Substituted Pyrazinecarboxamides: Synthesis and Biological Evaluation †

Condensation of the corresponding chlorides of some substituted pyrazine-2-carboxylic acids (pyrazine-2-carboxylic acid, 6-chloropyrazine-2-carboxylic acid, 5-tert-butylpyrazine-2-carboxylic acid or 5-tert-butyl-6-chloropyrazine-2-carboxylic acid) with various ring-substituted aminothiazoles or anilines yielded a series of amides. The syntheses, analytical and spectroscopic data of thirty newly prepared compounds are presented. Structure-activity relationships between the chemical structures and the anti-mycobacterial, antifungal and photosynthesis-inhibiting activity of the evaluated compounds are discussed. 3,5-Bromo-4-hydroxyphenyl derivatives of substituted pyrazinecarboxylic acid, 16-18, have shown the highest activity against Mycobacterium tuberculosis H 37 Rv (54-72% inhibition). The highest antifungal effect against Trichophyton mentagrophytes, the most susceptible fungal strain tested, was found for


Introduction
One third of the world's population is infected with tuberculosis (TB), therefore today TB still represents one of the major worldwide public health problems.The current recommended strategy is facing two problems: multidrug resistance and HIV/AIDS pandemic [1].There is an urgent need for new antimycobacterial drugs, especially for treatment of multi-drug resistant tuberculosis (MDR-TB), a growing problem among HIV-infected patients [2].Additionally, in patients with impaired cellular immunity, mycobacterial and fungal (Aspergillus, Histoplasma, etc.) infections predominate and may coexist [3].Pyrazinamide (PZA) is an important sterilising tuberculosis drug that helps to shorten the duration of current chemotherapy regimens for tuberculosis.PZA enters Mycobacterium tuberculosis by passive diffusion, is converted to pyrazinoic acid by nicotinamidase (pyrazinamidase) and is then excreted by a weak efflux pump [4].
One of the major goals for the physico-chemical characterisation of drugs is the prediction and/or measurement of their lipophilicity.The logarithm of the octanol-water partition coefficient (log P) has become the most widely used parameter for defining lipophilicity and various in silico calculation software packages have made possible the use of log P values in predictive models for absorption, distribution, excretion and metabolism properties of drugs [17,18].Reversed phase high-performance liquid chromatography (RP-HPLC) provides an easy, reliable and accurate way to determine the concentration of a compound in solvents used for the measurement of partition coefficients.The chromatographic retention time directly relates to the compound's distribution between the mobile and the stationary phases.The retention factor (K) determined from the retention time (T R ) and death time (T D ) as (T R -T D ) is equal to the ratio of the average number of analyte molecules in the stationary phase to the average number of molecules in the mobile phase (cf.Eq. 1) during the elution process.Log K, calculated from the capacity factor K, is used as the lipophilicity index converted to log P scale [19].
The aim of this work was to establish the structure-activity relationships in the mentioned series, i.e. to continue in studying of the substituent variability influence on the biological effect, and to determine the importance of increased hydrophobic properties for antimycobacterial, antifungal and photosynthesis-inhibiting activity of newly prepared substituted pyrazinecarboxamides.
Scheme 1: Synthesis of some substituted pyrazine-2-carboxamides 1-30.All compounds prepared were evaluated for their in vitro antimycobacterial activity.Both the highest activity (72% inhibition) against M. tuberculosis and the highest lipophilicity (log P = 6.00) of all compounds studied was found for 5-tert-butyl-6-chloro-N-(3,5-dibromo-4-hydroxyphenyl)pyrazine-2-carboxamide (18).Two other compounds, 16, 17, with the identical substitution on the aromatic part of the molecule, exert a comparable activity.The majority of compounds exhibited only modest antimycobacterial activity (see Table 1 and Figure 1).In the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) program compounds effecting <90% inhibition in this primary screen (i.e.MIC > 6.25 mg mL -1 ) are generally not evaluated further [22].On the other hand, such "inactive" compounds may still have significant inhibitory activity and this data should not be ignored; analogues, derivatives, and alterations in physical properties may confer some positive changes in biological effects.Therefore synthesis and evaluation of other pyrazinecarboxylic acid derivatives is necessary to round out the structure-activity data.The evaluation of in vitro antifungal activity of the synthesized compounds was performed against eight fungal strains.The results revealed no interesting activity against the majority of strains tested.Only the compounds 5-tert-butyl-6-chloro-N-(5-methyl-1,3-thiazol-2-yl)pyrazine-2-carboxamide (12) and especially 5-tert-butyl-6-chloro-N-(4-methyl-1,3-thiazol-2-yl)pyrazine-2-carboxamide (8) showed some promising in vitro antifungal activity against Trichophyton mentagrophytes, the most susceptible fungal strain evaluated, (MIC = 31.25 -62.5 µmol•mL -1 ), although this activity is only modest in comparison with fluconazole, the standard (MIC = 3.91 µmol•mL -1 after 120 h, see Table 1).The negative antifungal screening results do not allow us to draw detailed conclusions on potential structure-activity relationships.On the other hand, the influence of an increasing lipophilicity parameter on the increasing in vitro antifungal activity in the series of compounds evaluated is remarkable.
The majority of the thirty compounds studied inhibited photosynthetic electron transport in spinach chloroplasts (see Table 1 and Figure 1; compounds 1 and 3 were not tested for their photosynthesisinhibition activity due to their low solubility in DMSO).The IC 50 values varied in the range 41.9 to 1589 µmol•L -1 .The inhibitory activity of the studied compounds was relatively low, the most efficient inhibitors were compounds 8 (IC 50 = 88.8 µmol•L -1 ), 4 (IC 50 = 49.5 µmol•L -1 ), and mainly 5-tert-butyl-6-chloro-N-(5-bromo-2-hydroxyphenyl)pyrazine-2-carboxamide (27, IC 50 = 41.9 µmol•L -1 ).For the series of compounds 5-8 and 9-12 the biological activity showed a linear increase with increasing lipophilicity of the compounds within these series.In both series of anilides 13-15 and 16-18, in the case of the lipophilic compounds 15 (log P = 4.63) and/or 18 (log P = 5.28) a significant activity decrease was observed.Results from previous observations have exposed the importance of the phenolic moiety for the photosynthesis-inhibiting activity in the previously studied series of substituted pyrazine-2-carboxamides [7,8].However, the biological activity of compounds 16-18 was lower than that of compounds 13-15.We assume that this activity decrease was connected with the increased lipophilicity of the compounds due to the presence of two bromine atoms.
Hydrophobicity parameters (log P values) of compounds 1-30 were calculated and measured by means of RP-HPLC determination of capacity factor K and subsequently calculated log K.The values of calculated lipophilicity (log P) of compounds ranged from 0.31 to 6.00.It can be assumed that the computed log P values and the calculated log K values correspond relatively with expected lipophilicity increases within individual series of compounds (pyrazine < 6-chloropyrazine < 5-tertbutylpyrazine < 6-chloro-5-tert-butylpyrazine).Capacity factor K/calculated log K values specify lipophilicity within individual series of compounds.Results are shown in Table 1.
The lower antimycobacterial activities of the compounds presented do not allow us to draw final conclusions on structure-activity relationships (SAR).Better SAR results are expressed in Figure 1, where the quasi-parabolic dependence between the logarithm of the retention factors (log K) and photosynthesis-inhibiting activity {log (1/IC 50 [mol/L])} of all studied compounds is shown.Lipophilicity expressed as log K values ranged from 1.10 to 1.55.
From the point of view of the chemical structure, all compounds can be divided into two groups: (i) compounds with an aminothiazole moiety (1-12, triangles in Figure 1) and (ii) compounds with an aniline moiety (13-30, lozenges in Figure 1).The optimal substitution for the first group of compounds was found to be the methyl group on C (5) of the thiazole ring.The optimal substitution in the second group was found, in agreement with our previous results [7], to be the phenol and halogen (bromine) moieties.The compound 5-tert-butyl-6-chloro-N-(4-methyl-1,3-thiazol-2-yl)pyrazine-2-carboxamide (8) was identified as the most active one in the three different biological assays.However, there is no general trend in the SAR of the compounds evaluated.

Conclusions
In summary, the synthesis and biological evaluation of thirty new substituted amides of pyrazinecarboxylic acid are described.In the first series, among the compounds with substituted 2aminothiazoles, the highest antifungal effect was found for 5-tert-butyl-6-chloro-N-(4-methyl-1,3thiazol-2-yl)pyrazine-2-carboxamide (8).In the second series, among the compounds bearing ringsubstituted anilines, the bromohydroxyphenyl derivatives of substituted pyrazinecarboxylic acid (16)(17)(18)27) have shown the highest biological activity, i.e. against Mycobacterium tuberculosis H 37 Rv and inhibition of oxygen evolution rate in spinach chloroplasts, respectively.

General
All organic solvents used for the synthesis were of analytical grade.The solvents were dried and freshly distilled under argon atmosphere.TLC was performed on Silufol UV 254 plates (Kavalier, Votice, Czech Republic) in the following solvent systems: acetone-toluene (1:1) and petroleum etherethyl acetate (9:1).The plates were detected in UV (254 nm).Melting points were determined on Boetius PHMK 05 (VEB Kombinat Nagema, Radebeul, Germany).Infrared spectra were recorded using KBr pellets on an Nicolet Impact 400 IR-spectrometer. 1 H and 13 C-NMR Spectra were recorded on a Varian Mercury -Vx BB 300 (Varian, Palo Alto, CA, USA; 299.95 MHz for 1 H and 75.43 MHz for 13 C).Chemical shifts are given relative to the internal Si(CH 3 ) 4 .Log P values were computed using the CS ChemOffice Ultra ver.7.0 program (CambridgeSoft, Cambridge, MA, USA) and are summarized in Table 1.

Lipophilicity HPLC determination (capacity factor K/calculated log K)
The HPLC separation module Waters Alliance 2695 XE and Waters Photodiode Array Detector 2996 (Waters Corp., Milford, MA, U.S.A.) were used.The chromatographic column Symmetry ® C 18 5 µm, 4.6 × 250 mm, Part No. WAT054275, (Waters Corp., Milford, MA, U.S.A.) was used.The mixture of MeOH p.a. (70.0%) and H 2 O-HPLC -Mili-Q Grade (30.0%) was used as a mobile phase.The total flow of the column was 1.0 mL/min, injection 30 µL, column temperature 30 °C and sample temperature 10 °C.The detection wavelength 223 nm was chosen.The retention time (dead time) of the KI methanol solution was T D = 2.382 min.Retention times (T R ) was measured in minutes, capacity factors were calculated (K).The HPLC separation process was monitored using Millennium 32® Chromatography Manager Software, Waters 2004 (Waters Corp., Milford, MA, U.S.A.).The calculated log K values of all compounds are shown in Table 1.