(Pyrazin-2-Yl)Benzenesulfonamides; Synthesis, Anti-Infective Evaluation, Cytotoxicity, and in Silico Studies

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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, OndˇrejJand’ourek 1 , Klára Koneˇcná 1 , Pavla Paterová 2, Vladimír Kubíˇcek 1, Jiˇrí 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; [email protected] (M.J.); [email protected] (L.P.O.); [email protected] (C.P.d.l.R.); [email protected] (O.J.); [email protected] (K.K.); [email protected] (V.K.); [email protected] (J.J.); [email protected] (M.D.) 2 Department of Clinical Microbiology, University Hospital, Sokolská 581, 500 05 Hradec Králové, Czech Republic; [email protected] * Correspondence: [email protected] (G.B.); [email protected] (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

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 µg/mL, 22 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 [1]. Inaccessibility to treatment, poor adherence to the complex and lengthy treatment regimen, co-infection with HIV, and drug resistance all contribute to the lethality of this infection [1]. As a part of our long-term research 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 (Figure1c). Some of our previously prepared pyrazinecarboxamides exerted promising antimycobacterial activity (Figure1a) [ 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 (Figure1b) [ 4] by the sulfonamide moiety in title compounds. The detailed activity comparison between pyrazinecarboxamides (Figure1a) and N-(pyrazin-2-yl)benzamides (Figure1b) is mentioned elsewhere [4]. Generally, sulfonamides exert a wide range of biological activities, including anti-tumoral [5], anti-inﬂammatory [6], anti-convulsant [7], and anti-infective [8], among others. Nevertheless,

Molecules 2020, 25, 138; doi:10.3390/molecules25010138 www.mdpi.com/journal/molecules Molecules 2020, 25, 138 2 of 20 several compoundsMolecules 2020 containing, 25, x a pyrazine core directly connected to a sulfonamide2 of 20 moiety—like in our case—are already documented in the literature with a wide range of pharmacological applications [9]. Examples (Figure2) 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 sulfamethoxazoleFigure 1. Design [4-amino- rationale:N -(5-methylisoxazol-3-yl)benzenesulfonamide]—trimethoprimgeneral structure of (a) pyrazinecarboxamides = amides; (b) [5-(3,4,5-trimethoxybenzyl)pyrimidine-2,4-diamine],N-pyrazinylbenzamides = retro-amides; and (c) title compounds also known = sulfonamides as co-trimoxazole,. has in vitro and in vivo antimycobacterialGenerally, sulfonamides activity againstexert a wide drug-resistant range of biological strains activities, of Mycobacteriumincluding anti-tumoral tuberculosis [5], (Mtb), and it is usedanti for-inflammatory such cases [6] [13, anti,14-convulsant]. Sulfamethoxazole [7], and anti-infective is a known [8], among inhibitor others. Nevertheless, of bacterial several dihydropteroate compounds containing a pyrazine core directly connected to a sulfonamide moiety—like in our synthase (DHPS),Moleculescase—are while2020 already, 25, xtrimethoprim documented in isthe an literature inhibitor with ofa wide dihydrofolate range of pharmacological reductase applications (DHFR)2 of 20 [15,16]. [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 Figure 1.usedFigure Design for such 1. casesDesign rationale: [13,14] rationale:. Sulfamethoxazole general general structure structure is a of knownof (a) (a pyrazinecarboxamides) inhibitor pyrazinecarboxamides of bacterial = dihydropteroate amides; = (b) amides; (b) N-pyrazinylbenzamidessynthaseN-pyrazinylbenzamides (DHPS), while= retro-amides; trimethoprim = retro-amides; andis anand inhibitor ( c(c)) titletitle compounds compoundsof dihydrofolate = sulfonamides= reductasesulfonamides.. (DHFR) [15,16].

Generally, sulfonamides exert a wide range of biological activities, including anti-tumoral [5], anti-inflammatory [6], anti-convulsant [7], and anti-infective [8], among others. Nevertheless, 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 Figure 2.antimycobacterialTheFigure chemical 2. The chemical structureactivity structure against of (a ofdrug) zibotentan,(a) zibotentan,-resistant strains( (bb) halogenated) halogenated of Myco Nbacterium-(pyrazinyl)benzensulfonamides,N-(pyrazinyl)benzensulfonamides, tuberculosis (Mtb), and it is used for such cases [13,14]. Sulfamethoxazole is a known inhibitor of bacterial dihydropteroate c and (c) sulfamethoxypyrazine (sulfalene). and ( ) sulfamethoxypyrazinesynthase (DHPS), while trimethoprim (sulfalene). is an inhibitor of dihydrofolate reductase (DHFR) [15,16]. 2. Results and Discussion 2. Results and Discussion 2.1. Chemistry 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– The chemical13b), at room synthesis temperature entailed overnight a simple (Scheme 1). one-step Compounds reaction 4a and 4b in were acetone obtained with by reducing pyridine between different sulfonylcompounds chlorides 3a and and3b, aminopyrazinerespectively. The purpose (series of1a – attempting14a), or 6-chloroaminopyrazinethe synthesis with 6- (series 1b–13b), at roomchloroaminopyrazine temperature, using overnight the same sulfonyl (Scheme chloride1). was Compounds to evaluate the effect4a ofand the chlorine4b were atom obtained by on anti-infective activity. It is common knowledge that the introduction of a chlorine atom to a reducing compoundsmolecule increases3a andits lipophilicity.3b, respectively. For antimycobacterial The purposeactivity, the lipophilicity of attempting of a compound the synthesis is with 6-chloroaminopyrazine,an essential factor using since the mycobacteria same sulfonyl have thick, chloride lipid-rich was mycolic to evaluate cell walls the [17] e.ff Besides,ect of the chlorine atom on anti-infectivechloropyrazine activity. derivatives, It isspecifically common anilides knowledge of 6-chloropyrazine that the-2 introduction-carboxylic acid, ofwere a chlorineproven atom to a previouslyFigure 2.to The possess chemical in vitro structure antimycobacterial of (a) zibotentan, activity (b) halogenated [18,19]. N-(pyrazinyl)benzensulfonamides, molecule increases and its(c) sulfamethoxypyrazine lipophilicity. For (sulfalene). antimycobacterial activity, the lipophilicity of a compound is an essential factor since mycobacteria have thick, lipid-rich mycolic cell walls [17]. Besides, chloropyrazine derivatives,2. specifically Results and Discussion anilides of 6-chloropyrazine-2-carboxylic acid, were proven previously to Molecules 2020, 25, x 3 of 20 possess in vitro2.1. Chemistry antimycobacterial activity [18,19]. 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 essentialScheme factor 1. sinceGeneral mycobacteria synthetic have reaction. thick, lipid R1:H-richCl; mycolic R2: refer cell to walls Table [17]1.. Besides, Scheme 1. General synthetic reaction. R1: H/Cl;/ R2: refer to Table 1. chloropyrazine derivatives, specifically anilides of 6-chloropyrazine-2-carboxylic acid, were proven previously to possess in vitro antimycobacterial activity [18,19]. 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 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–13b), or a singlet and two doublets (J = 2.8–2.4 Hz) at 8.40–7.89 ppm in the case of non-chlorinated compounds (series 1a–14a). 1H-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.

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 No. Mtb M. M. M. M. Attachment Point) H37Rv kansasii avium smeg aurum 1a >100 100 >100 ≥250 ≥500 >1000

1b 50 100 >100 250 ≥500 731

2a >100 25 >100 ≥500 ≥500 >500 *

2b >100 25 >100 250 ≥500 >500 *

3a >100 >100 >100 >500 >500 >1000

3b >100 >100 >100 250 125 >1000

4a 6.25 1.56 100 ≥500 250 775.6 * 4b 6.25 12.5 25 ≥500 250 >500 *** 5a >100 50 >100 250 250 >1000

5b 100 >100 >100 250 250 364

6a >100 >100 >100 ≥500 ≥500 >1000

Molecules 2020, 25, x 3 of 21

Molecules 2020, 25, x Molecules 2020, 25, x 3 of 21 3 of 21

Scheme 1. General synthetic reaction. R1: H/Cl; R2: refer to Table 1.

Scheme 1. General synthetic reaction. R1: H/Cl; R2: refer to Table 1. 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 The final products were purified using flash chromatography. They were isolated as solid compounds (series 1b–13b) had lower yields than the non-chlorinated ones (series 1a–14a ) due to the compounds, in yields ranging from 12–70% of chromatographically pure products. Chlorinated electron-withdrawing property of the chlorine atom that reduces the strength of the primary amine compounds (seriesScheme 1b–13b 1. General) had lower synthetic yields reaction. than the R1: non-chlorinatedSchemeH/Cl; R2 :1. refer General to Table ones synthetic 1.(series reaction. 1a–14a R)1 :due H/Cl; to R the2: refer to Table 1. as a nucleophile. The final products were characterized by melting points, 1H- and 13C-NMR spectra, electron-withdrawing property of the chlorine atom that reduces the strength of the primary amine IR spectroscopy, and elemental analysis. The obtained analytical data fully supported the asThe a nucleophile. final products The finalwere productspurified wereusingThe characterized finalflash productschromatography. by meltingwere purified points,They were using1H- and isolatedflash 13C-NMR chromatography. as solid spectra, They were isolated as solid corresponding proposed structures. In the 1H-NMR spectra, the signal of the sulfonamidic hydrogen compounds,IR spectroscopy, in yields andranging elemental fromcompounds, 12–70%analysis. of The chromatographicallyin yieldsobtained ranging analytical from pure 12–70%data products. fully of chromatographically Chlorinatedsupported the pure products. Chlorinated appeared as a broad singlet at 12.10–10.27 ppm in DMSO-d6. Pyrazine hydrogens appeared at 8.98– compoundscorresponding (series proposed 1b–13b) had structures. lowercompounds yields In the than 1H-NMR (seriesthe non-chlorinated spectra, 1b–13b )the had signal lower ones of (seriesyields the sulfonamidic than1a–14a the) due non-chlorinated hydrogen to the ones (series 1a–14a) due to the 8.04 as two singlets in the case of chlorinated compounds (series 1b–13b), or a singlet and two electron-withdrawingappeared as a broad property singlet ofat 12.10–10.27theelectron-withdrawing chlorine ppm atom in that DMSO- reduces propertyd6. Pyrazine the ofstrength the hydrogens chlorine of the atom primary appeared that amine reduces at 8.98– the strength of the primary amine doublets (J = 2.8–2.4 Hz) at 8.40–7.89 ppm in the case of non-chlorinated1 compounds13 (series 1a–14a). as a8.04 nucleophile. as two singlets The final in products the case were asof achlorinated nucleophile.characterized compounds The by finalmelting products (series points, 1b were H-–13b and characterized), orC-NMR a singlet spectra,by and melting two points, 1H- and 13C-NMR spectra, 1H-NMR and 13C-NMR spectra of compounds 6a and 6b are presented in supplementary materials. IR doubletsspectroscopy, (J = 2.8–2.4 and Hz)elemental at 8.40–7.89 analysis.IR ppmspectroscopy, inThe the ob casetained and of non-chlorinated elementalanalytical dataanalysis. compounds fully The supported ob(seriestained 1athe –analytical14a ). data fully supported the −1 Regarding the IR spectra, the final compounds1 showed signals at 3105–28961 cm attributed to the corresponding1H-NMR and proposed 13C-NMR structures. spectra of Incorresponding compounds the H-NMR 6a spectra,proposed and 6b the are structures. signal presented of theIn in the sulfonamidic supplementary H-NMR spectra, hydrogen materials. the signal of the sulfonamidic hydrogen sulfonamidic N–H stretch, at 1609–1460 cm−1 attributed to the aromatic C=C stretch, at 1406–1324 cm−1 appearedRegarding as a broadthe IR singletspectra, at the 12.10–10.27 finalappeared compounds ppm as in a DMSO- broadshowed singletd6 .signals Pyrazine at 12.10–10.27at 3105–2896hydrogens ppm cmappeared −in1 attributed DMSO- at 8.98–d6 .to Pyrazine the hydrogens appeared at 8.98– attributed to the S=O unsymmetrical stretch, and at 1189–1092 cm−1 attributed to the S=O symmetrical 8.04sulfonamidic as two singlets N–H in stretch, the case at 1609–1460of chlorinated8.04 as cm two−1 attributedcompounds singlets into thethe(series aromatic case 1b of– 13b chlorinatedC=C), orstretch, a singlet compoundsat 1406–1324 and two (series cm −1 1b–13b), or a singlet and two stretch. doubletsattributed (J = 2.8–2.4 to the S=OHz) unsymmetricalat 8.40–7.89 ppmdoublets stretch, in the (J and=case 2.8–2.4 at of 1189–1092 non-chlorinated Hz) at 8.40–7.89 cm−1 attributed compounds ppm in to the the (series case S=O of symmetrical 1a non-chlorinated–14a). compounds (series 1a–14a). 1H-NMRstretch. and 13C-NMR spectra of compounds1H-NMR and6a and 13C-NMR 6b are spectrapresented of compoundsin supplementary 6a and materials. 6b are presented in supplementary materials. Table 1. Structure of prepared compounds, antimycobacterial activity expressed by minimum MoleculesRegarding2020 ,the25 IR, 138spectra, the final compoundsRegarding theshowed IR spectra, signals the at 3105–2896final compounds cm−1 attributed showed signalsto the at 3105–2896 cm−1 attributed to the 3 of 20 inhibitory concentrations (MIC), and HepG2 cytotoxicity expressed by IC50 values. sulfonamidicTable N–H 1. Structure stretch, atof 1609–1460preparedsulfonamidic compounds, cm−1 attributed N–Hantimycobacterial to stretch, the aromatic at 1609–1460 activity C=C stretch, expressedcm−1 attributed at 1406–1324 by minimum to the cm aromatic− 1 C=C stretch, at 1406–1324 cm−1 attributedinhibitory to the S=O concentrations unsymmetrical (MIC), stretch,attributed and HepG2 and to atcytotoxicity the 1189–1092 S=O unsymmetrical expressed cm−1 attributed by IC stretch,50 values. to the and S=O at symmetrical1189–1092 cm −1 attributed to the S=O symmetrical stretch. a stretch. b a b TableTable 1. 1. StructureStructure of prepared of prepared compounds,Table antimycobacterial1. compounds, Structure of prepared activity antimycobacterial compounds,expressed by antimycobacterial minimum activity expressed expressed by minimum by minimum inhibitory

inhibitory concentrations (MIC), and HepG2inhibitory cytotoxicity concentrations expressed (MIC), by IC50 an values.d HepG2 cytotoxicity expressed by IC50 values. concentrations (MIC), and HepG2 cytotoxicity expressed by IC50 values.

a a b b IC50 a b (µM) IC50

Molecules 2020, 25, x (µM)4 of 21 IC50 (µM) Antimycobacterial Activity MIC (µg/mL) Molecules 2020, 25, x 4 of 21 R (the Cut Line Represents the Antimycobacterial Activity MIC (µg/mL) No. Molecules3a 2020, 25, Attachmentx Point) >100 >100 >100 >500 >500 IC50>1000 4 of 21 IC50 R (the Cut Line Represents NO2 the Mtb M. M. M. M. No.3a >100 >100 >100 >500 >500 (µM)>1000 (µM) Attachment Point)NO H37Rv kansasii avium smeg aurum 2 Mtb M. M. M. M. Molecules3b3aR (the2020, 25 Cut, x Line Represents >100 >100 >100 Antimycobacterial>500250 >500125 >10004 of Activity 21 MIC (µg/mL) NO H37RvAntimycobacterial kansasii Activity avium MIC (µg/mL)smeg Antimycobacterial aurum Activity MIC (µg/mL) No. 2 Molecules3b 2020the, 25 Attachment, x Point) >100 >100 >100 250 125 >10004 of 21 Molecules1a R (the 2020 Cut, 25, Linex Represents the R>100 (the CutMtb Line100H37Rv Represents >100 the M.≥250 kansasii≥500 4 of 21>1000 M. avium M. smeg M. aurum No. No. 3a3b4a1a Attachment Point) >1006.25 Attachment>1001.56100 Point)>100100 >500≥250500250 >500≥250125500 775.6>1000 * Molecules 2020, 25, x NO2 Mtb M. M. M. MtbM. M. 4 of 21M. M. M. 1a 4a 6.25 >1.56100 100 ≥500 100250 775.6 * >100 250 500 >1000 3a1b3a H37Rv>100>10050 kansasii>100>100100 avium>100>100 >500smeg >500 250H37Rv >500aurum >500≥500 kansasii>1000 >1000731 avium smeg aurum NONO22 ≥ ≥

1b Molecules3b4b4a 2020, 25, x >1006.25 >1001.5650 >100100 ≥250500 100125250 775.6>10004 of * 21 >100 250 500 731 1b3a >1006.2550 >10012.5100 >10025 ≥>500250500 ≥>500250500 >1000 >500731 ≥ ***4b3b NO2 >100 >100 >100 250 125 >1000 1a 3b2a 1a >100>1006.25 100 >10012.525 >100>100 25 ≥250≥ 250500>100 ≥500 ≥ 125250500 100>1000 >500>1000 >500 * >100 ≥250 ≥500 >1000 2a Molecules*** 2020, 25, x >100 25 4 of 21 >100 500 500 >500 * 4a4b3a >1006.25 >1001.56 >100100 ≥>500500 >500250 775.6>1000 * ≥ ≥ 2a3b NO >1006.25 >10012.525 >10025 ≥250500 ≥250125500 >500>1000 >500 * 2b ***5a4a 2 6.25>100 1.56>50 100 100>100 ≥500 250 250 25 250 775.6 * >1000 >100 250 500 >500 * 1b 4a2b 1b 50 >1006.25 100 1.5625 >100>100 100 250 ≥250500 50 ≥ 500≥ 250500 100731 775.6>500 * >100 250 ≥500 731 ≥ 5a3a >100 >10050 >100 >500250 >500250 >1000 NO 3a 4b2b3b 2 >100 >>10025100 >100 250 >100≥125500 >500>1000 * >100 >500 >500 >1000 4a4b 6.25 12.51.56 10025 ≥500 250 775.6 >500 * ***5b5a 6.25>100100 12.5>10050 25 >100 ≥500 250 250 250 >500 >1000364 2a 4b*** 2a >100 25 >100 ≥500 >100≥500 >50025 * >100 ≥500 ≥500 >500 * 6.25 12.5 25 ≥500 250 >500 3b ***5b3b >100100 >>100100 >100 250 >100250125 >1000364 >100 250 125 >1000 4a 6.25 1.56 100 ≥500 250 775.6 * 4b 4a 5a5b6a >1006.25100 >10012.56.2550 >10025 ≥250500 1.56≥250500 >1000 >500364 100 500 250 775.6 * 2b ***5a 2b >100>100 2550 >100>100 250250 >100250≥500 >1000>50025 * >100 250 ≥500≥ >500 * 5a6a4a >1006.25 >1001.5650 >100100 ≥250500 ≥250500 775.6>1000 * 4b *** 4b 6.25 12.5 25 500 250 >500 6.25 12.5 25 ≥500 250 >500 5b6a >100100 >100 >100 ≥250500 ≥250500 >1000364 ≥ ***6b5a5b 100>100 >10010050 >100>100 250 ≥250500 250 ≥250500 364 >1000 5a 6b4b >100 >100100 >100 ≥500 50≥500 >1000 >100 250 250 >1000 5b 6.25100 >10012.5 >10025 ≥250500 250 >500364 *** 6a6b6a >100>100 >100>100100 >100>100 ≥500 ≥500 ≥500 ≥500>1000 >1000 5b 5b5a >100100 >10010050 >100 250 >100250 >1000364 >100 250 250 364 7b6a >100100 >10050 >100 ≥125500 ≥125500 >1000308 6a 7b5a >100100 >50100 >100 125250 >100125250 >1000308 >100 500 500 >1000 6b >100 100 >100 ≥500 ≥500 >1000 6b5b >100100 >100100 >100 ≥250500 ≥250500 >1000364 ≥ ≥ 6a >100 >100 >100 ≥500 ≥500 >1000 7b 100 50 >100 125 125 308 6b 6b >100 >100100 >100 ≥500 100≥500 >1000 >100 500 500 >1000 8a5b >100100 >10050 >100 ≥250500 250 >500364 * ≥ ≥ 6b6a8a7b 100>100 50>100 10050 >100>100 125 ≥500 125 ≥250500 308 >500>1000 * 7b 100 50 >100 125 125 308 7b 100 50 >100 125 125 308 8a >100 50 >100 ≥500 250 >500 * 8b7b6a >10010050 >10050 >10050 ≥125500 ≥125500 >1000523.3308

6b8b >10050 >100100 >10050 ≥500 ≥500 >1000523.3

8a 7b8a >100100 50> 50100 >100>100 ≥500 125 250 50 125 >500 * 308 >100 500 250 >500 * 8a8b9a6b >100>10050 >10010050 >100>10050 ≥≥125500500 ≥250500 >500>250>1000523.3 * ≥

8b 8a9a >100 >1005050 >100 ≥125500 >100≥250500 >500>250 * 50 500 500 523.3 7b8b 50100 >10050 50 >100 ≥500 125 ≥500 125 523.3 308 ≥ ≥ 8b9a >10050 >100 >10050 ≥125500 ≥500 >250523.3 * 9a 9b8a >100 >50100 >100 ≥500 >100≥250500 >500179.5 * >100 125 500 >250 * 9b7b 100 50 >100 125 125 308 ≥ 8b9a >100>10050 >100>10050 >100>100 50 125 ≥500 ≥500 ≥500>250 * 523.3179.5 9b >100 50 >100 500 500 179.5 9a9b8a >100 >10050 >100 ≥125500 ≥250500 >250>500179.5 * 8b 50 >100 50 ≥500 ≥500 523.3 ≥ ≥ 9a9b >100>100 50>100 >100>100 ≥500 125 ≥500 ≥500 179.5 >250 * 10a8a >100 50 >100 ≥500 ≥250500 >250>500 * 9b >100 50 >100 ≥500 ≥500 179.5 10a 10a8b9a >10050 >>10050100 >10050 ≥125500 50≥500 >250523.3 * >100 500 500 >250 *

9b >100 50 >100 ≥500 ≥500 179.5 ≥ ≥ 10a >100 50 >100 ≥500 ≥500 >250 * 8b 50 >100 50 ≥500 ≥500 523.3 Molecules9a10a 2020, 25, x >100>100 50>100 >100>100 ≥500 125 ≥500 ≥500>250 * >2505 of * 21 9b >100 50 >100 ≥500 ≥500 179.5 11a >100 >100 >100 ≥500 250 >1000 11a Molecules 2020, 25, x >100 >100 5 of 21 >100 500 250 >1000 10a >100 50 >100 ≥500 ≥500 >250 * Molecules11a9a 2020, 25, x >100 >100 >100 ≥125500 ≥250500 >250>10005 of * 21 ≥ 11b 10a9b >100 >50100 >100 ≥500 25≥500 >250179.5 * >100 500 500 778.5 11b12b11a >100100 >10012.525 >100 ≥500 ≥250500 >1000778.5302 11a >100 >100 >100 ≥500 250 >1000 ≥ ≥ 11b >100 25 >100 ≥500 ≥500 778.5 12a 12b10a9b >100100 12.510050 >100 ≥500 100≥500 >250179.5302 * >100 500 500 >250 * 12b 100 12.5 >100 ≥500 ≥500 302 ≥ ≥ 11b11a12a >100100 >10010025 >100 ≥500 ≥250500 >250>1000778.5 * 12b 13a11b >100>100 25 10050 >100>100 ≥500 62.5 ≥ 12.5500 ≥500 778.5 >500 * >100 500 500 302 11a10a12a >100100 >10010050 >100 ≥500 ≥250500 >250>1000 * ≥ ≥ 13a >100 50 >100 62.5 ≥500 >500 *

13a 11b13a12a >100100 >1002550100 >100 ≥62.5500 50≥500 >500>250778.5 * >100 62.5 500 >500 * 11a10a12a 100>100 100>100 50 >100>100 ≥500 ≥500 ≥500 ≥250500 >250 * >250>1000 * 13b 50 50 >100 125 250 >1000 ≥ 11b >100 25 >100 ≥500 ≥500 778.5 13b 13b 50 5050 >100 125 50250 >1000 >100 125 250 >1000

13b12a 10050 10050 >100 ≥125500 ≥250500 >250>1000 * 11b11a >100 >10025 >100 ≥500 ≥250500 >1000778.5 14a 14a12a >100100 >100100 >100 ≥500 100≥500 >250 * >100 500 500 >250 * 11a >100 >100 >100 ≥500 250 >1000 ≥ ≥ 11b14a12a >100100 10025 >100 ≥500 ≥500 >250778.5 * 14a >100 100 >100 ≥500 ≥500 >250 *

Sulfamethoxazole 3.13 3.13 12.5 250 500 n.t. 11b Sulfamethoxazole >1003.13 3.1325 >10012.5 ≥250500 ≥500 778.5n.t. 12a 100 100 >100 ≥500 ≥500 >250 * ≥ SulfaclozineSulfamethoxazole 3.13 3.13 6.25 12.5 250 12.5≥500 n.t. 25 250 250 n.t. Sulfamethoxazole 3.13 3.13 12.5 250 ≥500 n.t. 12aSulfaquinoxaline Sulfaclozine 6.25100 12.5 3.13100 >10025 ≥250500 12.5≥250500 >250n.t. * 50 125 250 n.t. Sulfaclozine 6.25 12.5 25 250 250 n.t. BenzenesulfonamideSulfaclozine 6.25 >12.5100 25 250 >100250 n.t. >100 31.25 125 n.t. Sulfaquinoxaline 3.13 12.5 50 125 250 n.t. 4 PZASulfaquinoxaline** 3.13 >12.5100 50 125 >100250 n.t. >100 500 500 >10 [20] Sulfaquinoxaline 3.13 12.5 50 125 250 n.t. ≥ ≥ BenzenesulfonamideINH >100 >1000.2 >100 31.25 25125 n.t. 12.5 15.625 3.91 79 103 [20] Benzenesulfonamide >100 >100 >100 31.25 125 n.t. × BenzenesulfonamideRFM >100 >100n.t. >100 31.25 n.t.125 n.t. n.t. 25 1.56 n.t. >104 PZA ** >100 >100 >100 ≥500 ≥500 [20] CPX n.t. n.t.>104 n.t. 0.125 0.008 n.t. PZA ** >100 >100 >100 ≥500 ≥500 >10[20]4 PZA ** >100 >100 >100 ≥500 ≥500 [20] * Measurement at higher concentrations was not possible due to79 × precipitation 103 of the tested compound in the cell INH 0.2 25 12.5 15.625 3.91 culture medium. ** MIC value from testing at pH = 5.6 (acidic) is[20] 6.25–12.5 µg/mL [2]. The value stated in the table 79 × 103 INH 0.2 25 12.5 15.625 3.91 79[20] × 10 3 is from testing atINH pH = 6.6 (neutral).0.2 *** Compound25 12.5 4b15.625 is structurally3.91 identical to the sulfaclozine standard. n.t.—not [20] tested. For structuresRFM of control drugsn.t. ofn.t. the sulfonamiden.t. 25 type1.56 refern.t. to Figure4.

RFM n.t. n.t. n.t. 25 1.56 n.t. RFM n.t. n.t. n.t. 25 1.56 n.t. CPX n.t. n.t. n.t. 0.125 0.008 n.t. The final productsCPX weren.t. purified n.t. usingn.t. 0.125 flash 0.008 chromatography. n.t. They were isolated as solid * MeasurementCPX at higher concentrations wasn.t. not possiblen.t. due to precipitationn.t. 0.125of the tested0.008 compound n.t. in the cell culture medium. ** MIC value from testing at pH = 5.6 (acidic) is 6.25–12.5 μg/mL [2]. The compounds,* Measurement in yields at higher concentrations ranging was not from possible due 12–70% to precipitation of the tested chromatographically compound pure products. Chlorinated value stated in the table is from testing at pH = 6.6 (neutral). *** Compound 4b is structurally identical in* Measurement the cell culture at highermedium. concentrations ** MIC value was from not testing possible at pHdue = to 5.6 precipitation (acidic) is 6.25–12.5 of the tested μg/mL compound [2]. The to the sulfaclozine standard. n.t.—not tested. For structures of control drugs of the sulfonamide type compoundsvaluein the(series statedcell culture in the medium. table1b is from– **13b MIC testing value) at had pHfrom = 6.testing6 lower(neutral). at pH *** = 5.6Compound yields (acidic) is 4b 6.25–12.5 is than structurally μg/mL the identical [2]. The non-chlorinated ones (series 1a–14a) due to refer to Figure 4. tovalue the statedsulfaclozine in the tablestandard. is from n.t.—n testingot tested. at pH =For 6.6 structures (neutral). ***of controlCompound drugs 4b of is the structurally sulfonamide identical type referto the to sulfaclozine Figure 4. standard. n.t.—not tested. For structures of control drugs of the sulfonamide type 2.2. Lipophilicity refer to Figure 4. 2.2. LipophilicityLipophilicity is a significant physico-chemical property in medicinal chemistry and drug design in2.2. general. Lipophilicity As stated earlier, lipophilicity plays a significant role specifically in the development of Lipophilicity is a significant physico-chemical property in medicinal chemistry and drug design new antituberculars. The log P values of prepared compounds were calculated using ChemDraw 18.1 in general.Lipophilicity As stated is a earlier, significant lipophilicity physico-chemical plays a significant property in role medicinal specifically chemistry in the anddevelopment drug design of (CambridgeSoft, Cambridge, MA, USA). Calculated lipophilicity (log P) values were plotted against newin general. antituberculars. As stated Theearlier, log Plipophilicity values of prepared plays a compoundssignificant role were specifically calculated inusing the ChemDrawdevelopment 18.1 of experimentally determined lipophilicity measures (log k). The measures were derived from retention (CambridgeSoft,new antituberculars. Cambridge, The log P MA, values USA). of prepared Calculated compounds lipophilicity were (log calculated P) values using were ChemDraw plotted against 18.1 times measured using reverse-phase HPLC. Both values agreed as shown in the graph below (Figure experimentally(CambridgeSoft, determined Cambridge, lipophilicity MA, USA). measures Calculated (log lipophilicity k). The measures (log P) were values derived were plottedfrom retention against 3). timesexperimentally measured determined using reverse-phase lipophilicity HPLC. measures Both valu (loges k ).agreed The measures as shown were in the derived graph from below retention (Figure 3).times measured using reverse-phase HPLC. Both values agreed as shown in the graph below (Figure 3).

Molecules 2020, 25, 138 4 of 20 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–13b), or a singlet and two doublets (J = 2.8–2.4 Hz) at 8.40–7.89 ppm in the case of non-chlorinated compounds (series 1a–14a). 1H-NMR and 13C-NMR spectra of compounds 6a and 6b are presented in supplementary materials. Regarding 1 the IR spectra, the ﬁnal compounds showed signals at 3105–2896 cm− attributed to the sulfonamidic 1 1 N–H stretch, at 1609–1460 cm− attributed to the aromatic C=C stretch, at 1406–1324 cm− attributed to 1 the S=O unsymmetrical stretch, and at 1189–1092 cm− attributed to the S=O symmetrical stretch.

2.2. Lipophilicity Lipophilicity is a significant physico-chemical property in medicinal chemistry and drug design inMolecules general. 2020 As, 25, statedx earlier, lipophilicity plays a significant role specifically in the development5 of of20 new antituberculars. The log P values of prepared compounds were calculated using ChemDraw 18.1 (CambridgeSoft,experimentally determined Cambridge, lipophilicity MA, USA). measures Calculated (log lipophilicity k). The measures (log P) were values derived were plotted from retention against experimentallytimes measured determined using reverse-phase lipophilicity HPLC. measures Both valu (logesk). agreed The measures as shown were in the derived graph frombelow retention (Figure times3). measured using reverse-phase HPLC. Both values agreed as shown in the graph below (Figure3).

Figure 3. Linear regression of log P (calculated) plotted against log k (experimental). Equation: log PFigure= 1.127 3. logLineark regression0.1149. (R2 of= 0.9664,log P (calculated)n = 25). plotted against log k (experimental). Equation: log P − = 1.127 log k − 0.1149. (R2 = 0.9664, n = 25). 2.3. Acidobasic Properties 2.3. AcidobasicThe acidic Properties character of sulfonamide moiety determines the ionization state of title compounds at physiologicalThe acidic character pH and inside of sulfonamide the mycobacteria. moiety dete It furtherrmines a fftheects ionization lipophilicity state and of title transport compounds across membranes.at physiological Three pH compounds and inside the6b, mycobacteria.9b and 13b were It further selected affects and their lipophilicity pKa values and were transport determined across experimentallymembranes. Three using compounds potentiometric 6b, 9b and and absorbance 13b were selected methods and (Table their2). p TheKa values values were of p Kadeterminedin water wereexperimentally then predicted using using potentiometric different software and absorbance to find the methods best value (Table that 2). gave The the values closest of predictions pKa in water to thewere actual then experimentallypredicted using determined different software values. ChemDrawto find the best was notvalue able that to gave predict the p Kaclosestvalues, predictions whereas valuesto the fromactual the experimentally Molecular Operating determined Environment values. (MOE,ChemDraw Chemical was Computingnot able to Group, predict Montreal, pKa values, QC Canada)—softwarewhereas values from is the available Molecular with Operating a paid license Environment only [21], were(MOE, far Chemical from the Computing experimental Group, ones. ChemicalizeMontreal, QC was Canada)—software then used for the prediction is available of pwithKa values a paid (September license only 2019, [21], https: were//chemicalize.com far from the/ developedexperimental by ones. ChemAxon Chemicalize http:// www.chemaxon.comwas then used for the). Theprediction latter values of pKa proved values to (September be the closest 2019, to thehttps://chemicalize.com/ experiments (Table2), developed and therefore by ChemAxon Chemicalize http://www.chemaxon.com). was selected to predict the pTheKa valueslatter values of the remainingproved to compoundsbe the closest (Table to 3the). Theexperiments experimental (Tab pleKa 2),value and oftherefore compound Chemicalize4a was documented was selected in the to literaturepredict the (p KapKa= 6.04)values [22 of]. the This remaining value was compounds close to our predicted(Table 3). valueThe experimental from the Chemicalize pKa value web of applicationcompound 4a (p Kawas= documented6.61). in the literature (pKa = 6.04) [22]. This value was close to our predicted value from the Chemicalize web application (pKa = 6.61).

Table 2. Experimental and calculated pKa values of compounds 6b, 9b, and 13b.

Experimental Calculated No. Absorbance Method Potentiometric Method ChemAxon MOE ChemDraw 6b 6.95 7.21 6.21 8.30 NA 9b 6.32 6.38 6.29 8.30 NA 13b 5.63 5.75 6.01 8.30 NA

Table 3. pKa values of title compounds predicted using the Chemicalize web application.

No. pKa No. pKa No. pKa No. pKa No. pKa 1a 6.11 3b 5.76 6a 6.30 9a 6.39 12a 6.11 1b 6.05 4a 6.61 6b 6.21 9b 6.29 12b 6.05 2a 6.16 4b 6.49 7b 6.03 10a 6.38 13a 6.07 2b 6.09 5a 6.10 8a 6.12 11a 6.16 13b 6.01 3a 5.80 5b 6.03 8b 6.05 11b 6.09 14a 6.07

Molecules 2020, 25, 138 5 of 20

Table 2. Experimental and calculated pKa values of compounds 6b, 9b, and 13b.

Experimental Calculated Potentiometric No. Absorbance Method ChemAxon MOE ChemDraw Method 6b 6.95 7.21 6.21 8.30 NA 9b 6.32 6.38 6.29 8.30 NA 13b 5.63 5.75 6.01 8.30 NA

Table 3. pKa values of title compounds predicted using the Chemicalize web application.

No. pKa No. pKa No. pKa No. pKa No. pKa 1a 6.11 3b 5.76 6a 6.30 9a 6.39 12a 6.11 1b 6.05 4a 6.61 6b 6.21 9b 6.29 12b 6.05 2a 6.16 4b 6.49 7b 6.03 10a 6.38 13a 6.07 Molecules2b 2020, 26.095, x 5a 6.10 8a 6.12 11a 6.16 13b 6.016 of 20 3a 5.80 5b 6.03 8b 6.05 11b 6.09 14a 6.07 2.4. Biological Activity

2.4. BiologicalSix of our Activity 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 evaluatedSix of ouras a titlephosphoinositide compounds, namely 3-kinase1a, 1b(PI3K), 2a, 3a inhibitor, 4a, and [24],4b, have and beencompound previously 3a possessed mentioned potent in the 1a 1b literature.in vitro and Compound in vivo nucleasewas evaluated activity as against an inhibitor Leishmania of B-raf infantum kinase [23 promastigotes], compound [25]. was Furthermore, evaluated ascompound a phosphoinositide 4a—also known 3-kinase as sulfapyrazine (PI3K) inhibitor—has [24 an], and antibacterial compound profile3a possessed comparable potent to sulfadiazine in vitro and in[26], vivo a protectivenuclease activityeffect against against sporozoiteLeishmania induced infantum falciparum promastigotes malaria[25 ].[27], Furthermore, and sperm production compound 4aand—also gonadal known development as sulfapyrazine—has stimulation in ananimals antibacterial [28]. Compound proﬁle comparable 4b—also known to sulfadiazine as sulfaclozin [26],e— a protectiveis a commonly eﬀect used against antiprotozoal sporozoite inducedto treat poultry falciparum coccidiosis malaria [29]. [27], It and is important sperm production to note andthat gonadalcompounds development 4a and 4b stimulationwere not tested in animals previously [28]. against Compound mycobacteria.4b—also knownSulfaclozine as sulfaclozine—is was purchased a commonlylater as standard. used antiprotozoal All title compounds to treat poultry were coccidiosis checked for [29 the]. It presence is important of PAINSto note that and compoundsaggregator 4afeaturesand 4b usingwere the not ZINC15 tested previously utility (http://z againstinc15.docking.org/patterns/home). mycobacteria. Sulfaclozine was All purchased the compounds later as standard.were clear. 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 2.4.1. Antimycobacterial Activity Evaluation against Mycobacterium tuberculosis, Mycobacterium kansasii, andkansasiiMycobacterium, and Mycobacterium avium avium All synthesized compounds were evaluated for for inin vitro antimycobacterial activity against M. tuberculosis H37Rv,H37Rv,M. kansasiiM. kansasii, and,M. and avium M.using avium microplate using alamarmicroplate blue assay.alamar The b antimycobacteriallue assay. The activityantimycobacterial results were activity expressed results as were a minimum expressed inhibitory as a minimum concentration inhibitory (MIC) concentration in µg/mL (MIC) against in isoniazidμg/mL against (INH) as isoniazidstandard. Structurally (INH) as related standard. sulfonamides, Structurally namely, related sulfamethoxazole, sulfonamides, sulfaclozine, namely, sulfaquinoxaline,sulfamethoxazole, and sulfaclozine, benzenesulfonamide sulfaquinoxaline, were and purchased benzenesulfonamide and subjected were to all purchased anti-infective and evaluationssubjected to (Figureall anti4-infective). evaluations (Figure 4).

Figure 4. Chemical structures of standards (a) sulfamethoxazole, (b) sulfaclozine, (c) sulfaquinoxaline, Figure 4. Chemical structures of standards (a) sulfamethoxazole, (b) sulfaclozine, (c) sulfaquinoxaline, and (d) benzenesulfonamide. and (d) benzenesulfonamide.

Based on the obtained data presented in Table 1, compounds 4a (MICMtb = 6.25 μg/mL, 25 μM) and 4b (MICMtb = 6.25 μg/mL, 22 μM) showed good antimycobacterial activity against Mtb. None 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

Molecules 2020, 25, 138 6 of 20

Based on the obtained data presented in Table1, compounds 4a (MICMtb = 6.25 µg/mL, 25 µM) and 4b (MICMtb = 6.25 µg/mL, 22 µM) showed good antimycobacterial activity against Mtb. None 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, Figure5) 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) containingMolecules 2020 condensed, 25, x sulfathiazole products [33], where it had also been rationalized using quantum7 of 20 mechanics/molecular mechanics simulations [35]. For more details on the docking experiments, refer tosection. in silico With studies regards from to thethe materialspKa values and of methodsthe sulfonamide section. moiety With regards (Tables to2 theand p3),Ka asvalues well as of the sulfonamideproposed mechanism moiety (Tablesof action2 and (competitive3), as well inhibitio as the proposedn with DHPS mechanism natural substrate of action— (competitivedeprotonated inhibitionPABA), the with hypothetical DHPS natural products substrate—deprotonated (Figure 5) of compounds PABA), 4a theand hypothetical 4b were docked products as deprotonated (Figure5) of compoundssulfonamides.4a and 4b were docked as deprotonated sulfonamides.

Figure 5. StructuresStructures of of ( (aa)) the the hypothetic hypothetic product product of of compound compound 4a4a and (b) 66-hydroxymethyl-7,8--hydroxymethyl-7,8- dihydropterin diphosphate (HMDPdiP) used for the docking experiments. Fragment of HMDPdiP in the condensed product is in thethe rectangle.

The docking experiments showed two main potential positions with the poses scoring from 7.9 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 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 Figure6) had a slightly better score than the positions with the pyrazine 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 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 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 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 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 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 Figure6, favorably positioned for hydrogen bonding with the guanidine group of Arg233. As seen in Figure substitution of the pyrazine ring with chlorine does not have a significant effect on the predicted 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 binding mode, which justifies the similar antimycobacterial activity of the non-chlorinated 4a (MICMtb = 6.25 µg/mL, 25 µM) to the chlorinated compound 4b (MICMtb = 6.25 µg/mL, 22 µM) compound 4a (MICMtb = 6.25 μg/mL, 25 μM) to the chlorinated compound 4b (MICMtb = 6.25 μg/mL, against Mtb. 22 μM) against Mtb.

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 μg/mL, 85.5 μM), 2b (MICM. kansasii = 25 μg/mL, 76.5 μM), 11b (MICM. kansasii = 25 μg/mL, 76.3 μM), and 12b (MICM kansasii = 12.5 μg/mL, 36.1 μM), had selective antitubercular activity against M. kansasii, and it was higher than INH (MIC of INH was 25 μg/mL, 182.3 μM). For compounds 2a and 2b, particularly, this activity was relatable to its chemical structure. The acetamido moiety may have worked as a pro-drug that was later hydrolyzed (supposedly by M.

Molecules 2020, 25, x 7 of 20 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.

O N N S O O O O N O N P P N N NH HO O O NH H OH OH

N N NH2 N NNH2 H H

(a) (b) 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 μg/mL, 25 μM) to the chlorinated compound 4b (MICMtb = 6.25 μg/mL, Molecules 2020, 25, 138 7 of 20 22 μM) against Mtb.

Figure 6. ProductProduct of of 4a4a (orange)(orange) and and product product of of 4b4b (green)(green) main main poses poses from from the the docking docking experiments. experiments. The pose ofof 7,8-dihydropteroate7,8-dihydropteroate (grey)(grey) corresponds corresponds to to the the experimentally experimentally determined determined position position seen seen in inbacterial bacterial DHPS DHPS [e.g., [e.g.,Burkholderia Burkholderia cenocepacia cenocepaciaDHPS DHPS (PDB (PDB ID: ID: 2y5s) 2y5s)or orBacillus Bacillus anthracisanthracis DHPS (PDB ID: 3tya)].

Compounds 2a (MIC = 25 µg/mL, 85.5 µM), 2b (MIC = 25 µg/mL, 76.5 µM), 11b Compounds 2a (MICM.M. kansasii = 25 μg/mL, 85.5 μM), 2b (MICM.M. kansasii = 25 μg/mL, 76.5 μM), 11b (MIC = 25 µg/mL, 76.3 µM), and 12b (MIC = 12.5 µg/mL, 36.1 µM), had selective (MICM. kansasii kansasii = 25 μg/mL, 76.3 μM), and 12b (MICM kansasii = 12.5 μg/mL, 36.1 μM), had selective antitubercular activity against against M.M. kansasii, kansasii, and and it it was was higher higher than than INH INH (MIC (MIC of of INH INH was was 25 25 μµg/mL,g/mL, 182.3 μµM). For compounds 2a andand 2b2b,, particularly, particularly, this this activity activity was was relatable relatable to to its its chemical chemical structure. structure. The acetamido moiety moiety may may have have worked worked as as a pro-drug a pro-drug that that was was later later hydrolyzed hydrolyzed (supposedly (supposedly by M. 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 [37,38]. It must be noted that the 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 µg/mL, 6.2 µM) and 4b (MICM. kansasii = 12.5 µg/mL, 44 µM) were also found to be more potent than INH against M. 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 µg/mL, 305.1 µM) vs. 11b (active; MICM. kansasii = 25 µg/mL, 76.3 µM), and 12a (inactive; MICM. kansasii = 100 µg/mL, 321.2 µM) vs. 12b (active; MICM. kansasii = 12.5 µg/mL, 36.1 µM). Compound 4b (MICM. avium = 25 µg/mL, 88 µM) was the only compound to exert antimycobacterial activity against 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 µg/mL, 12 µM, less potent than INH), M. kansasii (MICM. kansasii = 3.13 µg/mL, 12 µM, more potent than INH), and M. avium (MICM. avium = 12.5 µg/mL, 49 µM, more potent than INH). As for sulfaclozine, its MIC values 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 µg/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 8 of 20

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 (Table4). 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 Retro-amide Sulfonamide H H 100 >100 >100 (1a)

H 4-OCH3 25 >100 >100 (6a) 6-Cl H >100 25 50 (1b) 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 µg/mL, 199 µM), which was inactive against the slow-growing strains of mycobacteria. Some 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 Molecules 2020, 25, 138 9 of 20 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 (Table1). 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 µM). Compounds with the chlorine atom (series 1b–13b) were usually more cytotoxic than their corresponding non-chlorinated homologs (series 1a–14a). The most toxic compound among all series was compound 9b (IC50 = 179.5 µM) (Figure7). When compared to amides and retro-amides, sulfonamidesMolecules 2020, exerted 25, x a lesser extent of in vitro cytotoxicity [2–4]. 10 of 20

Figure 7. Cytotoxic effect of different incubation concentrations of compound 9b on HepG2 cells. 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 2.4.5.Although Exploring designed Potential as potentialTargets and antimycobacterial Activities using compounds,Docking Studies the sulfonamides presented in this paperAlthough only exerted designed low as antimycobacterial potential antimycobacter activity (withial compounds, the exception the of sulfonamides4a and 4b, as presented discussed in above).this paper To discover only exerted new potential low antimycobacterial uses for our compounds, activity (with we ranthe aexception target fishing of 4a campaign and 4b, as based discussed on structuralabove). similarityTo discover between new potential our N-(pyrazin-2-yl)benzensulfonamides uses for our compounds, we ran and a target ligands fishing from campaign the RCSB PDB based database.on structural First, similarity potential targetsbetween were our N searched-(pyrazin-2-yl)benzensulfonamide in the RCSB database, ands then and theligands title from compounds the RCSB werePDB docked database. into First, the selected potential enzymes, targets afterwere whichsearched poses in ofthe the RCSB molecules database, were and investigated then the totitle establishcompounds their possiblewere docked activities. into For the details selected of theenzymes, target fishing after which and docking poses procedures,of the molecules refer to were in silicoinvestigated studies from to theestablish materials their and possible methods activities. section. For details of the target fishing and docking procedures,The search refer yielded to in sixsilico targets, studies as fr presentedom the materials in Table and S1 methods in supplementary section. materials. Among these wasThe DHPS, search whichyielded we six have targets, already as presented investigated in whenTable determiningS1 in supplementary the mechanism materials. of actionAmong ofthese compounds was DHPS,4a and which4b. Thenwe have we already selected investigat three targetsed when from determining the remaining the five mechanism to explore of further. action of Compoundcompounds1a and4a and its derivatives 4b. Then we have selected already three been targets patented from as potential the remaining inhibitors five of to B-raf explore kinase further. [23]. WeCompound then docked 1a and our its compound derivatives into have GTPase already KRas, been withpatent noed interesting as potential results. inhibitors Finally, of B-raf matrix kinase metalloproteinase-8[23]. We then docked (MMP-8; our compound also known into as neutrophilGTPase KRas, collagenase; with no PDBinteresting ID: 5h8x), results. was Finally, investigated matrix inmetalloproteinase-8 more detail, where (MMP-8; its recently also described known as inhibitors neutrophil are collagenase; of the above-mentioned PDB ID: 5h8x), structuralwas investigated type (Ar-SONHin more detail,2-Ar) [where43]. This its recently endopeptidase described is inhibito a partrs of are a veryof the complex above-mentioned proteolytic structural MMP enzyme type (Ar- family, where its overexpression was linked to several pathological processes [43]. As several articles SONH2-Ar) [43]. This endopeptidase is a part of a very complex proteolytic MMP enzyme family, havewhere already its overexpression described the was coordination linked to ofseveral metals pathol by pyrazinesogical processes [44,45], [43]. we investigatedAs several articles whether have 2+ thealready pyrazine described in our compounds the coordination could coordinateof metals by the pyrazines Zn ion in[44,45], the MMP-8. we investigated This could whether promote the potentialpyrazine inhibitory in our compounds activity on could MMP-8, coordinate as presented the Zn2+ by ion Tauro in the et MMP-8. al. [43]. This We testedcould promote this hypothesis potential inhibitory activity on MMP-8, as presented by Tauro et al. [43]. We tested this hypothesis 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, N1′ was interacting with the metal ion, as seen in Figure 8. The interaction of Zn2+ and N4′ 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.

Molecules 2020, 25, 138 10 of 20 using molecular docking studies. The results showed that in compounds 2a (Figure8) 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 2+ coordinate the Zn in the receptor cavity (Figure8). In the majority of poses, N1 0 was interacting with 2+ the metal ion, as seen in Figure8. The interaction of Zn 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 theirMolecules further 2020, 25 evaluation, x as inhibitors of MMP-8 or other enzymes of the MMP family. 11 of 20

(a) (b)

Figure 8. (a) Compound 2a (green carbons) docked to MMP-8 (PDB(PDB ID: 5h8x) in comparison to the co-crystalized pose of the originaloriginal catecholcatechol ligandligand (grey(grey carbons).carbons). (b) DetailDetail presentingpresenting interactionsinteractions 22++ between pyrazine nitrogen N1N10′ and Zn ionion coordinated coordinated by by His His residu residues.es. (Measurements (Measurements are are in in Å). Å).

3. Materials and Methods 3.1. General Information 3.1. General Information All chemicals were of reagent or higher grade of purity. They were purchased from Sigma-Aldrich All chemicals were of reagent or higher grade of purity. They were purchased from Sigma- (Steinheim, Germany) unless stated otherwise. The progress of reactions was checked using Merck Aldrich (Steinheim, Germany) unless stated otherwise. The progress of reactions was checked using Silica 60 F254 TLC plates (Merck, Darmstadt, Germany) with UV detection at wavelength 254 nm. Merck Silica 60 F254 TLC plates (Merck, Darmstadt, Germany) with UV detection at wavelength 254 Microwave-assisted reactions were performed in a CEM Discover microwave reactor with a focused field nm. Microwave-assisted reactions were performed in a CEM Discover microwave reactor with a (CEM Corporation, Matthews, NC, USA) connected to an Explorer 24 autosampler (CEM Corporation). focused field (CEM Corporation, Matthews, NC, USA) connected to an Explorer 24 autosampler This equipment was running under CEM’s SynergyTM software for setting and monitoring the (CEM Corporation). This equipment was running under CEM’s SynergyTM software for setting and conditions of reactions. An internal infrared sensor monitored the temperature of the reaction mixture. monitoring the conditions of reactions. An internal infrared sensor monitored the temperature of the All obtained products were purified by a preparative flash chromatograph CombiFlash® Rf (Teledyne reaction mixture. All obtained products were purified by a preparative flash chromatograph Isco Inc., Lincoln, NE, USA) or preparative flash chromatograph Puriflash 420 XS (Intechim Chemicals, CombiFlash® Rf (Teledyne Isco Inc., Lincoln, NE, USA) or preparative flash chromatograph Puriflash Montlucon, France). The type of elution was gradient, using a mixture of hexane (LachNer, Neratovice, 420 XS (Intechim Chemicals, Montlucon, France). The type of elution was gradient, using a mixture Czech Republic) and ethyl acetate (Penta, Prague, Czech Republic) as the mobile phase. Silica gel of hexane (LachNer, Neratovice, Czech Republic) and ethyl acetate (Penta, Prague, Czech Republic) (0.040–0.063 mm, Merck, Darmstadt, Germany) was used as the stationary phase. NMR spectra were as the mobile phase. Silica gel (0.040–0.063 mm, Merck, Darmstadt, Germany) was used as the recorded on a Varian VNMR S500 (Varian, Palo Alto, CA, USA) at 500 MHz for 1H and 125 MHz for 13stationary phase. NMR spectra were recorded on a Varian VNMR S500 (Varian, Palo Alto, CA, USA) C. Chemical shifts1 were reported in13 ppm (δ) and were referred indirectly to tetramethylsilane via at 500 MHz for H and 125 MHz1 for C. Chemical13 shifts were reported in ppm (δ) and were referred a signal of solvent (2.49 for H and 39.7 for C in DMSO-d6). Infrared spectra were recorded by a indirectly to tetramethylsilane via a signal of solvent (2.49 for 1H and 39.7 for 13C in DMSO-d6). spectrometer FT-IR Nicolet 6700 (Thermo Scientific, Waltham, MA, USA) using the attenuated total Infrared spectra were recorded by a spectrometer FT-IR Nicolet 6700 (Thermo Scientific, Waltham, reflectance (ATR) methodology on a germanium crystal. Elemental analysis was performed on a vario MA, USA) using the attenuated total reflectance (ATR) methodology on a germanium crystal. MICRO cube element analyzer (Elementar Analysensysteme, Hanau, Germany). All values regarding Elemental analysis was performed on a vario MICRO cube element analyzer (Elementar elemental analyses were given as percentages. Melting points were determined in open capillary Analysensysteme, Hanau, Germany). All values regarding elemental analyses were given as on the Stuart SMP30 melting point apparatus (Bibby Scientific Limited, Staffordshire, UK) and were percentages. Melting points were determined in open capillary on the Stuart SMP30 melting point apparatus (Bibby Scientific Limited, Staffordshire, UK) and were uncorrected. Yields were expressed as percentages of the theoretical yield, and they referred to the isolated products after all purification steps.

3.2. Synthesis

Molecules 2020, 25, 138 11 of 20 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 1 204–208 ◦C[25]}; IR (ATR-Ge, cm− ): 2956 (NH stretch), 1587, 1558, 1532 (arom. stretch), 1345 (S=O 1 unsymmetrical stretch), 1092 (S=O symmetrical stretch); H-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.), 13 7.14 (d, J = 8.5 Hz, 2H, arom.), 7.01 (t, J = 7.4 Hz, 2H, arom.). C-NMR (125 MHz, DMSO-d6) δ 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, Molecules 2020, 25, 138 12 of 20

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 1 (ATR-Ge, cm− ): 2940 (NH stretch), 1570, 1526, 1499 (arom. stretch), 1398 (S=O unsymmetrical stretch), 1 1163 (S=O symmetrical stretch); H-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 13 (m, 2H, arom.). C-NMR (125 MHz, DMSO-d6) δ 147.5, 145.6, 139.4, 137.5, 133.8, 132.6, 129.5, 127.5. 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; 1 IR (ATR-Ge, cm− ): 3105 (NH stretch), 1592, 1558, 1532 (arom. stretch), 1406 (S=O unsymmetrical 1 stretch), 1162 (S=O symmetrical stretch); H-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, 13 arom.), 7.74 (d, J = 8.5 Hz, 2H, arom.), 2.06 (s, 3H, –CH3). C-NMR (125 MHz, DMSO-d6) δ 169.2, 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. 1 217.9–219.9 ◦C; IR (ATR-Ge, cm− ): 3043 (NH stretch), 1592, 1527, 1501 (arom. stretch), 1400 (S=O 1 unsymmetrical stretch), 1164 (S=O symmetrical stretch); H-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, 13 –CH3). C-NMR (125 MHz, DMSO-d6) δ 169.4, 147.6, 145.6, 144.0, 137.1, 132.7, 132.5, 129.0, 118.6, 24.3. 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 1 233–237 ◦C[25]}; IR (ATR-Ge, cm− ): 2990 (NH stretch), 1565, 1544, 1524 (arom. stretch), 1371 (S=O 1 unsymmetrical stretch), 1160 (S=O symmetrical stretch); H-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, 13 arom.), 7.14 (d, J = 9.2 Hz, 2H, arom.). C-NMR (125 MHz, DMSO-d6) δ 151.2, 147.9, 145.9, 141.4, 134.1, 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, 1 cm− ): 2899 (NH stretch), 1535, 1514, 1494 (arom. stretch), 1391 (S=O unsymmetrical stretch), 1105 1 (S=O symmetrical stretch); H-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) δ 156.1, 151.7, 145.7, 144, 135.4, 134.1, 128, 128, 124.5, 124.5. Elemental analysis 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 1 the literature 257–259 ◦C[46]}; IR (ATR-Ge, cm− ): 2961 (NH stretch), 1575, 1561, 1520 (arom. stretch), 1 1354 (S=O unsymmetrical stretch), 1164 (S=O symmetrical stretch); H-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 13 Hz, 2H, arom.), 6.52 (d, J = 9.1 Hz, 2H, arom.), 5.6 (bs, 2H, amino). C-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. Molecules 2020, 25, 138 13 of 20

4-amino-N-(6-chloropyrazin-2-yl)benzenesulfonamide (4b). Light yellow solid. Yield 81%; M.p. 1 234.7–236.1 ◦C; IR (ATR-Ge, cm− ): 3442, 3365 (NH2 stretch), 2929 (NH stretch), 1585, 1564, 1524 (arom. stertch), 1361 (S=O unsymmetrical stretch), 1150 (S=O symmetrical stretch); 1H-NMR (500 MHz, DMSO-d6) δ 11.06 (bs, 1H, sulfonamide), 8.22 (s, 1H, pyrazine), 8.14 (s, 1H, pyrazine), 7.48 (d, 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) δ 152.1, 148.9, 145.5, 142.4, 139.2, 134.9, 128.4, 123.9. Elemental analysis found: C, 41.88%; 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 1 ◦C; IR (ATR-Ge, cm− ): 2926 (NH stretch), 1609, 1590, 1532 (arom. stretch), 1324 (S=O unsymmetrical 1 stretch), 1145 (S=O symmetrical stretch); H-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, 13 J = 8.7, 7.5, 0.7 Hz, 2H, arom.). C-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. 1 125–127.2 ◦C; IR (ATR-Ge, cm− ): 2930 (NH stretch), 1589, 1525, 1495 (arom. stretch), 1397 (S=O 1 unsymmetrical stretch), 1159 (S=O symmetrical stretch); H-NMR (500 MHz, DMSO-d6) δ 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, 13 J = 9.2 Hz, 2H, arom.). C-NMR (125 MHz, DMSO-d6) δ 151.7, 147.3, 145.6, 138.4, 137.6, 132.8 (q, J = 31 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 1 (ATR-Ge, cm− ): 2924 (NH stretch), 1596, 1578, 1531 (arom. stretch), 1344 (S=O unsymmetrical stretch), 1 1158 (S=O symmetrical stretch); H-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, 13 arom.), 4.12 (s, 3H, –OCH3). C-NMR (125 MHz, DMSO-d6) δ 162.9, 148.4, 142.3, 138.9, 134.9, 131.6, 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 1 ◦C; IR (ATR-Ge, cm− ): 2994 (NH stretch), 1579, 1524, 1501 (arom. stretch), 1398 (S=O unsymmetrical 1 stretch), 1164 (S=O symmetrical stretch); H-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.), 13 3.81–3.79 (m, 3H, -OCH3-). C-NMR (125 MHz, DMSO-d6) δ 163.2, 147.6, 145.7, 137.1, 132.4, 130.9, 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 1 ◦C; IR (ATR-Ge, cm− ): 2925 (NH stretch), 1574, 1525, 1497 (arom. stretch), 1396 (S=O unsymmetrical 1 stretch), 1153 (S=O symmetrical stretch); H-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) δ 147.3, 145.6, 138.8, 137.6, 132.7, 132.5, 129.6, 127.7. Elemental analysis found: C, 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; 1 IR (ATR-Ge, cm− ): 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), 13 8.30 (s, 1H, pyrazine), 7.88 (s, 1H, arom.), 7.74 (s, 2H, arom.), 2.34 (s, 6H, –CH3). C-NMR (125 MHz, DMSO-d6) δ 148.3, 142.4, 140.1, 139.8, 139, 135, 134.8, 124.6, 20.9. Elemental analysis found: C, 55.34%; 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%. Molecules 2020, 25, 138 14 of 20

N-(6-chloropyrazin-2-yl)-3,5-dimethylbenzenesulfonamide (8b). Dark yellow-brownish solid. Yield 1 25%; M.p. 148.1–150.1 ◦C; IR (ATR-Ge, cm− ): 2930 (NH stretch), 1573, 1525, 1494 (arom. stretch), 1 1394 (S=O unsymmetrical stretch), 1159 (S=O symmetrical stretch); H-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, 13 6H, –CH3). C-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; 1 IR (ATR-Ge, cm− ): 2949 (NH stretch), 1601, 1586, 1530 (arom. stretch), 1337 (S=O unsymmetrical 1 stretch), 1154 (S=O symmetrical stretch); H-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, 13 3H, -CH3). C-NMR (125 MHz, DMSO-d6) δ 148.9, 142.5, 142.0, 139.4, 138.1, 134.2, 134.1, 131.8, 22.4, 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. 1 179.7–180.2 ◦C; IR (ATR-Ge, cm− ): 2925 (NH stretch), 1566, 1525, 1498 (arom. stretch), 1331 (S=O 1 unsymmetrical stretch), 1186 (S=O symmetrical stretch); H-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), 13 2.24 (s, 3H, –CH3). C-NMR (125 MHz, DMSO-d6) δ 156.8, 145.1, 144.9, 141.1, 137.2, 135.1, 134.9, 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 1 ◦C; IR (ATR-Ge, cm− ): 2955 (NH stretch), 1585, 1526, 1500 (arom. stretch), 1362 (S=O unsymmetrical 1 stretch), 1174 (S=O symmetrical stretch); H-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, 13 6H, –CH3). C-NMR (125 MHz, DMSO-d6) δ 148.9, 142.1, 138.1, 135.9, 135.7, 135.3, 134.6, 134.0, 20.7, 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%; 1 M.p. 264–267.1 ◦C; IR (ATR-Ge, cm− ): 2930 (NH stretch), 1591, 1569, 1540 (arom. stretch), 1350 (S=O 1 unsymmetrical stretch), 1161 (S=O symmetrical stretch); H-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 13 (d, J = 8.4 Hz, 2H, arom.), 4.33–4.25 (m, 4H, –CH2–). C-NMR (125 MHz, DMSO-d6) δ 156.2, 153.9, 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%; 1 M.p. 244.6–247◦C; IR (ATR-Ge, cm− ): 2896 (NH stretch), 1580, 1529, 1498 (arom. stretch), 1323 (S=O 1 unsymmetrical stretch), 1162 (S=O symmetrical stretch); H-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 13 (m, 4H, –CH2–). C-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 1 ◦C; IR (ATR-Ge, cm− ): 3070 (NH stretch), 1594, 1566, 1531 (arom. stretch), 1345 (S=O unsymmetrical 1 stretch), 1172 (S=O symmetrical stretch); H-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, 13 2H, arom.), 7.39 (t, J = 9 Hz, 3H, arom.). C-NMR (125 MHz, DMSO-d6) δ 148.3, 144.9, 142.4, 139.1, Molecules 2020, 25, 138 15 of 20

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 1 ◦C; IR (ATR-Ge, cm− ): 2937 (NH stretch), 1596, 1566, 1525 (arom.), 1397 (S=O unsymmetrical stretch), 1 1159 (S=O symmetrical stretch); H-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, 13 J = 9 Hz, 3H, arom.). C-NMR (125 MHz, DMSO-d6) δ 147.5, 145.7, 145.2, 138.4, 138.2, 137.5, 132.6, 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 1 (ATR-Ge, cm− ): 2925 (stretch NH), 1589, 1576, 1530 (arom. stretch), 1341 (S=O unsymmetrical stretch), 1 1160 (S=O symmetrical stretch); H-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) δ 148.3, 142.3, 139.1, 137.1, 135.1, 134.6, 131.7, 129.6, 129.5, 129.3, 128.8, 128.0, 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 1 ◦C; IR (ATR-Ge, cm− ): 3056 (NH stretch), 1522, 1505, 1460 (arom. stretch), 1398 (S=O unsymmetrical 1 stretch), 1169 (S=O symmetrical stretch); H-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, 13 arom.). C-NMR (125 MHz, DMSO-d6) δ 156.0, 147.5, 146.0, 145.6, 137.4, 136.3, 134.7, 132.5, 131.6, 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, 1 cm− ): 2927 (NH stretch), 1585, 1552, 1533 (arom. stretch), 1339 (S=O unsymmetrical stretch), 1159 1 (S=O symmetrical stretch); H-NMR (500 MHz, DMSO-d6) δ 11.53 (bs, 1H, sulfonamide), 8.87 (s, 1H, 13 pyrazine), 8.66 (d, J = 2.5 Hz, 2H, pyrazine), 7.99–7.14 (m, 6H, arom.). C-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 µm, 4 mm 4 mm and column ZORBAX Eclipse XDB-C18 5 µm, 4.6 × 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 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 Dt)/Dt. Capacity factor k was converted to the log scale and (log k), − and it was used as a measure of lipophilicity.

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 16 of 20 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 pKa = pH + log − A Au − 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 µL) 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 17 of 20

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 µg/mL) and 12b (MICM. kansasii = 12.5 µg/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 µg/mL, 219 µM). 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 µg/mL) and moderate activity against M. kansasii (MIC = 12.5 µg/mL), M. avium (MIC = 25 µg/mL), and M. aurum (MIC = 250 µg/mL). No antibacterial or antifungal activity was observed for any of the prepared 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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