3-substituted N-benzylpyrazine-2-carboxamide derivatives: Synthesis, antimycobacterial and antibacterial evaluation

Article abstract of DOI:10.3390/molecules22030495

A series of substituted N-benzyl-3-chloropyrazine-2-carboxamides were prepared as positional isomers of 5-chloro and 6-chloro derivatives, prepared previously. During the aminolysis of the acyl chloride, the simultaneous substitution of chlorine with benzylamino moiety gave rise to N-benzyl-3-(benzylamino)pyrazine-2-carboxamides as side products, in some cases. Although not initially planned, the reaction conditions were modified to populate this double substituted series. The final compounds were tested against four mycobacterial strains. N-(2-methylbenzyl) -3-((2-methylbenzyl)amino)pyrazine-2-carboxamide (1a) and N-(3,4-dichlorobenzyl)-3-((3,4-dichlorobenzyl)amino)pyrazine-2-carboxamide (9a) proved to be the most effective against Mycobacterium tuberculosis H37Rv, with MIC = 12.5 μg·mL^-1. Compounds were screened for antibacterial activity. The most active compound was 3-chloro-N-(2-chlorobenzyl) pyrazine-2-carboxamide (5) against Staphylococcus aureus with MIC = 7.81 μM, and Staphylococcus epidermidis with MIC = 15.62 μ·M. HepG2 in vitro cytotoxicity was evaluated for the most active compounds; however, no significant toxicity was detected. Compound 9a was docked to several conformations of the enoyl-ACP-reductase of Mycobacterium tuberculosis. In some cases, it was capable of H-bond interactions, typical for most of the known inhibitors.

Full text of DOI:10.3390/molecules22030495

molecules
Article
3-Substituted N-Benzylpyrazine-2-carboxamide
Derivatives: Synthesis, Antimycobacterial and
Antibacterial Evaluation
Lucia Semelková 1,*, Ondrˇej Jand’ourek 1, Klára Konecˇná 1, Pavla Paterová 2, Lucie Navrátilová 1,
František Trejtnar 1, Vladimír Kubícˇek 1, Jirˇí Kuneš 1, Martin Doležal 1 and Jan Zitko 1,
*
1
Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203, 500 05 Hradec Králové,
Czech Republic; JANDO6AA@faf.cuni.cz (O.J.); konecna@faf.cuni.cz (K.K.); navratl2@faf.cuni.cz (L.N.);
trejtnarf@faf.cuni.cz (F.T.); kubicek@faf.cuni.cz (V.K.); kunes@faf.cuni.cz (J.K.); dolezalm@faf.cuni.cz (M.D.)
Department of Clinical Microbiology, University Hospital, Sokolská 581, 500 05 Hradec Králové,
Czech Republic; pavla.paterova@fnhk.cz
2
*
Correspondence: semelkol@faf.cuni.cz (L.S.); jan.zitko@faf.cuni.cz (J.Z.); Tel.: +420-495-067-275 (L.S.)
Academic Editor: Derek J. McPhee
Received: 6 February 2017; Accepted: 17 March 2017; Published: 21 March 2017
Abstract: A series of substituted N-benzyl-3-chloropyrazine-2-carboxamides were prepared
as positional isomers of 5-chloro and 6-chloro derivatives, prepared previously. During the
aminolysis of the acyl chloride, the simultaneous substitution of chlorine with benzylamino moiety
gave rise to N-benzyl-3-(benzylamino)pyrazine-2-carboxamides as side products, in some cases
Although not initially planned, the reaction conditions were modified to populate this
double substituted series. The final compounds were tested against four mycobacterial
strains. N-(2-methylbenzyl) -3-((2-methylbenzyl)amino)pyrazine-2-carboxamide (1a) and N-(3,4-
dichlorobenzyl)-3-((3,4-dichlorobenzyl)amino)pyrazine-2-carboxamide (9a) proved to be the most
effective against Mycobacterium tuberculosis H37Rv, with MIC = 12.5 µg·mL−1. Compounds were
screened for antibacterial activity. The most active compound was 3-chloro-N-(2-chlorobenzyl)
.
pyrazine-2-carboxamide (5) against Staphylococcus aureus with MIC = 7.81 µM, and Staphylococcus
epidermidis with MIC = 15.62
µM. HepG2 in vitro cytotoxicity was evaluated for the most active
compounds; however, no significant toxicity was detected. Compound 9a was docked to several
conformations of the enoyl-ACP-reductase of Mycobacterium tuberculosis. In some cases, it was capable
of H-bond interactions, typical for most of the known inhibitors.
Keywords: pyrazinamide derivatives; benzylamines; antibacterial activity; antimycobacterial activity;
cytotoxicity; enoyl-ACP-reductase; molecular docking
1. Introduction
Every living organism is bound to evolve in order to survive. Although it is a natural process
spanning millions of years, in the case of pathogenic microorganisms, this adaptation for survival
is rather undesirable from a human point of view. Microorganisms have developed a range of
protective mechanisms to deactivate, remove, or otherwise circumvent the toxicity of anti-infective
compounds [
anti-infective. Practically, resistance to any clinically used anti-infective drug will develop sooner or
later [ ]. Increasing rates of microbial resistance to both commonly used and reserved agents highlight
1]. The appearance of resistance is positively linked to the frequency of use of such an
2
the necessity to develop new effective compounds.
According to the WHO Global Tuberculosis Report 2016, it was estimated that there were
10.4 million new tuberculosis (TB) cases and 1.4 million TB associated deaths in 2015. Although TB
Molecules 2017, 22, 495; doi:10.3390/molecules22030495
www.mdpi.com/journal/molecules

Molecules 2017, 22, 495
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incidence rates have been slowly declining for several consecutive years and an estimated 43 million
lives were saved between 2000 and 2014 through effective diagnosis and treatment, the presence of
resistant forms and co-infection with HIV pose a considerable threat to successful treatment. The
percentage of new TB cases that were multidrug-resistant (MDR-TB) was 3.3% in 2014, whereas
HIV-positive patients represented 12% of the 9.6 million people who developed TB [
(PZA), as a fist-line antitubercular drug, has gained an irreplaceable role in TB therapy and has been
subjected to various chemical modifications [ ]. Although PZA has been used for over sixty years [ ],
it is still not completely known how PZA acts in a mycobacterial cell [8–12].
3]. Pyrazinamide
4–
7
8
5-Chloropyrazinamide, as a fatty acid synthase I (FAS I, involved in mycolic acids biosynthesis)
inhibitor [10], was a pattern for a series of 5- or 6-chloropyrazine-2-carboxamides with N-benzylamino
substitution (Figure 1), prepared and previously reported by Servusová et al. [13
with 4-Cl, 4-Br, or 2,4-diCl substitution on the phenyl ring exerted antimycobacterial activity with a
minimum inhibitory concentration (MIC) of 12.5
mL−1, and 6-Cl derivatives with 4-OCH3, 3-CF3,
or 4-Cl substitution on the phenyl ring showed an MIC of 6.25–12.5 13 14]. As reported in
mL−1
this article, compounds 13 comprising 3-Cl-pyrazine moiety were originally designed as positional
,14]. 5-Cl derivatives
µg
·
µg
·
[ ,
1
–
isomers with respect to the chloro substitution on the pyrazine ring, in order to study the effect of
positional isomerism on anti-infective activity.
Figure 1. Structures of previously reported chloropyrazine-2-carboxamide derivatives with in vitro
antimycobacterial activity (M. tuberculosis H3Rv).
However, dibenzyl substituted derivatives 1a–13a, which initially originated as side-products of
intended monosubstituted derivatives, turned out to be more effective compounds.
As a complementary test, all of our compounds were subject to initial screening for in vitro activity
against clinically important Gram-positive and Gram-negative bacteria, including methicillin-resistant
Staphylococcus aureus (MRSA). Any potential hit compound against MRSA would be highly appreciated
as MRSA has become one of the most feared pathogens in the last few decades [14].
2. Results and Discussion
2.1. Chemistry
Substituted N-benzyl-3-chloropyrazine-2-carboxamides 1–13 were prepared by a three-step
procedure (Scheme 1), according to Ref. [15]. The initial 3-chloropyrazine-2-carbonitrile (Fluorochem
Ltd., Derbysshire, UK) was hydrolysed in 10% (m/m) aqueous NaOH solution. After 7 h, the reaction
mixture was acidified with 10% HCl to pH 3, to obtain crystals of 3-chloropyrazine-2-carboxylic acid
(3-Cl-POA), which were then filtered and dried. In the following step, 3-Cl-POA was treated with
SOCl2 in the presence of a catalytic amount of N,N-dimethylformamide (DMF) in toluene under reflux
for one hour, producing 3-chloropyrazine-2-carbonyl chloride, which was reacted with substituted
benzylamine in the presence of triethylamine (TEA) in acetone at room temperature overnight, to obtain
1
–
13. In the process of amide bond formation with 2-methyl-, 4-methyl-, and 4-methoxybenzylamine,
corresponding N-benzyl-3-(benzylamino)pyrazine-2-carboxamides 1a 3a were formed simultaneously
with as desired side-products in a molar ratio of approximately 2:3, with an excess of 1a–3a.
–
1–
3
The side-products could be easily detected by TLC and easily separated by flash-chromatography.
We decided to complete the disubstituted series. N-Benzyl-3-(benzylamino)pyrazine-2-carboxamides

Molecules 2017, 22, 495
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5a
–
13a were afforded via the aminodehalogenation of
5–
13, with the corresponding substituted
benzylamine and pyridine in methanol under microwave conditions (150 ◦C, 30 min, power output
100 W, focused field) (Scheme 1d). However, compounds 4a (R1,2 = 2,4-OCH3), 8a (R1,2 = 2,4-Cl) and
12a (R1,2 = 3-CF3) could not be prepared under the chosen conditions. This might have been due to the
electron-withdrawing effects of CF3 substitution and/or the steric hindrance of large substituents in
the ortho position of the phenyl ring. All prepared compounds (Table 1) were characterized by 1H-,
13C-NMR, and IR spectroscopy, their melting point, and elemental analysis. All results were fully in
accordance with the proposed structures.
Scheme 1. Synthesis of compounds
reflux 7 h, then 10% aq. sol. HCl; (
acetone, RT, overnight; (d) substituted benzylamine, pyridine, methanol, MW: 150 C, 30 min, 100 W.
1
–
13 and 1a
–
13a. Reagents and conditions: (
a
) 10% aq. sol. NaOH,
◦
b
) SOCl2, DMF, toluene, 95 C, 1 h; (
c) substituted benzylamine, TEA,
◦
Table 1. Prepared structures, calculated (ClogP) and measured (log k) lipophilicity parameters, and
antimycobacterial activity against M. tuberculosis H37Rv and M. smegmatis.
MIC (µg·mL−1) (µM)
R1
R2
ClogP
Log k
No.
MW
M. tuberculosis
M. smegmatis
1
1a
2
2a
3
3a
4
5
5a
6
6a
7
7a
8
9
9a
10
10a
11
11a
12
13
13a
2-CH3
2-CH3
4-CH3
4-CH3
4-OCH3
4-OCH3
2,4-diOCH3
2-Cl
-
261.71
346.43
261.71
346.43
277.71
2.004
4.862
2.054
4.962
1.474
3.802
1.563
2.268
5.390
2.268
5.390
2.268
5.390
2.981
2.861
6.576
1.698
4.250
1.698
4.250
2.438
2.438
5.730
0.009
1.459
0.033
1.517
−0.202
0.981
−0.095
0.033
1.542
0.060
1.515
0.082
1.533
0.375
0.309
1.997
−0.154
1.108
−0.139
1.058
0.110
0.145
1.593
>100
12.5 (36)
>100
25 (72)
25 (90)
50 (132)
>100
>100
>100
100
>100
>100
>50
>100
100
12.5 (27)
>100
>100
>100
>100
250
> 500
>500
>250
125
>500
>500
250
>500
125
>500
500
>250
125
250
>500
125
>500
250
2-CH3
-
4-CH3
-
4-OCH3 378.43
-
-
307.73
282.12
387.26
282.12
387.26
282.12
387.26
316.57
316.57
2-Cl
3-Cl
3-Cl
4-Cl
2-Cl
-
3-Cl
-
4-Cl
-
-
4-Cl
2,4-diCl
3,4-diCl
3,4-diCl
2-F
2-F
4-F
3,4-diCl 456.15
-
265.67
354.36
265.67
354.36
315.68
315.68
454.38
2-F
-
4-F
-
-
4-F
>500
125
>500
>500
3-CF3
4-CF3
4-CF3
>100
>100
>100
4-CF3
INH
-
-
-
-
-
0.2
7.8–15.6

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2.2. Lipophilicity
The lipophilicity parameters log P and ClogP were calculated for compounds by ChemDraw
Ultra, ver. 14.0. Capacity factor k, expressed as log k, was experimentally measured using RP-HPLC.
The values of the lipophilicity parameters ClogP and log k are presented in Table 1. The relationship
between the calculated ClogP and measured log k is linear, with a regression equation:
ClogP = (2.149 ± 0.053) log k + (2.29 ± 0.052); R2 = 0.987; s = 0.185; F = 1622; n = 23
(1)
see Figure 2. ClogP takes into consideration the intramolecular hydrogen bond formed between
carbonyl oxygen and amine hydrogen (Figure 3). Although the precise algorithm of ClogP calculation
is not publically known, its consideration of intramolecular H-bond formation can be documented
by shifting the benzylamino substituent to various positions of the pyrazine core and comparing the
obtained ClogP values. For example, in the case of compound 3a (3-benzylamino derivative) and its 5-
and 6-positional isomers, the calculated ClogP values are 3.802, 3.725, and 3.752. The increased value
for the vicinal disubstituted derivative is caused by the intramolecular H-bonding, which decreases
the probability to form compound-water H-bonds, and therefore, lowers its hydrophilicity. On the
contrary, the intramolecular interactions are not recognized by log P and all the three positional isomers
appeared to share the same predicted lipophilicity of log P = 2.35. As a result, the calculated ClogP
values of the presented compounds have a stronger correlation with the measured log k than the
log P values.
Figure 2. Plot of calculated ClogP on experimentally measured log k values.
Figure 3. Visualization of the intramolecular hydrogen bond (compound 3a).
2.3. Biological Evaluation
2.3.1. In Vitro Antimycobacterial Activity
All prepared compounds were tested against Mycobacterium tuberculosis H37Rv (M. tbc), as well as
non-tuberculous strains of M. avium and M. kansasii. Additionally, M. smegmatis was used as a fast
growing model organism with a highly lipophilic cell wall, similar in composition to the one of M. tbc.
In vitro screening was performed by a Microplate Alamar Blue Assay (MABA) on whole cells. Results

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were expressed as the minimum inhibitory concentration (MIC) in
M. smegmatis are shown in Table 1.
µg
·
mL−1. The results for M. tbc and
From the series of 3-chloro derivatives (
1–
13), the only substance exhibiting significant
activity against M. tbc H37Rv was compound
µM). Relative to the 5-Cl and 6-Cl derivatives published
before [13,14] (Figure 1), the reposition of chlorine to position 3 led to a decrease or loss
3
, with a 4-OCH3 substitution on the benzene
ring with MIC = 25 µg·mL−1 (90
of antimycobacterial activity against M. tbc H37Rv. The most effective compounds from the
3-benzylamino-N-benzylpyrazine-2-carboxamide series (1a
–
13a) were structures 1a (R1,2 = 2-CH3)
M; 9a 27 M) against M. tbc. The
9a) often led to an increase of antimycobacterial
). The presence of
and 9a (R1,2 = 3,4-diCl) with MIC = 12.5
introduction of the second phenyl ring (1a
µ
g
·
mL−1
2a
(
1a 36
µ
µ
,
,
activity when compared to the corresponding single phenyl derivatives (1, 2, 9
electron-donating substituents (2-CH3, 4-CH3, 4-OCH3) proved advantageous for the biological effect.
Electron-withdrawing substituents (F, Cl, CF3) did not produce active compounds, even in double
phenyl compounds, except for 9a (2,4-diCl).
The presented compounds were inactive against M. kansasii and M. avium, even at the highest
tested concentration of 100 µg·mL−1 (or 50 µg·mL−1 for 7a, due to its lower solubility in DMSO).
Five compounds, namely
M. smegmatis with MIC = 125
N-benzyl-3-chloropyrazine-2-carboxamides. On the contrary to the activity against M. tbc, the activity
against M. smegmatis was mutilated by the insertion of the second aromatic ring.
3, 6, 8, 10, and 12, showed modest activity against fast growing
µg
·
mL−1
.
All these compounds belong to the first series of
2.3.2. Antibacterial Activity
Antibacterial assays were performed against eight clinically significant strains (see Experimental
Section). Interesting antibacterial activity against gram-positive Staphylococcus aureus, methicillin-resistant
S. aureus (MRSA), and S. epidermidis, was found in the series of 3-chloro derivatives with a single
benzene core (see Table 2). Compound
MIC = 7.81 M, and against S. epidermidis with MIC = 15.62
positional isomers did not show any antibacterial activity in tested concentrations up to 500
5
(R1 = 2-Cl) exerted the highest activity against S. aureus with
M. Previously published 5-Cl and 6-Cl
M [13 14].
µ
µ
µ
,
Table 2. Notable antibacterial activities of the effective compounds and standards neomycin
and phenoxymethylpenicillin (Penicillin V) against Staphylococcus aureus, methicillin-resistant
Staphyloc-occus aureus (MRSA), and Staphylococcus epidermidis.
MIC (µM) after 24 h
Compound
S. aureus
MRSA
S. epidermidis
2
3
3a
4
5
8
125
31.25
15.62
125
7.81
125
>500
125
62.5
>500
500
500
250
>500
>500
15.62
125
250
9
13
13a
62.5
31.25
62.5
0.12
1.95
250
500
>500
62.5
0.98
31.25
62.5
>500
3.9
Penicillin V
Neomycin
7.81
The insertion of the second aromatic ring generally decreased the antibacterial activity (with
the exception of compound 3a). From the series containing two aromatic moieties (1a 13a), only
–
compounds with 4-OCH3 (3a) and 4-CF3 (13a) displayed antibacterial activity. The most effective
compound against MRSA was 3a (R1,2 = 4-OCH3), with MIC = 62.5 µM.

Molecules 2017, 22, 495
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Compounds not shown in Table 2 did not produce any significant activity against tested
bacterial strains.
2.3.3. Antifungal Activity
Additionally, the compounds were tested against eight fungal strains (see Experimental Section).
No compounds from the presented series showed any activity in antifungal assays up to the highest
tested concentration (500 µM).
2.3.4. Cytotoxicity
The in vitro cytotoxicity of the most active compounds 5, 1a, 3a, and 9a, was evaluated using
the standard human hepatocellular carcinoma cell line HepG2. The results of the experiments are
presented as a concentration, which reduces the viability of the cell population to 50% of the maximal
viability, IC50 (Table 3). The limited solubility of the compounds 1a
medium, did not allow a valid calculation of the IC50 value. Compound
active compound against methicillin sensitive S. aureus, exerted IC50 = 847.2
,
3a, and 9a in the cell culture
(R1 = 2-Cl), the most
M, leading to a superior
5
µ
selectivity index, SI = IC50/MIC = 108.
Table 3. Cytotoxicity of tested substances in HepG2 cells.
Compound
IC50 (µM)
5
847.2
>250 *
>100 *
>100 *
1a
3a
9a
* Measurement at higher concentration not reproducible due to the precipitation of the tested compound in the cell
culture medium.
2.4. Docking Studies
Based on molecular docking studies, various N-benzylpyrazine-2-carboxamides have been
previously suggested as potential inhibitors of mycobacterial enoyl-ACP-reductase (InhA) [ ],
4
an essential enzyme in the synthesis of mycolic acids and the primary target of the first-line
antitubercular isoniazid. In such PZA derivatives, the carbonyl oxygen of the carboxamide moiety
was predicted to form an H-bond network to Tyr158 and 20-OH of the ribose of the NAD+ cofactor,
which is a typical interaction pattern for most of the direct InhA inhibitors derived from triclosan [16].
Most of the compounds presented in this paper with in vitro activity against M. tuberculosis were
disubstituted derivatives with two large benzyl substituents on two adjacent positions of the pyrazine
core. We were interested to find out whether such sterically demanding derivatives would be able
to fit in the active site of InhA in a manner similar to smaller PZA derivatives with a single aryl
substituent. Therefore, we performed molecular docking of the most active dibenzyl derivative 9a
into various conformations of InhA, differing in the size of the active site cavity, which is formed by
the highly flexible substrate-binding loop (Figure S1, Supplementary Materials). Not surprisingly,
9a was not able to fit into closed conformations of InhA (pdb: 2X23; 3FNF) and did not show the
expected ligand-receptor interactions. On the other hand, when an opened conformation of the InhA
receptor was used (pdb: 4R9S, 4TZK, or 5G0S), we were able to identify two different binding modes
for 9a, with scores similar to the score of the co-crystalized ligands and, more importantly, with
ligand-receptor interactions known to be typical for InhA inhibitors. For a detailed depiction of the
predicted binding modes of 9a to InhA and experimental details on the computational procedures, see
the Supplementary Materials.
Although the results of molecular docking are not enough to confirm the inhibition of InhA as
the mechanism of the action of derivative 9a, we have shown that even such sterically demanding

Molecules 2017, 22, 495
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derivatives are able to mimic the poses and interactions of known InhA inhibitors. The selection of a
suitable structure of InhA with an open conformation was crucial in this docking study.
3. Experimental Section
3.1. General
The starting 3-chloropyrazine-2-carbonitrile was purchased from Fluorochem Ltd. (Hadfield,
Derbysshire, UK). Other chemicals were of a reagent or higher grade of purity and were purchased
from Sigma-Aldrich (Steinheim, Germany). The progress of the reactions was monitored by Thin
Layer Chromatography (TLC) (TLC Silica gel 60 F254, Merck, Darmstadt, Germany) with UV detection
using wavelength of 254 nm. Microwave-assisted reactions were performed in a CEM Discover
microwave reactor with a focused field (CEM Corporation, Matthews, NC, USA) connected to an
Explorer 24 autosampler (CEM Corporation), and the equipment was run under CEM’s Synergy
™
software (version 1.38, CEM Corporation) for setting and monitoring the conditions of the reactions.
The temperature of the reaction mixture was monitored indirectly, by an internal infrared sensor. All
obtained products were purified by a preparative flash chromatograph CombiFlash® Rf (Teledyne
Isco Inc., Lincoln, NE, USA). The type of elution was gradient, using a mixture of hexane (LachNer,
Neratovice, Czech Republic) and ethyl acetate (Penta, Prague, Czech Republic) as the mobile phase.
Silica gel (0.040–0.063 nm, Merck, Darmstadt, Germany) was used as the stationary phase. NMR
spectra were taken with spectrometers Varian Mercury-VxBB 300, with frequencies 299.95 MHz for
1
1H and 75.43 MHz for 13C or Varian VNMR S500 (499.87 MHz for H and 125.71 MHz for 13C)
(Varian Corporation, Palo Alto, CA, USA). Chemical shifts were reported in ppm (δ) and were referred
indirectly to tetramethylsilane via the solvent signal. Infrared spectra were recorded with a FT-IR
Nicolet 6700 spectrometer (Thermo Scientific, Waltham, MA, USA), using attenuated total reflectance
(ATR) on Ge crystal. Elemental analyses were performed using a Micro Cube Elemental Analyzer
(Elementar Analysensysteme GmbH, Hanau, Germany). Melting points were assessed by SMP3
Stuart Scientific (Bibby Sterling Ltd., Staffordshire, UK) in an open capillary and are uncorrected. The
lipophilicity parameters log P and ClogP were calculated by software CS ChemBioDraw Ultra 14.0
(CambridgeSoft, Cambridge, MA, USA). The yields of the products refer to chromatographically pure
products after all purification steps and are given in percentage to theoretical yield related to the
starting acid.
3.2. Chemistry
3.2.1. 3-Chloropyrazine-2-carboxylic Acid (3-Cl-POA)
The starting compound 3-chloropyrazine-2-carbonitrile (35.8 mmol) was dispersed in a 10%
solution of sodium hydroxide (125 mmol, 3.5 equiv.) in a round bottom flask. The reaction mixture
was stirred and heated under reflux in an oil bath for 7 h. The progress of the reaction was monitored
by TLC in the system butanol/acetic acid/water (4:1:5). The reaction mixture was acidified by 10%
solution of hydrochloric acid to pH 3. Resulting crystals of 3-chloropyrazine-2-carboxylic acid were
suctioned. The melting point and 1H-NMR corresponded with the literature [17].
3.2.2. General Synthetic Procedure for N-Benzyl-3-chloropyrazine-2-carboxamides 1–13
A total of 0.3 g of 3-Cl-POA (1.9 mmol) was dispersed in dry toluene with thionyl chloride
(0.4 mL, 5.7 mmol, 3 equiv.) and 1–2 drops of N,N-dimethylformamide (DMF) as a catalyst. The
reaction mixture was stirred and heated in a round bottom flask in an oil bath under a condenser,
at 95 ◦C for 1 h. The solvent was evaporated in vacuo and the residue was azeotroped with dry toluene
(3 × 20 mL). The acyl chloride was used for the following step, without purification.
The whole amount of the 3-chloropyrazine-2-carbonyl chloride prepared in the previous
step was dissolved in dry acetone. An appropriate benzylamine (5.7 mmol, 3 equiv., with

Molecules 2017, 22, 495
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respect to the starting acid), along with triethylamine (1.9 mmol, 1 equiv.), were added to the
reaction mixture and stirred at laboratory temperature overnight. The progress of the reaction
was checked by TLC in system hexane/ethyl acetate (1:1 or 2:1). The reaction mixture was
adsorbed to silica by removing the solvents in vacuo and the product was purified by flash
chromatography using gradient elution with ethyl acetate in hexane. For compounds
corresponding N-benzyl-3-(benzylamino)pyrazine-2-carboxamides 1a 3a were formed simultaneously
in a molar ratio of approximately 2:3, with an excess of 1a 3a. Rf in hexane/ethyl acetate 1:1 mobile
phase: 1a—0.85, —0.49, 2a—0.84, —0.51, 3a—0.71, and —0.40. After flash-chromatography,
1–3,
–
–
1
2
3
compounds 2, 2a, 3, 3a, 4, 8, and 13 were recrystallized from EtOH/H2O.
3.2.3. General Synthetic Procedure for N-Benzyl-3-(benzylamino)pyrazine-2-carboxamides (5a–7a,
9a–11a and 13a)
Compounds 5a–7a, 9a–11a, and 13a were synthetized using a microwave reactor with a focused
field. Corresponding N-benzyl-3-chloropyrazine-2-carboxamide (0.6 mmol) was dissolved in methanol
(3 mL) and appropriate benzylamine (1.8 mmol, 3 equiv.), along with pyridine (40 mg, 0.6 mmol,
1 equiv.) as a base, were added. Our previous observations revealed that triethylamine (TEA) as a base
cannot be used for microwave reactions. During the procedure, TEA is partially decomposed (probably
to diethylamine and similar species, which may act as undesired nucleophiles in the dehalogenation
reaction). The use of pyridine as the base combined with benz◦ylamines was experimentally verified
in previous projects [18]. Conditions for synthesis were 150 C, 30 min, and 100 W. The progress
of the reactions was monitored by TLC in system hexane/ethyl acetate 1:1. The reaction mixture
was adsorbed on silica by removing the solvents in vacuo and the product was purified by flash
chromatography using gradient elution with ethyl acetate (0–100%) in hexane. Products 7a and 10a
were recrystallized from EtOH/H2O.
3.3. Analytical Data of Compounds
3-Chloro-N-(2-methylbenzyl)pyrazine-2-carboxamide (
(ATR-Ge, cm−1): 3292, 1652 (C=O, CONH); 1H-NMR (500 MHz, CDCl3)
8.44 (d, J = 2.3 Hz, 1H, pyr.), 7.73 (bs, 1H, NH), 7.34–7.17 (m, 4H, Ar), 4.65 (d, J = 5.7 Hz, 2H, CH2), 2.39
(s, 3H, CH3); 13C-NMR (126 MHz, CDCl3)
161.67, 148.25, 145.62, 143.10, 140.51, 136.48, 135.17, 130.54,
1
). Ochre solid. Yield 18%; m.p. 94.5–96.5 ◦C; IR
8.52 (d, J = 2.3 Hz, 1H, pyr.),
δ
δ
128.65, 127.91, 126.22, 41.89, 19.04; Elemental analysis: calc. for C13H12ClN3O (MW 261.71): 59.66% C,
4.62% H, 16.06% N; found 59.69% C, 4.91% H, 15.85% N.
N-(2-Methylbenzyl)-3-((2-methylbenzyl)amino)pyrazine-2-carboxamide (1a). Yellow-brown solid. Yield 26%;
m.p. 89.6–91.4 ◦C; IR (ATR-Ge, cm−1): 3395, 3321, 1659 (C=O, CONH); 1H-NMR (500 MHz, CDCl3)
δ
8.96 (bs, 1H, NH), 8.21 (d, J = 2.4 Hz, 1H, pyr.), 8.13 (t, J = 5.8 Hz, 1H, NH), 7.67 (d, J = 2.4 Hz, 1H, pyr.),
7.39–7.16 (m, 8H, Ar), 4.71 (d, J = 5.5 Hz, 2H, CH2), 4.60 (d, J = 5.8 Hz, 2H, CH2), 2.40 (d, J = 17.6 Hz,
6H, CH3; 13C-NMR (126 MHz, CDCl3)
δ 166.21, 154.53, 146.49, 136.47, 136.32, 136.22, 135.54, 130.49,
130.32, 129.52, 128.26, 128.04, 127.73, 127.28, 126.42, 126.20, 126.02, 42.51, 41.30, 19.11, 19.03; Elemental
analysis: calc. for C21H22N4O (MW 346.43): 72.81% C, 6.40% H, 16.17% N; found 72.44% C, 6.49% H,
16.35% N.
3-Chloro-N-(4-methylbenzyl)pyrazine-2-carboxamide (
IR (ATR-Ge, cm−1): 3303, 1655 (C=O, CONH); 1H-NMR (500 MHz, CDCl3)
pyr.), 8.45 (d, J = 2.3 Hz, 1H, pyr.), 7.86 (bs, 1H, NH), 7.30–7.24 (m, 2H, Ar), 7.21–7.13 (m, 2H, Ar), 4.61
(d, J = 5.9 Hz, 2H, CH2, 2.35 (s, 3H, CH3); 13C-NMR (126 MHz, CDCl3)
161.77, 148.37, 145.63, 143.12,
2
). Pale brown solid. Yield 17%; m.p. 126.2–129.1 ◦C
8.52 (d, J = 2.3 Hz, 1H,
;
δ
δ
140.49, 137.43, 134.53, 129.42, 127.94, 43.54, 21.07; Elemental analysis: calc. for C13H12ClN3O (MW
261.71): 59.66% C, 4.62% H, 16.06% N; found 59.47% C, 5.07% H, 15.69% N.
N-(4-Methylbenzyl)-3-((4-methylbenzyl)amino)pyrazine-2-carboxamide (2a). Pale yellow solid. Yield 25%;
m.p. 145.8–147.6 ◦C; IR (ATR-Ge, cm−1): 3373, 3344, 1650 (C=O, CONH); 1H-NMR (300 MHz, CDCl3)
δ
9.03 (t, J = 5.7 Hz, 1H, NH), 8.22 (s, 1H, NH), 8.18 (d, J = 2.4 Hz, 1H, pyr.), 7.64 (d, J = 2.4 Hz, 1H,

Molecules 2017, 22, 495
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pyr.), 7.30–7.21 (m, 4H, Ar), 7.18–7.12 (m, 4H, Ar), 4.67 (d, J = 5.7 Hz, 2H, CH2), 4.55 (d, J = 6.0 Hz, 2H,
CH2), 2.34 (d, J = 2.9 Hz, 6H, CH3); 13C-NMR (75 MHz, CDCl3)
166.28, 154.52, 146.45, 137.17, 136.66,
δ
135.80, 134.93, 129.52, 129.35, 129.19, 127.68, 127.49, 126.46, 44.10, 42.89, 21.07; Elemental analysis: calc.
for C21H22N4O (MW 346.43): 72.81% C, 6.40% H, 16.17% N; found 72.47% C, 6.40% H, 15.73% N.
◦
3-Chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (
(ATR-Ge, cm−1): 3276, 1648 (C=O, CONH); 1H-NMR (300 MHz, CDCl3)
8.43 (d, J = 2.3 Hz, 1H, pyr.), 7.82 (bs, 1H, NH), 7.32–7.24 (m, 2H, Ar), 6.91–6.84 (m, 2H, Ar), 4.58 (d, J =
5.8 Hz, 2H, CH2), 3.79 (s, 3H, CH3); 13C-NMR (75 MHz, CDCl3)
161.76, 159.16, 148.37, 145.65, 143.14,
3
). Brown solid. Yield 11%; m.p. 107.7–109.3 C; IR
δ
8.51 (d, J = 2.3 Hz, 1H, pyr.),
δ
140.51, 129.65, 129.34, 114.15, 55.28, 43.26.; Elemental analysis: calc. for C13H12ClN3O2 (MW 277.71):
56.23% C, 4.36% H, 15.13% N; found 56.64% C, 4.84% H, 14.82% N.
N-(4-Methoxybenzyl)-3-((4-methoxybenzyl)amino)pyrazine-2-carboxamide (3a). Yellow-brown solid. Yield
◦
1
15%; m.p. 83.1–85.7 C; IR (ATR-Ge, cm−1): 3392, 3335, 1652 (C=O, CONH); H-NMR (300 MHz,
CDCl3)
7.34–7.22 (m, 4H, Ar), 6.91–6.82 (m, 4H, Ar), 4.63 (d, J = 5.6 Hz, 2H, CH2), 4.51 (d, J = 5.9 Hz, 2H, CH2),
3.79 (s, 6H, CH3); 13C-NMR (75 MHz, CDCl3)
166.28, 159.03, 158.76, 154.52, 146.46, 131.00, 130.09,
δ 8.99 (bs, 1H, NH), 8.19 (bs, 1H, NH), 8.17 (d, J = 2.4 Hz, 1H, pyr.), 7.64 (d, J = 2.4 Hz, 1H, pyr.),
δ
129.55, 129.06, 129.06, 128.87, 126.51, 114.11, 113.97, 55.28, 43.86, 42.63; Elemental analysis: calc. for
C21H22N4O3 (MW 378.43): 66.65% C, 5.86% H, 14.81% N; found 66.95% C, 6.12% H, 14.35% N.
3-Chloro-N-(2,4-dimethoxybenzyl)pyrazine-2-carboxamide (
4
). Pale brown solid. Yield 14%; m.p.
115.2–117.3 ◦C; IR (ATR-Ge, cm−1): 3275, 1640 (C=O, CONH); 1H-NMR (300 MHz, DMSO)
δ
8.97 (t,
J = 5.7 Hz, 1H, NH), 8.68 (d, J = 2.5 Hz, 1H, pyr.), 8.61 (d, J = 2.5 Hz, 1H, pyr.), 7.19 (d, J = 8.3 Hz, 1H,
Ar), 6.58–6.47 (m, 2H, Ar), 4.37 (d, J = 5.8 Hz, 2H, CH2), 3.79 (s, 3H, CH3), 3.74 (s, 3H, CH3); 13C-NMR
(75 MHz, DMSO) δ 163.74, 160.09, 157.95, 148.54, 145.32, 145.16, 142.61, 129.04, 118.24, 104.54, 98.46,
55.63, 55.40, 37.43.; Elemental analysis: calc. for C14H14ClN3O3 (MW 307.73): 54.64% C, 4.59% H,
13.65% N; found 54.95% C, 4.96% H, 13.66% N.
3-Chloro-N-(2-chlorobenzyl)pyrazine-2-carboxamide (
IR (ATR-Ge, cm−1): 3362, 1672 (C=O, CONH); 1H-NMR (500 MHz, CDCl3)
pyr.), 8.48 (d, J = 2.3 Hz, 1H, pyr.), 8.10 (bs, 1H, NH), 7.51–7.46 (m, 1H, Ar), 7.41–7.37 (m, 1H, Ar),
7.28–7.23 (m, 2H, Ar), 4.74 (d, J = 6.3 Hz, 2H, CH2); 13C-NMR (126 MHz, CDCl3)
161.84, 148.37,
5
). Pale brown solid. Yield 46%; m.p. 101.7–102.8 ◦C
8.53 (d, J = 2.3 Hz, 1H,
;
δ
δ
145.74, 142.83, 140.55, 135.03, 133.73, 130.39, 129.54, 129.13, 127.14, 41.67; Elemental analysis: calc. for
C12H9Cl2N3O (MW 282.12): 51.09% C, 3.22% H, 14.89% N; found 50.85% C, 3.58% H, 14.53% N.
3-Chloro-N-(3-chlorobenzyl)pyrazine-2-carboxamide (
(ATR-Ge, cm−1): 3279, 1660 (C=O, CONH); 1H-NMR (500 MHz, CDCl3)
8.47 (d, J = 2.3 Hz, 1H, pyr.), 8.01 (bs, 1H, NH), 7.34 (d, J = 2.0 Hz, 1H, Ar), 7.31–7.22 (m, 3H, Ar), 4.62
(d, J = 6.1 Hz, 2H, CH2); 13C-NMR (126 MHz, CDCl3)
161.92, 148.40, 145.83, 142.74, 140.54, 139.66,
6
). Brown solid. Yield 56%; m.p. 92.5–95.0 ◦C; IR
8.54 (d, J = 2.3 Hz, 1H, pyr.),
δ
δ
134.53, 130.00, 127.82, 127.80, 125.94, 43.07; Elemental analysis: calc. for C12H9Cl2N3O (MW 282.12):
51.09% C, 3.22% H, 14.89% N; found 51.42% C, 3.69% H, 14.61% N.
3-Chloro-N-(4-chlorobenzyl)pyrazine-2-carboxamide (
(ATR-Ge, cm−1): 3382, 1669 (C=O, CONH); 1H-NMR (300 MHz, CDCl3)
8.45 (d, J = 2.3 Hz, 1H, pyr.), 7.94 (bs, 1H, NH), 7.34–7.24 (m, 4H, Ar), 4.61 (d, J = 6.1 Hz, 2H, CH2);
13C-NMR (75 MHz, CDCl3)
161.90, 148.42, 145.81, 142.80, 140.52, 136.15, 133.48, 129.22, 128.87, 43.01;
7
). Brown solid. Yield 63%; m.p. 133.5–135.2 ◦C; IR
8.53 (d, J = 2.4 Hz, 1H, pyr.),
δ
δ
Elemental analysis: calc. for C12H9Cl2N3O (MW 282.12): 51.09% C, 3.22% H, 14.89% N; found 50.81%
C, 3.50% H, 14.74% N.
◦
3-Chloro-N-(2,4-dichlorobenzyl)pyrazine-2-carboxamide (
(ATR-Ge, cm−1): 3295, 1655 (C=O, CONH); 1H-NMR (300 MHz, DMSO)
8.73 (d, J = 2.5 Hz, 1H, pyr.), 8.66 (d, J = 2.4 Hz, 1H, pyr.), 7.63 (t, J = 1.2 Hz, 1H, Ar), 7.46 (d, J = 1.3 Hz
2H, Ar), 4.53 (d, J = 5.9 Hz, 2H, CH2); 13C-NMR (75 MHz, DMSO)
163.93, 147.60, 145.72, 145.33,
8). Ochre solid. Yield 58%; m.p. 99.5–100.4 C; IR
δ
9.38 (t, J = 6.0 Hz, 1H, NH),
,
δ

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142.63, 134.94, 133.25, 132.69, 130.38, 128.85, 127.59, 40.53; Elemental analysis: calc. for C12H8Cl3N3O
(MW 316.57): 45.53% C, 2.55% H, 13.27% N; found 45.72% C, 2.79% H, 12.96% N.
3-Chloro-N-(3,4-dichlorobenzyl)pyrazine-2-carboxamide (9). Brown solid. Yield 49%; m.p. 121.5–124.5 ◦C;
IR (ATR-Ge, cm−1): 3241, 1648 (C=O, CONH); 1H-NMR (300 MHz, CDCl3)
δ
8.55 (d, J = 2.3 Hz, 1H,
pyr.), 8.47 (d, J = 2.3 Hz, 1H, pyr.), 8.02 (bs, 1H, NH), 7.47–7.36 (m, 2H, Ar), 7.28–7.16 (m, 1H, Ar), 4.59
(d, J = 6.1 Hz, 2H, CH2); 13C-NMR (75 MHz, CDCl3)
161.96, 148.48, 145.97, 142.51, 140.54, 137.96,
δ
132.73, 131.69, 130.67, 129.67, 127.16, 42.54; Elemental analysis: calc. for C12H8Cl3N3O (MW 316.57):
45.53% C, 2.55% H, 13.27% N; found 45.41% C, 2.77% H, 12.87% N.
3-Chloro-N-(2-fluorobenzyl)pyrazine-2-carboxamide (10). Pale brown solid. Yield 46%; m.p. 116.7–117.9 ◦C
IR (ATR-Ge, cm−1): 3320, 1661 (C=O, CONH); 1H-NMR (500 MHz, CDCl3)
8.53 (d, J = 2.2 Hz, 1H,
;
δ
pyr.), 8.47 (d, J = 2.2 Hz, 1H, pyr.), 8.01 (bs, 1H, NH), 7.44 (dt, J = 7.6 Hz J = 2.0 Hz, 1H, Ar), 7.32–7.26
(m, 1H, Ar), 7.15–7.10 (m, 1H, Ar), 7.10–7.05 (m, 1H, Ar), 4.70 (d, J = 5.9 Hz, 2H, CH2); 13C-NMR
(125 MHz, CDCl3) δ 161.9, 161.1 (d, J = 247.1 Hz), 148.4, 145.8, 142.9, 140. 5, 130.4 (d, J = 3.9 Hz), 129.5
(d, J = 7.7 Hz), 124.6 (d, J = 15.3 Hz), 124.4 (d, J = 3.9 Hz), 115.4 (d, J = 21.0 Hz), 37.7 (d, J = 3.9 Hz);
Elemental analysis: calc. for C12H9ClFN3O (MW 265.67): 54.25% C, 3.41% H, 15.82% N; found 54.61%
C, 3.89% H, 15.59% N.
3-Chloro-N-(4-fluorobenzyl)pyrazine-2-carboxamide (11). Brown solid. Yield 55%; m.p. 115.0–117.9 ◦C;
IR (ATR-Ge, cm−1): 3403, 1661 (C=O, CONH); 1H-NMR (300 MHz, CDCl3)
δ
8.53–8.51 (m, 1H, pyr.),
8.45 (d, J = 2.4 Hz, 1H, pyr.), 7.92 (bs, 1H, NH), 7.37–7.28 (m, 2H, Ar), 7.07–6.97 (m, 2H, Ar), 4.60 (d,
J = 6.5 Hz, 2H, CH2); 13C-NMR (75 MHz, CDCl3)
162.2 (d, J = 243.1 Hz), 161.9, 148.4, 145.9, 142.9,
δ
140.6, 133.4 (d, J = 3.5 Hz), 129.6 (d, J = 9.2 Hz), 115.6 (d, J = 21.9 Hz), 43.0; Elemental analysis: calc. for
C12H9ClFN3O (MW 265.67): 54.25% C, 3.41% H, 15.82% N; found 54.71% C, 3.76% H, 15.78% N.
3-Chloro-N-(3-(trifluoromethyl)benzyl)pyrazine-2-carboxamide (12). Pale yellow solid. Yield 55%; m.p.
◦
79.2–81.5 C; IR (ATR-Ge, cm−1): 3292, 1662 (C=O, CONH); 1H-NMR (500 MHz, DMSO)
δ
9.44 (t,
J = 6.0 Hz, 1H, NH), 8.72 (d, J = 2.4 Hz, 1H, pyr.), 8.65 (d, J = 2.4 Hz, 1H, pyr.), 7.74–7.72 (1H, m, Ar),
7.68–7.57 (m, 3H, Ar), 4.60 (d, J = 6.0 Hz, 2H, CH2); 13C-NMR (125 MHz, DMSO)
164.0, 147.9, 145.7,
δ
145.3, 142.7, 140.4, 131.6, 129.6, 129.3 (q, J = 31.4 Hz), 124.4 (q, J = 272.5 Hz), 123.9 (q, J = 3.8 Hz), 42.0;
Elemental analysis: calc. for C13H9ClF3N3O (MW 315.68): 49.46% C, 2.87% H, 13.31% N; found 49.12%
C, 3.17% H, 12.89% N.
3-Chloro-N-(4-(trifluoromethyl)benzyl)pyrazine-2-carboxamide (13). White solid. Yield 67%; m.p.
123.7–125.7 ◦C; IR (ATR-Ge, cm−1): 3299, 1660 (C=O, CONH); 1H-NMR (500 MHz, CDCl3)
δ
8.54 (d,
J = 2.4 Hz, 1H, pyr.), 8.46 (d, J = 2.4 Hz, 1H, pyr.), 8.05 (bs, 1H, NH), 7.62–7.57 (m, 2H, Ar), 7.50–7.45 (m,
2H, AA , BB 162.1, 148.5, 145.9, 142.7,
, Ar), 4.70 (d, 2H, J = 6.4 Hz, CH2); 13C-NMR (125 MHz, CDCl3)
´
´
δ
141.7, 140.6, 129.9 (q, J = 32.4 Hz), 128.0, 125.7 (q, J = 3.9 Hz), 124.0 (q, J = 271.9 Hz), 43.2; Elemental
analysis: calc. for C13H9ClF3N3O (MW 315.68): 49.46% C, 2.87% H, 13.31% N; found 49.01% C, 3.34%
H, 12.95% N.
N-(2-Chlorobenzyl)-3-((2-chlorobenzyl)amino)pyrazine-2-carboxamide (5a). White solid. Yield 39%; m.p.
90.8–91.9 ◦C; IR (ATR-Ge, cm−1): 3382, 3353, 1663 (C=O, CONH); 1H-NMR (500 MHz, CDCl3)
δ 9.09
(bs, 1H, NH), 8.39 (t, J = 6.1 Hz, 1H, NH), 8.19 (d, J = 2.4 Hz, 1H, pyr.), 7.71 (d, J = 2.4 Hz, 1H, pyr.),
7.45–7.36 (m, 4H, Ar), 7.28–7.23 (m, 2H, Ar), 7.23–7.18 (m, 2H, Ar), 4.82 (d, J = 6.1 Hz, 2H, CH2), 4.72 (d,
J = 6.3 Hz, 2H, CH2); 13C-NMR (126 MHz, CDCl3)
δ 166.40, 154.52, 146.50, 136.27, 135.40, 133.64, 133.57,
129.96, 129.70, 129.58, 129.45, 129.17, 128.90, 128.35, 127.06, 126.73, 126.46, 42.12, 41.04; Elemental
analysis: calc. for C19H16Cl2N4O (MW 387.26): 58.93% C, 4.16% H, 14.47% N; found 58.69% C, 4.07%
H, 14.21% N.
N-(3-Chlorobenzyl)-3-((3-chlorobenzyl)amino)pyrazine-2-carboxamide (6a). Yellow solid. Yield 62%; m.p.
68.7–70.4 ◦C; IR (ATR-Ge, cm−1): 3377, 3337, 1652 (C=O, CONH); 1H-NMR (500 MHz, CDCl3)
δ
9.04
(t, J = 6.3 Hz, 1H, NH), 8.32 (bs, 1H, NH), 8.20 (d, J = 2.4 Hz, 1H, pyr.), 7.71 (d, J = 2.4 Hz, 1H, pyr.),

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7.37–7.33 (m, 2H, Ar), 7.31–7.21 (m, 6H, Ar), 4.71 (d, J = 6.0 Hz, 2H, CH2), 4.58 (d, J = 6.3 Hz, 2H,
CH2); 13C-NMR (126 MHz, CDCl3)
166.45, 154.46, 146.66, 141.14, 140.07, 134.57, 134.34, 130.09, 129.98,
δ
129.76, 127.69, 127.65, 127.56, 127.24, 126.25, 125.70, 125.53, 43.73, 42.55; Elemental analysis: calc. for
C19H16Cl2N4O (MW 387.26): 58.93% C, 4.16% H, 14.47% N; found 59.16% C, 4.17% H, 14.20% N.
N-(4-Chlorobenzyl)-3-((4-chlorobenzyl)amino)pyrazine-2-carboxamide (7a). Pale yellow solid. Yield 77%;
m.p. 150.1–151.4 ◦C; IR (ATR-Ge, cm−1): 3375, 3346, 1651 (C=O, CONH); 1H-NMR (300 MHz, CDCl3)
δ
8.94 (bs, 1H, NH), 8.20 (bs, 1H, NH), 8.10 (s, 1H, pyr.), 7.60 (s, 1H, pyr.), 7.32–7.12 (m, 8H, Ar), 4.59
(d, J = 5.9 Hz, 2H, CH2), 4.47 (d, J = 5.9 Hz, 2H, CH2); 13C-NMR (75 MHz, CDCl3)
δ
166.4, 154.5,
146.6, 137.5, 136.5, 133.3, 132.8, 130.0, 129.0, 128.8, 128.6, 126.3, 43.6, 42.4; Elemental analysis: calc. for
C19H16Cl2N4O (MW 387.26): 58.93% C, 4.16% H, 14.47% N; found 58.68% C, 4.24% H, 14.16% N.
N-(3,4-Dichlorobenzyl)-3-((3,4-dichlorobenzyl)amino)pyrazine-2-carboxamide (9a). Yellow solid. Yield 67%;
◦
m.p. 101.9–104.7 C; IR (ATR-Ge, cm−1): 3368, 3317, 1666 (C=O, CONH); 1H-NMR (500 MHz, CDCl3)
δ
9.03 (bs, 1H, NH), 8.34 (bs, 1H, NH), 8.20 (d, J = 2.4 Hz, 1H, pyr.), 7.74 (d, J = 2.5 Hz, 1H, pyr.), 7.46–7.37
(m, 4H, Ar), 7.28–7.18 (m, 2H, Ar), 4.68 (d, J = 6.0 Hz, 2H, CH2), 4.56 (d, J = 6.3 Hz, 2H, CH2); 13C-NMR
(126 MHz, CDCl3)
δ 166.46, 154.29, 146.61, 139.40, 138.30, 132.78, 132.49, 131.59, 131.00, 130.66, 130.42,
130.34, 129.48, 129.38, 126.91, 126.77, 126.22, 43.24, 42.06; Elemental analysis: calc. for C19H14 Cl4N4O
(MW 456.15): 50.03% C, 3.09% H, 12.28% N; found 50.09% C, 3.22% H, 11.84% N.
N-(2-Fluorobenzyl)-3-((2-fluorobenzyl)amino)pyrazine-2-carboxamide (10a). Pale yellow solid. Yield 84%;
m.p. 94.8–95.9 ◦C; IR (ATR-Ge, cm−1): 3375, 3349, 1652 (C=O, CONH); 1H-NMR (500 MHz, CDCl3)
δ
9.00 (t, J = 5.5 Hz, 1H, NH), 8.30 (t, J = 6.1 Hz, 1H, NH), 8.19 (d, J = 2.2 Hz, 1H, pyr.), 7.69 (d, J = 2.2 Hz
1H, pyr.), 7.42–7.36 (m, 2H, Ar), 7.31–7.21 (m, 2H, Ar), 7.15–7.08 (m, 4H, Ar), 4.78 (d, J = 5.5 Hz, 2H,
CH2), 4.66 (d, J = 6.1 Hz, 2H, CH2); 13C-NMR (125 MHz, CDCl3)
166.4, 161.0 (d, J = 247.0 Hz), 154.5,
,
δ
146.5, 130.0 (d, J = 3.9 Hz), 129.9, 129.5 (d, J = 3.9 Hz), 129.3 (d, J = 7.7 Hz), 128.7 (d, J = 7.7 Hz),
126.4, 125.9 (d, J = 14.3 Hz), 125.0 (d, J = 14.3 Hz), 124.3 (d, J = 3.8 Hz), 124.0 (d, J = 2.9 Hz), 115.5 (d,
J = 19.1 Hz), 115.3 (d, J = 19.1 Hz), 38.1 (d, J = 4.8 Hz), 37.1 (d, J = 3.9 Hz); Elemental analysis: calc. for
C19H16F2N4O (MW 354.36): 64.40% C, 4.55% H, 15.81% N; found 64.24% C, 4.49% H, 15.97% N.
N-(4-Fluorobenzyl)-3-((4-fluorobenzyl)amino)pyrazine-2-carboxamide (11a). Yellow-brown solid. Yield 75%;
m.p. 97.1–99.1 ◦C; IR (ATR-Ge, cm−1): 3378, 3345, 1650 (C=O, CONH); 1H-NMR (500 MHz, CDCl3)
δ
9.06–9.00 (m, 1H, NH), 8.31–8.25 (m, 1H, NH), 8.20 (d, J = 2.4 Hz, 1H, pyr.), 8.69 (d, J = 2.4 Hz, 1H,
pyr.), 7.37–7.29 (m, 4H, Ar), 7.06–6.99 (m, 4H, Ar), 4.69 (d, J = 6.1 Hz, 2H, CH2), 4.56 (d, J = 6.1 Hz, 2H,
CH2); 13C-NMR (125 MHz, CDCl3)
166.4, 162.2 (d, J = 246.1 Hz), 162.0 (d, J = 245.1 Hz), 154.5, 146.5,
δ
134.6 (d, J = 3.9 Hz), 133.8 (d, J = 2.9 Hz), 129.7, 129.3 (d, J = 8.7 Hz), 129.1 (d, J = 8.5 Hz), 126.4, 115.6 (d,
J = 22.0 Hz), 115.3 (d, J = 21.0 Hz), 43.6, 42.4; Elemental analysis: calc. for C19H16F2N4O (MW 354.36):
64.40% C, 4.55% H, 15.81% N; found 64.55% C, 4.41% H, 15.63% N.
N-(4-(Trifluoromethyl)benzyl)-3-((4-(trifluoromethyl)benzyl)amino)pyrazine-2-carboxamide (13a). Yellow
solid. Yield 86%; m.p. 99.7–102.5 ◦C; IR (ATR-Ge, cm−1): 3388, 3334, 1652 (C=O, CONH); 1H-NMR
(500 MHz, CDCl3)
pyr.), 7.73 (d, J = 2.4 Hz, 1H, pyr.), 7.64–7.57 (m, 4H, Ar), 7.50–7.45 (m, 4H, Ar), 4.79 (d, J = 5.6 Hz, 2H,
CH2), 4.67 (d, J = 6.3 Hz, 2H, CH2); 13C-NMR (125 MHz, CDCl3)
166.6, 154.5 146.8, 143.2, 142.1, 130.3,
δ 9.08 (t, J = 5.6 Hz, 1H, NH), 8.38 (t, J = 6.3 Hz, 1H, NH), 8.20 (d, J = 2.4 Hz, 1H,
δ
129.6 (q, J = 32.4 Hz), 129.6 (q, J = 32.4 Hz), 127.8, 127.6, 126.2, 125.7 (q, J = 3.8 Hz), 125.5 (q, J = 3.8 Hz),
121.1 (q, J = 276.9 Hz), 124.0 (q, J = 271.8 Hz), 43.9, 42.7; Elemental analysis: calc. for C21H16F6N4O
(MW 454.38): 55.51% C, 3.55% H, 12.33% N; found 55.47% C, 3.68% H, 12.17% N.
3.4. Methods
3.4.1. Determination of Lipophilicity by HPLC (log k)
Log k was determined using an Agilent Technologies 1200 SL liquid chromatograph with
Diode-array Detector SL G1315C (Agilent Technologies Inc., Colorado Springs, CO, USA), with

Molecules 2017, 22, 495
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pre-column ZORBAX XDB-C18 5
Inc.) and column ZORBAX Eclipse XDB-C18 5
Technologies Inc.). The separation process was controlled by Agilent ChemStation, version B.04.02,
extended by a spectral module (Agilent Technologies Inc.). The mobile phase consisted of MeOH
(HPLC grade, 70%) and H2O (HPLC-Milli-Q Grade, 30%).
µ
m, 4 mm
×
4 mm, Part No. 7995118-504 (Agilent Technologies
µ
m, 4.6 mm
×
250 mm, Part No. 7995118-585 (Agilent
The flow rate was 1.0 mL/min, samples were injected in a volume of 20 µL, and the column
temperature was 30 ◦C. A value of 210 nm as the detection wavelength and 270 nm as the monitor
wavelength were used. Retention times (tR) were measured in minutes. The dead time of the system
(tD) was determined as the retention time of the KI methanol solution. Capacity factors k for individual
compounds were calculated according to the formula k = (tR
capacity factor k, as used as the lipophilicity index converted to log scale.
−
tD)/tD. Log k, calculated from the
3.4.2. Evaluation of In Vitro Antimycobacterial Activity
Microdilution panel method. 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 the Czech National Collection of Type Cultures (CNCTC), National Institute of
Public Health, Prague, Czech Republic. Middlebrook 7H9 broth (Sigma-Aldrich, Steinheim, Germany)
enriched with the 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
4.475 mL of broth) and placed (100 L) into microplate 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
of the DMSO/broth solution of tested compound. Final concentrations of the tested compounds in
wells were 100, 50, 25, 12.5, 6.25, 3.13, and 1.56
mL−1. Isoniazid was used as the positive control
(inhibition of growth). The negative control consisted of broth plus DMSO. A total of 30 L of Alamar
µL of DMSO solution in
µ
µ
µL
µg
·
µ
Blue working solution (1:1 mixture of 0.1% resazurin sodium salt (aq. sol.) and 10% Tween 80) was
added after five days of incubation. Results were then determined after 24 h of incubation. The
minimum inhibitory concentration (MIC;
µg
mL−1) was determined as the lowest concentration which
·
prevented the blue to pink colour change. MIC values of isoniazid were 6.25–12.5
M. avium, 3.13–12.5 µg·mL−1 against M. kansasii, and 0.2 µg·mL−1 against M. tbc.
µg
mL−1 against
·
3.4.3. Antimycobacterial In Vitro Activity Screening Against Mycobacterium Smegmatis
An antimycobacterial assay was performed with fast growing Mycobacterium smegmatis CCM
4622 (ATCC 607) from the Czech Collection of Microorganisms (Brno, Czech Republic). The technique
used for activity determination was a microdilution broth panel method using 96-well microtitration
plates. The 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), and the MB broth was then
added to obtain a concentration of 2000
µg
mL−1. Standards used for activity determination were
·
isoniazid (INH), rifampicin (RIF), and ciprofloxacin (CPX) (Sigma-Aldrich). Final concentrations were
reached by binary dilution and the addition of mycobacterial suspension, and were set as 500, 250, 125,
62.5, 31.25, 15.625, 7.81, and 3.91
where the final concentrations were 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, and 0.098
µg
mL−1, except for the standards of ciprofloxacin and rifampicin,
·
µg
mL−1. The
·
final concentration of DMSO did not exceeded 2.5% (v/v) and did not affect the growth of M. smegmatis.
Plates were also 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, producing
a 0.02% solution. Then, a 10% aqueous solution of Tween 80 (Sigma-Aldrich) was prepared. Both
liquids were mixed up making use of the same volumes and filtered through a syringe membrane

Molecules 2017, 22, 495
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filter. Microtitration panels were then incubated for a further 4 h. Antimycobacterial activity was
expressed as the minimal inhibition concentration (MIC) and the value was read on the basis of stain
colour change (blue colour—active compound; pink colour—not active compound). The MIC values
for the standards were in the range of 7.81–15.625
µ
g
·
mL−1 for INH, 0.78–1.56
µg
mL−1 for RIF, and
·
0.098–0.195 µg·mL−1 for CPX. All experiments were conducted in duplicate.
3.4.4. Evaluation of In Vitro Antibacterial Activity
Microdilution broth method. Antibacterial evaluation was performed against eight bacterial
strains from the Czech Collection of Microorganisms (Brno, Czech Republic) (Staphylococcus aureus
CCM 4516/08, Escherichia coli CCM 4517, Pseudomonas aeruginosa CCM 1961) or clinical isolates from the
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. The compounds were dissolved in DMSO, and the antibacterial activity
was determined in Mueller-Hinton liquid broth (Difco/Becton Dickinson), and 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 the minimum concentration to prevent the visible growth compared to control, was
determined after 24 and 48 h of static incubation at 35 ◦C. The standards were neomycin, bacitracin,
penicillin G, ciprofloxacin, and phenoxymethylpenicillin.
3.4.5. Evaluation of In Vitro Antifungal Activity
Antifungal evaluation was performed using a microdilution broth method against eight fungal
strains (Candida albicans ATCC 44859, C. tropicalis 156, C. krusei E28, C. glabrata 20/I, Trichosporon asahii
1188, Aspergillus fumigatus 231, Lichtheimia corymbifera 272 and Trichophyton mentagrophytes 445).
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. The standards were amphotericin
B, voriconazole, nystatin, and fluconazole.
3.4.6. Cytotoxicity Assays
The human liver hepatocellular carcinoma cell line HepG2 (passage 35–36), purchased from
Health Protection Agency Culture Collections (ECACC, Salisbury, UK), was routinely cultured in
Minimum Essential Eagle Medium MEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum
(PAA Laboratories GmbH, Pasching, Austria), 2mM L-glutamine solution (Sigma-Aldrich), and 1%
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. To evaluate the cytotoxicity, the HepG2 cells treated with the tested substances were used as
experimental groups, whereas untreated HepG2 cells served as control groups.
The HepG2 cells were seeded in a density of 1
following day (24 h after seeding), they were treated with tested substances dissolved in DMSO
(maximal incubation concentration of DMSO was 1%). The tested substances were prepared according
×
104 cells per well on a 96-well plate. The
to their solubility in DMSO, at incubation concentrations of 1–2500
out in a humidified atmosphere containing 5% CO2 at 37 C, in triplicate, for 24 h. The controls
representing 100% cell viability, 0% cell viability (the cells treated with 10% DMSO), no-cell controls,
µ
M. The treatment was carried
◦

Molecules 2017, 22, 495
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and vehiculum controls were incubated in triplicate, simultaneously. After 24 h exposure, the reagent
from the kit CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega) was added,
according to the recommendation of the manufacturer. After 2 h incubation at 37 ◦C in humidified,
5% CO2 atmosphere, the absorbance was recorded at 490 nm. Inhibitory curves were constructed for
each compound, plotting incubation concentrations vs. percentage of absorbance relative to untreated
control. The standard toxicological parameter IC50 was calculated by a nonlinear regression analysis
of the inhibitory curves using GraphPad Prism software (version 6, GraphPad Software, Inc., La Jolla,
CA, USA).
4. Conclusions
Thirteen new N-benzyl-3-chloropyrazine-2-carboxamide derivatives and ten N-benzyl-3-
(benzylamino)pyrazine-2-carboxamide derivatives were prepared and analytically characterized.
Further, antimycobacterial, antibacterial, and antifungal assays were performed. Compounds 1–13
presented in this paper were prepared for the purpose of completing series of 5- and 6-positional
isomers, as reported previously. However, the chlorine shift to position 3 on the pyrazine ring led
to the decrease of antimycobacterial activity compared to 5- or 6-positional isomers. Contrary to
5-Cl and 6-Cl positional isomers, N-benzyl-3-chloropyrazine-2-carboxamides exerted antibacterial
activity against staphylococcal strains. The most effective compound was 5, with micromolar activity
against Staphylococcus aureus and Staphylococcus epidermidis. Interestingly, some initially unplanned side
products with double benzyl substitution possessed better antimycobacterial activity than the intended
N-benzyl-3-chloropyrazine-2-carboxamides. From compounds with double phenyl substitution, 1a
and 9a exerted moderate antimycobacterial activity (tens of
µM) and 3a was moderately active against
methicillin-resistant Staphylococcus aureus (tens of M). Generally, the insertion of the second aromatic
µ
ring was beneficial to the activity against M. tuberculosis H37Rv, but contra-productive for activity
against M. smegmatis and other tested bacteria. Importantly, the discussed active compounds were
assayed for in vitro cytotoxicity with plausible results.
Supplementary Materials: The docking studies are available online.
Acknowledgments: This study was supported by the Grant Agency of Charles University B-CH/1594214, SVV
260 401 and by the Czech Science Foundation project No. 17-27514Y.
Author Contributions: L.S. synthetized the compounds; L.S. and J.Z. designed the structures, evaluated the
biological activity, and wrote the paper; O.J. and P.P. performed the antimycobacterial assays; K.K. performed
the antibacterial and antifungal assays; L.N. and F.T. conceived and performed the cytotoxicity; V.K. performed
and evaluated the lipophilicity measurements; J.K. interpreted the NMR spectra; and M.D. participated in the
structure design.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2017 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
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).