 400 in KBr blocks
(Nicolet, Madison, WI, USA). The purity of the compounds was
checked by HPLC. The detection wavelength of 210 nm was
chosen. Peaks in the chromatogram of the solvent (blank) were
deducted from peaks in the chromatogram of the sample solu-
tion. The purity of the individual compounds was determined
from the area peaks in the chromatogram of the sample solu-
tion. UV spectra (k, nm) were determined on a Waters Photo-
diode Array Detector 2996 (Waters Corp., Milford, MA, USA) in
acetonitrile solution (approx. 3 N 10– 4 M).
Compound Inhibition Assay
(%)
MIC
IC50
SI
(lg/mL) (lg/mL)
11
94
Alamar 6.25
2.86
0,45
All prepared compounds underwent biological activity
screening. The compounds 1 and 12 showed the best anti-
fungal activities whereas compound 11 reached the best
anti-tuberculosis activity (Table 2). Structure 11 was
released into level 2 of anti-tuberculosis screening. It was
found that this compound has a high cytotoxicity
(Table 3) and a low selectivity index, therefore it was not
evaluated further. More structures have to be described
to conclude more structure-activity relationships.
This study was supported by the Ministry of Education of the
Czech Republic (MSM002160822). Antimycobacterial data were
provided by the Tuberculosis Antimicrobial Acquisition and
Coordinating Facility (TAACF) through a research and develop-
ment contract with the U. S. National Institute of Allergy and
Infectious Diseases.
HPLC lipophilicity determination (capacity factor K/
calculated log K)
The HPLC separation module Waters Alliance 2695 XE and
Waters Photodiode Array Detector 2996 (Waters Corp., Milford,
MA, USA) were used. The chromatographic column Symmetrym
C18 5 lm, 4.66250 mm, Part No. WAT054275, (Waters Corp.) was
used. The HPLC separation process was monitored by Millen-
nium32m Chromatography Manager Software, Waters 2004
(Waters Corp.). A mixture of MeOH (p.a., 70%) and H2O-HPLC Mili-
Q Grade (30.0%) was used as mobile phase. The total flow of the
column was 1.0 mL/min, injection 30 lL, column temperature
The authors have declared no conflict of interest.
Experimental
General experimental details
All reactions were run with newly purchased chemicals; where
relevant, a protective atmosphere of argon was used. All used sol-
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64
L. Palek et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 61–65
308C and sample temperature 108C; as detection wavelength,
210 nm was chosen. The KI acetonitrile solution was used for the
dead time (TD) determination. Retention times (TR) were meas-
ured in minutes.
fied using flash chromatography with gradient of elution from
0% to 20% ethyl acetate in hexane. Finally, the compound was
crystallized from water/ethanol.
The capacity factors K were calculated using the Millen-
nium32m Chromatography Manager Software according to the
formula K = (TR –TD)/TD, where TR is the retention time of the sol-
ute, and TD denotes the dead time obtained via an unretained
analyte. Log K, calculated from the capacity factor K, is used as
the lipophilicity index converted to log P scale. Distributive
parameters p have been firmly established as the parameter of
choice for correlating both binding to biological macromole-
cules and transport through a biological system [11]. Constant p
describes the lipophilicity contribution of individual moieties
substituted in the same skeleton. These p parameters character-
izing hydrophobicity of the individual substituents were calcu-
lated according to the formula p = log KS – log KU, where log KS is
the determined capacity-factor logarithm of the individual sub-
stituted compounds, and log KU denotes the determined
capacity-factor logarithm of the unsubstituted compound 3phe-
nylaminopyrazine-2,5-dicarbonitrile.
3-(3-Chlorophenylamino)pyrazine-2,5-dicarbonitrile 5
General coupling procedure was used. Yield: 25%; mp. 197–
1998C; 1H-NMR (DMSO): d 9.52 (s, 1H), 8.68 (s, 1H), 7.65 (s, 1H),
7.51 (d, 1H, J = 7.8 Hz), 7.4 (t, 1H, J = 2.06 Hz), 7.1 (d, 1H, J =
7,6 Hz); 13C-NMR (CDCl3): d 154.05, 147.15, 139.56, 132.13,
130.88, 130.55, 122.46, 121.52, 121.05, 120.88, 115.64, 114.78; IR
(KBr): 3305, 2258, 1662, 1510; LRMS (m/z) 254 [M – 1]. Anal. Calcd
for C12H6ClN5 (255.7): C, 56.37; H, 2.37; N, 27.39. Found: C, 56.4;
H, 2.35; N, 27.4.
3-(3,5-Dibromo-4-hydroxyphenylamino)pyrazine-2,5-
dicarbonitrile 6
General coupling procedure was used. Yield: 40%; mp. 199–
2018C; 1H-NMR (DMSO): d 9.95 (s, 1H), 9.22 (s, 1H), 8.6 (s, 1H), 6.9
(s, 2H); 13C-NMR (DMSO): d 158.54, 150.08, 145.15, 139.17, 130.00,
120.79, 120.70, 115.70, 114.35, 112.95, 110.65, 110.55; IR (KBr)
3510, 3295, 2265, 1655, 1530; LRMS (m/z) 394 [M – 1]. Anal. Calcd
for C12H5Br2N5O (395.01): C, 36.49; H, 1.28; N, 17.73. Found: C,
36.50; H, 1.29; N, 17.71.
Lipophilicity calculations
Log P, i. e. the logarithm of the partition coefficient for n-octa-
nol/water, was calculated using the programs CS ChemOffice
Ultra ver. 7.0 (CambridgeSoft, Cambridge, MA, USA) and ACD/
Log P ver. 1.0 (Advanced Chemistry Development Inc., Toronto,
Canada). Clog P values (the logarithm of n-octanol/water parti-
tion coefficient based on established chemical interactions)
were generated by means of CS ChemOffice Ultra ver. 7.0 (Cam-
bridgeSoft, Cambridge, MA, USA) software.
3-(4-Fluorophenylamino)pyrazine-2,5-dicarbonitrile 7
General coupling procedure was used. Yield: 65%; mp. 182–
1848C; 1H-NMR (DMSO): d 9.25 (s, 1H), 8.25 (s, 1H), 7.35 (dd, 2H, J =
8.2 Hz, J = 2.9 Hz), 7.25 (dd, 2H, J = 8.2 Hz, J = 2.9 Hz); 13C-NMR
(DMSO): d 159.23, 156.15 (d, J = 239.1 Hz), 145.00 (d, J = 2.9 Hz),
140.03, 127.25 (d, J = 7.8 Hz), 122.05, 122.05 (d, J = 7.5 Hz), 115.78,
114.95, 114.74, 114.87 (d, J = 27.9 Hz), 114.28; IR (KBr): 3310,
2245, 1665, 1515; LRMS (m/z) 238 [M – 1]. Anal Calcd for
C12H6FN5 (239.21): C, 60.25; H, 2.53; N, 29.28. Found: C, 60.23; H,
2.52; N, 29.26.
Synthesis
3-Chloro-5-cyanopyrazine-2-carboxamide 3
Pyrazinamide was used as a starting material. The method used
is described elsewhere [16], briefly, pyrazinamide was oxidized
to give pyrazinamide-N-oxide 1a–1c, the oxide was then reduced
to 6-chloropyrazine-2-carbonitrile 2a–2c and further reacted
using formamide and ammonium persulfate to give 3-chloro-5-
cyanopyrazine-2-carboxamide.
3-(2-Hydroxy-4-nitrophenylamino)pyrazine-2,5-
dicarbonitrile 8
To a solution of 6-chloropyrazine-2,5-dicarbonitrile in dimethyl
formamide was added one equivalent of 4-amino-3-nitrophenol
and 1.1 equivalent of pyridine. The reaction was heated to reflux
overnight. Then, the solvent was removed, the reaction mixture
was dissolved in ethyl acetate, and dried over Na2SO4. The sol-
vent was removed and the compound was purified using flash
chromatography with a gradient of elution from 0% to 15% ethyl
acetate in hexane. The pure compound was crystallized from
water. Yield: 58%; mp. 148–1518C; 1H-NMR (DMSO): d 9.82 (s, 1H),
8.25 (s, 2H), 8.14 (dd, 1H, J = 8.3 Hz, J = 2.1 Hz), 7.8 (s, 1H), 7.62 (d,
1H, J = 8.3 Hz); 13C-NMR (DMSO): d 152.23, 150.95, 144.04, 141.02,
139.87, 129.10, 124.63, 120.96, 117.24, 115.07, 114.96, 102.45; IR
(KBr): 3298, 2258, 1660, 1523; LRMS (m/z) 281 [M – 1]; Anal. Calcd
for C12H6N6O3 (282.21): C, 51.07; H, 2.14; N, 29.78. Found: C,
51.08; H, 2.13; N, 29.79.
6-Chloropyrazine-2,5-dicarbonitrile 4
3-Chloro-5-cyano-pyrazine-2-carboxamide was dissolved in phos-
phoroxychloride and heated to 908C. The mixture was kept at
this temperature for one hour, than cooled and poured drop-by-
drop onto ice. Formed crystals were filtered off and the solution
extracted with chloroform. The pure compound was crystallized
from water/ethanol. Yield: 66%; mp. 116–1188C; 1H-NMR
(DMSO): 9.41 (s, 1H); 13C-NMR (DMSO): 153.24, 148.34, 135.65,
133.30, 117.14, 115.67; IR (KBr): 3310, 2260, 1660, 1525; LRMS
(m/z) 165 [M + 1]; Anal. Calcd for C6HClN4 (164.55): C, 43.79; H,
0.61; Cl, 21.55; N, 34.05. Found: C, 43.70; H, 0.55; N, 34.08.
General coupling method (final compound formation)
To a solution of 6-chloropyrazine-2,5-dicarbonitrile in toluene
was added one equivalent of ring-substituted aniline and 1,1
equivalent of pyridine as a base. The reaction mixture was
heated to reflux overnight. Then, the solvent was removed, the
crude compound was dissolved in ethyl acetate and dried over
Na2SO4 The solvent was then removed and the mixture was puri-
3-(2-Mercaptophenylamino)pyrazine-2,5-dicarbonitrile 9
General coupling procedure was used. Yield: 80%; mp. 227–
2308C; 1H-NMR (DMSO): d 9.15 (s, 1H), 8.08 (s, 1H), 7.60 (d, 1H, J =
8.1 Hz), 7.05 (m, 2H), 6.65 (d, 1H, J = 8.2 Hz), 3.50 (s, 1H); 13C-NMR
(DMSO): d 157.08, 149.90, 140.95, 138.18, 130.50, 130.05, 123.15,
121.04, 116.55, 116.05, 115.94, 115.05; IR (KBr): 3355, 3015,
2570, 2262, 1650, 1520; LRMS (m/z) 252 [M – 1]; Anal. Calcd for
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Arch. Pharm. Chem. Life Sci. 2008, 341, 61–65
New Pyrazinamide Analogues as Potential Anti-Infectives
65
C12H7N5S (253.28): C, 56.90; H, 2.79; N, 27.65. Found: C, 56.92; H,
2.80; N, 27.64.
Used strains were: Mycobacterium tuberculosis H37Rv, Mycobacte-
rium kansasii CNCTC My 235/80, and Mycobacterium avium My
CNCTC 80/72. The culture were ten days old and the culture
medium used was ꢂula's medium at pH6.0 and 378C; microdilu-
tion panel method. [14].
The Department of Medical and Biological Sciences at the Fac-
ulty of Pharmacy, Charles University, Prague, performs antifun-
gal susceptibility assays. The method used is microdilution
panel bouillon method with RPMI medium with glutamine as a
growth medium. Incubation takes 28–48 h at atmospheric pres-
sure, temperature 358C, and darkness [15]. Tested strains were:
Candida albicans ATCC 44859, C. tropicalis 156, C. krusei E28, C.
glabrata 20/I, Trichosporon beigelii 1188, Trichophyton mentagro-
phytes 445, Aspergillus fumigantus 231, and Absidia corymbifera
272.
3-{[2-(Trifluoromethyl)phenyl]amino}pyrazine-2,5-
dicarbonitrile 10
General coupling procedure was used. Yield: 83%; mp. 185–
1878C; 1H-NMR (DMSO): d 9.65 (s, 1H), 8.82 (s, 1H), 7.85 (d, 1H, J =
7.9 Hz), 7.5 (d, 1H, J = 7.7 Hz), 7.35 (t, 1H, J = 1.9 Hz) 7.25 (t, 1H, J =
1.7 Hz); 13C-NMR (DMSO): d 165.15, 143.20 (q, J = 22,4 Hz), 141.25,
130.45, 128.05, 126.16 (q, J = 15.2 Hz), 124.62 (q, J = 272.1 Hz),
121.95 (q, J = 6.1 Hz), 118.15 (q, J = 3.0 Hz), 117,55 (q, J = 75.4 Hz),
115.62, 115.24, 112.25; IR (KBr): 3300, 2250, 1660, 1500; LRMS
(m/z) 288 [M – 1]; Anal. Calcd for C12H6F3N5 (289.22): C, 53.99; H,
2.09; N, 24.21. Found: C, 54.00; H, 2.08; N, 24.23.
3-{[3-(Trifluoromethyl)phenyl]amino}pyrazine-2,5-
dicarbonitrile 11
References
General coupling procedure was used. Yield 63%; mp. 185–
1
1888C; H-NMR (CDCl3), d: 9.35 (s, 1H), 8.45 (s, 1H), 7.95 (s, 1H),
7.85 (t, 1H, J = 2.0 Hz), 7.60 (d, 1H, J = 7.9 Hz), 7.25 (d, 1H, J =
7.8 Hz); 13C-NMR (CDCl3), d: 165.44, 152.2 (q, J = 8.5 Hz), 141.95,
130.05, 128.21 (q, J = 68.4 Hz), 126.25 (q, J = 8.7 Hz), 124.50,
122.16 (q, J = 275.2 Hz), 121.05 (q, J = 32 Hz), 118.05, 116.15,
115.00, 114.45; IR (KBr): 3230, 2260,1655, 1510; LRMS (m/z) 288
[M – 1]; Anal. Calcd for C12H6F3N5 (289.22): C, 53.99; H, 2.09; N,
24.21. Found: C, 54.09; H, 2.01; N, 24.28.
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xdr/dir_stb_message_25sep06/en/index.html (12. June
2007).
[2] EU determined to eradicate TB: http://cordis.europa.eu/
fetch?CALLER=EN_NEWS&ACTION=D&SESSION=&RCN=
21787 (12. October 2006).
[3] Treatment of tuberculosis: http://www.cdc.gov/MMWR/
preview/mmwrhtml/rr5211a1.htm (12. June 2007).
3-(2-Cyano-4-nitrophenylamino)pyrazine-2,5-
dicarbonitrile 12
[4] M. Doleˇzal, M. Miletꢃn, J. KuneÐ, K. Krꢀlovꢀ, Molecules
2002, 7, 363–373.
General coupling procedure was used. Yield 42%; mp. 195–
1978C; 1H-NMR (DMSO): d 9.65 (s, 1H), 9.05 (d, 1H, J = 8.4 Hz), 8.25
(s, 1H), 8.15 (s, 1H), 7.65 (d, 1H, J = 8.3 Hz); 13C-NMR (DMSO): d
165.87, 148.25, 142.28, 140.97, 135.64, 129.27, 125.74, 121.07,
118.45, 115.15, 114.19, 112.25, 104.85; IR (KBr) 3225, 2250, 1650,
1505; LRMS (m/z) 290 [M – 1]; Anal. Calcd for C13H5N7O2 (291.22):
C, 53.61; H, 1.73; N, 33.67. Found: C, 53.48; H, 1.76; N, 33.54.
[5] M. Doleˇzal, J. Jampꢃlek, Z. Osicka, J. KuneÐ, V. Buchta, P.
Vꢃchovꢀ, Farmaco 2003, 58, 1105–1111.
[6] M. A. Hassan, S. E. Zayed, W. N. El-Gaziri, S. Metwally,
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[7] M. H. Cynamon, R. J. Speirs, J. T. Welch, Antimicrob. Agents
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[8] K. Dlabal, K. Palat, A. Lycka, Z. Odlerova, Collect. Czech.
Chem. Commun. 1990, 55, 2493–2501.
Biological evaluation
We used two antituberculosis assays and one antifungal assay.
All compounds were screened at the TAACF screening (Tuber-
culosis Antimicrobial Acquisition and Coordinating Facility by
The National Institute of Health of the US government) [12]. This
screening has three levels: To release a compound into a higher
level of assays a successful result in previous level is necessary.
The primary screen is conducted at 6.25 lg/mL (or molar equiva-
lent of the highest molecular-weight compound in a series of
congeners) against Mycobacterium tuberculosis H37Rv (ATCC
27294) in BACTEC 12B medium using the Microplate Alamar
Blue Assay (MABA) [13]. Compounds exhibiting fluorescence are
tested in the BACTEC 460-radiometric system. In general, com-
pounds effecting a90% inhibition in the primary screen (MIC A
6.25 lg/mL) were not further evaluated. Compounds relieved
into level 2 underwent MIC and IC50 determination followed by
Selectivity Index calculation (SI, ratio of measured IC50 to MIC).
To be relieved to level 3 (in-vivo screening) the compound must
exhibit SI>10. None of the prepared structures was relieved to
level 3.
[9] J. Jampꢃlek, M. Doleˇzal, J. KuneÐ, D. ꢂatꢃnsky´, I. Raich,
Curr. Org. Chem. 2005, 9, 49–60.
[10] J. Jampꢃlek, L. Palek, M. Doleˇzal, ECSOC–10, 2006, Novem-
ber 1–30 http://www.usc.es/congresos/ecsoc/10/GOS/
a019/index.htm.
[11] F. E. Norrington, R. M. Hyde, S. G. Williams, R. J. Woot-
ton, Med. Chem. 1975, 18, 604–607.
[12] http://www.taacf.org/about-TAACF.htm (12. June 2007).
[13] L. Collins, S. G. Franzblau, Antimicrob. Agents. Chemother.
1997, 41, 1004–1009.
[14] W. McDermott, R. Tompsett, Am. Rev. Tuberc. 1954, 70,
748–754.
[15] National Committee for Clinical Laboratory Standards.
Reference method for broth dilution antifungal suscepti-
bility testing of yeast: Approved Standard, NCCLS docu-
ment, M27–A; NCCLS: Villanova, PA, USA, 1997.
Another antituberculosis assay was provided by the Faculty
Hospital in Hradec Kralove and the assays were run under differ-
ent conditions (different growth medium, more TBC genuses).
[16] S. Kushner, H. Dalalian, J. L. Sanjurjo, F. L. Bach Jr., et al.,
J. Am. Chem. Soc. 1952, 74, 3617–3621.
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