Design, synthesis, antimycobacterial evaluation, and in silico studies of 3-(phenylcarbamoyl)-pyrazine-2-carboxylic acids

Article abstract of DOI:10.3390/molecules22091491

Pyrazinamide, the first-line antitubercular drug, has been regarded the basic component of tuberculosis treatment for over sixty years. Researchers have investigated its effect on Mycobacterium tuberculosis for this long time, and as a result, new potential targets of pyrazinamide or its active form, pyrazinoic acid, have been found. We have designed and prepared 3-(phenyl-carbamoyl)pyrazine-2-carboxylic acids as more lipophilic derivatives of pyrazinoic acid. We also prepared methyl and propyl derivatives as prodrugs with further increased lipophilicity. Antimycobacterial, antibacterial and antifungal growth inhibiting activity was investigated in all prepared compounds. 3-[(4-Nitrophenyl)carbamoyl]pyrazine-2-carboxylic acid (16) exerted high antimycobacterial activity against Mycobacterium tuberculosis H37Rv with MIC = 1.56 μg·mL^?1 (5 μM). Propyl 3-{[4-(trifluoromethyl)phenyl]carbamoyl}pyrazine-2-carboxylate (18a) showed also high antimycobacterial activity against Mycobacterium tuberculosis H37Rv with MIC = 3.13 μg·mL^?1. In vitro cytotoxicity of the active compounds was investigated and no significant cytotoxic effect was observed. Based to structural similarity to known inhibitors of decaprenylphosphoryl-β-D-ribose oxidase, DprE1, we performed molecular docking of the prepared acids to DprE1. These in silico experiments indicate that modification of the linker connecting aromatic parts of molecule does not have any negative influence on the binding.

Full text of DOI:10.3390/molecules22091491

molecules
Article
Design, Synthesis, Antimycobacterial Evaluation,
and In Silico Studies of 3-(Phenylcarbamoyl)-
pyrazine-2-carboxylic Acids
Lucia Semelková 1,*, Petra Janošcová 1, Carlos Fernandes 1, Ghada Bouz 1, Ondrˇej Jand’ourek 1,
Klára Konecˇná 1, Pavla Paterová 2, Lucie Navrátilová 1, Jirˇí Kuneš 1, Martin Doležal 1
and
ID
Jan Zitko 1,
*
ID
1
Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203, Hradec Králové 500 05,
Czech Republic; Petra.Janoscova@seznam.cz (P.J.); oaksdonsad@gmail.com (C.F.); bouzg@faf.cuni.cz (G.B.);
JANDO6AA@faf.cuni.cz (O.J.); konecna@faf.cuni.cz (K.K.); navratl2@faf.cuni.cz (L.N.);
kunes@faf.cuni.cz (J.K.); dolezalm@faf.cuni.cz (M.D.)
Department of Clinical Microbiology, University Hospital, Sokolská 581, Hradec Králové 500 05,
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.)
Received: 15 August 2017; Accepted: 5 September 2017; Published: 7 September 2017
Abstract: Pyrazinamide, the first-line antitubercular drug, has been regarded the basic component
of tuberculosis treatment for over sixty years. Researchers have investigated its effect on
Mycobacterium tuberculosis for this long time, and as a result, new potential targets of pyrazinamide
or its active form, pyrazinoic acid, have been found. We have designed and prepared
3-(phenyl-carbamoyl)pyrazine-2-carboxylic acids as more lipophilic derivatives of pyrazinoic acid.
We also prepared methyl and propyl derivatives as prodrugs with further increased lipophilicity.
Antimycobacterial, antibacterial and antifungal growth inhibiting activity was investigated in all
prepared compounds. 3-[(4-Nitrophenyl)carbamoyl]pyrazine-2-carboxylic acid (16) exerted high
antimycobacterial activity against Mycobacterium tuberculosis H37Rv with MIC = 1.56
(5 M). Propyl 3-{[4-(trifluoromethyl)phenyl]carbamoyl}pyrazine-2-carboxylate (18a) showed also
high antimycobacterial activity against Mycobacterium tuberculosis H37Rv with MIC = 3.13
mL−1
In vitro cytotoxicity of the active compounds was investigated and no significant cytotoxic effect was
observed. Based to structural similarity to known inhibitors of decaprenylphosphoryl- -D-ribose
µg
·
mL−1
µ
µg
·
.
β
oxidase, DprE1, we performed molecular docking of the prepared acids to DprE1. These in silico
experiments indicate that modification of the linker connecting aromatic parts of molecule does not
have any negative influence on the binding.
Keywords: anilides; antimycobacterial activity; cytotoxicity; DprE1; pyrazinamide; pyrazinoic
acid; RpsA
1. Introduction
Pyrazinamide (PZA, Figure 1) is one of the fist-line drugs used in the treatment of tuberculosis (TB).
Due to its unique sterilizing effect on semi-dormant tubercle bacilli and ability to shorten the duration
of therapy, PZA has been used for over sixty years in combination with other antituberculotics [1–3].
Although PZA has been applied for such a long time, the mechanism of action is still not yet completely
understood. PZA is known to be a prodrug that is converted to its active form, pyrazinoic acid (POA),
by the enzyme pyrazinamidase/nicotinamidase inside the mycobacterial cell [4].
Its non-specific mechanism of action is based on a cyclic expulsion of POA anion to the
extracellular space, protonation and penetration of the uncharged POA back to the cell. This cycle
Molecules 2017, 22, 1491; doi:10.3390/molecules22091491
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1491
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results in proton accumulation and potentially to cytoplasmic acidification along with the possible
collapse of membrane potential and membrane transport [ ]. With respect to previous observations
and the pKa of POA, an acidic pH is considered important for the PZA/POA activity. However,
recent experiments by Peterson et al. [ ] disputed the acidification of cytoplasm as a mechanism of
5,6
7
action of PZA and POA and showed that the acidification of intracellular compartment required
PZA/POA concentrations at least 1–2 orders higher than minimum inhibitory concentrations (MICs).
Specific targets of PZA/POA were suggested as Fatty Acid Synthase (FAS) I (involved in mycolic acid
biosynthesis) [
8], aspartate decarboxylase (biosynthetic pathway of coenzyme A) [9,10] and ribosomal
protein S1 (RpsA, important for trans-translation) [11].
Figure 1. Structures of pyrazinamide and its active form, pyrazinoic acid.
RpsA, a 30S ribosomal protein S1, is essential for translation and is involved in trans-translation,
which is the process of rescuing ribosomes stalled in the translation of mRNA [12]. Shi et al. [11
]
proposed that POA binds directly to the C-terminus region and disrupts the formation of RpsA-tmRNA
complex. Experiments of Yang et al. [13] have pointed to the fourth S1 domain of RpsA as the binding
site for POA, where the surface is supposed to interact with tmRNA [13,14].
POA itself cannot penetrate through the lipid-rich mycobacterial cell wall easily due to its
low lipophilicity and significant acidity (pKa = 2.9) [5] According to the performed experiments,
Yang et al. [13] suggested a substitution with any lipophilic moiety in the position 5 or 6 on the
pyrazine ring. This hypothesis is based on the interactions observed in a co-crystallized complex of
POA-RpsA, where the substituents at C-5 and C-6 of the pyrazine ring seem to be less critical for POA
binding to S1 domain.
Our research group has focused on the synthesis of N-phenylpyrazine-2-carboxamides for
several years. Many of these compounds exerted antimycobacterial effect comparable to PZA
(MIC = 6.25–12.5
µg ´s medium, pH = 5.6 [15–17]). Considering the previous results
mL−1, Šula
·
of N-phenylcarboxamides (anilides), we decided to choose the phenylcarbamoyl moiety as a lipophilic
substituent for the synthesis of more lipophilic POA derivatives as Yang et al. have suggested. We
intend to prepare derivatives substituted in positions 5, 6 and even 3 to study the influence of positional
isomerism. First, we decided to prepare substituted derivatives of POA with phenylcarbamoyl
substituent in position 3.
We thus prepared a series of 18 substituted POA derivatives and also 17 propyl esters
and 12 methyl esters from the synthesized acids to increase their lipophilicity and possibly
enhance the penetration through the lipid-rich mycobacterial cell wall. After synthesis of these
derivatives, we went through literature and found an article of Neres et al. [18] describing
2-carboxyquinoxalines as inhibitors of mycobacterial decaprenylphosphoryl-
(DprE1). DprE1 is an essential enzyme involved in the biosynthesis of arabinogalactan, a basic
component of mycobacterial cell wall [19 20]. Nitrobenzothiazinones have become the first
β-D-ribose oxidase
,
effective inhibitors of DprE1 [21]. Since then, the modification and design of new potential
inhibitors continue. Discovered inhibitors belong to many structural classes, namely azaindoles [22],
4-aminoquinolone piperidine amides [23], pyrazolopyridones [24], 8-pyrrolobenzothiazinones [25],
benzothiazolylpyrimidine-5-carboxamides [26], and 2-carboxyquinoxalines [18], among others.
Although the 3-substituted derivatives may fail to bind to the RpsA for the reasons mentioned
above, their structural similarity to known inhibitors of DprE1 (Figure 2) led us to perform a molecular
docking to DprE1. We intended to study the effect of structural differences between reported

Molecules 2017, 22, 1491
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inhibitors and our compounds, that is, the absence of the second condensed ring and alteration
of -NH-CH2- linker.
Figure 2. Comparison of the basic scaffold of previously prepared DprE1 inhibitors and structures
presented in this paper.
2. Results and Discussion
2.1. Chemistry
In this project 18 compounds
carboxylic acids, 17 related propyl esters 1a
and 17b 18b were prepared. Compounds
semi-products—see Section 3.2.3 for references.
1
–
18 from a series of substituted 3-(phenylcarbamoyl)pyrazine-2-
18a and 12 related methyl esters 1b 4b 6b 8b 11b 15b
11 16 and 18 were published previously as
–
,
–
,
,
–
,
1
,
7,
9,
,
The majority of compounds from the acid series were prepared starting from commercially
available pyrazine-2,3-dicarboxylic anhydride and the synthetic procedure was performed according
to [27] with some modifications, see Scheme 1. Several compounds were prepared using
pyrazine-2,3-dicarboxylic acid as a starting compound. Pyrazine-2,3-dicarboxylic acid was treated in
acetic acid anhydride at reflux to convert to pyrazine-2,3-dicarboxylic anhydride; the procedure was
modified on the basis of the same literature. The analytical characteristics of pyrazine-2,3-dicarboxylic
anhydride were in accordance with published literature data. Subsequently the anhydride was
dissolved in tetrahydrofuran and a corresponding substituted aniline was added. The reaction
mixture was stirred for 1 h at RT. Afterwards water was poured in and a saturated aqueous solution
of NaHCO3 was added dropwise to pH 6 in order to form crystals of the desired substituted
3-(phenylcarbamoyl)pyrazine-2-carboxylic acids
prepared via esterification in propanol with a catalytic amount of H2SO4 in 35–85% yield. Methyl
esters 1b 18b of several of the acids ( 11 15 and 17 18) were also prepared using the same
1–18 in 58–98% yield. Propyl esters 1a–18a were
–
1,
4–
6
,
8,
–
,
procedure as propyl esters with methanol instead as the solvent in 52–76% yield. The esterification
was carried out under microwave irradiation to accelerate the progress of the reaction. The reaction
was performed under mild conditions in a microwave reactor to facilitate the forming of the ester bond
and to prevent a decomposition of the carboxylic moiety. Despite the mild conditions and an effort to
avoid decarboxylation, we have not been successful in the synthesis of propyl ester of compound 16
(the presence of the decarboxylation product was confirmed by NMR). Subsequently, synthesis of the
ester 16a was attempted by conversion of the acid 16 to its acyl chloride by conventional addition of
SOCl2, followed by alcoholysis by propanol in the presence of pyridine as a base. However, this was
not successful either, due to the low yields.
All compounds were characterized by 1H- and 13C-NMR, IR, elemental analysis, and melting
1
point. The H-NMR spectra showed the proton peaks of the carboxylic groups in the range of
13.91–13.73 ppm. In case of some acids, this peak was not visible due to exchange with D2O. Peaks for
the pyrazine hydrogens were in the range of 8.99–8.86 in DMSO-d6 and 8.82–8.68 in CDCl3. They were
detected as one peak or as two peaks with coupling constant J ranging between 2.2–2.7 Hz, which is
in accordance with the literature [28]. The IR spectroscopy confirmed the presence of −th1e expected
characteristic functional groups. The C=O stretching bands were located at 1697–1611 cm for amidic
moiety, 1757–1701 cm−1 for carboxylic acids, and 1748–1727 cm−1 for esters. Amidic N-H stretching

Molecules 2017, 22, 1491
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bands were at 3379–3106 cm−1 and carboxylic O-H stretching at 3187–2584 cm−1. Esters showed an
alkyl C-H stretching in the range of 2983–2919 cm−1
.
The stability of the prepared esters in DMSO solution was studied. Samples were kept in the
refrigerator (7 ◦C) for one month and subsequently the stability was verified by TLC (mobile phase:
propanol/30% aq. sol. of ammonia 3:1) in comparison with the original acid. The temperature used
for stability validation was based on storage conditions of dissolved samples before biological testing.
No presence of the original acid was observed in the studied samples.
Scheme 1. Synthetic procedures of prepared series. Reagents and Conditions: (
a
) acetic anhydride,
reflux, 1 h; (
b
) 1. tetrahydrofuran, RT, 1 h, 2. water, NaHCO3 sol.; (
c
) propanol, H2SO4, MW: 120 ◦C,
◦
20 min, 50 W; (d) methanol, H2SO4, MW: 120 C, 20 min, 50 W.
2.2. Lipophilicity
Lipophilicity is an important physico-chemical property of drugs or biologically active
compounds. This property determines the penetration through biological membranes by passive
diffusion. A successful biological effect depends on an appropriate ratio between the hydrophilic and
hydrophobic properties of the substance. This aspect is very important especially for antitubercular
drugs due to the presence of the lipid-rich mycobacterial wall. Log P values, mentioned in Table 1,
were calculated by ChemBioDraw Ultra 14.0. The log P value of POA is
lipophilicity of acids 18, compared to POA, was 1.59 0.58 (n = 18). Further esterification of
increased log P by 0.28 0.06 (n = 12) in the case of methyl esters (1b 4b 6b 8b 11b 15b, 17b, 18b)
−
0.66. The average increase of
1
–
±
1–18
±
,
–
,
,
–
and 1.09 ± 0.03 (n = 17) for propyl esters 1a–18a.
2.3. Antimycobacterial Evaluation
Antimycobacterial in vitro screening was performed on four mycobacterial strains—Mtb,
M. avium, M. kansasii and fast growing M. smegmatis—using a Microplate Alamar Blue Assay (MABA;
see the Supplementary Material for the experimental details). The antimycobacterial activity results
were expressed as the minimum inhibitory concentration (MIC) in
used—POA, PZA and isoniazid (INH).
µg
mL−1. Three standards were
·
Only six compounds from
are presented in Table 1. Compounds 10
(MIC = 100
mL−1). The antimycobacterial activity of PZA and POA is dependent on the conditions
of biological assays; the activity increases with a decrease of the environmental pH [29 30]. For example,
mL−1
in acidic pH = 5.6 PZA exerts MIC of 12.5–25 16] yet is only weakly active at pH = 6 with
mL−1 mL−1
MIC = 200 30]. Compounds and 11 showed moderate activity with MIC = 50 .
1–
18 exerted certain antimycobacterial activity against Mtb, the results
,
14 and 17 showed activity comparable with PZA and POA
µg
·
,
µg
·
[
µ
g
·
[
2
µ
g
·
The most active compound from the series of acids was compound 16 with 4-NO2 substitution on
−1
phenyl ring with MIC = 1.56
not achieve the antimycobacterial activity of isoniazid (MIC = 0.1–0.2 µg·mL−1; 0.7–1.5 µM).
µg
·
mL (5
µ
M). Although this activity can be considered as high, it does

Molecules 2017, 22, 1491
5 of 17
In general, the increase of lipophilicity via conversion of the acids to their propyl or methyl
esters did not cause any significant improvement in antimycobacterial activity against Mtb. The
only exception was compound 18a, which displayed activity with MIC = 3.13
µg µM).
mL−1 (10
·
Compound
ester 2a
2 with 2-OH substitution kept its antimycobacterial effect after conversion to the propyl
(
MIC = 50 µg·mL−1). Methyl esters did not exert any antimycobacterial activity at the tested
concentrations. None of the presented compounds proved any significant activity against M. kansasii,
M. avium and M. smegmatis.
Table 1. Prepared compounds with lipophilicity parameter log P and antimycobacterial activity against
−1
Mtb in µg·mL and in µM in brackets.
MIC Mtb
MIC Mtb
MIC Mtb
R
Cpd.
Log P
Cpd.
Log P
Cpd.
Log P
−1
−1
−1
(µg·mL
)
(µg·mL
)
(µg·mL
)
H
2-OH
1
2
3
4
5
6
7
8
9
0.57
0.18
0.44
0.32
1.54
1.47
0.73
0.89
1.13
1.69
1.40
1.21
1.61
0.74
−0.26
0.13
1.49
1.49
−1.31
>100
50 (193)
>100
>100
>100
>100
>100
>100
>100
100
50 (155)
>100
>100
100
>100
1.56 (5)
100
1a
2a
3a
4a
5a
6a
7a
8a
9a
10a
11a
12a
13a
14a
15a
-
17a
18a
POA
1.66
1.27
1.53
1.40
2.63
2.56
1.81
1.97
2.21
2.77
2.49
2.30
2.70
1.83
0.96
-
>100
50 (160)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
-
1b
-
-
4b
5b
6b
-
8b
-
0.83
-
-
0.58
1.81
1.74
-
1.15
-
>100
-
-
>100
>100
>100
-
>100
-
4-OCH3
2,4-diOCH3
2,5-diCH3
4-CH2CH3
4-F
2,4-diF
4-Cl
3,4-diCl
4-Br
5-F-2-CH3
2-Cl-5-CH3
5-Cl-2-OH
2-OH-5-NO2
4-NO2
10
11
12
13
14
15
16
17
18
PZA
-
-
-
11b
12b
13b
14b
15b
-
17b
18b
INH
1.66
1.48
1.88
1.00
0.21
-
1.75
1.75
−0.64
>100
>100
>100
>100
>100
-
3-CF3
4-CF3
-
2.58
2.58
−0.66
>100
3.13 (10)
100
>100
>100
>100
100
0.1–0.2 (0.7–1.5)
INH—isoniazid; PZA—pyrazinamide.
2.4. Antibacterial and Antifungal Evaluation
All prepared compounds were tested for antibacterial and antifungal activity using the
microdilution broth method. These biological assays were performed on eight bacterial and eight
fungal strains of clinical importance. See the Supplementary Material for the experimental details.
None of compounds
concentration of 500
1
–
18 displayed any antibacterial or antifungal activity up to the highest tested
µM. Propyl esters 1a 18a did not show any antifungal and antibacterial activity,
–
except for compound 11a. This compound had low antibacterial activity against Staphylococcus aureus
and Pseudomonas aeruginosa with MIC = 250 M. Only one compound among the methyl esters, 8b
µ
,
showed antibacterial activity against Staphylococcus aureus with MIC = 250 µM, and the rest of the
methyl esters were inactive against the tested bacterial and fungal strains.
2.5. Cytotoxicity
The active compounds 16 and 18a were studied for their in vitro cytotoxic effect in the HepG2
cell line. The results of the experiments are presented as the inhibitory concentration that reduces the
viability of the cell population to 50% of the maximal viability, IC50. None of the tested compounds
(16: 4-NO2; 18a: 4-CF3) exerted significant cytotoxic effect. Compound 16 showed IC50 > 750
µM
(precipitation at higher concentration) and ester 18a showed IC50 = 252.4 M. The values of selectivity
µ
index (SI = IC50 / MIC) related to Mtb were SI > 150 for compound 16 and for 18a SI = 25.24.
2.6. In Silico Docking Study
We performed molecular docking studies on DrpE1 of Mtb H37Rv. DprE1 was chosen as a
potential new target involved in mycobacterial cell wall synthesis. We studied only acids 18, as
1–

Molecules 2017, 22, 1491
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the methyl and propyl esters are considered as prodrugs which are hydrolyzed in mycobacterium.
Molecular Operating Environment (MOE) 2016.08 (Chemical Computing Group, Montreal, QC,
Canada) was used to conduct the in silico study. To verify the docking procedure, the originally
co-crystalized ligand (quinoxaline) was removed and redocked again with RMSD = 0.24 Å.
PDB structure 4P8N (chain A) was chosen for in silico study of DprE1. The predicted poses of
individual ligands
original ligand. Eight ligands (
1
–
18 were evaluated with regard to the ligand-receptor interactions and position of
10 11 16 18) combining the best docking score, similarity in
3,
4
,
6,
9
,
,
,
,
interactions to the original ligand, and overlapping with the original ligand were considered as the
best candidates for DprE1 inhibition. In comparison to the original structure of 2-carboxyquinoxalines,
the replacement of -NH-CH2- linker by -CONH- group does not radically change the character of
binding mode. On the other hand, the loss of the condensed ring along with large CF3 substituent
seems to decrease the antimycobacterial activity. Probably the large lipophilic substituent is needed
for the filling of the hydrophobic binding sub-pocket and will be considered for further investigation.
The interactions and binding scores are described and depicted in Supplementary Material.
3. Experimental
3.1. General Information
All chemicals were of reagent or higher grade of purity and were purchased from Sigma-Aldrich
(Steinheim, Germany). The progress of reactions was monitored by Thin Layer Chromatography
(TLC; TLC Silica gel 60 F254, Merck, Darmstadt, Germany) with UV detection using a 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 this equipment was run under CEM’s Synergy
™ software (version 1.38)
for setting and monitoring the reaction conditions. The temperature of reactions was monitored
by an internal infrared sensor. Propyl esters and methyl esters were purified by preparative flash
chromatograph CombiFlash® Rf (Teledyne Isco Inc., Lincoln, NE, USA). The type of elution was
gradient, using the mixture of hexane (LachNer, Neratovice, Czech Republic) and ethyl acetate (Penta,
Prague, Czech Republic) as mobile phase. Silica gel (0.040–0.063 nm, Merck) was used as the stationary
phase. NMR spectra were taken in DMSO-d6 with a Varian VNMR S500 spectrometer (499.87 MHz for
1H 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. 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 measured using 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.
Lipophilicity parameter log P was calculated by software CS ChemBioDraw Ultra 14.0 (CambridgeSoft,
Cambridge, MA, USA).
3.2. Chemistry
3.2.1. General Procedure for the Synthesis of Acids 1–18
Pyrazine-2,3-dicarboxylic acid (4.0 g, 23.8 mmol) was dissolved in acetic anhydride (30 mL).
The reaction mixture was refluxed for one hour, and subsequently cooled down to 0 ◦C in ice bath.
The obtained crystals of pyrazine-2,3-dicarboxylic anhydride were filtered off (yield 70%).
Pyrazine-2,3-dicarboxylic anhydride (1.0 g, 6.7 mmol) was dissolved in tetrahydrofuran (40 mL)
in an Erlenmeyer flask and the corresponding substituted aniline (6.7 mmol, 1 equiv.) was added in
one dose. The reaction mixture was stirred for 1 hour at laboratory temperature. Water (30 mL) was
added into the mixture followed by the saturated aqueous solution of NaHCO3 until pH 6 to form the
corresponding 3-(phenylcarbamoyl)pyrazine-2-carboxylic acid 1–18. Obtained crystals were filtered

Molecules 2017, 22, 1491
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off and washed with water. The progress of the procedure was monitored by TLC eluted with the
system water/butanol/acetic acid 5:4:1.
3.2.2. General Procedure for the Synthesis of Propyl and Methyl Esters
Propyl esters 1a–15a, 17a and 18a were prepared using microwave irradiation. Substituted acid
1–18 (300 mg), propanol (3 mL) and one drop of H2SO4 were put to a thick-wall vial for the microwave
reactions. Conditions for the synthesis were 120 ◦C, 20 min, and 50 W in the pressurized vial. The
progress of the reactions was monitored by TLC in system propanol/30% aq. sol. of ammonia 3:1.
The reaction mixture was adsorbed on silica and purified by flash chromatography using gradient
elution with ethyl-acetate (50–90%) in hexane. Methyl esters 1b
,
4b–
6b
,
8b
,
11b
–15b and 17b
,
18b were
prepared using the same procedure as propyl esters using methanol instead as the solvent.
3.2.3. Analytical Data of Prepared Compounds
◦
3-(Phenylcarbamoyl)pyrazine-2-carboxylic acid (
1
) [31]. Pale yellow solid. Yield 91%; m.p. 163.1–164.6 C;
IR (cm−1): 3341 (N-H, CONH), 3051 (O-H, COOH), 1712 (C=O, COOH), 1682 (C=O, CONH); 1H-NMR
13.77 (bs, 1H, COOH), 10.75 (s, 1H, NH), 8.92–8.86 (m, 2H, pyr.), 7.80–7.75 (m, 2H, Ar), 7.40–7.34 (m,
2H, Ar), 7.16–7.11 (m, 1H, Ar); 13C-NMR
166.45, 162.61, 146.40, 145.85, 145.80, 144.69, 138.57, 128.98,
δ
δ
124.39, 120.25; Elemental analysis: calc. for C12H9N3O3 (MW 243.22): 59.26% C, 3.73% H, 17.28% N;
found 59.34% C, 3.61% H, 17.24% N.
3-[(2-Hydroxyphenyl)carbamoyl]pyrazine-2-carboxylic acid (
IR (cm−1): 3278 (O-H, OH), 3106 (N-H, CONH), 2854 (O-H, COOH), 1713 (C=O, COOH), 1611 (C=O,
CONH); 1H-NMR
13.74 (bs, 1H, COOH), 10.31 (s, 1H, OH), 10.15 (s, 1H, NH), 8.93 (d, J = 2.5 Hz, 1H,
pyr.), 8.90 (d, J = 2.5 Hz, 1H, pyr.), 8.24–8.20 (m, 1H, Ar), 7.03–6.93 (m, 2H, Ar), 6.88–6.83 (m, 1H, Ar);
13C-NMR
166.92, 160.10, 147.86, 147.12, 146.85, 144.27, 141.64, 125.81, 125.03, 119.93, 119.48, 115.13;
2
). Yellow solid. Yield 95%; m.p. 266.4–269.3 ◦C;
δ
δ
Elemental analysis: calc. for C12H9N3O4 (MW 259.22): 55.60% C, 3.50% H, 16.21% N; found 55.77% C,
3.39% H, 16.07% N.
3-[(4-Methoxyphenyl)carbamoyl]pyrazine-2-carboxylic acid (
IR (cm−1): 3330 (N-H, CONH), 3169 (O-H, COOH), 1757 (C=O, COOH), 1665 (C=O, CONH), 1243
3
). Yellow solid. Yield 98%; m.p. 167.1–168.0 ◦C;
(C-O, OCH3); 1H-NMR
δ 13.74 (bs, 1H, COOH), 10.64 (s, 1H, CONH), 8.89–8.86 (m, 2H, pyr.), 7.73–7.68
(m, 2H, Ar), 6.96–6.92 (m, 2H, Ar), 3.74 (s, 3H, CH3); 13C-NMR δ 166.56, 161.97, 156.06, 146.67, 145.76,
145.47, 144.51, 131.65, 121.79, 114.08, 55.40; Elemental analysis: calc. for C13H11N3O4 (MW 273.25):
57.14% C, 4.06% H, 15.38% N; found 56.68% C, 3.93% H, 15.13% N.
◦
3-[(2,4-Dimethoxyphenyl)carbamoyl]pyrazine-2-carboxylic acid (
4
). Yellow solid. Yield 85%; m.p. 199.6–200.3 C;
IR (cm−1): 3309 (N-H, CONH), 3129 (O-H, COOH), 1741 (C=O, COOH), 1667 (C=O, CONH); 1H-NMR
13.73 (bs, 1H, COOH), 9.95 (s, 1H, NH), 8.91 (d, J = 2.4 Hz, 1H, pyr.), 8.87 (d, J = 2.4 Hz, 1H, pyr.), 8.11
(d, J = 8.8 Hz, 1H, Ar), 6.70 (d, J = 2.6 Hz, 1H, Ar), 6.58–6.55 (m, 1H, Ar), 3.89 (s, 3H, CH3), 3.77 (s, 3H,
CH3); 13C-NMR
166.87, 160.06, 157.18, 150.63, 147.69, 146.60, 144.22, 142.07, 121.24, 119.89, 104.50,
δ
δ
99.14, 56.27, 55.56; Elemental analysis: calc. for C14H13N3O5 (MW 303.27): 55.45% C, 4.32% H, 13.86%
N; found 55.46% C, 4.31% H, 13.84% N.
3-[(2,5-Dimethylphenyl)carbamoyl]pyrazine-2-carboxylic acid (
(cm−1): 3339 (N-H, CONH), 2918 (O-H, COOH), 1729 (C=O, COOH),1687 (C=O, CONH); 1H-NMR
13.75 (bs, 1H, COOH), 10.15 (s, 1H, NH), 8.91–8.87 (m, 2H, pyr.), 7.40–7.38 (m, 1H, Ar), 7.14 (d,
J = 7.7 Hz, 1H, Ar), 6.99–6.95 (m, 1H, Ar), 2.28 (s, 3H, CH3), 2.22 (s, 3H, CH3); 13C-NMR
166.53,
5
). Grey solid. Yield 81%; m.p. 177.4–178.8 ◦C; IR
δ
δ
162.37, 146.50, 145.90, 145.30, 144.69, 135.48, 135.36, 130.39, 129.06, 126.63, 125.43, 20.80, 17.39; Elemental
analysis: calc. for C14H13N3O3 (MW 271.28): 61.99% C, 4.83% H, 15.49% N; found 62.46% C, 4.73% H,
15.53% N.

Molecules 2017, 22, 1491
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◦
3-[(4-Ethylphenyl)carbamoyl]pyrazine-2-carboxylic acid (6). Beige solid. Yield 98%; m.p. 159.1–159.9 C; IR
(cm−1): 3314 (N-H, CONH), 2972 (O-H, COOH), 1704 (C=O, COOH), 1664 (C=O, CONH); 1H-NMR
δ
10.68 (s, 1H, NH), 8.90–86 (m, 2H, pyr.), 7.71–7.65 (m, 2H, Ar), 7.23–7.16 (m, 2H, Ar), 2.58 (q, J = 7.6 Hz,
2H, CH2), 1.17 (t, J = 7.6 Hz, 3H, CH3); 13C-NMR
δ
166.50, 162.32, 146.52, 145.79, 145.67, 144.60, 139.87,
136.24, 128.16, 120.32, 27.85, 15.88; Elemental analysis: calc. for C14H13N3O3 (MW 271.28): 61.99% C,
4.83% H, 15.49% N; found 61.51% C, 5.24% H, 15.08% N.
◦
3-[(4-Fluorophenyl)carbamoyl]pyrazine-2-carboxylic acid (
7
) [27]. Grey solid. Yield 90%; m.p. 168.7–169.6 C;
IR (cm−1): 3358 (N-H, CONH), 3069 (O-H, COOH), 1730 (C=O, COOH), 1683 (C=O, CONH); 1H-NMR
13.79 (bs, 1H, COOH), 10.85 (s, 1H, CONH), 8.92–8.87 (m, 2H, pyr.), 7.84–7.78 (m, 2H, Ar), 7.24–7.18
(m, 2H, Ar); 13C-NMR
166.46, 162.48, 158.75 (d, J = 240.8 Hz), 146.53, 145.93, 145.51, 144.66, 134.95
δ
δ
(d, J = 2.5 Hz), 122.16 (d, J = 8.0 Hz), 115.59 (d, J = 22.3 Hz); Elemental analysis: calc. for C12H8FN3O3
(MW 261.21): 55.18% C, 3.09% H, 16.09% N; found 54.96% C, 3.15% H, 15.85% N.
◦
3-[(2,4-Difluorophenyl)carbamoyl]pyrazine-2-carboxylic acid (
8
). Grey solid. Yield 94%; m.p. 187.0–188.0 C;
IR (cm−1): 3346 (N-H, CONH), 2906 (O-H, COOH), 1702 (C=O, COOH), 1654 (C=O, CONH);1H-NMR
δ
13.79 (bs, 1H, COOH), 10.54 (s, 1H, NH), 8.91 (d, 1H, J = 2.7 Hz, pyr.), 8.89 (d, 1H, J = 2.7 Hz, pyr.),
7.86–7.79 (m, 1H, Ar), 7.42–7.35 (m, 1H, Ar), 7.17–7.12 (m, 1H, Ar); 13C-NMR δ 165.4, 162.7, 159.5 (dd,
J = 245.1 Hz, J = 11.4 Hz), 155.1 (dd, J = 249.9 Hz, J = 12.4 Hz), 146.4, 146.2, 144.8, 144.6, 126.7 (dd,
J = 9.6 Hz, J = 2.9 Hz), 122.0 (dd, J = 12.4 Hz, J = 3.9 Hz), 111.5 (dd, J = 21.9 Hz, J = 3.8 Hz), 104.6 (dd,
J = 26.6 Hz, J = 23.9 Hz); Elemental analysis: calc. for C12H7F2N3O3 (MW 279.20): 51.62% C, 2.53% H,
15.05% N; found 51.41% C, 2.05% H, 14.72% N.
3-[(4-Chlorophenyl)carbamoyl]pyrazine-2-carboxylic acid (
IR (cm−1): 3331 (N-H, CONH), 2584 (O-H, COOH), 1708 (C=O, COOH), 1692 (C=O, CONH); 1H-NMR
13.81 (bs, 1H, COOH), 10.92 (s, 1H, CONH), 8.91–8.88 (m, 2H, pyr.), 7.84–7.80 (m, 2H, Ar), 7.45–7.41
(m, 2H, Ar); 13C-NMR
166.38, 162.70, 146.41, 145.97, 145.52, 144.70, 137.53, 128.90, 128.05, 121.83;
9
) [32]. White solid. Yield 98%; m.p. 171.1–173.2 ◦C;
δ
δ
Elemental analysis: calc. for C12H8ClN3O3 (MW 277.66): 51.91% C, 2.90% H, 15.13% N; found 52.24%
C, 2.83% H, 15.25% N.
◦
3-[(3,4-Dichlorophenyl)carbamoyl]pyrazine-2-carboxylic acid (10). White solid. Yield 85%; m.p. 290.0–292.1 C;
IR (cm−1): 3274 (N-H, CONH), 2898 (O-H, COOH), 1724 (C=O, COOH), 1671 (C=O, CONH); 1H-NMR
δ
11.10 (s, 1H, NH), 8.92 (d, J = 2.5 Hz, 1H, pyr.), 8.90 (d, J = 2.5 Hz, 1H, pyr.), 8.16 (d, J = 2.4 Hz, 1H,
Ar), 7.77–7.71 (m, 1H, Ar), 7.63 (d, J = 8.8 Hz, 1H, Ar); 13C-NMR
δ
166.35, 162.94, 146.51, 146.23, 145.03,
144.76, 138.65, 131.26, 130.96, 126.00, 121.49, 120.37; Elemental analysis: calc. for C12H7Cl2N3O3 (MW
312.11): 46.18% C, 2.26% H, 13.46% N; found 46.18% C, 2.24% H, 13.32% N.
◦
3-[(4-Bromophenyl)carbamoyl]pyrazine-2-carboxylic acid (11) [33]. White solid. Yield 84%; m.p. 171.1–171.9 C;
IR (cm−1): 3312 (N-H, CONH), 2973 (O-H, COOH), 1706 (C=O, COOH), 1662 (C=O, CONH); 1H-NMR
δ
10.92 (s, 1H, NH), 8.92–8.86 (m, 2H, pyr.), 7.80–7.72 (m, 2H, Ar), 7.59–7.52 (m, 2H, Ar); 13C-NMR
δ
166.39, 162.75, 146.43, 145.98, 145.55, 144.71, 137.96, 131.82, 122.20, 116.16; Elemental analysis: calc. for
C12H8BrN3O3 (MW 322.12): 44.75% C, 2.50% H, 13.05% N; found 44.30% C, 2.62% H, 12.55% N.
◦
3-[(5-Fluoro-2-methylphenyl)carbamoyl]pyrazine-2-carboxylic acid (12). Beige solid. Yield 92%; m.p. 185.0–186.1 C;
IR (cm−1): 3301 (N-H, CONH), 3187 (O-H, COOH), 1746 (C=O, COOH), 1670 (C=O, CONH); 1H-NMR
δ
13.83 (bs, 1H, COOH), 10.26 (s, 1H, NH), 8.92 (d, 1H, J = 2.4 Hz, pyr.), 8.90 (d, 1H, J = 2.4 Hz, pyr.),
7.51 (dd, 1H, J = 9.1 Hz J = 2.9 Hz, Ar), 7.01 (dd, 1H, J = 9.1 Hz J = 6.9 Hz, Ar), 7.00 (dt, 1H, J = 9.1 Hz
J = 2.9 Hz, Ar), 2.25 (s, 3H, CH3); 13C-NMR
166.4, 162.7, 160.4 (d, J = 240.3 Hz), 146.3, 146.1, 145.2,
δ
144.9, 136.9 (d, J = 10.4 Hz), 131.8 (d, J = 8.5 Hz), 127.5 (d, J = 2.9 Hz), 112.3 (d, J = 21.0 Hz), 111.0 (d,
J = 24.8 Hz), 17.1; Elemental analysis: calc. for C13H10FN3O3 (MW 275.24): 56.73% C, 3.66% H, 15.27%
N; found 56.84% C, 3.81% H, 14.93% N.

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3-[(2-Chloro-5-methylphenyl)carbamoyl]pyrazine-2-carboxylic acid (13). Yellow solid. Yield 90%; m.p.
190.1–191.2 ◦C; IR (cm−1): 3342 (N-H, CONH), 2964 (O-H, COOH), 1701 (C=O, COOH), 1664 (C=O,
CONH); 1H-NMR
δ
13.79 (bs, 1H, COOH), 10.34 (s, 1H, NH), 8.94 (d, J = 2.5 Hz, 1H, pyr.), 8.91 (d, J =
2.5 Hz, 1H, pyr.), 7.91 (d, J = 2.0 Hz, 1H, Ar), 7.43 (d, J = 8.2 Hz, 1H, Ar), 7.09–7.05 (m, 1H, Ar), 2.33 (s,
3H, CH3); 13C-NMR
166.56, 161.54, 147.20, 146.73, 144.57, 142.86, 137.65, 133.83, 129.34, 127.35, 124.58,
δ
122.67, 20.86; Elemental analysis: calc. for C13H10ClN3O3 (MW 291.69): 53.53% C, 3.46% H, 14.41% N;
found 53.88% C, 3.37% H, 14.32% N.
3-[(5-Chloro-◦2-hydroxyphenyl)carbamoyl]pyrazine-2-carboxylic acid (14). Yellow solid. Yield 97%; m.p.
263.1–265.1 C; IR (cm−1): 3271 (O-H, OH), 3116 (N-H, CONH), 3048 (O-H, COOH), 1709 (C=O,
COOH), 1610 (C=O, CONH); 1H-NMR
δ
13.78 (bs, 1H, COOH), 10.68 (s, 1H, OH), 10.16 (s, 1H, NH),
8.94 (d, J = 2.5 Hz, 1H, pyr.), 8.90 (d, J = 2.4 Hz, 1H, pyr.), 8.29 (d, J = 2.6 Hz, 1H, Ar), 7.07–7.03 (m, 1H,
Ar), 6.95 (d, J = 8.7 Hz, 1H, Ar); 13C-NMR
166.74, 160.59, 147.63, 146.97, 146.02, 144.37, 141.58, 126.94,
δ
124.38, 122.70, 119.30, 116.21; Elemental analysis: calc. for C12H8ClN3O4 (MW 293.66): 49.08% C, 2.75%
H, 14.31% N; found 49.57% C, 2.69% H, 14.17% N.
3-[(2-Hydroxy-5-nitrophenyl)carbamoyl]pyrazine-2-carboxylic acid (15). Pale yellow solid. Yield 90%; m.p.
◦
290.0–291.3 C; IR (cm−1): 3566 (O-H, OH), 3363 (N-H, CONH), 2972 (O-H, COOH), 1744 (C=O,
COOH), 1676 (C=O, CONH), 1545 (N-O, NO2), 1349 (N-O, NO2); 1H-NMR
12.08 (bs, 1H, OH), 10.25 (s, 1H, NH), 9.16 (d, J = 2.8 Hz, 1H, Ar), 8.95 (d, J = 2.4 Hz, 1H, pyr.), 8.91
(d, J = 2.4 Hz, 1H, pyr.), 8.00–7.96 (m, 1H, Ar), 7.11 (d, J = 9.0 Hz, 1H, Ar); 13C-NMR
166.66, 161.15,
δ 13.73 (bs, 1H, COOH),
δ
153.61, 147.46, 147.04, 144.49, 141.76, 139.49, 126.02, 121.49, 115.12, 114.78; Elemental analysis: calc. for
C12H8N4O6 (MW 304.22): 47.38% C, 2.65% H, 18.42% N; found 46.99% C, 3.13% H, 18.15% N.
3-[(4-Nitrophenyl)carbamoyl]pyrazine-2-carboxylic acid (16) [34]. Yellow solid. Yield 58%; m.p.
224.5–227.2◦C; IR (cm−1): 3281 (N-H, CONH), 3065 (O-H, COOH), 1713 (C=O, COOH), 1693 (C=O,
CONH), 1543 (N-O, NO2), 1339 (N-O, NO2); 1H-NMR
δ
13.91 (bs, 1H, COOH), 11.36 (s, 1H, CONH),
8.95–8.92 (m, 2H, pyr.), 8.30–8.25 (m, 2H, Ar), 8.06–8.02 (m, 2H, Ar); 13C-NMR
δ
166.18, 163.52, 146.24,
146.06, 145.61, 144.96, 144.69, 143.13, 125.12, 120.06; Elemental analysis: calc. for C12H8N4O5 (MW
288.22): 50.01% C, 2.80% H, 19.44% N; found 50.28% C, 2.82% H, 19.35% N.
3-{[3-(Trifluoromethyl)phenyl]carbamoyl}pyrazine-2-carboxylic acid (17). Pale yellow solid. Yield 97%;
m.p. 176.3–177.1◦C; IR (cm−1): 3356 (N-H, CONH), 3064 (O-H, COOH), 1718 (C=O, COOH), 1694
1
(C=O, CONH); H-NMR
δ 11.14 (s, 1H, NH), 8.93 (d, 1H, J = 2.4 Hz, pyr.), 8.91 (d, 1H, J = 2.4 Hz,
pyr.), 8.30–8.28 (m, 1H, Ar), 8.01 (d, 1H, J = 7.9 Hz, Ar), 7.62 (t, 1H, J = 7.9 Hz, Ar), 7.50 (d, 1H,
J = 7.9 Hz, Ar); 13C-NMR
δ
166.4, 163.1, 146.5, 146.2, 145.2, 144.8, 139.4, 130.3, 129.7 (q, J = 31.4 Hz),
124.3 (q, J = 271.8 Hz), 123.9, 120.8 (q, J = 3.8 Hz), 116.4 (q, J = 3.8 Hz); Elemental analysis: calc. for
C13H8F3N3O3 (MW 311.22): 50.17% C, 2.59% H, 13.50% N; found 49.86% C, 2.75% H, 13.42% N.
3-{[4-(Trifluoromethyl)phenyl]carbamoyl}pyrazine-2-carboxylic acid (18) [35]. White solid. Yield 90%; m.p.
◦
153.5–154.7 C; IR (cm−1): 3306, 1706 (C=O, COOH), 1669 (C=O, CONH); 1H-NMR
δ
11.13 (s, 1H, NH),
8.92–8.91 (m 1H, pyr.), 8.91–8.89 (m, 1H, pyr.), 8.08–7.96 (m, 2H, Ar), 7.77–7.70 (m, 2H, Ar); 13C-NMR
166.4, 163.3, 146.4, 146.1, 145.7, 144.8, 142.2, 126.3 (q, J = 3.8 Hz), 124.5 (q, J = 270.9 Hz), 124.4 (q,
δ
J = 31.4 Hz), 120.2; Elemental analysis: calc. for C13H8F3N3O3 (MW 311.22): 50.17% C, 2.59% H, 13.50%
N; found 50.42% C, 2.21% H, 13.84% N.
◦
Propyl 3-(phenylcarbamoyl)pyrazine-2-carboxylate (1a). White solid. Yield 85%; m.p. 74.9–76.5 C; IR
(cm−1): 3355 (N-H, CONH), 2961 (C-H), 1734 (C=O, COO), 1686 (C=O, CONH), 1112, 1076 (C-O,
COO); 1H-NMR
δ
10.82 (s, 1H, CONH), 8.94 (s, 2H, pyr.), 7.80–7.77 (m, 2H, Ar), 7.407.35 (m, 2H, Ar),
7.17–7.12 (m, 1H, Ar), 4.26 (t, J = 6.6 Hz, 2H, CH2), 1.70–1.61 (m, 2H, CH2), 0.89 (t, J = 7.4 Hz, 3H, CH3);
13C-NMR
165.04, 162.19, 146.14, 145.68, 145.49, 145.11, 138.36, 128.94, 124.47, 120.32, 67.46, 21.45,
δ

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10.38; Elemental analysis: calc. for C15H15N3O3 (MW 285.30): 63.15% C, 5.30% H, 14.73% N; found
63.41% C, 5.40% H, 14.61% N.
Propyl 3-[(2-hydroxyphenyl)carbamoyl]pyrazine-2-carboxylate (2a). Pale yellow solid. Yield 64%; m.p.
156.8–159.6 ◦C; IR (cm−1): 3348 (N-H, CONH), 2924 (C-H), 1741 (C=O, COO), 1678 (C=O, CONH),
1111, 1086 (C-O, COO); 1H-NMR
δ
10.33 (s, 1H, CONH), 10.16 (s, 1H, OH), 8.96 (d, J = 2.5 Hz, 1H, pyr.),
8.95 (d, J = 2.4 Hz, 1H, pyr.), 8.22–8.17 (m, 1H, Ar), 7.04–6.93 (m, 2H, Ar), 6.89–6.82 (m, 1H, Ar), 4.31 (t,
J = 6.6 Hz, 2H, CH2), 1.76–1.67 (m, 2H, CH2), 0.93 (t, J = 7.4 Hz, 3H, CH3); 13C-NMR
165.50, 159.83,
δ
147.16, 146.98, 146.63, 144.83, 142.09, 125.63, 125.11, 120.01, 119.45, 115.10, 67.41, 21.49, 10.41; Elemental
analysis: calc. for C15H15N3O4 (MW 301.30): 59.80% C, 5.02% H, 13.95% N; found 59.83% C, 5.15% H,
13.52% N.
Propyl 3-[(4-methoxyphenyl)carbamoyl]pyrazine-2-carboxylate (3a). White solid. Yield 43%; m.p.
102.0–102.9 ◦C; IR (cm−1): 3284 (N-H, CONH), 2968 (C-H), 1745 (C=O, COO), 1652 (C=O, CONH),
1
1159, 1089 (C-O, COO); H-NMR
δ 10.71 (s, 1H, CONH), 8.92 (s, 2H, pyr.), 7.72–7.68 (m, 2H, Ar),
6.96–6.92 (m, 2H, Ar), 4.26 (t, J = 6.6 Hz, 2H, CH2), 3.75 (s, 3H, CH3), 1.70–1.62 (m, 2H, CH2), 0.89 (t,
J = 7.4 Hz, 3H, CH3); 13C-NMR
δ
165.18, 161.58, 156.12, 146.06, 145.74, 145.43, 144.97, 131.44, 121.88,
114.06, 67.40, 55.37, 21.46, 10.39; Elemental analysis: calc. for C16H17N3O4 (MW 315.33): 60.94% C,
5.43% H, 13.33% N; found 60.90% C, 5.47% H, 13.21% N.
Propyl 3-[(2,4-dimethoxyphenyl)carbamoyl]pyrazine-2-carboxylate (4a). Yellow solid. Yield 75%; m.p.
116.4–118.5 ◦C; IR (cm−1): 3371 (N-H, CONH), 2962 (C-H), 1736 (C=O, COO), 1677 (C=O, CONH) ),
112 , 1084 (C-O, COO); 1H-NMR
δ
9.97 (s, 1H, CONH), 8.76 (d, J = 2.4 Hz, 1H, pyr.), 8.68 (d, J = 2.4 Hz,
1H, pyr.), 8.43 (d, J = 8.6 Hz, 1H, Ar), 6.54–6.50 (m, 2H, Ar), 4.47 (t, J = 6.8 Hz, 2H, CH2), 3.93 (s, 3H,
CH3), 3.82 (s, 3H, CH3), 1.89–1.80 (m, 2H, CH2), 1.03 (t, J = 7.5 Hz, 3H, CH3); 13C-NMR
166.02, 158.94,
δ
157.01, 149.92, 148.00, 145.69, 143.06, 142.49, 120.76, 120.42, 103.79, 98.67, 68.05, 55.84, 55.51, 21.77, 10.31;
Elemental analysis: calc. for C17H19N3O5 (MW 345.36): 59.12% C, 5.55% H, 12.17% N; found 58.70% C,
5.42% H, 11.87% N.
Propyl 3-[(2,5-dimethylphenyl)carbamoyl]pyrazine-2-carboxylate (5a). White solid. Yield 78%; m.p.
◦
111.8–112.9 C; IR (cm−1): 3354 (N-H, CONH), 2976 (C-H), 1741 (C=O, COO), 1686 (C=O, CONH), 1145,
1079 (C-O, COO); 1H-NMR
pyr.), 8.02–8.00 (m, 1H, Ar), 7.11 (d, J = 7.6 Hz, 1H, Ar), 6.93 (d, J = 7.4 Hz, 1H, Ar), 4.47 (t, J = 6.8 Hz
2H, CH2), 2.35 (d, J = 6.1 Hz, 6H, CH3), 1.90–1.80 (m, 2H, CH2), 1.03 (t, J = 7.5 Hz, 3H, CH3); 13C-NMR
165.87, 159.33, 148.23, 146.08, 143.00, 142.07, 136.74, 134.76, 130.23, 126.04, 125.13, 122.31, 68.13, 21.76,
δ 9.54 (s, 1H, CONH), 8.80 (d, J = 2.4 Hz, 1H, pyr.), 8.68 (d, J = 2.5 Hz, 1H,
,
δ
21.17, 17.16, 10.29; Elemental analysis: calc. for C17H19N3O3 (MW 313.36): 65.16% C, 6.11% H, 13.41%
N; found 64.93% C, 5.91% H, 13.47% N.
Propyl 3-[(4-ethylphenyl)carbamoyl]pyrazine-2-carboxylate (6a). White solid. Yield 80%; m.p. 59.8–61.9 ◦C;
IR (cm−1): 3332 (N-H, CONH), 2971 (C-H), 1743 (C=O, COO), 1659 (C=O, CONH), 1154, 1090 (C-O,
1
COO); H-NMR
δ 10.75 (s, 1H, CONH), 8.93 (s, 2H, pyr.), 7.69 (d, J = 8.1 Hz, 2H, Ar), 7.20 (d, J =
8.1 Hz, 2H, Ar), 4.26 (t, J = 6.6 Hz, 2H, CH2), 2.58 (q, J = 7.6 Hz, 2H, CH2), 1.74–1.61 (m, 2H, CH2),
1.17 (t, J = 7.6 Hz, 3H, CH3), 0.89 (t, J = 7.4 Hz, 3H, CH3); 13C-NMR
δ
165.11, 161.90, 146.10, 145.62,
145.56, 145.03, 139.97, 136.04, 128.13, 120.41, 67.42, 27.83, 21.46, 15.82, 10.38; Elemental analysis: calc.
for C17H19N3O3 (MW 313.36): 65.16% C, 6.11% H, 13.41% N; found 64.97% C, 6.02% H, 12.95% N.
Propyl 3-[(4-fluorophenyl)carbamoyl]pyrazine-2-carboxylate (7a). Beige solid. Yield 72%; m.p. 100.9–103.1 ◦C;
IR (cm−1): 3351 (N-H, CONH), 2981 (C-H), 1736 (C=O, COO), 1690 (C=O, CONH), 1159, 1103 (C-O,
COO); 1H-NMR
δ
10.92 (s, 1H, CONH), 8.94 (s, 2H, pyr.), 7.84–7.80 (m, 2H, Ar), 7.24–7.19 (m, 2H,
Ar), 4.26 (t, J = 6.5 Hz, 2H, CH2), 1.70–1.62 (m, 2H, CH2), 0.88 (t, J = 7.4 Hz, 3H, CH3); 13C-NMR
165.07, 162.08, 158.80 (d, J = 241.2 Hz), 146.24, 145.63, 145.37, 145.09, 134.76 (d, J = 2.5 Hz), 122.25 (d,
δ

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J = 8.0 Hz), 115.57 (d, J = 22.3 Hz), 67.46, 21.46, 10.37; Elemental analysis: calc. for C15H14FN3O3 (MW
303.29): 59.40% C, 4.65% H, 13.85% N; found 59.04% C, 4.51% H, 13.59% N.
◦
Propyl 3-[(2,4-difluorophenyl)carbamoyl]pyrazine-2-carboxylate (8a). White solid. Yield 74%; m.p. 93.3–96.1 C;
IR (cm−1): 3358 (N-H, CONH), 2976 (C-H), 1742 (C=O, COO), 1694 (C=O, CONH), 1141, 1099 (C-O,
COO); 1H-NMR
δ
10.61 (bs, 1H, CONH), 8.97–8.93 (m, 2H, pyr.), 7.84–7.77 (m, 1H, Ar), 7.42–7.36 (m,
1H, Ar), 7.18–7.12 (m, 1H, Ar), 4.25 (t, J = 6.6 Hz, 2H, CH2), 1.71–1.62 (m, 2H, CH2), 0.89 (t, J = 7.4 Hz,
3H, CH3); 13C-NMR
165.00, 162.29, 159.62 (dd, J = 244.9, 11.6 Hz), 155.18 (dd, J = 249.7, 12.7 Hz),
δ
146.54, 145.58, 145.24, 144.32, 126.84 (dd, J = 9.9, 2.7 Hz), 121.79 (dd, J = 12.3, 3.7 Hz), 111.54 (dd, J = 22.0,
3.9 Hz), 104.61 (dd, J = 27.0, 23.8 Hz), 67.46, 21.45, 10.35.; Elemental analysis: calc. for C15H13F2N3O3
(MW 321.28): 56.08% C, 4.08% H, 13.08% N; found 55.94% C, 4.12% H, 13.21% N.
◦
Propyl 3-[(4-chlorophenyl)carbamoyl]pyrazine-2-carboxylate (9a). White solid. Yield 65%; m.p. 121.0–122.1 C; IR
(cm−1): 3352 (N-H, CONH), 2981 (C-H), 1737 (C=O, COO), 1690 (C=O, CONH), 1146, 1104 (C-O, COO);
1H-NMR
δ
10.99 (s, 1H, CONH), 8.94 (s, 2H, pyr.), 7.83 (d, J = 8.6 Hz, 2H, Ar), 7.44 (d, J = 8.5 Hz, 2H,
Ar), 4.26 (t, J = 6.5 Hz, 2H, CH2), 1.65 (h, J = 7.0 Hz, 2H, CH2), 0.88 (t, J = 7.4 Hz, 3H, CH3); 13C-NMR
δ
164.98, 162.31, 146.29, 145.50, 145.40, 145.15, 137.34, 128.89, 128.18, 121.91, 67.49, 21.45, 10.37; Elemental
analysis: calc. for C15H14ClN3O3 (MW 319.75): 56.35% C, 4.41% H, 13.14% N; found 56.50% C, 4.39%
H, 13.20% N.
Propyl 3-[(3,4-dichlorophenyl)carbamoyl]pyrazine-2-carboxylate (10a). White solid. Yield 34%; m.p.
101.4–102.2 ◦C; IR (cm−1): 3336 (N-H, CONH), 2975 (C-H), 1735 (C=O, COO), 1697 (C=O, CONH),
1148, 1082 (C-O, COO); 1H-NMR
δ
11.17 (s, 1H, CONH), 8.97–8.95 (m, 2H, pyr.), 8.16 (d, J = 2.5 Hz,
1H, Ar), 7.77 (dd, J = 8.8, 2.5 Hz, 1H, Ar), 7.64 (d, J = 8.8 Hz, 1H, Ar), 4.27 (t, J = 6.5 Hz, 2H, CH2),
1.72–1.60 (m, 2H, CH2), 0.89 (t, J = 7.4 Hz, 3H, CH3); 13C-NMR
164.92, 162.54, 146.52, 145.56, 145.18,
δ
144.91, 138.45, 131.20, 130.93, 126.11, 121.58, 120.43, 67.53, 21.45, 10.36; Elemental analysis: calc. for
C15H13Cl2N3O3 (MW 354.19): 50.87% C, 3.70% H, 11.86% N; found 50.77% C, 3.71% H, 11.49% N.
◦
Propyl 3-[(4-bromophenyl)carbamoyl]pyrazine-2-carboxylate (11a). White solid. Yield 68%; m.p. 86.6–88.6 C; IR
(cm−1): 3337 (N-H, CONH), 2966 (C-H), 1740 (C=O, COO), 1693 (C=O, CONH), 1142, 1083 (C-O, COO);
1H-NMR
δ
10.99 (s, 1H, CONH), 8.94 (s, 2H, pyr.), 7.79–7.75 (m, 2H, Ar), 7.58–7.54 (m, 2H, Ar.), 4.26 (t,
J = 6.5 Hz, 2H, CH2), 1.70–1.61 (m, 2H, CH2), 0.88 (t, J = 7.4 Hz, 3H, CH3); 13C-NMR
δ
164.97, 162.33,
146.29, 145.48, 145.41, 145.15, 137.76, 131.80, 122.26, 116.30, 67.49, 21.45, 10.38; Elemental analysis: calc.
for C13H14BrN3O3 (MW 364.20): 49.47% C, 3.87% H, 11.54% N; found 49.01% C, 3.75% H, 11.18% N.
Propyl 3-[(5-fluoro-2-methylphenyl)carbamoyl]pyrazine-2-carboxylate (12a). White solid. Yield 33%; m.p.
◦
124.8–127.0 C; IR (cm−1): 3358 (N-H, CONH), 2973 (C-H), 1739 (C=O, COO), 1690 (C=O, CONH), 1135,
1106 (C-O, COO); 1H-NMR
1H, Ar), 7.03–6.98 (m, 1H, Ar), 4.28 (t, J = 6.6 Hz, 2H, CH2), 2.24 (s, 3H, CH3), 1.72–1.64 (m, 2H, CH2),
0.91 (t, J = 7.4 Hz, 3H, CH3); 13C-NMR
165.09, 162.07, 160.31 (d, J = 240.5 Hz), 146.48, 145.64, 145.20,
δ 10.34 (s, 1H, CONH), 8.96 (s, 2H, pyr.), 7.53–7.49 (m, 1H, Ar), 7.33–7.28 (m,
δ
144.59, 136.76 (d, J = 10.7 Hz), 131.77 (d, J = 9.0 Hz), 127.50 (d, J = 3.2 Hz), 112.38 (d, J = 20.7 Hz), 111.04
(d, J = 24.7 Hz), 67.47, 21.49, 17.06, 10.38; Elemental analysis: calc. for C16H16FN3O3 (MW 317.32):
60.56% C, 5.08% H, 13.24% N; found 60.14% C, 5.01% H, 12.82% N.
Propyl 3-[(2-chloro-5-methylphenyl)carbamoyl]pyrazine-2-carboxylate (13a). White solid. Yield 53%; m.p.
110.9–111.4 ◦C; IR (cm−1): 3323 (N-H, CONH), 2974 (C-H), 1736 (C=O, COO), 1697 (C=O, CONH),
1141, 1087 (C-O, COO); 1H-NMR
δ
10.21 (s, 1H, CONH), 8.82 (d, J = 2.4 Hz, 1H, pyr.), 8.73 (d, J = 2.4 Hz
1H, pyr.), 8.42–8.40 (m, 1H, Ar), 7.32–7.26 (m, 1H, Ar), 6.94–6.90 (m, 1H, Ar), 4.49 (t, J = 6.8 Hz, 2H,
CH2), 2.38 (s, 3H, CH3), 1.90–1.82 (m, 2H, CH2), 1.04 (t, J = 7.4 Hz, 3H, CH3); 13C-NMR
165.79, 159.54,
148.23, 146.28, 143.18, 141.68, 138.03, 133.47, 128.72, 126.10, 121.84, 120.47, 68.17, 21.78, 21.28, 10.30;
,
δ

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Elemental analysis: calc. for C16H16ClN3O3 (MW 333.77): 57.58% C, 4.83% H, 12.59% N; found 57.72%
C, 4.87% H, 12.62% N.
Propyl 3-[(5-chloro-2-hydroxyphenyl)carbamoyl]pyrazine-2-carboxylate (14a). Beige solid. Yield 43%; m.p.
196.6–199.2 ◦C; IR (cm−1): 3369 (N-H, CONH), 2983 (C-H), 1689, 1542, 1339 (N-O, NO2), 1156, 1116
(C-O, COO); 1H-NMR
δ
10.70 (bs, 1H, OH), 10.18 (s, 1H, CONH), 8.97 (d, J = 2.4 Hz, 1H, pyr.), 8.95 (d,
J = 2.5 Hz, 1H, pyr.), 8.25 (d, J = 2.7 Hz, 1H, Ar), 7.07–7.04 (m, 1H, Ar), 6.95 (d, J = 8.6 Hz, 1H, Ar), 4.32
(t, J = 6.6 Hz, 2H, CH2), 1.75–1.68 (m, 2H, CH2), 0.93 (t, J = 7.4 Hz, 3H, CH3); 13C-NMR
165.34, 160.33,
δ
147.13, 146.43, 146.09, 144.94, 141.99, 126.78, 124.50, 122.65, 119.33, 116.22, 67.48, 21.50, 10.40; Elemental
analysis: calc. for C15H14ClN3O4 (MW 335.74): 53.66% C, 4.20% H, 12.52% N; found 53.24% C, 3.85%
H, 12.39% N.
Propy◦l 3-[(2-hydroxy-5-nitrophenyl)carbamoyl]pyrazine-2-carboxylate (15a). Beige solid. Yield 58%; m.p.
171.4 C decomposition; IR (cm−1): 3324 (N-H, CONH), 3208 (O-H, OH), 2968 (C-H), 1727 (C=O, COO),
1678 (C=O, CONH), 1094, 1071 (C-O, COO); 1H-NMR
δ 12.16 (bs, 1H, OH), 10.29 (s, 1H, CONH), 9.12
(d, J = 2.9 Hz, 1H, Ar), 8.99 (d, J = 2.4 Hz, 1H, pyr.), 8.96 (d, J = 2.4 Hz, 1H, pyr.), 8.01–7.97 (m, 1H, Ar),
7.11 (d, J = 9.0 Hz, 1H, Ar), 4.33 (t, J = 6.6 Hz, 2H, CH2), 1.77–1.67 (m, 2H, CH2), 0.94 (t, J = 7.4 Hz, 3H,
CH3); 13C-NMR
δ 165.23, 160.90, 153.75, 147.20, 146.22, 145.06, 142.17, 139.44, 125.87, 121.63, 115.21,
114.81, 67.55, 21.52, 10.41; Elemental analysis: calc. for C15H14N4O6 (MW 346.30): 52.03% C, 4.08% H,
16.18% N; found 51.60% C, 4.31% H, 16.60% N.
Propyl 3-{[3-(trifluoromethyl)phenyl]carbamoyl}pyrazine-2-carboxylate (17a). White solid. Yield 53%; m.p.
80.7–83.3 ◦C; IR (cm−1): 3347 (N-H, CONH), 2974 (C-H), 1745 (C=O, COO), 1697 (C=O, CONH), 1148,
1105 (C-O, COO); 1H-NMR
δ
9.69 (s, 1H, CONH), 8.82 (d, J = 2.4 Hz, 1H, pyr.), 8.68 (d, J = 2.4 Hz, 1H,
pyr.), 8.02 (d, J = 1.9 Hz, 1H, Ar), 7.96–7.93 (m, 1H, Ar), 7.51 (t, J = 8.0 Hz, 1H, Ar), 7.45–7.42 (m, 1H, Ar),
4.48 (t, J = 6.8 Hz, 2H, CH2), 1.90–1.82 (m, 2H, CH2), 1.04 (t, J = 7.4 Hz, 3H, CH3); 13C-NMR
165.68,
δ
159.76, 148.33, 146.49, 142.98, 141.19, 137.48, 131.55 (q, J = 32.7 Hz), 129.70, 123.74 (q, J = 272.4 Hz),
122.90, 121.48 (q, J = 3.8 Hz), 120.49, 116.62 (q, J = 4.1 Hz), 68.27, 21.76, 10.29; Elemental analysis: calc.
for C16H14F3N3O3 (MW 353.30): 54.39% C, 3.99% H, 11.89% N; found 53.95% C, 4.03% H, 11.65% N.
Propyl 3-{[4-(trifluoromethyl)phenyl]carbamoyl}pyrazine-2-carboxylate (18a). White solid. Yield 60%; m.p.
102.0–102.9 ◦C; IR (cm−1): 3308 (N-H, CONH), 2965 (C-H), 1741 (C=O, COO), 1677 (C=O, CONH),
1110, 1070 (C-O, COO); 1H-NMR
δ
11.20 (s, 1H, CONH), 8.97 (s, 2H, pyr.), 8.01 (d, J = 8.4 Hz, 2H, Ar),
7.75 (d, J = 8.6 Hz, 2H, Ar), 4.27 (t, J = 6.6 Hz, 2H, CH2), 1.69–1.61 (m, 2H, CH2), 0.88 (t, J = 7.4 Hz
3H, CH3); 13C-NMR
165.16, 163.12, 146.70, 145.75, 145.62, 145.56, 142.25, 126.56 (q, J = 3.8 Hz),
,
δ
124.78 (q, J = 32.1 Hz), 124.72 (q, J = 271.4 Hz), 120.61, 67.82, 21.73, 10.63; Elemental analysis: calc. for
C16H14F3N3O3 (MW 353.30): 54.39% C, 3.99% H, 11.89% N; found 54.06% C, 4.05% H, 11.97% N.
Methyl 3-(phenylcarbamoyl)pyrazine-2-carboxylate (1b). Pale yellow solid. Yield 71%; m.p. 81.8–82.7 ◦C;
IR (cm−1): 3349 (N-H, CONH), 2960 (C-H), 1735 (C=O, COO), 1678 (C=O, CONH), 1142, 1087 (C-O,
COO); 1H-NMR
δ
10.83 (s, 1H, NH), 8.96–8.93 (m, 2H, pyr.), 7.81–7.76 (m, 2H, Ar), 7.40–7.35 (m, 2H,
Ar), 7.18–7.12 (m, 1H, Ar), 3.89 (s, 3H, CH3); 13C-NMR
δ
165.58, 161.96, 146.65, 146.24, 145.52, 145.15,
138.24, 128.95, 124.56, 120.52, 53.07; Elemental analysis: calc. for C13H11N3O3 (MW 257.25): 60.70% C,
4.31% H, 16.33% N; found 60.35% C, 4.55% H, 16.79% N.
Methyl 3-[(2,4-dimethoxyphenyl)carbamoyl]pyrazine-2-carboxylate (4b). Yellow solid. Yield 59%; m.p.
192.5–194.2 ◦C; IR (cm−1): 3367 (N-H, CONH), 2965 (C-H), 1740 (C=O, COO), 1676 (C=O, CONH),
1124, 1084 (C-O, COO); 1H-NMR
δ
9.99 (s, 1H, NH), 8.95 (d, J = 2.4 Hz, 1H, pyr.), 8.94 (d, J = 2.4 Hz, 1H,
pyr.), 8.06 (d, J = 8.8 Hz, 1H, Ar), 6.71 (d, J = 2.6 Hz, 1H, Ar), 6.59–6.55 (m, 1H, Ar), 3.90 (d, J = 6.9 Hz
6H, CH 3), 3.77 (s, 3H, CH 3); 13C-NMR
165.88, 159.72, 157.33, 150.75, 146.81, 146.26, 144.91, 142.39,
,
δ

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121.52, 119.60, 104.56, 99.12, 56.27, 55.56, 53.00; Elemental analysis: calc. for C15H15N3O5 (MW 317.30):
56.78% C, 4.77% H, 13.24% N; found 56.83% C, 4.56% H, 13.44% N.
Methyl 3-[(2,5-dimethylphenyl)carbamoyl]pyrazine-2-carboxylate (5b). Pale yellow solid. Yield 67%; m.p.
132.1–134.6 ◦C; IR (cm−1): 3379 (N-H, CONH), 2953 (C-H), 1735 (C=O, COO), 1683 (C=O, CONH),
1121, 1082 (C-O, COO); 1H-NMR
δ
10.26 (s, 1H, NH), 8.96–8.93 (m, 2H, pyr.), 7.38–7.35 (m, 1H, Ar),
7.15 (d, J = 7.7 Hz, 1H, Ar), 7.01–6.96 (m, 1H, Ar), 3.88 (s, 3H, CH3), 2.29 (s, 3H, CH3), 2.21 (s, 3H, CH3);
13C-NMR
165.65, 161.75, 146.33, 145.63, 145.19, 144.69, 135.44, 135.29, 130.39, 129.19, 126.81, 125.52,
δ
53.02, 20.77, 17.34; Elemental analysis: calc. for C15H15N3O3 (MW 285.30): 63.15% C, 5.30% H, 14.73%
N; found 62.71% C, 5.49% H, 14.92% N.
Methyl 3-[(4-ethylphenyl)carbamoyl]pyrazine-2-carboxylate (6b). Pale brown solid. Yield 58%; m.p.
◦
89.1–90.7 C; IR (cm−1): 3356 (N-H, CONH), 2962 (C-H), 1737 (C=O, COO), 1686 (C=O, CONH),
1
1147, 1083 (C-O, COO); H-NMR
δ 10.76 (s, 1H, NH), 8.95–8.92 (m, 2H, pyr.), 7.72–7.67 (m, 2H,
Ar), 7.23–7.18 (m, 2H, Ar), 3.88 (s, 3H, CH3), 2.58 (q, J = 7.6 Hz, 2H, CH2), 1.19–1.14 (m, 3H, CH3);
13C-NMR
165.63, 161.69, 146.19, 145.62, 145.12, 145.08, 140.07, 135.92, 128.14, 120.59, 53.04, 27.84,
δ
15.84; Elemental analysis: calc. for C15H15N3O3 (MW 285.30): 63.15% C, 5.30% H, 14.73% N; found
62.88% C, 5.23% H, 14.92% N.
Methyl 3-[(2,4-difluorophenyl)carbamoyl]pyrazine-2-carboxylate (8b). White solid. Yield 72%; m.p.
165.9–167.4 ◦C; IR (cm−1): 3348 (N-H, CONH), 2965 (C-H), 1739 (C=O, COO), 1693 (C=O, CONH),
1143, 1103, 1082 (C-O, COO); 1H-NMR
δ
10.63 (bs, 1H, NH), 8.96 (s, 2H, pyr.), 7.82–7.74 (m, 1H, Ar),
7.43–7.36 (m, 1H, Ar), 7.18–7.11 (m, 1H, Ar), 3.87 (s, 3H, CH3); 13C-NMR
δ
165.5, 162.2, 159.7 (dd,
J = 245.1 Hz, J = 13.7 Hz), 155.4 (dd, J = 249.9 Hz, J = 13.5 Hz), 146.7, 145.5, 143.4, 144.1, 127.2 (dd,
J = 9.6 Hz, J = 1.9 Hz), 121.7 (dd, J = 12.4 Hz, J = 3.9 Hz), 111.6 (dd, J = 22.0 Hz, J = 3.9 Hz), 104.7 (dd,
J = 26.6 Hz, J = 23.8 Hz), 53.1; Elemental analysis: calc. for C13H9F2N3O3 (MW 293.23): 53.25% C,
3.09% H, 14.33% N; found 53.36% C, 2.76% H, 14.53% N.
Methyl 3-[(4-bromophenyl)carbamoyl]pyrazine-2-carboxylate (11b). White solid. Yield 66%; m.p.
128.5–130.4 ◦C; IR (cm−1): 3326 (N-H, CONH), 2956 (C-H), 1742 (C=O, COO), 1686 (C=O, CONH),
1174, 1071 (C-O, COO); 1H-NMR
δ
10.99 (s, 1H, NH), 8.96–8.94 (m, 2H, pyr.), 7.80–7.76 (m, 2H, Ar),
7.58–7.55 (m, 2H, Ar), 3.88 (s, 3H, CH 3); 13C-NMR
δ
165.51, 162.07, 146.64, 146.39, 145.18, 144.87,
137.64, 131.79, 122.45, 116.39, 53.10; Elemental analysis: calc. for C13H10BrN3O3 (MW 336.15): 46.45%
C, 3.00% H, 12.50% N; found 46.11% C, 2.76% H, 12.19% N.
Methyl 3-[(5-fluoro-2-methylphenyl)carbamoyl]pyrazine-2-carboxylate (12b). White solid. Yield 76%; m.p.
174.2–176.9 ◦C; IR (cm−1): 3375 (N-H, CONH), 2955 (C-H), 1741 (C=O, COO), 1692 (C=O, CONH),
1142, 1112 (C-O, COO); 1H-NMR
δ
10.36 (bs, 1H, NH), 8.97–8.95 (m, 2H, pyr.), 7.49 (dd, 1H, J = 9.7 Hz,
J = 2.9 Hz, Ar), 7.33–7.28 (1H, m, Ar), 7.01 (dt, 1H, J = 9.7 Hz J = 2.9 Hz, Ar), 3.89 (s, 3H, CH3), 2.24 (s,
3H, CH3); 13C-NMR
165.5, 162.0, 160.3 (d, J = 240.3 Hz), 146.5, 145.3, 144.5, 144.1, 136.7 (d, J = 10.4
δ
Hz), 131.8 (d, J = 9.4 Hz), 127.7 (d, J = 2.9 Hz), 112.5 (d, J = 21.0 Hz), 111.2 (d, J = 24.8 Hz), 53.1, 17.0;
Elemental analysis: calc. for C14H12FN3O3 (MW 289.27): 58.13% C, 4.18% H, 14.53% N; found 57.74%
C, 3.97% H, 14.26% N.
Methyl 3-[(2-chloro-5-methylphenyl)carbamoyl]pyrazine-2-carboxylate (13b). Pale yellow solid. Yield 74%;
m.p. 166.7–168.8 C; IR (cm−1): 3349 (N-H, CONH), 2956 (C-H), 1737 (C=O, COO), 1689 (C=O, CONH),
◦
1144, 1088 (C-O, COO); 1H-NMR
δ 10.43 (s, 1H, NH), 8.99–8.97 (m, 2H, pyr.), 7.86 (d, J = 2.0 Hz, 1H,
Ar), 7.44 (d, J = 8.1 Hz, 1H, Ar), 7.12–7.05 (m, 1H, Ar), 3.91 (s, 3H, CH3), 2.33 (s, 3H, CH3); 13C-NMR
δ
165.63, 161.15, 147.02, 146.00, 145.19, 142.85, 137.73, 133.66, 129.36, 127.60, 124.87, 123.02, 53.13, 20.83;
Elemental analysis: calc. for C14H12ClN3O3 (MW 305.72): 55.00% C, 3.96% H, 13.75% N; found 55.43%
C, 3.85% H, 13.88% N.

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Methyl 3-[(5-chloro-2-hydroxyphenyl)carbamoyl]pyrazine-2-carboxylate (14b). Yellow solid. Yield 72%; m.p.
174.2–176.9 ◦C; IR (cm−1): 3316 (N-H, CONH), 2963 (C-H), 1735 (C=O, COO), 1681 (C=O, CONH),
1148, 1091 (C-O, COO); 1H-NMR
δ
10.71 (s, 1H, OH), 10.19 (s, 1H, NH), 8.98 (d, J = 2.4 Hz, 1H, pyr.),
8.96 (d, J = 2.5 Hz, 1H, pyr.), 8.25 (d, J = 2.5 Hz, 1H, Ar), 7.09–7.03 (m, 1H, Ar), 6.95 (d, 1H, Ar), 3.92 (d,
J = 9.0 Hz, 3H, CH3); 13C-NMR
165.80, 160.17, 147.20, 146.66, 146.10, 145.02, 141.77, 126.72, 124.55,
δ
122.69, 119.34, 116.23, 53.13; Elemental analysis: calc. for C13H10ClN3O4 (MW 307.69): 50.75% C, 3.28%
H, 13.66% N; found 50.39% C, 3.19% H, 13.72% N.
Methyl 3-[(2-hydr◦oxy-5-nitrophenyl)carbamoyl]pyrazine-2-carboxylate (15b). Pale yellow solid. Yield 60%;
m.p. 226.4–228.6 C; IR (cm−1): 3317 (N-H, CONH), 2969 (C-H), 1732 (C=O, COO), 1683 (C=O, CONH),
1267, 1095 (C-O, COO); 1H-NMR
δ
12.10 (bs, 1H, OH), 10.29 (s, 1H, NH), 9.10 (d, J = 2.9 Hz, 1H, Ar),
8.99 (d, J = 2.4 Hz, 1H, pyr.), 8.97 (d, J = 2.4 Hz, 1H, pyr.), 8.01–7.94 (m, 1H, Ar), 7.10 (d, J = 9.0 Hz, 1H,
Ar), 3.95 (s, 3H, CH3); 13C-NMR
165.71, 160.66, 153.69, 147.28, 146.16, 145.12, 141.83, 139.46, 125.81,
δ
121.65, 115.15, 114.79, 53.19; Elemental analysis: calc. for C13H10N4O6 (MW 318.25): 49.06% C, 3.17%
H, 17.61% N; found 48.76% C, 3.05% H, 17.52% N.
Methyl 3-{[3-(trifluoromethyl)phenyl]carbamoyl}pyrazine-2-carboxylate (17b). Yellow solid. Yield 52%; m.p.
◦
106.3–108.5 C; IR (cm−1): 3357 (N-H, CONH), 2919 (C-H), 1748 (C=O, COO), 1693 (C=O, CONH), 1110,
1081 (C-O, COO); 1H-NMR
pyr.), 8.08 (s, 1H, Ar), 7.93 (d, 1H, J = 8.0 Hz, Ar), 7.51 (t, 1H, J = 8.0 Hz, Ar), 7.43 (d, 1H, J = 8.0 Hz, Ar),
4.10 (s, 3H, CH3); 13C-NMR
166.0, 159.8, 148.0, 146.5, 145.5, 143.1, 137.4, 131.6 (q, J = 33.3 Hz), 129.7,
δ 9.71 (bs, 1H, NH), 8.82 (d, 1H, J = 2.2 Hz, pyr.), 8.69 (d, 1H, J = 2.2 Hz,
δ
123.0, 123.7 (q, J = 271.9 Hz), 121.6 (q, J = 3.8 Hz), 116.7 (q, J = 3.9 Hz), 53.4.
Methyl 3-{[4◦-(trifluoromethyl)phenyl]carbamoyl}pyrazine-2-carboxylate (18b). White solid. Yield 50%; m.p.
146.8–148.2 C; IR (cm−1): 3297 (N-H, CONH), 2961 (C-H), 1746 (C=O, COO), 1691 (C=O, CONH), 1112,
1063 (C-O, COO); 1H-NMR
δ
9.73 (bs, 1H, NH), 8.82 (d, 1H, J = 2.2 Hz, pyr.), 8.69 (d, 1H, J = 2.2 Hz
,
pyr.), 7.87–7.84 (m, 2H, Ar), 7.65–7.62 (m, 2H, Ar), 4.09 (s, 3H, CH3); 13C-NMR
δ
166.0, 159.8, 148.0,
146.5, 145.5, 143.1, 141.2, 126.8 (q, J = 33.8 Hz), 126.4 (q, J = 3.8 Hz), 123.9 (q, J = 270.9 Hz), 119.7, 53.4;
Elemental analysis: calc. for C14H10F3N3O3 (MW 325.25): 51.70% C, 3.10% H, 12.92% N; found 51.95%
C, 3.41% H, 12.83% N.
4. Conclusions
In this research project, we focused on the synthesis of 18 POA derivatives with increased
lipophilicity achieved by substitution with phenylcarbamoyl moiety in position 3 of the pyrazine
ring. Furthermore, we prepared 17 propyl esters and 12 methyl esters as prodrugs to further increase
the lipophilicity and enhance the penetration of these derivatives through the mycobacterial cell
wall. The recently uncovered antimycobacterial target, DprE1, was evaluated as potential target for
our new series of POA derivatives, on the basis of structural similarities with previously published
2-carboxyquinoxalines (Figure 2). The results from a molecular docking study to DprE1 showed that
the alteration of -NH-CH2- linker of the original scaffold to -CONH- group (title compounds of this
study) did not alter the position of the scaffold in DprE1.
Prepared compounds were tested for in vitro growth inhibition activity against M. tuberculosis
H37Rv and three nontuberculous strains. Five compounds showed moderate activity against Mtb
(MIC = 50 or 100 µg·mL−1) and compound 16 exerted high efficiency with MIC = 1.56 µg·mL−1. The
whole cell activity of 16 (MIC = 5.0
µM) was fully comparable with the activity of the best template
2-carboxyquinoxalines (Figure 1, MIC = 3.1
µM). With respect to our docking and biological results and
the activities of 2-carboxyquinoxalines published previously (Figure 2), a bulky lipophilic substituent
on the pyrazine ring, complimentary to the lipophilic cavity, is suggested to be important for the
inhibition of DprE1. The lowered antimycobacterial activity of our compounds in whole cell assay
could be caused by insufficient penetration through the cell wall (insufficient lipophilicity), low intrinsic

Molecules 2017, 22, 1491
15 of 17
affinity to the enzyme, or both. The most lipophilic compound from our series was propyl ester 10a
with log P = 2.77, which is significantly lower in comparison with the model 2-carboxyquinoxaline
derivative (log P = 4.05).
The increase of lipophilicity by the conversion to propyl and methyl esters did not produce
any improvement in biological activity in general. Only propyl ester 18a showed antimycobacterial
activity against Mtb with MIC = 3.13
µg
mL−1, a value similar to its parent acid 18. Antibacterial and
·
antifungal assays did not reveal any compound with significant activity. As next research, we will
focus on the design of compounds with lipophilic/bulky substituents at C-5 or C-6 of the pyrazine
core as an attempt to fill in the lipophilic cavity of DprE1.
Supplementary Materials: Supplementary Materials are available online. Docking study and methodologies of
biological assays.
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: M.D. set the topic and designed the study. J.Z. designed the study and assisted to the
molecular docking studies. L.S. designed the structures, evaluated the biological activity, and wrote the paper.
L.S., P.J., C.F. and G.B. synthetized the compounds. O.J. and P.P. performed the antimycobacterial assays. K.K.
performed the antibacterial and antifungal assays. L.N. conceived and performed the cytotoxicity. J.K. interpreted
the NMR spectra.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Singh, P.; Mishra, A.K.; Malonia, S.K.; Chauhan, D.S.; Sharma, V.D.; Venkatesan, K.; Katoch, V.M. The paradox
of pyrazinamide: An update on the molecular mechanisms of pyrazinamide resistance in Mycobacteria.
J. Commun. Dis. 2006, 38, 288–298. [PubMed]
2.
3.
World Health Organization. Global Tuberculosis Report 2016; WHA68/2015/REC/1; World Health
Organization: Geneva, Switzerland, 2016.
Heifets, L.; Lindholm-Levy, P. Pyrazinamide sterilizing activity in vitro against semidormant
Mycobacterium tuberculosis bacterial populations. Am. Rev. Respir. Dis. 1992, 145, 1223–1225. [CrossRef]
[PubMed]
4.
5.
6.
Stehr, M.; Elamin, A.A.; Singh, M. Filling the pipeline—New drugs for an old disease. Curr. Top. Med. Chem.
2014, 14, 110–129. [CrossRef] [PubMed]
Zhang, Y.; Mitchison, D. The curious characteristics of pyrazinamide: A review. Int. J. Tuberc. Lung Dis. 2003
7, 6–21. [PubMed]
,
Zhang, Y.; Wade, M.M.; Scorpio, A.; Zhang, H.; Sun, Z. Mode of action of pyrazinamide: Disruption of
Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic acid. J. Antimicrob. Chemother.
2003, 52, 790–795. [CrossRef] [PubMed]
7.
8.
9.
Peterson, N.D.; Rosen, B.C.; Dillon, N.A.; Baughn, A.D. Uncoupling environmental pH and intrabacterial
acidification from pyrazinamide susceptibility in Mycobacterium tuberculosis. Antimicrob. Agents Chemother.
2015, 59, 7320–7326. [CrossRef] [PubMed]
Sayahi, H.; Pugliese, K.M.; Zimhony, O.; Jacobs, W.R., Jr.; Shekhtman, A.; Welch, J.T. Analogs of the
antituberculous agent pyrazinamide are competitive inhibitors of NADPH binding to M. tuberculosis fatty
acid synthase I. Chem. Biodivers. 2012, 9, 2582–2596. [CrossRef] [PubMed]
Shi, W.; Chen, J.; Feng, J.; Cui, P.; Zhang, S.; Weng, X.; Zhang, W.; Zhang, Y. Aspartate decarboxylase (PanD)
as a new target of pyrazinamide in Mycobacterium tuberculosis. Emerg. Microbes Infect. 2014, 3, e58. [CrossRef]
[PubMed]
10. Dillon, N.A.; Peterson, N.D.; Rosen, B.C.; Baughn, A.D. Pantothenate and pantetheine antagonize the
antitubercular activity of pyrazinamide. Antimicrob. Agents Chemother. 2014, 58, 7258–7263. [CrossRef]
[PubMed]
11. Shi, W.; Zhang, X.; Jiang, X.; Yuan, H.; Lee, J.S.; Barry, C.E., III; Wang, H.; Zhang, W.; Zhang, Y. Pyrazinamide
inhibits trans-translation in Mycobacterium tuberculosis. Science 2011, 333, 1630–1632. [CrossRef] [PubMed]
12. Keiler, K.C. Biology of trans-translation. Annu. Rev. Microbiol. 2008, 62, 133–151. [CrossRef] [PubMed]

Molecules 2017, 22, 1491
16 of 17
13. Yang, J.; Liu, Y.; Bi, J.; Cai, Q.; Liao, X.; Li, W.; Guo, C.; Zhang, Q.; Lin, T.; Zhao, Y.; et al. Structural basis for
targeting the ribosomal protein S1 of Mycobacterium tuberculosis by pyrazinamide. Mol. Microbiol. 2015, 95,
791–803. [CrossRef] [PubMed]
14. Salah, P.; Bisaglia, M.; Aliprandi, P.; Uzan, M.; Sizun, C.; Bontems, F. Probing the relationship between
Gram-negative and Gram-positive S1 proteins by sequence analysis. Nucleic Acids Res. 2009, 37, 5578–5588.
[CrossRef] [PubMed]
15. Dolezal, M.; Zitko, J.; Kesetovicova, D.; Kunes, J.; Svobodova, M. Substituted N-phenylpyrazine-2-
carboxamides: Synthesis and antimycobacterial evaluation. Molecules 2009, 14, 4180–4189. [CrossRef]
[PubMed]
16. Servusova, B.; Vobickova, J.; Paterova, P.; Kubicek, V.; Kunes, J.; Dolezal, M.; Zitko, J. Synthesis and
antimycobacterial evaluation of N-substituted 5-chloropyrazine-2-carboxamides. Bioorg. Med. Chem. Lett.
2013, 23, 3589–3591. [CrossRef] [PubMed]
17. Zitko, J.; Franco, F.; Paterova, P. Synthesis and anti-infective evaluation of 5-amino-N-phenylpyrazine-2
-carboxamides. Ceska Slov. Farm. 2015, 64, 19–24. [PubMed]
18. Neres, J.; Hartkoorn, R.C.; Chiarelli, L.R.; Gadupudi, R.; Pasca, M.R.; Mori, G.; Venturelli, A.; Savina, S.;
Makarov, V.; Kolly, G.S.; et al. 2-Carboxyquinoxalines kill Mycobacterium tuberculosis through noncovalent
inhibition of DprE1. ACS Chem. Biol. 2015, 10, 705–714. [CrossRef] [PubMed]
19. Riccardi, G.; Pasca, M.R.; Chiarelli, L.R.; Manina, G.; Mattevi, A.; Binda, C. The DprE1 enzyme, one of
the most vulnerable targets of Mycobacterium tuberculosis. Appl. Microbiol. Biotechnol. 2013, 97, 8841–8848.
[CrossRef] [PubMed]
20. Wolucka, B.A. Biosynthesis of D-arabinose in mycobacteria—A novel bacterial pathway with implications
for antimycobacterial therapy. FEBS J. 2008, 275, 2691–2711. [CrossRef] [PubMed]
21. Makarov, V.; Manina, G.; Mikusova, K.; Mollmann, U.; Ryabova, O.; Saint-Joanis, B.; Dhar, N.; Pasca, M.R.;
Buroni, S.; Lucarelli, A.P.; et al. Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan
synthesis. Science 2009, 324, 801–804. [CrossRef] [PubMed]
22. Shirude, P.S.; Shandil, R.; Sadler, C.; Naik, M.; Hosagrahara, V.; Hameed, S.; Shinde, V.; Bathula, C.;
Humnabadkar, V.; Kumar, N.; et al. Azaindoles: Noncovalent DprE1 inhibitors from scaffold morphing
efforts, kill Mycobacterium tuberculosis and are efficacious in vivo. J. Med. Chem. 2013, 56, 9701–9708.
[CrossRef] [PubMed]
23. Naik, M.; Humnabadkar, V.; Tantry, S.J.; Panda, M.; Narayan, A.; Guptha, S.; Panduga, V.; Manjrekar, P.;
Jena, L.K.; Koushik, K.; et al. 4-Aminoquinolone piperidine amides: Noncovalent inhibitors of DprE1 with
long residence time and potent antimycobacterial activity. J. Med. Chem. 2014, 57, 5419–5434. [CrossRef]
[PubMed]
24. Panda, M.; Ramachandran, S.; Ramachandran, V.; Shirude, P.S.; Humnabadkar, V.; Nagalapur, K.; Sharma, S.;
Kaur, P.; Guptha, S.; Narayan, A.; et al. Discovery of pyrazolopyridones as a novel class of noncovalent
DprE1 inhibitor with potent anti-mycobacterial activity. J. Med. Chem. 2014, 57, 4761–4771. [CrossRef]
[PubMed]
25. Makarov, V.; Neres, J.; Hartkoorn, R.C.; Ryabova, O.B.; Kazakova, E.; Sarkan, M.; Huszar, S.; Piton, J.;
Kolly, G.S.; Vocat, A.; et al. The 8-pyrrole-benzothiazinones are noncovalent inhibitors of DprE1 from
Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 2015, 59, 4446–4452. [CrossRef] [PubMed]
26. Chikhale, R.; Menghani, S.; Babu, R.; Bansode, R.; Bhargavi, G.; Karodia, N.; Rajasekharan, M.V.;
Paradkar, A.; Khedekar, P. Development of selective DprE1 inhibitors: Design, synthesis, crystal structure
and antitubercular activity of benzothiazolylpyrimidine-5-carboxamides. Eur. J. Med. Chem. 2015, 96, 30–46.
[CrossRef] [PubMed]
27. Saudi, M.; Zmurko, J.; Kaptein, S.; Rozenski, J.; Neyts, J.; Van Aerschot, A. Synthesis and evaluation of
imidazole-4,5-and pyrazine-2,3-dicarboxamides targeting dengue and yellow fever virus. Eur. J. Med. Chem.
2014, 87, 529–539. [CrossRef] [PubMed]
28. Holzer, W.; Eller, G.A.; Datterl, B.; Habicht, D. Derivatives of pyrazinecarboxylic acid: H-1, C-13 and N-15
NMR spectroscopic investigations. Magn. Reson. Chem. 2009, 47, 617–624. [CrossRef] [PubMed]
29. Salfinger, M.; Heifets, L.B. Determination of Pyrazinamide MICs for Mycobacterium-tuberculosis at different
pHs by the radiometric method. Antimicrob. Agents Chemother. 1988, 32, 1002–1004. [CrossRef] [PubMed]
30. Zhang, Y.; Permar, S.; Sun, Z.H. Conditions that may affect the results of susceptibility testing of
Mycobacterium tuberculosis to pyrazinamide. J. Med. Microbiol. 2002, 51, 42–49. [CrossRef] [PubMed]

Molecules 2017, 22, 1491
17 of 17
31. Vaughan, G.B.; Rose, J.C.; Brown, G.P. Polyimides Based on Pyrazinetetracarboxylic dianhydride and some
related model compounds. J. Polym. Sci. A1 1971, 9, 1117–1138. [CrossRef]
32. Roehrig, S.; Jeske, M.; Akbaba, M.; Rosentreter, U.; Boyer, S.; Fischer, K.; Pohlmann, J.; Tuch, A.; Perzborn, E.;
Gerdes, C.; et al. Pyrazine Dicarboxamides and the Use Thereof. Patent WO2006061116 (A1), 15 June 2006.
33. Mackerell, A., Jr.; Zhang, H.; Osterman, A.; Kolhatkar, R. Targeting NAD Biosynthesis in Bacterial Pathogens.
Patent WO2011006158 (A2), 13 January 2011.
34. Lui, Y.; Li, J.; Bi, K.; Liu, L. Preparing Method for N-substituted Pyrrolo [3,4-B] pyrazine-5,7(6H)-diketone.
Patent CN106220630 (A), 14 December 2016.
35. Leban, J.; Kramer, B.; Saeb, W.; Garcia, G. Novel Compounds as Anti-Inflammatory, Immunomodulatory
and Anti-Proliferatory Agents. U.S. Patent 2003203951 (A1), 23 January 2003.
Sample Availability: Samples of the compounds are available from the authors.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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