Ureidopyrazine derivatives: Synthesis and biological evaluation as Anti-infectives and abiotic elicitors

Article abstract of DOI:10.3390/molecules22101797

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) has become a frequently deadly infection due to increasing antimicrobial resistance. This serious issue has driven efforts worldwide to discover new drugs effective against Mtb. One research area is the synthesis and evaluation of pyrazinamide derivatives as potential anti-TB drugs. In this paper we report the synthesis and biological evaluations of a series of ureidopyrazines. Compounds were synthesized by reacting alkyl/aryl isocyanates with aminopyrazine or with propyl 5-aminopyrazine-2-carboxylate. Reactions were performed in pressurized vials using a CEM Discover microwave reactor with a focused field. Purity and chemical structures of products were assessed, and the final compounds were tested in vitro for their antimycobacterial, antibacterial, and antifungal activities. Propyl 5-(3-phenylureido)pyrazine-2-carboxylate (compound 4, MIC_Mtb = 1.56 μg/mL, 5.19 μM) and propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (compound 6, MIC_Mtb = 6.25 μg/mL, 18.91 μM) had high antimycobacterial activity against Mtb H37Rv with no in vitro cytotoxicity on HepG2 cell line. Therefore 4 and 6 are suitable for further structural modifications that might improve their biological activity and physicochemical properties. Based on the structural similarity to 1-(2-chloropyridin-4-yl)-3-phenylurea, a known plant growth regulator, two selected compounds were evaluated for similar activity as abiotic elicitors.

Full text of DOI:10.3390/molecules22101797

molecules
Article
Ureidopyrazine Derivatives: Synthesis and Biological
Evaluation as Anti-Infectives and Abiotic Elicitors
Ghada Bouz * ID , Martin Juhás, Pavlína Niklová, Ondrˇej Jand’ourek, Pavla Paterová,
ID
Jirˇí Janoušek, Lenka Tu˚mová, Zuzana Kovalíková, Petr Kastner, Martin Doležal
and Jan Zitko *
ID
Faculty of Pharmacy in Hradec Kralove, Charles University, Heyrovskeho 1203,
Hradec Kralove 50005, Czech Republic; juhasm@faf.cuni.cz (M.J.); niklovp@faf.cuni.cz (P.N.);
jando6aa@faf.cuni.cz (O.J.); pavla.paterova@fnhk.cz (P.P.); janousj2@faf.cuni.cz (J.J.); tumova@faf.cuni.cz (L.T.);
kovalikz@faf.cuni.cz (Z.K.); kastner@faf.cuni.cz (P.K.); dolezalm@faf.cuni.cz (M.D.)
* Correspondence: bouzg@faf.cuni.cz (G.B.); jan.zitko@faf.cuni.cz (J.Z.); Tel.: +420-495-067-275 (G.B.);
+420-495-067-272 (J.Z.)
Received: 29 September 2017; Accepted: 20 October 2017; Published: 23 October 2017
Abstract: Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) has become a frequently
deadly infection due to increasing antimicrobial resistance. This serious issue has driven efforts
worldwide to discover new drugs effective against Mtb. One research area is the synthesis and
evaluation of pyrazinamide derivatives as potential anti-TB drugs. In this paper we report the
synthesis and biological evaluations of a series of ureidopyrazines. Compounds were synthesized by
reacting alkyl/aryl isocyanates with aminopyrazine or with propyl 5-aminopyrazine-2-carboxylate.
Reactions were performed in pressurized vials using a CEM Discover microwave reactor with a
focused field. Purity and chemical structures of products were assessed, and the final compounds
were tested in vitro for their antimycobacterial, antibacterial, and antifungal activities. Propyl 5-
(3-phenylureido)pyrazine-2-carboxylate (compound
5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (compound
had high antimycobacterial activity against Mtb H37Rv with no in vitro cytotoxicity on HepG2
cell line. Therefore and are suitable for further structural modifications that might improve
4, MICMtb = 1.56
µg/mL, 5.19
µM) and propyl
6, MICMtb = 6.25
µ
g/mL, 18.91 M)
µ
4
6
their biological activity and physicochemical properties. Based on the structural similarity to
1-(2-chloropyridin-4-yl)-3-phenylurea, a known plant growth regulator, two selected compounds
were evaluated for similar activity as abiotic elicitors.
Keywords: abiotic elicitors; anti-infectives; callus culture; ester; Mycobacterium tuberculosis; pyrazinoic
acid; ureidopyrazine
1. Introduction
Tuberculosis (TB) is a common infection that had been successfully treated with appropriate first
line anti-TB drugs, including isoniazid (INH), rifampicin, pyrazinamide (PZA), and ethambutol [
Yet in the last few years, this curable infection has become a frequently deadly illness due to the
raising issue of antimicrobial resistance (AMR) [ ]. AMR includes multi-drug resistant TB (MDR-TB),
1].
1
when the bacteria is resistant to both INH and rifampicin, and extensively drug resistant TB (XDR-TB),
when the bacteria is resistant to INH, rifampicin, fluoroquinolones, and one of the three parenteral
second line drugs (amikacin, kanamycin, or capreomycin) [
of deaths due to TB has fallen from the year 2000 till today, TB remains one of the top ten causes of
death worldwide and the leading cause of death from infectious diseases [ ]. According to the World
Health Organization (WHO) annual report, there were 10.4 million new cases of TB worldwide in
2]. Despite the fact that the annual number
3
2015, out of which 480,000 cases were MDR-TB [
3]. The number of deaths attributed to TB in that year
Molecules 2017, 22, 1797; doi:10.3390/molecules22101797
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1797
2 of 16
was 1.4 million, whether due to inaccessibility to treatment or treatment failure [
2
]. It is estimated
that by 2050 deaths due to AMR will reach 10 million deaths/year in comparison to 8.2 million
deaths/year due to all types of cancer combined if no measures are implemented to stop resistance
spread [4]. In 2015 alone, the number of deaths of TB due to AMR was 250,000 [3]. The serious
issue of AMR has driven efforts worldwide to find new therapeutic drugs Mycobacterium tuberculosis
(Mtb) bacteria are sensitive to. An ongoing research area is the synthesis and evaluation of PZA
derivatives as potential anti-TB drugs [5–10]. PZA is considered to be an analogue of nicotinamide and
its chemical structure is closely related to INH (Figure 1). PZA plays an important role in conventional
(drug-sensitive) TB treatment regimen as it shortens the duration of therapy from 9–12 months to
6 months [11]. It is converted to its active form, pyrazinoic acid (POA, Figure 1), intracellularly in
acidic pH by a hydrolytic enzyme known as pyrazinamidase (PZase) encoded by the Mtb bacterium
itself [12]. Several new specific mechanisms were identified by which PZA or POA exerts its anti-TB
activity. Those include interference with ribosomal protein S1 (RpsA) [12], inhibition of quinolinic acid
phosphoribosyl transferase (QAPRTase) [13], inhibition of aspartate decarboxylase [14], and inhibition
of Fatty acid synthase I (FAS I) [15,16]. The knowledge of such new targets will help to design new
potentially active PZA derivatives that may overcome the issue of AMR.
Figure 1. The chemical structure of (a) INH; (b) nicotinamide; (c) PZA; and POA (d).
In this paper, we focus on the design, synthesis, and anti-infective evaluation of ureidopyrazine
derivatives. In general, urea derivatives have shown wide range of pharmacological activities,
including hypoglycemic, anti-cancer, anticonvulsant, antiviral, and antimicrobial activities, as reviewed
elsewhere [17]. For instance, N-alkylurea hydroxamic acids showed potent antibacterial activity against
both Gram-positive and Gram-negative bacteria by inhibiting peptide deformylase (PDF) that is essential
for bacterial growth [18]. Furthermore, one compound from this series (compound 19) has been already
proved to be a Chk-1 kinase inhibitor with anticancer activity [19], yet have not been evaluated for
any anti-infective property. Title compounds of this study were evaluated for their antimycobacterial,
antibacterial, and antifungal activities in vitro. Two of the prepared compounds,
8 and 18, were further
assessed as plant abiotic elicitors since similar pyrazinecarboxylic acid derivatives were proved to exert
such activity. The chemical structures of the latter two compounds also resemble that of a commercially
available plant growth promotor, 1-(2-chloropyridin-4-yl)-3-phenylurea (for its structure readers may
refer to Section 2.2.5).
2. Results and Discussion
2.1. Chemistry
2.1.1. Compounds 1–9
The starting 5-aminopyrazine-2-carboxylic acid (1) was prepared by reacting 5-chloropyrazine-
2-carboxylic acid with an aqueous solution of ammonia to replace the chlorine atom with amino
group (Scheme 1, step a). Then, it was esterified (Fischer esterification) with propanol in the presence
of catalytic amounts of concentrated sulfuric acid to yield propyl 5-aminopyrazine-2-carboxylate
(2
) (Scheme 1, step b). The obtained ester was then reacted with six different aromatic substituted
isocyanates in hexane (Scheme 1, step c), resulting in six different aryl substituted ureidopyrazine

Molecules 2017, 22, 1797
3 of 16
propyl esters (Group B). The esterification step preceded the introduction of the urea moiety to
the molecule in order to prevent potential unwanted side-reactions between the isocyanates and
the free carboxylic acid moiety that results in amide formation [20], and also to prevent possible
decarboxylation at the high reaction temperature. The most biologically active ester
hydrolyzed to in methanol and potassium carbonate as a base to compare the biological activity of
the ester to the corresponding free carboxylic acid.
4 was then
5
Scheme 1. Synthetic procedures of compounds
1
–
9
. Reagents and Conditions: (
a
) NH3 (25% aq.sol),
◦
◦
MW: 100 C, 30 min, 80 W; (
b
) esterification with propanol, H2SO4, MW: 100 C, 1 h, 80 W; (
c) hexane,
◦
MW: 120 C, 1 h, 80 W.
2.1.2. Compounds 10–20
Aminopyrazine was reacted with five different alkyl substituted isocyanates (Group C) and six
different substituted phenyl or benzyl isocyanates (Group D) in one step reaction, in the presence
of hexane as solvent, to yield the corresponding ureidopyrazine derivatives lacking the ester
moiety of group B compounds (Scheme 2). We also attempted reacting 3-chloropyrazin-2-amine
and 6-chloro-pyrazin-2-amine with different isocyanates under same conditions but no product
was detected.
◦
Scheme 2. Synthetic procedure of compounds 10–20. Reagents and Conditions: hexane, MW: 120 C, 1 h, 80 W.
All chemical reactions were performed in pressurized test vials in a CEM Discover microwave
reactor with a focused field. The over-pressurized system of microwave test tubes is essential to
achieve higher temperatures than the boiling points of solvents (propanol and hexane). Moreover,
the closed system prevents the escape of ammonia from the reaction mixture.
Final ureido derivatives were purified using flash chromatography. They were isolated as solids
compounds of white to yellow color, in yields ranging from 12% to 70% of chromatographically
pure products. Then, they were characterized by their melting points, 1H- and 13C-NMR spectra, IR
spectroscopy, and elemental analysis. The acquired analytical data fully supported the corresponding
proposed structures.

Molecules 2017, 22, 1797
4 of 16
2.2. Biological Activity
2.2.1. Antimycobacterial Activity Evaluation against Mycobacterium tuberculosis, Mycobacterium kansasii,
and Mycobacterium avium
All prepared compounds, including the starting acid and ester that lack the urea moiety,
were screened for in vitro activity against Mtb H37Rv, Mycobacterium kansasii (M. kansasii) and
Mycobacterium avium (M. avium) using a Microplate Alamar Blue Assay [21]. Antimycobacterial activity
results were expressed as minimum inhibitory concentration (MIC) in
INH as standard (Table 1).
µg
mL−1 in comparison with
·
Table 1. Structure of prepared compounds, antimycobacterial activity expressed by minimum
inhibitory concentrations (MIC), and cytotoxicity expressed by IC50 values.
A
B
C & D
Antimycobacterial Activity MIC (µg/mL)
log P
No.
R
Mtb
M. kansasii
M. avium
1
2
H
propyl
−0.75
>100
>100
>100
>100
>100
>100
A
B
0.34
3
4
benzyl
phenyl
phenyl
1.69
1.62
0.54
1.50
2.18
2.18
2.74
>100
1.56
>100
6.25
>100
25
>100
>100
>100
25
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
5 a
6
4-methoxyphenyl
2-chlorophenyl
4-chlorophenyl
3,4-dichlorophenyl
7
8
9
>100
10
11
12
13
14
propyl
butyl
pentyl
octyl
−0.28
0.13
0.55
1.8
>100
>100
100
25
>100
>100
>100
>100
25
>100
>100
>100
>100
>100
C
D
decyl
2.64
>100
15
16
17
18
19
20
benzyl
0.62
0.43
1.11
1.11
1.67
0.14
>100
>100
>100
12.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4-methoxyphenyl
2-chlorophenyl
4-chlorophenyl
3,4-dichlorophenyl
2-chlorobenzyl
>100 b
0.2–0.4
PZA
INH
−1.31
−0.64
>100
6.25–12.5
>100
6.25–12.5
a
Free acid form of compound
4
prepared by base catalyzed hydrolysis; b MIC value from testing at pH = 5.6 (acidic)
is 6.25–12.5 µg/mL [22]. The value stated in the table is from testing at pH = 6.6 (neutral).
Five of the twenty prepared compounds had anti-TB activity within the range of tested
concentrations. Based on the results of the biological evaluation (Table 1), we can conclude that in this
series the ester moiety is important for anti-TB activity. When compound (MICMtb = 1.56 g/mL,
5.19 M) (log P = 1.62) was hydrolyzed to compound (MICMtb > 100 g/mL, >387.23 M)
4
µ
µ
5
µ
µ
(log P = 0.54), it lost its biological activity, suggesting that the free carboxylic moiety significantly
reduced the lipophilicity and hence could have impaired penetration through the highly lipophilic
mycobacterial cell wall. Lipophilicity is an important aspect of activity against Mtb. The five
active compounds had log P values ranging from 1.11 to 2.18. Compounds with either lower or
higher lipophilicity had diminished activity. However, lipophilicity is not the only determinant of

Molecules 2017, 22, 1797
5 of 16
anti-TB activity; compounds
7
and
8
share the same log P value, yet compound
8
is active while
7
is not. We also found that non-substituted phenyl (compound
4
) or 4-monosubstituted phenyl
(compound
6) derivatives of ureidopyrazine esters had higher anti-TB activity than other substituents.
Interestingly, 4-chloro substitution on the phenyl core resulted in active compounds in the series
with ester moiety (Compound , MICMtb = 25 g/mL), as well as in the series lacking the ester
moiety (compound 18, MICMtb = 12.5 g/mL). When comparing the chemical structure and anti-TB
activity of compounds and , we found that the introduction of a -CH2- bridge resulted in loss
8
µ
µ
3
4
of biological activity. Aryl substituted ureidopyrazine esters (Group B) had better activity than
the corresponding non-ester compounds of the same substituent (Group D). When comparing the
non-ester compounds (10
aryl derivatives (Group D). Among the aliphatic series (Group C), compound 13 (MICMtb = 25
99.86 M) exerted the highest anti-TB activity. This result is consistent with pervious findings in
–
20), alkyl substituted ureidopyrazines (Group C) had inferior activity to the
µg/mL,
µ
our research group with N-Alkyl-3-(alkylamino)-pyrazine-2-carboxamides of 6–8 carbon alkyl chain
having the best activity [23]. Since the starting acid and ester had no urea moiety, we can infer that
urea is an important pharmacophore for the activity of such designed compounds. Compounds
MICMtb = 1.56 µg/mL, 5.19 M) and (MICMtb = 6.25 g/mL, 18.91 M) are suitable for further
structural modifications that might improve their anti-TB activity and physicochemical properties.
Compound MICM.kansasii = 25 µg/mL, 75.67 M) and compound 13 MICM.kansasii = 25 µg/mL
89.79 M) were the only two compounds with moderate activity against M. kansasii. None of the tested
4
(
µ
6
µ
µ
6
(
µ
(
,
µ
compounds showed activity against M. avium.
2.2.2. Antimycobacterial Activity Evaluation against Mycobacterium smegmatis and
Mycobacterium aurum
In order to compare the activity of prepared compounds on fast growing mycobacteria to that on
previously mentioned slow growing Mtb, M. kansasii, and M. avium, this complementary screening
was conducted. This test has several advantages since M. smegmatis and M. aurum are avirulent
surrogate organisms [24]. Unlike slow growing mycobacteria that cause serious courses of infection in
humans, M. smegmatis and M. aurum result in soft tissue infections in humans [25–27]. The two latter
mycobacteria also have similar antibiotic susceptibility profile to Mtb [28]. Microplate Alamar Blue
assays with INH, rifampicin, and ciprofloxacin as standards was used in performing this test. None of
the tested compounds exerted activity against the mentioned fast growing mycobacteria up to the
highest tested concentration of 125
µg/mL for compounds 9, 17, 20, 250 µg/mL for compounds 8, 14,
19, and 500 g/mL for the remaining compounds (Supplementary Materials, Table S1), suggesting
µ
that those compounds active against Mtb might work through a pathway not shared among all species
of mycobacteria.
2.2.3. Antibacterial and Antifungal Activity Evaluation
Most compounds were tested in vitro for their biological activity against eight common bacterial
strains and eight fungal strains of clinical importance using standard methods [29]. The test excluded
compounds 3, 6, 7, 9, and 18 due to precipitation in the testing media upon dilution. None of the
tested compounds exerted antibacterial or antifungal activity up to the highest tested concentrations of
125 µM for compounds 4, 8, 16, 250 µM for compound 14, and 500 µM for the remaining compounds
µM (Supplementary Materials, Tables S2 and S3).
2.2.4. In Vitro Cytotoxicty Assays
TB treatment regimens are known to carry a risk of hepatotoxicity [30]. Therefore, it is important
when developing new anti TB drugs test them for this crucial side effect. The most active compounds,
4
and 6, were further evaluated for any possible cytotoxicity. For this screening, standard hepatic
cell line HepG2 (hepatocellular carcinoma) was used, and results were expressed by the inhibitory
concentration required to decrease the viability of cell population to 50% (IC50) compared to a control

Molecules 2017, 22, 1797
6 of 16
of 100% cell viability. The used a CellTiter 96 assay is based on the reduction of tetrazolium dye MTS
in living cells to formazan, which is then determined colorimetrically. Due to low solubility at higher
concentrations in cell culture medium, it was difficult to determine the exact IC50 of compounds
and . However, it can be stated that those two compounds were nontoxic at their highest tested
concentrations (Table 2, Figure 2).
4
6
Table 2. Cytotoxicity of the tested substances in HepG2 cells.
Compound
IC50 (µM)
4
6
>25 * (>16 × MICMtb
)
)
>50 * (>8 × MICMtb
>104 [31]
PZA
INH
79 × 103 [31]
* Measurement at higher concentrations was not possible due to the precipitation of the tested compounds in cell
culture medium.
Figure 2. Cytotoxic effect of different concentrations of the tested substances on HepG2 cells.
Data points from precipitated concentrations are not shown.
2.2.5. Plant Growth Regulation Activity Evaluation
Previous studies showed that derivatives of pyrazine-2-carboxylic acid used as elicitors were
able to increase secondary metabolites production in in vitro plant cultures. Compounds and
8
18 structurally resemble a plant growth regulator, 1-(2-chloropyridin-4-yl)-3-phenylurea (Figure 3).
This chemical promotes cell division and growth, leading to an increase in fruit size. Its use has been
approved in the USA on kiwi and grapes [32]. Based on those two facts, the effect of compounds
8 and
18 on rutin production in Fagopyrum esculentum var. Bamby callus culture was assessed.
O
Cl
N
Cl
N
N
O
N
O
O
O
c
N
N
Cl
N
HN
N
N
HN
H
H
H
H
a
b
Figure 3. The chemical structure of (a) 1-(2-chloropyridin-4-yl)-3-phenylurea; (b) compound 8; and (c) compound 18.
Full numerical data regarding the content of rutin in callus cultures of F. esculentum after treatment
with compounds and 18 separately can be found in Supplementary Materials, Table S4. For compound
, the highest rutin levels, 0.80 (70.2% increase) and 0.83 (76.6% increase)
−1 DW, were reached
6 and 12 h respectively post treatment in comparison with control (24 K). Gradual decrease in rutin
8
8
µg g
·

Molecules 2017, 22, 1797
7 of 16
content was observed after 48 and 72 h of compound
8
application. On the other hand, compound
18 resulted in a more significant increase in rutin level 6 h post application (115% increase) when
compared to control (24 K, control after 24 h). After 12 and 24 h of elicitor application, rutin content
is increased only about 12.8% and 21.2%. The two compounds decreased rutin production in similar
fashion after 48 and 72 h; however, compound 18 further decreased rutin content in comparison with
control (168 K, control after 168 h).
These results indicate that the two proposed elicitors are able to increase rutin production in
callus culture of Fagopyrum esculetum var. Bamby. This increase in rutin production level is mainly
affected by the time of elicitor application. According to literature, secondary metabolite production in
general is affected by various factors, including the type of elicitor, its concentrations, and the time
of administration. In our previous studies we declared the positive effect of various concentrations
of pyrazinecarboxamide derivatives on the flavonoids and flavonolignans production in callus and
suspension cultures of Ononis arvensis and Silybum marianum [33,34] and the current results support
ureidopyrazines as well.
3. Materials and Methods
3.1. General Information
All chemicals were of reagent or higher grade of purity. They were purchased from Sigma-Aldrich
(Steinheim, Germany), unless stated otherwise. Progress of reactions was checked by using Merck
Silica 60 F254 TLC plates (Merck, Darmstadt, Germany) with UV detection using 254 nm wavelength.
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 running under CEM’s SynergyTM software for setting and
monitoring the conditions of reactions. The temperature of the reaction mixture was monitored by
internal infrared sensor. All obtained products 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, Darmstadt, Germany) was used as
the stationary phase. NMR spectra were recorded on Varian VNMR S500 (Varian, Palo Alto, CA,
USA) at 500 MHz for 1H and 125 MHz for 13C. Chemical shifts were reported in ppm (
δ) and were
referred indirectly to tetramethylsilane via signal of solvent (2.49 for 1H and 39.7 for 13C in DMSO-d6;
7.26 for 1H and 77.2 for 13C in CDCl3). Infrared spectra were recorded with spectrometer FT-IR Nicolet
6700 (Thermo Scientific, Waltham, MA, USA) using attenuated total reflectance (ATR) methodology
on germanium crystal. Elemental analysis was performed on vario MICRO cube Element Analyzer
(Elementar Analysensysteme, Hanau, Germany). All values regarding elemental analyses are given
as percentages. Melting points were determined in open capillary on Stuart SMP30 melting point
apparatus (Bibby Scientific Limited, Staffordshire, UK) and are uncorrected. Yields are expressed as
percentages of theoretical yields and refer to the isolated products (chromatographically pure) after all
purification steps. Theoretical lipophilicity parameters log P was calculated by algorithms of program
CS ChemBioDraw Ultra 16.0 (CambridgeSoft, Cambridge, MA, USA).
3.2. Synthesis
3.2.1. Compounds 1 and 2
The starting 5-chloropyrazine-2-carboxylic acid (317 mg, 2 mmol) was converted to
5-aminopyrazine-2-carboxylic acid (1) by substitution reaction with 25% (m/m) aqueous solution
of ammonia (3 mL). The reaction was carried out 10 mL microwave pressurized vials with stirring
(reaction temperature: 100 ◦C, reaction time: 30 min, power output: 80 W). The reaction was repeated
20 times to yield reasonable quantity of the starting acid. Once the reaction was completed, the vials

Molecules 2017, 22, 1797
8 of 16
content was put onto Petri dish and heated above a water bath with intermittent stirring until a dry
solid was obtained (ammonium salt of the product). To get the free acid form, the ammonium salt was
dissolved in water and drop-wise acidified with 10% hydrochloric acid to reach pH of 4. The mixture
was then left to cool down in room temperature for 5 min then kept in the fridge for 15 min. The formed
free acid crystals were filtered off by filtration paper with suction and left to dry overnight. After it
was dried, the resulting 5-aminopyrazine-2-carboxylic acid (1) was esterified in several microwave
pressurized vials; 3 mL of anhydrous propanol and 2 drops of concentrated sulfuric acid were added to
278 mg (2 mmol) of compound
1 in each vial. The esterification was carried out in microwave reactor
◦
(reaction temperature: 100 C, reaction time: 1 h, power output: 80 W). The completion of reaction was
monitored by TLC in system hexane/ethyl acetate (EtOAc) (1:3). The ester was then purified by flash
chromatography using gradient elution 40 to 100% EtOAc in hexane.
3.2.2. Compounds 3–9
The resulted propyl 5-aminopyrazine-2-carboxylate (2) (360 mg, 2 mmol) was reacted with
different aryl substituted isocyanates (2.2 mmol) in hexane as solvent (3 mL). The reaction was carried
out in 10 mL microwave pressurized vials with stirring (reaction temperature: 120 ◦C, reaction time:
1 h, power output: 80 W). Reactions were monitored by TLC in system hexane/EtOAc (1:3). The
reaction mixture was adsorbed to silica and then purified by flash chromatography using gradient
elution 0 to 100% EtOAc in hexane.
Compound
4 (130 mg, 0.5 mmol) was hydrolyzed to 5 by base catalyzed hydrolysis, using
anhydrous potassium carbonate (3 g, 20 mmol) as base and methanol (approximately 20 mL) as solvent.
◦
The reaction mixture was stirred and heated to 85 C under reflux in an oil bath for approximately 6 h.
Potassium carbonate was then filtered off and the mixture was acidified with 10% hydrochloric acid
to reach a pH of 3. The solution was evaporated and the product washed with water to remove any
remnants of potassium carbonate and then left to dry.
3.2.3. Compounds 10–20
In a 10 mL microwave pressurized vial, commercially available aminopyrazine (190 mg, 2
mmol) was reacted with different aryl/alky substituted isocyanates (2.2 mmol) in hexane as solvent
(3 mL) with a magnetic stirrer. The reaction proceeded in a microwave reactor with stirring (reaction
temperature: 120 ◦C, reaction time: 1 h, power output: 80 W). Reaction process was monitored by
TLC in system hexane/EtOAc (1:3). The reaction mixture was adsorbed to silica and then purified
by flash chromatography using gradient elution 20 to 100% EtOAc in hexane for alkyl substituted
ureidopyrazines and 0 to 70% EtOAc in hexane for aryl substituted ureidopyrazines. N.B.: Alkyl
substituted ureidopyrazines 10–14 needed some time to solidify after the evaporation of solvents after
the flash chromatography.
3.3. Analytical Data of the Prepared Compounds
5-Aminopyrazine-2-carboxylic acid (
). Light pinkish solid. Yield 70%; m.p. 238–245 ◦C; IR (ATR-Ge,
cm−1): 3316 (-NH-), 1643 (-C=O), 1621, 1591 (arom.); 1H-NMR (DMSO-d6)
12.58 (s, 1H,-COOH), 8.51
(d, J = 1.3 Hz, 1H, arom.), 7.91 (d, J = 1.3 Hz, 1H, arom.), 7.27 (s, 2H, -NH2). 13C-NMR (DMSO-d6)
1
δ
δ
165.8, 157.4, 145.7, 131.9, 130.8. Elemental analysis found: C, 42.82%; H, 3.51%; N, 29.64%. Calculated
for C5H5N3O2 (MW 139.11): C, 43.17%; H, 3.62%; N, 30.21%.
Propyl 5-aminopyrazine-2-carboxylate (
3317 (-NH-), 1722 (-C=O), 1661, 1584 (arom.); 1H-NMR (DMSO-d6)
(d, J = 1.3 Hz, 1H, arom.), 7.32 (s, 2H, -NH2), 4.16 (t, J = 6.7 Hz, 2H, -CH2-), 1.73–1.62 (m, 2H, -CH2-),
0.93 (t, J = 7.4 Hz, 3H, -CH3). 13C-NMR (DMSO-d6)
164.4, 157.5, 145.7, 132.3, 130.2, 65.8, 21.8, 10.5.
2
). Yellow solid. Yield 45%; m.p. 135–138 ◦C; IR (ATR-Ge, cm−1):
8.53 (d, J = 1.3 Hz, 1H, arom.), 7.91
δ
δ
Elemental analysis found: C, 53.44%; H, 6.26%; N, 22.71%. Calculated for C8H11N3O2 (MW 181.20): C,
53.03%; H, 6.12%; N, 23.19%.

Molecules 2017, 22, 1797
9 of 16
◦
Propyl 5-(3-benzylureido)pyrazine-2-carboxylate (3). White solid. Yield 65%; m.p. 190.5–193.3 C; IR
(ATR-Ge, cm−1): 3329 (-NH-), 1717 (-C=O, ester), 1705 (-C=O, urea), 1625, 1539, 1471, 1456 (arom.);
1H-NMR (CDCl3)
δ 10.67 (s, 1H, urea), 9.36 (s, 1H, urea), 8.82 (d, J = 1.3 Hz, 1H, pyrazine), 8.54–8.50
(m, 1H, pyrazine), 7.40–7.28 (m, 4H, arom.), 7.32–7.26 (m, 1H, arom.), 4.62 (d, J = 5.8 Hz, 2H, -OCH2-),
4.43–4.33 (m, 2H, -NCH2-), 1.92–1.78 (m, 2H, -CH2-), 1.04 (t, J = 14.8, 7.4 Hz, 3H, -CH3). 13C-NMR
(CDCl3)
δ 163.9, 155.8, 151.2, 142.7, 138.9, 138.3, 136.0, 135.7, 128.8, 128.7, 127.5, 127.5, 127.4, 67.6,
67.4, 44.7, 44.0, 22.0, 10.4. Elemental analysis found: C, 60.73%; H, 5.66%; N, 17.58%. Calculated for
C16H18N4O3 (MW 314.35): C, 61.14%; H, 5.77%; N, 17.82%.
◦
Propyl 5-(3-phenylureido)pyrazine-2-carboxylate (
4
). White solid. Yield 17%; m.p. 217.9–219.9 C; IR
(ATR-Ge, cm−1): 3302 (-NH-), 1721 (-C=O, ester), 1704 (-C=O, urea), 1604, 1557, 1540, 1506, 1499 (arom.);
1H-NMR (DMSO-d6)
10.02 (s, 1H, urea), 9.69 (s, 1H, urea), 9.13–9.08 (m, 1H, pyrazine), 8.90–8.81 (m,
1H, pyrazine), 7.54–7.49 (m, 1H, arom.), 7.36–7.29 (m, 2H, arom.), 7.06–7.02 (m, 2H, arom.), 4.28–4.19
(m, 2H, -OCH2-), 1.72–1.68 (m, 2H, -CH2-), 0.96 (t, 3H, -CH3).13C-NMR (DMSO-d6)
163.6, 151.4, 144.0,
δ
δ
138.6, 136.0, 134.7, 129.1, 123.3, 119.1, 66.6, 21.8, 10.5. Elemental analysis found: C, 60.43%; H, 5.59%; N,
18.38%. Calculated for C15H16N4O3 (MW 300.32): C, 59.99%; H, 5.37%; N, 18.66%.
Propyl 5-(3-Phenylureido)pyrazine-2-carboxylic acid (
(ATR-Ge, cm−1): 3569 (-NH-), 1728 (-C=O, acid), 1685 (-C=O, urea), 1596, 1567, 1552, 1500 (arom.);
1H-NMR (DMSO-d6)
13.27 (s, 1H, -COOH), 9.98 (s, 1H, urea), 9.73 (s, 1H, urea), 9.12 (d, J = 1.4 Hz,
1H, pyrazine), 8.87 (d, J = 1.4 Hz, 1H, pyrazine), 7.52 (d, J = 8.0 Hz, 2H, arom.), 7.33 (t, J = 7.8 Hz,
2H, arom.). 13C-NMR (DMSO-d6)
165.1, 151.5, 151.2, 144.0, 138.6, 136.8, 134.4, 129.1, 123.2, 119.1.
5
). White solid. Yield 35%; m.p. 228–229.3 ◦C; IR
δ
δ
Elemental analysis found: C, 62.88%; H, 5.50%; N, 24.16%. Calculated for C12H10N4O3 (MW 258.24):
C, 63.15%; H, 5.30%; N, 24.55%.
Propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (
◦C; IR (ATR-Ge, cm−1): 3333 (-NH-), 1718 (-C=O, ester), 1702 (-C=O, urea), 1610, 1546, 1508, 1404
(arom.), 1231(-C-O); 1H-NMR (CDCl3)
11.05 (s, 1H, urea), 10.87 (s, 1H, urea), 8.90 (d, J = 1.3 Hz, 1H,
6). White solid. Yield 39%; m.p. 189–193
δ
pyrazine), 8.56 (s, 1H, pyrazine), 7.54–7.42 (m, 2H, arom.), 6.97–6.84 (m, 2H, arom.), 4.39 (t, J = 6.9 Hz,
2H, -OCH2-), 3.84 (s, 3H, -OCH3), 1.92–1.80 (m, 2H, -CH2-), 1.06 (t, J = 7.4 Hz, 3H, -CH3). 13C-NMR
(CDCl3) δ 163.8, 156.5, 153.4, 150.8, 142.4, 135.9, 135.8, 130.2, 122.0, 114.3, 67.4, 55.5, 22.0, 10.3. Elemental
analysis found: C, 58.58%; H, 5.62%; N, 16.62%. Calculated for C16H18N4O4 (MW 330.34): C, 58.17%;
H, 5.49%; N, 16.96%.
Propyl 5-(3-(2-chlorophenyl)ureido)pyrazine-2-carboxylate (
IR (ATR-Ge, cm−1): 3567 (-NH-), 1720 (-C=O, ester), 1689 (-C=O, urea), 1636, 1591, 1549, 1511, 1473
(arom.); 1H-NMR (CDCl3)
11.86 (s, 1H, urea), 10.90 (s, 1H, urea), 8.86 (d, J = 1.4 Hz, 1H, pyrazine),
7
). White solid. Yield 28%; m.p. 195–197 ◦C;
δ
8.52 (d, J = 1.4 Hz, 1H, pyrazine), 8.32 (dd, J = 1.5, 8.3 Hz, 1H, arom.), 7.38 (dd, J = 1.5, 8.0 Hz, 1H,
arom.), 7.31–7.23 (m, 1H, arom.), 7.09–6.99 (m, 1H, arom.), 4.39 (t, J = 6.9 Hz, 2H, -OCH2-), 1.94–1.81
(m, 2H. -CH2-), 1.06 (t, J = 7.4 Hz, 3H, -CH3). 13C-NMR (CDCl3)
δ 163.7, 153.3, 150.4, 142.4, 136.3, 135.4,
134.9, 129.3, 127.8, 124.6, 123.4, 121.3, 67.5, 22.1, 10.4. Elemental analysis found: C, 54.12%; H, 4.59%; N,
16.58%. Calculated for C15H15ClN4O3 (MW 334.76): C, 53.82%; H, 4.52%; N, 16.74%.
◦
Propyl 5-(3-(4-chlorophenyl)ureido)pyrazine-2-carboxylate (
8
). White solid. Yield 57%; m.p. 240.6–243.8 C;
IR (ATR-Ge, cm−1): 3328 (-NH-), 1720 (-C=O, ester), 1704 (-C=O, urea), 1606, 1540, 1493, 1471, 1454
1
(arom.); H-NMR (DMSO-d6)
δ 10.17 (s, 1H, urea), 10.02 (s, 1H, urea), 9.11–8.99 (m, 1H, pyrazine),
8.92–8.84 (m, 1H, pyrazine), 7.93 (d, J = 2.5 Hz, 1H, arom.), 7.56 (d, J = 8.8 Hz, 1H, arom.), 7.45 (dd, J =
8.8, 2.5 Hz, 2H, arom.), 4.26 (t, J = 6.6 Hz, 2H, -OCH2-), 1.78–1.67 (m, 2H, -CH2-), 0.96 (t, J = 7.4 Hz, 3H,
-CH3). 13C-NMR (DMSO-d6)
δ 163.6, 151.4, 151.3, 143.9, 137.6, 136.2, 134.7, 128.9, 126.9, 120.7, 66.6, 21.7,
10.5. Elemental analysis found: C, 53.39%; H, 4.39%; N, 16.53%. Calculated for C15H15ClN4O3 (MW
334.76): C, 53.82%; H, 4.52%; N, 16.74%.
Propyl 5-(3-(2,3-dichlorophenyl)ureido)pyrazine-2-carboxylate (
9). White solid. Yield 70%; m.p. 242.6–243.9
◦C; IR (ATR-Ge, cm−1): 3336 (-NH-), 1705 (-C=O, ester), 1655 (-C=O, urea), 1612, 1537, 1481, 1378

Molecules 2017, 22, 1797
10 of 16
1
(arom.); H-NMR (DMSO-d6)
δ
10.17 (s, 1H, urea), 10.02 (s, 1H, urea), 9.11–8.99 (m, 1H, pyrazine),
8.92–8.84 (m, 1H, pyrazine), 7.93 (d, J = 2.5 Hz, 1H, arom.), 7.56 (d, J = 8.8 Hz, 1H, arom.), 7.45 (dd,
J = 8.8, 2.5 Hz, 1H, arom.), 4.26 (t, J = 6.6 Hz, 2H, -OCH2-), 1.78–1.67 (m, 2H, -CH2-), 0.96 (t, J = 7.4
Hz, 3H, -CH3). 13C-NMR (DMSO-d6)
δ 163.6, 151.4, 151.0, 143.9, 138.8, 136.3, 134.8, 131.4, 130.9, 124.7,
120.3, 119.3, 66.7, 21.7, 10.5. Elemental analysis found: C, 48.65%; H, 3.56%; N, 14.84%. Calculated for
C15H14Cl2N4O3 (MW 369.20): C, 48.80%; H, 3.82%; N, 15.18%.
◦
1-Propyl-3-(pyrazin-2-yl)urea (10). Light yellow solid. Yield 36%; m.p. 104.6–107.1 C; IR (ATR-Ge,
cm−1): 3274 (-NH-), 2963 (-CH2-), 1694 (-C=O), 1586, 1536, 1499 (arom.); 1H-NMR (CDCl3)
δ
10.19 (s,
1H, urea), 8.90 (s, 1H, urea), 8.44 (d, J = 1.4 Hz, 1H, pyrazine), 8.14–8.04 (m, 2H, pyrazine), 3.44–3.33 (m,
2H, -CH2-), 1.70–1.61 (m, 2H, -CH2-) 0.99 (t, J = 7.4 Hz, 3H, -CH3). 13C-NMR (CDCl3)
154.5, 153, 139.5,
δ
137.3, 136.1, 41.3, 22.7, 10.9. Elemental analysis found: C, 53.68%; H, 6.72%; N, 30.89%. Calculated for
C8H12N4O (MW 180.21): C, 53.32%; H, 6.71%; N, 31.09%.
◦
1-Butyl-3-(pyrazin-2-yl)urea (11). Yellow-light brown solid. Yield 29%; m.p. 107.8–108.5 C; IR (ATR-Ge,
cm−1): 3276 (-NH-), 2953 (-CH2-), 1688 (-C=O), 1600, 1553, 1502 (arom.); 1H-NMR (CDCl3)
δ 10.16 (s,
1H, urea), 8.91 (s, 1H, urea), 8.42 (d, J = 1.5 Hz, 1H, pyrazine), 8.14–8.04 (m, 2H, pyrazine), 3.45–3.37 (m,
2H, -CH2-), 1.66–1.57 (m, 2H, -CH2-), 1.53–1.33 (m, 2H, -CH2-), 0.97 (t, J = 7.4 Hz, 3H, -CH3). 13C-NMR
(CDCl3) δ 156.2, 145, 139.3, 136.4, 136.3, 39.7, 31.9, 20.1, 13.8. Elemental analysis found: C, 55.34%; H,
7.21%; N, 28.31%. Calculated for C9H14N4O (MW 194.24): C, 55.65%; H, 7.27%; N, 28.85%.
1-Pentyl-3-(pyrazin-2-yl)urea (12). Yellow solid. Yield 51%; m.p. 108–110 ◦C; IR (ATR-Ge, cm−1): 3258
(-NH-), 2955 (-CH2-), 1686 (-C=O), 1618, 1599, 1501 (arom.); 1H-NMR (CDCl3)
δ
10.19 (s, 1H, urea), 8.90
(s, 1H, urea), 8.43 (d, J = 8.2 Hz, 1H, pyrazine), 8.13–8.04 (m, 2H, pyrazine), 3.44–3.36 (m, 2H, -CH2-),
1.44–1.31 (m, 6H, -C3H6-), 0.92 (t, 3H, -CH3). 13C-NMR (CDCl3)
156.2, 150, 139.3, 136.4, 136.3, 40, 29.6,
δ
29.1, 22.3, 14. Elemental analysis found: C, 58.78%; H, 7.95%; N, 26.62%. Calculated for C10H16N4O
(MW 208.27): C, 57.67%; H, 7.74%; N, 26.91%.
◦
1-Octyl-3-(pyrazin-2-yl)urea (13). White solid. Yield 43%; m.p. 115.6–117.2 C; IR (ATR-Ge, cm−1): 3276
(-NH-), 2929 (-CH2-), 1699 (-C=O), 1552, 1535, 1503 (arom.); 1H-NMR (CDCl3)
δ
10.21 (s, 1H, urea), 8.91
(s, 1H, urea), 8.43 (d, J = 2.8 Hz, 1H, pyrazine), 8.11 (d, J = 2.8 Hz, 1H, pyrazine), 8.07 (dd, J = 2.8, 1.4
Hz, 1H, pyrazine), 3.40 (td, J = 7.1, 5.5 Hz, 2H, -CH2-), 1.67–1.57 (m, 4H, -C2H4-), 1.43–1.37 (m, 2H,
-CH2-), 1.40–1.26 (m, 4H, -C2H4-), 1.30–1.21 (m, 2H, -CH2-), 0.91–0.84 (m, 3H, -CH3).13C-NMR (CDCl3)
δ
156.2, 150, 139.3, 136.3, 136.4, 136.3, 40, 31.8, 29.8, 29.2, 29.1, 27, 22.6, 14. Elemental analysis found: C,
60.39%; H, 8.42%; N, 23.23%. Calculated for C13H22N4O (MW 250.35): C, 60.99%; H, 8.53%; N, 23.71%.
◦
1-Decyl-3-(pyrazin-2-yl)urea (14). Yellow solid. Yield 24%; m.p. 121.3–123.5 C; IR (ATR-Ge, cm−1): 3270
(-NH-), 2926 (-CH2-), 1693 (-C=O), 1600, 1552, 1504 (arom.); 1H-NMR (CDCl3)
δ
10.08 (s, 1H, urea),
8.91 (s, 1H, urea), 8.91 (d, J = 2.9 Hz, 1H, pyrazine), 8.91 (d, J = 2.9 Hz, 1H, pyrazine), 8.09–8.04 (m,
1H, pyrazine), 3.44–3.36 (m, 2H, -CH2-), 1.36–1.21 (m, 16H, -C8H16-), 0.88 (t, J = 6.9 Hz, 3H, -CH3).
13C-NMR (CDCl3) 156.1, 150, 139.3, 136.4, 136.2, 40, 31.9, 29.9, 29.6, 29.5, 29.3, 27, 22.6, 14.1. Elemental
analysis found: C, 64.82%; H, 9.37%; N, 19.77%. Calculated for C15H26N4O (MW 278.40): C, 64.71%; H,
9.41%; N, 20.12%.
◦
1-Benzyl-3-(pyrazin-2-yl)urea (15). Beige solid. Yield 32%; m.p. 176.5–179.3 C; IR (ATR-Ge, cm−1): 3258
(-NH-), 2895 (-CH2-), 1694 (-C=O), 1566, 1540, 1508, 1476, 1448 (arom.); 1H-NMR (DMSO-d6)
9.51 (s,
1H, urea), 8.91 (s, 1H, urea), 8.26–8.11 (m, 2H, pyrazine), 7.93–7.85 (m, 1H, pyrazine), 7.41–7.27 (m, 5H,
arom.), 4.46–4.31 (m, 2H, -CH2). 13C-NMR (DMSO-d6)
154.8, 150.3, 141.3, 140.1, 137.5, 135.6, 128.9,
δ
δ
127.6, 127.3, 43.2. Elemental analysis found: C, 61.35%; H, 5.30%; N, 24.15%.Calculated for C12H12N4O
(MW 228.26): C, 61.67%; H, 5.19%; N, 23.74%.
1-(4-Methoxyphenyl)-3-(pyrazin-2-yl)urea (16). White solid. Yield 12%; m.p. 225.5–227.4 ◦C; IR (ATR-Ge,
cm−1): 3023 (-NH-), 1682 (-C=O), 1614, 1597, 1567, 1551, 1513, 1501 (arom.), 1247 (-C-O); 1H-NMR
(DMSO-d6)
δ 9.49 (s, 1H, urea), 8.99 (s, 1H, urea), 8.29 (dd, J = 2.7, 1.5 Hz, 1H, pyrazine), 8.22

Molecules 2017, 22, 1797
11 of 16
(d, J = 2.7 Hz, 2H, pyrazine), 7.44–7.36 (m, 2H, arom.), 6.93–6.86 (m, 2H, arom.), 3.72 (s, 3H,
-OCH3).13C-NMR (DMSO-d6)
155.3, 152.0, 149.7, 141.8, 137.8, 135.4, 131.8, 120.9, 114.3, 55.4. Elemental
δ
analysis found: C, 59.01%; H, 4.95%; N, 22.14%. Calculated for C12H12N4O2 (MW 244.25): C, 60.34%;
H, 5.30%; N, 21.97%.
1-(2-Chlorophenyl)-3-(pyrazin-2-yl)urea (17). White solid. Yield 26%; m.p. 137.5–240.9 ◦C; IR (ATR-Ge,
cm−1): 3129 (-NH-), 1699 (-C=O), 1593, 1552, 1509, 1482, 1442 (arom.); 1H-NMR (DMSO-d6)
δ
10.35 (s,
1H, urea), 10.28 (s, 1H, urea), 8.85–8.80 (m, 1H, pyrazine), 8.35–8.30 (m, 2H, pyrazine), 7.52–7.46 (m,
2H, arom.), 7.36–7.29 (m, 1H, arom.), 7.11–7.04 (m, 1H, arom.). 13C-NMR (DMSO-d6)
151.8, 149.3,
δ
141.1, 137.9, 135.7, 135.6, 129.4, 127.8, 124.2, 122.5, 121.7. Elemental analysis found: C, 53.13%; H, 3.65%;
N, 22.53%. Calculated for C11H9ClN4O (MW 248.67): C, 53.22%; H, 3.70%; N, 22.17%.
1-(4-Chlorophenyl)-3-(pyrazin-2-yl)urea (18). White solid. Yield 29%; m.p. 211–213.7 ◦C; IR (ATR-Ge,
cm−1): 3116 (-NH-), 1690 (-C=O), 1603, 1590, 1556, 1504, 1492 (arom.); 1H-NMR (DMSO-d6)
δ 9.71 (s,
1H, urea), 9.60 (s, 1H, urea), 9.01 (d, J = 1.5 Hz, 1H, pyrazine), 8.30 (dd, J = 1.5 Hz, 1H, pyrazine), 8.25
(d, J = 1.5 Hz, 1H, pyrazine), 7.60–7.42 (m, 2H, arom.), 7.44–7.27 (m, 2H, arom.). 13C-NMR (DMSO-d6)
δ
151.9, 149.4, 141.9, 138.2, 137.9, 135.4, 129.0, 126.6, 120.6. Elemental analysis found: C, 53.13%; H,
3.65%; N, 22.53%. Calculated for C11H9ClN4O (MW 248.67): C, 53.15%; H, 3.61%; N, 22.18%.
1-(2,3-Dichlorophenyl)-3-(pyrazin-2-yl)urea (19). White solid. Yield 17%; m.p. 246–247.8 ◦C; IR (ATR-Ge,
cm−1): 3037 (-NH-), 1734 (-C=O), 1647, 1609, 1593, 1550, 1500 (arom.); 1H-NMR (DMSO-d6)
δ
9.89 (s,
1H, urea), 9.67 (s, 1H, urea), 9.06–8.92 (m, 1H, pyrazine), 8.38–8.19 (m, 2H, pyrazine), 7.98–7.84 (m, 1H,
arom.), 7.59–7.46 (m, 1H, arom.), 7.42–7.33 (m, 1H, arom.). 13C-NMR (DMSO-d6)
151.8, 149.2, 141.8,
δ
139.1, 138.3, 135.4, 131.3, 130.8, 124.3, 120.1, 119.1. Elemental analysis found: C, 46.67%; H, 2.95%; N,
18.19%. Calculated for C11H8Cl2N4O (MW 283.11): C, 47.02%; H, 3.42%; N, 17.64%.
1-(2-Chlorobenzyl)-3-(pyrazin-2-yl)urea (20). White solid. Yield 33%; m.p. 204.6–207.6 ◦C; IR (ATR-Ge,
cm−1): 3240 (-NH-), 1681(-C=O), 1567, 1580, 1542, 1505 (arom.); 1H-NMR (DMSO-d6)
δ 9.59 (s, 1H,
urea), 8.88 (s, 1H, urea), 8.27–8.12 (m, 2H, pyrazine), 7.99 (t, J = 5.9 Hz, 1H, pyrazine), 7.50–7.25 (m,
4H, arom.). 13C-NMR (DMSO-d6) 154.7, 150.2, 141.8, 137.6, 137.1, 135.5, 132.6, 129.7, 129.4, 129.2, 127.8.
Elemental analysis found: C, 54.87%; H, 4.22%; N, 21.33%. Calculated for C12H11ClN4O (MW 262.70):
C, 54.98%; H, 4.15%; N, 21.35%.
3.4. Biological Assays
3.4.1. In Vitro Activity Evaluation against Mycobacterium tuberculosis, Mycobacterium kansasii, and
Mycobacterium avium
Microdilution panel method. Tested strains M. tuberculosis H37Rv CNCTC My 331/88 (ATCC
27294), M. kansasii Hauduroy CNCTC My 235/80 (ATCC 12478), M. avium ssp. Avium Chester CNCTC
My 80/72 (ATCC 15769) were obtained from Czech National Collection of Type Cultures (CNCTC),
National Institute of Public Health, Prague, Czech Republic. Middlebrook 7H9 broth (Sigma-Aldrich)
enriched with 0.4% (v/v) of glycerol (Sigma-Aldrich) and 10% (v/v) of OADC supplement (oleic acid,
albumin, dextrose, catalase; Himedia, Mumbai, India) of declared pH = 6.6. Tested compounds were
dissolved and diluted in DMSO, mixed with broth (25
and placed (100 L) into microplate wells. Mycobacterial inocula were suspended in isotonic saline
solution and the density was adjusted to 0.5–1.0 McFarland scale. These suspensions were diluted
by 10−1 and used to inoculate the testing wells, adding 100
L of mycobacterial suspension per well.
Final concentrations of the tested compounds in wells were 100, 50, 25, 12.5, 6.25, 3.13, and 1.56 g/mL.
µL) of DMSO solution in 4.475 mL of broth
µ
µ
µ
INH and PZA were used as positive controls (inhibition of growth). Negative control (mycobacterial
growth control) consisted of broth plus DMSO. Plates were statically incubated in a dark, humid
◦
atmosphere at 37 C. After five days of incubation, 30
µL of Alamar Blue working solution (1:1 mixture
of 0.1% resazurin sodium salt (aq. sol.) and 10% Tween 80) was added per well. Results were then
determined after 24 h of incubation and interpreted according to Franzblau et al. [21]. The minimum

Molecules 2017, 22, 1797
12 of 16
inhibition concentration (MIC,
µg/mL) was determined as the lowest concentration that prevented
the blue to pink colour change as indicated by visual inspection. The experiments were conducted in
duplicates. For the results to be valid, the difference in MIC for one compound determined from two
parallel measurements must not be greater than one step on the dilution scale.
3.4.2. In Vitro Activity Evaluation against Mycobacterium smegmatis and Mycobacterium aurum
Antimycobacterial assay was performed on fast growing M. smegmatis DSM 43465 (ATCC
607) and M. aurum DSM 43999 (ATCC 23366) from German Collection of Microorganisms and Cell
Cultures (Braunschweig, Germany). The technique used for activity determination was microdilution
broth panel method using 96-well microtitration plates. Culturing medium was Middlebrook 7H9
broth (Sigma-Aldrich) enriched with 0.4% of glycerol (Sigma-Aldrich) and 10% of Middlebrook
OADC growth supplement (Himedia). Mycobacterial strains were cultured on Middlebrook 7H9
agar and suspensions were prepared in Middlebrook 7H9 broth. Final density was adjusted to
value ranging from 0.5 to 1.0 according to McFarland scale and diluted in ratio 1:20 with broth.
Tested compounds were dissolved in DMSO (Sigma-Aldrich) then MB broth was added to obtain
concentration of 2000 µg/mL. Standards used for activity determination were 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
µ
g/mL, except to standards rifampicin, where the final concentrations were 12.5, 6.25, 3.125, 1.56, 0.78,
0.39, 0.195, 0.098 g/mL, and ciprofloxacin, where the final concentrations were 1, 0.5, 0.25, 0.125,
0.0625, 0.0313, 0.0156, 0.0078 g/mL. The final concentration of DMSO did not exceeded 2.5% (v/v)
µ
µ
and did not affect the growth of M. smegmatis or M. aurum. 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, and after 72 h of incubation for M. aurum.
Stain was prepared by dissolving resazurin sodium salt (Sigma-Aldrich) in deionised water to get
0.02% solution. Then 10% aqueous solution of Tween 80 (Sigma-Aldrich) was prepared. Equal volumes
of both liquids were mixed and filtered a through syringe membrane filter. Microtitration panels
were then incubated for further 2.5 h for determination of activity against M. smegmatis, and 4 h
for M. aurum. Antimycobacterial activity was expressed as minimal inhibition concentration (MIC)
and the value was read on the basis of stain colour change (blue colour—active compound; pink
colour—inactive compound). MIC values for standards were in ranges 7.81–15.625
12.5–25 g/mL for RIF, and 0.0625–0.125 g/mL for CPX against M. smegmatis, 1.95–3.91
INH, 0.78–1.56 g/mL for RIF, and 0.00781–0.01563 g/mL for CPX against M. aurum, respectively.
µg/mL for INH,
µ
µ
µg/mL for
µ
µ
All experiments were conducted in duplicate. For the results to be valid, the difference in MIC for
one compound determined from two parallel measurements must not be greater than one step on the
dilution scale.
3.4.3. In Vitro Antibacterial Activity Evaluation
Microdilution broth method was used [35]. Antibacterial evaluation was performed against
eight bacterial strains from the Czech Collection of Microorganisms (CCM, Brno, Czech Republic)
(Staphylococcus aureus CCM 4223 (ATCC 29213), Staphylococcus aureus methicilin resistant CCM 4750
(ATCC 43300), Enterococcus faecalis CCM 4224 (ATCC 29212), Escherichia coli CCM 3954 (ATCC
25922), Pseudomonas aeruginosa CCM 3955 (ATCC 27853)) or clinical isolates from the Department of
Clinical Microbiology, University Hospital and Faculty of Medicine in Hradec Králové, Charles
University in Prague, Czech Republic (Staphylococcus epidermidis 112-2016, Klebsiella pneumoniae
64-2016, Serratia marcescens 62-2016). All strains◦were subcultured on Mueller-Hinton agar ◦(MHA)
(Difco/Becton Dickinson, Detroit, MI, USA) at 35 C and maintained on the same medium at 4 C. The
compounds were dissolved in DMSO, and the antibacterial activity was determined in cation adjusted
Mueller-Hinton liquid broth (Difco/Becton Dickinson) buffered to pH 7.0. Controls consisted of

Molecules 2017, 22, 1797
13 of 16
medium and DMSO solely. The final concentration of DMSO in the test medium did not exceed 1% (v/v)
of the total solution composition. The minimum inhibitory concentration (MIC) was determined after
◦
24 and 48 h of static incubation at 35 C by visual inspection or using Alamar Blue dye. The standards
were gentamicin and ciprofloxacin. All experiments were conducted in duplicate. For the results to be
valid, the difference in MIC for one compound determined from two parallel measurements must not
be greater than one step on the dilution scale.
3.4.4. In Vitro Antifungal Activity Evaluation
Antifungal evaluation was performed using a microdilution broth method [29] against eight
fungal strains from the Czech Collection of Microorganisms (CCM) (Candida albicans CCM 8320 (ATCC
24433), C. krusei CCM 8271 (ATCC 6258), C. parapsilosis CCM 8260 (ATCC 22019), C. tropicalis CCM 8264
(ATCC 750), Aspergillus flavus CCM 8363, Absidia/Lichtheimia corymbifera CCM 8077 and Trichophyton
interdigitale CCM 8377 (ATCC 9533) or the American Type Collection Cultures (ATCC, Mannasas,
VA, USA) (Aspergillus fumigatus ATCC 204305). Compounds were dissolved in DMSO and diluted
in a twofold manner with RPMI 1640 medium, with glutamine and 2% glucose, buffered to pH 7.0
(3-morpholinopropane-1-sulfonic acid). The final concentration of DMSO in the tested medium did not
exceed 2.5% (v/v) of the total solution composition. Static incubation was performed in the dark and
◦
in humid atmosphere, at 35 C, for 24 and 48 h (72 and 120 h for Trichophyton interdigitale respectively).
Drug-free controls were included. MIC was inspected visually or making use of Alamar Blue staining.
The standards were amphotericin B and fluconazole. All experiments were conducted in duplicate.
For the results to be valid, the difference in MIC for one compound determined from two parallel
measurements must not be greater than one step on the dilution scale.
3.4.5. Cytotoxicity Determination
Human hepatocellular liver carcinoma cell line HepG2 (passage 9) purchased from Health
Protection Agency Culture Collections (ECACC, Salisbury, UK) was cultured in MEM (Minimum
Essentials Eagle Medium) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal
bovine serum (PAA), 1% L-Glutamine solution (Sigma-Aldrich) and non-essential amino acid solution
(Sigma-Aldrich) in a humidified atmosphere containing 5% CO2 at 37 ◦C. For subculturing, the cells
were harvested after trypsin/EDTA (Sigma-Aldrich) treatment at 37 ◦C. 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 of 10,000 cells per well in a 96-well plate.
On the following day, the cells were treated with each of the tested substances dissolved in DMSO.
The tested substances were prepared at different incubation concentrations (Table 2) 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, MA, USA) was added. After 2 h incubation at 37%, absorbance of samples was recorded
at 490 nm (TECAN, Infinita M200, Sydney, 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 7 software (GraphPad
Software, La Jolla, CA, USA).
3.4.6. Plant Growth Regulation Activity Evaluation
Callus cultures were derived from germinating seeds of Fagypyrum esculentum var. Bamby.
Seeds were obtained from Crop Research Institute (Piešt’any, Slovak Republic). F. esculentum callus
cultures in the 23–25th passages were used. Calluses were cultivated on Murashige-Skoog medium [36
]
supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at a concentration of 1 mg/L as growth

Molecules 2017, 22, 1797
14 of 16
regulator. Callus cultures were cultivated on paper bridges in Erlenmeyer flasks for 4 weeks in growth
chambers at 26 ± 1 ◦C for 16 h photoperiod. White light intensity of 3.500 lux was used.
The abiotic elicitors,
8
at the concentration of 2.993
×
10−3 mol/L and 18 at a concentration of
4.056
×
10−3 mol/L, were tested. Proposed elicitors (1 mL) were added to the callus cultures on the
21st day of cultivation. After 6, 12, 24, 48, 72, and 168 h of elicitor treatment the calluses were sampled
and dried, and then the content of rutin was determined. Simultaneously, controls without elicitor
application were run after 24 and 168 h. Rutin content was estimated according to Kreft et al. [37].
All experiments were conducted in triplets.
4. Conclusions
In this research project we prepared eighteen different ureidopyrazine derivatives. We focused on
testing the anti-infective activity of the prepared compounds against Mycobacterium tuberculosis and five
other nontubercular strains, along with antibacterial and antifungal activity evaluation. We found five
compounds active against Mtb out of the twenty tested, and we have started an initial structure-activity
relationships (SAR) study. According to the results of antimycobacterial assays, the two most active
compounds, compound
4 (MICMtb = 1.56 µg/mL, 5.19 µM) and compound 6 (MICMtb = 6.25 µg/mL,
18.91 M), were aryl substituted ureidopyrazine propyl esters. Those two compounds were proven
µ
to be nontoxic on HepG2 cancer cells. All tested compounds had neither antibacterial nor antifungal
activity. Two of the prepared compounds, compounds 8 and 18, structurally resemble a known plant
growth regulator and other pyrazinecarboxamides that were proved to be abiotic elicitors. Those two
compounds were assessed for similar activity and were found to be promising elicitors by stimulating
rutin production in plant cultures. Compounds
4 and 6 can be possible starting points for future
research that can improve both biological activity and physicochemical properties. Their anti-TB
activity will be further evaluated against resistant strains of Mtb and their mechanism of action shall
be investigated.
Supplementary Materials: Supplementary materials are available online at www.mdpi.com/1420-3049/22/10/
1797/s1. Table S1: Structure of prepared compounds with their activity against fast growing M. smegmatis and
M. aurum, Table S2: Antibacterial assay results of prepared compounds, Table S3: Antifungal assay results of
prepared compounds, Table S4: Rutin content (µ
g g−1 DW) in Fagopyrum esculentum var. Bamby callus culture
after treatment with compounds 8 and 18, 1H- and 13C-NMR spectra of the most active compound 4.
Acknowledgments: This study was supported by the Ministry of Education, Youth and Sports of the Czech
Republic (SVV 260 401) and (SVV 260 416), as well as by Grant Agency of Charles University (project
C-C3/1572317) and Czech Science Foundation (project No. 17-27514Y).
Author Contributions: M.D., J.Z., and G.B. conceived and designed the experiments; G.B, M.J., and P.N.
performed the experiments; G.B. and J.Z. interpreted analytical data and results of anti-infective screening;
P.P. and O.J. performed biological assays (antimycobacterial, antibacterial, antifungal); J.J. carried out cytotoxicity
screening and interpreted its results; L.T., Z.K., and P.K. performed plant growth regulation activity evaluation.
G.B. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
Sia, I.G.; Wieland, M.L. Current Concepts in the Management of Tuberculosis. Mayo Clin. Proc. 2011, 86,
348–361. [CrossRef] [PubMed]
Shenoi, S.; Heysell, S.; Moll, A.; Friedland, G. Multidrug-resistant and extensively drug-resistant tuberculosis:
Consequences for the global HIV community. Curr. Opin. Infect. Dis. 2009, 22, 11–17. [CrossRef] [PubMed]
World Health Organization. Global Tuberculosis Report 2016. WHO/HTM/TB/2016.13. Available online:
http://www.who.int/tb/publications/global_report/en/ (accessed on 2 February 2017).
Review on Antimicrobial Resistance. Antimicrobial Resistance: Tackling a Crisis for the Health and Wealth of
Nations. Available online: http://www.his.org.uk/files/4514/1829/6668/AMR_Review_Paper_-_Tackling_
a_crisis_for_the_health_and_wealth_of_nations_1.pdf (accessed on 27 November 2016).

Molecules 2017, 22, 1797
15 of 16
5.
6.
Chaluvaraju, K.C.; Bhat, K.I. Synthesis and antimicrobial activities of amino benzylated mannich bases of
pyrazinamide. Int. J. ChemTech Res. 2010, 2, 1368–1371.
Jandourek, O.; Tauchman, M.; Paterova, P.; Konecna, K.; Navratilova, L.; Kubicek, V.; Holas, O.; Zitko, J.;
Dolezal, M. Synthesis of Novel Pyrazinamide Derivatives Based on 3-Chloropyrazine-2-carboxamide and
Their Antimicrobial Evaluation. Molecules 2017, 22, 223. [CrossRef] [PubMed]
7.
8.
9.
Kucerova-Chlupacova, M.; Vyskovska-Tyllova, V.; Richterova-Finkova, L.; Kunes, J.; Buchta, V.; Vejsova, M.;
Paterova, P.; Semelkova, L.; Jandourek, O.; Opletalova, V. Novel Halogenated Pyrazine-Based Chalcones as
Potential Antimicrobial Drugs. Molecules 2016, 21, 1421. [CrossRef] [PubMed]
Semelkova, L.; Janoscova, P.; Fernandes, C.; Bouz, G.; Jandourek, O.; Konecna, K.; Paterova, P.; Navratilova, L.;
Kunes, J.; Dolezal, M.; Zitko, J. Design, Synthesis, Antimycobacterial Evaluation, and In Silico Studies of
3-(Phenylcarbamoyl)-pyrazine-2-carboxylic Acids. Molecules 2017, 22, 1491. [CrossRef] [PubMed]
Zhou, S.; Yang, S.; Huang, G. Design, synthesis and biological activity of pyrazinamide derivatives for
anti-Mycobacterium tuberculosis. J. Enzym. Inhibit. Med. Chem. 2017, 32, 1183–1186. [CrossRef] [PubMed]
10. Zitko, J.; Barbora, S.-V.; Paterová, P.; Navrátilová, L.; Trejtnar, F.; Kuneš, J.; Doležal, M. Design, synthesis and
anti-mycobacterial evaluation of some new iV-phenylpyrazine-2-carboxamides. Chem. Pap. 2016, 70, 649.
11. Conde, M.B.; Lapa, E.S.J.R. New regimens for reducing the duration of the treatment of drug-susceptible
pulmonary tuberculosis. Drug Dev. Res. 2011, 72, 501–508. [CrossRef] [PubMed]
12. Shi, W.; Zhang, X.; Jiang, X.; Yuan, H.; Lee, J.S.; Barry, C.E., 3rd; Wang, H.; Zhang, W.; Zhang, Y. Pyrazinamide
inhibits trans-translation in Mycobacterium tuberculosis. Science 2011, 333, 1630–1632. [CrossRef] [PubMed]
13. Kim, H.; Shibayama, K.; Rimbara, E.; Mori, S. Biochemical Characterization of Quinolinic Acid
Phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv and Inhibition of Its Activity by
Pyrazinamide. PLoS ONE 2014, 9, e100062. [CrossRef] [PubMed]
14. Shi, W.L.; Chen, J.Z.; Feng, J.; Cui, P.; Zhang, S.; Weng, X.H.; Zhang, W.H.; Zhang, Y. Aspartate decarboxylase
(PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis. Emerg. Microbes Infect. 2014, 3, e58.
[CrossRef] [PubMed]
15. Boehringer, D.; Ban, N.; Leibundgut, M. 7.5-Å Cryo-EM Structure of the Mycobacterial Fatty Acid Synthase.
J. Mol. Biol. 2013, 425, 841–849. [CrossRef] [PubMed]
16. Ciccarelli, L.; Connell, S.R.; Enderle, M.; Mills, D.J.; Vonck, J.; Grininger, M. Structure and Conformational
Variability of the Mycobacterium tuberculosis Fatty Acid Synthase Multienzyme Complex. Structure 2013
21, 1251–1257. [CrossRef] [PubMed]
,
17. Imaizumi, T.; Nakagawa, H.; Hori, R.; Watanabe, Y.; Soga, S.; Iida, K.; Onodera, H. The synthesis and
evaluation of the antiproliferative activity of deacidified GEX1A analogues. J. Antibiot. 2017, 70, 675–679.
[CrossRef] [PubMed]
18. Hackbarth, C.J.; Chen, D.Z.; Lewis, J.G.; Clark, K.; Mangold, J.B.; Cramer, J.A.; Margolis, P.S.; Wang, W.;
Koehn, J.; Wu, C.; et al. N-Alkyl Urea Hydroxamic Acids as a New Class of Peptide Deformylase Inhibitors
with Antibacterial Activity. Antimicrob. Agents Chemother. 2002, 46, 2752–2764. [CrossRef] [PubMed]
19. Boyle, R.G.; Imogai, H.J.; Cherry, M. Preparation of Diarylureas as Chk-1 Kinase Inhibitors for the Treatment
of Cancer. Patent WO200,310,144,4A1, 11 August 2005.
20. Sorenson, W.R. Reaction of an Isocyanate and a Carboxylic Acid in Dimethyl Sulfoxide. J. Org. Chem. 1959
24, 978–980. [CrossRef]
,
21. Franzblau, S.G.; Witzig, R.S.; McLaughlin, J.C.; Torres, P.; Madico, G.; Hernandez, A.; Degnan, M.T.;
Cook, M.B.; Quenzer, V.K.; Ferguson, R.M.; et al. Rapid, low-technology MIC determination with clinical
Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay. J. Clin. Microbiol. 1998, 36,
362–366. [PubMed]
22. Zitko, J.; Servusová, B.; Paterová, P.; Mandíková, J.; Kubícˇek, V.; Kucˇera, R.; Hrabcová, V.;
Kuneš, J.; Soukup, O.; Doležal, M. Synthesis, Antimycobacterial Activity and In Vitro Cytotoxicity of
5-Chloro-N-phenylpyrazine-2-carboxamides. Molecules 2013, 18, 14807. [CrossRef] [PubMed]
23. Semelkova, L.; Konecna, K.; Paterova, P.; Kubicek, V.; Kunes, J.; Novakova, L.; Marek, J.; Naesens, L.;
Pesko, M.; Kralova, K.; et al. Synthesis and Biological Evaluation of N-Alkyl-3-(alkylamino)-pyrazine-2-
carboxamides. Molecules 2015, 20, 8687–8711. [CrossRef] [PubMed]
24. Nguta, J.M.; Appiah-Opong, R.; Nyarko, A.K.; Yeboah-Manu, D.; Addo, P.G. Current perspectives in drug
discovery against tuberculosis from natural products. Int. J. Mycobacteriol. 2015, 4, 165–183. [CrossRef]
[PubMed]

Molecules 2017, 22, 1797
16 of 16
25. Hsiao, C.H.; Tsai, T.F.; Hsueh, P.R. Characteristics of skin and soft tissue infection caused by non-tuberculous
mycobacteria in Taiwan. Int. J. Tuberc. Lung Dis. 2011, 15, 811–817. [CrossRef] [PubMed]
26. Katoch, V.M. Infections due to non-tuberculous mycobacteria (NTM). Indian J. Med. Res. 2004, 120, 290–304.
[PubMed]
27. Shimizu, F.; Hatano, Y.; Okamoto, O.; Katagiri, K.; Fujiwara, S.; Sato, S.; Kato, A.; Uezato, H.; Asato, Y.;
Takahashi, K. Mycobacterium smegmatis soft tissue infection. Int. J. Dermatol. 2012, 51, 1518–1520. [CrossRef]
[PubMed]
28. Gupta, A.; Bhakta, S. An integrated surrogate model for screening of drugs against Mycobacterium
tuberculosis. J. Antimicrob. Chemother. 2012, 67, 1380–1391. [CrossRef] [PubMed]
29. Espinel-Ingroff, A.; Fothergill, A.; Ghannoum, M.; Manavathu, E.; Ostrosky-Zeichner, L.; Pfaller, M.;
Rinaldi, M.; Schell, W.; Walsh, T. Quality control and reference guidelines for CLSI broth microdilution
susceptibility method (M 38-A document) for amphotericin B, itraconazole, posaconazole, and voriconazole.
J. Clin. Microbiol. 2005, 43, 5243–5246. [CrossRef] [PubMed]
30. Yew, W.W.; Leung, C.C. Antituberculosis drugs and hepatotoxicity. Respirology 2006, 11, 699–707. [CrossRef]
[PubMed]
31. Tostmann, A.; Boeree, M.J.; Peters, W.H.M.; Roelofs, H.M.J.; Aarnoutse, R.E.; van der Ven, A.J.A.M.;
Dekhuijzen, P.N.R. Isoniazid and its toxic metabolite hydrazine induce in vitro pyrazinamide toxicity.
Int. J. Antimicrob. Agents 2008, 31, 577–580. [CrossRef] [PubMed]
32. Zhang, Z.; Guo, K.; Bai, Y.; Dong, J.; Gao, Z.; Yuan, Y.; Wang, Y.; Liu, L.; Yue, T. Identification, Synthesis,
and Safety Assessment of Forchlorfenuron (1-(2-Chloro-4-pyridyl)-3-phenylurea) and Its Metabolites in
Kiwifruits. J. Agric. Food. Chem. 2015, 63, 3059–3066. [CrossRef] [PubMed]
33. Dolezal, M.; Tumova, L.; Kesetovicova, D.; Tuma, J.; Kral‘ova, K. Substituted N-phenylpyrazine-2-
carboxamides, their synthesis and evaluation as herbicides and abiotic elicitors. Molecules 2007, 12, 2589–2598.
[CrossRef] [PubMed]
34. Tumova, L.; Tuma, J.; Dolezal, M. Pyrazinecarboxamides as Potential Elicitors of Flavonolignan and
Flavonoid Production in Silybum marianum and Ononis arvensis Cultures In Vitro. Molecules 2011, 16,
9142–9152. [CrossRef] [PubMed]
35. Jones, R.N.; Barry, A.L. Optimal dilution susceptibility testing conditions, recommendations for MIC
interpretation, and quality control guidelines for the ampicillin-sulbactam combination. J. Clin. Microbiol.
1987, 25, 1920–1925. [PubMed]
36. Murashige, T.; Skoog, F. A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures.
Physiol. Plant. 1962, 15, 473–497. [CrossRef]
37. Kreft, I.; Fabjan, N.; Yasumoto, K. Rutin content in buckwheat (Fagopyrum esculentum Moench) food
materials and products. Food Chem. 2006, 98, 508–512. [CrossRef]
Sample Availability: Samples of the title compounds are available from the authors.
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).