Synthesis, antimycobacterial activity and in vitro cytotoxicity of 5-chloro-N-phenylpyrazine-2-carboxamides

Article abstract of DOI:10.3390/molecules181214807

5-Chloropyrazinamide (5-Cl-PZA) is an inhibitor of mycobacterial fatty acid synthase I with a broad spectrum of antimycobacterial activity in vitro. Some N-phenylpyrazine-2-carboxamides with different substituents on both the pyrazine and phenyl core possess significant in vitro activity against Mycobacterium tuberculosis. To test the activity of structures combining both the 5-Cl-PZA and anilide motifs a series of thirty 5-chloro-N-phenylpyrazine-2-carboxamides with various substituents R on the phenyl ring were synthesized and screened against M. tuberculosis H37Rv, M. kansasii and two strains of M. avium. Most of the compounds exerted activity against M. tuberculosis H37Rv in the range of MIC = 1.56-6.25 μg/mL and only three derivatives were inactive. The phenyl part of the molecule tolerated many different substituents while maintaining the activity. In vitro cytotoxicity was decreased in compounds with hydroxyl substituents, preferably combined with other hydrophilic substituents. 5-Chloro-N-(5- chloro-2-hydroxyphenyl)pyrazine-2-carboxamide (21) inhibited all of the tested strains (MIC = 1.56 μg/mL for M. tuberculosis; 12.5 μg/mL for other strains). 4-(5-Chloropyrazine-2- carboxamido)-2-hydroxybenzoic acid (30) preserved good activity (MIC = 3.13 μg/mL M. tuberculosis) and was rated as non-toxic in two in vitro models (Chinese hamster ovary and renal cell adenocarcinoma cell lines; SI = 47 and 35, respectively).

Full text of DOI:10.3390/molecules181214807

Molecules 2013, 18, 14807-14825; doi:10.3390/molecules181214807
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Antimycobacterial Activity and In Vitro Cytotoxicity
of 5-Chloro-N-phenylpyrazine-2-carboxamides
Jan Zitko 1,*, Barbora Servusová 1, Pavla Paterová 2, Jana Mandíková 1, Vladimír Kubíček 1,
Radim Kučera 1, Veronika Hrabcová 3,4, Jiří Kuneš 1, Ondřej Soukup 3 and Martin Doležal 1
1
Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Heyrovského 1203,
Hradec Králové 500 05, Czech Republic; E-Mails: barbora.servusova@faf.cuni.cz (B.S.);
jana.mandikova@faf.cuni.cz (J.M.); kubicek@faf.cuni.cz (V.K.); kucera@faf.cuni.cz (R.K.);
kunes@faf.cuni.cz (J.K.); dolezalm@faf.cuni.cz (M.D.)
2
Department of Clinical Microbiology, University Hospital, Sokolská 581, Hradec Králové 500 05,
Czech Republic; E-Mail: pavla.paterova@fnhk.cz
3
Biomedical Research Center, Sokolská 581, Hradec Králové 500 05, Czech Republic;
E-Mails: veronika.hrabcova@fnhk.cz (V.H.); ondrej.soukup@fnhk.cz (O.S.)
4
Department of Chemistry, Faculty of Science, University of Hradec Králové, Jana Koziny 1237,
Hradec Králové 500 05, Czech Republic
* Author to whom correspondence should be addressed; E-Mail: jan.zitko@faf.cuni.cz;
Tel.: +420-495-067-272; Fax: +420-495-512-423.
Received: 15 November 2013; in revised form: 26 November 2013 / Accepted: 26 November 2013 /
Published: 2 December 2013
Abstract: 5-Chloropyrazinamide (5-Cl-PZA) is an inhibitor of mycobacterial fatty acid
synthase I with a broad spectrum of antimycobacterial activity in vitro. Some
N-phenylpyrazine-2-carboxamides with different substituents on both the pyrazine and
phenyl core possess significant in vitro activity against Mycobacterium tuberculosis. To
test the activity of structures combining both the 5-Cl-PZA and anilide motifs a series of
thirty 5-chloro-N-phenylpyrazine-2-carboxamides with various substituents R on the
phenyl ring were synthesized and screened against M. tuberculosis H37Rv, M. kansasii and
two strains of M. avium. Most of the compounds exerted activity against M. tuberculosis
H37Rv in the range of MIC = 1.56–6.25 µg/mL and only three derivatives were inactive.
The phenyl part of the molecule tolerated many different substituents while maintaining
the activity. In vitro cytotoxicity was decreased in compounds with hydroxyl
substituents, preferably combined with other hydrophilic substituents. 5-Chloro-N-(5-
chloro-2-hydroxyphenyl)pyrazine-2-carboxamide (21) inhibited all of the tested strains

Molecules 2013, 18
14808
(MIC = 1.56 µg/mL for M. tuberculosis; 12.5 µg/mL for other strains). 4-(5-Chloropyrazine-2-
carboxamido)-2-hydroxybenzoic acid (30) preserved good activity (MIC = 3.13 µg/mL
M. tuberculosis) and was rated as non-toxic in two in vitro models (Chinese hamster ovary
and renal cell adenocarcinoma cell lines; SI = 47 and 35, respectively).
Keywords: pyrazinamide; 5-chloropyrazinamide; anilides; antimycobacterial activity;
cytotoxicity
1. Introduction
Although both relative and absolute incidence of tuberculosis (TB) have been decreasing globally
since approximately the beginning of the millennium, tuberculosis remains a serious threat to public
health and is the second leading cause of death from infectious diseases. According to the WHO
Global Tuberculosis Report 2013 estimates 8.6 million of people developed active form of TB in 2012
and 1.3 million died from the disease (including 320,000 deaths among HIV-positive people) [1].
Besides the HIV co-infection, TB control policy is endangered mainly by increasing resistance to
current clinically used antitubercular drugs. In 2012, there were 450,000 new cases of multidrug
resistant tuberculosis (MDR-TB) and 170,000 deaths from MDR-TB [1]. Therefore, there is still a
need for the development of new antitubercular medicines, especially those active against resistant
strains of mycobacteria.
5-Chloropyrazine-2-carboxamide (5-Cl-PZA) was previously shown to possess in vitro
antimycobacterial activity, not only against M. tuberculosis (M. tbc), but also against pyrazinamide
(PZA)-resistant strains and against atypical mycobacteria naturally resistant to PZA—M. kansasii,
M. smegmatis, M. fortuitum, M. avium [2]. The Fatty Acid Synthase I (FAS I) system of mycobacteria
was proposed to be the possible target of 5-Cl-PZA based on the observation that overexpression of
fas1 gene in M. smegmatis conferred resistance to 5-Cl-PZA [3]. Indeed, later studies confirmed that
5-Cl-PZA inhibited the FAS I in both whole cell [4] and isolated enzyme [4,5] assays. Sayahi et al.
showed by Saturation Difference Transfer NMR experiments that 5-Cl-PZA binds competitively to the
NADPH binding site of FAS I and that the affinity of 5-Cl-PZA is superior to that of non-substituted
PZA [6]. Little is known about the possible in vivo activity of 5-Cl-PZA. A recent study using a
chronic murine TB-model failed to confirm the in vivo activity of 5-Cl-PZA [7]. The possible
explanation could be its in vivo metabolic instability [8] and/or poor pharmacokinetics. If so, these
issues might be solved by proper structural modifications of 5-Cl-PZA.
Some N-phenylpyrazine-2-carboxamides, i.e., anilides of pyrazine-2-carboxylic acid (POA), with
various substituents both on the pyrazine and phenyl core were previously described to possess
significant antimycobacterial activity [9,10]. The published compounds were anilides of non-substituted
pyrazine-2-carboxylic acid, 6-chloro-POA, 5-tert-butyl-POA; and 5-tert-butyl-6-chloro-POA. Based
on the facts describe above, we decided to synthesize and probe the potential antimycobacterial activity
of 5-chloro-N-phenylpyrazine-2-carboxamides, i.e., compounds combining the 5-Cl-PZA motif
possessing FAS I inhibiting activity and the anilide motif. The preliminary data of first six compounds
from this series were published recently [11] and showed a significant level of antimycobacterial

Molecules 2013, 18
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activity in low micromolar concentration against M. tuberculosis H37Rv. This paper presents the
extended study of 5-chloro-N-phenylpyrazine-2-carboxamides, their antimycobacterial activity against
four different mycobacterial strains and in vitro cytotoxicity of the compounds with significant
activity. The basic insights into the structure-activity and structure-toxicity relationships of these
compounds is presented too.
2. Results and Discussion
2.1. Chemistry
The title 5-chloro-N-phenylpyrazine-2-carboxamide compounds 1–30 were synthesized from
commercially available 5-hydroxypyrazine-2-carobxylic acid (5-OH-POA). The formation of the
corresponding acyl chloride was simultaneously accompanied with the substitution of 5-OH group
with chlorine. For reagents and conditions see Scheme 1. The overall yields of the two-step reaction
ranged from 18% to 89% (of chromatographically pure product). Final products were isolated as white,
beige, pale yellow or yellow solids.
Scheme 1. Synthesis of the title compounds.
Reagents and conditions: (a) SOCl2, DMF, anhydrous PhMe, 100 °C, 1 h; (b) TEA,
anhydrous acetone, RT.
The compounds were characterized by 1H-NMR, 13C-NMR, FT-IR spectroscopy, melting point and
elemental analysis. The analytical data were fully consistent with proposed structures. The results of the
elemental analyses were in the range of ±0.3% relative to calculated values. The parent compound 1
(with a non-substituted benzene ring) was also analysed by ion-trap MS. The full-scan spectrum
contained the [M+H]+ ion at m/z 234. The loss of water was observed in the MS2 spectrum and ion at
m/z 216 was found. The further fragmentation of ion at m/z 216 produced the main fragments
corresponding to: (a) the loss of HCl (m/z 180), and (b) the benzene ring (m/z 77). NMR and MS
spectra of compound 1 (internal laboratory code JZ-90) are included in the Supplementary Material.
2.2. Lipophilicity
Lipophilicity is one of the most important physico-chemical properties determining the biological
activity of small molecules, affecting the non-specific diffusion through biological membranes. It is
well known that antimycobacterial activity is often enhanced by increased lipophilicity, which
facilitates the penetration through highly lipophilic mycobacterial outer envelope and cell wall. The
lipophilicity of the prepared compounds was predicted as logP using commercially available software
CS ChemDraw Ultra ver. 12.0 (CambridgeSoft, Cambridge, MA, USA). Additionally, the lipophilicity

Molecules 2013, 18
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was measured experimentally by RP-HPLC and expressed as logk derived from retention times of
individual compounds (see Table 1). The plot of computer predicted logP vs. measured logk (Figure 1)
indicated a linear correlation, although a few of the compounds were apparently below the expected
line of regression, indicating that the computational algorithm underestimated their lipophilicity. Most
of these mispredicted compounds had an ortho substituent capable of H-bond formation (2-OH,
2-OCH3, 2-NO2 in compounds 2, 5, 21, 23, and 24). Chem3D Pro’s (CambridgeSoft) built-in MM2
energy minimization function with default parameters was used to study the possibility of
intramolecular H-bond formation. Indeed, all of these compounds were capable of forming the H-bond
between the ortho substituent on the phenyl ring (lone electron pair as acceptor) and the hydrogen of
the amide function (donor). The amide bond was in the trans configuration. See the model of
compound 5 in Figure 1B for a representative example. As found in literature, the existence of this
H-bond was confirmed experimentally for some similar anilides of POA (e.g., with ortho-NO2
substituent) [12].
Figure 1. (A) Correlation of predicted lipophilicity parameter logP and experimentally
determined logk. Linear regression parameters: R2 = 0.823, s = 0.2142, F = 93.03, n = 22.
Compounds capable of H-bonds formation by their ortho substituents on the phenyl ring
are indicated by triangle marks and were omitted from regression analysis. (B) Model of
compound 5 including the intramolecular H-bond.
(A)
(B)
2.3. Biological Activity
2.3.1. In Vitro Antimycobacterial Activity
The prepared compounds were screened for in vitro whole cell antimycobacterial activity against
M. tbc H37Rv, M. kansasii and two strains of M. avium using a micro-plate dilution method [13].
Firstly, the compounds were tested in concentrations 100–50–25–12.5–6.25–3.13–1.56 µg/mL. Based
on the results selected compounds with MIC ≤ 3.13 µg/mL were retested using extended dilution scale
up to 0.39 µg/mL (Table 1, values in parentheses). The differences between MIC values obtained in
this retest and original values were less or equal to two steps on the dilution scale, which is a usual

Molecules 2013, 18
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error for this type of assay. The MIC values detected for standard 5-chloropyrazine-2-carboxamide
(5-Cl-PZA; see Table 1) were in good agreement with values reported in literature (MIC = 8-32 μg/mL
for PZA-sensitive M. tbc strains, MIC = 8–64 μg/mL for PZA-resistant strains) [2].
Table 1. Summary of prepared compounds, their antimycobacterial activity expressed as
MIC (μg/mL), HepG2 cytotoxicity, and calculated and measured lipophilicity parameters a.
Compound
Antimycobacterial activity MIC (μg/mL)
Cytotoxicity
Lipophilicity
No.
R
M. tbc b
3.13 (1.56)
3.13 (0.78)
6.25
M.kansasii b M.avium b M. avium b IC50 (μM)
SI c
0.19
2.39
1.28
5.47
n.a.
logP
1.49
1.10
1.10
1.10
1.24
2.46
2.39
2.72
1.65
1.65
1.81
2.05
2.05
2.61
3.16
2.32
2.32
3.41
2.13
2.53
1.66
1.66
1.01
1.39
1.39
1.52
1.52
2.41
2.41
0.66
−0.41
−1.31
−0.64
logk
0.286
0.248
−0.053
−0.138
0.579
0.618
0.685
0.859
0.490
0.389
0.477
0.819
0.587
0.881
n.a.
1
2
H
25
n.d.
>100
>100
>100
>100
>50
50
>100
100
2.55
30.00
32.10
68.60
n.a.
2-OH
3-OH
3
50
4
4-OH
3.13 (12.5)
>50
100
50
5
2,4-(OCH3)2
2,5-(CH3)2
4-C2H5
4-i-Pr
>50
>100
n.d.
>50
6
1.56 (1.56)
1.56 (0.78)
1.56
>100
>100
>100
>100
>100
>50
>100
>100
>100
>100
>100
>50
11.66
7.17
14.44
n.a.
1.96
2.41
2.55
n.a.
7
8
n.d.
9
2-F
6.25
12.5
12.5
6.25
n.d.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5-Cl-PZA
PZA
INH
3-F
6.25
n.a.
n.a.
2,4-F2
3.13
n.a.
n.a.
2-Cl
3.13 (0.78)
6.25 (3.13)
3.13
>100
25
>100
25
6.72
n.a.
0.58
n.a.
3-Cl
25
3,4-Cl2
2,4,5-Cl3
3-Br
>100
>100
25
>100
>100
>100
>100
>50
>100
>100
>100
>100
>50
9.10
n.a.
0.88
n.a.
>100
25
n.a.
n.a.
0.646
0.660
n.a.
4-Br
3.13
6.25
1.56
25
n.a.
n.a.
2-Cl-4-I
2-CH3-5-F
2-Cl-5-CH3
5-Cl-2-OH
3-Cl-4-OH
2-OH-5-NO2
2-NO2
3-NO2
3-CN
12.5
n.a.
n.a.
3.13 (6.25)
1.56
>100
>100
12.5
>100
>100
12.5
25
n.a.
n.a.
0.525
1.061
0.602
0.070
0.230
0.674
0.344
0.192
0.201
0.643
0.704
n.a.
25
15.84
40.59
12.9
1.52
n.a.
2.86
7.39
1.17
0.29
n.a.
1.56 (6.25)
3.13 (0.39)
1.56 (1.56)
12.5
12.5
>100
n.d.
50
50
50
n.d.
>100
>100
>50
>100
>100
>50
3.13
n.d.
32.70
n.a.
2.91
n.a.
25
3.13
>100
12.5
12.5
n.d.
4-CN
>100
>100
>100
>100
>100
>100
>100
12.5–25
>100
>100
>100
>100
>100
>100
3.13–6.25
n.a.
n.a.
3-CF3
3.13 (6.25)
1.56 (3.13)
3.13
41.39
8.50
n.a.
3.99
1.64
n.a.
4-CF3
4-COOH-3-OH
−
25
12.5
>100
12.5–25
1594
>104
79 × 103 d
10.0
>196
n.a.
n.a.
−
6.25–12.5
−0.687
−
0.39–0.78
−0.743
a Data in parentheses represent the MIC values in confirmation retest; n.d.—not detected due to decreased viability of the
strain, data not reproducible; n.a.—not available; 5-Cl-PZA—5-chloropyrazine-2-carboxamide; PZA—pyrazinamide;
b
INH—isoniazid; Tested strains from left to right M. tuberculosis H37Rv, M. kansasii Hauduroy CNCTC My 235/80,
c
M. avium ssp. avium Chester CNCTC My 80/72, M. avium CNCTC My 152/73; SI values calculated for M. tbc as
d
IC50/MIC (in μM) using the lower MIC values; Data from literature [14] in a comparable HepG2 cytotoxicity assay:
PZA—IC50 = 79.1 mM, INH—IC50 = 78.8 mM.

Molecules 2013, 18
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As seen in Table 1, most of the compounds exerted antimycobacterial activity against M. tbc H37Rv
in the range of MIC = 1.56–6.25 µg/mL. The aniline part of the molecule tolerated many different
substituents R while maintaining the activity—both electron-donating (-OH, alkyl substituents)
and electron-withdrawing substituents (3-NO2, 3-CF3, 4-CF3). All compounds with simple alkyl
substituent R (6–8) exerted MIC = 1.56 µg/mL or lower (M. tbc H37Rv). The combination of halogen
substituent with methyl (compounds 19, 20), hydroxyl (21, 22) or nitro substituent (23) seemed to be
advantageous and produced compounds with MIC = 3.13 µg/mL or lower. Compounds with R = CN
were inactive (27) or of low activity (26).
The lipophilicity (expressed as logk) did not correlate with antimycobacterial activity. However,
highly lipophilic compounds with multiple halogen substituents (compounds 15, 18) suffered from low
solubility in testing medium and were inactive or weakly active. Insufficient solubility in testing
medium was observed also with inactive compound 5 (2,4-dimethoxy derivative), despite of its rather
low lipophilicity.
The activity against M. kansasii was generally lower in comparison to the activity against M. tbc
H37Rv. According to incomplete results, the fluorinated (9–11) and brominated (16, 17) compounds
preserved the same or similar level of MIC against M. kansasii and M. tbc H37Rv. With the exception
of 3-CN derivative 26, compounds with significant activity against M. kansasii (MIC ≤ 6.25 µg/mL)
had a halogen substituent R. The most lipophilic (logP) compound 18 with two halogen substituents on
the phenyl ring (R = 2-Cl-4-I) was the most active against M. kansasii. On the contrary hydrophilic
substituents R (hydroxyl in 3 and 4) lead to inactive derivatives. We suggest that in this series
halogenation on the phenyl ring and increased lipophilicity are advantageous with the respect to
activity against M. kansasii.
Only three of the tested compounds (13, 21, 22) were active against M. avium strains (weak activity,
MIC = 12.5–25 µg/mL). Interestingly, all of them had chlorine substitution in meta position of the
phenyl ring. This could indicate a steric need for a large (and hydrophobic) substituent in this position.
Doležal et al. published several papers on synthesis and antimycobacterial activity of substituted
anilides of POA, 6-Cl-POA, 5-tert-Bu-POA, and 5-tert-Bu-6-Cl-POA. The summary of structure-activity
relationships within these series was published recently [9,10], including the references to original
articles. The antimycobacterial activity (M. tbc H37Rv) was indicated as percent of growth inhibition
at fixed concentration of 6.25 µg/mL. Only six out of 91 anilides exerted the inhibition of 80% or
higher and 21 compounds were completely inactive [10]. The best reported MIC values (measured
only for compounds with inhibition over 90%) were from 3.13 to 12.5 µg/mL [10]. Judged from the
relatively large portion of inactive anilides from the previous series (21/91) compared with the number
of inactive anilides of the title series of N-phenyl-5-chloropyrazine-2-carboxamides (three out of 30),
we conclude that the 5-chloro substitution of the pyrazine nucleus is the most advantageous from all of
the discussed series. This is supported by another study which found only moderate to weak activity
(MIC = 50–100 µg/mL) for some anilides of non-substituted POA [15].
Recently we have published a letter [11] on the antimycobacterial activity of N-benzyl-5-
chloropyrazine-2-carboxamides, i.e., the methylene homologues of the anilides discussed herein. The
direct comparison of anilides 5, 9, 12, 13, 17, and 25 with the respective N-benzyl derivatives with
identical substitution patterns on the benzene ring clearly reveals that all of the compared anilides
possess significantly better activity against M. tbc H37Rv. Similarly, anilides 1 and 28 had better

Molecules 2013, 18
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activity compared with their N-benzyl homologues presented in another publication [16]. Generally,
the MIC values for N-benzyl-5-chloropyrazine-2-carboxamides ranged from 12.5 to 25 µg/mL [11,16],
whereas 20 of 30 anilides of 5-Cl-POA discussed in this article reached MIC ≤ 3.13 µg/mL (in primary
or repeated testing). As all of the compounds discussed in this paragraph were tested by the same
methodology and by the same researcher, the comparison is of a significant value. We conclude that
antimycobacterial activity of N-phenyl-5-chloropyrazine-2-carboxamides is superior to the activity of
N-benzyl-5-chloropyrazine-2-carboxamides, i.e., that incorporation of the -CH2– bridge leads to
significant decrease of antimycobacterial activity.
2.3.2. In Vitro Cytotoxicity
Drug-induced hepatotoxicity is a common side-effect of many of the clinically used antitubercular
agents (PZA, INH, rifampicin) [17]. Tuberculosis treatment regimens are always multi-drug; therefore,
newly introduced antitubercular agent would be probably used in a combination with at least some of
the classic antituberculars with significant hepatotoxicity. Thus the hepatotoxicity of new compounds
being developed as potential antituberculars should be considered very carefully.
To evaluate the potential hepatotoxicity, the IC50 of selected title compounds with promising
antitubercular activity were determined in a hepatocellular carcinoma cell line (HepG2) in vitro model.
This model has been widely used to study the hepatotoxicity of various antitubercular drugs
before [18,19]. The decrease of viability of HepG2 cells was measured using a standard protocol [20]
based on colorimetric method measuring reduction of tetrazolium salt.
The tested compounds exerted significant hepatotoxicity with IC50 values from units to tens of µM.
With the exception of compound 23 where combined with aromatic nitro group, the presence of
hydroxyl on the phenyl ring lead to relative decrease of hepatotoxicity, as in compounds 2–4, and 21.
Four of six compounds with the highest IC50 values (lowest toxicity) bore a hydroxyl. The -OH group
is probably a conjugation site involved in the drug metabolism - detoxification process.
The obtained IC50 values were used to calculate the selectivity indexes (SI) related to antimycobacterial
activity against M. tbc H37Rv. None of the compounds had SI > 10, which is a limit considered safe
for further development. Promising SI values (SI > 5) were obtained for compounds 4 and 21, both of
them with R = OH.
To evaluate the in vitro cytotoxicity more comprehensively, we chose the afforementioned
compunds 4 and 21 with the lowest IC50 values in HepG2 model, together with compound 30
(R = 4-COOH 3-OH), which we presumed could also possess diminished cytotoxicity because of its
low hydrophobicity and hydroxyl substitution, and assessed them for in vitro cytotoxicity on renal cell
adenocarcinoma (ACHN) and Chinese hamster ovary (CHO-K1) cell lines. Table 2 presents the
comparison of IC50 values for HepG2, CHO-K1 and ACHN cell lines accompanied by antimycobacterial
activity of individual compounds against M. tuberculosis H37Rv (MIC converted to molar concentrations).
The level of cytotoxicity was similar among all three cell lines, for compouds 4 and 21 the IC50 was at
tens of µM, leading to SI = 3.8–9.3. Notably, compound 30 can be designated as non-toxic for both
CHO-K1 and ACHN cell lines with IC50 at hundreds of µM. Selectivity indexes of compund 30 are of
value which is favourable for furhter development. This finding confirms that hydroxyl substitution of

Molecules 2013, 18
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the phenyl ring, preferrably combined with other lipophilicity decreasing substituents, leads to less
toxic or non-toxic derivatives in the series of 5-chloro-N-phenylpyrazine-2-carboxamides.
Table 2. Cytotoxic effect of selected compounds on different cell lines expressed as IC50 and SI.
M. tbc H37Rv
MIC (µM)
12.5
HepG2
IC50 (µM)
CHO-K1
ACHN
IC50 (µM)
Compound
SI
5.5
7.4
IC50 (µM) SI
SI
8.0
9.3
4
21
69
41
48 ± 4
39 ± 2
3.8
7.1
100 ± 39
51 ± 14
5.5
30
10.7
n.a.
1594
n.a. 502 ± 122 47.1 371 ± 96 34.8
10.0 290 ± 43 0.9 540 ± 120 1.7
5-Cl PZA
158.7
Values are expressed as the IC50: Mean ± SEM (µM) (n = 3) where applicable. SI = IC50/MIC.
2.3.3. In Vitro Antibacterial and Antifungal Activity
As a complementary screening test, all of the final compounds were tested for activity against
selected pathogenic bacterial and fungal species, but no significant activity compared with standards
was detected.
3. Experimental
3.1. General
All chemicals (unless stated otherwise) were purchased from Sigma-Aldrich (Schnelldorf, Germany).
The reaction process and the purity of final compounds were checked using Merck Silica 60 F254 TLC
plates (Merck, Darmstadt, Germany). Flash chromatography of the final compounds was run on
automated chromatograph CombiFlash Rf (Teledyne Isco, Lincoln, NE, USA) using columns filled
with Kieselgel 60, 0.040–0.063 mm (Merck), detection wavelength 280 nm. NMR spectra were
recorded on Varian VNMR S500 (Varian, Palo Alto, CA, USA) at 500 MHz for 1H and 125 MHz for
1
13C or at Varian Mercury VX-BB 300 at 300 MHz for H and 75 MHz for 13C. The spectra were
recorded in DMSO-d6 or CDCl3 at ambient temperature. The chemical shifts as δ values in ppm are
indirectly referenced to tetramethylsilane (TMS) via the solvent signal. IR spectra were recorded on
Nicolet Impact 400 (Nicolet, Madison, WI, USA) using ATR Ge method. Elemental analysis was
performed on CE Instruments EA-1110 CHN analyser (CE Instruments, Wigan, UK). All values are
given as percentages. Melting points were determined in open capillary on Stuart SMP30 melting point
apparatus (Bibby Scientific Limited, Staffordshire, UK) and are uncorrected. The mass spectra were
recorded in the mixture of MeOH, water, formic acid (80:20:0.01 v/v/v) using LCQ Advantage Max
ion-trap mass spectrometer (Thermo Finnigan, San Jose, CA, USA). The sample was ionised using
APCI probe in a positive mode. Yields are given as percentages and refer to the amount of
chromatographically pure product after all purification steps.

Molecules 2013, 18
14815
3.2. Synthesis and Purification of Final Compounds
General procedure: 5-Hydroxypyrazine-2-carboxylic acid (5-OH-POA, 300 mg, 2.14 mmol) was
dispersed in dry toluene (approx. 30 mL). Thionyl chloride (SOCl2, approximately 1.0 mL, 14 mmol)
was added to the reaction mixture, followed by a catalytic amount (1–2 drops) of N,N-dimethylformamide
(DMF). The reaction mixture was heated and stirred in an oil bath under a condenser at 100 °C for
approx. 1 h. During the course of reaction the starting solid 5-OH-POA dissolved (chemically
changed) and the reaction mixture turned brown-red. When no further conversion of the solid could be
observed (usually there was a small amount of dark solid particles left), the solvents were decanted
from the dark residue and concentrated in vacuo. To remove the unreacted SOCl2, the residue was
azeotroped with dry toluene (3 × 20 mL). The crude 5-chloropyrazine-2-carbonyl chloride product ,
obtained in the form of brown-red viscous liquid, was diluted with dry acetone (10 mL) and added
dropwise to the stirred solution of respective aniline (1.71 mmol, 0.8 molar equivalents) and
triethylamine (433 mg, 4.18 mmol, 2 molar equivalents) in dry acetone (20 mL). The product
precipitated from the reaction mixture. The mixture was stirred at laboratory temperature for 30 min
and the completeness was checked by TLC (silica 60 F254, hexane–EtOAc 3:1). The reaction mixture
was adsorbed to silica by removing the solvents in vacuo and the product was purified by flash
chromatography (silica, 0%–25% EtOAc in hexane gradient elution) and recrystallized from
EtOH/H20 if needed. Note: For compounds with higher polarity, e.g., compounds 2–4 and 30, it was
necessary to increase the strength of the mobile phase for flash chromatography. Usually gradient
elution 0%–60% EtOAc in hexane was sufficient, although for compound 30 we had to use EtOAc
with 10% of MeOH.
3.3. Data of the Prepared Target Compounds
Analytical data of compounds 5, 9, 12, 13, 17, and 25 were published previously in a preliminary
study [11].
5-Chloro-N-phenylpyrazine-2-carboxamide (1). White solid. Yield: 43%. mp 157.2–158.1 °C. 1H-NMR
(300 MHz, CDCl3) δ 9.48 (1H, bs, NH), 9.27 (1H, d, J = 1.4 Hz, H3), 8.57 (1H, d, J = 1.4 Hz, H6),
7.76–7.70 (2H, m, H2', H6'), 7.44–7.36 (2H, m, H3', H5'), 7.22–7.15 (1H, m, H4'). 13C-NMR (75 MHz,
CDCl3) δ 159.7, 152.3, 144.2, 142.6, 142.3, 137.0, 129.2, 125.0, 119.8. IR (ATR Ge, cm−1):
3323 (NH, CONH), 2357, 1667 (C=O, CONH), 1598, 1536, 1444, 1310, 1137, 1115, 1024, 897, 754,
694. Anal. Calcd. for C11H8Cl1N3O1 (MW 233.65): C, 56.55; H, 3.45; N, 17.98. Found: C, 56.38;
H, 3.57; N, 18.05.
5-Chloro-N-(2-hydroxyphenyl)pyrazine-2-carboxamide (2). Pale yellow solid. Yield: 46%.
1
mp 220.9–221.6 °C. H-NMR (300 MHz, DMSO-d6) δ 10.35 (1H, s, OH), 10.07 (1H, bs, NH),
9.13–9.11 (1H, m, H3), 8.95–8.93 (1H, m, H6), 8.28 (1H, dd, J = 8.0 Hz, J = 1.4 Hz, H3'),
7.02–6.81 (3H, m, H4', H5', H6').13C-NMR (75 MHz, DMSO-d6) δ 159.5, 151.4, 146.9, 143.6, 143.4,
143.1, 125.8, 124.9, 119.6, 119.5, 115.0. IR (ATR Ge, cm−1): 3319 (NH, CONH), 3090,
1655 (C=O, CONH), 1551, 1454, 1373, 1314, 1284, 1241, 1150, 1116, 1020, 907, 856, 758, 699.

Molecules 2013, 18
14816
Anal. Calcd. for C11H8Cl1N3O2 (MW 249.65): C, 52.92; H, 3.23; N, 16.83. Found: C, 53.01; H, 3.40;
N, 16.98.
5-Chloro-N-(3-hydroxyphenyl)pyrazine-2-carboxamide (3). Pale yellow solid. Yield: 39%.
1
mp 225.1–226.3 °C. H-NMR (300 MHz, DMSO-d6) δ 10.63 (1H, bs, NH), 9.53 (1H, s, OH),
9.16 (1H, d, J = 1.4 Hz, H3), 8.98 (1H, d, J = 1.4 Hz, H6), 7.50 (1H, t, J = 2.1 Hz, H2'),
13
7.34–7.28 (1H, m, H4'), 7.19 (1H, t, J = 8.1 Hz, H5'), 6.65–6.57 (1H, m, H6'). C-NMR (75 MHz,
DMSO-d6) δ 160.9, 157.7, 151.0, 144.2, 144.2, 143.1, 139.2, 129.5, 111.7, 111.6, 107.8.
IR (ATR Ge, cm−1): 3318 (NH, CONH), 3268, 1682, 1666 (C=O, CONH), 1615, 1544, 1451, 1279,
1196, 1137, 1116, 1022, 895, 784, 685. Anal. Calcd. for C11H8Cl1N3O2 (MW 249.65): C, 52.92;
H, 3.23; N, 16.83. Found: C, 53.13; H, 3.22; N, 16.72.
5-Chloro-N-(4-hydroxyphenyl)pyrazine-2-carboxamide (4). Yellow solid. Yield: 45%. mp 204.8–206.7 °C.
1H-NMR (300 MHz, DMSO-d6) δ 10.49 (1H, bs, NH), 9.32 (1H, s, OH), 9.06 (1H, d, J = 1.4 Hz, H3),
8.87 (1H, d, J = 1.4 Hz, H6), 7.67–7.58 (2H, m, AA', BB', H2', H6'), 6.77–6.69 (2H, m, AA', BB', H3',
13
H5'). C-NMR (75 MHz, DMSO-d6) δ 160.4, 154.4, 150.9, 144.3, 144.0, 143.0, 129.8, 122.5, 115.2.
IR (ATR Ge, cm−1): 3339 (NH, CONH), 3291, 1689 (C=O, CONH), 1639, 1602, 1554, 1510, 1444,
1264, 1219, 1142, 1115, 1026, 901, 836, 809, 668. Anal. Calcd. for C11H8Cl1N3O2 (MW 249.65):
C, 52.92; H, 3.23; N, 16.83. Found: C, 53.07; H, 3.11; N, 16.57.
5-Chloro-N-(2,5-dimethylphenyl)pyrazine-2-carboxamide
(6).
White
solid.
Yield:
77%.
1
mp 139.7–140.5 °C. H-NMR (300 MHz, CDCl3) δ 9.46 (1H, bs, NH), 9.27 (1H, d, J = 1.4 Hz, H3),
8.58 (1H, d, J = 1.4 Hz, H6), 8.02 (1H, s, H6'), 7.11 (1H, d, J = 7.7 Hz, H3'), 6.93 (1H, d, J = 7.7 Hz,
13
H4'), 2.37 (3H, s, CH3), 2.34 (3H, s, CH3). C-NMR (75 MHz, CDCl3) δ 159.6, 152.3, 144.1, 142.9,
142.4, 136.8, 134.8, 130.3, 126.0, 125.0, 122.1, 21.2, 17.1. IR (ATR Ge, cm−1): 3370 (NH, CONH),
1697 (C=O, CONH), 1583, 1541, 1450, 1256, 1146, 1127, 1023, 897, 811. Anal. Calcd.
for C13H12Cl1N3O1 (MW 261.71): C, 59.66; H, 4.62; N, 16.06. Found: C, 59.38; H, 4.89; N, 16.13.
5-Chloro-N-(4-ethylphenyl)pyrazine-2-carboxamide
(7).
White
crystalline.
Yield:
51%.
1
mp 155.6–156.5 °C. H-NMR (300 MHz, CDCl3) δ 9.44 (1H, bs, NH), 9.26 (1H, d, J = 1.4 Hz, H3),
8.56 (1H, d, J = 1.4 Hz, H6), 7.67–7.60 (2H, m, AA', BB', H2', H6'), 7.26–7.19 (2H, m, AA', BB', H3',
H5'), 2.65 (2H, q, J = 7.7 Hz, CH2), 1.24 (3H, t, J = 7.7 Hz, CH3). 13C-NMR (75 MHz, CDCl3)
δ 159.6, 152.2, 144.2, 142.7, 142.3, 141.2, 134.6, 128.5, 119.9, 28.3, 15.6. IR (ATR Ge, cm−1):
3355 (NH, CONH), 2967 (CH3), 1673 (C=O, CONH), 1595, 1530, 1518, 1459, 1414, 1311, 1137,
1126, 1024, 901, 834, 660. Anal. Calcd. for C13H12Cl1N3O1 (MW 261.71): C, 59.66; H, 4.62; N, 16.06.
Found: C, 59.70; H, 4.58; N, 15.93.
5-Chloro-N-(4-isopropylphenyl)pyrazine-2-carboxamide (8). White crystalline. Yield: 44%.
1
mp 151.1–152.8 °C. H-NMR (300 MHz, CDCl3) δ 9.44 (1H, bs, NH), 9.27 (1H, d, J = 1.0 Hz, H3),
8.57 (1H, d, J = 1.0 Hz, H6), 7.69–7.61 (2H, m, AA', BB', H2', H6'), 7.30–7.22 (2H, m, AA', BB', H3',
13
H5'), 3.01–2.83 (1H, m, CH), 1.26 (6H, d, J = 6.9 Hz, CH3). C-NMR (75 MHz, CDCl3) δ 159.6,
152.2, 145.8, 144.2, 142.7, 142.3, 134.7, 127.1, 119.9, 33.6, 24.0. IR (ATR Ge, cm−1):
3360 (NH, CONH), 2959 (CH3), 2360, 1677 (C=O, CONH), 1594, 1518, 1415, 1310, 1134, 1021,

Molecules 2013, 18
14817
902, 831, 660. Anal. Calcd. for C14H14Cl1N3O1 (MW 275.73): C, 60.99; H, 5.12; N, 15.24. Found: C,
61.11; H, 5.27; N, 15.41.
5-Chloro-N-(3-fluorophenyl)pyrazine-2-carboxamide (10). White to pale yellow crystalline. Yield: 56%.
1
mp 144.5–145.5 °C. H-NMR (300 MHz, CDCl3) δ 9.53 (1H, bs, NH), 9.27 (1H, d, J = 1.4 Hz, H3),
8.58 (1H, d, J = 1.4 Hz, H6), 7.74–7.67 (1H, m, H2'), 7.42–7.29 (2H, m, H5', H6'), 6.94–6.84 (1H, m,
H4'). 13C-NMR (75 MHz, CDCl3) δ 163.0 (d, J = 245.4 Hz), 159.9, 152.6, 144.3, 142.4, 142.2, 138.4 (d,
J = 10.9 Hz), 130.3 (d, J = 9.4 Hz), 115.2 (d, J = 3.2 Hz), 111.8 (d, J = 21.4 Hz), 107.4 (d, J = 26.6 Hz).
IR (ATR Ge, cm−1): 3364 (NH, CONH), 1679 (C=O, CONH), 1616, 1532, 1442, 1273, 1153, 1132,
1024, 875, 785, 682, 663. Anal. Calcd. for C11H7Cl1F1N3O1 (MW 251.64): C, 52.50; H, 2.80; N, 16.70.
Found: C, 52.33; H, 2.84; N, 16.55.
5-Chloro-N-(2,4-difluorophenyl)pyrazine-2-carboxamide (11). White to pale yellow solid. Yield: 49%.
1
mp 176.6–178.1 °C. H-NMR (300 MHz, CDCl3) δ 9.67 (1H, bs, NH), 9.25 (1H, d, J = 1.8 Hz, H3),
8.60 (1H, d, J = 1.8 Hz, H6), 8.53–8.40 (1H, m, H3'), 7.00–6.88 (2H, m, H5', H6'). 13C-NMR
(75 MHz, CDCl3) δ 159.8, 159.0 (d, J = 247.6 Hz), 158.9 (d, J = 247.7 Hz), 154.5 (d, J = 12.7 Hz),
152.7, 144.1, 142.6, 142.2, 122.4 (d, J = 9.2 Hz), 111.4 (dd, J = 21.9 Hz, J = 3.5 Hz), 103.9 (dd,
J = 26.5 Hz, J = 23.0 Hz). IR (ATR Ge, cm−1): 3362 (NH, CONH), 3057, 1694 (C=O, CONH), 1533,
1428, 1255, 1146, 1138, 1118, 1023, 963, 871, 842, 653. Anal. Calcd. for C11H6Cl1F2N3O1
(MW 269.64): C, 49.00; H, 2.24; N, 15.58. Found: C, 48.94; H, 2.03; N, 15.71.
5-Chloro-N-(3,4-dichlorophenyl)pyrazine-2-carboxamide (14). White solid. Yield: 87%.
1
mp 185.5–186.9 °C. H-NMR (500 MHz, CDCl3) δ 9.49 (1H, bs, NH), 9.25 (1H, d, J = 1.4 Hz, H3),
8.57 (1H, d, J = 1.5 Hz, H6), 7.97 (1H, d, J = 2.5 Hz, H2'), 7.57 (1H, dd, J = 8.7, 2.6 Hz, H6'),
7.44 (1H, d, J = 8.7 Hz, H5'). 13C-NMR (126 MHz, CDCl3) δ 159.87, 152.77, 144.25, 142.43, 141.95,
136.40, 133.08, 130.74, 128.29, 121.49, 119.01. IR (ATR Ge, cm−1): 3354 (NH, CONH), 2360, 2342,
1692 (C=O, CONH), 1578, 1520, 1478, 1463, 1458, 1388, 1133, 1024, 919, 882, 824, 668.
Anal. Calcd. for C11H6Cl3N3O1 (MW 302.54): C, 43.67; H, 2.00; N, 13.89. Found: C, 43.81; H, 2.24;
N, 14.03.
5-Chloro-N-(2,4,5-trichlorophenyl)pyrazine-2-carboxamide (15). White solid. Yield: 73%.
mp 189.3–192.4 °C. 1H-NMR (300 MHz, CDCl3) δ 10.13 (1H, bs, NH), 9.25 (1H, d, J = 1.2 Hz, H3),
8.82 (1H, s, H3'), 8.63 (1H, d, J = 1.2 Hz, H6), 7.54 (1H, s, H6'). 13C-NMR (75 MHz, CDCl3) δ 160.0,
153.0, 144.3, 142.8, 141.9, 133.3, 132.1, 130.1, 128.1, 122.0, 121.9. IR (ATR Ge, cm−1):
3332 (NH, CONH), 2360, 2342, 1690 (C=O, CONH), 1574, 1511, 1459, 1366, 1258, 1143, 1074,
1022, 901, 887, 669. Anal. Calcd. for C11H5Cl4N3O1 (MW 336.99): C, 39.21; H, 1.50; N, 12.47.
Found: C, 39.49; H, 1.57; N, 12.42.
5-Chloro-N-(3-bromophenyl)pyrazine-2-carboxamide (16). White solid. Yield: 56%. mp 135.3–136.0 °C.
1H-NMR (500 MHz, CDCl3) δ 9.48 (1H, bs, NH), 9.26 (1H, d, J = 1.5 Hz, H3), 8.58 (1H, d, J = 1.5 Hz,
H6), 7.99 (1H, t, J = 2.0 Hz, H2'), 7.65 (1H, d, J = 8.1 Hz, H6'), 7.32 (1H, d, J = 8.1 Hz, H4'),
7.26 (1H, t, J = 8.1 Hz, H5'). 13C-NMR (125 MHz, CDCl3) δ 159.8, 152.6, 144.3, 142.4, 142.2, 138.2,
130.5, 128.0, 122.8, 122.8, 118.3. IR (ATR Ge, cm−1): 3360 (NH, CONH), 1693 (C=O, CONH), 1586,

Molecules 2013, 18
14818
1521, 1455, 1421, 1273, 1251, 1135, 1024, 906, 788, 776, 681. Anal. Calcd. for C11H7Br1Cl1N3O1
(MW 312.55): C, 42.27; H, 2.26; N, 13.44. Found: C, 41.93; H, 2.07; N, 13.28.
5-Chloro-N-(2-chloro-4-iodophenyl)pyrazine-2-carboxamide (18). Pale beige solid. Yield: 55%.
mp 175.6–177.3 °C. 1H-NMR (300 MHz, CDCl3) δ 10.13 (1H, bs, NH), 9.25 (1H, d, J = 1.2 Hz, H3),
8.62 (1H, d, J = 1.2 Hz, H6), 8.37 (1H, d, J = 8.8 Hz, H6'), 7.76 (1H, d, J = 1.8 Hz, H3'), 7.64 (1H, dd,
13
J = 8.8 Hz, J = 1.8 Hz, H5'). C-NMR (75 MHz, CDCl3) δ 159.9, 152.8, 144.2, 142.7, 142.2, 137.3,
136.9, 133.9, 124.1, 122.4, 87.3. IR (ATR Ge, cm−1): 3326 (NH, CONH), 3079, 2360, 1687 (C=O,
CONH), 1586, 1521, 1463, 1378, 1320, 1254, 1135, 1023, 901, 871, 832, 719, 705, 680. Anal. Calcd.
for C11H6Cl2I1N3O1 (MW 394.00): C, 33.53; H, 1.54; N, 10.67. Found: C, 33.50; H, 1.49; N, 10.82.
5-Chloro-N-(5-fluoro-2-methylphenyl)pyrazine-2-carboxamide (19). White crystalline needles. Yield:
1
40%. mp 138.0–138.7 °C. H-NMR (300 MHz, CDCl3) δ 9.56 (1H, bs, NH), 9.26 (1H, d, J = 1.4 Hz,
H3), 8.59 (1H, d, J = 1.4 Hz, H6), 8.12 (1H, dd, J = 11.0 Hz, J = 2.8 Hz, H6'), 7.15 (1H, dd, J = 8.2 Hz,
J = 6.3 Hz, H3'), 6.80 (1H, dt, J = 8.2 Hz, J = 2.8 Hz, H4'), 2.35 (3H, s, CH3). 13C-NMR (75 MHz, CDCl3)
δ 161.4 (d, J = 242.8 Hz), 159.6, 152.6, 144.2, 142.5, 136.1 (d, J = 11.2 Hz), 131.2 (d, J = 9.1 Hz),
122.6 (d, J = 3.1 Hz), 111.5 (d, J = 21.2 Hz), 108.3 (d, J = 27.2 Hz), 16.9. IR (ATR Ge, cm−1):
3364 (NH, CONH), 1696 (C=O, CONH), 1602, 1540, 1450, 1252, 1165, 1140, 1117, 1022, 899, 888,
818, 726, 662. Anal. Calcd. for C12H9Cl1F1N3O1 (MW 265.67): C, 54.25; H, 3.41; N, 15.82. Found:
C, 54.37; H, 3.44; N, 15.99.
5-Chloro-N-(2-chloro-5-methylphenyl)pyrazine-2-carboxamide (20). White solid. Yield: 53%.
mp 130.8–131.9 °C. 1H-NMR (300 MHz, CDCl3) δ 10.11 (1H, bs, NH), 9.25 (1H, d, J = 1.4 Hz, H3),
8.61 (1H, d, J = 1.4 Hz, H6), 8.44–8.38 (1H, m, H6'), 7.29 (1H, d, J = 8.2 Hz, H3'), 6.95–6.89 (1H, m, H4'),
2.38 (3H, s, CH3). 13C-NMR (75 MHz, CDCl3) δ 159.8, 152.5, 144.1, 142.6, 138.1, 133.5, 128.8,
126.1, 121.7, 120.5, 21.3. IR (ATR Ge, cm−1): 3319 (NH, CONH), 2360, 1689 (C=O, CONH), 1588,
1530, 1452, 1300, 1256, 1143, 1128, 1112, 1046, 1022, 895, 805, 690. Anal. Calcd. for C12H9Cl2N3O1
(MW 282.13): C, 51.09; H, 3.22; N, 14.89. Found: C, 50.85; H, 3.35; N, 14.94.
5-Chloro-N-(5-chloro-2-hydroxyphenyl)pyrazine-2-carboxamide (21). Pale yellow solid. Yield: 40%.
1
mp 221.4–222.2 °C. H-NMR (300 MHz, DMSO-d6) δ 10.72 (1H, bs, NH), 10.05 (1H, s, OH),
9.11 (1H, d, J = 1.2 Hz, H3), 8.94 (1H, d, J = 1.2 Hz, H6), 8.33 (1H, d, J = 2.5 Hz, H6'), 7.02 (1H, dd,
13
J = 8.7 Hz, J = 2.5 Hz, H4'), 6.93 (1H, d, J = 8.7 Hz, H3'). C-NMR (75 MHz, DMSO-d6) δ 159.8,
151.6, 145.8, 143.7, 143.4, 142.7, 126.9, 124.3, 122.8, 118.9, 116.1. IR (ATR Ge, cm−1):
3326 (NH, CONH), 3118, 2359, 2342, 1660 (C=O, CONH), 1543, 1453, 1426, 1257, 1196, 1148,
1122, 1020, 921, 875, 810, 740, 701, 652. Anal. Calcd. for C11H7Cl2N3O2 (MW 284.10): C, 46.51; H,
2.48; N, 14.79. Found: C, 46.42; H, 2.42; N, 14.68.
5-Chloro-N-(3-chloro-4-hydroxyphenyl)pyrazine-2-carboxamide (22). Pale yellow solid. Yield: 40%.
1
mp 200.7–203.5 °C. H-NMR (300 MHz, DMSO-d6) δ 10.67 (1H, bs, NH), 10.04 (s, 1H, OH),
9.07 (1H, d, J = 1.4 Hz, H3), 8.89 (1H, d, J = 1.4 Hz, H6), 7.92 (1H, d, J = 2.2 Hz, H2'), 7.62 (1H, dd,
13
J = 8.8 Hz, J = 2.2 Hz, H6'), 6.94 (1H, d, J = 8.8 Hz, H5'). C-NMR (75 MHz, DMSO-d6) δ 160.7,
151.0, 150.0, 144.1, 144.0, 143.1, 130.6, 122.4, 121.0, 119.2, 116.5. IR (ATR Ge, cm−1): 3343 (NH,

Molecules 2013, 18
14819
CONH), 3233, 1761, 1649 (C=O, CONH), 1600, 1552, 1519, 1429, 1310, 1275, 1251, 1200, 1133,
1023, 895, 878, 828, 694, 665. Anal. Calcd. for C11H7Cl2N3O2 (MW 284.10): C, 46.51; H, 2.48;
N, 14.79. Found: C, 46.69; H, 2.57; N, 14.61.
5-Chloro-N-(2-hydroxy-5-nitrophenyl)pyrazine-2-carboxamide (23). Pale yellow solid. Yield: 42%.
1
mp 235.0–236.3 °C. H-NMR (300 MHz, DMSO-d6) δ 10.07 (1H, bs, NH), 9.17–9.13 (1H, m, H6'),
9.11–9.09 (1H, m, H3), 8.97–8.92 (1H, m, H6), 7.93 (1H, dd, J = 9.1 Hz, J = 2.8 Hz, H4'), 7.06 (1H, d,
13
J = 9.1 Hz, H3'). C-NMR (75 MHz, DMSO-d6) δ 160.2, 153.3, 151.8, 143.8, 143.5, 142.4, 139.5,
125.9, 121.4, 114.6. IR (ATR Ge, cm−1): 3338 (NH, CONH), 3091, 2360, 2342, 1663 (C=O, CONH),
1593, 1544, 1520, 1497, 1456, 1337, 1289, 1262, 1149, 1117, 1074, 1022, 905, 746, 736, 703.
Anal. Calcd. for C11H7Cl1N4O4 (MW 294.65): C, 44.84; H, 2.39; N, 19.02. Found: C, 44.59; H, 2.62;
N, 19.12.
5-Chloro-N-(2-nitrophenyl)pyrazine-2-carboxamide (24). Yellow solid. Yield: 33%. mp 156.3–156.8 °C.
1H-NMR (300 MHz, CDCl3) δ 12.36 (1H, bs, NH), 9.19 (1H, d, J = 1.4 Hz, H3), 8.90 (1H, dd, J = 8.5 Hz,
J = 1.5 Hz, H3'), 8.62 (1H, d, J = 1.4 Hz, H6), 8.21 (1H, dd, J = 8.5 Hz, J = 1.5 Hz, H6'),
13
7.70–7.62 (1H, m, H4'), 7.25–7.16 (1H, m, H5'). C-NMR (75 MHz, CDCl3) δ 161.2, 152.9, 144.4,
143.0, 142.3, 137.0, 136.0, 133.9, 126.0, 124.1, 122.1. IR (ATR Ge, cm−1): 3308 (NH, CONH),
1687 (C=O, CONH), 1610, 1584, 1502, 1428, 1340, 1312, 1275, 1256, 1139, 1022, 906, 862, 731,
688. Anal. Calcd. for C11H7Cl1N4O3 (MW 278.65): C, 47.41; H, 2.53; N, 20.11. Found: C, 47.13;
H, 2.57; N, 20.31.
5-Chloro-N-(3-cyanophenyl)pyrazine-2-carboxamide (26). Pale beige solid. Yield: 45%.
1
mp 206.8–208.3 °C. H-NMR (300 MHz, DMSO-d6) δ 11.1 (1H, bs, NH), 9.11 (1H, s, H3),
8.93 (1H, s, H6), 8.32 (1H, s, H2'), 8.23–8.14 (1H, m, H6'), 7.62–7.55 (2H, m, H4', H5').
13C-NMR (75 MHz, DMSO-d6) δ 161.6, 151.4, 144.4, 143.5, 143.2, 139.2, 130.3, 128.0, 125.5, 123.6,
118.8, 111.7. IR (ATR Ge, cm−1): 3304 (NH, CONH), 3073, 2360, 2241 (CN, nitrile),
1683 (C=O, CONH), 1589, 1552, 1437, 1300, 1254, 1134, 1023, 894, 882, 797, 789, 679. Anal. Calcd.
for C12H7Cl1N4O1 (MW 258.66): C, 55.72; H, 2.73; N, 21.66. Found: C, 55.83; H, 2.81; N, 21.52.
5-Chloro-N-(4-cyanophenyl)pyrazine-2-carboxamide (27). White solid. Yield: 42%. mp 225.4–226.8 °C.
1H-NMR (300 MHz, DMSO-d6) δ 11.15 (1H, bs, NH), 9.11 (1H, d, J = 1.4 Hz, H3), 8.93 (1H, d,
J = 1.4 Hz, H6), 8.14–8.07 (2H, m, AA', BB', H3', H5'), 7.86–7.79 (2H, m, AA', BB', H2', H6').
13C-NMR (75 MHz, DMSO-d6) δ 161.8, 151.4, 144.5, 143.5, 143.2, 142.6, 133.3, 120.9, 119.1, 106.3.
IR (ATR Ge, cm−1): 3348 (NH, CONH), 2360, 2228 (CN, nitrile), 1700 (C=O, CONH), 1587, 1518,
1455, 1409, 1313, 1245, 1173, 1132, 1023, 897, 862, 823, 665. Anal. Calcd. for C12H7Cl1N4O1
(MW 258.66): C, 55.72; H, 2.73; N, 21.66. Found: C, 55.49; H, 2.67; N, 21.71.
5-Chloro-N-(3-(trifluoromethyl)phenyl)pyrazine-2-carboxamide (28). White solid. Yield: 71%.
1
mp 121.5–122.5 °C. H-NMR (300 MHz, CDCl3) δ 9.60 (1H, bs, NH), 9.27 (1H, d, J = 1.4 Hz, H3),
8.59 (1H, d, J = 1.4 Hz, H6), 8.03 (1H, bs, H2'), 7.95 (1H, d, J = 8.0 Hz, H4'), 7.52 (1H, t, J = 8.0 Hz, H5'),
13
7.44 (1H, d, J = 8.0 Hz, H6'). C-NMR (75 MHz, CDCl3) δ 160.1, 152.8, 144.3, 142.5, 142.1, 137.5,
131.7 (q, J = 32.4 Hz), 129.8, 123.7 (q, J = 272.6 Hz), 122.9, 121.6 (q, J = 3.7 Hz), 116.6 (q, J = 4.0 Hz).

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IR (ATR Ge, cm−1): 3367 (NH, CONH), 2359, 1686 (C=O, CONH), 1608, 1544, 1453, 1340, 1325,
1136, 1119, 1071, 1024, 918, 903, 802, 699, 660. Anal. Calcd. for C12H7Cl1F3N3O1 (MW 301.65):
C, 47.78; H, 2.34; N, 13.93. Found: C, 47.95; H, 2.42; N, 13.80.
5-Chloro-N-(4-(trifluoromethyl)phenyl)pyrazine-2-carboxamide (29). White solid. Yield: 78%.
mp 179.8–181.1 °C. 1H-NMR (300 MHz, CDCl3) δ 9.63 (1H, bs, NH), 9.28 (1H, d, J = 1.2 Hz, H3), 8.59
(1H, d, J = 1.2 Hz, H6), 7.90–7.84 (2H, m, AA', BB', H3', H5'), 7.69–7.62 (2H, m, AA', BB', H2', H6').
13C-NMR (75 MHz, CDCl3) δ 160.1, 152.8, 144.3, 142.4, 142.1, 140.0, 126.8 (q, J = 32.9 Hz),
126.5 (q, J = 4.0 Hz), 123.9 (q, J = 271.7 Hz), 119.5. IR (ATR Ge, cm−1): 3372 (NH, CONH),
2359, 1690 (C=O, CONH), 1530, 1412, 1322, 1315, 1162, 1143, 1119, 1066, 1028, 902, 846, 666.
Anal. Calcd. for C12H7Cl1F3N3O1 (MW 301.65): C, 47.78; H, 2.34; N, 13.93. Found: C, 47.58; H, 2.28;
N, 13.71.
4-(5-Chloropyrazine-2-carboxamido)-2-hydroxybenzoic acid (30). Pale yellow solid. Yield: 18%.
1
mp 240.0–244.0 dec °C. H-NMR (300 MHz, DMSO-d6) δ 10.62 (1H, bs, NH), 9.09 (1H, d,
J = 1.1 Hz, H3), 8.91 (1H, d, J = 1.1 Hz, H6), 7.66 (1H, d, J = 8.5 Hz, H5'), 7.36 (1H, d, J = 2.1 Hz,
H2'), 7.21 (1H, dd, J = 8.5 Hz, J = 2.1 Hz, H6'). 13C-NMR (75 MHz, DMSO-d6) δ 171.9, 163.0, 161.2,
151.1, 145.4, 144.3, 144.1, 143.1, 130.6, 109.6, 107.6. IR (ATR Ge, cm−1): 3389 (NH, CONH),
1694 (C=O, CONH), 1600, 1133, 1024, 785. Anal. Calcd. for C12H8Cl1N3O4 (MW 293.66): C, 49.08;
H, 2.75; N, 14.31. Found: C, 48.86; H, 2.79; N, 14.17.
3.4. Determination of Lipophilicity by HPLC (Logk)
Instrumentation: Agilent Technologies 1200 SL liquid chromatograph with Diode-array Detector
SL G1315C (Agilent Technologies Inc., Colorado Springs, CO, USA); pre-column ZORBAX XDB-C18
5 µm, 4 × 4 mm, Part No. 7995118-504 (Agilent Technologies Inc.) and column ZORBAX Eclipse
XDB-C18 5 µm, 4.6 × 250 mm, Part No. 7995118-585 (Agilent Technologies Inc.). The separation
process was controlled by Agilent ChemStation, version B.04.02 extended by spectral module
(Agilent Technologies Inc.). Mobile phase consisted of MeOH (HPLC grade, 70%) and H2O
(HPLC-Milli-Q Grade, 30%).
Conditions: Flow rate 1.0 mL/min, sample injection volume 20 µL, column temperature 30 °C,
detection wavelength 210 nm, monitor wavelength 270 nm. Retention times (tR) were measured in
minutes. The dead time of the system (tD) was determined as the retention time of KI methanol
solution. Capacity factors k for individual compounds were calculated according to the formula
k = (tR − tD)/tD. Logk, calculated from the capacity factor k, is used as the lipophilicity index converted
to log scale.
3.5. Biological Methods
3.5.1. Evaluation of In Vitro Antimycobacterial Activity
Microdilution panel method. Tested strains M. tuberculosis H37Rv CNCTC My 331/88,
M. kansasii Hauduroy CNCTC My 235/80, M. avium ssp. avium Chester CNCTC My 80/72 and

Molecules 2013, 18
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M. avium CNCTC My 152/73 were obtained from Czech National Collection of Type Cultures
(CNCTC), National Institute of Public Health, Prague, Czech Republic. Tested compounds were
dissolved and diluted in DMSO and mixed with growth media (Šula's semisynthetic medium, pH = 5.6,
Trios, Prague, Czech Republic) to final concentrations of 100–50–25–12.5–6.25–3.13–1.56 μg/mL.
The mycobacterial suspensions for each strain were prepared by dilution of the basic isotonic saline
suspension (McFarland 0.5–1.0) by 10−1 and 10−3. These suspensions were used to inoculate the testing
wells so each compound was tested in duplicates at two different concentrations of mycobacterial
suspension. The assay involved positive controls of mycobacterial growth (DMSO plus broth).
Pyrazinamide (PZA) and isoniazid (INH) were used as standards. The testing plates were incubated
at 36 ± 1 °C until the growth of mycobacteria was visually evident in positive control wells
(usually 10–14 days). The MIC (μg/mL) was determined visually as the lowest concentration of tested
compound that inhibited the growth of mycobacteria. The difference in MIC of a compound read from
the parallel lines with different concentrations of mycobacterial suspension must not exceed one step
on the dilution scale.
3.5.2. HepG2 Cytotoxicity Determination
The human hepatocellular liver carcinoma cell line HepG2 (p 26–27, p 32–33) purchased from
Health Protection Agency Culture Collections (ECACC, Salisbury, UK) was routinely cultured in
MEM (Minimum Essentials Eagle Medium) (Sigma-Aldrich) supplemented with 10% foetal bovine
serum (PAA), 1% L-Glutamine solution (Sigma-Aldrich) and non-essential amino acid solution
(Sigma-Aldrich) in a humidified atmosphere containing 5% CO2 at 37 °C. For subculturing, the cells
were harvested after trypsin/EDTA (Sigma-Aldrich) treatment at 37 °C. The cells treated with the
tested substances were used as experimental groups. Untreated HepG2 cells were used as control
groups. The cells were seeded in density 1 × 104 cells per well in a 96-well plate. Next day, the cells
were treated with each of the tested substances dissolved in DMSO by dilution so that a final solution
contained less than 1% of DMSO in the medium. The tested compounds were prepared in incubation
concentrations 0–100 µM in triplicates. The controls: 100% cell viability, 0% cell viability (the cells
treated with 10% DMSO), no cell control and vehiculum controls were also prepared in triplicates.
After 24 h of incubation in a humidified atmosphere containing 5% CO2 at 37 °C, the reagent from the
kit CellTiter 96 AQueous One Solution Cell Proliferation Assay (CellTiter 96; Promega) was added.
After 2 h incubation at 37 °C the absorbance was recorded at 490 nm. A standard toxicological parameter
IC50 was calculated in each of the tested substances using GraphPad Prism software (version 5.02).
3.5.3. CHO-K1 and ACHN Cytotoxicity Determination
The standard MTT assay (Sigma Aldrich) was applied according to the manufacturer’s protocol on
renal cell adenocarcinoma (ACHN) and Chinese hamster ovary (CHO-K1) cell lines (all from
ECACC, Salisbury, UK). The cells were cultured according to ECACC recommended conditions and
seeded in a density of 8 × 103, 12 × 103 per well respectively for CHO-K1, ACHN cells. Cells were
exposed for tested compounds for 24 h, then the medium was replaced for a medium containing 10 μM
of MTT and cells were allowed to produce formazan for another approximately 1 h under surveillance.
Then, medium with MTT was sucked out and crystals of formazan were dissolved in DMSO. Cell

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viability was assessed spectrophotometrically by the amount of formazan produced. Absorbance was
measured at 570 nm with 650 nm reference wavelength on Synergy HT (BioTek, Winooski, VT,
USA). IC50 was then calculated from the triplicates using non-linear regression (four parameters) of
GraphPad Prism 5 software. Final IC50 value was obtained as a mean of at least three independent
measurements.
3.5.4. Evaluation of In Vitro Antibacterial Activity
Microdilution broth method [21]. The organisms examined included strains from Czech Collection
of Microorganisms (Brno, Czech Republic): Staphylococcus aureus CCM 4516/08, Escherichia coli
CCM 4517, Pseudomonas aeruginosa CCM 1961. These strains are recommended as standards for
testing of antibacterial activities. Other strains were clinical isolates (Department of Clinical
Microbiology, University Hospital and Faculty of Medicine in Hradec Králové, Charles University
in Prague, Czech Republic): Staphylococcus aureus H 5996/08-methicilin resistant (MRSA),
Staphylococcus epidermidis H 6966/08, Enterococcus sp. J 14365/08, Klebsiella pneumoniae D 11750/08,
Klebsiella pneumoniae J 14368/08-ESBL positive. All strains were subcultured on Mueller-Hinton
agar (MHA) (Difco/Becton Dickinson, Detroit, MI, USA) at 35 °C and maintained on the same
medium at 4 °C. Prior to testing, each strain was passaged onto MHA. Bacterial inocula were prepared
by suspending in sterile 0.85% saline. The cell density of the inoculum was adjusted to yield
suspension of density equivalent 0.5 McFarland scale (1.5 × 108 viable CFU/mL). The compounds
were dissolved in DMSO, and the antibacterial activity was determined in Mueller-Hinton liquid broth
(Difco/Becton Dickinson, Detroit, MI, USA), buffered to pH 7.0. Controls consisted of medium and
DMSO alone. The final concentration of DMSO in the test medium did not exceed 1% (v/v) of the
total solution composition. The minimum inhibitory concentration (MIC), defined as 95% inhibition of
bacterial growth as compared to control, was determined after 24 and 48 h of static incubation at 35 °C.
3.5.5. Evaluation of In Vitro Antifungal Activity
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,
which was carried out using microdilution broth method [22]. Compounds were dissolved in DMSO
and diluted in a twofold manner with RPMI 1640 medium with glutamine buffered to pH 7.0
(3-morpholinopropane-1-sulfonic acid). The final concentration of DMSO in the tested medium did
not exceed 2.5% (v/v) of the total solution composition. Static incubation was performed in the dark
and humid, at 35 °C for 24 and 48 h (respectively 72 and 120 h for Trichophyton mentagrophytes).
Drug-free controls were included. Fluconazole was used as standard. Tested species: Candida albicans
ATCC 44859, C. tropicalis 156, C. krusei E28, C. glabrata 20/I, Trichosporon asahii 1188,
Aspergillus fumigates 231, Absidia corymbifera 272 and Trichophyton mentagrophytes 445.
4. Conclusions
To conclude, we have successfully demonstrated that the anilides of 5-Cl-POA (5-chloro-N-
phenylpyrazine-2-carboxamides) possess significant antimycobacterial activity, especially against

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M. tuberculosis H37Rv. We determined the basic structure-activity and structure-toxicity relationships.
The phenyl part tolerated many various substituents while maintaining the activity. Most of the
compounds exerted significant in vitro hepatotoxicity as evaluated by HepG2 cell line model—however,
hydroxyl and other hydrophilic substituents decreased the cytotoxicity. 5-Chloro-N-(5-chloro-2-
hydroxyphenyl)pyrazine-2-carboxamide (21) possessed the broadest spectrum of antimycobacterial
activity and inhibited all of the tested strains (including the strains resistant to PZA), and at the same
time had one of the lowest HepG2 cytotoxicity. 4-(5-Chloropyrazine-2-carboxamido)-2-
hydroxybenzoic acid (30), although not the absolutely most active in the presented series, was
approximately 5–10 times more active in comparison with first-line antitubercular agent PZA (MIC for
M. tbc H37Rv—10 µM for 30, 51–102 µM for PZA). Notably, compound 30 was rated as non-toxic in
CHO-K1 and ACHN cell line models. Therefore, compounds 21 (for its broad spectrum of activity) and 30
(for its non-toxicity) can be highlighted as the lead structures for further development. The 5-chloro
substituent on the pyrazine core will allow easy structural modifications via nucleophilic substitution.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/12/14807/s1.
Acknowledgments
This publication is a result of the project implementation: ‘Support of establishment, development,
and mobility of quality research teams at the Charles University’, project number CZ.1.07/2.3.00/30.0022,
supported by The Education for Competitiveness Operational Programme (ECOP) and co-financed by
the European Social Fund and the state budget of the Czech Republic.
Additional support was provided by the Ministry of Education, Youth and Sports of the Czech
Republic (SVV-2013-267-001), GAUK B-CH/710312, and Ministry of Health of the Czech Republic
IGA NT 13346 (2012) and DRO (UHHK, 00179906).
The authors wish to thank Ida Dufková for performing the antibacterial and antifungal assessment
and Lenka Slavětinská for her participation in the synthesis of some of the title compounds.
Conflicts of Interest
The authors declare no conflict of interest.
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© 2013 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
(http://creativecommons.org/licenses/by/3.0/).