Novel halogenated pyrazine-based chalcones as potential antimicrobial drugs

Article abstract of DOI:10.3390/molecules21111421

Chalcones, i.e., compounds with the chemical pattern of 1,3-diphenylprop-2-en-1-ones, exert a wide range of bio-activities, e.g., antioxidant, anti-inflammatory, anticancer, anti-infective etc. Our research group has been focused on pyrazine analogues of

Full text of DOI:10.3390/molecules21111421

molecules
Article
Novel Halogenated Pyrazine-Based Chalcones as
Potential Antimicrobial Drugs †
Marta Kucerova-Chlupacova 1,*, Veronika Vyskovska-Tyllova 1, Lenka Richterova-Finkova 1,
Jiri Kunes 2, Vladimir Buchta 3,4, Marcela Vejsova 3,4, Pavla Paterova 3,4, Lucia Semelkova 1,
Ondrej Jandourek 1 and Veronika Opletalova 1
1
Department of Pharmaceutical Chemistry and Pharmaceutical Analysis,
Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203,
500 05 Hradec Kralove, Czech Republic; good-message@seznam.cz (V.V.-T.);
lenka.finkova@seznam.cz (L.R.-F.); semelkol@faf.cuni.cz (L.S.); jando6aa@faf.cuni.cz (O.J.);
opletalova@faf.cuni.cz (V.O.)
2
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy in Hradec Kralove,
Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic;
kunes@faf.cuni.cz
Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Kralove,
3
Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic;
vejsova@faf.cuni.cz
Department of Clinical Microbiology, University Hospital Hradec Kralove, Sokolska 581,
4
500 05 Hradec Kralove, Czech Republic; vladimir.buchta@fnhk.cz (V.B.); pavla.paterova@fnhk.cz (P.P.)
*
Correspondence: kucerom@faf.cuni.cz; Tel.: +420-495-067-373
†
Preliminary Results were Presented at the 8th Central European Conference “Chemistry towards Biology”
(CTB-2016), Brno, Czech Republic, 28 August–1 September 2016 (Paper P-38).
Academic Editor: Josef Jampilek
Received: 23 September 2016; Accepted: 14 October 2016; Published: 27 October 2016
Abstract: Chalcones, i.e., compounds with the chemical pattern of 1,3-diphenylprop-2-en-1-ones,
exert a wide range of bio-activities, e.g., antioxidant, anti-inflammatory, anticancer, anti-infective etc.
Our research group has been focused on pyrazine analogues of chalcones; several series have been
synthesized and tested in vitro on antifungal and antimycobacterial activity. The highest potency was
exhibited by derivatives with electron withdrawing groups (EWG) in positions 2 and 4 of the ring B.
As halogens also have electron withdrawing properties, novel halogenated derivatives were prepared
by Claisen-Schmidt condensation. All compounds were submitted for evaluation of their antifungal
and antibacterial activity, including their antimycobacterial effect. In the antifungal assay against
eight strains of selected fungi, growth inhibition of Candida glabrata and Trichophyton interdigitale
(formerly T. mentagrophytes) was shown by non-alkylated derivatives with 2-bromo or 2-chloro
substitution. In the panel of selected bacteria, 2-chloro derivatives showed the highest inhibitory
effect on Staphylococcus sp. In addition, all products were also screened for their antimycobacterial
activity against Mycobacterium tuberculosis H37RV My 331/88, M. kansasii My 235/80, M. avium 152/80
and M. smegmatis CCM 4622. Some of the examined compounds, inhibited growth of M. kansasii and
M. smegmatis with minimum inhibitory concentrations (MICs) comparable with those of isoniazid.
Keywords: pyrazine; chalcone; halogenated; antifungal; antibacterial; antimycobacterial
1. Introduction
Chalcones are compounds with the basic scaffold of 1,3-diphenylprop-2-en-1-one, containing ring
A and ring B connected by an
α,β-unsaturated keto linker (indicated in red in Figure 1, structure 1).
Naturally-occurring chalcones (in the plant kingdom) usually bear hydroxy-, methoxy- or prenyl
Molecules 2016, 21, 1421; doi:10.3390/molecules21111421
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1421
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substitutions and might serve as precursors of other flavonoid groups. Chalcones exert a wide range
of bio-activities (e.g., antioxidant, anti-inflammatory, anticancer, anti-infective etc.), which were several
times reviewed [1–3]. In synthetic chalcones, substitutents can be more varied and benzene rings have
been many times replaced with other aryls or heteroaryls [2,3].
Figure 1. Examples of antifungal and antibacterial chalcone derivatives.
Based on our previous results concerning chalcones and the positive effect of
electron-withdrawing group (EWG) substitution on their antimicrobial effects [ ], we decided
to prepare pyrazine analogues of chalcones halogenated in the ring B. Substitution of chalcones
with halogens emerges also in studies of other authors, e.g., compound inhibited growth of
Trichophyton rubrum at minimum inhibitory concentrations (MIC) 12.5 g/mL [ ]. Compound
exerted better antifungal activity against dermatophytes (MIC 0.5–25 g/mL) than amphotericin
B and ketoconazole [ ]. In a study comparing antifungal activity of mono- and dihalogenated
1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-ones (3a) and their corresponding flavonols (3b) [ ],
4,5
1
µ
6
2
µ
7
8
it was found that chalcones are more active against Trichophyton longifusus, Aspergillus flavus and
Microsporum canis than the flavonols. The most active compound was fluorinated in position 4 of the
ring B. Chalcones 4a–4c with halogen substitution in position 4 either in the ring A or in the ring B
inhibited growth of Candida tropicalis and Aspergillus flavus at a concentration of 2 mg/mL at least
by 98% [9].
Antibacterial effects of chalcones have also been reviewed [10–13] and importance of searching
for new antimicrobial agents due to increasing antibiotic resistance of bacteria and fungi has been
highlighted [13]. The substitution of aromatic rings with EWG was emphasized in a quinoline-based
series of chalcones in association with antibacterial activity. The most active compound, inhibiting
growth of Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Klebsiella aerogenes
and Salmonella typhimurium, better than chloramphenicol or ciprofloxacin, is depicted in structure 5.
Supposed mechanism of action was explored in a bacterial gyrase assay [14]. Two potentially
antibacterial entities were combined in several series containing halogenated chalcones and variously
substituted 2-thioxothiazolidin-4-ones (rhodanines) [15
that inhibited Staphylococcus sp. comparably to norfloxacin, are drawn as structure
in Figure 2. In the same research group, analogical chalcones with substituted thiazolidinediones 8a
,
16] or thiazolidin-2,4-dione [17]. Compounds,
6
[
15] and 7 [16]

Molecules 2016, 21, 1421
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and 8b were synthesized (Figure 2). They inhibited growth of two strains of S. aureus similarly to
oxacillin and norfloxacin [17].
Figure 2. Examples of antibacterial chalcone derivatives.
Halogen substitution has been found to be favorable in antimycobacterial diphenylpropenones [18
]
and 3-phenyl-1-pyridylpropenones [19]. Chalcones including halogenated derivatives have been
described as inhibitors of Mycobacterium tuberculosis phosphatases [20] or inhibitors of fatty acid
synthase II [21].
Halogenated chalcones are further mentioned in literature as potential antimalarial [22],
anti-inflammatory [23–25], anti-cancer [26] agents, and as potential modulators of multidrug resistance [27].
Sofalcone, explored and marketed in Japan, is a chalcone derivative. Mechanism of its action in
ulcer disease is complex, but it possesses bactericidal effect against Helicobacter pylori as well [28].
2. Results and Discussion
2.1. Synthesis
Preparation of the title compounds is shown in Scheme 1. Pyrazine-2-carbonitrile (10) was used
as the starting material. Syntheses of intermediates 11a
formerly described methods [29 30]. Acetylpyrazine 12a and its 5-alkylated congeners 12b
as starting materials for Claisen-Schmidt condensation with 2- or 4-halogenated aromatic aldehydes
affording (2E)-1-(5-unsubstituted or 5-alkyl-pyrazin-2-yl)-3-(halogenphenyl)prop-2-en-1-ones 13a 18g
–
11g and 12a
–
12g were accomplished by
,
–
12g served
–
(substitutions indicated in Table 1), according to a previously reported method [31].
Column chromatography, in several cases repeated, was necessary for the separation of the product
from the reaction mixture. To obtain analytically pure crystals, recrystallizations from absolute ethanol
were needed, which finally resulted in quite low yields.
Scheme 1. Synthesis of halogenated pyrazine-based chalcones. Reagents and conditions: (a) aliphatic acid,
◦
AgNO3, (NH4)2S2O8, water, 80 C; (b) CH3MgI, Et2O; (c) halogenated benzaldehyde, pyridine, Et2NH.

Molecules 2016, 21, 1421
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Due to difficult purification, only 5 fluorinated derivatives (13a
,
13b
,
13e
,
14a and 14b) were
obtained in sufficient amounts. In 2-chloro substituted series, two compounds 15a and 15b were
successfully purified and available for biological evaluation. Compounds with chlorine in position 4 of
the ring B (16a–16f) were synthesized earlier [5,31]. Influence of compounds 16a–16e on M. tuberculosis
H37RV (ATCC 27294) and photosynthetic processes has already been published [32]. However, the
accomplishment of their antifungal and antibacterial tests failed due to poor solubility in testing media.
For the purpose of the present study, these compounds were newly adjusted by rubbing to be able to
pass biological assays. Compound 16f was reported in our previous paper [
antifungal nor antimycobacterial activity against M. tuberculosis H37RV (ATCC 27294) [
present study, we report the results of its antibacterial testing and its effects on the growth of other
mycobacteria. All 2-brominated compounds 17a 17f were obtained without difficulties in acceptable
yields (20%–50%). In contrast, 4-brominated derivatives 18a 18g were mostly obtained in small
5
]. It displayed neither
5]. In the
–
–
amounts (yields: 2%–6%). (2E)-3-(4-bromophenyl)-1-(5-pentylpyrazin-2-yl)prop-2-en-1-one 18g is the
only representative with a longer alkyl in the pyrazine ring since the longer alkyl the more difficult
separation of the product from the reaction mixture. This has been observed for both 2-alkylated
pyrazine-2-carbonitriles 12 [29,30] and halogenated (2E)-3-phenyl-1-(pyrazin-2-yl)prop-2-en-1-ones
reported here.
Table 1. Halogenated pyrazine-based chalcones 13a–18g tested within this work.
R1
R2
H
tert-butyl isobutyl
butyl
propyl isopropyl pentyl
2-F
4-F
2-Cl 15a
13a
14a
13b
14b
15b
-
-
-
-
-
-
13e
-
-
-
-
-
-
-
-
16a
16b a
16c a
16d a
16e a
-
16f b
4-Cl
a
2-Br 17a
4-Br 18a
17b
18b
17c
18c
17d
18d
17e
18e
17f
18f
-
18g
a
Synthesis, antimycobacterial activity against M. tuberculosis H37RV (ATCC 27294) and influence on
photosynthetic processes published previously [32]; b synthesis, antimycobacterial activity against M. tuberculosis
H37RV (ATCC 27294) and antifungal activity published previously [5].
Identity of compounds was confirmed by melting points, NMR and IR spectra. Proton NMR
spectra clearly showed (JHαHβ = xx
the compounds was verified by TLC and elemental analysis.
−
yy Hz) that E-isomers have been obtained in all cases. Purity of
2.2. Evaluation of In Vitro Antifungal Activity
All prepared compounds, including 16a–16e, were subjected to antifungal assay, only the most
active ones are displayed in Table 2. In the panel of selected fungi, inhibition of growth of Trichophyton
interdigitale 445 by the non-alkylated compounds was the most notable. That is in accordance with
the previous results of our research group in the field of antifungal activity of pyrazine analogues of
chalcones [
the best activity (MIC 3.9
used antimycotic fluconazole (MIC 6.51
did not reach the activity of terbinafine (MIC 0.01–1.72
dermatomycosis. 4-Fluoro, 2-bromo or 4-bromo substitution (14a
effect on growth of T. interdigitale (MIC 3.9–7.81 mol/L after 24 h incubation). Halogenated derivatives
suppressed the growth of Candida spp. as well. In case of 4-fluoro derivative (14a), it was a moderate,
non-specific inhibition (MIC 31.25–62.5 mol/L after 24 h incubation), whereas chlorinated derivatives
15a and 16a inhibited specifically growth of Candida glabrata 20/I and Candida krusei E 28 (in both cases
MIC 7.81 mol/L after 24 h incubation). The effect of 15a on C. glabrata (MIC 7.81 mol/L after 24 h
4
,5,
31]. The derivative with 2-chloro substitution in the phenyl, i.e., ring B (15a) proved
mol/L after 24 h incubation), comparable with that of systematically
mol/L after 24 h incubation). However, the derivative 15a
mol/L), that is usually used for therapy of
17a and 18a) had also inhibiting
µ
µ
µ
,
µ
µ
µ
µ

Molecules 2016, 21, 1421
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incubation) is better than the effect of clinically used antimycotics listed in Table 2. Comparison with
previously prepared substances (19 22) is provided as well. Upper mentioned active halogenated
derivatives were in common more antifungal effective than hydroxy (20a and 20b), nitro (21a and 21b
–
)
or methoxy (22a and 22b) derivatives, with the exception of 2-nitro derivative (21a) inhibiting strongly
Candida spp. (antifungal activity in Table 2, structures in Figure 3).
Table 2. Comparison of antifungal activity of selected halogenated 3-phenyl-1-pyrazin-2-ylprop-2-en-1-
ones with ring B-variously substituted analogues and standard antimycotics.
MIC (µmol/L) *
CA
24 h
48 h
CT
24 h
48 h
CK
24 h
48 h
CG
24 h
48 h
TA
AF
LC
TI
Substituent
in the Ring B
Compd.
24 h
48 h
24 h
48 h
24 h
48 h
72 h
120 h
15.63
62.5
250
>500
15.62
125
62.5
125
125
125
250
>500
>500
>500
250
250
13a
14a
15a
16a
17a
18a
19 a
20a b
20b
21a
2-F
4-F
31.25
62.5
62.5
125
31.25
62.5
62.5
125
62.5
250
250
>500
>500
>500
7.81
15.62
>125
>125
>125
>125
31.25
62.5
7.81
15.62
62.5
62.5
>125
>125
>125
>125
3.9
3.9
2-Cl
125
>125
125
>125
7.81
15.62
31.25
62.5
31.25
125
62.5
>125
>125
>125
>125
>125
4-Cl
62.5
>125
>125
>125
62.5
>125
7.81
31.25
62.5
125
>125
>125
>125
>125
7.81
7.81
2-Br
15.62
31.25
31.25
125
32.6
>125
>125
>125
>125
>125
125
>125
>125
>125
3.9
7.81
4-Br
15.63
31.25
31.25
62.5
31.25
62.5
31.25
62.5
250
250
125
125
250
500
7.81
15.63
H
62.5
62.5
>250 c
>250 c
125
250
>250 c
>250 c
125
250
>250 c
>250 c
125
250
>250 c
>250 c
125
250
>250 c
>250 c
125
250
>250 c
>250 c
125
250
>250 c
>250 c
15.62
31.25
2-OH
4-OH
2-NO2
4-NO2
2-OCH3
2-OCH3
62.5 b
125 b
31.25 e
31.25 e
7.81 d
7.81 d
7.81 d
7.81 d
7.81 d
7.81 d
>125 d
>125 d
31.25 d
>125 d
>125 d
>125 d
15.63 d
15.63 d
15.63 e
15.63 e
31.25 f
62.5 f
>62.5 f
>62.5 f
31.25 f
31.25 f
>62.5 f
>62.5 f
>62.5 f
>62.5 f
>62.5 f
>62.5 f
>62.5 f
>62.5 f
21b
31.25
125
31.25
62.5
>125
>125
31.25
125
>125
>125
>125
>125
>125
>125
31.25
125
22a d
62.5
125
125
250
125
250
125
125
500
>500
62.5
125
500
>500
31.25
62.5
22b d
0.24
0.24
>500
>500
125
250
41.64
250
250
500
>500
>500
>500
>500
6.51
104
Fluconazole
0.005
0.007
6.86 g
125
250
6.86 g
0.65
1.95
6.86 g
83.58
250
6.86 g
3.26
14.32
0.49
1.3
208
250
0.08
0.12
0.01–0.86 g
Voriconazole
Terbinafine
-
-
-
* MIC defined as IC80 for yeasts and yeast-like organisms and IC50 for molds; Notes: CA = Candida albicans
ATCC 44859; CT = Candida tropicalis 156; CK = Candida krusei E 28; CG = Candida glabrata 20/I; TA = Trichosporon
asahii 1188; AF = Aspergillus fumigatus 231; LC = Lichtheimia corymbifera 272; TI = Trichophyton interdigitale 445;
a
ref. [32]; b ref. [31]; c ref. [33]; d ref. [34]; e ref. [ ]; f ref. [35]; g IC50 after 48 h of incubation for yeasts and
4
7 days of incubation for T. interdigitale [36].
Figure 3. Structure of pyrazine-based chalcones comprised in Table 2 for comparison of antifungal activity.

Molecules 2016, 21, 1421
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2.3. Evaluation of In Vitro Antibacterial Activity
All prepared compounds, including 16a
–16f were subjected to antibacterial assay, and only the
most active ones are displayed in Table 3. From the panel of tested bacteria, Staphylococcus spp.
was the most susceptible. Growth of Staphylococcus aureus CCM 4516/08 and S. aureus H 5996/08
was inhibited by most compounds listed in Table 2 with moderate effect, but growth of
Staphylococcus epidermidis H6966/08 was inhibited more significantly by 2-chlorinated derivatives
(MIC of 15a: 3.9 µmol/L after 24 h incubation and MIC of 15b: 7.81 µmol/L after 24 h incubation)
than by standard antibiotics. Comparing the character of halogen substitution, fluorine does not
seem to be as important in our series as in diphenylpropenones [37]. In another study, focused on
diphenylpropenones, derivatives with 3,5-dibromo or 3,5-bis(trifluoromethyl) substitution were the
most active compounds [38]. In agreement with the positive effect of chlorine substitution in our series,
the most antifungal and antibacterial substance in a 3-quinolinyl-1-thienyl propenones had so far three
chlorine atoms [39]. Polyhalogenation of 3-phenyl-1-pyrrol-2-ylpropenones also positively reflected in
their antimicrobial activity [40]. In a thiazole-based series of chalcones, derivatives chlorinated in the
ring B exerted also better antibacterial and antifungal activity than unsubstituted derivative or nitro
derivatives [41]. However, pyrimidine analogues of chalcones, variable halogenated in position 2 of
the ring B, exert no remarkable antibacterial or antifungal activity [42].
Table 3. Comparison of antibacterial activity of selected novel halogenated 3-phenyl-1-pyrazin-2-
ylprop-2-en-1-ones with standard antibacterial agents.
IC95 (µmol/L)
Substituent in the Ring
SA
24 h
48 h
MRSA SE
EF
EC
KP
24 h
48 h
KP-E
24 h
48 h
PA
Compd.
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
B
A
125
250
250
250
31.25
125
500
500
>500
>500
>500
>500
>500
>500
>500
>500
13a
13e
H
2-F
2-F
125
>500
>500
>500
31.25
125
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
5-pro
125
125
125
500
31.25
62.5
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
14a
H
H
4-F
31.25
62.5
62.5
250
3.9
15.62
62.5
250
>500
>500
>500
>500
>500
>500
>500
>500
15a
2-Cl
2-Cl
2-Br
125
500
>500
>500
7.81
31.25
125
500
>500
>500
>500
>500
>500
>500
>500
>500
15b
5-t-bu
H
31.25
31.25
62.5
250
62.5
250
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
17a
2.60
3.25
1.95
4.23
9.11
13.02
291.67 2.28
291.67 2.28
1.30
1.30
2.28
2.28
7.81
15.62
Neomycin
10.41
18.23
13.02
26.04
83.33
15.62
31.25
-
52.08
-
-
-
Bacitracin
31.25
0.57
0.73
135.42 7.81
125.00 333.33
125.00 416.67
-
-
Penicillin
104.17 208.33 15.62
SA = Staphylococcus aureus CCM 4516/08; MRSA = Staphylococcus aureus H 5996/08 methicillin-resistant;
SE = Staphylococcus epidermidis H6966/08; EF = Enterococcus sp. J 14365/08; EC = Escherichia coli CCM4517;
KP = Klebsiella pneumoniae D 11750/08; KP-E = Klebsiella pneumoniae J 14368/08 (ESBL positive, KP-E);
PA = Pseudomonas aeruginosa CCM 1961.
2.4. Evaluation of In Vitro Antimycobacterial Activity
As far as the antimycobacterial screening is concerned, four strains were tested in total (all
results are shown Table 4). The most susceptible strain to inhibition by halogenated pyrazine-based
analogues of chalcones seems to be Mycobacterium kansasii Hauduroy CNCTC My 235/80. However,

Molecules 2016, 21, 1421
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the lowest MICs have been achieved in 2-chlorinated compounds (15a and 15b) during testing on
Mycobacterium tuberculosis H37RV CNCTC My 331/88 (MIC 6.25 g/mL for 15a and 3.13 g/mL
for 15b). The efficacy on M. tuberculosis in these two cases correlates well with results of in-house
testing on Mycobacterium smegmatis CCM 4622 (ATCC 607) (MIC 7.81 g/mL for 15a and 3.9 g/mL
for 15b). In comparison with our previous results [ 31], positive influence of tert-butyl substitution
µ
µ
µ
µ
4,5,
in the position 5 of ring A has been confirmed again during testing on M. tuberculosis, which is the
most pathogenic strain. As for substitution of ring B, 2-halogen substituent seems to be of importance,
as products 13a
, 15b and 17a showed the lowest MICs (3.13–6.25 µg/mL against M. tuberculosis H37RV
CNCTC My 331/88).
In Table 4, calculated lipophilicities of all tested compounds are provided. They do not seem to
correlate with biological activity of the compounds. Conformation of particular molecules influenced
by halogen position may play a more important role.
Table 4. Comparison of antimycobacterial activity of halogenated 3-phenyl-1-pyrazin-2-ylprop-2-en-
1-ones with standard antimycobacterial agents and calculated lipophilicities of the compounds.
Substituent in the Ring
MIC (µg/mL)
Lipophilicity a
Compd.
A
B
MK
MA
MT
MS
LogP
ClogP
13a
13b
13e
14a
14b
15a
15b
16a
16b
16c
16d
16e
16f
17a
17b
17c
17d
17e
17f
18a
18b
18c
18d
18e
18f
H
5-t-bu
5-pro
H
5-t-bu
H
5-t-bu
H
5-t-bu
5-isobu
5-bu
5-pro
5-isopro
H
5-t-bu
5-isobu
5-bu
5-pro
5-isopro
H
5-t-bu
5-isobu
5-bu
2-F
2-F
2-F
4-F
4-F
12.5
12.5
12.5
12.5
>25
25
100
>100
>100
100
12.5
6.25
12.5
25
>25
6.25
3.13
50
>50
>25
>50
25
12.5
25
6.25
>100
25
12.5
12.5
12.5
>50
>100
>50
50
15.625
≥500
15.625
15.625
≥125
7.81
1.49
3.62
3.1
2.11066
3.93666
3.66767
2.11066
3.93666
2.68067
4.50666
2.68067
4.50666
4.63666
4.76666
4.23767
4.10767
2.83067
4.65667
4.78667
4.91667
4.38767
4.25767
2.83067
4.65667
4.78667
4.91667
4.38767
4.25767
5.44566
1.49
3.62
1.89
4.02
1.89
4.02
3.83
3.92
3.50
3.48
2.16
4.39
4.10
4.19
3.77
3.75
2.16
4.29
4.10
4.19
3.77
3.75
4.61
>25
2-Cl
2-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
2-Br
2-Br
2-Br
2-Br
2-Br
2-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
>100
>100
100
>50
>25
12.5
12.5
>50
100
100
>100
>100
>100
50
>100
>50
>100
>50
>50
>100
>50
3.13–6.25
-
25
3.9
6.25
>50
12.5
12.5
12.5
6.25
12.5
12.5
100
25
6.25
12.5
12.5
12.5
25
>50
12.5
6.25
>50
3.13–12.5
-
15.625
≥250
≥125
≥250
≥500
≥125
15.625
62.5
62.5
15.625
≥500
15.625
62.5
≥250
250
125
5-pro
5-isopro
5-pent
≥250
≥500
≥250
7.81–15.625
0.78–1.56
0.098–0.195
25
18g
>50
0.1–0.39
-
Isoniazid
Rifampicin
Ciprofloxacin
-
-
-
MK = Mycobacterium kansasii Hauduroy CNCTC My 235/80; MA = M. avium ssp. avium Chester CNCTC My
80/72; MT = M. tuberculosis H37RV CNCTC My 331/88; MS = Mycobacterium smegmatis CCM 4622 (ATCC
607); a lipophilicity calculated by ChemDraw Professional 15.0, part of ChemOffice (Perkin Elmer, Waltham,
MA, USA).
3. Experimental Section Materials and Methods
3.1. Chemistry
3.1.1. Materials and Methods
Pyrazine-2-carbonitrile, 2-fluorobenzaldehyde, 4-fluorobenzaldehyde, 2-chlorobenzaldehyde,
2-bromobenzaldehyde and 4-bromobenzaldehyde were used as starting compounds and were
purchased from Sigma-Aldrich (Prague, Czech Republic).

Molecules 2016, 21, 1421
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5-Alkylated pyrazine-2-carbonitriles and 5-alkylated pyrazin-2-ylethan-1-ones were prepared
from pyrazinecarbonitrile according to previously published procedures [29
intermediates was confirmed by TLC.
,30] and identity of these
Silica gel 0.040–0.063 nm (Merck, Darmstadt, Germany) and Silpearl (Kavalier, Votice,
Czech Republic) were used for flash column chromatography. The purity of the products was checked
by TLC on aluminium sheets, silica gel 60 F254 (Merck). Mixtures of hexane and ethyl acetate were used
for TLC and column chromatography. Analytical samples were dried over anhydrous phosphorus
pentoxide under reduced pressure at room temperature. Melting points were determined either
on a Boëtius apparatus or on Stuart SMP 20 (Bibby Scientific Ltd., Stone, UK) and are uncorrected.
Elemental analyses were performed on an EA 1110 CHNS instrument (CE Instruments, Milano, Italy)
or on a Vario Micro Cube Elemental Analyzer (Elementar Analysensysteme GmbH, Hanau, Germany).
Infrared spectra were recorded either in KBr pellets on a Nicolet Impact 400 IR spectrophotometer
(Thermo Scientific, Waltham, MA, USA) or on germanium crystal using ATR method (indicated at
particular compound spectra) on a Nicolet Impact 6700 IR spectrophotometer (Thermo Scientific).
1
Characteristic wavenumbers are given in cm−1. H- and 13C-NMR spectra were recorded at ambient
temperature on a Varian Mercury-Vx BB 300 spectrometer (Varian Corp., Palo Alto, CA, USA) operating
at 300 MHz for 1H and 75 MHz for 13C or on a VNMR S500 (500 MHz for 1H-NMR a 125 MHz for
13C-NMR). Chemical shifts were recorded as
δ values in ppm, and were indirectly referenced to
tetramethylsilane (TMS) via the solvent signal (2.49 for 1H, 39.7 for 13C in DMSO-d6 and 7.26 for 1H,
77.0 for 13C in CDCl3). Coupling constants J are given in Hz.
3.1.2. Synthesis of Halogenated (E)-1-(Pyrazin-2-yl)-3-phenylprop-2-en-1-ones
1-(Pyrazin-2-yl)ethan-1-one or 1-(5-alkylpyrazin-2-yl)ethan-1-one (0.01 mol) and the
corresponding halogenated benzaldehyde (0.01 mol) were dissolved in pyridine (4.4 mL).
Diethylamine (0.01 mol) was added, and the reaction mixture was stirred at 80–120 ◦C for 1 h. After
cooling, the mixture was poured into ice water (200 mL), acidified to pH 3 with a few drops of acetic
acid, and then refrigerated for 24 h. The separation of crude products from water depended on their
character. Solids were filtered off and crystallized from anhydrous ethanol, while oily mixtures were
extracted with diethyl ether and subjected to flash chromatography on silica gel. Hexane–ethyl acetate
was used as the eluent in an appropriate ratio (70:30 (v/v), 80:20 (v/v) or 90:10 (v/v)). The fractions
containing the desired compounds were combined and crystallized from absolute ethanol to obtain
analytically pure crystals. Using this procedure, the following compounds were obtained:
(2E)-3-(2-Fluorophenyl)-1-(pyrazin-2-yl)prop-2-en-1-one (13a). Light yellow powder; yield 18%, m.p.
110–111 ◦C (subl.) (114–115 ◦C [43]); IR (ATR-Ge) 1015, 1056, 1334 (pyrazine) 1597 (C=C), 1670 (C=O);
1H-NMR (300 MHz, CDCl3)
H-50), 8.23 (d, 1H, J = 16.2,
1H, H-4), 7.24–7.08 (2H, H3, H-5); 13C-NMR (75 MHz, CDCl3)
δ
9.36 (d, 1H, J = 1.4, H-30), 8.37 (d, 1H, J = 2.5, H-60), 8.70–8.68 (m, 1H,
β
-CH), 8.10 (d, 1H, J = 16.2,
α
-CH), 7.80–7.70 (m, 1H, H-6), 7.45–7.35 (m,
188.6, 161.9 (d, J = 255.1), 148.3, 147.5,
δ
144.9, 143.4, 138.0 (d, J = 3.2), 132.4 (d, J = 8.6), 129.5 (d, J = 2.6), 124.5 (d, J = 3.7), 122.9 (d, J = 11.4),
123.3 (d, J = 6.3), 116.3 (d, J = 22.1); elem. anal. calcd. for C13H9FN2O (228.23) 68.42% C; 3.97% H;
12.27% N, found 68.20% C; 4.09% H; 12.55% N.
(2E)-1-(5-tert-Butylpyrazin-2-yl)-3-(2-fluorophenyl)prop-2-en-1-one (13b). Light yellow powder; yield 13%;
m.p. 97–99◦C; IR (ATR-Ge) 1014, 1237, 1282 (pyrazine) 1604 (C=C), 1671 (C=O); 1H-NMR (500 MHz,
CDCl3)
7.74 (d, 1H, J = 7.6, H-6), 7.42–7.35 (m, 1H, H-4), 7.19 (t, 1H, J = 7.6, H-5), 7.015–7.09 (m, 1H, H-3), 1.44
(s, 9H, CH3); 13C-NMR (125 MHz, CDCl3)
188.6, 167.7, 161.9 (d, J = 255.3), 145.6, 143.3, 139.9, 137.4
δ β-CH), 8.08 (d, 1H, J = 16.1, α-CH),
9.27 (s, 1H, H-30), 8.73 (s, 1H, H-60), 8.23 (d, 1H, J = 16.1,
δ
(d, J = 2.9), 132.1 (d, J = 8.9), 129.4 (d, J = 3.0), 124.4 (d, J = 3.9), 123.0 (d, J = 11.8), 122.7 (d, J = 5.8), 116.2
(d, J = 21.6), 37.1, 30.9; elem. anal. calcd. for C17H17FN2O (284.33) 71.81% C; 6.03% H; 9.85% N, found
71.72% C; 6.36% H; 10.28% N.

Molecules 2016, 21, 1421
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(2E)-3-(2-Fluorophenyl)-1-(5-propylpyrazin-2-yl)prop-2-en-1-one (13e). Light yellow crystals; yield 20%;
m.p. 66–67◦C; IR (ATR-Ge) 1018, 1232, 1321 (pyrazine) 1603 (C=C), 1672 (C=O), 1H-NMR (500 MHz,
CDCl3)
7.77–7.72 (m, 1H, H-6), 7.41–7.35 (m, 1H, H-4), 7.18 (t, 1H, J = 7.3, H-5), 7.14–7.09 (m, 1H, H-3), 2.88
(t, 2H, J = 7.5, CH2), 1.87–1.76 (m, 2H, CH2), 0.99 (t, 3H, J = 7.5, CH3), 13C-NMR (125 MHz, CDCl3)
δ β-CH), 8.08 (d, 1H, J = 16.1, α-CH),
9.26 (s, 1H, H-30), 8.52 (s, 1H, H-60), 8.22 (d, 1H, J = 16.1,
δ
188.6, 161.9 (d, J = 255.1), 161.1, 146.0, 144.1, 142.8, 137.5 (d, J = 3.0), 132.2 (d, J = 8.8), 129.4 (d, J = 2.9),
124.4 (d, J = 3.9), 123.0 (d, J = 10.8), 122.6 (d, J = 5.9), 116.2 (d, J = 21.5), 37.7, 22.5, 13.7; elem. anal. calcd.
for C16H15FN2O (270.30) 71.10% C; 5.59% H; 10.36% N, found 71.15% C; 5.88% H; 10.89% N.
◦
(2E)-3-(4-Fluorophenyl)-1-(pyrazin-2-yl)prop-2-en-1-one (14a). Yellow crystals; yield 6%; m.p. 120–124 C;
IR (ATR-Ge) 1016, 1058, 1231 (pyrazine), 1589 (C=C), 1671 (C=O), 1H-NMR (300 MHz, CDCl3)
δ
9.37 (s,
-CH),
188.4, 164.3
1H, H-30), 8.77 (s, 1H, H-50), 8.68 (s, 1H, H-60), 8.11 (d, 1H, J = 15.9,
β-CH), 7.93 (d, 1H, J = 15.9, α
7.78–7.65 (m, 2H, H-2, H-6), 7.11 (t, 2H, J = 8.2, H-3, H-5), 13C-NMR (75 MHz, CDCl3)
δ
(d, J = 252.8), 148.3, 147.5, 144.8, 144.3, 143.3, 131.0 (d, J = 3.4), 130.9 (d, J = 8.6), 119.7 (d, J = 2.3), 116.2
(d, J = 22.0); elem. anal. calcd. for C13H9FN2O (228.23) 68.42% C; 3.97% H; 12.27% N, found 67.98% C;
4.12% H; 12.17% N.
(2E)-1-(5-tert-Butylpyrazin-2-yl)-3-(4-fluorophenyl)prop-2-en-1-one (14b). Light yellow powder; yield 15%;
◦
m.p. 136–138 C; IR (ATR-Ge) 1011, 1139, 1228 (pyrazine), 1595 (C=C), 1669 (C=O); 1H-NMR (300 MHz,
CDCl3)
J = 16.0, α-CH), 7.75–7.66 (m, 2H, H-2, H-6), 7.11 (t, 2H, J = 8.7, H-3, H-5), 1.45 (s, 9H, CH3); 13C-NMR
(75 MHz, DMSO) 188.5, 167.7, 164.2 (d, J = 252.3), 145.6, 143.8, 143.3, 139.8, 131.2 (d, J = 3.5), 130.8
δ β-CH), 7.90 (d, 1H,
9.28 (d, 1H, J = 1.5, H-30), 8.73 (d, 1H, J = 1.5, H-60), 8.10 (d, 1H, J = 16.0,
δ
(d, J = 8.6), 120.1 (d, J = 2.3), 116.1 (d, J = 21.7), 37.1, 29.7; elem. anal. calcd. for. C17H17FN2O (M 284.33)
71.81% C; 6.03% H; 9.85% N, found 71.54% C; 6.22% H; 10.16% N.
(2E)-3-(2-Chlorophenyl)-1-(pyrazin-2-yl)prop-2-en-1-one (15a). Light yellow needles; yield 38%; m.p.
132.4–134.2 ◦C (127–129 ◦C [43]; IR (ATR-Ge) 1015, 1060, 1330 (pyrazine), 1599 (C=C), 1668 (C=O),
1H-NMR (500 MHz, CDCl3)
δ
9.39 (d, 1H, J = 1.5, H-30), 8.79 (d, 1H, J = 2.5, H-50), 8.71–8.69 (m, 1H,
0
H-6 ), 8.40 (d, 1H, J = 16.1,
β-CH), 8.17 (d, 1H, J = 16.1,
α
-CH), 7.88 (dd, 1H, J = 7.8, J=2.0, H-6), 7.47–7.44
188.3, 148.3, 147.5, 144.9,
(m, 1H, H-3), 7.38–7.30 (m, 2H, H-4, H-5), 13C-NMR (125 MHz, CDCl3)
δ
143.3, 141.3, 136.0, 133.0, 131.6, 130.3, 128.0, 127.1, 122.4 elem. anal. calcd. for C13H9ClN2O (M 244.68)
63.82% C; 3.71% H; 11.45% N, found 63.76% C; 3.78% H; 11.42% N.
(2E)-1-(5-tert-Butylpyrazin-2-yl)-3-(2-chlorophenyl)prop-2-en-1-one (15b). Light yellow powder; yield
16%; m.p. 99.8–101.0 ◦C IR (ATR-Ge) 1014, 1139, 1272 (pyrazine), 1598 (C=C), 1670 (C=O); 1H-NMR
(300 MHz, CDCl3)
8.16 (d, 1H, J = 15.9,
H-4, H-5), 1.49 (s, 9H, CH3); 13C-NMR (75 MHz, DMSO)
δ
9.30 (d, 1H, J = 1.5, H-30), 8.74 (d, 1H, J = 1.5, H-60), 8.38 (d, 1H, J = 15.9,
α
β
-CH),
-CH), 7.87 (d, 1H, J = 7.3, J = 2.0, H-6), 7.47–7.43 (m, 1H, H-3), 7.37–7.29 (m, 2H,
188.4, 167.8, 145.6, 143.4, 140.7, 139.8, 135.9,
δ
133.1, 131.4, 130.3, 128.0, 127.0, 122.8, 37.1, 29.7; elem. anal. calcd for C17H17ClN2O (300.79) 67.88% C;
5.70% H; 9.31% N, found 68.18% C; 5.65% H; 9.42% N.
(2E)-3-(2-Bromophenyl)-1-pyrazin-2-yl)prop-2-en-1-one (17a). Light yellow crystals; yield 50%; m.p.
130.0–133.0 ◦C (128–130 ◦C [43]); IR (KBr) 1016, 1029, 1059, 1330 (pyrazine) 1601 (C=C), 1665 (C=O);
1H-NMR (300 MHz, CDCl3)
J = 1.5, H-60), 8.35 (1H, d, J = 16.1,
7.64 (1H, dd, J = 8.0, J = 1.6, H-6), 7.40–7.33 (1H, m, H-4), 7.30–7.22 (1H, m, H-5); 13C-NMR (75 MHz,
CDCl3) 188.2, 148.2, 147.6, 144.9, 143.9, 143.3, 134.7, 133.6, 131.7, 128.1, 127.7, 126.5, 122.6; elem. anal.
δ
9.38 (1H, d, J = 1.5, H-30), 8.78 (1H, d, J = 2.5, H-50), 8.69 (1H, dd, J = 2.5,
β-H), 8.11 (1H, d, J = 16.1, α-H), 7.85 (1H, dd, J = 8.0, J = 1.6, H-3),
δ
calcd. for C13H9BrN2O (M 289.13) 54.00% C; 3.14% H; 9.69% N, found 53.59% C; 3.27% H; 9.74% N.
(2E)-3-(2-Bromophenyl)-1-(5-tert-butylpyrazin-2-yl)prop-2-en-1-one (17b). Light yellow crystals; yield 40%;
m.p. 91.0–94.5 ◦C; IR (KBr) 1015, 1040, 1316 (pyrazine), 1597 (C=C), 1669 (C=O); 1H-NMR (300 MHz,
CDCl3)
J = 16.1,
δ
9.29 (1H, d, J = 1.5, H-30), 8.73 (1H, d, J = 1.5, H-60), 8.33 (1H, d, J = 16.1,
β
-H), 8.11 (1H, d,
-H), 7.85 (1H, dd, J = 7.7, J = 1.5, H-3), 7.64 (1H, dd, J = 7.7, J = 1.5, H-6), 7.40–7.32 (1H, m,
188.3, 167.8,
α
H-4), 7.25 (1H, td, J = 7.7, J = 1.5, H-5), 1.45 (9H, s, CH3); 13C-NMR (75 MHz, CDCl3)
δ

Molecules 2016, 21, 1421
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145.6, 143.4, 143.3, 139.8, 134.9, 133.6, 131.6, 128.1, 127.7, 126.4, 123.0, 37.1, 29.7; elem. anal. calcd. for
C17H17BrN2O (345.23) 59.14% C; 4.96% H; 8.11% N, found 59.05% C; 5.13% H; 8.21% N.
(2E)-3-(2-Bromophenyl)-1-(5-isobutylpyrazin-2-yl)prop-2-en-1-one (17c). Light yellow crystals; yield 29%;
m.p. 96.0–100.0 ◦C; IR (KBr) 1019, 1049, 1309 (pyrazine), 1598 (C=C), 1698 (C=O); 1H-NMR (300 MHz,
CDCl3)
δ β-H), 8.11 (1H, d,
9.29 (1H, d, J = 1.4, H-30), 8.49 (1H, d, J = 1.4, H-60), 8.33 (1H, d, J = 15.9,
J = 15.9, α-H), 7.85 (1H, dd, J = 7.9, J = 1.7, H-3), 7.64 (1H, dd, J = 7.9, J = 1.7, H-6), 7.40–7.31 (1H, m,
H-4), 7.25 (1H, td, J = 7.9, J = 1.7, H-5), 2.78 (2H, d, J = 6.9, CH2), 2.27–2.08 (1H, m, CH), 0.97 (6H, d,
J = 6.9, CH3); 13C-NMR (75 MHz, CDCl3)
δ 188.3, 160.6, 145.9, 144.2, 143.4, 143.3, 134.9, 133.6, 131.6,
128.2, 127.6, 126.4, 123.0, 44.8, 29.1, 22.3; elem. anal. calcd. for C17H17BrN2O (M 345.24): 59.14% C;
4.96% H; 8.11% N, found 59.31% C; 5.02% H; 8.42% N.
(2E)-3-(2-Bromophenyl)-1-(5-butylpyrazin-2-yl)prop-2-en-1-one (17d). Light yellow crystals; yield 20%;
m.p. 83.0–88.0 ◦C; IR (KBr) 1019, 1051, 1320 (pyrazine), 1602 (C=C), 1674 (C=O); 1H-NMR (300 MHz,
CDCl3)
δ
9.28 (1H, d, J = 1.2, H-30), 8.52 (1H, d, J = 1.2, H-60), 8.33 (1H, d, J = 15.8,
β-H), 8.10 (1H, d,
J = 15.8,
α-H), 7.85 (1H, dd, J = 7.6, J = 1.3, H-3), 7.63 (1H, dd, J = 7.6, J = 1.3, H-6), 7.39–7.32 (1H, m,
H-4), 7.25 (1H, dt, J = 7.6, J = 1.3, H-5), 2.91 (2H, t, J = 7.6, CH2), 1.85–1.69 (2H, m, CH2), 1.50–1.33 (2H,
m, CH2), 0.96 (3H, t, J = 7.6, CH3); 13C-NMR (75 MHz, CDCl3)
δ
188.3, 161.5, 145.9, 144.1, 143.4, 142.8,
134.9, 133.5, 131.6, 128.1, 127.6, 126.4, 123.0, 35.5, 31.4, 22.4, 13.8; elem. anal. calcd. for C17H17BrN2O
(M 345.24) 59.14% C; 4.96% H; 8.11% N, found 59.20% C; 5.34% H; 8.29% N.
(2E)-3-(2-Bromophenyl)-1-(5-propylpyrazin-2-yl)prop-2-en-1-one (17e). Light yellow crystals; yield 24%;
m.p. 105.0–107.0 C; IR (KBr) 1018, 1059, 1317 (pyrazine) 1603 (C=C), 1672 (C=O); 1H-NMR (300 MHz,
◦
CDCl3)
δ β-H), 8.10 (1H, d,
9.28 (1H, d, J = 1.4, H-30), 8.52 (1H, d, J = 1.4, H-60), 8.32 (1H, d, J = 15.9,
J = 15.9, α-H), 7.85 (1H, dd, J = 7.8, J = 1.6, H-3), 7.63 (1H, dd, J = 7.8, J = 1.6, H-6), 7.39–7.32 (1H, m,
H-400), 7.25 (1H, td, J = 7.8, J = 1.6, H-5), 2.89 (2H, t, J = 7.4, CH2), 1.91–1.74 (2H, m, CH2), 1.00 (3H, d,
J = 7.4, CH3); 13C-NMR (75 MHz, CDCl3)
δ 188.3, 161.2, 146.0, 144.1, 143.4, 142.8, 134.9, 133.5, 131.6,
128.1, 127.6, 126.4, 123.0, 37.7, 22.6, 13.8; elem. anal. calcd. for C16H15BrN2O (M 331.21) 58.02% C;
4.57% H; 8.46% N, found 58.01% C; 4.70% H; 8.49% N.
(2E)-3-(2-Bromophenyl)-1-(5-isopropylpyrazin-2-yl)prop-2-en-1-one (17f). Light yellow needles; yield 33%;
◦
m.p. 71.0–73.0 C; IR (KBr) 1016, 1313 (pyrazine) 1602 (C=C), 1668 (C=O); 1H-NMR (300 MHz, CDCl3)
δ
9.29 (1H, d, J = 1.4, H-30), 8.55 (1H, d, J = 1.4, H-60), 8.33 (1H, d, J = 15.9,
-H), 7.85 (1H, dd, J = 7.8, J = 1.6, H-3), 7.64 (1H, dd, J = 7.8, J = 1.6, H-6), 7.39–7.32 (1H, m, H-400),
β-H), 8.11 (1H, d, J = 15.9,
α
7.25 (1H, td, J = 7.8, J = 1.6, H-5), 3.32–3.12 (1H, m, CH), 1.38 (6H, d, J = 6.9, CH3); 13C-NMR (75 MHz,
CDCl3) 188.3, 165.8, 146.1, 144.0, 143.4, 141.5, 134.9, 133.6, 131.6, 128.1, 127.7, 126.4, 123.0, 34.4, 22.0;
δ
elem. anal. calcd. for C16H15BrN2O (M 331.21) 58.02% C; 4.57% H; 8.46% N, found 58.11% C; 4.57% H;
8.81% N.
(2E)-3-(4-Bromophenyl)-1-(pyrazin-2-yl)prop-2-en-1-one (18a). Light yellow crystals; yield 5%; m.p.
◦
1
163.0–168.0 C; IR (KBr) 1017, 1059, 1332 (pyrazine) 1605 (C=C), 1671 (C=O); H-NMR 0(300 MHz,
0
0
CDCl3)
d, J = 16.1,
CDCl3) 188.4, 148.2, 147.6, 144.9, 144.2, 143.3, 133.6, 132.2, 130.2, 125.3, 120.5; elem. anal. calcd for
δ 9.37 (1H, d, J = 1.5, H-3 ), 8.78 (1H, d, J = 2.5, H-6 ), 8.69 (1H, dd, J = 2.5, J = 1.5, H-5 ), 8.17 (1H,
β
-H), 7.89 (1H, d, J = 16.1, α
-H), 7.61–7.52 (4H, m, H-2, H-3, H-5, H-6); 13C-NMR (75 MHz,
δ
C13H9BrN2O (289.13) 54.00% C; 3.14% H; 9.69% N, found 53.19% C; 3.58% H; 9.21% N.
(2E)-3-(4-Bromophenyl)-1-(5-tert-butylpyrazin-2-yl)prop-2-en-1-one (18b). Light yellow powder; yield 2%;
◦
m.p. 139.0–142.0 C; IR (KBr) 1020, 1041, 1302 (pyrazine), 1603 (C=C), 1669 (C=O); 1H-NMR (300 MHz,
CDCl3)
δ β-H), 7.87 (1H, d,
9.28 (1H, d, J = 1.4, H-30), 8.73 (1H, d, J = 1.4, H-60), 8.17 (1H, d, J = 16.1,
J = 16.1, α-H), 7.62–7.51 (4H, m, H-2, H-3, H-5, H-6), 1.45 (9H, s, CH3); 13C-NMR (75 MHz, CDCl3)
δ
188.5, 167.8, 145.6, 143.6, 143.3, 139.9, 133.8, 132.2, 130.2, 125.1, 120.9, 37.1, 29.7; elem. anal. calcd for
C17H17BrN2O (345.24) 59.14% C; 4.96% H; 8.11% N, found 59.34% C; 5.14% H; 8.24% N.

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(2E)-3-(4-Bromophenyl)-1-(5-isobutylpyrazin-2-yl)prop-2-en-1-one (18c). Light yellow needles; yield 3%;
◦
m.p. 109.0–111.0 C; IR (KBr) 1020, 1043, 1304 (pyrazine) 1602 (C=C), 1668 (C=O); 1H-NMR (300 MHz,
CDCl3) -H), 7.87 (1H, d,
9.28 (1H, d, J = 1.4, H-30), 8.49 (1H, d, J = 1.4, H-60), 8.17 (1H, d, J = 16.1,
J = 16.1, α-H), 7.61–7.51 (4H, m, H-2, H-3, H-5, H-6), 2.79 (2H, d, J = 6.9, CH2), 2.27–2.08 (1H, m, CH),
0.97 (6H, d, J = 6.9, CH3); 13C-NMR (75 MHz, CDCl3)
188.4, 160.6, 146.0, 144.0, 143.8, 143.3, 133.7,
δ
β
δ
132.2, 130.2, 125.2, 120.8, 44.7, 29.2, 22.3; elem. anal. calcd. for C17H17BrN2O (345.24) 59.14% C; 4.96%
H; 8.11% N, found 58.96% C; 5.21% H; 8.21% N.
(2E)-3-(4-Bromophenyl)-1-(5-butylpyrazin-2-yl)prop-2-en-1-one (18d). Light yellow crystals; yield 2%; m.p.
◦
115.0–116.0 C; IR (KBr) 1009, 1071, 1330 (pyrazine), 1600 (C=C), 1669 (C=O); 1H-NMR (300 MHz,
CDCl3)
J = 16.2, α-H), 7.62–7.51 (4H, m, H-2, H-3, H-5, H-6), 2.92 (2H, t, J = 7.5, CH2), 1.85–1.70 (2H, m, CH2),
1.50–1.34 (2H, m, CH2), 0.96 (3H, t, J = 7.5, CH3); 13C-NMR (75 MHz, CDCl3)
188.4, 161.5, 145.9, 144.1,
δ β-H), 7.87 (1H, d,
9.27 (1H, d, J = 1.4, H-30), 8.52 (1H, d, J = 1.4, H-60), 8.16 (1H, d, J = 16.2,
δ
143.7, 142.8, 133.8, 132.2, 130.2, 125.1, 120.9, 35.5, 31.4, 22.4, 13.82; elem. anal. calcd. for C17H17BrN2O
(345.24) 59.14% C; 4.96% H; 8.11% N, found 59.05% C; 5.33% H; 8.40% N.
(2E)-3-(4-Bromophenyl)-1-(5-propylpyrazin-2-yl)prop-2-en-1-one (18e). Light yellow crystals; yield 4%; m.p.
99.0–100.0 ◦C; IR (KBr) 1009, 1045, 1071, 1321 (pyrazine) 1600 (C=C), 1670 (C=O); 1H-NMR (300 MHz,
CDCl3)
J = 15.9, α-H), 7.61–7.51 (4H, m, H-2, H-3, H-5, H-6), 2.90 (2H, d, J = 7.6, CH2), 1.91–1.75 (1H, m, CH2),
1.01 (3H, t, J = 7.6, CH3); 13C-NMR (75 MHz, CDCl3)
188.4, 161.2, 146.0, 144.0, 143.7, 142.9, 133.8,
δ β-H), 7.87 (1H, d,
9.27 (1H, d, J = 1.4, H-30), 8.52 (1H, d, J = 1.4, H-60), 8.16 (1H, d, J = 15.9,
δ
132.2, 130.2, 125.1, 120.8, 37.7, 22.6, 13.8; elem. anal. calcd. for C16H15BrN2O (331.21) 58.02% C; 4.57%
H; 8.46% N, found 57.63% C; 4.80% H; 8.57% N.
(2E)-3-(4-Bromophenyl)-1-(5-isopropylpyrazin-2-yl)prop-2-en-1-one (18f). Light yellow crystals; yield 4%;
◦
1
m.p. 106.0–108.0 C; IR (KBr) 1017, 1040, 1068, 1334 (pyrazine) 1600 (C=C), 1667 (C=O); H-NMR
(300 MHz, CDCl3) -H),
9.27 (1H, d, J = 1.5, H-30), 8.56 (1H, d, J = 1.5, H-60), 8.16 (1H, d, J = 15.8,
7.87 (1H, d, J = 15.8, -H), 7.61–7.52 (4H, m, H-2, H-3, H-5, H-6), 3.31–3.15 (1H, m, CH), 1.38 (6H, d,
J = 7.0, CH3);13C-NMR (75 MHz, CDCl3)
188.4, 165.8, 146.2, 143.9, 143.7, 141.5, 133.8, 132.2, 130.2,
δ
β
α
δ
125.1, 120.9, 34.3, 22.0; elem. anal. calcd. for C16H15BrN2O (331.21) 58.02% C; 4.57% H; 8.46% N, found
57.92% C; 4.75% H; 8.52% N.
(2E)-3-(4-Bromophenyl)-1-(5-pentylpyrazin-2-yl)prop-2-en-1-one (18g). Light yellow needles; yield 6%; m.p.
98.0–100.0 ◦C; IR (KBr) 1010, 1036, 1075, 1320 (pyrazine) 1603 (C=C), 1669 (C=O); 1H-NMR (300 MHz,
CDCl3)
J = 16.2 Hz,
CH2), 1.46–1.28 (4H, m, CH2), 0.90 (3H, t, J = 7.4, CH3); 13C-NMR (75 MHz, CDCl3)
δ
9.27 (1H, d, J = 1.1, H-30), 8.52 (1H, d, J = 1.1, H-60), 8.16 (1H, d, J = 16.2,
β
-H), 7.87 (1H, d,
-H), 7.62–7.51 (4H, m, H-2, H-3, H-5, H-6), 2.91 (2H, t, J = 7.4, CH2), 1.87–1.71 (2H, m,
188.4, 161.5,
α
δ
145.9, 144.1, 143.7, 142.8, 133.8, 132.2, 130.2, 125.1, 120.9, 35.8, 31.4, 29.0, 22.4, 13.9; elem. anal. calcd. for
C18H19BrN2O (359.27) 60.18% C; 5.33% H; 7.80% N, found 59.72% C; 5.58% H; 7.88% N.
3.2. Biological Evaluation
3.2.1. Evaluation of In Vitro Antifungal Activity
The antifungal activity of all compounds was evaluated by the modified microdilution
broth CSLI standards [44,45]. The organisms examined included Candida albicans ATCC 44859
(American Type Culture Collection, Manassas, VA, USA), Candida tropicalis 156, Candida krusei E 28,
Candida glabrata 20/I, Trichosporon asahii 1188, Aspergillus fumigatus 231, Lichtheimia corymbifera 272, and
Trichophyton interdigitale 445. All strains, except ATCC one tested, are clinical isolates obtained from
the Department of Clinical Microbiology, University Hospital and Faculty of Medicine, Charles
University, Prague, Czech Republic. Before testing, each strain was subcultured on Sabouraud
dextrose agar (SDA; Difco/Becton Dickinson, Detroit, MI, USA) and maintained on the same
medium at 4 ◦C. Fungal inocula were prepared by suspending yeasts, conidia, or sporangiospores
in sterile 0.85% saline. The cell density was adjusted using a Bürker’s chamber to yield a stock

Molecules 2016, 21, 1421
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suspension of 1.0 ± 0.2 × 105 colony forming units (CFU)/mL and 1.0
±
0.2
×
106 CFU/mL for
yeasts and molds, respectively. The final inoculum was made by 1:20 dilution of the stock suspension
with the test medium. The compounds were dissolved in DMSO, and the antifungal activity was
determined in RPMI 1640 media (KlinLab, Prague, Czech Republic) buffered to pH 7.0 with 0.165 M
3-morpholinopropane-1-sulfonic acid (Sigma-Aldrich, St. Louis, MO, USA). 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. The concentrations of the studied substances ranged from 500 to
0.488 µmol/L. The minimum inhibitory concentration (MIC), was defined as 80% or greater (for
yeasts and yeast-like organisms—IC80), resp. 50% or greater (for molds—IC50) reduction of growth in
comparison with the control. The values of MICs were determined after 24 and 48 h of static incubation
◦
at 35 C. In the case of T. interdigitale, the MICs were recorded after 72 and 120 h due to its slow growth
rate. Fluconazole and voriconazole were used as reference antifungal drugs.
3.2.2. Evaluation of In Vitro Antibacterial Activity
The antibacterial activity of all compounds was evaluated by the microdilution broth method [46].
The organisms examined included strains from Czech Collection of Microorganisms (Brno, Czech
Republic): Staphylococcus aureus CCM 4516/08 (SA), Escherichia coli CCM 4517 (EC), Pseudomonas
aeruginosa CCM 1961 (PA). 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 (methicillin resistant, MRSA), Staphylococcus epidermidis H 6966/08 (SE),
Enterococcus sp. J 14365/08 (EF), Klebsiella pneumoniae D11750/08 (KP), Klebsiella pneumoniae J 14368/08
(ESBL positive, KP-E). All strains were subcultured on Mueller-Hinton agar (MHA) (Difco/Becton
Dickinson, Detroit, MI) 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 using densitometer to yield suspension of density
equivalent 0.5 McFarland scale which is equal to a number of 1. 5 × 108 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. Neomycin, bacitracin and penicillin have been used as reference
antibacterial drugs.
3.2.3. Evaluation of In Vitro Antimycobacterial Activity on Mycobacterium tuberculosis H37RV CNCTC
My 331/88, M. kansasii Hauduroy CNCTC My 235/80 and M. avium ssp. avium Chester CNCTC
My 80/72
Microdilution panel method was used. Tested strains Mycobacterium tuberculosis H37RV CNCTC
My 331/88, M. kansasii Hauduroy CNCTC My 235/80 and M. avium ssp. avium Chester CNCTC
My 80/72 were obtained from Czech National Collection of Type Cultures (CNCTC), National
Institute of Public Health, Prague, Czech Republic. Middlebrook 7H9 broth (Sigma-Aldrich, Steinheim,
Germany) enriched with 0.4% of glycerol (Sigma-Aldrich) and 10% of OADC supplement (oleic acid,
albumin, dextrose, catalase; Himedia, Mumbai, India) of declared pH 6.6 was used for cultivation.
Tested compounds were dissolved and diluted in DMSO and mixed with broth (25
solution in 4.475 mL of broth) and placed (100 L) into microtitration plate wells. Mycobacterial
inocula were suspended in isotonic saline solution and the density was adjusted to 0.5–1.0 McFarland.
These suspensions were diluted by 10−1 and used to inoculate the testing wells, adding 100
L of
suspension to 100 L of the DMSO/broth solution of tested compound. Final concentrations of tested
compounds in wells were 100, 50, 25, 12.5, 6.25, 3.13 and 1.56 g/mL. Isoniazid was used as positive
µL of DMSO
µ
µ
µ
µ

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control (inhibition of growth). Negative control consisted of broth plus DMSO. 30
µL of Alamar Blue
working solution (1:1 mixture of 0.01% resazurin sodium salt (aq. sol.) and 10% aqueous solution of
Tween 80) was added usually after 5 days of incubation. Results were then determined after 24 h of
incubation and interpreted according to Franzblau [47]. The MIC (
µg/mL) was determined as the
lowest concentration which prevented the blue to pink colour change.
3.2.4. Evaluation of In Vitro Antimycobacterial Activity on Mycobacterium smegmatis
An antimycobacterial assay was performed with fast growing Mycobacterium smegmatis CCM 4622
(ATCC 607) from Czech Collection of Microorganisms (Brno, Czech Republic). The technique used
for activity determination was microdilution broth panel method using 96-well microtitration plates.
Culturing medium was Middlebrook 7H9 (MB) broth (Sigma-Aldrich) enriched with 0.4% of glycerol
(Sigma-Aldrich) and 10% of Middlebrook OADC growth supplement (Himedia, Mumbai, India).
Tested compounds were dissolved in DMSO (Sigma-Aldrich) then MB broth was added to obtain
concentration 2000 µg/mL. Standards used for activity determination were isoniazid (INH), rifampicin
(RIF) and ciprofloxacin (CPX) (Sigma-Aldrich). Final concentrations were reached by twofold dilution
and addition of mycobacterial suspension and were set as 500, 250, 125, 62.5, 31.25, 15.625, 7.81 and
3.91
µg/mL except to standards ciprofloxacin and rifampicin where the final concentrations were
12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195 and 0.098
µg/mL. Drug-free controls consisted of broth and
DMSO and were used as a growth control. The final concentration of DMSO did not exceeded 2.5%
(v/v) to not affect the growth of M. smegmatis. Plates were sealed with polyester adhesive film and
incubated in the dark at 37 ◦C without agitation. The addition of 0.01% solution of resazurin sodium
salt followed after 48 h. This stain was prepared by dissolving resazurin sodium salt (Sigma-Aldrich)
in deionised water to get 0.02% solution. Then 10% aqueous solution of Tween 80 (Sigma-Aldrich)
was prepared. Both liquids were mixed up making use of the same volumes and filtered through
syringe membrane filter. Microtitration plates were then incubated for further 4 h. Antimycobacterial
activity was expressed as MIC and the value was determined on the basis of stain color change (blue
color—active compound; pink color—non active compound). MIC values for standards were within
the ranges 7.81–15.625 µg/mL for INH, 0.78–1.56 µg/mL for RIF and 0.098–0.195 µg/mL for CPX.
All experiments were conducted in duplicate.
3.3. Calculation of Lipophilicity
Theoretical lipophilicities logP and corrigated values ClogP of all compounds have been calculated
in ChemDraw Professional 15.0, part of ChemOffice (Perkin Elmer, Waltham, MA, USA).
4. Conclusions
Chalcones have a very simple chemistry, which enables multiplicity of substitutions with
easy synthesis and different pharmacological potential in dependence on particular structural
modification [13]. Within this work, twenty halogenated pyrazine-based compounds have been
prepared and characterized. They are all novel compounds with exception of three compounds.
They were tested on antifungal, antibacterial, as well as on antimycobacterial effects. 4-Chlorinated
series, prepared earlier, have been included in the assays. The results of biological screenings have
been compared with our previously published compounds. Importance of an EWG substitution has
been confirmed and some compounds displayed comparable or even better inhibitory activity than
standard antifungals, antibiotics and antimycobacterial agents.
As far as the structure-activity relationships are concerned, halogen substitution in the ring B of
pyrazine-based chalcones proved to have positive influence on antifungal effect against Candida spp.
and Trichophyton interdigitale in comparison with our previously prepared series of pyrazine-based
chalcones [4,5,31]. Generally, chlorinated and brominated series inhibited growth of fungi more
significantly than fluorinated derivatives.

Molecules 2016, 21, 1421
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Halogen substitution in the ring B had positive impact on inhibition of Staphylococcus spp., whereas
it did not influence inhibition of other bacteria from the panel. As for specific substitution, 2-chlorinated
derivative inhibited markedly growth of S. epidermidis. Concerning mycobacteria, halogenated series
inhibited significantly growth of M. krusei, M. tuberculosis and M. smegmatis, but they did not strongly
influence the growth of M. avium. The inhibiting activity of halogenated series was comparable with
that of nitro series prepared previously [4,5]. Derivatives substituted by halogen in position 2 of the
ring B and concurrently substituted by tert-butyl in the position 5 of the pyrazine ring showed the
highest inhibition of M. tuberculosis. Importance of tert-butyl substitution has been observed previously
in other series [5]. Presence of isopropyl in position 5 of the pyrazine ring seems to be important in
chlorinated or brominated series in the test against M. krusei.
Acknowledgments: The study was supported by Ministry of Education, Youth and Sports of the Czech Republic,
projects SVV 260 291 and SVV 260 289.
Author Contributions: Marta Kucerova-Chlupacova, Veronika Vyskovska-Tyllova and Lenka Richterova-Finkova
carried out syntheses, interpreted yields, elemental analysis, IR spectra, and antimycobacterial data; Jiri Kunes
recorded and interpreted NMR data; Vladimir Buchta designed antifungal and antibacterial assays and interpreted
their results; Marcela Vejsova performed antifungal and antibacterial assays; Pavla Paterova, Lucia Semelkova and
Ondrej Jandourek carried out the antimycobacterial assays, Veronika Opletalova proposed the subject, designed
the study and together with Marta Kucerova-Chlupacova wrote the paper. All the authors read and approved the
final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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