5-Alkylamino-N-phenylpyrazine-2-carboxamides: Design, preparation, and antimycobacterial evaluation

Article abstract of DOI:10.3390/molecules25071561

According to the World Health Organization, tuberculosis is still in the top ten causes of death from a single infectious agent, killing more than 1.7 million people worldwide each year. The rising resistance developed by Mycobacterium tuberculosis against currently used antituberculars is an imperative to develop new compounds with potential antimycobacterial activity. As a part of our continuous research on structural derivatives of the first-line antitubercular pyrazinamide, we have designed, prepared, and assessed the in vitro whole cell growth inhibition activity of forty-two novel 5-alkylamino-N-phenylpyrazine-2-carboxamides with various length of the alkylamino chain (propylamino to octylamino) and various simple substituents on the benzene ring. Final compounds were tested against Mycobacterium tuberculosis H37Ra and four other mycobacterial strains (M. aurum, M. smegmatis, M. kansasii, M. avium) in a modified Microplate Alamar Blue Assay. We identified several candidate molecules with micromolar MIC against M. tuberculosis H37Ra and low in vitro cytotoxicity in HepG2 cell line, for example, N-(4-hydroxyphenyl)-5-(pentylamino)pyrazine-2-carboxamide (3c, MIC = 3.91 μg/mL or 13.02 μM, SI > 38) and 5-(heptylamino)-N-(p-tolyl)pyrazine-2-carboxamide (4e, MIC = 0.78 μg/mL or 2.39 μM, SI > 20). In a complementary screening, we evaluated the in vitro activity against bacterial and fungal strains of clinical importance. We observed no antibacterial activity and sporadic antifungal activity against the Candida genus.

Full text of DOI:10.3390/molecules25071561

molecules
Article
5-Alkylamino-N-phenylpyrazine-2-carboxamides:
Design, Preparation,
and Antimycobacterial Evaluation
Weronika Ambroz˙kiewicz 1,
* ,
, Marta Kucˇerová-Chlupácˇová 1 , Ondrˇej Jand’ourek 1
Klára Konecˇná 1 , Pavla Paterová 2, Pavel Bárta 1 , Jarmila Vinšová 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; kucerom@faf.cuni.cz (M.K.-C.); jando6aa@faf.cuni.cz (O.J.); konecna@faf.cuni.cz (K.K.);
bartp7aa@faf.cuni.cz (P.B.); vinsova@faf.cuni.cz (J.V.); dolezalm@faf.cuni.cz (M.D.)
Department of Clinical Microbiology, University Hospital, Sokolská 581,
2
500 05 Hradec Králové, Czech Republic; pavla.paterova@fnhk.cz
*
Correspondence: weronika.ambrozkiewicz@gmail.com (W.A.); jan.zitko@faf.cuni.cz (J.Z.);
Tel.: +420-495-067-409 (J.Z.); Fax: +420-495-518-002 (J.Z.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 9 March 2020; Accepted: 27 March 2020; Published: 28 March 2020
Abstract: According to the World Health Organization, tuberculosis is still in the top ten causes
of death from a single infectious agent, killing more than 1.7 million people worldwide each year.
The rising resistance developed by Mycobacterium tuberculosis against currently used antituberculars is
an imperative to develop new compounds with potential antimycobacterial activity. As a part of our
continuous research on structural derivatives of the first-line antitubercular pyrazinamide, we have
designed, prepared, and assessed the in vitro whole cell growth inhibition activity of forty-two
novel 5-alkylamino-N-phenylpyrazine-2-carboxamides with various length of the alkylamino chain
(propylamino to octylamino) and various simple substituents on the benzene ring. Final compounds
were tested against Mycobacterium tuberculosis H37Ra and four other mycobacterial strains (M. aurum,
M. smegmatis, M. kansasii, M. avium) in a modified Microplate Alamar Blue Assay. We identified several
candidate molecules with micromolar MIC against M. tuberculosis H37Ra and low in vitro cytotoxicity
in HepG2 cell line, for example, N-(4-hydroxyphenyl)-5-(pentylamino)pyrazine-2-carboxamide
(
(
3c, MIC = 3.91
4e, MIC = 0.78
µ
g/mL or 13.02
µ
M, SI > 38) and 5-(heptylamino)-N-(p-tolyl)pyrazine-2-carboxamide
µ
g/mL or 2.39
µM, SI > 20). In a complementary screening, we evaluated the in vitro
activity against bacterial and fungal strains of clinical importance. We observed no antibacterial
activity and sporadic antifungal activity against the Candida genus.
Keywords: alkylamino derivatives; antibacterial; antifungal; antimycobacterial; cytotoxicity;
pyrazinamide
1. Introduction
According to the World Health Organization (WHO) [1] recommendations, tuberculosis (TB) is
already a globally established healthcare priority for which innovative new treatments are urgently
needed. Human TB is mainly caused by members of the Mycobacterium tuberculosis complex, a group of
acid-fast bacilli. It is estimated that one-quarter of the global human population (~1.7 billion of people)
is sub-clinically (latently) infected by Mycobacterium tuberculosis (Mtb); however, only 5–15% of them
will develop active TB disease during their lifetime [
developed TB and 1.7 million deaths (including deaths from TB among HIV positives) [
makes TB the number one cause of death due to the fact of a single infectious agent [
2
]. In 2018, there were 10.4 million new cases of
]. Such statistics
]. Due to the
3
4
Molecules 2020, 25, 1561; doi:10.3390/molecules25071561
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1561
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deficiency of rapid and sensitive diagnostic methods, an outdated vaccine (Bacille Calmette–Guérin;
BCG) with limited effectiveness, and difficulty in identifying latent cases, global TB control is
still questionable. Moreover, sub-treatment increases resistance to currently used antitubercular
drugs [
immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS) and the
cause of death among a quarter of them [ ]. Non-tuberculous mycobacteria (NTM) can cause infection
in immune-compromised individuals [ ]. Mycobacterium avium and Mycobacterium kansasii belong to
the subset of NTM frequently associated with opportunistic human infections, both pulmonary and
extrapulmonary [ ]. Disseminated NTM infections occur mainly in immunocompromised people;
2,4]. Tuberculosis is one of the most frequent opportunistic infections for patients with human
2
4
5
however, the incidence of NTM lung infections is rising globally [6]. Colonization of lungs by NTM,
especially M. avium complex, is connected with bronchiectasis [7].
The group of first-line anti-TB drugs includes isoniazid (INH), rifampicin (RIF), ethambutol (EMB)
and pyrazinamide (PZA). Pyrazinamide is an indispensable component of all basic TB treatment
regimens, and it is also used in the treatment of extrapulmonary tuberculosis and pulmonary
tuberculosis resistant to rifampicin or isoniazid [8].
Many different PZA derivatives have been studied since the 1970s when PZA was established
as an anti-TB drug [9]. Cynamon et al. [10] presented in vitro antimycobacterial activity of
5-chloropyrazine-2-carboxamide (5-Cl-PZA) against PZA-resistant strains and against atypical
mycobacteria naturally resistant to PZA (M. kansasii, M. smegmatis, M. fortuitum, and M. avium).
Our research team has already published several studies focused on structural modifications of
PZA. One of our previous publication concerned a series of N-phenylpyrazine-2-carboxamides
variously substituted on both aromatic rings which exerted significant antimycobacterial
activity [11,12]. Further studies on 5-chloro-N-phenylpyrazine-2-carboxamides [13] and 5-alkylamino-
N-phenylpyrazine-2-carboxamides [14] also showed promising results. Most of the tested 5-chloro-
N-phenylpyrazine-2-carboxamides exerted activity expressed as minimum inhibitory concentration
(MIC) in the range of 1.56–6.25
substitution in the phenyl ring exerted activity in the range of MIC = 0.78–3.13
M. tuberculosis H37Rv in both cases) and exerted low in vitro cytotoxicity (Figure 1, the left structure.).
µg/mL [13] and alkylamino-N- phenylpyrazine-2-carboxamides without
µg/mL [14] (against
Figure 1. 5-Alkylamino-N-phenylpyrazine-2-carboxamides [14] as a starting point for this study.
The current study is an extension to the previously published series of 5-alkylamino-
N-phenylpyrazine-2-carboxamides [14] (see the original publication for the essential points which led
us to the design of this core structure). In the previous publication, we focused mainly on the role of the
substituent on C-5 of the pyrazine ring along with simple alkylamino substituents, and we probed the
substituents with terminal aryl, hydroxy or methoxy group in the alkylamino substituent and find out
that these modifications of the alkylamino chain were mostly undesirable for antimycobacterial activity.
We now present derivatives with increased variability in the substituents on the benzene ring,
including electron-donating substituents (–OH, alkyl) and electron-withdrawing substituents (–CF3,

Molecules 2020, 25, 1561
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–Cl), both lipophilic and hydrophilic. The length of the alkylamino chain attached at position 5 in the
pyrazine ring was varied the same way as in the original publication (n-propylamino to n-octylamino).
In this study, we present forty-two new 5-alkylamino-N-phenylpyrazine-2-carboxamides,
their synthesis, in vitro whole cell antimycobacterial activity, and in vitro cytotoxicity.
As a primary test, we employed the in vitro growth inhibition assay with attenuated M. tuberculosis
(Mtb) H37Ra (avirulent strain). This avirulent strain is a widely used surrogate organism for the
virulent Mtb H37Rv. It has been shown that there are only 244 non-identical proteins between Mtb
H37Ra and Mtb H37Rv (virulent strain) out of more than 4000 proteins [15]. Also, the susceptibility
to the majority of clinically used antimycobacterial compounds is equal or very similar between
H37Ra and H37Rv [16]. These facts, together with lower costs of testing and decreased biosafety level
requirements for the attenuated strain, make Mtb H37Ra a valuable surrogate organism for virulent
Mtb H37Rv.
It is a common practice to use fast-growing mycobacterial species, such as M. aurum or M. smegmatis,
as models for rapid screening of drugs instead of Mtb, which requires long incubation time and a
higher biosafety level. The usage of surrogate organisms has many benefits, like being easy to handle
due to the fact of its non-infectious nature and being good models of latent infection. For example,
M. aurum can survive in macrophages and possesses drug resistance patterns and a cell wall profile
similar to M. tuberculosis [17]. All compounds presented in this article were tested in vitro against Mtb
H37Ra, M. smegmatis, and M. aurum. The majority of compounds were also tested against two other
strains of NTM: M. avium and M. kansasii.
Additionally, final compounds were also screened for their antibacterial and antifungal activity
in vitro against pathogens of clinical importance.
2. Results and Discussion
2.1. Chemistry
2.1.1. Synthesis of Variously Substituted 5-chloro-N-phenylpyrazine-2-carboxamides as Intermediates
Compounds 1–7 (Scheme 1, a) were prepared following the previously described procedure [18]
from commercially available 5-chloropyrazine-2-carboxylic acid (5-Cl-POA, Fluorochem, Hadfield,
UK). The 5-Cl-POA was activated by thionyl chloride in the presence of catalytic amount of
N,N-dimethylformamide to yield the acyl chloride. The crude acyl chloride was used immediately for
subsequent reaction with the respective substituted aniline, using pyridine as a base. Compounds
were isolated as solids in 70–90% of theoretical yield (relative to 5-Cl-POA). Five of the intermediate
compounds (1–3, 5, 6) were reported previously [13]. Analytical data were fully consistent both with
proposed structures and with previously reported values if available.
Scheme 1. Synthesis of title compounds. Solvents and conditions: a-1—PhMe, 100 ◦C, 1 h; a-2—dry
acetone, pyridine, RT, 1 h; b—EtOH, refluxing 7 h.

Molecules 2020, 25, 1561
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2.1.2. Synthesis of Variously Substituted 5-alkylamino-N-phenylpyrazine-2-carboxamides
The 5-alkylmino-N-phenylpyrazine-2-carboxamides (1a–7f) were prepared by nucleophilic
substitution of the chloropyrazine derivative by alkylamine following the previously described
procedure [14] (Scheme 1, b). The relative excess (three equivalents relative to the chloropyrazine
derivative) of amine was used to increase the rate of conversion. As indicated by TLC, the conversions
were complete or close to complete after 3–7 h of stirring at ambient temperature. The products
were purified by flash-chromatography, followed by crystallization from hot ethanol, and isolated
as white, cream, beige or yellow solids with yields ranging from 15% to 98% of theoretical yield
(related to the amount of starting 5-chloro-N-phenylpyrazine-2-carboxamide, and after all purification
steps). The compounds were characterized by 1H-NMR, 13C-NMR, FT-IR spectroscopy, and melting
point. The purity of the products was evaluated by elemental analysis. The analytical data were fully
consistent with proposed structures. The results of the elemental analyses were in the range of 0.5%
±
relative to calculated values. The IR spectroscopy confirmed the presence of characteristic functional
groups. In the IR spectra, the final compounds showed a signal at 1672–1639 cm−1, attributed to the
amidic carbonyl. According to the literature data, it was expected in the regions 1750–1600 cm−1
(stretching) [19]. The N–H stretching bonds from the amide group and from the 5-amino group were
expected in the range 3500–3000 cm−1
[
19]. In the present case, those signals were assigned in the
range 3495–3301 cm−1. In the H-NMR spectra (in DMSO-d6), the signal of the amidic hydrogen
appeared as a singlet at 10.93–9.84 ppm. The two signals of pyrazine hydrogens H3 and H6 appeared
as doublets at 7.92–8.66 ppm with coupling constant J in the range of 1.3–1.4 Hz which is in accordance
with literature [20]. The signal of the 5-amino hydrogen appeared usually as triplet at 7.98–7.76 ppm
with coupling constant J in the range 5.5–5.9 Hz. In the 13C-NMR spectra, the signal of the amidic
carbon appeared at 163.46–161.64 ppm. Representative 1H NMR and 13C NMR spectra in graphic form
(of selected final compounds) are attached in the Supplementary Materials.
1
2.2. Biological Activity
2.2.1. Antimycobacterial Activity
All prepared compounds were screened for in vitro whole cell growth inhibitory activity against
avirulent strain of Mtb H37Ra, fast growing M. smegmatis and M. aurum. Most of compounds were
tested also for in vitro whole cell activity M. kansasii and M. avium. Tests were performed by modified
Microplate Alamar Blue Assay (MABA) [21]. Antimycobacterial activity results were expressed as MIC
in µg/mL in comparison with isoniazid (INH), rifampicin (RIF), and ciprofloxacin (CPX) as standards.
The results are presented in Table 1.
Substitution of chlorine in the pyrazine ring by alkylamino substituents brought an increase in the
growth inhibition of Mtb H37Ra. Compound 4e showed an MIC value in the same order of magnitude
as ciprofloxacin against Mtb H37Ra. Among alkylamino derivatives, monosubstitution on the benzene
ring was more favorable (series 1–4) compared to disubstituted derivatives (series 6, 7).

Molecules 2020, 25, 1561
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Table 1. In vitro antimycobacterial activity against Mtb H37Ra, Mycobacterium aurum and M. smegmatis
(MIC in g/mL), cytotoxicity (IC50), selectivity index (SI), and calculated lipophilicity (logP) of
prepared compounds.
µ
Mtb
H37Ra
Mycobacterium Mycobacterium
HepG2 IC50
No.
R1
R2
SI a
logP
aurum
smegmatis
(µM)
1
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-CH3
4-CH3
4-CH3
4-CH3
-
7.81
6.25
3.13
≥500
≥500
6.25
3.13
25
≥500
≥500
3.13
123.8
>25
125.6
18.9
>25
222.9
66.2
>500
>100
>25
>10
>10
>10
>50
350.2
410.5
377.9
>500
58.6
44.8
60.2
>50
>100
>50
>25
>50
>50
>25
>50
494.6
>50
>25
>25
>25
>10
>250
n.a.
4.78
>1.3
13.6
2.13
n.a.
n.a.
n.a.
>3.99 1.10
>3.5 1.24
>0.92 1.65
n.a.
n.a.
n.a.
n.a.
1.40
7.18
3.46
2.41
2.55
2.96
3.38
3.80
4.22
4.63
1a
1b
1c
1d
1e
1f
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
-
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
-
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
-
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
-
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
-
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
-
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
3.13
1.56
≥500
≥500
≥500
31.25
7.81
≥500
≥500
≥500
≥500
≥500
≥500
≥500
≥500
250
62.5
≥500
≥500
≥500
≥500
≥500
≥500
≥500
≥500
≥250
≥500
≥500
≥500
≥500
≥500
≥250
≥500
≥500
≥250
≥500
≥500
≥500
≥250
≥500
≥500
≥500
≥500
≥500
≥500
≥250
≥500
≥500
≥500
≥500
≥500
≥500
≥500
31.25
250
2
2a
2b
2c
2d
2e
2f
7.81
≥500
≥500
≥500
≥500
62.50
15.63
31.25
3.91
3.91
3.91
3.13
≥500
15.63
≥ 250
≥500
1.56
2.07
2.49
2.91
3.32
1.10
1.24
1.65
250
3
≥250
≥500
≥500
31.25
250
31.25
≥250
≥500
31.25
≥250
≥500
≥500
≥500
≥250
≥500
7.81
≥500
≥500
≥500
≥500
≥250
≥500
3.91
3a
3b
3c
3d
3e
3f
>38.4 2.07
4.71
3.76
6.6
n.a.
>1.73 2.11
n.a.
n.a.
>10.01 3.36
>20.91 3.78
n.a.
n.a.
18.01 2.53
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
2.49
2.91
3.32
1.98
4
4a
4b
4c
4d
4e
4f
2.53
2.95
4-CH3
4-CH3
4-CH3
0.78
≥250
4.20
3.29
5
4-C2H5
4-C2H5
4-C2H5
4-C2H5
4-C2H5
4-C2H5
4-C2H5
4-Cl-2-OH
4-Cl-2-OH
4-Cl-2-OH
4-Cl-2-OH
4-Cl-2-OH
4-Cl-2-OH
4-Cl-2-OH
5-Cl-2-OH
5-Cl-2-OH
5-Cl-2-OH
5-Cl-2-OH
5-Cl-2-OH
5-Cl-2-OH
5-Cl-2-OH
250
5a
5b
5c
5d
5e
5f
7.81
≥500
≥500
≥500
≥500
≥250
≥500
≥500
≥500
≥500
3.13
≥500
≥500
15.63
≥500
≥500
≥500
≥500
≥500
≥500
2.95
3.36
3.78
4.20
4.62
1.66
1.79
2.21
2.63
6
6a
6b
6c
6d
6e
6f
3.91
3.91
3.91
>50
81.1
>50
>25
>50
151.2
>25
>25
>25
>25
>10
>50
>5.27 3.05
≥500
n.a.
n.a.
2.75
n.a.
n.a.
n.a.
n.a.
3.46
3.88
1.66
1.79
2.21
2.63
3.05
7.81
7
15.63
<3.91
3.91
≥500
≥500
31.25
250
7a
7b
7c
7d
7e
7f
≥500
≥500
≥500
≥500
≥500
>0.32 3.46
n.a. 3.88
INH
RIF
CPX
0.125–0.250
0.0039–0.0078 0.39–0.78
0.125–0.25 0.0156–0.0313
3.91–7.81
7.81–15.63
6.25–12.5
0.0625–0.125
a
SI—Selectivity index, values calculated from MIC against Mtb H37Ra as IC50/MIC (in
µM); n.a.—not available;
INH—isoniazid; RIF—rifampicin; CPX—ciprofloxacin. In the column HepG2 IC50 (µM), symbol “>” means that
measurement at higher concentration was not reproducible due to the precipitation of the tested compound in the
cell culture medium.

Molecules 2020, 25, 1561
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Regarding the type of substituents on the benzene ring, the highest number of active alkylamino
derivatives was found in series
(R1 = 4-OH; six derivatives), followed by series (R1 = 3-CF3)
and
3
1
4
(R1 = 4-CH3) with three active derivatives. This finding denotes possible variability in
the substituents, regarding both electronic properties (electron-donating, R1 = 4-OH, R1 = 4-CH3;
vs. electron-withdrawing, R1 = 3-CF3) and hydrophobic properties (lipophilic versus hydrophilic).
Introduction of chlorine substituent in para (series 6) or meta (series 7) position decreased the
antimycobacterial activity. This is in concordance with previous a study, where alkylamino derivatives
(C3-C8) with R1 = 2-Cl were inactive against Mtb H37Rv [14].
We also observed that minute changes in the substitution of the benzene ring led to decrease
of activity in terms of higher MIC values and/or lower number of active derivatives in the series,
for example, methyl versus ethyl substitution (series
(series versus , positional isomerism). The fact that small structural changes on the benzene ring
sometimes lead to significant differences in biological activity indicates that a specific target might be
involved. This is exemplified by series
(R1 = 4-OH) in which the activity grew from propylamino to
octylamino substituent, in comparison with series
(R1 = 3-OH), where alkylamino derivatives longer
4 versus 5, homology) or 4-OH versus 3-OH
3
2
3
2
than C4 were inactive. In this case, the differences in activity of positional isomers cannot be explained
by modified physico-chemical properties, such as lipophilicity, which, at least by calculated logP, is
the same. Neither in 3-OH nor 4-OH isomers do we expect any significant intramolecular H-bonds
affecting the actual lipophilicity or solubility (contrary to 2-OH isomers as we showed before [13]).
In all series with monosubstitution on the benzene ring, the structural exchange of the 5-Cl of the
parent compound for propylamino moiety led to preservation or increase of the activity (1a
, 2a, 3a,
4a 5a). In general, the propylamino substituent can be considered an activity-enhancing substituent
,
which can introduce activity even to inactive or weakly active 5-Cl parental compounds (compare
4
versus 4a and 5 versus 5a).
Activity against M. aurum was noted only for alkylamino derivatives from series
C4–C7 sidechain. The activity here culminated in pentylamino derivative 1c with MIC = 3.13 µg/mL.
Alkylamino derivatives from series 1b 1c) were active also against M. smegmatis with similar
MIC values. Isolated activities against M. smegmatis were also found in series 3 (R1 = 4-CH3, 3c
3e).
In series 5a) were active.
(R1 = 4-CH3) and (R1 = 4-C2H5) only the propylamino derivatives (4a
The most successful substitution on the benzene ring enhancing the activity against M. smegmatis were
1
(R1 = 3-CF3) with
1
(
,
,
4
5
,
the combinations of chloro and hydroxy as seen in series
compounds).
6 (five active compounds) and 7 (four active
Compounds with activity against non-tuberculous mycobacterial strains M. kansasii and M. avium
are presented in Table 2. The activity against M. kansasii was noted only for alkylamino derivatives
with C5 or longer chains. The activity was equal to or higher than INH. None of the tested compounds
exerted significant activity against M. avium.
Table 2. In vitro antimycobacterial activity (expressed as MIC in
mycobacterial strains of M. kansasii and M. avium.a
µg/mL) against non-tuberculous
No.
R1
R2
Mycobacterium kansasii Mycobacterium avium
3c
3f
4-OH
4-OH
4-CH3
Pentyl
Octyl
12.5
6.25
12.5
12.5
25
25
25
25
50
>100
>100
>100
>100
>100
>100
25
4d
4e
6c
6d
6e
Hexyl
Heptyl
Pentyl
Hexyl
Heptyl
4-CH3
4-Cl-2-OH
4-Cl-2-OH
4-Cl-2-OH
INH
a
Compounds 1, 1a, 1c, 1f, 2, 3, 3d, 3e, 4, 5, 5f, 6, 6f, and 7 were not tested in this study. Other compounds not
mentioned in the table were inactive against both strains (MIC > 100 µg/mL).

Molecules 2020, 25, 1561
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No direct correlation between calculated lipophilicity logP and antimycobacterial activity was
observed. However, there was a slight increase in activity against Mtb H37Ra along with increasing
lipophilicity within active compounds (Figure 2). Active compounds had logP values ranging from 1.24
to 3.78. Lipophilicity was not the sole determinant of antimycobacterial activity in the presented series.
Figure 2. Plot of antimycobacterial activity against Mtb H37Ra expressed as log (1/MIC [M]) and
calculated lipophilicity logP. Inactive compounds had an arbitrary value of activity of 3.0.
2.2.2. Antifungal and Antibacterial Activity
As a complementary test, final compounds were tested for in vitro activity against selected fungal
and bacterial pathogens of clinical importance. The microdilution broth method was performed
according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST) instructions
with slight modifications [22–24]. The minimum inhibitory concentration was expressed in µM. Not all
compounds were evaluated due to the fact of a problem with solubility which occurred for 18 samples.
Compounds 2c
,
2d
,
2e
,
4a
,
5b
7a
,
,
5c, and 5f precipitated in both growth media (MHB, RPMI) and
7b 7c, 7d 7e, and 7f did not dissolve in DMSO. See Supplementary
compounds 3f 6c
,
,
6d
,
6e
,
6f
,
,
,
Materials for the list of tested strains, methodology, and full results.
None of the evaluated compounds revealed any significant activity against the selected
bacterial strains.
Five compounds revealed moderate activity against various strains of the Candida genus (Table 3).
Table 3. Significant anti-candidal activity of final compounds expressed as MIC in µM.
No.
R1
R2
CA
CK
CP
1c
2a
3b
3c
4d
3-CF3
3-OH
4-OH
4-OH
4-CH3
Pentyl
Propyl
Butyl
Pentyl
Hexyl
>125
>125
62.5
62.5
>125
62.5
62.5
>250
>125
125
125
>125
250
>125
125
Amphotericin B
0.5 µg/mL (0.54 µM)
1 µg/mL (1.08 µM)
0.5 µg/mL (0.54 µM)
CA—Candida albicans; CK—Candida krusei; CP—Candida parapsilosis.

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2.2.3. In Vitro Cytotoxicity
Results of the experiments were presented as inhibitory concentration which reduced viability of
the cell population to 50% from the maximal viability (IC50). Cytotoxicity of the tested compounds
was measured using the standard hepatic cell line HepG2. The used CellTiter 96 assay is based on the
reduction of tetrazolium dye MTS in living cells to formazan, which is then determined colorimetrically.
The reduction of the reagent is related to availability of NADH and NADPH. The decline in levels of
these metabolically important compounds in the cell leads to reduced production of formazan.
It was not possible to determine the IC50 values for two-thirds of the compounds due to the
fact of their low solubility in the cell culture medium. The effective cytotoxic concentrations of all
tested compounds are listed in Table 1. Among these compounds, 1c exhibited the highest cytotoxicity
(IC50 = 18.9
(IC50 = 66.2
µ
M). Other compounds with relatively high cytotoxicity were 1f
, 3d, 3e, 3f, and 6c
µM, 58.6 M, 44.8 M, 60.2 M, and 81.1 M, respectively). Compound 3c was relatively
µ
µ
µ
µ
non-cytotoxic, as its IC50 value was above the tested range of concentrations. Compounds 3a
, 3b,
and 5a exhibited low cytotoxicity (IC50 = 410.5 µM, 377.9 µM and 494.6 µM, respectively).
3. Materials and Methods
3.1. General
Reagents and solvents were obtained from Sigma–Aldrich (Schnelldorf, Germany) if not stated
otherwise and were used without further purification. The reaction progress was checked using Merck
Silica 60 F254 TLC plates (Merck, Darmstadt, Germany). Flash chromatography of the final compounds
was run on an automated chromatograph puriFlash 5.125 (INTERCHIM, Montluçon, France) using
original puriFlash columns with 50 µm silica and a mixture of hexane and ethyl acetate (gradient
mode), detection wavelength 254 nm. The NMR spectra were recorded on a Varian VNMR S500
(Varian, Palo Alto, CA, USA) at 500 MHz for 1H and 126 MHz for 13C. The spectra were recorded in
DMSO-d6 or pyridine at ambient temperature. The chemical shifts as δ values in ppm are indirectly
referenced to tetramethylsilane (TMS) via the solvent signal (2.49 for 1H and 39.7 for 13C in DMSO-d6;
8.74, 7.58, 7.22 for 1H and 150.35, 135.91, 123.87 for 13C in pyridine-d5). The IR spectra were recorded on
a Nicolet Impact 400 (Nicolet, Madison, WI, USA) using the ATR-Ge method. Elemental analysis was
performed on a Vario MICRO cube Element Analyzer (Elementar Analysensysteme, Hanau, Germany)
and the values are given as percentages. Melting points were determined in open capillaries on a Stuart
SMP20 melting point apparatus (Bibby Scientific Limited, Staffordshire, UK) and are uncorrected.
Relative yields are given as percentages (of theoretical yield) and refer to the amount of product after
all purification steps. The logP was calculated using the program CS ChemDraw Professional version
18.1.2 (PerkinElmer, Waltham, MA, USA).
3.2. Chemistry
3.2.1. General Synthetic Procedure for Preparation of 5-chloro-N-phenylpyrazine-2-carboxamides
5-Chloro-N-phenylpyrazine-2-carboxamides (1–7) were prepared by a published procedure [13].
A mixture of 5-chloro-N-phenylpyrazine-2-carboxylic acid (5-Cl-POA, 10 mmol), thionyl chloride
(SOCl2, 3 mL, 41 mmol) and N,N-dimethylformamide (3 drops) in dry toluene (20 mL) was stirred
intensively under a reflux condenser for 1 h at 100 ◦C. The process turned the suspension into clear
burgundy solution. After the completion of the reaction, the liquids were evaporated under reduced
pressure upon addition of dry toluene (3
×
20 mL) to remove the unreacted thionyl chloride. The crude
acyl chloride in the form of viscous liquid was diluted with dry acetone (20 mL) and added dropwise to
the stirred and cooled (ice bath) mixture of respective aniline (9 mmol) and pyridine (1.6 mL, 20 mmol)
in dry acetone (20 mL). The mixture was stirred at room temperature for 1 h and during the reaction
the product precipitated from solution. Solvents were evaporated under reduced pressure and the
crude product was washed with distilled water.

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3.2.2. General Synthetic Procedure for Preparation of 5-alkylamino-N-phenylpyrazine-2-carboxamides
The 5-alkylamino-N-phenylpyrazine-2-carboxamides (1a 2a 3a 4a 5a 6a 7a ) were
–
f,
–
f,
–f
,
–
f,
–f
,
–
f,
–f
prepared by a published procedure [14]. A mixture of 5-chloro-N-phenylpyrazine-2-carboxamide
(1 mmol), triethylamine (TEA, 3 mmol) and respective aliphatic amine (3 mmol) in ethanol (20 mL)
was stirred intensively at 90 ◦C under a reflux condenser for 7 h. The progress of the reaction was
monitored by TLC. To remove excess of TEA the reaction mixture was evaporated under reduced
pressure with ethanol (3 × 20 mL).
Final products were purified by flash chromatography (silica, EtOAc in hexane, gradient elution)
and recrystallized from hot ethanol. Prepared compounds were characterized with melting point, 1H
NMR, 13C NMR, and IR spectroscopy. The purity was confirmed by elemental analysis.
3.3. Analytical Data of Prepared Compounds
Compounds
full analytical data.
5-Chloro-N-(3-(trifluoromethyl)phenyl)pyrazine-2-carboxamide (1)
1–3, 5, and 6 were prepared and reported previously in a preliminary study [13] with
◦
◦
Beige solid. Yield: 86%. mp 124–125 C (lit. mp 121.5–122.5 C [13]). 1H NMR (500 MHz,
DMSO-d6) 10.43 (s, 1H, CONH), 9.19 (d, J = 1.4 Hz, 1H, H3), 8.81 (d, J = 1.4 Hz, 1H, H6), 8.43 (s, 1H,
H20), 8.21 (d, J = 8.0 Hz, 1H, H40), 7.66 (t, J = 8.0 Hz, 1H, H50), 7.52 (d, J = 8.0 Hz, 1H, H60). 13C NMR
(126 MHz, DMSO-d6) 161.84, 152.84, 144.91, 144.04, 143.71, 139.83, 131.57 (q, J = 31.50 Hz), 130.70,
δ
δ
126.22 (q, J = 3.78 Hz), 124.57, 121.63 (q, J = 3.78 Hz), 117.59 (q, J = 3.78 Hz).
5-Chloro-N-(3-hydroxyphenyl)pyrazine-2-carboxamide (2)
◦
◦
Beige solid. Yield: 76%. mp 222–224 C (lit. mp 225.1–226.3 C [13]). 1H NMR (500 MHz,
DMSO-d6)
δ 10.64 (s, 1H, CONH), 9.52 (s, 1H, OH), 9.09 (d, J = 1.4 Hz, 1H, H3), 8.89 (d, J = 1.4 Hz, 1H,
H6), 7.45 (t, J = 2.1 Hz, 1H, H20), 7.24–7.20 (m, 1H, H40), 7.17 (t, J = 8.1 Hz, 1H, H50), 6.58–6.53 (m, 1H,
H60). 13C NMR (126 MHz, DMSO-d6)
δ 160.90, 157.73, 151.01, 144.17, 144.14, 143.05, 139.20, 129.51,
111.69, 111.56, 107.86.
5-Chloro-N-(4-hydroxyphenyl)pyrazine◦-2-carboxamide (3)
◦
Yellow solid. Yield: 70%. mp 199–201 C (lit. mp 204.8–206.7 C [13]). 1H NMR (500 MHz,
DMSO-d6)
δ 10.49 (s, 1H, CONH), 9.32 (s, 1H, OH), 9.06 (d, J = 1.4 Hz, 1H, H3), 8.87 (d, J = 1.4 Hz, 1H,
H6), 7.67–7.58 (m, 2H, H10, H60), 6.77–6.69 (m, 2H, H30, H50). 13C NMR (126 MHz, DMSO-d6)
154.39, 150.88, 144.32, 144.02, 143.04, 129.78, 122.51, 115.18.
δ 160.41,
5-Chloro-N-(p-tolyl)pyrazine-2-carboxamide (4)
Beige solid. Yield: 90%. mp 179–180 ◦C. 1H NMR (500 MHz, DMSO-d6)
δ
10.63 (s, 1H, CONH),
9.09 (d, J = 1.4 Hz, 1H, H3), 8.91 (d, J = 1.4 Hz, 1H, H6), 7.76–7.73 (m, 2H, H20, H60), 7.18–7.15 (m,
2H, H30, H50), 2.28 (s, 1H, CH3). 13C NMR (126 MHz, DMSO-d6)
160.78, 150.98, 144.11, 144.08,
δ
143.03, 135.70, 133.57, 129.23, 120.72, 20.69. Elemental analysis found: C, 57.85%; H, 4.11%; N, 16.88%.
Calculated for C12H10N3O (MW 247.68): C, 58.19%; H, 4.07%; N, 16.97%.
5-Chloro-N-(4-ethylphenyl)pyrazine-2-carboxamide (5)
1
Beige solid. Yield: 88%. mp 156–157 ◦C (lit. mp 155–156.5 ◦C [13]). H NMR (500 MHz, DMSO-d6)
δ
10.62 (s, 1H, CONH), 9.11 (d, J = 1.4 Hz, 1H, H3), 8.91 (d, J = 1.4 Hz, 1H, H6), 7.79–7.76 (m, 2H, H20,
H60), 7.21–7.18 (m, 2H, H30, H50), 2.57–2.61 (m, 1H, CH2), 1.25 (t, J = 7.7 Hz, 1H, CH3). 13C NMR
(126 MHz, DMSO-d6) δ 160.80, 151.00, 144.13, 144.08, 143.04, 135.89, 128.05, 120.84, 27.83, 15.86.
5-Chloro-N-(5-chloro-2-hydroxyphenyl)pyrazine-2-carboxamide (6)
Yellow solid. Yield: 73.1%. mp 220–223 ◦C (lit. mp 221.4–222.2 ◦C [13]). 1H NMR (500 MHz,
DMSO-d6)
H6), 8.33 (d, J = 2.5 Hz, 1H, H60), 7.03 (dd, J = 8.7, 2.5 Hz, 1H, H40), 6.92 (d, J = 8.7, 2.5 Hz, 1H, H30). 13C
NMR (126 MHz, DMSO-d6) 159.83, 151.59, 145.79, 143.69, 143.44, 142.68, 126.86, 124.27, 122.76, 118.94,
δ 10.73 (s, 1H, CONH), 10.06 (s, 1H, OH), 9.07 (d, J = 1.3 Hz, 1H, H3), 8.93 (d, J = 1.3 Hz, 1H,
δ
116.08. Elemental analysis found: C, 46.08%; H, 2.50%; N, 14.61%. Calculated for C11H7Cl2N3O2 (MW
284.10): C, 46.51%; H, 2.48%; N, 14.79%.

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5-Chloro-N-(4-chloro-2-hydroxyphenyl)pyrazine-2-carboxamide (7)
Beige solid. Yield: 96%. mp 247.7–249 ◦C. 1H NMR (500 MHz, DMSO-d6)
δ
10.93 (s, 1H, CONH),
10.02 (s, 1H, OH), 9.11 (s, 1H, H3), 8.94 (s, 1H, H6), 8.27 (d, J = 8.6 Hz, 1H, H60), 6.96 (d, J = 2.3 Hz, 1H,
H30), 6.92 (dd, J = 8.6, 2.4 Hz, 1H, H50). 13C NMR (126 MHz, DMSO-d6)
δ 161.65, 156.45, 147.45, 142.92,
131.70, 131.13, 126.78, 125.98, 119.89, 119.10, 114.55. Elemental analysis found: C, 46.08%; H, 2.50%; N,
14.61%. Calculated for C11H7Cl2N3O2 (MW 284.10): C, 46.51%; H, 2.48%; N, 14.79%.
5-(Propylamino)-N-(3-(trifluoromethyl)phenyl)pyrazine-2-carboxamide (1a)
White solid. Yield: 87%. mp 155–156 ◦C. IR (ATR-Ge, cm−1) 3495 (N-H, NH), 3343 (N-H, CONH),
1
2933, 2879 (C-H, alkyl), 1670 (C=O, CONH), 1591, 1529 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
10.48 (s, 1H, CONH), 8.66 (d, J = 1.3 Hz, 1H, H3), 8.40 (d, J = 2.1 Hz, 1H, H20), 8.11 (dt, J = 7.9, 2.1 Hz,
1H, H60), 7.97 (d, J = 1.3 Hz, 1H, H6), 7.91 (t, J = 5.6 Hz, 1H, NH), 7.55 (t, J = 7.9 Hz, 1H, H50), 7.40
(dd, J = 7.9, 2.1, 1H, H40), 3.33–3.27 (m, 2H, CH2), 1.63–1.52 (m, 2H, CH2), 0.92 (t, J = 7.4 Hz, 3H, CH3).
13C NMR (126 MHz, DMSO-d6)
δ 163.17, 156.45, 143.51, 139.86, 131.49, 129.84, 129.49 (q, J = 31,50 Hz),
125.44, 123.78 (q, J = 3.78 Hz), 123.28, 119.70 (q, J = 3.78 Hz), 116.35 (q, J = 3.78 Hz), 42.27, 21.97, 11.61.
Elemental analysis found: C, 55.23%; H, 4.71%; N, 17.53%. Calculated for C15H15F3N4O (MW 324.31):
C, 55.55%; H, 4.66%; N, 17.28%.
5-(Butylamino)-N-(3-(trifluoromethyl)phenyl)pyrazine-2-carboxamide (1b)
White solid. Yield: 90%. mp 141–142 ◦C. IR (ATR-Ge, cm−1) 3385 (N-H, NH), 3348 (N-H, CONH),
1
2933, 2877 (C-H, alkyl), 1669 (C=O, CONH), 1590, 1529 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
10.48 (s, 1H, CONH), 8.66 (d, J = 1.4 Hz, 1H, H3), 8.40 (d, J = 2.1 Hz, 1H, H20), 8.11 (dt, J = 8.3, 2.1 Hz,
1H, H60), 7.96 (d, J = 1.4 Hz, 1H, H6), 7.88 (t, J = 5.6 Hz, 1H, NH), 7.55 (t, J = 8.3 Hz, 1H, H50), 7.39
(dd, J = 8.3, 2.1 Hz, 1H, H40), 3.34 (td, J = 7.7, 5.5 Hz, 2H, CH2), 1.54 (dd, J = 7.7, 6.6 Hz, 2H, CH2),
1.42–1.31 (m, 2H, CH2), 0.90 (t, J = 7.3 Hz, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 163.17, 156.43,
143.51, 139.87, 131.46, 129.84, 129.49 (q, J = 31.50 Hz), 125.44, 123.77 (q, J = 3.78 Hz), 123.28, 119.70 (q,
J = 3.78 Hz), 116.37 (q, J = 3.78 Hz), 40.15, 30.78, 19.82, 13.83. Elemental analysis found: C, 56.55%; H,
5.13%; N, 16.93%. Calculated for C16H17F3N4O (MW 338.33): C, 56.80%; H, 5.06%; N, 16.56%.
5-(Pentylamino)-N-(3-(trifluoromethyl)phenyl)pyrazine-2-carboxamide (1c)
White solid. Yield: 98%. mp 128–129 ◦C. IR (ATR-Ge, cm−1) 3307 (N-H, NH, CONH), 2933, 2875
(C-H, alkyl), 1666 (C=O, CONH), 1595, 1527 (C=C, Ar). 1H NMR (500 MHz, DMSO-d6)
δ 10.47 (s,
1H, CONH), 8.65 (d, J = 1.3 Hz, 1H, H3), 8.39 (t, J = 2.0 Hz, 1H, H20), 8.14–8.08 (m, 1H, H60), 7.96 (d,
J = 1.3 Hz ,1H, H6), 7.88 (t, J = 5.5 Hz, 1H, NH), 7.54 (t, J = 8.0 Hz, 1H, H50), 7.42–7.36 (m, 1H, H40),
3.33 (td, J = 7.1, 5.5 Hz, 2H, CH2), 1.55 (q, J = 7.1 Hz, 2H, CH2), 1.31 (m, 4H, (CH2)2), 0.91–0.84 (m,
3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 163.18, 156.42, 143.52, 139.88, 131.46, 129.88, 129.51 (q,
J = 31.50 Hz), 125.45, 123.77 (q, J = 3.78 Hz), 123.28, 119.70 (q, J = 3.78 Hz), 116.35 (q, J = 3.78 Hz), 40.44,
28.85, 28.37, 22.06, 14.06. Elemental analysis found: C, 57.55%; H, 5.50%; N, 16.02%. Calculated for
C17H19F3N4O (MW 352.36): C, 57.95%; H, 5.44%; N, 15.90%.
5-(Hexylamino)-N-(3-(trifluoromethyl)phenyl)pyrazine-2-carboxamide (1d)
White solid. Yield: 92%. mp 148–149 ◦C. IR (ATR-Ge, cm−1) 3301 (N-H, NH, CONH), 2934, 2875
1
(C-H, alkyl), 1665 (C=O, CONH), 1596, 1529 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ 10.48 (s, 1H,
CONH), 8.65 (d, J = 1.4 Hz, 1H, H3), 8.40 (d, J = 2.1 Hz, 1H, H20), 8.11 (dd, J = 8.0, 2.1 Hz, 1H, H60),
7.96 (d, J = 1.4 Hz, 1H, H6), 7.88 (t, J = 5.6 Hz, 1H, NH), 7.54 (t, J = 8.0 Hz, 1H, H50), 7.39 (dd, J = 8.0,
2.1 Hz, 1H, H40), 3.36–3.29 (m, 2H, CH2), 1.54 (p, J = 7.1 Hz, 2H, CH2), 1.34 (m, 2H, CH2), 1.32–1.22 (m,
4H, (CH2)2), 0.89–0.81 (m, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 163.17, 156.41, 143.51, 139.87,
131.46, 129.83, 129.49 (q, J = 31.50 Hz), 125.45, 123.77 (q, J = 3.78 Hz), 123.28, 119.70 (q, J = 3.78 Hz),
116.34 (q, J = 3.78 Hz), 40.47, 31.19, 28.65, 26.34, 22.24, 14.06. Elemental analysis found: C, 58.96%; H,
5.90%; N, 15.33%. Calculated for C18H21F3N4O (MW 366.39): C, 59.01%; H, 5.78%; N, 15.29%.
5-(Heptylamino)-N-(3-(trifluoromethyl)phenyl)pyrazine-2-carboxamide (1e)
White solid. Yield: 76%. mp 148–149 ◦C. IR (ATR-Ge, cm−1) 3302 (N-H, NH, CONH), 2933, 2856
1
(C-H, alkyl), 1664 (C=O, CONH), 1596, 1529 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ 10.48 (s, 1H,
CONH), 8.65 (d, J = 1.4 Hz, 1H, H3), 8.39 (t, J = 2.1 Hz, 1H, H20), 8.11 (dt, J = 8.4, 2.1 Hz, 1H, H60), 7.96

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(d, J = 1.4 Hz, 1H, H6), 7.88 (t, J = 5.5 Hz, 1H, NH), 7.54 (t, J = 8.4 Hz, 1H, H50), 7.39 (dd, J = 8.4, 2.1 Hz,
1H, H40), 3.36–3.29 (m, 2H, CH2), 1.54 (h, J = 6.7 Hz, 2H, CH2), 1.38–1.17 (m, 8H, (CH2)4), 0.88–0.81
(m, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 163.46, 156.70, 143.80, 140.16, 131.74, 130.12, 129.58
(q, J = 31.5 Hz), 125.74, 124.06 (q, J = 3.78 Hz), 123.57, 120.00 (q, J = 3.78 Hz), 116.66 (q, J = 3.78 Hz),
40.75, 31.71, 28.97, 28.91, 26.92, 22.51, 14.38. Elemental analysis found: C, 59.76%; H, 6.14%; N, 14.90%.
Calculated for C19H23F3N4O (MW 380.42): C, 59.99%; H, 6.09%; N, 14.73%.
5-(Octylamino)-N-(3-(trifluoromethyl)phenyl)pyrazine-2-carboxamide (1f)
White solid. Yield: 97%. mp 135–136 ◦C. IR (ATR-Ge, cm−1) 3405 (N-H, NH), 3340 (N-H, CONH),
1
2926, 2854 (C-H, alkyl), 1671 (C=O, CONH), 1591, 1529 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
10.47 (s, 1H, CONH), 8.65 (d, J = 1.4 Hz, 1H, H3), 8.40 (t, J = 2.0 Hz, 1H, H20), 8.14–8.08 (m, 1H,
H60), 7.96 (d, J = 1.4 Hz, 1H, H6), 7.87 (t, J = 5.5 Hz, 1H, NH), 7.54 (t, J = 8.0 Hz, 1H, H50), 7.42–7.36
(m, 1H, H40), 3.32 (td, J = 7.1, 5.5 Hz, 2H, CH2), 1.54 (h, J = 6.7 Hz, 2H, CH2), 1.37–1.29 (m, 2H, CH2),
1.28–1.20 (m, 8H, (CH2)4), 0.87–0.80 (m, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 163.16, 156.41,
143.50, 139.87, 131.45, 129.81, 129.49 (q, J = 31.50 Hz), 125.44, 123.75 (q, J = 3.78 Hz), 123.28, 119.68
(q, J = 3.78 Hz), 116.33 (q, J = 3.78 Hz), 40.45, 31.40, 28.91, 28.84, 28.67, 26.66, 22.24, 14.07. Elemental
analysis found: C, 60.57%; H, 6.45%; N, 14.25%. Calculated for C20H25F3N4O (MW 394.44): C, 60.90%;
H, 6.39%; N, 14.20%.
N-(3-hydroxyphenyl)-5-(propylamino)pyrazine-2-carboxamide (2a)
White solid. Yield: 68%. mp 194–195 ◦C. IR (ATR-Ge, cm−1) 3338 (N-H, NH), 3272 (N-H, CONH),
1
2955, 2874 (C-H, alkyl), 1651 (C=O, CONH), 1599, 1531 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
9.89 (s, 1H, CONH), 9.37 (s, 1H, OH), 8.63 (d, J = 1.4 Hz, 1H, H3), 7.95 (d, J = 1.4 Hz, 1H, H6), 7.84 (t,
J = 5.6 Hz, 1H, NH), 7.43 (t, J = 2.2 Hz, 1H, H20), 7.17 (dd, J = 8.1, 2.2 Hz, 1H, H60), 7.08 (t, J = 8.0 Hz, 1H,
H50), 6.48 (dd, J = 8.0, 2.2 Hz, 1H, H40), 3.30 (td, J = 7.3, 5.6 Hz, 2H, CH2), 1.57 (h, J = 7.3 Hz, 2H, CH2),
0.92 (t, J = 7.3 Hz, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 162.40, 157.72, 156.35, 143.08, 139.89,
131.93, 131.26, 129.39, 110.93, 110.71, 107.20, 42.28, 21.99, 11.64. Elemental analysis found: C, 61.83%; H,
5.97%; N, 20.24%. Calculated for C14H16N4O2 (MW 272.31): C, 61.75%; H, 5.92%; N, 20.58%.
5-(Butylamino)pyrazine-N-(3-hydroxyphenyl)-2-carboxamide (2b)
Cream solid. Yield: 84%. mp 203–204 ◦C. IR (ATR-Ge, cm−1) 3353 (N-H, NH), 3297 (N-H, CONH),
1
2954, 2871 (C-H, alkyl), 1655 (C=O, CONH), 1601, 1532 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
9.89 (s, 1H, CONH), 9.37 (s, 1H, OH), 8.63 (d, J = 1.3 Hz, 1H, H3), 7.94 (d, J = 1.3 Hz, 1H, H6), 7.81 (t,
J = 5.5 Hz, 1H, NH), 7.43 (t, J = 2.2 Hz, 1H, H20), 7.17 (dd, J = 8.1, 2.2 Hz, 1H, H60), 7.08 (t, J = 8.1 Hz,
1H, H50), 6.48 (dt, J = 8.1, 2.2 Hz, 1H, H40), 3.33 (td, J = 7.2, 5.5 Hz, 2H, CH2), 1.58–1.49 (m, 2H, CH2),
1.36 (h, J = 7.2 Hz, 2H, CH2), 0.90 (t, J = 7.2 Hz, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 162.41,
157.73, 156.34, 143.09, 139.90, 131.91, 131.15, 129.40, 110.93, 110.71, 107.20, 40.18, 30.82, 19.84, 13.87.
Elemental analysis found: C, 63.14%; H, 6.48%; N, 19.14%. Calculated for C15H18N4O2 (MW 286.34):
C, 62.92%; H, 6.34%; N, 19.57%.
N-(3-hydroxyphenyl)-5-(pentylamino)pyrazine-2-carboxamide (2c)
◦
Light cream solid. Yield: 75%. mp 224.8–226.1 C. IR (ATR-Ge, cm−1) 3352 (N-H, NH), 3290
1
(N-H, CONH), 2934, 2873 (C-H, alkyl), 1654 (C=O, CONH), 1602, 1529 (C=C, Ar). H NMR (500 MHz,
DMSO-d6) δ 9.87 (s, 1H, CONH), 9.41 (s, 1H, OH), 8.61 (d, J = 1.4 Hz, 1H, H3), 8.03–7.94 (m, 1H, H6),
7.94 (d, J = 5.6 Hz, 1H, NH), 7.41 (t, J = 2.2 Hz, 1H, H20), 7.17 (dd, J = 8.2, 2.2 Hz, 1H, H60), 7.07 (t,
J = 8.2 Hz, 1H, H50), 6.49 (dd, J = 8.2, 2.2 Hz, 1H, H40), 3.32 (m, 2H, CH2), 2.76–2.69 (m, 2H, CH2), 1.55
(m, 2H, CH2), 1.29 (m, 2H, CH2), 0.86 (q, J = 6.7 Hz, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 162.40,
157.76, 156.34, 143.06, 139.86, 131.84, 131.37, 129.35, 110.88, 110.73, 107.20, 40.43, 28.86, 26.78, 22.06,
14.09. Elemental analysis found: C, 63.50%; H, 6.72%; N, 18.18%. Calculated for C16H20N4O2 (MW
300.36): C, 63.98%; H, 6.71%; N, 18.65%.
5-(Hexylamino)pyrazine-N-(3-hydroxyphenyl)-2-carboxamide (2d)
Light cream solid. Yield: 69%. mp 225–226 ◦C. IR (ATR-Ge, cm−1) 3351 (N-H, NH), 3290 (N-H,
CONH), 2934, 2858 (C-H, alkyl), 1655 (C=O, CONH), 1602, 1529 (C=C, Ar). 1H NMR (500 MHz,
DMSO-d6) δ 9.89 (s, 1H, CONH), 9.37 (s, 1H, OH), 8.62 (d, J = 1.4 Hz, 1H, H3), 7.94 (d, J = 1.4 Hz, 1H,

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H6), 7.82 (t, J = 5.5 Hz, 1H, NH), 7.42 (t, J = 2.2 Hz, 1H, H20), 7.17 (dd, J = 8.1, 2.2 Hz, 1H, H60), 7.08
(t, J = 8.1 Hz, 1H, H50), 6.48 (dd, J = 8.1, 2.2 Hz, 1H, H40), 3.32 (td, J = 7.1, 5.5 Hz, 2H, CH2), 1.54 (p,
J = 7.1 Hz, 2H, CH2), 1.38–1.19 (m, 6H, (CH2)3), 0.86 (t, J = 6.9 Hz, 3H, CH3). 13C NMR (126 MHz,
DMSO-d6) δ 162.39, 157.72, 156.32, 143.08, 139.89, 131.90, 131.19, 129.38, 110.92, 110.70, 107.19, 40.48,
31.20, 28.67, 26.35, 22.26, 14.09. Elemental analysis found: C, 65.18%; H, 7.32%; N, 17.58%. Calculated
for C17H22N4O2 (MW 314.39): C, 64.95%; H, 7.05%; N, 17.82%.
5-(Heptylamino)pyrazine-N-(3-hydroxyphenyl)-2-carboxamide (2e)
Light cream solid. Yield: 47%. mp 222–223 ◦C. IR (ATR-Ge, cm−1) 3349 (N-H, NH), 3291 (N-H,
CONH), 2955, 2857 (C-H, alkyl), 1653 (C=O, CONH), 1603, 1529 (C=C, Ar). 1H NMR (500 MHz,
DMSO-d6) δ 9.89 (s, 1H, CONH), 9.36 (s, 1H, OH), 8.62 (d, J = 1.4 Hz, 1H, H3), 7.94 (d, J = 1.4 Hz, 1H,
H6), 7.82 (t, J = 5.6 Hz, 1H, NH), 7.42 (t, J = 2.2 Hz, 1H, H20), 7.17 (dd, J = 8.0, 2.2 Hz, 1H, H60), 7.08 (t,
J = 8.0 Hz, 1H, H50), 6.48 (dd, J = 8.0, 2.2 Hz, 1H, H40), 3.31 (t, J = 7.0 Hz, 2H, CH2), 1.55 (p, J = 7.0 Hz,
2H, CH2), 1.37–1.21 (m, 8H, (CH2)4), 0.85 (t, J = 7.0 Hz, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ
162.39, 157.72, 156.32, 143.08, 139.89, 131.90, 131.29, 129.38, 110.91, 110.70, 107.18, 40.47, 31.43, 28.71,
28.64, 26.64, 22.23, 14.11. Elemental analysis found: C, 65.68%; H, 7.43%; N, 16.93%. Calculated for
C18H24N4O2 (MW 328.42): C, 65.83%; H, 7.37%; N, 17.06%.
N-(3-hydroxyphenyl)-5-(octylamino)pyrazine-2-◦carboxamide (2f)
Light cream solid. Yield: 65%. mp 223.0–225.9 C. IR (ATR-Ge, cm−1) 3357 (N-H, NH), 3283
1
(N-H, CONH), 2955, 2857 (C-H, alkyl), 1652 (C=O, CONH), 1602, 1528 (C=C, Ar). H NMR (500 MHz,
DMSO-d6) δ 9.89 (s, 1H, CONH), 9.37 (s, 1H, OH), 8.62 (d, J = 1.4 Hz, 1H, H3), 7.94 (d, J = 1.4 Hz, 1H,
H6), 7.82 (t, J = 5.5 Hz, 1H, NH), 7.43 (t, J = 2.2 Hz, 1H, H20), 7.17 (dd, J = 8.2, 2.2 Hz, 1H, H60), 7.08
(t, J = 8.2 Hz, 1H, H50), 6.48 (dd, J = 8.2, 2.2 Hz, 1H, H40), 3.31 (dd, J = 7.1, 5.5 Hz, 2H, CH2), 1.54
(p, J = 7.1 Hz, 2H, CH2), 1.38–1.29 (m, 2H, CH2), 1.32 – 1.21 (m, 8H, (CH2)4), 0.87–0.81 (m, 3H, CH3).
13C NMR (126 MHz, DMSO-d6)
δ 162.39, 157.72, 156.32, 143.08, 139.89, 131.89, 131.21, 129.38, 110.92,
110.70, 107.19, 40.47, 31.42, 28.93, 28.85, 28.71, 26.68, 22.26, 14.11. Elemental analysis found: C, 66.66%;
H, 7.75%; N, 16.45%. Calculated for C19H26N4O2 (MW 342.44): C, 66.64%; H, 7.65%; N, 16.36%.
N-(4-hydroxyphenyl)-5-(propylamino)pyrazine-2-carboxamide (3a)
White solid. Yield: 85%. mp 209.3–215.6 ◦C. IR (ATR-Ge, cm−1) 3307, (N-H, O-H, CONH, NH,
1
OH), 2971 (C-H, alkyl),1638 (C=O, CONH), 1571, 1527 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
9.85 (s, 1H, CONH), 9.21 (s, 1H, OH), 8.60 (d, J = 1.4 Hz, 1H, H3), 7.94 (d, J = 1.4 Hz, 1H, H6), 7.79 (t,
J = 5.6 Hz, 1H, NH), 7.63–7.56 (m, 2H, H20, H60), 6.75–6.68 (m, 2H, H30, H50), 3.33–3.22 (m, 2H, CH2),
1.57 (h, J = 7.3 Hz, 2H, CH2), 0.92 (t, J = 7.3 Hz, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 162.04,
156.29, 153.68, 142.80, 132.19, 131.19, 130.58, 121.89, 115.17, 42.30, 22.03, 11.67. Elemental analysis
found: C, 61.44%; H, 6.04%; N, 20.47%. Calculated for C14H16N4O2 (MW 272.31): C, 61.75%; H, 5.92%;
N, 20.58%.
5-(Butylamino)pyrazine-N-(4-hydroxyphenyl)-2-carboxamide (3b)
White solid. Yield: 51%. mp 208.0–211.4 ◦C. IR (ATR-Ge, cm−1) 3323, (N-H, O-H, CONH, NH,
1
OH), 2969, 2874 (C-H, alkyl),1639 (C=O, CONH), 1573, 1524 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
9.84 (s, 1H, CONH), 9.20 (s, 1H, OH), 8.60 (d, J = 1.4 Hz, 1H, H3), 7.93 (d, J = 1.4 Hz, 1H, H6), 7.76 (t,
J = 5.5 Hz, 1H, NH), 7.67–7.56 (m, 2H, H20, H60), 6.75–6.68 (m, 2H, H30, H50), 3.33 (td, J = 7.1, 5.5 Hz,
2H, CH2), 1.54 (p, J = 7.1 Hz, 2H, CH2), 1.36 (h, J = 7.1 Hz, 2H, CH2), 0.90 (t, J = 7.1 Hz, 3H, CH3).
13C NMR (126 MHz, DMSO-d6)
δ 162.02, 156.26, 153.67, 142.79, 132.15, 131.26, 130.57, 121.86, 115.15,
40.16, 30.84, 19.85, 13.88. Elemental analysis found: C, 62.56%; H, 6.41%; N, 19.48%. Calculated for
C15H18N4O2 (MW 286.34): C, 62.92%; H, 6.34%; N, 19.57%.
N-(4-hydroxyphenyl)-5-(pentylamino)pyrazine-2-carboxamide (3c)
White solid. Yield: 76%. mp 162.6–166.8 ◦C. IR (ATR-Ge, cm−1) 3336, (N-H, O-H, CONH, NH,
1
OH), 2956, 2869 (C-H, alkyl), 1655 (C=O, CONH), 1601, 1576, 1537, 1513 (C=C, Ar). H NMR (500 MHz,
DMSO-d6) δ 9.84 (s, 1H, CONH), 9.19 (s, 1H, OH), 8.60 (d, J = 1.4 Hz, 1H, H3), 7.93 (d, J = 1.4 Hz, 1H,
H6), 7.77 (t, J = 5.6 Hz, 1H, NH), 7.62–7.56 (m, 2H, H20, H60), 6.74–6.68 (m, 2H, H30, H50), 3.34–3.28
(m, 2H, CH2), 1.55 (q, J = 7.1 Hz, 2H, CH2), 1.38–1.30 (m, 4H, (CH2)2), 0.91–0.84 (m, 3H, CH3). 13C

Molecules 2020, 25, 1561
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NMR (126 MHz, DMSO-d6)
δ
161.99, 156.24, 153.65, 142.78, 132.15, 131.18, 130.56, 121.84, 115.13, 40.43,
28.86, 28.40, 22.07, 14.09. Elemental analysis found: C, 63.56%; H, 6.57%; N, 18.54%. Calculated for
C16H20N4O2 (MW 300.36): C, 63.98%; H, 6.71%; N, 18.65%.
5-(Hexylamino)pyrazine-N-(4-hydroxyphenyl)-2-carboxamide (3d)
White solid. Yield: 34%. mp 157.7–161.5 ◦C. IR (ATR-Ge, cm−1) 3352 (N-H, NH), 3263 (N-H, O-H,
1
CONH, OH), 2932, 2860 (C-H, alkyl),1657 (C=O, CONH), 1603, 1582, 1532, 1515 (C=C, Ar). H NMR
(500 MHz, DMSO-d6)
δ 9.84 (s, 1H, CONH), 9.19 (s, 1H, OH), 8.60 (d, J = 1.3 Hz, 1H, H3), 7.93 (d,
J = 1.3 Hz, 1H, H6), 7.77 (t, J = 5.6 Hz, 1H, NH), 7.62–7.57 (m, 2H, H20, H60), 6.74–6.68 (m, 2H, H30,
H50), 3.34–3.28 (m, 2H, CH2), 1.54 (p, J = 7.1 Hz, 2H, CH2), 1.34 (m, 2H, CH2), 1.27 (dt, J = 7.1, 3.8 Hz,
4H, (CH2)2), 0.89–0.83 (m, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 162.00, 156.24, 153.66, 142.78,
132.15, 131.09, 130.57, 121.84, 115.14, 40.47, 31.21, 28.69, 26.35, 22.26, 14.09. Elemental analysis found: C,
64.48%; H, 7.02%; N, 17.72%. Calculated for C17H22N4O2 (MW 314.39): C, 64.95%; H, 7.05%; N, 17.82%.
5-(Heptylamino)pyrazine-N-(4-hydroxyphenyl-2-carboxamide (3e)
White solid. Yield: 63%. mp 151.4–155.4 ◦C. IR (ATR-Ge, cm−1) 3357(N-H, NH), 3268 (N-H, O-H,
1
CONH, OH), 2931, 2858 (C-H, alkyl), 1655 (C=O, CONH), 1602, 1582, 1531, 1513 (C=C, Ar). H NMR
(500 MHz, DMSO-d6)
δ 9.84 (s, 1H, CONH), 9.20 (s, 1H, OH), 8.60 (d, J = 1.4 Hz, 1H, H3), 7.93 (d,
J = 1.4 Hz, 1H, H6), 7.76 (t, J = 5.6 Hz, 1H, NH), 7.63–7.56 (m, 2H, H20, H60), 6.74–6.68 (m, 2H, H30,
H50), 3.32 (q, J = 6.7 Hz, 2H, CH2), 1.54 (p, J = 6.7 Hz, 2H, CH2), 1.37 – 1.21 (m, 8H, (CH2)4), 0.85 (t,
J = 6.7 Hz, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 162.01, 156.25, 153.67, 142.79, 131.26, 132.15,
130.57, 121.85, 115.14, 40.47, 31.44, 28.74, 28.65, 26.65, 22.24, 14.12. Elemental analysis found: C, 65.51%;
H, 7.40%; N, 17.07%. Calculated for C18H24N4O2 (MW 328.42): C, 65.83%; H, 7.37%; N, 17.06%.
N-(4-hydroxyphenyl)-5-(octylamino)pyrazine-2-carboxamide (3f)
White solid. Yield: 60%. mp 142–143 ◦C. IR (ATR-Ge, cm−1) 3360 (N-H, NH), 3272 (N-H, O-H,
1
CONH, OH), 2928, 2856 (C-H, alkyl), 1645 (C=O, CONH), 1601, 1582, 1529, 1512 (C=C, Ar). H NMR
(500 MHz, DMSO-d6)
δ 9.84 (s, 1H, CONH), 9.19 (s, 1H, OH), 8.60 (d, J = 1.3 Hz, 1H, H3), 7.93 (d,
J = 1.3 Hz, 1H, H6), 7.76 (t, J = 5.5 Hz, 1H, NH), 7.63 – 7.56 (m, 2H, H20, H60), 6.75 – 6.67 (m, 2H,
H30, H50), 3.31 (q, J = 6.8 Hz, 2H, CH2), 1.54 (p, J = 6.8 Hz, 2H, CH2), 1.33 (h, J = 6.8 Hz, 2H, CH2),
1.31 – 1.18 (m, 8H, (CH2)4), 0.87 – 0.81 (m, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 161.99, 156.24,
153.66, 142.77, 132.14, 131.16, 130.57, 121.83, 115.13, 40.46, 31.42, 28.93, 28.85, 28.71, 26.68, 22.26, 14.11.
Elemental analysis found: C, 66.77%; H, 7.73%; N, 16.26%. Calculated for C19H26N4O2 (MW 342.44):
C, 66.64%; H, 7.65%; N, 16.36%.
5-(Propylamino)-N-(p-tolyl)pyrazine-2-carboxamide (4a)
Cream solid. Yield: 83%. mp 168–169 ◦C. IR (ATR-Ge, cm−1) 3356 (N-H, NH), 3290 (N-H, CONH),
1
2927, 2875 (C-H, alkyl), 1655 (C=O, CONH), 1591, 1518 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
9.98 (s, 1H, CONH), 8.63 (d, J = 1.3 Hz, 1H, H3), 7.95 (d, J = 1.3 Hz, 1H, H6), 7.83 (t, J = 5.6 Hz, 1H,
NH), 7.74 – 7.68 (m, 2H, H20, H60), 7.12 (d, J = 8.2 Hz, 2H, H30, H50), 3.34–3.26 (m, 2H, CH2), 2.26 (s,
3H, CH3), 1.57 (h, J = 7.3 Hz, 2H, CH2), 0.92 (t, J = 7.3 Hz, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ
162.38, 156.34, 143.02, 136.42, 132.45, 131.98, 131.04, 129.15, 120.10, 42.28, 22.00, 20.64, 11.65. Elemental
analysis found: C, 66.81%; H, 6.72%; N, 20.61%. Calculated for C15H18N4O (MW 270.34): C, 66.64%; H,
6.71%; N, 20.73%.
5-(Butylamino)-N-(p-tolyl)pyrazine-2-carboxamide (4b)
Yellow crystalline. Yield: 32%. mp 162.6–163.6 ◦C. IR (ATR-Ge, cm−1) 3358 (N-H, NH), 3268 (N-H,
1
CONH), 2956, 2869 (C-H, alkyl), 1667 (C=O, CONH), 1591, 1570, 1517 (C=C, Ar). H NMR (500 MHz,
Pyridine-d5)
δ 10.27 (s, 1H, CONH), 9.28 (d, J = 1.4 Hz, 1H, H3), 8.36 (t, J = 5.6 Hz, 1H, NH), 8.13–8.07
(m, 2H, H20, H60), 7.96 (d, J = 1.4 Hz, 1H, H6), 7.22–7.17 (m, 2H, H30, H50), 3.51 (q, J = 7.4 Hz, 2H, CH2),
2.22 (s, 3H, CH3), 1.66–1.56 (m, 2H, CH2), 1.34 (h, J = 7.4 Hz, 2H, CH2), 0.84 (t, J = 7.4 Hz, 3H, CH3).
13C NMR (126 MHz, Pyridine-d5)
δ 163.02, 157.12, 143.87, 137.21, 133.17, 132.93, 130.87, 129.80, 120.38,
41.10, 31.48, 20.78, 20.43, 13.89. Elemental analysis found: C, 67.57%; H, 7.18%; N, 19.63%. Calculated
for C16H20N4O (MW 284.36): C, 67.58%; H, 7.09%; N, 19.70%.
5-(Pentylamino)-N-(p-tolyl)pyrazine-2-carboxamide (4c)

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Cream solid. Yield: 52%. mp 155.9–156.6 ◦C. IR (ATR-Ge, cm−1) 3344 (N-H, NH), 3283 (N-H,
1
CONH), 2929, 2872 (C-H, alkyl), 1654 (C=O, CONH), 1590, 1547, 1519 (C=C, Ar). H NMR (500 MHz,
DMSO-d6)
δ 9.98 (s, 1H, CONH), 8.64–8.60 (m, 1H, H3), 7.95 (d, J = 1.4 Hz, 1H, H6), 7.82 (t, J = 5.5 Hz,
1H, NH), 7.74–7.68 (m, 2H, H20, H60), 7.12 (d, J = 8.3 Hz, 2H, H30, H50), 3.36–3.29 (m, 2H, CH2), 2.26 (s,
3H, CH3), 1.60–1.51 (m, 2H, CH2), 1.37–1.26 (m, J = 3.8, 3.2 Hz, 4H, (CH2)2), 0.91–0.84 (m, 3H, CH3). 13C
NMR (126 MHz, DMSO-d6)
δ 162.37, 156.30, 143.02, 136.42, 132.43, 131.95, 131.05, 129.14, 120.09, 40.44,
28.86, 28.39, 22.06, 20.63, 14.08. Elemental analysis found: C, 68.95%; H, 7.49%; N, 18.94%. Calculated
for C17H22N4O (MW 298.39): C, 68.43%; H, 7.43%; N, 18.78%.
5-(Hexylamino)-N-(p-tolyl)pyrazine-2-carboxamide (4d)
Cream solid. Yield: 66%. mp 163–164 ◦C. IR (ATR-Ge, cm−1) 3344 (N-H, NH), 3287 (N-H, CONH),
1
2926, 2857 (C-H, alkyl), 1655 (C=O, CONH), 1590, 1547, 1520 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
9.98 (s, 1H, CONH), 8.62 (d, J = 1.4 Hz, 1H, H3), 7.94 (d, J = 1.4 Hz, 1H, H6), 7.81 (t, J = 5.5 Hz, 1H,
NH), 7.74–7.68 (m, 2H, H20, H60), 7.12 (d, J = 8.3 Hz, 2H, H30, H50), 3.36–3.28 (m, 2H, CH2), 2.26 (s, 3H,
CH3), 1.59–1.50 (m, 2H, CH2), 1.38–1.22 (m, 6H, (CH2)3), 0.91–0.83 (m, 3H, CH3). 13C NMR (126 MHz,
DMSO-d6)
δ 162.36, 156.30, 143.01, 136.42, 132.43, 131.95, 131.27, 129.14, 120.08, 40.48, 31.20, 28.68,
26.35, 22.25, 20.63, 14.08. Elemental analysis found: C, 69.32%; H, 7.84%; N, 17.83%. Calculated for
C18H24N4O (MW 312.42): C, 69.20%; H, 7.74%; N, 17.93%.
5-(Heptylamino)-N-(p-tolyl)pyrazine-2-carboxamide (4e)
◦
White solid. Yield: 38%. mp 157.8–158.9 C. IR (ATR-Ge, cm−1) 3354 (N-H, NH), 3292 (N-H,
CONH), 2928, 2856 (C-H, alkyl), 1659 (C=O, CONH), 1592, 1521 (C=C, Ar). 1H NMR (500 MHz,
DMSO-d6)
δ 9.98 (s, 1H, CONH), 8.61 (d, J = 1.4 Hz, 1H, H3), 7.98 (d, J = 1.4 Hz, 1H, H6), 7.94 (t,
J = 5.5 Hz, 1H, NH), 7.74 – 7.69 (m, 2H, H20, H60), 7.11 (d, J = 8.2 Hz, 2H, H30, H50), 3.35–3.29 (m, 2H,
CH2), 2.26 (s, 3H, CH3), 1.59–1.49 (m, 2H, CH2), 1.37–1.18 (m, 8H, (CH2)4), 0.85 (td, J = 6.9, 2.1 Hz, 3H,
CH3). 13C NMR (126 MHz, DMSO-d6)
δ 162.38, 156.33, 142.99, 136.43, 132.41, 131.88, 131.22, 129.13,
120.08, 40.44, 31.33, 28.68, 27.12, 26.82, 22.18, 20.63, 14.09. Elemental analysis found: C, 69.61%; H,
8.08%; N, 17.07%. Calculated for C19H26N4O (MW 326.44): C, 69.91%; H, 8.03%; N, 17.16%.
5-(octylamino)-N-(p-tolyl)pyrazine-2-carboxamide (4f)
Cream solid. Yield: 66%. mp 155–156 ◦C. IR (ATR-Ge, cm−1) 3343 (N-H, NH), 3279 (N-H, CONH),
2928, 2854 (C-H, alkyl), 1655 (C=O, CONH). 1H NMR (500 MHz, Pyridine-d5) δ 10.27 (s, 1H, CONH),
9.30 (d, J = 1.2 Hz, 1H, H3), 8.40 (t, J = 5.6 Hz, 1H, NH), 8.13–8.07 (m, 2H, H20, H60), 7.99 (d, J = 1.2 Hz,
1H, H6), 7.19 (d, J = 8.2 Hz, 2H, H30, H50), 3.55 (q, J = 7.36 Hz, 2H, CH2), 2.22 (s, 3H, CH3), 1.67 (p,
J = 7.3 Hz, 2H, CH2), 1.39–1.29 (m, 2H, CH2), 1.27–1.11 (m, 8H, (CH2)4), 0.83 (td, J = 7.3, 1.0 Hz, 3H,
CH3). 13C NMR (126 MHz, Pyridine-d5)
δ 163.02, 157.16, 143.89, 137.21, 133.17, 132.95, 130.91, 129.80,
120.37, 41.46, 31.97, 29.54, 29.49, 29.46, 27.36, 22.85, 20.79, 14.23. Elemental analysis found: C, 70.27%;
H, 8.42%; N, 16.37%. Calculated for C20H28N4O (MW 340.47): C, 70.56%; H, 8.29%; N, 16.46%.
N-(4-ethylphenyl)-5-(propylamino)pyrazine-2-carboxamide (5a)
Cream solid. Yield: 69%. mp 141.8–142.8 ◦C. IR (ATR-Ge, cm−1) 3355 (N-H, NH), 3283 (N-H,
1
CONH), 2964, 2827 (C-H, alkyl), 1651 (C=O, CONH), 1589, 1548, 1518 (C=C, Ar). H NMR (500 MHz,
DMSO-d6)
δ 9.99 (s, 1H, CONH), 8.63 (d, J = 1.3 Hz, 1H, H3), 7.95 (d, J = 1.3 Hz, 1H, H6), 7.84 (t,
J = 5.6 Hz, 1H, NH), 7.77–7.70 (m, 2H, H20, H60), 7.18–7.12 (m, 2H, H30, H50), 3.30 (td, J = 7.3, 5.6 Hz,
2H, CH2), 2.56 (q, J = 7.6 Hz, 2H, CH2), 1.57 (h, J = 7.3 Hz, 2H, CH2), 1.16 (t, J = 7.6 Hz, 3H, CH3), 0.93
(t, J = 7.3 Hz, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 162.36, 156.33, 143.01, 138.92, 136.60, 131.97,
131.21, 127.94, 120.18, 42.27, 27.79, 21.99, 15.87, 11.63. Elemental analysis found: C, 67.55%; H, 7.17%;
N, 19.59%. Calculated for C16H20N4O (MW 284.36): C, 67.58%; H, 7.09%; N, 19.70%.
5-(Butylamino)pyrazine-N-(4-ethylphenyl)-2-carboxamide (5b)
Cream solid. Yield: 74%. mp 133.8–134.8 ◦C. IR (ATR-Ge, cm−1) 3354 (N-H, NH), 3265 (N-H,
1
CONH), 2955, 2871 (C-H, alkyl), 1664 (C=O, CONH), 1590, 1573, 1519 (C=C, Ar). H NMR (500 MHz,
DMSO-d6)
δ 9.99 (s, 1H, CONH), 8.63 (d, J = 1.3 Hz, 1H, H3), 7.95 (d, J = 1.3 Hz, 1H, H6), 7.81 (t,
J = 5.5 Hz, 1H, NH), 7.77–7.70 (m, 2H, H20, H60), 7.17–7.12 (m, 2H, H30, H50), 3.37–3.30 (m, 2H, CH2),
2.56 (q, J = 7.6 Hz, 2H, CH2), 1.59–1.49 (m, 2H, CH2), 1.36 (h, J = 7.3 Hz, 2H, CH2), 1.16 (t, J = 7.6 Hz,

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3H, CH3), 0.90 (t, J = 7.3 Hz, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ
162.36, 156.31, 143.02, 138.91,
136.60, 131.94, 131.20, 127.94, 120.17, 40.15, 30.81, 27.79, 19.83, 15.87, 13.85. Elemental analysis found: C,
68.38%; H, 7.63%; N, 18.69%. Calculated for C17H22N4O (MW 298.39): C, 68.43%; H, 7.43%; N, 18.78%.
N-(4-ethylphenyl)-5-(pentylamino)pyrazine-2-carboxamide (5c)
Cream/white solid. Yield: 58%. mp 136.1–137.1 ◦C. IR (ATR-Ge, cm−1) 3346 (N-H, NH), 3296
1
(N-H, CONH), 2930, 2871 (C-H, alkyl), 1662 (C=O, CONH), 1592, 1520 (C=C, Ar). H NMR (500 MHz,
DMSO-d6)
δ 9.99 (s, 1H, CONH), 8.63 (d, J = 1.3 Hz, 1H, H3), 7.95 (d, J = 1.3 Hz, 1H, H6), 7.81 (t,
J = 5.5 Hz, 1H, NH), 7.78–7.70 (m, 2H, H20, H60), 7.18–7.12 (m, 2H, H30, H50), 3.36–3.29 (m, 2H, CH2),
2.56 (q, J = 7.6 Hz, 2H, CH2), 1.60–1.51 (m, 2H, CH2), 1.38–1.27 (m, 4H, (CH2)2), 1.16 (dd, J = 8.0, 7.2 Hz,
3H, CH3), 0.91–0.84 (m, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 162.37, 156.30, 143.02, 138.92,
136.61, 131.95, 131.25, 127.95, 120.18, 40.44, 28.86, 28.39, 27.80, 22.07, 15.88, 14.08. Elemental analysis
found: C, 69.25%; H, 7.83%; N, 17.83%. Calculated for C18H24N4O (MW 312.42): C, 69.20%; H, 7.74%;
N, 17.93%.
N-(4-ethylphenyl)-5-(hexylamino)pyrazine-2-carboxamide (5d)
Cream solid. Yield: 82%. mp 138–139 ◦C. IR (ATR-Ge, cm−1) 3336 (N-H, NH), 3293 (N-H, CONH),
1
2929, 2869 (C-H, alkyl), 1662 (C=O, CONH), 1592, 1548, 1519 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
9.99 (s, 1H, CONH), 8.63 (d, J = 1.3 Hz, 1H, H3), 7.95 (d, J = 1.3 Hz, 1H, H6), 7.81 (t, J = 5.5 Hz,
1H, NH), 7.76–7.70 (m, 2H, H20, H60), 7.18–7.11 (m, 2H, H30, H40), 3.36–3.29 (m, 2H, CH2), 2.56 (q,
J = 7.6 Hz, 2H, CH2), 1.59–1.50 (m, 2H, CH2), 1.37–1.22 (m, 6H, (CH2)3), 1.16 (t, J = 7.6 Hz, 3H, CH3),
0.90–0.82 (m, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 162.36, 156.29, 143.01, 138.91, 136.60, 131.93,
131.23, 127.94, 120.17, 40.47, 31.19, 28.67, 27.79, 26.34, 22.25, 15.86, 14.07. Elemental analysis found: C,
69.93%; H, 8.02%; N, 17.05%. Calculated for C19H26N4O (MW 326.44): C, 69.91%; H, 8.03%; N, 17.16%.
N-(4-ethylphenyl)-5-(heptylamino)pyrazine-2-carboxamide (5e)
Cream solid. Yield: 71%. mp 131–132 ◦C. IR (ATR-Ge, cm−1) 3335 (N-H, NH), 3296 (N-H, CONH),
1
2928, 2856 (C-H, alkyl), 1662 (C=O, CONH), 1592, 1547, 1521 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
9.98 (s, 1H, CONH), 8.62 (d, J = 1.4 Hz, 1H, H3), 7.94 (d, J = 1.4 Hz, 1H, H6), 7.81 (t, J = 5.5 Hz, 1H,
NH), 7.76–7.70 (m, 2H, H20, H60), 7.18–7.11 (m, 2H, H30, H50), 3.36–3.28 (m, 2H, CH2), 2.56 (q, J = 7.6 Hz,
2H, CH2), 1.54 (h, J = 6.6 Hz, 2H, CH2), 1.38–1.21 (m, 8H, (CH2)4), 1.16 (t, J = 7.6 Hz, 3H, CH3), 0.88–0.82
(m, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 162.35, 156.29, 143.01, 138.91, 136.60, 131.93, 131.08,
127.93, 120.17, 40.45, 31.42, 28.71, 28.63, 27.79, 26.63, 22.22, 15.86, 14.10. Elemental analysis found: C,
70.26%; H, 8.36%; N, 16.25%. Calculated for C20H28N4O (MW 340.47): C, 70.56%; H, 8.29%; N, 16.46%
N-(4-ethylphenyl)-5-(octylamino)pyrazine-2-carboxamide (5f)
White solid. Yield: 77%. mp 131–132 ◦C. IR (ATR-Ge, cm−1) 3358 (N-H, NH), 3296 (N-H, CONH),
1
2928, 2852 (C-H, alkyl), 1662 (C=O, CONH), 1595, 1548, 1523 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
9.98 (s, 1H, CONH), 8.63 (d, J = 1.3 Hz, 1H, H3), 7.94 (d, J = 1.3 Hz, 1H, H6), 7.81 (t, J = 5.5 Hz,
1H, NH), 7.76–7.70 (m, 2H, H20, H60), 7.18–7.11 (m, 2H, , H30, H50), 3.36–3.28 (m, 2H, CH2), 2.56 (q,
J = 7.6 Hz, 2H, CH2), 1.54 (h, J = 6.6 Hz, 2H, CH2), 1.30–1.22 (m, 10H, (CH2)5), 1.16 (t, J = 7.6 Hz, 3H,
CH3), 0.88–0.81 (m, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 162.35, 156.30, 143.01, 138.91, 136.60,
131.93, 131.25, 127.93, 120.17, 40.46, 31.41, 28.93, 28.84, 28.70, 27.80, 26.67, 22.25, 15.87, 14.09. Elemental
analysis found: C, 71.25%; H, 8.69%; N, 15.74%. Calculated for C21H30N4O (MW 354.50): C, 71.15%; H,
8.53%; N, 15.80%.
N-(4-chloro-2-hydroxyphenyl)-5-(propylamino)pyrazine-2-carboxamide (6a)
Yellow solid. Yield: 44%. mp 248.9–250 ◦C. IR (ATR-Ge, cm−1) 3323 (N-H, CONH, NH), 2965,
1
2878 (C-H, alkyl), 1664 (C=O, CONH), 1603, 1528 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
10.75
(s, 1H, CONH), 9.81 (s, 1H, OH), 8.64 (d, J = 1.4 Hz, 1H, H3), 8.34 (d, J = 8.7 Hz, 1H, H60), 7.98–7.92
(m, 2H, H6, NH), 6.93 (d, J = 2.4 Hz, 1H, H30), 6.88 (dd, J = 8.7, 2.4 Hz, 1H, H50), 3.35–3.26 (m, 2H,
CH2), 1.57 (h, J = 7.3 Hz, 2H, CH2), 0.92 (t, J = 7.3 Hz, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ
161.65, 156.50, 147.46, 142.92, 131.67, 131.13, 126.80, 125.97, 119.91, 119.11, 114.57, 40.33, 21.94, 11.64.
Elemental analysis found: C, 54.50%; H, 4.76%; N, 17.99%. Calculated for C14H15ClN4O2 (MW 306.75):
C, 54.82%; H, 4.93%; N, 18.27%.

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5-(Butylamino)pyrazine-N-(4-chloro-2-hydroxyphenyl)-2-carboxamide (6b)
Pale yellow solid. Yield: 70%. mp 221.8–225.4 ◦C. IR (ATR-Ge, cm−1) 3315 (N-H, CONH, NH),
1
2930, 2874 (C-H, alkyl), 1663 (C=O, CONH), 1603, 1554, 1526 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
10.73 (s, 1H, CONH), 9.80 (s, 1H, OH), 8.63 (d, J = 1.3 Hz, 1H, H3), 8.34 (d, J = 8.7 Hz, 1H, H60),
7.95–7.89 (m, 2H, H6, NH), 6.93 (d, J = 2.3 Hz, 1H, H30), 6.87 (dd, J = 8.7, 2.3 Hz, 1H, H50), 3.33 (td,
J = 7.0, 5.4 Hz, 2H, CH2), 1.58–1.49 (m, 2H, CH2), 1.41–1.30 (m, 2H, CH2), 0.90 (t, J = 7.0 Hz, 3H, CH3).
13C NMR (126 MHz, DMSO-d6)
δ 161.65, 156.47, 147.45, 142.93, 131.67, 131.13, 126.79, 125.98, 119.91,
119.11, 114.56, 40.20, 30.76, 19.83, 13.85. Elemental analysis found: C, 56.32%; H, 5.30%; N, 17.55%.
Calculated for C15H17ClN4O2 (MW 320.78): C, 56.17%; H, 5.34%; N, 17.47%.
N-(4-chloro-2-hydroxyphenyl)-5-(pentylamino)pyrazine-2-carboxamide (6c)
Pale yellow solid. Yield: 23%. mp 206.5–208.7 ◦C. IR (ATR-Ge, cm−1) 3321 (N-H, CONH, NH),
1
2927, 2872 (C-H, alkyl), 1663 (C=O, CONH), 1604, 1554, 1529 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
10.77 (s, 1H, CONH), 9.81 (s, 1H, OH), 8.63 (d, J = 1.3 Hz, 1H, H3), 8.34 (d, J = 8.7 Hz, 1H, H60), 7.93
(s, 2H, H6, NH), 6.93 (d, J = 2.4 Hz, 1H, H30), 6.87 (dd, J = 8.7, 2.4 Hz, 1H, H50), 3.32 (q, J = 6.8 Hz,
2H, CH2), 1.55–1.44 (m, 2H, CH2), 1.38–1.23 (m, 4H, (CH2)2), 0.85 (d, J = 6.8 Hz, 3H, CH3). 13C NMR
(126 MHz, DMSO-d6)
δ 161.64, 156.47, 147.52, 142.91, 131.75, 131.13, 126.75, 125.97, 119.82, 119.02,
114.59, 40.49, 28.85, 28.33, 22.06, 14.07. Elemental analysis found: C, 56.94%; H, 5.68%; N, 16.48%.
Calculated for C16H19ClN4O2 (MW 334.8): C, 57.40%; H, 5.72%; N, 16.73%.
N-(4-chloro-2-hydroxyphenyl)-5-(hexylamino)pyrazine-2-carboxamide (6d)
Yellow solid. Yield: 40%. mp 201.7–204.9 ◦C. IR (ATR-Ge, cm−1) 3324 (N-H, CONH, NH), 2929,
1
2857 (C-H, alkyl), 1664 (C=O, CONH), 1603, 1554, 1527 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
10.76 (s, 1H, CONH), 9.81 (s, 1H, OH), 8.63 (s, 1H, H3), 8.34 (d, J = 8.7 Hz, 1H, H60), 7.93 (m, 2H,
H6, NH), 6.93 (d, J = 2.4 Hz, 1H, H30), 6.87 (dd, J = 8.7, 2.4 Hz, 1H, H50), 3.38–3.24 (m, 2H, CH2),
1.54 (p, J = 7.1 Hz, 2H, CH2), 1.38–1.23 (m, 6H, (CH2)3), 0.89–0.82 (m, 3H, CH3). 13C NMR (126 MHz,
DMSO-d6) δ 161.65, 156.46, 147.48, 142.92, 132.41, 131.13, 126.78, 125.98, 119.88, 119.08, 114.56, 40.54,
31.19, 28.62, 26.35, 22.25, 14.08. Elemental analysis found: C, 58.08%; H, 5.60%; N, 16.33%. Calculated
for C17H21ClN4O2 (MW 348.83): C, 58.53%; H, 6.07%; N, 16.06%.
N-(4-chloro-2-hydroxyphenyl)-5-(heptylamino)pyrazine-2-carboxamide (6e)
Pale yellow solid. Yield: 27%. mp 199.8–200.8 ◦C. IR (ATR-Ge, cm−1) 3317 (N-H, CONH, NH),
1
2930, 2858 (C-H, alkyl), 1664 (C=O, CONH), 1603, 1527 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
10.73 (s, 1H, CONH), 9.80 (s, 1H, OH), 8.63 (d, J = 1.3 Hz, 1H, H3), 8.34 (d, J = 8.7 Hz, 1H, H60),
7.94–7.89 (m, 2H, H6, NH), 6.92 (d, J = 2.4 Hz, 1H, H30), 6.87 (dd, J = 8.7, 2.4 Hz, 1H, H50), 3.34–3.28 (m,
2H, CH2), 1.54 (p, J = 7.1 Hz, 2H, CH2), 1.37–1.20 (m, 8H, (CH2)4), 0.84 (t, J = 6.8 Hz, 3H, CH3). 13C
NMR (126 MHz, DMSO-d6)
δ 161.65, 156.46, 147.46, 142.92, 131.75, 131.13, 126.79, 125.98, 119.90, 119.10,
114.56, 40.52, 31.43, 28.66, 28.63, 26.64, 22.23, 14.10. Elemental analysis found: C, 59.19%; H, 6.22%; N,
15.07%. Calculated for C18H23ClN4O2 (MW 362.86): C, 59.58%; H, 6.39%; N, 15.44%.
N-(4-chloro-2-hydroxyphenyl)-5-(octylamino)pyrazine-2-carboxamide (6f)
Yellow solid. Yield: 15%. mp 196.9–197.9 ◦C. IR (ATR-Ge, cm−1) 3315 (N-H, CONH, NH), 2926,
1
2855 (C-H, alkyl), 1663 (C=O, CONH), 1603, 1554, 1526 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
10.74 (s, 1H, CONH), 9.81 (s, 1H, OH), 8.63 (s, 1H, H3), 8.34 (d, J = 8.7 Hz, 1H, H60), 7.93 (s, 2H, NH,
H6), 6.92 (d, J = 2.4 Hz, 1H, H30), 6.87 (dd, J = 8.7, 2.4 Hz, 1H, H50), 3.35–3.29 (m, 2H, CH2), 1.54 (p,
J = 7.0 Hz, 2H, CH2), 1.32 (dt, J = 12.6, 7.0 Hz, 2H, CH2), 1.25 (tt, J = 12.6, 9.0, 7.7 Hz, 8H, (CH2)4), 0.84
(t, J = 7.0 Hz, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 161.64, 156.45, 147.46, 142.92, 131.69, 131.12,
126.78, 125.97, 119.89, 119.09, 114.55, 40.51, 31.40, 28.91, 28.83, 28.63, 26.66, 22.24, 14.09. Elemental
analysis found: C, 60.60%; H, 6.71%; N, 14.66%. Calculated for C19H25ClN4O2 (MW 376.89): C, 60.55%;
H, 6.69%; N, 14.87%.
N-(5-chloro-2-hydroxyphenyl)-5-(propylamino)pyrazine-2-carboxamide (7a)
Pale yellow solid. Yield: 45%. mp 254.0–259.0 ◦C. IR (ATR-Ge, cm−1) 3344 (N-H, NH), 3321 (N-H,
1
CONH), 2934, 2861 (C-H, alkyl), 1672 (C=O, CONH), 1604, 1560, 1530 (C=C, Ar). H NMR (500 MHz,
DMSO-d6)
δ 10.54 (s, 1H, CONH), 9.86 (s, 1H, OH), 8.64 (d, J = 1.4 Hz, 1H, H3), 8.41 (d, J = 2.6 Hz, 1H,

Molecules 2020, 25, 1561
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H60), 7.98 (t, J = 5.7 Hz, 1H, NH), 7.94 (d, J = 1.4 Hz, 1H, H6), 7.01–6.88 (m, 2H, H30, H40), 3.30 (td,
J = 7.2, 5.7 Hz, 2H, CH2), 1.57 (h, J = 7.2 Hz, 2H, CH2), 0.92 (t, J = 7.2 Hz, 3H, CH3). 13C NMR (126 MHz,
DMSO-d6) δ 161.83, 156.54, 145.25, 143.04, 131.72, 130.98, 127.94, 122.93, 122.76, 118.31, 115.77, 42.34,
21.93, 11.64. Elemental analysis found: C, 54.62%; H, 4.79%; N, 17.91%. Calculated for C14H15ClN4O2
(MW 306.75): C, 54.82%; H, 4.93%; N, 18.27%.
5-(Butylamino)pyrazine-N-(5-chloro-2-hydroxyphenyl)-2-carboxamide (7b)
Pale yellow solid. Yield: 40%. mp 257.9–262.1 ◦C. IR (ATR-Ge, cm−1) 3329 (N-H, CONH, NH),
1
2925, 2861 (C-H, alkyl), 1668 (C=O, CONH), 1600, 1558, 1528 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
10.54 (s, 1H, CONH), 9.85 (s, 1H, OH), 8.64 (d, J = 1.3 Hz, 1H, H3), 8.41 (d, J = 2.5 Hz, 1H, H60), 7.95
(t, J = 5.6 Hz, 1H, NH), 7.93 (d, J = 1.3 Hz, 1H, H6), 6.95 (dd, J = 8.6, 2.5 Hz, 1H, H40), 6.91 (d, J = 8.6 Hz,
1H, H30), 3.36–3,30 (m, 2H, CH2), 1.58–1.49 (m, 2H, CH2), 1.41–1.30 (m, 2H, CH2), 0.90 (t, J = 7.3 Hz,
3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 161.82, 156.51, 145.22, 143.05, 131.87, 130.95, 127.93, 122.92,
122.77, 118.30, 115.77, 40.52, 30.74, 19.83, 13.85. Elemental analysis found: C, 55.90%; H, 5.18%; N,
17.45%. Calculated for C15H17ClN4O2 (MW 320.78): C, 56.17%; H, 5.34%; N, 17.47%.
N-(5-chloro-2-hydroxyphenyl)-5-(pentylamino)pyrazine-2-carboxamide (7c)
Cream solid. Yield: 55%. mp 250.3–255.1 ◦C. IR (ATR-Ge, cm−1) 3328 (N-H, CONH, NH), 2929,
1
2861 (C-H, alkyl), 1667 (C=O, CONH), 1600, 1558, 1528 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
10.54 (s, 1H, CONH), 9.86 (s, 1H, OH), 8.64 (d, J = 1.4 Hz, 1H, H3), 8.41 (d, J = 2.5 Hz, 1H, H60),
7.95 (t, J = 5.5 Hz, 1H, NH), 7.93 (d, J = 1.4 Hz, 1H, H6), 6.95 (dd, J = 8.5, 2.5 Hz, 1H, H40), 6.91 (d,
J = 8.5 Hz, 1H, H30), 3.36–3.30 (m, 2H, CH2), 1.55 (p, J = 7.1 Hz, 2H, CH2), 1.31 (h, J = 7,1, 4.6, Hz,
4H, (CH2)2), 0.90–0.84 (m, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 161.82, 156.50, 145.22, 143.05,
131.82, 130.95, 127.93, 122.92, 122.77, 118.30, 115.76, 40.50, 28.85, 28.32, 22.06, 14.07. Elemental analysis
found: C, 57.18%; H, 5.64%; N, 16.79%. Calculated for C16H19ClN4O2 (MW 334.83): C, 57.40%; H,
5.72%; N, 16.73%
N-(5-chloro-2-hydroxyphenyl)-5-(hexylamino)pyrazine-2-carboxamide (7d)
Pale yellow solid. Yield: 53%. mp 249.3–251.9 ◦C. IR (ATR-Ge, cm−1) 3329 (N-H, CONH, NH),
1
2929, 2858 (C-H, alkyl), 1668 (C=O, CONH), 1600, 1558, 1529 (C=C, Ar). H NMR (500 MHz, DMSO-d6)
δ
10.16 (s, 1H, CONH), 9.87 (s, 1H, OH), 8.64 (d, J = 1.4 Hz, 1H, H3), 8.41 (d, J = 2.5 Hz, 1H, H60), 7.96
(t, J = 5.7 Hz, 1H, NH), 7.93 (J = 1.4 Hz, 1H, H6), 6.97–6.88 (m, 2H, H30, H40) 3.35–3.26 (m, 2H, CH2),
1.54 (p, J = 7.2 Hz, 2H, CH2), 1.36–1.30 (m, 2H, CH2), 1.32–1.21 (m, 4H, (CH2)2), 0.89–0.81 (m, 3H, CH3).
13C NMR (126 MHz, DMSO-d6)
δ 162.36, 156.51, 145.32, 143.07, 131.91, 131.06, 128.04, 123.08, 122.77,
118.32, 115.85, 40.55, 31.51, 28.78, 26.79, 22.27, 14.10. Elemental analysis found: C, 58.05%; H, 5.91%; N,
15.95%. Calculated for C17H21ClN4O2 (MW 348.83): C, 58.53%; H, 6.07%; N, 16.06%.
N-(5-chloro-2-hydroxyphenyl)-5-(heptylamino)pyrazine-2-carboxamide (7e)
Cream solid. Yield: 34%. mp 243.6–246.0 ◦C. IR (ATR-Ge, cm−1) 3354 (N-H, NH), 3331 (N-H,
1
CONH), 2927, 2856 (C-H, alkyl), 1668 (C=O, CONH), 1600, 1557, 1529 (C=C, Ar). H NMR (500 MHz,
DMSO-d6)
δ 10.56 (s, 1H, CONH), 9.85 (s, 1H, OH), 8.61 (d, J = 1.4 Hz ,1H, H3), 8.38 (d, J = 2.6 Hz,
1H, H60), 7.92 (d, J = 5.6 Hz, 1H, NH), 7.91 (d, J = 1.4 Hz, 1H, H6), 6.97–6.88 (m, 2H, H30, H40), 3.29
(t, J = 7.1 Hz, 2H, CH2), 1.52 (p, J = 7.1 Hz, 2H, CH2), 1.30 (d, J = 7.1 Hz, 2H, CH2), 1.32–1.21 (m,
6H, (CH2)3), 0.82 (t, J = 6.8 Hz, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 162.08, 156.66, 145.46,
143.21, 131.77, 131.09, 128.08, 123.18, 122.97, 118.52, 115.95, 40.74, 31.60, 28.80, 28.58, 26.80, 22.41, 14.28.
Elemental analysis found: C, 59.14%; H, 6.22%; N, 15.21%. Calculated for C18H23ClN4O2 (MW 362.86):
C, 59.58%; H, 6.39%; N, 15.44%.
N-(5-chloro-2-hydroxyphenyl)-5-(octylamino)pyrazine-2-carboxamide (7f)
Pale yellow solid. Yield: 25%. mp 239.7–242.4 ◦C. IR (ATR-Ge, cm−1) 3362 (N-H, NH), 3330 (N-H,
1
CONH), 2928, 2856 (C-H, alkyl), 1669 (C=O, CONH), 1600, 1558, 1529 (C=C, Ar). H NMR (500 MHz,
DMSO-d6)
δ 10.55 (s, 1H, CONH), 9.86 (s, 1H, OH), 8.64 (d, J = 1.4 Hz, 1H, H3), 8.41 (d, J = 2.4 Hz, 1H,
H60), 7.96 (d, J = 5.9 Hz, 1H, NH), 7.93 (d, J = 1.4 Hz, 1H, H6), 6.97–6.89 (m, 2H, H30, H40), 3.35–3.29
(m, 2H, CH2), 1.54 (p, J = 7.1 Hz, 2H, CH2), 1.37–1.21 (m, 10H, (CH2)5), 0.84 (t, J = 6.6 Hz, 3H, CH3).
13C NMR (126 MHz, DMSO-d6)
δ 161.82, 156.50, 145.28, 143.04, 133.82, 130.96, 127.94, 122.90, 122.72,

Molecules 2020, 25, 1561
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118.28, 115.76, 40.52, 31.41, 28.92, 28.84, 28.63, 26.67, 22.25, 14.09. Elemental analysis found: C, 60.17%;
H, 6.51%; N, 14.62%. Calculated for C19H25ClN4O2 (MW 376.89): C, 60.55%; H, 6.69%; N, 14.87%.
3.4. Biological Methods
3.4.1. Antimycobacterial In Vitro Activity Screening Against Mycobacterium tuberculosis H37Ra,
Mycobacterium smegmatis, and Mycobacterium aurum
Antimycobacterial assays were performed with fast growing Mycobacterium smegmatis DSM 43465
(ATCC 607), Mycobacterium aurum DSM 43999 (ATCC 23366) from German Collection of Microorganisms
and Cell Cultures (Braunschweig, Germany) and with an avirulent strain of Mycobacterium tuberculosis
H37Ra ITM-M006710 (ATCC 9431) from Belgian Co-ordinated Collections of Micro-organisms.
The technique used for activity determination was microdilution broth panel method using 96-well
microtitration plates. Culturing medium was Middlebrook 7H9 broth (Sigma–Aldrich, Steinheim,
Germany) enriched with 0.4% of glycerol (Sigma–Aldrich) and 10% of Middlebrook OADC growth
supplement (Himedia, Mumbai, India).
Mycobacterial strains were cultured on Middlebrook 7H9 agar and suspensions were prepared in
Middlebrook 7H9 broth. Final density was adjusted to value 1.0 according to McFarland scale and
diluted in ratio 1:20 (for fast growing mycobacteria) or 1:10 (for M. tbc) with broth.
Tested compounds were dissolved in DMSO (Sigma–Aldrich) then Middlebrook 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
binary dilution and addition of mycobacterial suspension and were set as 500, 250, 125, 62.5, 31.25,
15.625, 7.81, 3.91, 1.56, and 0.78
against fast growing mycobacteria and in range 1–0.0078
Rifampicin final concentrations ranged from 50 to 0.39 g/mL for fast growing mycobacteria and from
1 to 0.0078 g/mL for M. tuberculosis H37Ra. Ciprofloxacin was used for screening antimycobacterial
activity with the final concentrations 1, 0.5, 0.25, 0.125, 0.0625, 0.0313, 0.0156, and 0.0078 g/mL.
µg/mL. Isoniazid was diluted in range 500–3.91
µg/mL for screening
µg/mL for screening against M. tuberculosis.
µ
µ
µ
The final concentration of DMSO did neither exceeded 2.5% (v/v) and did not affect the growth of
M. smegmatis, M. aurum nor M. tuberculosis. Positive (broth, DMSO, bacteria) and negative (broth,
DMSO) controls were included.
Plates were sealed with polyester adhesive film and incubated in dark at 37 ◦C without agitation.
The addition of 0.01% solution of resazurin sodium salt followed after 48 h of incubation for M. smegmatis,
after 72 h of incubation for M. aurum and after 120 h of incubation for M. tuberculosis, respectively. Stain
was prepared by dissolving resazurin sodium salt (Sigma–Aldrich) in deionized 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
panels were then incubated for further 2.5 h for determination of activity against M. smegmatis, 4 h for
M. aurum and 24 h for M. tuberculosis, respectively.
Antimycobacterial activity was expressed as minimal inhibition concentration (MIC) and the
value was read based on stain color change (blue color—active compound; pink color—not active
compound). All experiments were conducted in duplicate.
3.4.2. Antimycobacterial In Vitro Activity Screening Against Mycobacterium kansasii and
Mycobacterium avium
A 96 well plate microdilution broth method was performed. Tested M. kansasii CNCTC My 235/80
(ATCC 12478) and M. avium ssp. avium CNCTC My 80/72 (ATCC 15769) were obtained from the
Czech National Collection of Type Cultures (CNCTC), National Institute of Public Health (Prague,
Czech Republic). Middlebrook 7H9 broth of declared pH = 6.6 (Sigma–Aldrich) enriched with 0.4% of
glycerol (Sigma–Aldrich) and 10% of OADC growth supplement (oleic acid, albumin, dextrose, catalase;
Himedia, Mumbai, India) was used for cultivation. Tested compounds were dissolved and diluted in
DMSO and mixed with broth (25 µL of DMSO solution in 4.475 mL of broth) and placed (100 µL) into

Molecules 2020, 25, 1561
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microplate wells. Mycobacterial inocula were suspended in isotonic saline solution and the density was
adjusted to 0.5–1.0 according to McFarland scale. These suspensions were diluted by 10−1 and used to
inoculate the testing wells, adding 100
of tested compounds in wells were 100, 50, 25, 12.5, 6.25, 3.13, and 1.56
as positive control (inhibition of growth). Negative control (visible growth) consisted of broth plus
mycobacterial suspension plus DMSO (purity of broth). A total of 30 L of Alamar Blue working
solution (1:1 mixture of 0.01% 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 MIC (in g/mL) was
µL of mycobacterial suspension per well. Final concentrations
µg/mL. The INH was used
µ
µ
determined as the lowest concentration that prevented the blue to pink color change. All experiments
were conducted in duplicates.
3.4.3. HepG2 Cytotoxicity Determination
The human hepatocellular liver carcinoma cell line HepG2 purchased from Health Protection
Agency Culture Collections (ECACC, Salisbury, UK) was cultured in MEM (Minimum Essentials
Eagle Medium, Sigma–Aldrich) supplemented with 10% fetal bovine serum (PAA Laboratories,
Pasching, Austria), 1% L-glutamine solution (Sigma–Aldrich) and non-essential amino acid solution
(Sigma–Aldrich) in a humidified atmosphere containing 5% CO2 at 37 ◦C.
For subculturing, the cells were harvested after trypsin/EDTA (Sigma–Aldrich) treatment at 37 ◦C.
To evaluate cytotoxicity, the cells treated with the tested substances were used as experimental groups
whereas untreated HepG2 cells served as controls.
The cells were seeded in density 10,000 cells per well in a 96 well plate. The next day the cells were
treated with each of the tested substances dissolved in DMSO. The tested substances were prepared
at different incubation concentrations in triplicates according to their solubility. Simultaneously,
the controls representing 100% cell viability, 0% cell viability (the cells treated with 10% DMSO),
no cell control and vehiculum controls were also prepared in triplicates. After 24 h incubation in a
humidified atmosphere containing 5% CO2 at 37%, the reagent from the kit CellTiter 96 AQueous
One Solution Cell Proliferation Assay (CellTiter 96; PROMEGA, Fitchburg, USA) was added. After 2h
incubation at 37% absorbance of samples was recorded at 490 nm (TECAN, Infinita M200, Austria).
A standard toxicological parameter IC50 was calculated by nonlinear regression from a semilogarithmic
plot of incubation concentration versus percentage of absorbance relative to untreated controls using
GraphPad Prism 8 software.
4. Conclusions
The present work reflects the successful design and synthesis of novel, potentially active
antimycobacterial agents structurally based on pyrazinamide. A library of forty-two compounds was
obtained. Final compounds were evaluated for in vitro growth inhibition activity against Mycobacterium
tuberculosis H37Ra and several non-tuberculous mycobacteria. As a complementary test, the final
compounds were evaluated in a panel of clinically important Gram-positive and Gram-negative
bacteria and fungal pathogens. No antibacterial and sporadic antifungal activity has been found,
but some of the compounds proved the same or even better in vitro antimycobacterial activity in
comparison with known antimycobacterial drugs.
From the structure–activity relationship point of view, we explored the chemical space around
previously evaluated antimycobacterial 5-alkylamino-N-phenylpyrazine-2-carboxamides [14] to find
out that the benzene ring tolerates different substitution patterns, including lipo/hydrophilic and
electron donating/withdrawing substituents.
Compounds with relatively good selectivity index (calculated for Mtb H37Ra and
cytotoxicity on HepG2 liver cancer cells) need to be highlighted: N-(4-hydroxyphenyl)-5-(pentylamino)
pyrazine-2-carboxamide (3c, MIC = 3.91
tolyl)pyrazine-2-carboxamide (4e, MIC = 0.78
(propylamino)pyrazine-2-carboxamide 5a MIC
µ
g/mL, 13.02
g/mL, 2.39
7.81
µ
M, SI >38), 5-(heptylamino)-N-(p-
M, SI >20), N-(4-ethylphenyl)-5-
g/mL, 27.47 M, SI 18),
µ
µ
(
,
=
µ
µ
=

Molecules 2020, 25, 1561
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5-(butylamino)-N-(3-(trifluoromethyl)phenyl)pyrazine-2-carboxamide (1b, MIC = 3.13
µg/mL,
9.24 µM, SI = 14) and 5-(hexylamino)-N-(p-tolyl)pyrazine-2-carboxamide (4d, MIC = 1.56 µg/mL,
4.99 µM, SI > 10).
Out of the derivatives mentioned in the previous paragraph, compounds 1b and 3d exerted a
broad spectrum of antimycobacterial activity, inhibiting the growth of at least three out of five tested
strains. We propose derivative 4e (for its potency against Mtb H37Ra and good SI) and derivatives 1b
and 3d (for their broad spectrum of antimycobacterial activity and favorable SI) as starting points for
further development.
Supplementary Materials: Representative 1H and 13C NMR spectra of selected compounds (all spectra in the
form of FID files can be retrieved from the corresponding author J.Z. upon request); methodology and full results
of antibacterial testing; methodology and full results of antifungal testing are available online.
Author Contributions: Conceptualization, J.Z.; Data curation, W.A.; Formal analysis, M.K.-C.; Investigation,
W.A., O.J., P.P. and P.B.; Methodology, O.J., K.K. and P.P.; Supervision, J.V., M.D. and J.Z.; Writing—original draft,
W.A., M.K.-C. and J.Z.; Writing—review and editing, M.K.-C., M.D. and J.Z. All authors have read and agreed to
the published version of the manuscript.
Funding: This work was supported by EFSA-CDN (No. CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by ERDF.
Additionally, the study was supported by the Czech Science Foundation project No. 20-19638Y.
Acknowledgments: The authors wish to thank Ida Dufkov
á for performing the antibacterial and
antifungal assessment.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2020 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/).