Molecules 2010, 15, 8567-8581; doi:10.3390/molecules15128567
Synthesis, Antimycobacterial, Antifungal and
Photosynthesis-Inhibiting Activity of Chlorinated
Martin Dolezal 1,*, Jan Zitko 1, Zdenek Osicka 2, Jiri Kunes 1, Marcela Vejsova 1,
Vladimir Buchta 3, Jiri Dohnal 4,5, Josef Jampilek 4,5 and Katarina Kralova 6
1 Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec
Kralove, 500 05, Czech Republic; E-Mails: email@example.com (J.Z.);
firstname.lastname@example.org (J.K.); email@example.com (M.V.)
2 Bioveta a.s., Komenskeho 212, 683 23 Ivanovice na Hane, Czech Republic;
E-Mail: firstname.lastname@example.org (Z.O.)
3 Department of Clinical Microbiology, Faculty of Medicine and University Hospital, Charles
University in Prague, Sokolska 581, Hradec Kralove, 500 05, Czech Republic;
E-Mail: email@example.com (V.B.)
4 Zentiva k.s., U Kabelovny 130, 102 37 Prague 10, Czech Republic; E-Mails: firstname.lastname@example.org
(J.D.); email@example.com (J.J.)
5 Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 61242
Brno, Czech Republic
6 Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska Dolina CH-2,
842 15 Bratislava, Slovak Republic; E-Mail: firstname.lastname@example.org (K.K.)
† Preliminary results were presented at the Fourteenth Electronic Conference on Synthetic Organic
Chemistry (ECSOC-14, http://www.sciforum.net/presentation/380), November 1-30, 2010.
* Author to whom correspondence should be addressed; E-Mail: email@example.com;
Tel.: +420-495-067-389; Fax: +420-495-067-167.
Received: 15 November 2010; in revised form: 22 November 2010 / Accepted: 25 November 2010 /
Published: 26 November 2010
Abstract: A series of sixteen pyrazinamide analogues with the -CONH- linker connecting
the pyrazine and benzene rings was synthesized by the condensation of chlorides of
substituted pyrazinecarboxylic acids with ring-substituted (chlorine) anilines. The prepared
compounds were characterized and evaluated for their antimycobacterial and antifungal
Molecules 2010, 15
activity, and for their ability to inhibit photosynthetic electron transport (PET). 6-Chloro-
N-(4-chlorophenyl)pyrazine-2-carboxamide manifested the highest activity against
Mycobacterium tuberculosis strain H37Rv (65% inhibition at 6.25 μg/mL). The highest
antifungal effect against Trichophyton mentagrophytes, the most susceptible fungal strain
tested, was found for 6-chloro-5-tert-butyl-N-(3,4-dichlorophenyl)pyrazine-2-carboxamide
(MIC = 62.5 μmol/L). 6-Chloro-5-tert-butyl-N-(4-chlorophenyl)pyrazine-2-carboxamide
showed the highest PET inhibition in spinach chloroplasts (Spinacia oleracea L.)
chloroplasts (IC50 = 43.0 μmol/L). For all the compounds, the relationships between the
lipophilicity and the chemical structure of the studied compounds as well as their structure-
activity relationships are discussed.
Keywords: pyrazinecarboxamides; lipophilicity; in vitro antimycobacterial activity; in
vitro antifungal activity; spinach chloroplasts; PET inhibition; structure–activity
Compounds possessing a -CONH- moiety simulating a peptide bond in their molecule show a broad
range of biological effects. Pyrazinamide, with its simple structure, provides a good opportunity for
further modification with a view to increasing its antimycobacterial activity. We have prepared and
studied several series of the pyrazinamide analogues with the -CONH- linker connecting the pyrazine
and benzene rings. All compounds were assayed in vitro against major Mycobacterium and various
fungal species [1-6]. Some compounds were found to exhibit photosynthesis-inhibiting activity
[2,5,7,8]. Various N-substituted amides of pyrazinecarboxylic acid were prepared and evaluated as
potential abiotic elicitors [9-12]. Introducing of halogens (-Cl, -F, -CF3) was the most successful
structural modification. N-(3-Trifluoromethylphenyl)pyrazine-2-carboxamide, 5-tert-butyl-6-chloro-N-
(3-trifluoromethylphenyl)pyrazine-2-carboxamide, and N-(3-iodo-4-methylphenyl)pyrazine-2-carbox-
amide have shown the highest activity against M. tuberculosis H37Rv (MIC = 3.13-6.25 μg/mL) .
This paper describes the preparation, biological evaluation and structure-activity relationship studies of
a series of chlorinated pyrazinamide analogues. We synthesized in preference compounds with
lipophilic and/or electron-withdrawing substituents on the benzene moiety (R3, chlorine), and the
compounds with the substitution on the pyrazine nucleus with R1 (hydrogen, chlorine) and/or R2
(hydrogen, tert-butyl) moiety (see Figure 1).
Many low molecular weight drugs cross biological membranes through passive transport, which
strongly depends on their lipophilicity, which is one of the most important physical properties of
biologically active compounds. It influences the transport of a molecule through cellular membranes,
because drugs cross biological barriers most frequently through passive transport, which strongly
depends on their lipophilicity. Lipophilicity is a property that has a major effect on absorption,
distribution, metabolism, excretion, and toxicity (ADME/Tox) properties as well as pharmacological
activity. Lipophilicity has been studied and applied as an important drug property for decades .
Molecules 2010, 15
The lipophilicity of pyrazinamide is quite low (log P = -1.31/CLogP = -0.67632), therefore in an
effort to increase it we have chosen hydrophobic electron-withdrawing (chlorine), and bulky
substitutents on the pyrazine (tert-butyl), and the combination of substituents (chlorine) on the benzene
part. Distributive π parameters are firmly established as the parameter of choice for correlating both
binding to biological macromolecules and transport through a biological system. The determined π
parameters of substituents can be used for describing relationships between physico-chemical
properties and biological activity of prepared ring-substituted pyrazine-based compounds [14,15]. The
distributive π parameters of individual substituents are listed for the mentioned studied compounds.
Although all the discussed compounds are relatively simple structures substituted within the series
only by chlorine, interesting intramolecular interactions influencing lipophilicity were observed,
probably due to the simultaneous presence of a pyrazine ring and a carboxamide moiety.
Figure 1. Pyrazinamide (red colour) structure modification (black colour).
The aim of this work was to examine the structure–activity relationships (SAR) in the mentioned
series, i.e. to continue in the study of the substituent variability influence on the biological activity, and
to determine the importance of increased lipophilic properties for biological effect of the newly
prepared substituted pyrazinecarboxamides.
2. Results and Discussion
Condensation of the chlorides of pyrazine-2-carboxylic, 6-chloropyrazine-2-carboxylic, 5-tert-
butyl-pyrazine-2-carboxylic or 5-tert-butyl-6-chloropyrazine-2-carboxylic acids with commercially
available ring-substituted anilines yielded a series of 16 substituted amides 1-16 [2,3,5]. All studied
compounds were prepared according to Scheme 1.
Scheme 1. Synthetic pathway and general formula of prepared amides 1-16.
R1 = H, Cl
R2 = H, tert-Butyl
R3 = 3-Cl, 4-Cl, 2,6-Cl, 3,4-Cl
Reagents and conditions: a) SOCl2, toluene, b) acetone, pyridine.
Molecules 2010, 15
Lipophilicity parameters (log P) of the compounds 1-16 were calculated using the commercially
available program ACD/LogP and also measured by means of the RP-HPLC determination of capacity
factors k with subsequent calculation of log k. The procedure was performed under isocratic conditions
with methanol as an organic modifier in the mobile phase using an end-capped non-polar C18
stationary RP column. The results are shown in Table 1 and illustrated in Figure 2.
Table 1. Comparison of the calculated lipophilicity (log P) with the determined log k
values of the discussed pyrazinecarboxamides 1-16, as well as the determined distributive
parameters π calculated from log k.
Comp. R1 R2 R3 log k
H H 3-Cl 0.4914 2.17 ± 0.41 0.00/0.08 0.373
Cl H 3-Cl 0.7864 3.29 ± 0.42 0.30/0.10 0.373
H (CH3)3C 3-Cl 1.0996 3.85 ± 0.41 0.61/0.26 0.373
Cl (CH3)3C 3-Cl 1.4896 4.98 ± 0.43 1.00/0.25 0.373
H H 4-Cl 0.4987 2.13 ± 0.41 0.00/0.09 0.227
Cl H 4-Cl 0.8185 3.25 ± 0.42 0.32/0.13 0.227
H (CH3)3C 4-Cl 1.1043 3.81 ± 0.41 0.61/0.16 0.227
Cl (CH3)3C 4-Cl 1.5015 4.91 ± 0.43 1.00/0.26 0.227
H H 2,6-Cl 0.6656 2.17 ± 0.41 0.00/0.25 0.40
Cl H 2,6-Cl 0.9456 3.29 ± 0.43 0.30/0.28 0.40
H (CH3)3C 2,6-Cl 1.2802 3.85 ± 0.42 0.61/0.34 0.40
Cl (CH3)3C 2,6-Cl 1.6631 4.97 ± 0.44 1.00/0.42 0.40
H H 3,4-Cl 0.6962 3.03 ± 0.42 0.00/0.30 0.60
Cl H 3,4-Cl 0.9950 4.15 ± 0.44 0.28/0.31 0.60
H (CH3)3C 3,4-Cl 1.3395 4.72 ± 0.43 0.62/0.40 0.60
Cl (CH3)3C 3,4-Cl 1.7563 5.84 ± 0.45 1.04/0.51 0.60
Compounds 1, 5, 9 show the lowest lipophilicity, whereas compound 16 possesses the highest
lipophilicity. The calculated log P data and the determined log k parameters correspond to the expected
lipophilicity increasing within individual series of compounds (pyrazine < 6-chloropyrazine < 5-tert-
butylpyrazine < 6-chloro-5-tert-butylpyrazine derivatives). This dependence is approximately linear.
Some significant differences between the experimental values log k and the calculated parameters
log P at compounds 9-12 with substitution in ortho-position (2,6-Cl) were observed. Better correlation
at derivatives with chloro substitution in position 3 or 4 was found. Lipophilicity increases according
to substitution in anilide part of the molecule this way: 3-Cl < 4-Cl < 2,6-Cl < 3,4-Cl. It can be
Molecules 2010, 15
assumed that log k values specify lipophilicity within the individual series of the studied compounds
more precisely than calculated log P data at compounds with the ortho substitution in the benzene part.
Figure 2. Match of the calculated log P data with the experimentally found log k values.
123456789 10 1112 13 1415 16
log klog P [ACD/LogP]
y = 0.3484x - 0.2446
R2 = 0.9297
2.02.5 3.0 3.54.04.5 5.0 5.5 6.0
log P [ACD/LogP]
The distributive parameter π describes the lipophilicity contribution of individual moieties
substituted on some skeleton. The distributive constants π of individual substituents are dependent on
the basic skeleton (aliphatic, aromatic, heteroaromatic), as well as on the character of the
heteroaromatic system. A number of distributive parameters π for various substituents for all three
substituent positions in the benzene ring has been described [14,15]. The determined π parameters of
substituents can be used for describing relationships between the physico-chemical properties and
activity of prepared compounds. Due to similarity of the determined π phenyl parameters for
compounds 1-4 (3-Cl) and 5-8 (4-Cl) it can be predicted that these individual/independent
positions/substitutions do not show any intramolecular interactions between chlorine and the pyrazine
core or carboxamide moiety contrary to disubstituted compounds 13-16 (3,4-Cl), where both chlorine
atoms interact with each other. Results from Table 1 show quite different behaviours of both chlorine
atoms in 9-12 (2,6-Cl).
Molecules 2010, 15
2.3. In vitro antimycobacterial evaluation
All compounds were assayed in vitro against M. tuberculosis H37Rv. In the tuberculosis
antimicrobial acquisition and coordinating facility (TAACF) program  the compounds showing
>90% inhibition in this preliminary screen (i.e. MIC < 6.25 µg/mL) are further evaluated to determine
their actual minimum inhibitory concentration (MIC) in the MABA. None of the tested derivatives
overcame this limit, see Table 2.
Table 2. The antimycobacterial activity (%) of the compounds in comparison with the
standard pyrazinamide (PZA), the in vitro antifungal (IC50) activity of the compounds
against Trichophyton mentagrophytes (determined after 72h/120h) compared with the
fluconazole (FLU) standard and IC50 values of compounds 1-16 related to photosynthetic
electron transport (PET) inhibition in spinach chloroplasts in comparison with the
3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) standard. (MIC = minimum inhibitory
concentration, ND = not determined due to their low solubility in the testing medium).
a The MIC determination for moulds-fungi is determined as IC50 value; b MIC = 12.5 µg/mL .
M. tuberculosis strain H37Rv 65% inhibition at 6.25 μg/mL and 6-chloro-N-(3,4-dichlorophenyl)
pyrazine-2-carboxamide (14) showed 61%. Although their activity did not warrant progression to
phase II screening, medium-active compounds such as 6, 14 should not be ignored, because their
chemical analogues and alterations in physico-chemical properties may confer some positive changes
in biological effects.
(6) possessed activity against
Molecules 2010, 15
With respect to the mostly low-active compounds, only some general structure-activity relationship
(SAR) aspects within this series of these specific substituted compounds can be proposed. There is
considered the positive effect of chlorine atom for both pyrazine (C(6) position) and benzene ring
(especially in C(4) position), and the negative influence of alkyl introduction to the pyrazine nucleus.
Lipophilicity of the compounds has an important role. The above discussed compounds 6 and 14
comply with both πPyr and πPh limits for antimycobacterial activity within this series of the chlorine
substituted N-phenylpyrazine-2-carboxamides 1-16.
2.4. In vitro antifungal susceptibility testing
The evaluation of in vitro antifungal activity of the synthesized compounds was performed against
eight fungal strains, but only moderate activity against Trichophyton mentagrophytes is showed in
Table 2. Generally, all the compounds afforded only slight antifungal activity caused by the low
solubility of compounds in the testing medium and their precipitation during the incubation period,
therefore no thorough structure-activity relationships could be established. More lipophilic
disubstituted compounds (log k < 1) with chlorine atoms especially in positions 3 and 4 on the benzene
part of molecule possessed some weak antifungal activity, and 6-chloro-5-tert-butyl-N-(3,4-dichloro-
phenyl)pyrazine-2-carboxamide (16) exhibited MIC = 62.5 μmol/L (log k = 1.7563) against
T. mentagrophytes, the most susceptible fungal strain tested within the discussed series of the
compound. This activity is only modest in comparison with fluconazole (MIC = 3.91 μmol/L after
120 h, see Table 2).
2.5. Inhibition of photosynthetic electron transport (PET) in spinach chloroplasts
Over 50% of commercially available herbicides act by reversibly binding to photosystem II (PS II),
a membrane-protein complex in the thylakoid membranes which catalyses the oxidation of water and
the reduction of plastoquinone  and thereby inhibit photosynthesis [20-22]. Some organic
compounds, e.g., substituted anilides of 2,6-disubstituted pyridine-4-thiocarboxamides  were found
to interact with tyrosine radicals TyrZ and TyrD which are situated in D1 and D2 proteins on the donor
side of PS II and due to this interaction interruption of the photosynthetic electron transport occurred.
On the other hand, 6-chloro-5-tert-butyl-N-(4-hydroxyphenyl)pyrazine-2carboxamide and 6-chloro-5-
tert-butyl-N-(5-chloro-3-hydroxyphenyl)pyrazine-2carboxamide interacted only with the D+
All the discussed compounds were tested for their photosynthetic electron transport (PET)
inhibition in spinach chloroplasts and they showed some wide-range activity, see Table 2. The IC50
values related to PET inhibition could not be determined for 7, 9, 13 and 15 due to precipitation of the
compounds during the experiments. The activity of the majority of the studied compounds was
moderate or low relative to the standard.
The most effective inhibitor from the series was 6-chloro-5-tert-butyl-N-(4-chlorophenyl)pyrazine-
2-carboxamide (8, IC50 = 43 μmol/L), as measured on photosynthetic electron transport (PET) in
spinach (Spinacia oleracea L.) chloroplasts, see Table 2. It can be again considered the positive effect
of chlorine atom for both pyrazine (C(6) position) and benzene ring (especially in C(4) position), as
discussed above. Influence of tert-butyl moiety introduction on pyrazine demonstrated the positive
Molecules 2010, 15
effect, contrary to negative influence on antimycobacterial activity. It is evident from Tables 1 and 2
that the lipophilicity of the compound is determining for PET inhibition. It seems to be fundamental
that PET inhibition is conditioned by high πPyr parameter (Table 1), which is around 1 (substituted both
by chlorine in the C(6) and by tert-butyl in the C(5) positions of pyrazine). Substitution of pyrazine core
completes an advantageous substitution of benzene ring, especially in the C(3) or C(4) positions.
Disubstitution of both C(3), C(4) positions on benzene increases lipophilicity and at the same time
depresses water-solubility. The sum of both πPyr, πPh of substituents present in each compound can be
de facto considered log k value. When values of PET inhibition with log k are compared, it can be
stated that an increase in lipophilicity to log k ~ 1.50 enhances the effectiveness of PET-inhibiting
activity, but subsequent increasing lipophilicity of the compounds decreases their activity.
Beside lipophilicity parameters, the contribution of electronic properties of phenyl substituents to
PET-inhibiting activity was investigated as well. These properties, expressed as Hammett's σ constants
are described in Table 1 [15,16].
Despite the relatively low inhibitory activity of the studied compounds, the correlations between
log (1/IC50) and lipophilicity characteristics (log k, log P, πPyr, πPh, (πpyr+πPh) or Hammett's constants
(σ) of the R3 substituent were calculated. The importance of compound lipophilicity was for the
inhibitory activity (IC50 in µmol/L) of compounds much more significant (Eqs. 1 and 3) than the
electronic properties of the R3 substituent. Introduction of σ parameter in the correlations did not
improve the results of statistical analysis (Eqs. 2 and 4) indicating that this parameter is not significant
for PET-inhibiting activity:
log (1/IC50) = 0.822 (± 0.225) log k + 2.808 (± 0.267)
r = 0.756, s = 0.321, F =13.31, n = 12 (1)
log (1/IC50) = 0.807 (± 0.254) log k + 0.137 (± 0.883) σ + 2.771 (± 0.363)
r = 0.756, s = 0.338, F =6.02, n = 12 (2)
log (1/IC50) = 0.314 (± 0.083) log P + 2.500 (± 0.334)
r = 0.769, s = 0.314, F =14.45, n = 12 (3)
log (1/IC50) = 3.254 (± 0.098) log P – 0.236 (± 0.903) σ + 2.544 (± 0.390)
r = 0.771, s = 0.330, F =6.59, n = 12 (4)
Similarly, correlations between PET-inhibiting activity and distributive lipophilicity parameters
πPyr, πPh as well as their sum [πpyr+πphenyl] (Eqs. 5-7) were performed:
log (1/IC50) = 0.920 (± 0.237) πpyr + 3.225 (± 0.156)
r = 0.775, s = 0.310, F = 15.06, n = 12 (5)
log (1/IC50) = 1.954 (± 0.904) πPh + 3.227 (± 0.257)
r = 0.564, s = 0.405, F = 4.67, n = 12 (6)
log (1/IC50) = 0.606 (± 0.223) (πPyr+πPh) + 3.291 (± 0.191)
r = 0.652, s = 0.372, F = 7.39, n = 12 (7)
From the results it is evident that for PET-inhibiting activity predominantly the lipophilicity of
substituents on the pyrazine ring (R1 and R2) is determinant. Lower values of correlation coefficients
Molecules 2010, 15
could be affected by relatively low inhibitory activity of the studied compound as well as with
decreased aqueous solubility of more lipophilic compounds.
All organic solvents used for the synthesis were of analytical 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 (Merck UV 254 TLC plates, Darmstadt, Germany) using developing solvents
hexane/ethyl acetate (9:1). Compounds were purified using a Flash Master Personal Chromatography
System (Argonaut Technologies, Redwood City, CA, USA), with hexane/ethyl acetate (9:1) as solvent
and Kieselgel 60, 0.040-0.063 mm (Merck, Darmstadt, Germany) as the column sorbent. The melting
points were determined using a Melting Point Apparatus SMP 3 (BIBBY Stuart Scientific, UK) and
are uncorrected. Elemental analyses were performed on an automatic microanalyser CHNS-O CE
instrument (FISONS EA 1110, Milano, Italy). Infrared spectra were recorded on a Nicolet™ Impact
400 FT-IR Spectrometer (Thermo Scientific, USA) in KBr pellets. All 1H- and 13C-NMR Spectra were
recorded on a Varian Mercury – Vx BB 300 (300 MHz for 1H and 75 MHz for 13C), Varian (Palo Alto
CA, USA) in CDCl3 solutions at ambient temperature. Chemical shifts are reported in ppm (δ) using
internal Si(CH3)4 as the reference, with diffuse, easily exchangeable signals being omitted.
3.2.1. General procedure for the synthesis of compounds 1-16
A mixture of acid, i.e., pyrazinecarboxylic, 6-chloropyrazine-2-carboxylic , 5-tert-butylpyrazine-
2-carboxylic  or 5-tert-butyl-6-chloropyrazine-2-carboxylic  acid, respectively, (50.0 mmol) and
thionyl chloride (5.5 mL, 75.0 mmol) in dry toluene (20 mL) was refluxed for about 1 h. 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 dropwise to a stirred solution of the
corresponding substituted amine (50.0 mmol) and pyridine (50.0 mmol) in 50 mL of dry acetone
keeping at the room temperature. After the addition was complete, stirring continued for the next
30 min. Then the reaction mixture was poured into 100 mL of cold water and the crude amide was
collected and purified by the column chromatography. The studied compounds 1-16 are presented in
the Table 1. The synthesis, physico-chemical data and analytical parameters of several of these
compounds were described elsewhere (derivatives 5-8  and 13-16 ).
Pyrazine-2-carboxylic acid (3-chlorophenyl)amide (1). Yield: 73%; m.p. 139.0-140.0 °C; Anal. Calcd.
for C11H8ClN3O (233.7): 56.54% C, 3.45% H, 17.98% N; found: 56.53% C, 3.51% H, 18.03% N; IR
(cm-1): 3435 (N-H), 1673 (C=O); 1H-NMR δ: 9.68 (bs, 1H, NH), 9.50 (s, 1H, H3), 8.83 (d, 1H,
J = 2.19 Hz, H6), 8.62-8.57 (m, 1H, H5), 7.92-7.86 (m, 1H, H2´), 7.65-7.56 (m, 1H, H6´), 7.31 (t, 1H,
J = 1.97 Hz, H5´), 7.18-7.11 (m, 1H, H4´); 13C-NMR δ: 160.7, 147.7, 144.7, 144.0, 142.4, 138.3,
134.8, 130.2, 124.9, 119.9, 117.7.
Molecules 2010, 15
6-Chloropyrazine-2-carboxylic acid (3-chlorophenyl)amide (2). Yield: 91%; m.p. 107.0-108.0 °C;
Anal. Calcd. for C11H7Cl2N3O (268.1): 49.28% C, 2.63% H, 15.67% N; found: 49.33% C, 2.61% H,
15.63% N; IR (cm-1): 3435 (N-H), 1676 (C=O); 1H-NMR δ: 9.44-9.35 (m, 2H, NH, H3), 8.82 (s, 1H,
H5), 7.88 (t, 1H, J = 1.93 Hz, H2´), 7.60 (ddd, 1H, J = 7.97 Hz, J = 1.93 Hz, J = 0.83 Hz, H6´), 7.32 (t,
1H, J = 7.96 Hz, H5´), 7.17 (ddd, 1H, J = 7.97 Hz, J = 1.92 Hz, J = 0.82 Hz, H4´). 13C-NMR δ: 159.4,
147.8, 147.5, 143.6, 142.2, 137.9, 134.9, 130.2, 125.2, 120.1, 118.0.
5-tert-Butylpyrazine-2-carboxylic acid (3-chlorophenyl)amide (3). Yield: 83%; m.p. 117.0-118.0 °C;
Anal. Calcd. for C15H16ClN3O (289.8): 62.18% C, 5.57% H, 14,50% N; found: 62.15% C, 5.51% H,
14.59% N; IR (cm-1): 3440 (N-H), 1685 (C=O);1H-NMR δ: 9.67 (bs, 1H, NH), 9.38 (d, 1H,
J = 1.37 Hz, H3), 8.62 (d, 1H, J = 1.37 Hz, H6), 7.89 (t, 1H, J = 2.07 Hz, H2´), 7.59 (ddd, 1H,
J = 7.96 Hz, J = 2.07 Hz, J = 1.10 Hz, H4´), 7.30 (t, 1H, J = 7.96 Hz, H5´), 7.13 (ddd, 1H, J = 7.96 Hz,
J = 2.07 Hz, J = 1.10 Hz, H6´), 1.45 (s, 9H, CH3); 13C-NMR δ: 168.0, 161.1, 143.0, 141.0, 139.0,
138.5, 134.8, 130.1, 124.6, 120.45, 119.8, 117.6, 64.29, 37.1, 29.7.
m.p. 86.0-87.0 °C; Anal. Calcd. for C15H15Cl2N3O (324.2): 55.57% C, 4.66% H, 12.96% N; found:
55.45% C, 4.63% H, 13.08% N; IR (KBr, cm-1): 3432 (N-H), 1678 (C=O); 1H-NMR δ: 9.39 (bs, 1H,
NH), 9.26 (s, 1H, H3), 7.88 (t, 1H, J = 2.07 Hz, H2´), 7.60 (ddd, 1H, J = 7.97 Hz, J = 2.07 Hz,
J = 1.10 Hz, H6´), 7.31 (t, 1H, J = 7.97 Hz, H5´), 7.15 (ddd, 1H, J = 7.97 Hz, J = 2.07 Hz, J = 1.10 Hz,
H4´), 1.55 (s, 9H, CH3); 13C-NMR δ: 164.9, 159.9, 145.8, 140.7, 140.3, 138.2, 134.8, 130.1, 125.0,
120.0, 117.9, 116.79, 64.07, 39.0, 28.20.
N-(2,6-dichlorophenyl)pyrazine-2-carboxamide (9). Yield 66%; m.p. 151.0-152.0 °C; Anal. Calcd. for
C11H7Cl2N3O (268.1): 49.28% C, 2.63% H, 15.67% N; found: 49.51% C, 2.68% H, 15.21% N; IR
(cm-1): 3377 (NH), 1685 (CO). 1H-NMR δ: 9.51 (bs, 1H, NH), 9.41 (s, 1H, H3), 8.85 (s, 1H, H5), 8.75
(s, 1H, H6), 7.10-7.61 (m, 3H, H3´, H4´, H5´); 13C-NMR δ: 160.8, 147.8, 144.8, 142.7, 134.2, 133.5,
132.4, 129.8, 128.8, 128.5, 123.5.
6-Chloro-N-(2,6-dichlorophenyl)pyrazine-2-carboxamide (10). Yield 78%; m.p. 178.0-179.2 °C; Anal.
Calcd. for C11H6Cl3N3O (302.6): 43.67% C, 2.00% H, 13.89% N; found: 43.51% C, 1.98% H, 13.91%
N; IR (cm-1): 3370 (NH), 1690 (CO); 1H-NMR δ: 9.41 (bs, 1H, NH), 9.38 (s, 1H, H3), 8.83 (s, 1H,
H5), 7.12-7.52 (m, 3H, H3´, H4´, H5´); 13C-NMR δ: 159.3, 147.8, 147.4, 143.2, 142.1, 136.1, 132.9,
130.7, 130.6, 128.3, 121.5.
5-tert-Butyl-N-(2,6-dichlorophenyl)pyrazine-2-carboxamide (11). Yield 43%; m.p. 53.5-55.0 °C. Anal.
Calcd. for C15H15Cl2N3O (324.2): 55.57% C, 4.66% H, 12.69% N; found: 55.63% C, 4.71% H, 13.08%
N; IR (cm-1): 3365 (NH), 1685 (CO); 1H-NMR δ: 9.67 (bs, 1H, NH), 9.37 (d, 1H, J = 1.37 Hz, H3),
8.61 (d, 1H, J = 1.37 Hz, H6), 7.12-7.48 (m, 3H, H3´, H4´, H5´), 1.45 (s, 9H, CH3); 13C-NMR δ:
168.2, 161.2, 143.2, 143.0, 142.1, 140.7, 139.0, 136.9, 133.0, 130.6, 127.7, 121.3, 118.9, 37.1, 29.7.
(3-chlorophenyl)amide (4). Yield: 97%;
Molecules 2010, 15
130.1-131.0 °C; Anal. Calcd. for C15H14Cl3N3O (358.7): 50.23% C, 3.93% H, 11.72% N; found:
50.33% C, 3.71% H, 12.08% N; IR (cm-1): 3390 (NH), 1685 (CO); 1H-NMR δ: 9.38 (bs, 1H, NH),
9.25 (s, 1H, H3), 7.12-7.48 (m, 3H, H3´, H4´, H5´), 1.55 (s, 9H, CH3); 13C-NMR δ: 165.1, 159.9,
145.8, 143.2, 142.1, 140.5, 140.3, 136.5, 133.0, 130.7, 128.2, 121.6, 119.1, 39.1, 28.2.
(12). Yield 77%; m.p.
3.3. Lipophilicity determination by HPLC (capacity factor k/calculated log k)
Waters Alliance 2695 XE HPLC separation module and Waters Photodiode Array Detector 2996
(Waters Corp., Milford, MA, USA) were used. Waters Symmetry® C18 5 μm, 4.6 × 250 mm, Part No.
WAT054275 (Waters Corp., Milford, MA, USA) chromatographic column was used. The HPLC
separation process was monitored by Empower™ 2 Chromatography Data Software, Waters 2009
(Waters Corp., Milford, MA, USA). The mixture of MeOH (HPLC grade, 70%) and H2O
(HPLC–Mili-Q Grade, 30%) was used as a mobile phase. The total flow rate of the column was 1.0
mL/min, injection volume 30 μL, column temperature 30 °C and sample temperature 10 °C were used.
The detection wavelength of 210 nm was chosen. The KI methanolic solution was used for the dead
time (tD) determination. Retention times (tR) were measured in minutes.
The capacity factors k were calculated using the Empower™ 2 Chromatography Data Software
according to formula k = (tR - tD)/tD, where tR is the retention time of the solute, whereas tD denotes the
dead time obtained using an unretained analyte. Log k, calculated from the capacity factor k, is used as
the lipophilicity index converted to log P scale. The log k values of the individual compounds are
shown in Table 1.
Distributive π parameters characterizing lipophilicity of the individual substituents were calculated
according to the formula π = log kS – log kU, where log kS is the determined capacity factor logarithm
of the individual substituted compounds, whereas log kU denotes the determined capacity factor
logarithm of the unsubstituted compound, it means π = 0. The determined pyrazine parameters πPyr of
compounds 1, 5, 9 and 13 can be used as πPyr = 0. The determined π parameters of
pyrazinecarboxamides with unsubstituted aniline (πH = 0.4119, π6-Cl = 0.6884, πt-Bu = 0.9439, π6-Cl+t-
Bu = 1.2432) were used as πPh reference values. The distributive π parameters of the individual
compounds are shown in Table 1.
3.4. Lipophilicity calculations
Log P, i.e., the logarithm of the partition coefficient for n-octanol/water, was calculated using the
program ACD/LogP ver. 1.0 (Advanced Chemistry Development Inc., Toronto, Canada). The results
are shown in Table 1.
3.5. In vitro antimycobacterial screening
Antimycobacterial evaluation was carried out in the tuberculosis 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 µg/mL against M. tuberculosis H37Rv (ATCC27294) in BACTEC 12B medium using both
Molecules 2010, 15
BACTEC 460 radiometric system and the Microplate Alamar Blue Assay (MABA) [17,24]. For the
results see Table 2.
3.6. In vitro antifungal susceptibility testing
The Department of Medical and Biological Sciences at the Faculty of Pharmacy in Hradec Králové,
Charles University in Prague, Czech Republic, performed the antifungal susceptibility assays. The
method used was the microdilution panel broth method with RPMI medium containing glutamine as a
growth medium. Tested strains: Candida albicans ATCC 44859, C. tropicalis 156, C. krusei E28,
C. glabrata 20/I, Trichosporon asahii 1188, Trichophyton mentagrophytes 445, Aspergillus fumigatus
231 and Absidia corymbifera 272. The values of the minimum inhibitory concentration (MICs) were
determined after 24 and 48 h of static incubation at 35 °C and darkness . For T. mentagrophytes,
the final MICs were determined after 72 and 120 h of incubation. For the results of the most sensitive
fungal strain T. mentagrophytes, see Table 2.
3.7. Study of inhibition photosynthetic electron transport (PET) in spinach chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovičová and
Kráľová . The inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was
determined spectrophotometrically (Genesys 6, Thermo Scientific, USA), using an artificial electron
acceptor 2,6-dichlorophenol-indophenol (DCIPP) according to Kráľová et al. , 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 chlorophyll content was 30 mg/L in these experiments and
the samples were irradiated (~100 W/m2) from 10 cm distance with a halogen lamp (250 W) using
a 4 cm water filter to prevent warming of the samples (suspension temperature 22 °C). The studied
compounds were dissolved in DMSO due to their limited water solubility. The applied DMSO
concentration (up to 4%) practically did not affect the photochemical activity in spinach chloroplasts
(observed differences in DCPIP photoreduction due DMSO addition were within experimental error).
The inhibitory efficiency of the studied compounds was manifested by IC50 values, i.e. by molar
concentration of the compounds causing 50% decrease in the oxygen evolution rate relative to the
untreated control. The comparable IC50 value for a selective herbicide 3-(3,4-dichlorophenyl)-1,1-
dimethylurea, DCMU (Diurone®) was about 1.9 μmol/L . The results are summarized in Table 2.
A series of sixteen ring-substituted N-phenylpyrazine-2-carboxamides were prepared by
condensation of the corresponding chlorides of some substituted pyrazinecarboxylic acids
(pyrazinecarboxylic acid, 6-chloropyrazine-2-carboxylic acid, 5-tert-butylpyrazine-2-carboxylic acid
or 5-tert-butyl-6-chloropyrazine-2-carboxylic acid) with ring-substituted (chlorinated) anilines. The
synthesis, analytical and spectroscopic data of newly prepared compounds are presented. Lipophilicity
of the compounds was determined using a well characterized RP-HPLC method. The prepared
compounds were tested for their ability to inhibit photosynthetic electron transport (PET) in spinach
Molecules 2010, 15
chloroplasts (Spinacia oleracea L.) and for their antifungal and antimycobacterial activity.
6-Chloro-N-(4-chlorophenyl)pyrazine-2-carboxamide (6) showed the highest activity against
M. tuberculosis strain H37Rv (65% inhibition at 6.25 μg/mL). The highest antifungal effect
(MIC = 62.5 μmol/L) against Trichophyton mentagrophytes was found for 6-chloro-5-tert-butyl-N-
(3,4-dichlorophenyl)pyrazine-2-carboxamide (16). 6-Chloro-5-tert-butyl-N-(4-chlorophenyl) pyrazine-
2-carboxamide (8) was the most active in the inhibition of photosynthetic electron transport (PET) in
spinach (Spinacia oleracea L.) chloroplasts (IC50 = 43.0 μmol/L). The relationships between the
lipophilicity and the chemical structure of the studied compounds as well as structure-activity
relationships between the chemical structures and the antimycobacterial, antifungal and
photosynthesis-inhibiting activities of the evaluated compounds are briefly discussed. The results of in
vitro antimycobacterial and antifungal screening indicated the significance of lipophilicity of
compounds. Correlations between PET-inhibiting activity and lipophilicity characteristics (log k, log
P, π) of the compounds and Hammett's constants (σ) of the substituents on phenyl ring were
performed. For the PET-inhibiting activity, the importance of compound lipophilicity was more
significant than the electronic properties of the substituents expressed by σ values. Predominantly, the
lipophilicity of substituents on the pyrazine was determinant.
This study was supported by the Ministry of Education of the Czech Republic (MSM0021620822),
by the Ministry of Health of the Czech Republic (IGA NS 10367-3), by the Grant Agency of Charles
University in Prague (B CH/120509), and by Sanofi-Aventis Pharma Slovakia. Antimycobacterial data
were provided by the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF)
through a research and development contract with the U.S. National Institute of Allergy and Infectious
Diseases. We take this opportunity to convey our sincere thanks to Dr. Joseph Maddry, the co-
ordinator of the TAACF project.
1. Dlabal, K.; Doležal, M.; Macháček, M. Preparation of some 6-substituted N-pyrazinyl-2-
pyrazinecarboxamides. Collect. Czech. Chem. Commun. 1993, 58, 452–454.
2. Doležal, M.; Vičík, R.; Miletín, M.; Kráľová, K. Synthesis and antimycobacterial, antifungal, and
photosynthesis-inhibiting evaluation of some anilides of substituted pyrazine-2-carboxylic acids.
Chem. Pap. 2000, 54, 245–248.
3. Doležal, M.; Miletín, M.; Kuneš, J.; Kráľová, K. Substituted amides of pyrazine-2-carboxylic
acids, their synthesis and biological activity. Molecules 2002, 7, 363–373.
4. Doležal, M.; Palek, L.; Vinšová, J.; Buchta, V.; Jampílek, J.; Kráľová, K. Substituted
pyrazinecarboxamides: Synthesis and their biological evaluation. Molecules 2006, 11, 242–256.
5. Doležal, M.; Čmedlová, P.; Palek, L.; Vinšová, J.; Kuneš, J.; Buchta, V.; Jampílek, J.; Kráľová,
K. Synthesis and biological evaluation of pyrazinecarboxamides. Eur. J. Med. Chem. 2008, 43,
Molecules 2010, 15
6. Doležal, M.; Zitko, J.; Kešetovičová, D.; Kuneš, J.; Svobodová, M. Substituted
N-phenylpyrazine-2-carboxamides: Synthesis and antimycobacterial evaluation. Molecules 2009,
7. Doležal, M.; Hartl, J.; Miletín, M.; Macháček, M.; Kráľová, K. Synthesis and photosynthesis-
inhibiting activity of some anilides of substituted pyrazine-2-carboxylic acids. Chem. Pap. 1999,
8. Doležal, M.; Kráľová, K.; Šeršeň, F.; Miletín, M. The site of action of some anilides of pyrazine-
2-carboxylic acids in the photosynthetic apparatus. Folia Pharm. Univ. Carol. 2001, 26, 13–20.
9. Tůmová, L.; Gallová, K.; Řimáková, J.; Doležal, M.; Tůma, J. The effect of substituted amides of
pyrazine-2-carboxylic acids on flavonolignan production in Silybum marianum culture in vitro.
Acta Physiol. Plant. 2005, 27, 357–362.
10. Doležal, M.; Tůmová, L.; Kešetovičová, D.; Tůma, J.; Kráľová, K. Substituted N-phenylpyrazine-
2-carboxamides, their synthesis and evaluation as herbicides and abiotic elicitors. Molecules
2007, 12, 2589–2598.
11. Tůmová, L.; Tůma, J.; Megušar, K.; Doležal, M. Substituted pyrazinecarboxamides as abiotic
elicitors of flavolignan production in Silybum marianum (L.) gaertn cultures in vitro. Molecules
2010, 15, 331–340.
12. Stancheva, I.; Georgiev, G.; Geneva, M.; Ivanova, A; Doležal, M.; Tůmová, L. Influence of foliar
fertilization and growth effector 5-tert-butyl-N-m-tolylpyrazine-2-carboxamide (MD 148/II) on
the milk thistle (Silybum marianum L.) seed yield and quality. J. Plant Nutr. 2010, 33, 818–830.
13. Kerns, E.H.; Li, D. Drug-like Properties: Concept, Structure Design and Methods; Elsevier: San
Diego, CA, USA, 2008; pp. 122–136.
14. Hansch, C.; Leo, A.; Unger, S.H.; Nikaitani, D.; Lien, E.J. Aromatic substituent constant for
structure-activity correlations. J. Med. Chem. 1973, 16, 1207–1216.
15. Norrington, F.E.; Hyde, R.M.; Williams, S.G.; Wotton, R. Physicochemical-activity relations in
practice. 1. Rational and self-consistent data bank. J. Med. Chem. 1975, 18, 604–607.
16. Fujita, T.; Nishioka, T. The analysis of the ortho effect. Prog. Phys. Org. Chem. 1976, 12, 49–89.
17. TAACF. Global discovery program for novel anti-tuberculosis drugs. http://www.taacf.org/about-
TAACF.htm /, (accessed on 10 October 2010).
18. Doležal, M.; Jampílek, J.; Osička, Z.; Kuneš, J.; Buchta, V.; Víchová, P. Substituted
5-aroylpyrazine-2-carboxylic acid derivatives: Synthesis and biological activity. Farmaco 2003,
19. Kráľová, K.; Šeršeň, F.; Miletín, M.; Doležal, M. Inhibition of photosynthetic electron transport
in spinach chloroplasts by 2,6-disubstituted pyridine-4-thiocarboxamides. Chem. Pap. 2002, 56,
20. Draber, W.; Tietjen, K.; Kluth, J.F.; Trebst, A. Herbicides in photosynthesis research. Angew.
Chem. 1991, 3, 1621–1633.
21. Tischer, W.; Strotmann, H. Relationship between inhibitor binding by chloroplasts and inhibition
of photosynthetic electron-transport. Biochim. Biophys. Acta 1977, 460, 113–125.
22. Trebst, A.; Draber, W. Structure activity correlations of recent herbicides in photosynthetic
reactions. In Advances in Pesticide Science; Greissbuehler, H., Ed.; Pergamon Press: Oxford, UK,
1979; pp. 223–234.
Molecules 2010, 15 Download full-text
23. Bowyer, J.R.; Camilleri, P.; Vermaas, W.F.J. Herbicides, Topics in Photosynthesis; Baker, N.R.,
Percival, M.P., Eds.; Elsevier: Amsterdam, The Netherlands, 1991; Volume 10, pp. 27–85.
24. Collins, L.A.; Franzblau, S.G. Microplate alamar blue assay versus BACTEC 460 system for
high-throughput screening of compounds
Mycobacterium avium. Antimicrob. Agents Chemother. 1997, 41, 1004–1009.
25. National Committee for Clinical Laboratory Standards. Method for Antifungal Disk Diffusion
Susceptibility Testing of Yeasts: Approved Guideline M44-A; National Committee for Clinical
Laboratory Standards: Wayne, PA, USA, 2004.
26. Masarovičová, E.; Kráľová, K. Approaches to measuring plant photosynthesis activity. In
Handbook of Photosynthesis, 2nd ed.; Pessarakli, M., Ed.; Taylor & Francis Group: Boca Raton,
FL, USA, 2005; pp. 617–656.
27. Kráľová, K.; Šeršeň, F.; Sidóová, E. Photosynthesis inhibition produced by 2-alkylthio-6-R-
benzothiazoles. Chem. Pap. 1992, 46, 348–350.
28. Fedke, C. Biochemistry and Physiology of Herbicide Action; Springer Verlag: New York, NY,
Sample Availability: Samples of the compounds are available from the authors.
Mycobacterium tuberculosis and
© 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license