Novel Halogenated Pyrazine-Based Chalcones as Potential Antimicrobial Drugs †

Chalcones, i.e., compounds with the chemical pattern of 1,3-diphenylprop-2-en-1-ones, exert a wide range of bio-activities, e.g., antioxidant, anti-inflammatory, anticancer, anti-infective etc. Our research group has been focused on pyrazine analogues of chalcones; several series have been synthesized and tested in vitro on antifungal and antimycobacterial activity. The highest potency was exhibited by derivatives with electron withdrawing groups (EWG) in positions 2 and 4 of the ring B. As halogens also have electron withdrawing properties, novel halogenated derivatives were prepared by Claisen-Schmidt condensation. All compounds were submitted for evaluation of their antifungal and antibacterial activity, including their antimycobacterial effect. In the antifungal assay against eight strains of selected fungi, growth inhibition of Candida glabrata and Trichophyton interdigitale (formerly T. mentagrophytes) was shown by non-alkylated derivatives with 2-bromo or 2-chloro substitution. In the panel of selected bacteria, 2-chloro derivatives showed the highest inhibitory effect on Staphylococcus sp. In addition, all products were also screened for their antimycobacterial activity against Mycobacterium tuberculosis H37RV My 331/88, M. kansasii My 235/80, M. avium 152/80 and M. smegmatis CCM 4622. Some of the examined compounds, inhibited growth of M. kansasii and M. smegmatis with minimum inhibitory concentrations (MICs) comparable with those of isoniazid.


Introduction
Chalcones are compounds with the basic scaffold of 1,3-diphenylprop-2-en-1-one, containing ring A and ring B connected by an α,β-unsaturated keto linker (indicated in red in Figure 1, structure 1). Naturally-occurring chalcones (in the plant kingdom) usually bear hydroxy-, methoxy-or prenyl substitutions and might serve as precursors of other flavonoid groups. Chalcones exert a wide range of bio-activities (e.g., antioxidant, anti-inflammatory, anticancer, anti-infective etc.), which were several times reviewed [1][2][3]. In synthetic chalcones, substitutents can be more varied and benzene rings have been many times replaced with other aryls or heteroaryls [2,3]. Based on our previous results concerning chalcones and the positive effect of electron-withdrawing group (EWG) substitution on their antimicrobial effects [4,5], we decided to prepare pyrazine analogues of chalcones halogenated in the ring B. Substitution of chalcones with halogens emerges also in studies of other authors, e.g., compound 1 inhibited growth of Trichophyton rubrum at minimum inhibitory concentrations (MIC) 12.5 μg/mL [6]. Compound 2 exerted better antifungal activity against dermatophytes (MIC 0.5-25 μg/mL) than amphotericin B and ketoconazole [7]. In a study comparing antifungal activity of mono-and dihalogenated 1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-ones (3a) and their corresponding flavonols (3b) [8], it was found that chalcones are more active against Trichophyton longifusus, Aspergillus flavus and Microsporum canis than the flavonols. The most active compound was fluorinated in position 4 of the ring B. Chalcones 4a-4c with halogen substitution in position 4 either in the ring A or in the ring B inhibited growth of Candida tropicalis and Aspergillus flavus at a concentration of 2 mg/mL at least by 98% [9].
Antibacterial effects of chalcones have also been reviewed [10][11][12][13] and importance of searching for new antimicrobial agents due to increasing antibiotic resistance of bacteria and fungi has been highlighted [13]. The substitution of aromatic rings with EWG was emphasized in a quinoline-based series of chalcones in association with antibacterial activity. The most active compound, inhibiting growth of Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Klebsiella aerogenes and Salmonella typhimurium, better than chloramphenicol or ciprofloxacin, is depicted in structure 5. Supposed mechanism of action was explored in a bacterial gyrase assay [14]. Two potentially antibacterial entities were combined in several series containing halogenated chalcones and variously substituted 2-thioxothiazolidin-4-ones (rhodanines) [15,16] or thiazolidin-2,4-dione [17]. Compounds, that inhibited Staphylococcus sp. comparably to norfloxacin, are drawn as structure 6 [15] and 7 [16] in Figure 2. In the same research group, analogical chalcones with substituted thiazolidinediones 8a and 8b were synthesized ( Figure 2). They inhibited growth of two strains of S. aureus similarly to oxacillin and norfloxacin [17]. Based on our previous results concerning chalcones and the positive effect of electron-withdrawing group (EWG) substitution on their antimicrobial effects [4,5], we decided to prepare pyrazine analogues of chalcones halogenated in the ring B. Substitution of chalcones with halogens emerges also in studies of other authors, e.g., compound 1 inhibited growth of Trichophyton rubrum at minimum inhibitory concentrations (MIC) 12.5 µg/mL [6]. Compound 2 exerted better antifungal activity against dermatophytes (MIC 0.5-25 µg/mL) than amphotericin B and ketoconazole [7]. In a study comparing antifungal activity of mono-and dihalogenated 1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-ones (3a) and their corresponding flavonols (3b) [8], it was found that chalcones are more active against Trichophyton longifusus, Aspergillus flavus and Microsporum canis than the flavonols. The most active compound was fluorinated in position 4 of the ring B. Chalcones 4a-4c with halogen substitution in position 4 either in the ring A or in the ring B inhibited growth of Candida tropicalis and Aspergillus flavus at a concentration of 2 mg/mL at least by 98% [9].
Antibacterial effects of chalcones have also been reviewed [10][11][12][13] and importance of searching for new antimicrobial agents due to increasing antibiotic resistance of bacteria and fungi has been highlighted [13]. The substitution of aromatic rings with EWG was emphasized in a quinoline-based series of chalcones in association with antibacterial activity. The most active compound, inhibiting growth of Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Klebsiella aerogenes and Salmonella typhimurium, better than chloramphenicol or ciprofloxacin, is depicted in structure 5. Supposed mechanism of action was explored in a bacterial gyrase assay [14]. Two potentially antibacterial entities were combined in several series containing halogenated chalcones and variously substituted 2-thioxothiazolidin-4-ones (rhodanines) [15,16] or thiazolidin-2,4-dione [17]. Compounds, that inhibited Staphylococcus sp. comparably to norfloxacin, are drawn as structure 6 [15] and 7 [16] in Figure 2. In the same research group, analogical chalcones with substituted thiazolidinediones 8a and 8b were synthesized ( Figure 2). They inhibited growth of two strains of S. aureus similarly to oxacillin and norfloxacin [17]. Halogen substitution has been found to be favorable in antimycobacterial diphenylpropenones [18] and 3-phenyl-1-pyridylpropenones [19]. Chalcones including halogenated derivatives have been described as inhibitors of Mycobacterium tuberculosis phosphatases [20] or inhibitors of fatty acid synthase II [21].
Sofalcone, explored and marketed in Japan, is a chalcone derivative. Mechanism of its action in ulcer disease is complex, but it possesses bactericidal effect against Helicobacter pylori as well [28].
Sofalcone, explored and marketed in Japan, is a chalcone derivative. Mechanism of its action in ulcer disease is complex, but it possesses bactericidal effect against Helicobacter pylori as well [28].
Sofalcone, explored and marketed in Japan, is a chalcone derivative. Mechanism of its action in ulcer disease is complex, but it possesses bactericidal effect against Helicobacter pylori as well [28].
Identity of compounds was confirmed by melting points, NMR and IR spectra. Proton NMR spectra clearly showed (J HαHβ = xx − yy Hz) that E-isomers have been obtained in all cases. Purity of the compounds was verified by TLC and elemental analysis.

Evaluation of In Vitro Antifungal Activity
All prepared compounds, including 16a-16e, were subjected to antifungal assay, only the most active ones are displayed in Table 2. In the panel of selected fungi, inhibition of growth of Trichophyton interdigitale 445 by the non-alkylated compounds was the most notable. That is in accordance with the previous results of our research group in the field of antifungal activity of pyrazine analogues of chalcones [4,5,31]. The derivative with 2-chloro substitution in the phenyl, i.e., ring B (15a) proved the best activity (MIC 3.9 µmol/L after 24 h incubation), comparable with that of systematically used antimycotic fluconazole (MIC 6.51 µmol/L after 24 h incubation). However, the derivative 15a did not reach the activity of terbinafine (MIC 0.01-1.72 µmol/L), that is usually used for therapy of dermatomycosis. 4-Fluoro, 2-bromo or 4-bromo substitution (14a, 17a and 18a) had also inhibiting effect on growth of T. interdigitale (MIC 3.9-7.81 µmol/L after 24 h incubation). Halogenated derivatives suppressed the growth of Candida spp. as well. In case of 4-fluoro derivative (14a), it was a moderate, non-specific inhibition (MIC 31.25-62.5 µmol/L after 24 h incubation), whereas chlorinated derivatives 15a and 16a inhibited specifically growth of Candida glabrata 20/I and Candida krusei E 28 (in both cases MIC 7.81 µmol/L after 24 h incubation). The effect of 15a on C. glabrata (MIC 7.81 µmol/L after 24 h incubation) is better than the effect of clinically used antimycotics listed in Table 2. Comparison with previously prepared substances (19)(20)(21)(22) is provided as well. Upper mentioned active halogenated derivatives were in common more antifungal effective than hydroxy (20a and 20b), nitro (21a and 21b) or methoxy (22a and 22b) derivatives, with the exception of 2-nitro derivative (21a) inhibiting strongly Candida spp. (antifungal activity in Table 2, structures in Figure 3). derivatives were in common more antifungal effective than hydroxy (20a and 20b), nitro (21a and 21b) or methoxy (22a and 22b) derivatives, with the exception of 2-nitro derivative (21a) inhibiting strongly Candida spp. (antifungal activity in Table 2, structures in Figure 3).    Table 2 for comparison of antifungal activity.  Table 2 for comparison of antifungal activity.

Evaluation of In Vitro Antibacterial Activity
All prepared compounds, including 16a-16f were subjected to antibacterial assay, and only the most active ones are displayed in Table 3. From the panel of tested bacteria, Staphylococcus spp. was the most susceptible. Growth of Staphylococcus aureus CCM 4516/08 and S. aureus H 5996/08 was inhibited by most compounds listed in Table 2 with moderate effect, but growth of Staphylococcus epidermidis H6966/08 was inhibited more significantly by 2-chlorinated derivatives (MIC of 15a: 3.9 µmol/L after 24 h incubation and MIC of 15b: 7.81 µmol/L after 24 h incubation) than by standard antibiotics. Comparing the character of halogen substitution, fluorine does not seem to be as important in our series as in diphenylpropenones [37]. In another study, focused on diphenylpropenones, derivatives with 3,5-dibromo or 3,5-bis(trifluoromethyl) substitution were the most active compounds [38]. In agreement with the positive effect of chlorine substitution in our series, the most antifungal and antibacterial substance in a 3-quinolinyl-1-thienyl propenones had so far three chlorine atoms [39]. Polyhalogenation of 3-phenyl-1-pyrrol-2-ylpropenones also positively reflected in their antimicrobial activity [40]. In a thiazole-based series of chalcones, derivatives chlorinated in the ring B exerted also better antibacterial and antifungal activity than unsubstituted derivative or nitro derivatives [41]. However, pyrimidine analogues of chalcones, variable halogenated in position 2 of the ring B, exert no remarkable antibacterial or antifungal activity [42].

Evaluation of In Vitro Antimycobacterial Activity
As far as the antimycobacterial screening is concerned, four strains were tested in total (all results are shown Table 4). The most susceptible strain to inhibition by halogenated pyrazine-based analogues of chalcones seems to be Mycobacterium kansasii Hauduroy CNCTC My 235/80. However, the lowest MICs have been achieved in 2-chlorinated compounds (15a and 15b) during testing on Mycobacterium tuberculosis H37RV CNCTC My 331/88 (MIC 6.25 µg/mL for 15a and 3.13 µg/mL for 15b). The efficacy on M. tuberculosis in these two cases correlates well with results of in-house testing on Mycobacterium smegmatis CCM 4622 (ATCC 607) (MIC 7.81 µg/mL for 15a and 3.9 µg/mL for 15b). In comparison with our previous results [4,5,31], positive influence of tert-butyl substitution in the position 5 of ring A has been confirmed again during testing on M. tuberculosis, which is the most pathogenic strain. As for substitution of ring B, 2-halogen substituent seems to be of importance, as products 13a, 15b and 17a showed the lowest MICs (3.13-6.25 µg/mL against M. tuberculosis H37RV CNCTC My 331/88).
In Table 4, calculated lipophilicities of all tested compounds are provided. They do not seem to correlate with biological activity of the compounds. Conformation of particular molecules influenced by halogen position may play a more important role.
Silica gel 0.040-0.063 nm (Merck, Darmstadt, Germany) and Silpearl (Kavalier, Votice, Czech Republic) were used for flash column chromatography. The purity of the products was checked by TLC on aluminium sheets, silica gel 60 F 254 (Merck). Mixtures of hexane and ethyl acetate were used for TLC and column chromatography. Analytical samples were dried over anhydrous phosphorus pentoxide under reduced pressure at room temperature. Melting points were determined either on a Boëtius apparatus or on Stuart SMP 20 (Bibby Scientific Ltd., Stone, UK) and are uncorrected. Elemental analyses were performed on an EA 1110 CHNS instrument (CE Instruments, Milano, Italy) or on a Vario Micro Cube Elemental Analyzer (Elementar Analysensysteme GmbH, Hanau, Germany). Infrared spectra were recorded either in KBr pellets on a Nicolet Impact 400 IR spectrophotometer (Thermo Scientific, Waltham, MA, USA) or on germanium crystal using ATR method (indicated at particular compound spectra) on a Nicolet Impact 6700 IR spectrophotometer (Thermo Scientific). Characteristic wavenumbers are given in cm −1 . 1 H-and 13 C-NMR spectra were recorded at ambient temperature on a Varian Mercury-Vx BB 300 spectrometer (Varian Corp., Palo Alto, CA, USA) operating at 300 MHz for 1 H and 75 MHz for 13 C or on a VNMR S500 (500 MHz for 1 H-NMR a 125 MHz for 13 C-NMR). Chemical shifts were recorded as δ values in ppm, and were indirectly referenced to tetramethylsilane (TMS) via the solvent signal (2.49 for 1 H, 39.7 for 13 C in DMSO-d6 and 7.26 for 1 H, 77.0 for 13 C in CDCl 3 ). Coupling constants J are given in Hz.

Synthesis of Halogenated (E)-1-(Pyrazin-2-yl)-3-phenylprop-2-en-1-ones
1-(Pyrazin-2-yl)ethan-1-one or 1-(5-alkylpyrazin-2-yl)ethan-1-one (0.01 mol) and the corresponding halogenated benzaldehyde (0.01 mol) were dissolved in pyridine (4.4 mL). Diethylamine (0.01 mol) was added, and the reaction mixture was stirred at 80-120 • C for 1 h. After cooling, the mixture was poured into ice water (200 mL), acidified to pH 3 with a few drops of acetic acid, and then refrigerated for 24 h. The separation of crude products from water depended on their character. Solids were filtered off and crystallized from anhydrous ethanol, while oily mixtures were extracted with diethyl ether and subjected to flash chromatography on silica gel. Hexane-ethyl acetate was used as the eluent in an appropriate ratio (70:30 (v/v), 80:20 (v/v) or 90:10 (v/v)). The fractions containing the desired compounds were combined and crystallized from absolute ethanol to obtain analytically pure crystals. Using this procedure, the following compounds were obtained: suspension of 1.0 ± 0.2 × 10 5 colony forming units (CFU)/mL and 1.0 ± 0.2 × 10 6 CFU/mL for yeasts and molds, respectively. The final inoculum was made by 1:20 dilution of the stock suspension with the test medium. The compounds were dissolved in DMSO, and the antifungal activity was determined in RPMI 1640 media (KlinLab, Prague, Czech Republic) buffered to pH 7.0 with 0.165 M 3-morpholinopropane-1-sulfonic acid (Sigma-Aldrich, St. Louis, MO, USA). Controls consisted of medium and DMSO alone. The final concentration of DMSO in the test medium did not exceed 1% (v/v) of the total solution. The concentrations of the studied substances ranged from 500 to 0.488 µmol/L. The minimum inhibitory concentration (MIC), was defined as 80% or greater (for yeasts and yeast-like organisms-IC 80 ), resp. 50% or greater (for molds-IC 50 ) reduction of growth in comparison with the control. The values of MICs were determined after 24 and 48 h of static incubation at 35 • C. In the case of T. interdigitale, the MICs were recorded after 72 and 120 h due to its slow growth rate. Fluconazole and voriconazole were used as reference antifungal drugs.

Evaluation of In Vitro Antibacterial Activity
The antibacterial activity of all compounds was evaluated by the microdilution broth method [46]. Institute of Public Health, Prague, Czech Republic. Middlebrook 7H9 broth (Sigma-Aldrich, Steinheim, Germany) enriched with 0.4% of glycerol (Sigma-Aldrich) and 10% of OADC supplement (oleic acid, albumin, dextrose, catalase; Himedia, Mumbai, India) of declared pH 6.6 was used for cultivation. Tested compounds were dissolved and diluted in DMSO and mixed with broth (25 µL of DMSO solution in 4.475 mL of broth) and placed (100 µL) into microtitration plate wells. Mycobacterial inocula were suspended in isotonic saline solution and the density was adjusted to 0.5-1.0 McFarland. These suspensions were diluted by 10 −1 and used to inoculate the testing wells, adding 100 µL of suspension to 100 µL of the DMSO/broth solution of tested compound. Final concentrations of tested compounds in wells were 100, 50, 25, 12.5, 6.25, 3.13 and 1.56 µg/mL. Isoniazid was used as positive control (inhibition of growth). Negative control consisted of broth plus DMSO. 30 µL of Alamar Blue working solution (1:1 mixture of 0.01% resazurin sodium salt (aq. sol.) and 10% aqueous solution of Tween 80) was added usually after 5 days of incubation. Results were then determined after 24 h of incubation and interpreted according to Franzblau [47]. The MIC (µg/mL) was determined as the lowest concentration which prevented the blue to pink colour change.

Evaluation of In Vitro Antimycobacterial Activity on Mycobacterium smegmatis
An antimycobacterial assay was performed with fast growing Mycobacterium smegmatis CCM 4622 (ATCC 607) from Czech Collection of Microorganisms (Brno, Czech Republic). The technique used for activity determination was microdilution broth panel method using 96-well microtitration plates. Culturing medium was Middlebrook 7H9 (MB) broth (Sigma-Aldrich) enriched with 0.4% of glycerol (Sigma-Aldrich) and 10% of Middlebrook OADC growth supplement (Himedia, Mumbai, India). Tested compounds were dissolved in DMSO (Sigma-Aldrich) then MB broth was added to obtain concentration 2000 µg/mL. Standards used for activity determination were isoniazid (INH), rifampicin (RIF) and ciprofloxacin (CPX) (Sigma-Aldrich). Final concentrations were reached by twofold dilution and addition of mycobacterial suspension and were set as 500, 250, 125, 62.5, 31.25, 15.625, 7.81 and 3.91 µg/mL except to standards ciprofloxacin and rifampicin where the final concentrations were 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195 and 0.098 µg/mL. Drug-free controls consisted of broth and DMSO and were used as a growth control. The final concentration of DMSO did not exceeded 2.5% (v/v) to not affect the growth of M. smegmatis. Plates were sealed with polyester adhesive film and incubated in the dark at 37 • C without agitation. The addition of 0.01% solution of resazurin sodium salt followed after 48 h. This stain was prepared by dissolving resazurin sodium salt (Sigma-Aldrich) in deionised water to get 0.02% solution. Then 10% aqueous solution of Tween 80 (Sigma-Aldrich) was prepared. Both liquids were mixed up making use of the same volumes and filtered through syringe membrane filter. Microtitration plates were then incubated for further 4 h. Antimycobacterial activity was expressed as MIC and the value was determined on the basis of stain color change (blue color-active compound; pink color-non active compound). MIC values for standards were within the ranges 7.81-15.625 µg/mL for INH, 0.78-1.56 µg/mL for RIF and 0.098-0.195 µg/mL for CPX. All experiments were conducted in duplicate.

Calculation of Lipophilicity
Theoretical lipophilicities logP and corrigated values ClogP of all compounds have been calculated in ChemDraw Professional 15.0, part of ChemOffice (Perkin Elmer, Waltham, MA, USA).

Conclusions
Chalcones have a very simple chemistry, which enables multiplicity of substitutions with easy synthesis and different pharmacological potential in dependence on particular structural modification [13]. Within this work, twenty halogenated pyrazine-based compounds have been prepared and characterized. They are all novel compounds with exception of three compounds. They were tested on antifungal, antibacterial, as well as on antimycobacterial effects. 4-Chlorinated series, prepared earlier, have been included in the assays. The results of biological screenings have been compared with our previously published compounds. Importance of an EWG substitution has been confirmed and some compounds displayed comparable or even better inhibitory activity than standard antifungals, antibiotics and antimycobacterial agents.
As far as the structure-activity relationships are concerned, halogen substitution in the ring B of pyrazine-based chalcones proved to have positive influence on antifungal effect against Candida spp. and Trichophyton interdigitale in comparison with our previously prepared series of pyrazine-based chalcones [4,5,31]. Generally, chlorinated and brominated series inhibited growth of fungi more significantly than fluorinated derivatives.
Halogen substitution in the ring B had positive impact on inhibition of Staphylococcus spp., whereas it did not influence inhibition of other bacteria from the panel. As for specific substitution, 2-chlorinated derivative inhibited markedly growth of S. epidermidis. Concerning mycobacteria, halogenated series inhibited significantly growth of M. krusei, M. tuberculosis and M. smegmatis, but they did not strongly influence the growth of M. avium. The inhibiting activity of halogenated series was comparable with that of nitro series prepared previously [4,5]. Derivatives substituted by halogen in position 2 of the ring B and concurrently substituted by tert-butyl in the position 5 of the pyrazine ring showed the highest inhibition of M. tuberculosis. Importance of tert-butyl substitution has been observed previously in other series [5]. Presence of isopropyl in position 5 of the pyrazine ring seems to be important in chlorinated or brominated series in the test against M. krusei.