Synthesis and Biological Evaluation of N-Alkyl-3-(alkylamino)-pyrazine-2-carboxamides

A series of N-alkyl-3-(alkylamino)pyrazine-2-carboxamides and their N-alkyl-3-chloropyrazine-2-carboxamide precursors were prepared. All compounds were characterized by analytical methods and tested for antimicrobial and antiviral activity. The antimycobacterial MIC values against Mycobacterium tuberculosis H37Rv of the most effective compounds, 3-(hexylamino)-, 3-(heptylamino)- and 3-(octylamino)-N-methyl-pyrazine-2-carboxamides 14‒16, was 25 μg/mL. The compounds inhibited photosystem 2 photosynthetic electron transport (PET) in spinach chloroplasts. This activity was strongly connected with the lipophilicity of the compounds. For effective PET inhibition longer alkyl chains in the 3-(alkylamino) substituent in the N-alkyl-3-(alkylamino)pyrazine-2-carboxamide molecule were more favourable than two shorter alkyl chains.


Introduction
Pyrazines are symmetrical heterocyclic aromatic organic compounds. Pyrazine is a weaker base than pyridine, pyridazine and pyrimidine. Pyrazine derivatives occur in many natural sources and can be synthesized chemically or biologically. In animals and plants, they are considered to be alert signal molecules, functioning as deterrents or attractants depending on the circumstances without having harmful or beneficial effect themselves. Pyrazines contribute to many aromas and flavours of heated foods such as beef products, toasted barley, cocoa, coffee, peanuts, popcorn, potato chips, rye crisp bread and roasted filberts, as well as in fresh foods like tomatoes, peas, green bell peppers, asparagus, kohlrabi and dairy products. The pyrazine moiety is an important part of many clinically used drugs, including anticancer, diuretic [1], antidiabetic, antithrombotic, antidepressants or anti-infective (antituberculotics, bactericides and fungicides) agents, and offers many possibilities in drug development [2][3][4].
Tuberculosis (TB) remains a serious global problem and the need for new drugs is still actual [5][6][7]. One of the most important drugs for TB treatment is pyrazinamide (PZA), which belongs to the first-line drugs for TB treatment and has been used extensively since 1980s. PZA has an excellent sterilizing effect on semi-dormant tubercle bacilli and, when used in combination with rifampicin, helps in shortening the duration of treatment [8]. One of the mechanisms of action is competitive inhibition of NADPH binding to Mycobacterium tuberculosis Fatty Acid Synthase I. This enzyme is devoted to the synthesis of common fatty acids and of specific mycolic acids, which constitute the major lipid component of the envelope and form the external mycomembrane. 5-Chloropyrazine-2-carboxamide analogues act as well as PZA [9,10]. 5-(Alkylamino)pyrazine-2-carboxamides and 6-(alkylamino)pyrazine-2-carboxamides prepared previously by Servusová et al. [11], showed an interesting in vitro whole cell antimycobacterial activity. Based on these results we decided to prepare a series of 3-(alkylamino)pyrazine-2-carboxamides, to evaluate the importance of positional isomerism in these derivatives.
Several herbicides acting as photosynthesis inhibitors (acylanilides, thioacylanilides, phenylcarbamates, ureas, etc.) have (thio)carbamoyl group, -NHCO-or -NHCS-, in their molecules [12][13][14]. Some commercially available herbicides act by reversible binding to photosystem 2 (PS2), a membrane-protein complex in the thylakoid membranes which catalyses the oxidation of water and the reduction of plastoquinone [15] and thereby inhibit photosynthetic electron transport (PET). Experimental studies have established that many PS 2 herbicides bind non-covalently to a 32 kDa protein in the PS2 complex and inhibit electron transfer between primary electron acceptor-quinone QA and the secondary electron acceptor-quinone QB on the reducing side of PS2 [16]. Numerous PS2 herbicides contain hydrophobic components as well as a flat polar component. The function of the hydrophobic components is to increase the lipid solubility of the entire herbicide molecule and to fit the hydrophobic surface of the herbicide binding site and it is assumed that the flat polar component binds electrostatically at a highly polar protein site [17]. Using EPR spectroscopy it was shown that tyrosine radicals TyrZ and TyrD which are situated in D1 and D2 proteins on the donor side of PS2 interacted with some organic compounds, e.g., substituted benzanilides [18] or substituted 2,6-disubstituted pyridine-4-thiocarboxamides [19] or anilides of pyrazine-2-carboxylic acids [14,20] and due to this interaction interruption of the photosynthetic electron transport occurred.
This study is focused on preparation of alkyl substituted derivatives of PZA. More specifically, it deals with the length of the alkylamino chain in position 3 and its influence on biological effects in comparison with previously evaluated 5-and 6-alkylamino isomers. Antimycobacterial activity of all prepared compounds was determined and compounds were evaluated also for inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. The structure-activity relationships between the chemical structure and in vitro biological activities of evaluated compounds are discussed.

Chemistry
We prepared a series of pyrazinamide alkylamino derivatives according to the general procedures shown in Scheme 1. 3-Chloro-N-methylpyrazine-2-carboxamide (1) was previously prepared by Allen et al. [21], 3-chloro-N-propylpyrazine-2-carboxamide (3) by Zhu et al. [22] and N-methyl-3-(methylamino)pyrazine-2-carboxamide (9) by Albert et al. [23], but they were not tested for any biological activity. The other 27 compounds are novel. Precursors 1-8 were synthesized from commercially available 3-chloropyrazine-2-carbonitrile by hydrolysis (Scheme 1, step a) to the corresponding acid, conversion to the acyl chloride (step b) and its aminolysis (step c) by the corresponding amine. Final products 9-30 were prepared in yields ranging from 41% to 98% by aminodehalogenation (step d) in a microwave reactor with a focused field. All compounds were characterized by 1 H-, 13 C-NMR and IR spectroscopy, solid compounds by melting points and elemental analysis, and liquid compounds by HRMS.

Lipophilicity
Lipophilicity is one of the most important drug characteristics. It plays a significant role in penetration through biological membranes. Experimentally determined values of lipophilicity log k and calculated values of log P of prepared compounds are summarized in Table 1 together with corresponding IC50 values related to PET inhibition in spinach chloroplasts and MIC values related to antimycobacterial activity against M. tuberculosis. According to their structure, the prepared compounds could be divided into four groups: N-alkyl-3chloropyrazine-2-carboxamides (group A; 1-8), 3-(alkylamino)-N-methylpyrazine-2-carboxamides (group B; 9-16), 3-(alkylamino)-N-ethylpyrazine-2-carboxamides (group C; 17-24) and N-alkyl-3-(alkylamino)pyrazine-2-carboxamides (group D; 25-30). At comparable log P values the experimentally determined log k values of N-alkyl-3-chloropyrazine-2-carboxamides (group A) were lower than the log k values estimated for compounds of groups B, C and D ( Figure 1). The dependences of log k vs. log P were linear for eight compounds of group A (1) as well as for 22 compounds belonging to groups B, C and D (2) and the corresponding correlations provided excellent results of statistical analysis: The antimycobacterial activity of compounds was closely connected with lipophilicity of the compounds (Table 1, Figure 2). From the series of N-alkyl-3-chloropyrazine-2-carboxamides (group A) the MIC values could be estimated only for three compounds and the results indicate that a strong increase in antimycobacterial activity was observed up to log k = 0.702. The dependence of log (1/MIC) on log k for 3-(alkylamino)-N-methylpyrazine-2-carboxamides (group B) was bilinear and strong activity increase was observed for log k from 0.401 (12, R 2 = C4H9) to 0.876 (14, C6H13), while with further lipophilicity increase up to log k = 1.359 the inhibitory activity expressed in molar concentrations slightly increased (14)(15)(16). Considerably lower antimycobacterial activity against M. tuberculosis of 3-(alkylamino)-Nethylpyrazine-2-carboxamides (group C) in comparison with 3-(alkylamino)-N-methylpyrazine-2carboxamides (group B) with comparable lipophilicity indicates that substitution of -CONHCH3 substituent in position 2 of the pyrazine ring with -CONHC2H5 accompanied with the reduction of alkyl chain length by one CH2 group in 3-alkylamino substituent led to significant activity decrease (Table 1). For example, compound 14 (group B, R 1 = CH3, R 2 = C6H13) has a total number of seven carbons in aliphatic alkyl chains, lipophilicity of log k = 0.876 and MIC = 25 μg/mL. Compound 21 (group C, R 1 = C2H5, R 2 = C5H11) has an equal number of carbons in aliphatic alkyl chains, similar lipophilicity of log k = 0.855, but MIC = 50 μg/mL, i.e., approx. half of the activity of compound 14. Consequently, from the aspect of antimycobacterial activity at comparable lipophilicity, longer alkyl chain in 3-(alkylamino) substituent is favourable.
Only one compound from group D containing two butyl groups in its molecule (26, R 1 = R 2 = C4H9) was found to be active against M. tuberculosis H37Rv and its activity was comparable with compound 22 from group C with similar lipophilicity (Figure 2).

Antibacterial and Antifungal Activity
This evaluation was performed in order to obtain results for antifungal and antibacterial activity against eight fungal strains and eight bacterial strains of clinical significance. The most effective compound against Trichophyton mentagrophytes was 8 (R 1 = C8H17) with MIC = 62.5 µmol/L, the results of other active compounds are shown in Table 2. The dependence of antifungal activity of tested compounds against T. mentagrophytes is shown in Figure 3. Although the MIC values were estimated only for two compounds of group A, it is evident that antifungal activity of these compounds was significantly higher than the activity of compounds from group B and C with comparable lipophilicity.

Antiviral Activity
All substances were tested for their activity against diverse DNA and RNA viruses. The virus panel included pathogens of medical importance such as herpesviruses, HIV and influenza virus. None of the prepared compounds displayed any antiviral activity.

Cytotoxicity Assay
In vitro cytotoxicity assays on HeLa and Vero cells were performed using standard assays. The results were expressed as minimum compound concentration (MCC) that causes a microscopically detectable alteration of normal cell morphology. No cytotoxicity was detected up to the highest tested concentration of 100 µmol/L, except for 30 (MCC = 100 µmol/L, Vero cells). The highest tested concentration for 28 in Vero cells assay was 4 µmol/L due to solubility issues.
The loss of a biological activity observed for amphiphilic compounds upon elongation of their hydrophobic (hydrocarbon) part is called 'cut-off' effect [25,26]. The hydrophobic parts of such compounds interact with lipidic parts of biological (including thylakoid) membranes. It could be noted that water solubility of compounds with longer alkyl chain is limited and too large values of compound partition coefficient did not enable the penetration of such molecules through hydrophilic (aqueous) regions of biological membranes. Thus, the final concentration of long-chain compounds in the membrane will be lower than that of compounds with shorter alkyl chain, and this phenomenon is connected with loss of biological activity. According to the free volume theory the extent of membrane disturbance due to incorporation of compound containing alkyl chain depends on the size of free volume created under its alkyl chain which can be then filled up with chains of neighbouring lipids as well as on the partition coefficient of the compounds [25][26][27][28]. Therefore the most effective disturbance of the membrane and thus the highest inhibitory activity is shown by compounds with middle alkyl chain length ensuring not only sufficiently high free volume under alkyl chain but also high concentration of the compound in the membrane due to suitable value of compound partition coefficient.
Moreover compounds with CONH group(s) in their molecules can interact with amino acid constituents in proteins resulting in the loss of their function. It should be stressed that the CONH group is characteristic of many herbicides [12,29] and it could also to a certain extent contribute to their inhibitory effects. For example, the determined IC50 value related to PET inhibition for N,N'-bis(2-dimethylaminoethyl)ethanediamide (which does not contain a long alkyl chain) was found to be 4.0 mmol/L, while the corresponding IC50 value estimated for 3,8-diaza-4,7-dioxodecane-1,10diylbis(nonyldimethyl)ammonium bromide was 1.74 mmol/L [30]. This indicates that CONH groups in spacer of this surfactant molecule participated on the resulting inhibitory effects. Because studied pyrazine-2-carboxamide derivatives do not contain in their structure longer alkyl chain than octyl, by analogy with above mentioned surfactants it could be assumed that CONH group also participates in PET inhibition, as it was shown previously by fluorescence experiments [24,31].
Strong dependence of the PET-inhibiting activity on the alkyl chain length of the alkoxy substituent was estimated previously for esters of 2-, 3-and 4-alkoxy substituted phenylcarbamic acids (alkyl = methyl -decyl). For these compounds the dependence of log (1/IC50) vs. alkyl chain length showed a typical quasi-parabolic course with maximum effect at 6-8 carbon atoms in the alkyl chain of piperidinoethylesters [13], 7-9 carbon atoms in the alkyl chain of dimethylaminoethylesters [32] and 8-9 carbon atoms in the alkyl chain of piperidinopropyl esters of alkoxyphenylcarbamic acids [33,34].
Because the tested pyrazine-2-carboxamides inhibited Hill reaction, similarly to previously studied pyrazine derivatives [14,20], they can be considered as photosystem 2 (PS2) inhibitors. The PS2 inhibitors can act on the donor and/or the acceptor side of PS2. Interaction of N-phenylpyrazine-2carboxamides with the D • intermediate which is situated at 161st position in D2 protein occurring on the donor side of PS2 was confirmed previously by EPR spectroscopy. Due to this interaction the photosynthetic electron transport from the oxygen evolving complex to the reaction centre of PS2 was impaired and consequently, the electron transport between PS2 and PS1 was inhibited.
2,5-Diphenylcarbazide (DPC) is an artificial electron donor acting in Z/D intermediate on the donor side of PS2 [35]. After addition of DPC to chloroplasts which activity had been inhibited by studied compounds to 75%, the PET was gradually restored with increasing DPC concentration. However, for complete restoration of PET, approximately five-fold higher DPC concentration was necessary compared to the applied concentration of inhibitor. This indicates that the studied compounds could damage PET also in the section between P680 and secondary quinone acceptor QB on the acceptor side of PS2. The binding of compounds with herbicidal activity, e.g., atrazine or metribuzine, was found to be altered by DPC presumably because of overlapping binding domain in the QB pocket, however DPC on the QB site affected plastoquinone reduction only at relatively high concentrations [36,37]. For PS2 hebicides such are DCMU or atrazin also a second binding site situated on the donor side of PS2 near Z/D intermediates and the high-affinity Mn-binding sites was described by several researchers [38][39][40]. Based on these finding as well as on above mentioned results related to interaction of N-phenylpyrazine-2-carboxamides with the D • intermediate on the donor side of PS2, we assume similar site of action also for the studied pyrazinamide alkylamino derivatives.
The three amino acids with aromatic ring side chains-phenylalanine, tyrosine and especially tryptophan-are sensitive to the local electrostatic environment in proteins and they will undergo fluorescence wavelength and/or intensity changes upon whatever functional process a protein performs [41]. Interaction of substituted pyrazine-2-carboxamides with residues of aromatic amino acids (AAA), mainly tryptophan and tyrosine occurring in photosynthetic proteins situated predominantly in PS2, was documented by the quenching of AAA fluorescence at 334 nm ( Figure 5A). Figure 5A   The quenching of the fluorescence of aromatic amino acids in the presence of 5-bromo-and 3,5-dibromo-2-hydroxy-N-phenylbenzamides [42] and ring-substituted 2-hydroxynaphthalene-1carboxanilides [31] was observed previously.

General Information
All chemicals were of reagent or higher grade of purity and were purchased from Sigma-Aldrich (Steinheim, Germany), unless stated otherwise. The progress of the reaction was checked by Thin Layer Chromatography (TLC) (Alugram ® Sil G/UV254, Machery-Nagel, Postfach, Germany) with UV detection using wavelength 254 nm. Microwave assisted reactions were performed in a CEM Discover microwave reactor with a focused field (CEM Corporation, Matthews, NC, USA) connected to an Explorer 24 autosampler (CEM Corporation) and this equipment was running under CEM's Synergy TM software for setting and monitoring the conditions of reactions. The temperature of the reaction mixture was monitored by internal infrared sensor. All obtained products were purified by preparative flash chromatograph CombiFlash ® Rf (Teledyne Isco Inc., Lincoln, NE, USA). The type of elution was gradient, using the mixture of hexane (LachNer, Neratovice, Czech Republic) and ethyl acetate (Penta, Prague, Czech Republic) as mobile phase. Silica gel (0.040-0.063 nm, Merck, Darmstadt, Germany) was used as the stationary phase. NMR spectra were recorded on Varian Mercury-VxBB 300 with frequencies 299.95 MHz for 1 H and 75.43 MHz for 13 C or Varian VNMR S500 (499.87 MHz for 1 H and 125.71 MHz for 13 C) spectrometers (Varian Corporation, Palo Alto, CA, USA). Chemical shifts were reported in ppm (δ) and were referred indirectly to tetramethylsilane via signal of solvent (2.49 for 1 H and 39.7 for 13 C in DMSO-d6). Infrared spectra were recorded with spectrometer FT-IR Nicolet 6700 (Thermo Scientific, Waltham, MA, USA) using attenuated total reflectance (ATR) methodology. Elemental analyses were measured with EA 1110 CHNS Analyzer (Fisons Instruments S. p. A., Carlo Erba, Milano, Italy). UHPLC system Acquity UPLC I-class (Waters, Millford, MA, USA) coupled to high resolution mass spectrometer (HRMS) Synapt G2Si (Waters, Manchester, UK) based on Q-TOF were used for HRMS spectra measurement. Chromatography was performed using Acquity UPLC BEH C18 (2.1 × 100 mm, 1.7 um) column using gradient elution with acetonitrile and 0.1% formic acid at flow-rate 0.4 mL/min. Electrospray ionization was operated in positive mode. The ESI spectra were recorded in the range 50-1200 m/z using leucine-enkefaline as a lock mass reference and sodium formate for mass calibration. Melting points were assessed by SMP3 Stuart Scientific (Bibby Sterling Ltd., Staffordshire, UK) in open capillary and are uncorrected. Lipophilicity parameter log P was calculated by software CS ChemBioDraw Ultra 13.0 (CambridgeSoft, Cambridge, MA, USA).
3-Cl-POA (1.0 g, 6.3 mmol) was dispersed in dry toluene (approx. 50 mL) with thionyl chloride (1.4 mL, 18.9 mmol, 3 equiv.) and a catalytic amount (1-2 drops) of N,N-dimethylformamide (DMF). The reaction mixture in round bottomed flask was stirred and heated in an oil bath under a condenser at 95 °C for approximately 1 h. Solvents were evaporated in vacuo and the residue was azeotroped with dry toluene (3 × 20 mL) to remove the unreacted SOCl2 to yield crude acyl chloride [43] as brown solid, which was used without further purification.
The whole amount of 3-chloropyrazine-2-carbonyl chloride prepared in the previous step was dissolved in dry acetone. An appropriate alkylamine (18.9 mmol, 3 equiv.) along with triethylamine (0.64 g, 6.3 mmol, 1 equiv.) were added to the reaction mixture and stirred at laboratory temperature overnight [43]. The progress of reaction was checked by TLC in system hexane/ethyl acetate (1:1 or 2:1). The reaction mixture was adsorbed to silica by removing the solvents in vacuo and the product was purified by flash chromatography using gradient elution with ethyl acetate in hexane. Precursors 1-8 were recrystallized from EtOH/H2O if needed.

Evaluation of in Vitro Antifungal Activity
Antifungal evaluation was performed using microdilution broth method [45] against 8 fungal strains (Candida albicans ATCC 44859, C. tropicalis 156, C. krusei E28, C. glabrata 20/I, Trichosporon asahii 1188, Aspergillus fumigates 231, Absidia corymbifera 272 and Trichophyton mentagrophytes 445). Compounds were dissolved in DMSO and diluted in a twofold manner with RPMI 1640 medium with glutamine buffered to pH 7.0 (3-morpholinopropane-1-sulfonic acid). The final concentration of DMSO in the tested medium did not exceed 2.5% (v/v) of the total solution composition. Static incubation was performed in the dark and humid, at 35 °C for 24 and 48 h (respectively 72 and 120 h for Trichophyton mentagrophytes). The MIC was defined as 80% inhibition of control (50% IC for filament fungi). Drug-free controls were included. The standards were amphotericin B, voriconazole, nystatin and fluconazole.

Antiviral Evaluation
Antiviral activity in cell culture assessed by cytopathic effect (CPE) reduction assays with a broad panel of viruses [46][47][48]. The following viruses were examined on human embryonic lung fibroblast cells: herpes simplex virus type 1 (HSV-1); a thymidine kinase-deficient (TK − ) HSV-1 KOS strain resistant to acyclovir; herpes simplex virus type 2 (HSV-2); vaccinia virus; human adenovirus type 2; and vesicular stomatitis virus (VSV). The viruses examined on human cervix carcinoma HeLa cells were: VSV; Coxsackie B4 virus; and respiratory syncytial virus (RSV). African Green Monkey Vero cells were used to determine the antiviral effect on para-influenza-3 virus; reovirus-1; Sindbis virus; Coxsackie B4 virus and Punta Toro virus. Human influenza A/H1N1, A/H3N2 and B viruses were assessed on Madin-Darby canine kidney (MDCK) cells. Activity against human immunofeficiency virus (HIV) type 1 and type 2 was studied in human MT-4 lymphoblast cells. To perform the tests, the virus was added to semiconfluent cell cultures in 96-well plates and simultaneously serial dilutions of the test compounds were added. The plates were incubated until clear CPE was reached (typically 3-6 days). Microscopic scoring was then performed to determine the antiviral activity [expressed as 50% effective concentration (EC50)]. In the case of HIV-1, HIV-2 and influenza virus, virus-induced CPE was determined by the colorimetric formazan-based MTS cell viability assay.

Study of Inhibition of Photosynthetic Electron Transport (PET) in Spinach Chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovicova and Kralova [49]. The inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was determined spectrophotometrically (Genesys 6, Thermo Electron Scientific Instruments, Madison, WI, USA), using an artificial electron acceptor 2,6-dichlorophenol-indophenol (DCPIP) according to Kralova et al. [50], and the rate of photosynthetic electron transport (PET) was monitored as a photoreduction of DCPIP. The measurements were carried out in phosphate buffer (0.02 mol/L, pH 7.2) containing sucrose (0.4 mol/L), MgCl2 (0.005 mol/L) and NaCl (0.015 mol/L). The chlorophyll content was 30 mg/L in these experiments and the samples were irradiated (~100 W/m 2 with 10 cm distance) with a halogen lamp (250 W) using a 4 cm water filter to prevent warming of the samples (suspension temperature 22 °C). The studied compounds were dissolved in DMSO due to their limited water solubility. The applied DMSO concentration (up to 4%) did not affect the photochemical activity in spinach chloroplasts. The inhibitory efficiency of the studied compounds was expressed by IC50 values, i.e., by molar concentration of the compounds causing 50% PET inhibition relative to the untreated control. The comparable IC50 value for a selective herbicide 3-(3,4-dichlorophenyl)-1,1dimethylurea (DCMU, Diuron ® ) was about 1.9 μmol/L.

Study of Fluorescence of Aromatic Amino Acids in Spinach Chloroplasts
The fluorescence emission spectra of aromatic amino acids (AAA) in spinach chloroplasts were recorded on fluorescence spectrophotometer F-2000 (Hitachi, Tokyo, Japan) using excitation wavelength λex = 275 nm for monitoring AAA fluorescence, excitation slit 20 nm and emission slit 10 nm. The samples were kept in the dark for 2 min before measuring. The phosphate buffer used for dilution of the chloroplast suspension was the same as described above. Due to low aqueous solubility the compounds were added to chloroplast suspension in DMSO solution. The DMSO concentration in all samples was the same as in the control (10%). The chlorophyll concentration in chloroplast suspension was 10 mg/L.