Substituted N-(Pyrazin-2-yl)benzenesulfonamides; Synthesis, anti-infective evaluation, cytotoxicity, and in silico studies

Article abstract of DOI:10.3390/molecules25010138

We prepared a series of substituted N-(pyrazin-2-yl)benzenesulfonamides as an attempt to investigate the effect of different linkers connecting pyrazine to benzene cores on antimicrobial activity when compared to our previous compounds of amide or retro-amide linker type. Only two compounds, 4-amino-N-(pyrazin-2-yl)benzenesulfonamide (MIC = 6.25 μg/mL, 25 μM) and 4-amino-N-(6-chloropyrazin-2-yl)benzenesulfonamide (MIC = 6.25 μg/mL, 22 μM) exerted good antitubercular activity against M. tuberculosis H37Rv. However, they were excluded fromthe comparison as they-unlike the other compounds-possessed the pharmacophore for the inhibition of folate pathway, which was proven by docking studies. We performed target fishing, where we identified matrixmetalloproteinase-8 as a promising target for our title compounds that isworth future exploration.

Full text of DOI:10.3390/molecules25010138

molecules
Article
Substituted N-(Pyrazin-2-yl)benzenesulfonamides;
Synthesis, Anti-Infective Evaluation, Cytotoxicity,
and In Silico Studies
Ghada Bouz 1,
*
, Martin Juhás 1 , Lluis Pausas Otero 1, Cristina Paredes de la Red 1,
Ondrˇej Jand’ourek 1 , Klára Konecˇná 1 , Pavla Paterová 2, Vladimír Kubícˇek 1, Jirˇí Janoušek 1
,
Martin Doležal 1 and Jan Zitko 1,
*
1
Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203, 50005 Hradec Králové,
Czech Republic; juhasm@faf.cuni.cz (M.J.); lluis_pausas@hotmail.com (L.P.O.);
cristinaparedesdelared@gmail.com (C.P.d.l.R.); JANDO6AA@faf.cuni.cz (O.J.); konecna@faf.cuni.cz (K.K.);
kubicek@faf.cuni.cz (V.K.); janousj2@faf.cuni.cz (J.J.); dolezalm@faf.cuni.cz (M.D.)
Department of Clinical Microbiology, University Hospital, Sokolská 581, 500 05 Hradec Králové, Czech
Republic; pavla.paterova@fnhk.cz
2
*
Correspondence: 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.)
Academic Editors: Maria João Queiroz and Derek J. McPhee
Received: 1 December 2019; Accepted: 26 December 2019; Published: 29 December 2019
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: We prepared a series of substituted N-(pyrazin-2-yl)benzenesulfonamides as an attempt
to investigate the effect of different linkers connecting pyrazine to benzene cores on antimicrobial
activity when compared to our previous compounds of amide or retro-amide linker type. Only
two compounds, 4-amino-N-(pyrazin-2-yl)benzenesulfonamide (MIC = 6.25
µg/mL, 25
µM) and
4-amino-N-(6-chloropyrazin-2-yl)benzenesulfonamide (MIC = 6.25 g/mL, 22
µ
µM) exerted good
antitubercular activity against M. tuberculosis H37Rv. However, they were excluded from the comparison
as they—unlike the other compounds—possessed the pharmacophore for the inhibition of folate
pathway, which was proven by docking studies. We performed target fishing, where we identified
matrix metalloproteinase-8 as a promising target for our title compounds that is worth future exploration.
Keywords: anti-infectives; Mycobacterium tuberculosis; pyrazinamide; sulfonamide; target fishing
1. Introduction
Despite the availability of an effective treatment regimen against tuberculosis (TB), this infection
remains the leading cause of death from infectious diseases worldwide [
poor adherence to the complex and lengthy treatment regimen, co-infection with HIV, and drug
resistance all contribute to the lethality of this infection [ ]. As a part of our long-term research
1]. Inaccessibility to treatment,
1
on the derivatives of pyrazinamide (a first-line antitubercular) as potential antimycobacterial agents, we
report the synthesis and anti-infective evaluation of a series of N-(pyrazin-2-yl)benzenesulfonamides
(Figure 1c). Some of our previously prepared pyrazinecarboxamides exerted promising antimycobacterial
activity (Figure 1a) [2,3]. As a follow-up, we are investigating different linkers connecting the pyrazine
core to the aryl fragment and their effect on in vitro anti-infective activity. In the current series, we aim to
study the influence of the isosteric replacement of the retro-amide moiety in our previously published
series of N-(pyrazin-2-yl)benzamides (Figure 1b) [4] by the sulfonamide moiety in title compounds. The
detailed activity comparison between pyrazinecarboxamides (Figure 1a) and N-(pyrazin-2-yl)benzamides
(Figure 1b) is mentioned elsewhere [4].
Generally, sulfonamides exert a wide range of biological activities, including anti-tumoral [
5],
anti-inflammatory [ ], anti-convulsant [ ], and anti-infective [ ], among others. Nevertheless,
6
7
8
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Molecules 2020, 25, 138
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several compounds containing a pyrazine core directly connected to a sulfonamide moiety—like
in our case—are already documented in the literature with a wide range of pharmacological
applications [9]. Examples (Figure 2) include zibotentan, an endothelin receptor antagonist with
antitumor activity for prostate cancer [10]; halogenated N-(pyrazinyl)benzenesulfonamides, which
are clathrin-coated pit (CCP) chemokine receptor antagonists used in treating chemokine mediated
diseases (such as asthma) [11]; and sulfamethoxypyrazine (sulfalene), which is an antibacterial
sulfonamide agent [12]. As for antitubercular activity of sulfonamides, a fixed combination
of sulfamethoxazole [4-amino-N-(5-methylisoxazol-3-yl)benzenesulfonamide]—trimethoprim
[5-(3,4,5-trimethoxybenzyl)pyrimidine-2,4-diamine], also known as co-trimoxazole, has in vitro and
in vivo antimycobacterial activity against drug-resistant strains of Mycobacterium tuberculosis (Mtb),
and it is used for such cases [13
,14]. Sulfamethoxazole is a known inhibitor of bacterial dihydropteroate
synthase (DHPS), while trimethoprim is an inhibitor of dihydrofolate reductase (DHFR) [15,16].
Figure 1. Design rationale: general structure of (
a) pyrazinecarboxamides = amides; (b)
N-pyrazinylbenzamides = retro-amides; and (c) title compounds = sulfonamides.
Figure 2. The chemical structure of (a) zibotentan, (b) halogenated N-(pyrazinyl)benzensulfonamides,
and (c) sulfamethoxypyrazine (sulfalene).
2. Results and Discussion
2.1. Chemistry
The chemical synthesis entailed a simple one-step reaction in acetone with pyridine between
different sulfonyl chlorides and aminopyrazine (series 1a 14a), or 6-chloroaminopyrazine (series
1b 13b), at room temperature overnight (Scheme 1). Compounds 4a and 4b were obtained by
–
–
reducing compounds 3a and 3b, respectively. The purpose of attempting the synthesis with
6-chloroaminopyrazine, using the same sulfonyl chloride was to evaluate the effect of the chlorine
atom on anti-infective activity. It is common knowledge that the introduction of a chlorine atom to a
molecule increases its lipophilicity. For antimycobacterial activity, the lipophilicity of a compound is an
essential factor since mycobacteria have thick, lipid-rich mycolic cell walls [17]. Besides, chloropyrazine
derivatives, specifically anilides of 6-chloropyrazine-2-carboxylic acid, were proven previously to
possess in vitro antimycobacterial activity [18,19].
Scheme 1. General synthetic reaction. R1: H/Cl; R2: refer to Table 1.

Molecules 2020, 25, 138
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Table 1. Structure of prepared compounds, antimycobacterial activity expressed by minimum inhibitory
concentrations (MIC), and HepG2 cytotoxicity expressed by IC50 values.
a
b
IC50 (µM)
Antimycobacterial Activity MIC (µg/mL)
R (the Cut Line Represents
the Attachment Point)
No.
Mtb H37Rv
>100
50
M. kansasii
100
M. avium
>100
>100
>100
>100
>100
>100
100
M. smeg
≥250
250
M. aurum
≥500
≥500
≥500
≥500
>500
125
1a
1b
>1000
731
100
2a
>100
>100
>100
>100
6.25
25
≥500
250
>500 *
>500 *
>1000
>1000
775.6 *
>500
2b
25
NO2
3a
>100
>100
1.56
12.5
50
>500
250
3b
4a
≥500
≥500
250
250
4b ***
5a
6.25
25
250
>100
100
>100
>100
>100
>100
250
>1000
364
5b
>100
>100
100
250
250
6a
>100
>100
≥500
≥500
≥500
≥500
>1000
>1000
6b
7b
100
50
>100
125
125
308
8a
8b
9a
9b
>100
50
50
>100
>100
50
>100
50
≥500
≥500
125
250
>500 *
523.3
≥500
≥500
≥500
>100
>100
>100
>100
>250 *
179.5
≥500
10a
>100
50
>100
≥500
≥500
>250 *
11a
11b
12a
12b
13a
13b
>100
>100
100
>100
25
>100
>100
>100
>100
>100
>100
≥500
≥500
≥500
≥500
62.5
250
≥500
≥500
≥500
≥500
250
>1000
778.5
>250 *
302
100
12.5
50
100
>100
50
>500 *
>1000
50
125
14a
>100
100
>100
≥500
≥500
>250 *
Sulfamethoxazole
Sulfaclozine
Sulfaquinoxaline
Benzenesulfonamide
PZA **
3.13
6.25
3.13
>100
>100
0.2
3.13
12.5
12.5
>100
>100
25
12.5
25
250
250
≥500
250
n.t.
n.t.
50
125
250
n.t.
>100
>100
12.5
n.t.
31.25
≥500
15.625
25
125
n.t.
>104 [20]
79 × 103 [20]
n.t.
≥500
3.91
1.56
0.008
INH
RFM
n.t.
n.t.
CPX
n.t.
n.t.
n.t.
0.125
n.t.
* Measurement at higher concentrations was not possible due to precipitation of the tested compound in the cell
culture medium. ** MIC value from testing at pH = 5.6 (acidic) is 6.25–12.5 g/mL [ ]. The value stated in the table
µ
2
is from testing at pH = 6.6 (neutral). *** Compound 4b is structurally identical to the sulfaclozine standard. n.t.—not
tested. For structures of control drugs of the sulfonamide type refer to Figure 4.
The final products were purified using flash chromatography. They were isolated as solid
compounds, in yields ranging from 12–70% of chromatographically pure products. Chlorinated
compounds (series 1b–13b) had lower yields than the non-chlorinated ones (series 1a–14a) due to

Molecules 2020, 25, 138
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the electron-withdrawing property of the chlorine atom that reduces the strength of the primary
amine as a nucleophile. The final products were characterized by melting points, 1H- and 13C-NMR
spectra, IR spectroscopy, and elemental analysis. The obtained analytical data fully supported the
corresponding proposed structures. In the 1H-NMR spectra, the signal of the sulfonamidic hydrogen
appeared as a broad singlet at 12.10–10.27 ppm in DMSO-d6. Pyrazine hydrogens appeared at 8.98–8.04
as two singlets in the case of chlorinated compounds (series 1b
(J = 2.8–2.4 Hz) at 8.40–7.89 ppm in the case of non-chlorinated compounds (series 1a
–
13b), or a singlet and two doublets
1
–
14a). H-NMR
and 13C-NMR spectra of compounds 6a and 6b are presented in supplementary materials. Regarding
the IR spectra, the final compounds showed signals at 3105–2896 cm−1 attributed to the sulfonamidic
N–H stretch, at 1609–1460 cm−1 attributed to the aromatic C=C stretch, at 1406–1324 cm−1 attributed to
the S=O unsymmetrical stretch, and at 1189–1092 cm−1 attributed to the S=O symmetrical stretch.
2.2. Lipophilicity
Lipophilicity is a significant physico-chemical property in medicinal chemistry and drug design
in general. As stated earlier, lipophilicity plays a significant role specifically in the development of
new antituberculars. The log P values of prepared compounds were calculated using ChemDraw 18.1
(CambridgeSoft, Cambridge, MA, USA). Calculated lipophilicity (log P) values were plotted against
experimentally determined lipophilicity measures (log k). The measures were derived from retention
times measured using reverse-phase HPLC. Both values agreed as shown in the graph below (Figure 3).
Figure 3. Linear regression of log P (calculated) plotted against log k (experimental). Equation: log
P = 1.127 log k − 0.1149. (R2 = 0.9664, n = 25).
2.3. Acidobasic Properties
The acidic character of sulfonamide moiety determines the ionization state of title compounds
at physiological pH and inside the mycobacteria. It further affects lipophilicity and transport across
membranes. Three compounds 6b, 9b and 13b were selected and their pKa values were determined
experimentally using potentiometric and absorbance methods (Table 2). The values of pKa in water
were then predicted using different software to find the best value that gave the closest predictions to
the actual experimentally determined values. ChemDraw was not able to predict pKa values, whereas
values from the Molecular Operating Environment (MOE, Chemical Computing Group, Montreal, QC
Canada)—software is available with a paid license only [21], were far from the experimental ones.
Chemicalize was then used for the prediction of pKa values (September 2019, https://chemicalize.com/
developed by ChemAxon http://www.chemaxon.com). The latter values proved to be the closest to
the experiments (Table 2), and therefore Chemicalize was selected to predict the pKa values of the
remaining compounds (Table 3). The experimental pKa value of compound 4a was documented in the
literature (pKa = 6.04) [22]. This value was close to our predicted value from the Chemicalize web
application (pKa = 6.61).

Molecules 2020, 25, 138
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Table 2. Experimental and calculated pKa values of compounds 6b, 9b, and 13b.
Experimental
Potentiometric
Calculated
No.
Absorbance Method
ChemAxon
MOE
ChemDraw
Method
6b
9b
6.95
6.32
5.63
7.21
6.21
6.29
6.01
8.30
8.30
8.30
NA
NA
NA
6.38
13b
5.75
Table 3. pKa values of title compounds predicted using the Chemicalize web application.
No.
1a
pKa
6.11
6.05
6.16
6.09
5.80
No.
3b
4a
pKa
5.76
6.61
6.49
6.10
6.03
No.
6a
pKa
6.30
6.21
6.03
6.12
6.05
No.
9a
pKa
6.39
6.29
6.38
6.16
6.09
No.
12a
12b
13a
13b
14a
pKa
6.11
6.05
6.07
6.01
6.07
1b
2a
6b
7b
8a
9b
4b
5a
10a
11a
11b
2b
3a
5b
8b
2.4. Biological Activity
Six of our title compounds, namely 1a
,
1b, 2a, 3a, 4a, and 4b, have been previously mentioned in the
literature. Compound 1a was evaluated as an inhibitor of B-raf kinase [23], compound 1b was evaluated
as a phosphoinositide 3-kinase (PI3K) inhibitor [24], and compound 3a possessed potent in vitro and
in vivo nuclease activity against Leishmania infantum promastigotes [25]. Furthermore, compound
4a—also known as sulfapyrazine—has an antibacterial profile comparable to sulfadiazine [26], a
protective effect against sporozoite induced falciparum malaria [27], and sperm production and
gonadal development stimulation in animals [28]. Compound 4b—also known as sulfaclozine—is a
commonly used antiprotozoal to treat poultry coccidiosis [29]. It is important to note that compounds
4a and 4b were not tested previously against mycobacteria. Sulfaclozine was purchased later as
standard. All title compounds were checked for the presence of PAINS and aggregator features using
the ZINC15 utility (http://zinc15.docking.org/patterns/home). All the compounds were clear.
2.4.1. Antimycobacterial Activity Evaluation against Mycobacterium tuberculosis, Mycobacterium kansasii,
and Mycobacterium avium
All synthesized compounds were evaluated for in vitro antimycobacterial activity against M.
tuberculosis H37Rv, M. kansasii, and M. avium using microplate alamar blue assay. The antimycobacterial
activity results were expressed as a minimum inhibitory concentration (MIC) in
µg/mL against
isoniazid (INH) as standard. Structurally related sulfonamides, namely, sulfamethoxazole, sulfaclozine,
sulfaquinoxaline, and benzenesulfonamide were purchased and subjected to all anti-infective
evaluations (Figure 4).
Figure 4. Chemical structures of standards (a) sulfamethoxazole, (b) sulfaclozine, (c) sulfaquinoxaline,
and (d) benzenesulfonamide.

Molecules 2020, 25, 138
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Based on the obtained data presented in Table 1, compounds 4a (MICMtb = 6.25
µg/mL, 25 µM)
and 4b (MICMtb = 6.25 g/mL, 22 M) showed good antimycobacterial activity against Mtb. None
µ
µ
of the other compounds had significant activity against Mtb. Compounds 4a and 4b are well-known
inhibitors of bacterial dihydropteroate synthase (DHPS) [30]. However, their antimycobacterial activity
has so far not been investigated. Due to the high similarity of the bacterial and mycobacterial DHPS in
the active sites [31], we concluded that the inhibition of Mtb growth might be due to the inhibition of
mycobacterial DHPS. To rationalize this hypothesis, we used molecular docking to investigate the
binding poses of these two compounds in the Mtb DHPS (PDB ID: 1eye). Even though mycobacterial
DHPS showed only moderate overall amino acid sequence similarity (35 to 39%) to bacterial DHPS [32],
the active sites in both DHPSs were highly conserved [31]. Since the only available structure of Mtb
DHPS (PDB ID: 1eye) misses one of the key conserved flexible loops forming the binding site for a
para-aminobenzoic acid (PABA) fragment, as discussed and reviewed further elsewhere [33,34], we
docked the hypothetical products of the DHPS-catalyzed condensation of studied compounds 4a and
4b with a natural substrate 6-hydroxymethyl-7,8-dihydropterin diphosphate (HMDPdiP, Figure 5)
This was done to overcome the inaccuracies that could arise from flexible loop homology modelling.
The formation of such a condensed product was described in Bacillus anthracis DHPS (PDB ID: 3tye)
containing condensed sulfathiazole products [33], where it had also been rationalized using quantum
mechanics/molecular mechanics simulations [35]. For more details on the docking experiments, refer
to in silico studies from the materials and methods section. With regards to the pKa values of the
sulfonamide moiety (Tables 2 and 3), as well as the proposed mechanism of action (competitive
inhibition with DHPS natural substrate—deprotonated PABA), the hypothetical products (Figure 5) of
compounds 4a and 4b were docked as deprotonated sulfonamides.
Figure 5. Structures of (a) the hypothetic product of compound 4a and (b) 6-hydroxymethyl-7,8-
dihydropterin diphosphate (HMDPdiP) used for the docking experiments. Fragment of HMDPdiP in
the condensed product is in the rectangle.
The docking experiments showed two main potential positions with the poses scoring from 7.9
−
to 8.3. Positions with the pyrazine core filling the cavity above the active site to the right (formed by
−
Gly181 and Arg214 as seen in Figure 6) had a slightly better score than the positions with the pyrazine
to the left. This outcome was probably due to the hydrogen bonds between sulfonamide oxygen and
the Arg214 backbone. Similar interactions to Ser221 (corresponding to Arg214 of the mycobacterial
DHPS) was also seen in the DHPS of Bacillus anthracis for 7,8-dihydropteroate (in this case to the
carbonyl oxygen of the carboxylate, PDB ID: 3tya), and it also occurred between the sulfathiazole
product (PDB ID: 3tye). On the other hand, poses pointing to the pyrazine to the left had the pyrazine
favorably positioned for hydrogen bonding with the guanidine group of Arg233. As seen in Figure 6,
substitution of the pyrazine ring with chlorine does not have a significant effect on the predicted
binding mode, which justifies the similar antimycobacterial activity of the non-chlorinated compound
4a (MICMtb = 6.25
against Mtb.
µg/mL, 25 µM) to the chlorinated compound 4b (MICMtb = 6.25 µg/mL, 22 µM)

Molecules 2020, 25, 138
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Figure 6. Product of 4a (orange) and product of 4b (green) main poses from the docking experiments.
The pose of 7,8-dihydropteroate (grey) corresponds to the experimentally determined position seen in
bacterial DHPS [e.g., Burkholderia cenocepacia DHPS (PDB ID: 2y5s) or Bacillus anthracis DHPS (PDB
ID: 3tya)].
Compounds 2a (MICM. kansasii = 25
(MICM. kansasii = 25 g/mL, 76.3 M), and 12b (MICM kansasii = 12.5
antitubercular activity against M. kansasii, and it was higher than INH (MIC of INH was 25
182.3 M). For compounds 2a and 2b, particularly, this activity was relatable to its chemical structure.
µg/mL, 85.5
µ
M), 2b (MICM. kansasii = 25
µ
g/mL, 76.5
M), had selective
g/mL,
µM), 11b
µ
µ
µg/mL, 36.1
µ
µ
µ
The acetamido moiety may have worked as a pro-drug that was later hydrolyzed (supposedly by
M. kansasii amidase) to the free amino group. This is essential for anti-infective activity based on
interference with the folate synthesis pathway [36]. The fact that compounds 2a and 2b were inactive
against other mycobacterial strains suggested that the hydrolysis machinery in the latter strains was
inefficient to liberate the free amino forms due to the difference between substrate specificity of the
mycobacterial amidases among different mycobacterial strains [37
pharmacokinetic profile of compound 4a in man is already established and it shows that compound
2a is the major metabolite of compound 4a 39]. Compounds 4a (MICM. kansasii = 1.56 g/mL, 6.2 M)
and 4b (MICM. kansasii = 12.5 g/mL, 44 M) were also found to be more potent than INH against M.
,38]. It must be noted that the
[
µ
µ
µ
µ
kansasii. The addition of the chlorine atom and subsequent increased lipophilicity had a positive effect
on antimycobacterial activity against M. kansasii, as seen with compounds 11a (inactive; MICM. kansasii
> 100
MICM. kansasii = 100
4b (MICM. avium = 25
M. avium, making it the broadest agent in the spectrum of activity among prepared compounds.
Sulfamethoxazole showed potent antimycobacterial activity against Mtb (MICMtb = 3.13 g/mL, 12 M,
less potent than INH), M. kansasii (MICM. kansasii = 3.13 g/mL, 12 M, more potent than INH), and M.
avium (MICM. avium = 12.5 g/mL, 49 M, more potent than INH). As for sulfaclozine, its MIC values
µ
g/mL, 305.1
µ
M) vs. 11b (active; MICM. kansasii = 25
g/mL, 321.2 M) vs. 12b (active; MICM. kansasii = 12.5
g/mL, 88 M) was the only compound to exert antimycobacterial activity against
µ
g/mL, 76.3
µ
M), and 12a (inactive;
µ
µ
µg/mL, 36.1
µM). Compound
µ
µ
µ
µ
µ
µ
µ
µ
fully matched that of our freshly prepared compound 4b, sulfaquinoxaline, which was similar to
sulfaclozine in possessing anticoccidiosis activity [40]. It also showed good antimycobacterial activity,
while benzenesulfonamide, a classic carbonic anhydrase inhibitor [41], was completely inactive against
these three strains.
Estimated pKa values showed no direct correlation with antimycobacterial activity. pKa values of
compounds that exhibited antimycobacterial activity were scattered and could not be put in range.
Despite having the same pKa value of 7.14, compound 13a was active against M. smegmatis (MIC = 62.5
µ
g/mL) while compound 1a was inactive (MIC
≥
250 µg/mL). The electron-withdrawing property of
the additional chlorine atom increased the acidity of the sulfonamidic hydrogen as indicated by lower
estimated pKa values, as compared to the corresponding non-chlorinated compounds.

Molecules 2020, 25, 138
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When we sought to compare title compounds to pyrazinecarboxamides (amides) and
N-pyrazinylbenzamides (retro-amides)—which was our main objective—we found that substitution
with the sulfonamide bridge, in general, did not improve the antimycobacterial activity. Compounds 4a
and 4b were excluded from this comparison as they probably exerted their antimycobacterial activity
through a different pathway (folate pathway) than the rest of the title compounds. To achieve a more
precise assessment, we compared the antimycobacterial activity against Mtb of title sulfonamides
to the activity of amides and retro-amides bearing the same substitution both on the pyrazine (R1)
and benzene (R2) ring. It was apparent that the sulfonamide bridge had no positive influence on
activity. Some of the antimycobacterial activity against other mycobacterial strains was missing from
previous compounds; therefore, we focused on the activity against Mtb (Table 4). We note that the
methodologies used for MIC determination of amides were not identical to the updated methods
used for the retro-amides and title sulfonamides. Therefore, the comparisons should not be taken as a
comparison of precise MIC values but as an assessment of general trends instead.
Table 4. Comparison between the antimycobacterial activity against Mtb H37Rv expressed by the
minimum inhibitory concentrations (MIC) in µg/mL.
Substituents
Antimycobacterial Activity MIC (µg/mL) against Mtb H37Rv
R1
R2
Amide
100
Retro-amide
>100
Sulfonamide
>100 (1a)
>100 (6a)
50 (1b)
H
H
H
4-OCH3
H
25
>100
6-Cl
>100
25
R1—substitution on pyrazine, R2—substitution on benzene.
2.4.2. Antimycobacterial Activity Evaluation against Mycobacterium smegmatis and
Mycobacterium aurum
The full series was evaluated for antimycobacterial activity against two fast-growing mycobacterial
strains; M. smegmatis and M. aurum. The latter two strains have the advantage of being avirulent
surrogate organisms with better safety profiles and shorter replication time in comparison to M.
tuberculosis [26]. Microplate alamar blue assay was used for this test against INH, rifampicin, and
ciprofloxacin as standards. The most active compounds against M. smegmatis were compounds 13a
(MICM. smegmatis = 62.5
µg/mL, 219 µM), and interestingly, benzenesulfonamide (MICM. kansasii = 31.25
µ
g/mL, 199 M), which was inactive against the slow-growing strains of mycobacteria. Some
µ
compounds besides sulfaclozine, sulfaquinoxaline, and benzenesulfonamide showed moderate to low
activity (MICM. aurum = 125–150 µg/mL) against M. aurum.
2.4.3. Antibacterial and Antifungal Activity Evaluation
All prepared compounds were screened in vitro for biological activity against four Gram-positive
(Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus
faecalis) and four Gram-negative (Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Pseudomonas
aeruginosa) bacterial strains and eight fungal strains (Candida albicans, Candida krusei, Candida parapsilosis,
Candida tropicalis, Aspergillus fumigatus, Aspergillus flavus, Absidia corymbifera, Trichophyton interdigitale)
using microplate dilution assay with MICs determined by the naked eye. No antibacterial or antifungal
activities were observed for any of the tested compounds up to the highest tested concentration
(500 µM). Compounds 4a and 4b satisfied the sulfonamide structural requirements for antibacterial
activity [36], yet in our testing, they exerted solely antimycobacterial activity. These findings were
consistent with the standard used for screening. Sulfamethoxazole had antimycobacterial activity, but
it was not active against any of the tested bacterial strains. The antibacterial activity of the mentioned
sulfonamides might be present at concentrations beyond the concentration scale (500–0.97
µM) that
we used for such evaluations. In general, the single use of sulfamethoxazole as a quality standard is

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discouraged due to the low or absence of activity. If it is to be used, then it shall be combined with
trimethoprim [42].
2.4.4. In Vitro Cytotoxicity Assays
Since zibotentan is a pyrazine containing sulfonamide with cytotoxic activity, our compounds
were evaluated for similar activity in standard HepG2 (hepatocellular carcinoma) cell line. Furthermore,
established antituberculars are known to carry a risk of hepatotoxicity; thus, such evaluations provide
an insight into the hepatotoxic effect of the intended compound. Obtained results were expressed by the
inhibitory concentration required to decrease the viability of the cell population to 50% (IC50) compared
to a control of 100% cell viability (Table 1). The used CellTiter 96 assay was based on the reduction of
tetrazolium dye MTS in living cells to formazan, which was then determined colorimetrically. Based
on the obtained results, none of the prepared compounds exerted significant in vitro cytotoxicity (IC50
> 100
their corresponding non-chlorinated homologs (series 1a
series was compound 9b (IC50 = 179.5 M) (Figure 7). When compared to amides and retro-amides,
µM). Compounds with the chlorine atom (series 1b
–13b) were usually more cytotoxic than
–14a). The most toxic compound among all
µ
sulfonamides exerted a lesser extent of in vitro cytotoxicity [2–4].
Figure 7. Cytotoxic effect of different incubation concentrations of compound 9b on HepG2 cells.
2.4.5. Exploring Potential Targets and Activities using Docking Studies
Although designed as potential antimycobacterial compounds, the sulfonamides presented in
this paper only exerted low antimycobacterial activity (with the exception of 4a and 4b, as discussed
above). To discover new potential uses for our compounds, we ran a target fishing campaign based on
structural similarity between our N-(pyrazin-2-yl)benzensulfonamides and ligands from the RCSB PDB
database. First, potential targets were searched in the RCSB database, and then the title compounds
were docked into the selected enzymes, after which poses of the molecules were investigated to
establish their possible activities. For details of the target fishing and docking procedures, refer to in
silico studies from the materials and methods section.
The search yielded six targets, as presented in Table S1 in supplementary materials. Among
these was DHPS, which we have already investigated when determining the mechanism of action
of compounds 4a and 4b. Then we selected three targets from the remaining five to explore further.
Compound 1a and its derivatives have already been patented as potential inhibitors of B-raf kinase [23].
We then docked our compound into GTPase KRas, with no interesting results. Finally, matrix
metalloproteinase-8 (MMP-8; also known as neutrophil collagenase; PDB ID: 5h8x), was investigated
in more detail, where its recently described inhibitors are of the above-mentioned structural type
(Ar-SONH2-Ar) [43]. This endopeptidase is a part of a very complex proteolytic MMP enzyme
family, where its overexpression was linked to several pathological processes [43]. As several articles
have already described the coordination of metals by pyrazines [44,45], we investigated whether
the pyrazine in our compounds could coordinate the Zn2+ ion in the MMP-8. This could promote
potential inhibitory activity on MMP-8, as presented by Tauro et al. [43]. We tested this hypothesis

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using molecular docking studies. The results showed that in compounds 2a (Figure 8) and 12a (not
presented), one of the pyrazine nitrogens was favorably positioned above the Zn2+ ion, at a distance of
2.19 Å (comparable to the distance of the His-Zn2+ coordinate), as we presumed. Thus, it could easily
coordinate the Zn2+ in the receptor cavity (Figure 8). In the majority of poses, N10 was interacting with
the metal ion, as seen in Figure 8. The interaction of Zn2+ and N40 was observed only in some poses of
12a. This interaction in addition to the interactions of the sulfonamide moiety was identical to the
interactions observed in the original catechol ligand (hydrogen bonds with backbones of Ala161 and
Leu160). This in turn gave the basis for the presumed inhibitory activity of our ligands and promoted
their further evaluation as inhibitors of MMP-8 or other enzymes of the MMP family.
(a)
(b)
Figure 8. (a) Compound 2a (green carbons) docked to MMP-8 (PDB ID: 5h8x) in comparison to the
co-crystalized pose of the original catechol ligand (grey carbons). (b) Detail presenting interactions
between pyrazine nitrogen N10 and Zn2+ ion coordinated by His residues. (Measurements are in Å).
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. The progress of reactions was checked using Merck
Silica 60 F254 TLC plates (Merck, Darmstadt, Germany) with UV detection at wavelength 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).
This equipment was running under CEM’s SynergyTM software for setting and monitoring the
conditions of reactions. An internal infrared sensor monitored the temperature of the reaction mixture.
All obtained products were purified by a preparative flash chromatograph CombiFlash® Rf (Teledyne
Isco Inc., Lincoln, NE, USA) or preparative flash chromatograph Puriflash 420 XS (Intechim Chemicals,
Montlucon, France). The type of elution was gradient, using a mixture of hexane (LachNer, Neratovice,
Czech Republic) and ethyl acetate (Penta, Prague, Czech Republic) as the mobile phase. Silica gel
(0.040–0.063 mm, Merck, Darmstadt, Germany) was used as the stationary phase. NMR spectra were
recorded on a 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
a signal of solvent (2.49 for 1H and 39.7 for 13C in DMSO-d6). Infrared spectra were recorded by a
spectrometer FT-IR Nicolet 6700 (Thermo Scientific, Waltham, MA, USA) using the attenuated total
reflectance (ATR) methodology on a germanium crystal. Elemental analysis was performed on a vario
MICRO cube element analyzer (Elementar Analysensysteme, Hanau, Germany). All values regarding
elemental analyses were given as percentages. Melting points were determined in open capillary
on the Stuart SMP30 melting point apparatus (Bibby Scientific Limited, Staffordshire, UK) and were

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uncorrected. Yields were expressed as percentages of the theoretical yield, and they referred to the
isolated products after all purification steps.
3.2. Synthesis
3.2.1. Synthesis of Final Compounds, General Procedure
In a 50 mL beaker, aminopyrazine (3 mmol, 285 mg) or 6-chloroaminopyrazine (3 mmol, 388 mg)
was dissolved in 1 mL anhydrous pyridine with stirring at room temperature. Pyridine was used
to neutralize the hydrochloric acid generated upon reacting the sulfonyl chloride reagent with the
corresponding aminopyrazine or 6-chloroaminopyrazine. Corresponding benzensulfonyl chloride (3
mmol) was dissolved in approximately 2 mL of acetone in a 50 mL pear-shaped beaker with stirring
at room temperature. Then the dissolved content in pyridine was added dropwise to the dissolved
benzensulfonyl chloride in acetone with stirring at room temperature. The system was then sealed
with an appropriate stopper and left to react overnight under the same conditions. The next day, the
completion of the reaction was checked by TLC in a 3:1 EtOAc/hexane system. The reaction mixture
was transferred to a 100 mL beaker and washed with distilled water to minimize any losses. Then
the content was acidified with 10% (m/m) HCl drop-wise until a solid precipitate was formed, which
represented the non-ionized form of the product. The acidification step was aimed at ionizing the
added pyridine, and any unreacted aminopyrazine or 6-chloroaminopyrazine, to form a salt that
dissolved in the aqueous layer during the subsequent extraction step. EtOAc was added as little as
needed (30 mL) to dissolve the formed solid precipitate. Distilled water was then added (30 mL)
and the two phases were mixed vigorously at room temperature and then transferred to a 500 mL
separating funnel. The two layers were then allowed to settle and were separated into two 250 mL
beakers. The aqueous layer was rewashed with EtOAc (2
×
30 mL). The combined organic layers from
all extractions were then washed one last time with distilled water (100 mL) and then with brine (30
mL). The final organic layer was then transferred to a 150 mL beaker (or less based on the obtained
overall volume) and stirred with magnesium sulfate (4 mmol, 500 mg) as a desiccant for 10 min at
room temperature. Finally, the dispersion was filtrated through cotton and the resulting filtrate was
adsorbed to silica gel to perform flash chromatography using gradient elution 0% to 100% EtOAc in
hexane with 0.01% acetic acid as the mobile phase modifier.
3.2.2. Compounds 4a and 4b
Compounds 3a and 3b (1 mmol) were reduced to the corresponding 4-amino-N-(pyrazin-2-yl)
benzenesulfonamides (4a and 4b, respectively) with SnCl2 dihydrate (1.8 g, 8 mmol) in 10 mL of
ethanol with stirring under reflux (80 ◦C, 30 min). The solvent was evaporated under vacuum and
the obtained solid was dissolved in EtOAc (10 mL), and then washed with water (2
×
10 mL) and
brine (1 × 5 mL). The final organic layer was then transferred to a 50 mL beaker (or less based on
the estimated overall volume) and stirred with magnesium sulfate (2 mmol, 250 mg) as a desiccant
for 10 min at room temperature. Finally, the dispersion was filtrated with cotton and the filtrate was
adsorbed to silica gel to perform flash chromatography using gradient elution 0% to 100% EtOAc
in hexane.
3.3. Analytical Data of the Prepared Compounds
N-(pyrazin-2-yl)benzenesulfonamide (1a). White solid. Yield 68%; M.p. 203–205 ◦C {in the literature
204–208 ◦C [25]}; IR (ATR-Ge, cm−1): 2956 (NH stretch), 1587, 1558, 1532 (arom. stretch), 1345 (S=O
unsymmetrical stretch), 1092 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.02 (bs,
1H, sulfonamide), 8.12 (s, 1H, pyrazine), 8.04 (d, J = 2.8, 2H, pyrazine), 7.47 (d, J = 8.5 Hz, 2H, arom.),
7.14 (d, J = 8.5 Hz, 2H, arom.), 7.01 (t, J = 7.4 Hz, 2H, arom.). 13C-NMR (125 MHz, DMSO-d6)
148.3, 142.3, 140.1, 139.1, 135.1, 133.4, 129.4, 127.2. Elemental analysis found: C, 51.33%; H, 3.87%; N,
δ

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17.60%; S, 13.98%. Calculated for C10H9N3O2S (MW 235.26): C, 51.05%; H, 3.86%; N, 17.86%; S, 13.63%.
CAS#7471-20-7.
N-(6-chloropyrazin-2-yl)benzenesulfonamide (1b). White solid. Yield 41%; M.p. 198–200.2 ◦C; IR
(ATR-Ge, cm−1): 2940 (NH stretch), 1570, 1526, 1499 (arom. stretch), 1398 (S=O unsymmetrical stretch),
1163 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.96 (bs, 1H, sulfonamide), 8.40
(s, 1H, pyrazine), 8.24 (s, 1H, pyrazine), 8.05–7.89 (m, 2H, arom.), 7.74–7.62 (m, 1H, arom.), 7.66–7.53
(m, 2H, arom.). 13C-NMR (125 MHz, DMSO-d6)
147.5, 145.6, 139.4, 137.5, 133.8, 132.6, 129.5, 127.5.
δ
Elemental analysis found: C, 44.23%; H, 2.98%; N, 15.37%; S, 11.76%. Calculated for C10H8ClN3O2S
(MW 269.70): C, 44.53%; H, 2.99%; N, 15.58%; S, 11.89%. CAS#887310-35-2.
N-(4-(N-(pyrazin-2-yl)sulfamoyl)phenyl)acetamide (2a). Beige solid. Yield 84%; M.p. 243.8–245.6 ◦C;
IR (ATR-Ge, cm−1): 3105 (NH stretch), 1592, 1558, 1532 (arom. stretch), 1406 (S=O unsymmetrical
stretch), 1162 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.42 (bs, 1H, sulfonamide),
10.32 (s, 1H, acetylamide), 8.34 (s, 1H, pyrazine), 8.21 (d, J = 2.8, 2H, pyrazine), 7.87 (d, J = 8.5 Hz, 2H,
arom.), 7.74 (d, J = 8.5 Hz, 2H, arom.), 2.06 (s, 3H, –CH3). 13C-NMR (125 MHz, DMSO-d6)
169.2,
δ
148.4, 143.7, 142.3, 138.9, 134.9, 133.5, 128.7, 118.7, 24.3. Elemental analysis found: C, 49.11%; H, 4.05%;
N, 18.98%; S, 10.90%. Calculated for C12H12N4O3S (MW 292.31): C, 49.31%; H, 4.14%; N, 19.17%, S,
10.97%.
N-(4-(N-(6-chloropyrazin-2-yl)sulfamoyl)phenyl)acetamide (2b). White solid. Yield 65%; M.p.
217.9–219.9 ◦C; IR (ATR-Ge, cm−1): 3043 (NH stretch), 1592, 1527, 1501 (arom. stretch), 1400 (S=O
unsymmetrical stretch), 1164 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.90 (bs,
1H, sulfonamide), 10.54 (bs, 1H, acetylamide), 8.30 (s, 2H, pyrazine), 7.89 (m, 4H, arom.), 2.07 (s, 3H,
–CH3).13C-NMR (125 MHz, DMSO-d6)
169.4, 147.6, 145.6, 144.0, 137.1, 132.7, 132.5, 129.0, 118.6, 24.3.
δ
Elemental analysis found: C, 44.50%; H, 3.41%; N, 17.24%; S, 9.99%. Calculated for C12H11ClN4O3S
(MW 326.76): C, 44.11%; H, 3.39%; N, 17.15%; S, 9.81%.
4-nitro-N-(pyrazin-2-yl)benzenesulfonamide (3a). Yield 69%; M.p. 230.8–231.8 ◦C {in the literature
233–237 ◦C [25]}; IR (ATR-Ge, cm−1): 2990 (NH stretch), 1565, 1544, 1524 (arom. stretch), 1371 (S=O
unsymmetrical stretch), 1160 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
12.10 (bs,
1H, sulfonamide), 8.98 (s, 1H, pyrazine), 8.35 (d, J = 2.7 Hz, 2H, pyrazine), 7.87 (d, J = 9.2 Hz, 2H,
arom.), 7.14 (d, J = 9.2 Hz, 2H, arom.). 13C-NMR (125 MHz, DMSO-d6)
151.2, 147.9, 145.9, 141.4, 134.1,
δ
135.6, 129.4, 124.8. Elemental analysis found: C, 42.80%; H, 2.65%; N, 19.79% S, 11.13%. Calculated for
C10H8N4O4S (MW 280.26): C, 42.86%; H, 2.88%; N, 19.99%; S, 11.44%.
N-(6-chloropyrazin-2-yl)-4-nitrobenzenesulfonamide (3b). Yield 57%; M.p. 218.1–219.8 ◦C; IR (ATR-Ge,
cm−1): 2899 (NH stretch), 1535, 1514, 1494 (arom. stretch), 1391 (S=O unsymmetrical stretch), 1105
(S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.89 (bs, 1H, sulfonamide), 8.47 (s, 1H,
pyrazine), 8.28 (s, 1H, pyrazine), 8.18 (d, J = 9.1 Hz, 2H, arom.), 7.89 (d, J = 9.1 Hz, 2H, arom.). 13C-NMR
(125 MHz, DMSO-d6) 156.1, 151.7, 145.7, 144, 135.4, 134.1, 128, 128, 124.5, 124.5. Elemental analysis
δ
found: C, 37.88%; H, 2.05%; N, 17.79% S, 10.13%. Calculated for C10H7ClN4O4S (MW 314.70): C,
38.17%; H, 2.24%; N, 17.80%; S, 10.19%.
4-amino-N-(pyrazin-2-yl)benzenesulfonamide (4a). White powder. Yield 87%; M.p. 254–255.8 ◦C {in
the literature 257–259 ◦C [46]}; IR (ATR-Ge, cm−1): 2961 (NH stretch), 1575, 1561, 1520 (arom. stretch),
1354 (S=O unsymmetrical stretch), 1164 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.96 (bs, 1H, sulfonamide), 8.62 (s, 1H, pyrazine), 8.14 (d, J = 2.7 Hz, 2H, pyrazine), 7.72 (d, J = 9.1
Hz, 2H, arom.), 6.52 (d, J = 9.1 Hz, 2H, arom.), 5.6 (bs, 2H, amino). 13C-NMR (125 MHz, DMSO-d6)
δ
150.1, 147.9, 145.7, 141.9, 139.3, 135.6, 129, 124.7. Elemental analysis found: C, 47.88%; H, 4.01%; N,
22.16% S, 12.62%. Calculated for C10H8N4O4S (MW 250.28): C, 47.99%; H, 4.03%; N, 22.39%; S, 12.81%.
CAS#116-44-9.

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4-amino-N-(◦6-chloropyrazin-2-yl)benzenesulfonamide (4b). Light yellow solid. Yield 81%; M.p.
234.7–236.1 C; IR (ATR-Ge, cm−1): 3442, 3365 (NH2 stretch), 2929 (NH stretch), 1585, 1564, 1524
1
(arom. stertch), 1361 (S=O unsymmetrical stretch), 1150 (S=O symmetrical stretch); H-NMR (500
MHz, DMSO-d6)
J = 9.1 Hz, 2H, arom.), 6.89 (d, J = 9.1 Hz, 2H, arom.), 6.02 (bs, 2H, amino). 13C-NMR (125 MHz,
DMSO-d6) 152.1, 148.9, 145.5, 142.4, 139.2, 134.9, 128.4, 123.9. Elemental analysis found: C, 41.88%;
δ 11.06 (bs, 1H, sulfonamide), 8.22 (s, 1H, pyrazine), 8.14 (s, 1H, pyrazine), 7.48 (d,
δ
H, 3.05%; N, 19.86% S, 11.23%. Calculated for C10H9ClN4O2S (MW 284.72): C, 42.19%; H, 3.19%; N,
19.68%; S, 11.26%. CAS#102-65-8.
N-(pyrazin-2-yl)-4-(trifluoromethyl)benzenesulfonamide (5a). White solid. Yield 41%; M.p. 154.3–155.5
◦C; IR (ATR-Ge, cm−1): 2926 (NH stretch), 1609, 1590, 1532 (arom. stretch), 1324 (S=O unsymmetrical
stretch), 1145 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ 10.27 (bs, 1H, sulfonamide),
8.53 (s, 1H, pyrazine), 8.40 (d, J = 2.5 Hz, 2H, pyrazine), 8.04 (m, J = 7.8, 1.8, 0.9 Hz, 2H, arom.), 7.89 (m,
J = 8.7, 7.5, 0.7 Hz, 2H, arom.). 13C-NMR (125 MHz, DMSO-d6)
δ 148.8, 142.8, 142.5, 140.5, 136.4, 132.3,
131.4, 130.8, 125.5.
N-(6-chloropyrazin-2-yl)-4-(trifluoromethyl)benzenesulfonamide (5b). Orange solid. Yield 29%; M.p.
◦
125–127.2 C; IR (ATR-Ge, cm−1): 2930 (NH stretch), 1589, 1525, 1495 (arom. stretch), 1397 (S=O
unsymmetrical stretch), 1159 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
12.10 (bs,
1H, sulfonamide), 8.31 (s, 1H, pyrazine), 8.26 (s, 1H, pyrazine), 8.18 (d, J = 9.2 Hz, 2H, arom.), 7.68 (d,
J = 9.2 Hz, 2H, arom.). 13C-NMR (125 MHz, DMSO-d6)
151.7, 147.3, 145.6, 138.4, 137.6, 132.8 (q, J = 31
δ
δ
Hz), 130.4, 121.5 (q, J = 272.1 Hz), 121.0 (q, J = 3.7 Hz), 118.9 (q, J = 4.2 Hz).
4-methoxy-N-(pyrazin-2-yl)benzenesulfonamide (6a). White solid. Yield 89%; M.p. 231.5–233.9 ◦C; IR
(ATR-Ge, cm−1): 2924 (NH stretch), 1596, 1578, 1531 (arom. stretch), 1344 (S=O unsymmetrical stretch),
1158 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.30 (bs, 1H, sulfonamide), 8.30 (s,
1H, pyrazine), 8.18 (d, J = 2.4 Hz, 2H, pyrazine), 7.78 (d, J = 7.8 Hz, 2H, arom.), 7.64 (d, J = 7.8 Hz, 2H,
arom.), 4.12 (s, 3H, –OCH3). 13C-NMR (125 MHz, DMSO-d6)
162.9, 148.4, 142.3, 138.9, 134.9, 131.6,
δ
129.6, 114.5, 55.9. Elemental analysis found: C, 51.12%; H, 4.20%; N, 15.98%; S, 12.12%. Calculated for
C11H11N3O3S (MW 265.29): C, 49.80%; H, 4.18%; N, 15.84%; S, 12.09%.
N-(6-chloropyrazin-2-yl)-4-methoxybenzenesulfonamide (6b). White solid. Yield 54%; 185.1–186.3
◦C; IR (ATR-Ge, cm−1): 2994 (NH stretch), 1579, 1524, 1501 (arom. stretch), 1398 (S=O unsymmetrical
stretch), 1164 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.91 (bs, 1H, sulfonamide),
8.31 (s, 1H, pyrazine), 7.95 (s, 1H, pyrazine), 7.45 (d, J = 9.2 Hz, 2H, arom.), 6.98 (d, J = 9.2 Hz, 2H, arom.),
3.81–3.79 (m, 3H, -OCH3-). 13C-NMR (125 MHz, DMSO-d6)
163.2, 147.6, 145.7, 137.1, 132.4, 130.9,
δ
123.0, 114.6, 55.9. Elemental analysis found: C, 44.48%; H, 3.30%; N, 14.14%; S, 10.56%. Calculated for
C11H10ClN3O3S (MW 299.73): C, 44.08%; H, 3.36%; N, 14.02%; S, 10.70%.
4-bromo-N-(6-chloropyrazin-2-yl)benzenesulfonamide (7b). Beige solid. Yield 41%; M.p. 176.5–179.3
◦C; IR (ATR-Ge, cm−1): 2925 (NH stretch), 1574, 1525, 1497 (arom. stretch), 1396 (S=O unsymmetrical
stretch), 1153 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
12.07 (bs, 1H, sulfonamide),
8.36 (s, 2H, pyrazine), 7.89 (d, J = 8.6 Hz, 2H, arom.), 7.84 (d, J = 8.6 Hz, 2H, arom.). 13C-NMR (125
MHz, DMSO-d6) 147.3, 145.6, 138.8, 137.6, 132.7, 132.5, 129.6, 127.7. Elemental analysis found: C,
δ
34.56%; H, 2.10%; N, 12.01%; S, 8.96%. Calculated for C10H7BrClN3O2S (MW 348.60): C, 34.46%; H,
2.02%; N, 12.05%; S, 9.20%.
3,5-dimethyl-N-(pyrazin-2-yl)benzenesulfonamide (8a). White solid. Yield 56%; M.p. 184–187 ◦C;
IR (ATR-Ge, cm−1): 2958 (NH stretch), 1588, 1530, 1504 (arom. stretch), 1345 (S=O unsymmetrical
1
stretch), 1189 (S=O symmetrical stretch). H-NMR (500 MHz, DMSO-d6)
δ 11.30 (bs, 1H, sulfonamide),
8.30 (s, 1H, pyrazine), 7.88 (s, 1H, arom.), 7.74 (s, 2H, arom.), 2.34 (s, 6H, –CH3). 13C-NMR (125 MHz,
DMSO-d6) 148.3, 142.4, 140.1, 139.8, 139, 135, 134.8, 124.6, 20.9. Elemental analysis found: C, 55.34%;
δ
H, 4.91%; N, 15.66%; S, 12.30%. Calculated for C12H13N3O2S (MW 263.32): C, 54.74%; H, 4.98%; N,
15.96%; S, 12.18%.

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N-(6-chloropyrazin-2-yl)-3,5-dimethylbenzenesulfonamide (8b). Dark yellow-brownish solid. Yield
25%; M.p. 148.1–150.1 ◦C; IR (ATR-Ge, cm−1): 2930 (NH stretch), 1573, 1525, 1494 (arom. stretch),
1394 (S=O unsymmetrical stretch), 1159 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.82 (bs, 1H, sulfonamide), 8.35 (s, 2H, pyrazine), 7.58 (s, 2H, arom.), 7.35 (s, 1H, arom.), 2.38 (s,
6H, –CH3).13C-NMR (125 MHz, DMSO-d6)
δ147.5, 145.6, 139.2, 138.9, 137.2, 135.1, 132.8, 125.2, 20.9.
Elemental analysis found: C, 48.35%; H, 4.49%; N, 14.15%; S, 10.05%. Calculated for C12H12ClN3O2S
(MW 297.03): C, 48.41%; H, 4.06%; N, 14.11%; S, 10.77%.
2,4,6-trimethyl-N-(pyrazin-2-yl)benzenesulfonamide (9a). White solid. Yield 67%; M.p. 200–201.9 ◦C;
IR (ATR-Ge, cm−1): 2949 (NH stretch), 1601, 1586, 1530 (arom. stretch), 1337 (S=O unsymmetrical
stretch), 1154 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.52 (bs, 1H, sulfonamide),
8.22 (s, 1H, pyrazine), 8.08 (d, J = 2.5 Hz, 2H, pyrazine), 7.01 (s, 2H, arom.), 2.64 (s, 6H, -CH3), 2.22 (s,
3H, -CH3).13C-NMR (125 MHz, DMSO-d6)
148.9, 142.5, 142.0, 139.4, 138.1, 134.2, 134.1, 131.8, 22.4,
δ
20.6. Elemental analysis found: C, 56.22%; H, 5.42%; N, 14.88%; S, 11.92%. Calculated for C13H15N3O2S
(MW 277.34): C, 56.30%; H, 5.45%; N, 15.15%; S, 11.56%.
N-(6-chloropyrazin-2-yl)-2,4,6-trimethylbenzenesulfonamide (9b). Beige solid. Yield 45%; M.p.
179.7–180.2 ◦C; IR (ATR-Ge, cm−1): 2925 (NH stretch), 1566, 1525, 1498 (arom. stretch), 1331 (S=O
unsymmetrical stretch), 1186 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.95 (bs,
1H, sulfonamide), 8.24 (s, 1H, pyrazine), 8.15 (s, 1H, pyrazine), 7.04 (s, 2H, arom.), 2.66 (s, 6H, –CH3),
2.24 (s, 3H, –CH3).13C-NMR (125 MHz, DMSO-d6)
156.8, 145.1, 144.9, 141.1, 137.2, 135.1, 134.9,
δ
130.4, 22.6, 21.9. Elemental analysis found: C, 49.87%; H, 4.51%; N, 13.11%; S, 9.99%. Calculated for
C13H14ClN3O2S (MW 311.78): C, 50.08%; H, 4.53%; N, 13.48%; S, 10.28%.
2,3,5,6-tetramethyl-N-(pyrazin-2-yl)benzenesulfonamide (10a). White solid. Yield 49%; M.p. 221–223.7
◦C; IR (ATR-Ge, cm−1): 2955 (NH stretch), 1585, 1526, 1500 (arom. stretch), 1362 (S=O unsymmetrical
stretch), 1174 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.55 (bs, 1H, sulfonamide),
8.18 (s, 1H, pyrazine), 8.09 (d, J = 2.5 Hz, 2H, pyrazine), 7.23 (s, 1H, arom.), 2.55 (s, 6H, –CH3), 2.21 (s,
6H, –CH3). 13C-NMR (125 MHz, DMSO-d6)
148.9, 142.1, 138.1, 135.9, 135.7, 135.3, 134.6, 134.0, 20.7,
δ
17.5. Elemental analysis found: C, 57.45%; H, 5.79%; N, 14.08%; S, 10.72%. Calculated for C14H17N3O2S
(MW 291.37): C, 57.71%; H, 5.88%; N, 14.42%; S, 11.00%.
N-(pyrazin-2-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide (11a). Light yellow solid. Yield 17%;
M.p. 264–267.1 ◦C; IR (ATR-Ge, cm−1): 2930 (NH stretch), 1591, 1569, 1540 (arom. stretch), 1350 (S=O
unsymmetrical stretch), 1161 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.41 (bs,
1H, sulfonamide), 8.35 (s, 1H, pyrazine), 8.21 (d, J = 2.4 Hz, 2H, pyrazine), 7.44 (s, 1H, arom.), 6.98
(d, J = 8.4 Hz, 2H, arom.), 4.33–4.25 (m, 4H, –CH2–). 13C-NMR (125 MHz, DMSO-d6)
156.2, 153.9,
δ
147.0, 144.5, 137.4, 136.1, 131.9, 117.3, 115.3, 111.9, 64.2. Elemental analysis found: C, 49.47%; H, 3.97%;
N, 14.55%; S, 10.87%. Calculated for C12H10N3O4S (MW 293.30): C, 49.14%; H, 3.78%; N, 14.33%; S,
10.93%.
N-(6-chloropyrazin-2-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide (11b). White solid. Yield 8%;
M.p. 244.6–247◦C; IR (ATR-Ge, cm−1): 2896 (NH stretch), 1580, 1529, 1498 (arom. stretch), 1323 (S=O
unsymmetrical stretch), 1162 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ 11.81 (bs,
1H, sulfonamide), 8.34 (s, 2H, pyrazine), 8.28 (s, 1H, arom.), 7.41 (d, J = 9 Hz, 2H, arom.), 4.42–4.26
(m, 4H, –CH2–). 13C-NMR (125 MHz, DMSO-d6) 156.8, 153.9, 147.0, 144.9, 135.1, 134.9, 131.9, 117.3,
115.3, 111.9, 64.2. Elemental analysis found: C, 43.74%; H, 3.01%; N, 12.54%; S, 9.54%. Calculated for
C12H10ClN3O4S (MW 327.74): C, 43.98%; H, 3.08%; N, 12.82%; S, 9.78%.
N-(pyrazin-2-yl)-[1,10-biphenyl]-4-sulfonamide (12a). Light yellow solid. Yield 79%; M.p. 242.2–244.3
◦C; IR (ATR-Ge, cm−1): 3070 (NH stretch), 1594, 1566, 1531 (arom. stretch), 1345 (S=O unsymmetrical
stretch), 1172 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.62 (bs, 1H, sulfonamide),
8.40 (s, 1H, pyrazine), 8.24 (d, J = 2.5 Hz, 2H, pyrazine), 7.79 (d, J = 9.2 Hz, 4H, arom.), 7.65 (d, J = 9 Hz,
2H, arom.), 7.39 (t, J = 9 Hz, 3H, arom.). 13C-NMR (125 MHz, DMSO-d6)
148.3, 144.9, 142.4, 139.1,
δ

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138.9, 138.5, 135.1, 129.3, 128.8, 128.0, 127.6, 127.3. Elemental analysis found: C, 61.32%; H, 4.21%;
N, 13.01%; S, 10.30%. Calculated for C16H13N3O2S (MW 311.36): C, 61.72%; H, 4.21%; N, 13.50%; S,
10.30%.
N-(6-chloropyrazin-2-yl)-[1,10-biphenyl]-4-sulfonamide (12b). White solid. Yield 40%; M.p. 229.3–230.3
◦C; IR (ATR-Ge, cm−1): 2937 (NH stretch), 1596, 1566, 1525 (arom.), 1397 (S=O unsymmetrical stretch),
1159 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
12.01 (bs, 1H, sulfonamide), 8.34 (s,
1H, pyrazine), 8.14 (s, 1H, pyrazine), 7.49 (d, J = 9.2 Hz, 4H, arom.), 7.25 (d, J = 9 Hz, 2H, arom.), 7.04 (t,
J = 9 Hz, 3H, arom.). 13C-NMR (125 MHz, DMSO-d6)
147.5, 145.7, 145.2, 138.4, 138.2, 137.5, 132.6,
δ
129.3, 128.9, 128.3, 127.6, 127.3. Elemental analysis found: C, 55.74%; H, 3.54%; N, 12.48%; S, 9.41%.
Calculated for C16H12ClN3O2S (MW 345.80): C, 55.57%; H, 3.50%; N, 12.15%; S, 9.27%.
N-(pyrazin-2-yl)naphthalene-2-sulfonamide (13a). White solid. Yield 57%; M.p. 223.5–224.7 ◦C; IR
(ATR-Ge, cm−1): 2925 (stretch NH), 1589, 1576, 1530 (arom. stretch), 1341 (S=O unsymmetrical stretch),
1160 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
11.67 (bs, 1H, sulfonamide), 8.77 (s,
1H, pyrazine), 8.65 (d, J = 2.4 Hz, 2H, pyrazine), 7.92 (m, 3H, arom.), 7.68 (m, 4H, arom.).13C-NMR
(125 MHz, DMSO-d6) 148.3, 142.3, 139.1, 137.1, 135.1, 134.6, 131.7, 129.6, 129.5, 129.3, 128.8, 128.0,
δ
127.9, 122.4. Elemental analysis found: C, 58.91%; H, 3.89%; N, 14.38%; S, 11.61%. Calculated for
C14H11N3O2S (MW 285.32): C, 58.94%; H, 3.89%; N, 14.73%; S, 11.24%.
N-(6-chloropyrazin-2-yl)naphthalene-2-sulfonamide (13b). Light yellow. Yield 33%; M.p. 204.6–207
◦C; IR (ATR-Ge, cm−1): 3056 (NH stretch), 1522, 1505, 1460 (arom. stretch), 1398 (S=O unsymmetrical
stretch), 1169 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ
12.05 (bs, 1H, sulfonamide),
8.35 (s, 1H, pyrazine), 8.30 (s, 1H, pyrazine), 8.20 (m, arom.), 7.93 (m, 3H, arom.), 7.76–7.64 (m, 4H,
arom.). 13C-NMR (125 MHz, DMSO-d6)
156.0, 147.5, 146.0, 145.6, 137.4, 136.3, 134.7, 132.5, 131.6,
δ
130.6, 129.6, 129.6, 128.7, 128.0, 128.0. Elemental analysis found: C, 52.89%; H, 3.10%; N, 13.07%; S,
10.04%. Calculated for C14H10ClN3O2S (MW 319.76): C, 52.59%; H, 3.15%; N, 13.14%; S, 10.03%.
N-(pyrazin-2-yl)quinoline-6-sulfonamide (14a). Beige solid. Yield 61%; M.p. 249.4–252 ◦C; IR (ATR-Ge,
cm−1): 2927 (NH stretch), 1585, 1552, 1533 (arom. stretch), 1339 (S=O unsymmetrical stretch), 1159
(S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6)
δ 11.53 (bs, 1H, sulfonamide), 8.87 (s, 1H,
pyrazine), 8.66 (d, J = 2.5 Hz, 2H, pyrazine), 7.99–7.14 (m, 6H, arom.). 13C-NMR (125 MHz, DMSO-d6)
153.1, 151.6, 147.5, 143.2, 137.1, 136.5, 136.0, 135.2, 131.7, 131.6, 128.6, 126.0, 122.8. Elemental analysis
found: C, 54.82%; H, 3.37%; N, 19.77%; S, 10.40%. Calculated for C13H10N4O2S (MW 286.31): C,
54.54%; H, 3.52%; N, 19.57%; S, 11.20%.
3.4. Log k Determination
Log k was determined using an Agilent Technologies 1200 SL liquid chromatograph with a
Diode-array Detector SL G1315C (Agilent Technologies Inc., Colorado Springs, CO, USA), with
pre-column ZORBAX XDB-C18 5
mm 250 mm (both Agilent Technologies Inc.). The mobile phase consisted of MeOH (HPLC grade,
70%) and H2O (HPLC-Milli-Q Grade, 30%) with 0.01% acetic acid as the mobile phase modifier. The flow
µm, 4 mm
×
4 mm and column ZORBAX Eclipse XDB-C18 5 µm, 4.6
×
rate was 1.0 mL/min, and samples were injected at a volume of 20 µL, where the column temperature
was 30 ◦C. The detection and monitoring wavelengths were 210 nm and 270 nm, respectively. Retention
times (Rt) were measured in minutes. The dead time of the system (Dt) was determined as the retention
time of the KI methanolic solution. Capacity factors k for individual compounds were calculated
according to the formula k = (Rt
and it was used as a measure of lipophilicity.
−
Dt)/Dt. Capacity factor k was converted to the log scale and (log k),
3.5. pKa Determination
For the spectrophotometric method, 1
µM solution of the compound in question was prepared in
methanol. The spectra of the sample at several (at least seven) different pH values were measured

Molecules 2020, 25, 138
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against blank (water) using a Helios spectrophotometer (Unicam Cambridge). The pH valued covered
the region in which all sample molecules were converted from ionized to molecular form or vice versa.
Each cuvette contained 200 µL of the sample solution and 1.8 mL of the corresponding buffer. The
pH values used and the absorbances allowed us to calculate the pKa of the sample according to the
following equation:
ꢀ
ꢁ
Ai − A
A − Au
pKa = pH + log
where A is the absorbance of the compound at the corresponding buffer, Ai is the absorbance of the
ionized state of the compound and Au is the absorbance of the unionized state. The final pKa was the
average of all the pKa at different pH values. For the potentiometric measurements, 1 µM solution of
the compound in question was prepared in 3:2 water:methanol, with the addition of 100
µL of 0.1%
NaOH. Then small amounts (100
µ
L) of 0.01% HCl were added repeatedly and the pH was measured
each time using a pH meter (inoLab® pH 7110 Czech Republic). The inflection point of the titration
curve determined the pKa. Each compound was measured two times and the resulting pKa was the
average of two values.
3.6. Biological Assays
For details, refer to the supplementary materials.
3.7. In Silico Studies
3.7.1. DHPS Docking
For the docking studies, we used the rigid receptor settings of the template docking feature
incorporated in MOE 2019.0101 (Chemical Computing Group, Montreal, Canada) [21]. For the template,
all heavy atoms of the pteridine core were selected as these atoms, in reality, were already inside
the enzyme. The compounds were docked to the publicly available structure of Mtb DHPS (PDB
ID: 1eye). Final refinement (Refinement: Rigid) of the pose was done using the default GBVI/WSA
dG scoring function. To test the ability of such settings to predict the correct binding poses, we
docked 7,8-dihydropteroate using the same settings, and we observed correct interactions of the
p-aminobenzoic acid carboxylic group. This was as seen in the crystallographic structures of such
products in, for example, Burkholderia cenocepacia DHPS (PDB ID: 2y5s) or Bacillus anthracis DHPS (PDB
ID: 3tya).
3.7.2. Target Fishing
For the target fishing, we searched the RCSB PDB database (https://www.rcsb.org/) for ligands
containing benzenesulfonamide moiety using the ‘Ligand search’ utility. The obtained ligands were
imported into the MOE database (MOE 2019.0101) [21] and then filtered using the SMARTS string
“cS(O)(O)[NH]c” to retrieve ligands that met the aromatic-SONH-aromatic scaffold. The resulting
database was used for similarity searching based on the MACCS structural keys (bit-packed) with
Tanimoto > 0.75, based on compound 1a as a template. The resulting compounds were inspected
visually to remove all unrelated compounds, e.g., compounds with a highly substituted aromatic ring.
Ligands in the final selection were searched in the RCSB PDB database for the associated co-crystalized
targets, which were considered candidate targets for the title molecules of this manuscript.
3.7.3. Other Targets Docking
For the docking study, we used the MOE 2019.0101 [21]. The docking was carried out on a rigid
receptor using default dock settings. The poses generated by the Triangle Matcher placement method
were scored using the London dG function. The best poses were refined (Refinement: Rigid) and scored
using the GBVI/WSA dG scoring function. Concerning the predicted pKa values of our compounds,
the deprotonated forms were used as ligands.

Molecules 2020, 25, 138
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4. Conclusions
To conclude, as a part of our ongoing research, we designed and synthesized 25 different
substituted N-(pyrazin-2-yl)benzenesulfonamides. This effort was an attempt to study the influence of
different linkers connecting the pyrazine core to different aromatic moieties on antimicrobial activity and
antitubercular activity in particular (amide vs. retro-amide vs. sulfonamide linkers). Nineteen of the title
compounds were not previously described in literature when we searched on the 5th of March 2019 using
SciFinder (Columbus, OH, USA) and Reaxys. Compounds 1a, 1b, 2a, 3a, 4a, and 4b were published
but not evaluated for antimycobacterial activity. We evaluated the prepared compounds for their
anti-infective activity against Mycobacterium tuberculosis and four other nontubercular mycobacterial
strains, along with antibacterial and antifungal activity evaluation. Based on the obtained results,
we concluded that the introduction of a sulfonamide linker did not bring significant improvement
to antimicrobial activity when compared to the other amide or retro-amide linkers. The exception
includes compounds 4a (MICMtb = 6.25 µg/mL, 25 µM) and 4b (MICMtb = 6.25 µg/mL, 22 µM), which
showed good antitubercular activity against Mtb. Such activity may be attributed to the presence
of a free amino group in the molecule, which characterizes antimicrobial sulfonamides, and not to
the sulfonamide linker itself. Compounds 2a (MICM. kansasii = 25
g/mL) selectively inhibited the growth of Mycobacterium kansasii, suggesting that these two compounds
targeted a certain pathway not common among all mycobacterial strains. Compound 13a showed
promising antimycobacterial activity against M. smegmatis (MICM. smegmatis = 62.5 g/mL, 219 M).
Some compounds showed moderate to weak activity against M. aurum. Among them all, compound 4a
had a broad spectrum of antimycobacterial activity with good activity against Mtb (MIC = 6.25 g/mL)
and moderate activity against M. kansasii (MIC = 12.5 g/mL), M. avium (MIC = 25 g/mL), and M.
aurum (MIC = 250 g/mL). No antibacterial or antifungal activity was observed for any of the prepared
µg/mL) and 12b (MICM. kansasii = 12.5
µ
µ
µ
µ
µ
µ
µ
compounds. Regarding in vitro cytotoxicity, when compared to reference amides or retro-amides, the
title compounds had a more favorable profile. The title compounds were explored for other activities
by docking studies. These studies further rationalized the antimycobacterial activity of the most active
compounds 4a and 4b as inhibitors of Mtb DHPS. Finally, we presented potentially active poses of the
compound 2a that could rationalize its inhibitory activity on MMP-8 or other MMP family enzymes
implicated in several pathological conditions.
Supplementary Materials: Biological assays; table of outcomes of target fishing; 1H-NMR and 13C-NMR spectra
of compounds 6a and 6b.
Author Contributions: G.B., M.D., and J.Z. conceived and designed the experiments; G.B., L.P.O., and C.P.d.l.r.
performed chemical synthesis and purifications; G.B. performed the pKa measurements by absorbance and
potentiometric methods, evaluated analytical data, and interpreted the results of anti-infective screening; M.J.
performed the pKa predictions and docking studies; V.K. performed lipophilicity measurements using HPLC; O.J.,
K.K., and P.P. performed biological assays (antimycobacterial, antibacterial, antifungal); J.J. carried out cytotoxicity
screening and interpreted the results; G.B. and M.J. wrote the paper. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was supported by the Ministry of Education, Youth and Sports of the Czech
Republic (SVV 260 401), the Grant Agency of Charles University (project C C3/1572317), EFSA-CDN (No.
CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by ERDF, and by CELSA—Project title: Structure-based design
of new antitubercular medicines—KU Leuven (Arthur Van Aerschot)—Charles University in Prague (Martin
Doležal).
Acknowledgments: Computational resources were supplied by the Ministry of Education, Youth and Sports of
the Czech Republic under the Projects CESNET (Project No. LM2015042) and CERIT-Scientific Cloud (Project No.
LM2015085) provided within the program Projects of Large Research, Development and Innovations Infrastructures.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of compounds are available from the authors.
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