Substituted Amides of Pyrazine-2-carboxylic acids: Synthesis and Biological Activity.

Condensation of 6-chloro-, 5-tert-butyl- or 6-chloro-5-tert-butylpyrazine-2-carboxylic acid chloride with ring substituted anilines yielded a series of amides, which were tested for their in vitro antimycobacterial, antifungal and photosynthesis-inhibiting activities. The highest antituberculotic activity (72% inhibition) against Mycobacterium tuberculosis and the highest lipophilicity (log P = 6.85) were shown by the 3,5-bis-trifluoromethylphenyl amide of 5-tert-butyl-6-chloropyrazine-2-carboxylic acid (2o). The 3-methylphenyl amides of 6-chloro- and 5-tert-butyl-6-chloro-pyrazine-2-carboxylic acid (2d and 2f) exhibited only a poor in vitro antifungal effect (MIC = 31.25-500 μmol·dm-3) against all strains tested, although the latter was the most active antialgal compound (IC50 = 0.063 mmol·dm-3). The most active inhibitor of oxygen evolution rate in spinach chloroplasts was the (3,5-bis-trifluoromethylphenyl)amide of 6-chloropyrazine-2-carboxylic acid (2m, IC50 = 0.026 mmol·dm-3).


Introduction
Recent years have seen increased incidence of tuberculosis in both developing and industrialized countries, the widespread emergence of drug-resistant strains and a deadly synergy with the human immunodeficiency virus (HIV) [1,2]. Pyrazinamide (PZA) is a nicotinamide analogue that has been used for almost 50 years as a first-line drug to treat tuberculosis [3]. PZA is bactericidal to semidormant mycobacteria and reduces total treatment time [4]. Although the exact biochemical basis of PZA activity in vivo is not known, under acidic conditions it is thought to be a prodrug of pyrazinoic acid, a compound with antimycobacterial activity [5]. The finding that PZA-resistant strains lose amidase (pyrazinamidase or nicotinamidase) activity and the hypothesis that amidase is required to convert PZA to pyrazinoic acid intracellularly led to the recent synthesis and study of various prodrugs of pyrazinoic acid [6]. Various compounds possessing -NHCO-grouping, e.g. substituted amides, acyl and thioacyl anilides, benzanilides, phenyl carbamates, etc., were found to inhibit photosynthetic electron transport [7][8][9][10]. Therefore, antifungal and photosynthesis-inhibiting evaluations of newly prepared pyrazine-2-carboxylic acid derivatives were additional areas of interest to us.
Amides of 2-alkylpyridine-4-carboxylic [11,12] and 2-alkylsulfanyl-4-pyridinecarboxylic [12,13] acids inhibited oxygen evolution rate in Chlorella vulgaris and their inhibitory activity depended on the lipophilicity of the compounds. Several esters of alkoxy substituted phenylcarbamic acids (APA) showed antialgal activity against Chlorella vulgaris [14][15][16]. The inhibitory efficiency of APA concerning chlorophyll production in Chlorella vulgaris depended on the lipophilicity of the alkoxy substituent and also on its position on the aromatic ring [14][15][16]. The antialgal activity of APA correlated with the antifungal activity of these compounds against Candida albicans [16]. We have recently reported the synthesis of a series of substituted amides prepared from some pyrazine-2carboxylic acids and some aminophenols [17], halogenated or alkylated anilines [18].

Results and Discussion
The synthesis of amides is shown in Scheme 1. Condensation of the chlorides of 6-chloropyrazine-2-carboxylic (1a) [19], 5-tert-butyl-pyrazine-2-carboxylic (1b) [17] or 5-tert-butyl-6-chloropyrazine-2carboxylic (1c) [17] acids with ring substituted anilines yielded a series of 18 substituted amides 2a-r of the aforementioned substituted pyrazine-2-carboxylic acids. Scheme 1. Preparation of substituted amides 2a-r of pyrazine-2-carboxylic acids The melting points, yields, and elemental analyses of the compounds prepared 2a-r are given in Table 3, and their spectral data in Tables 4 and 5. The structures were corroborated by 2D NMR spectroscopy using gHSQC and gHMBC experiments. The biological activities of the prepared amides 2a-r with regards to in vitro antimycobacterial, antifungal and inhibition of oxygen evolution rate in spinach chloroplasts were investigated. The highest antituberculotic activity (72% inhibition) against Mycobacterium tuberculosis and also the highest lipophilicity (log P = 6,85) was shown by the 3,5-bistrifluoromethylphenyl amide of 5-tert-butyl-6-chloropyrazine-2-carboxylic acid (2o). Some other amides (2d, 2f, 2k, 2l) with higher than 20% inhibition were investigated. Three of them contain a tertbutyl moiety in position 5 of the pyrazine ring. The negative results of antimycobacterial screening allow us to make no conclusions regarding potential structure-activity relationships. Results of their antimycobacterial activity (MIC, % Inhibition) and calculated log P values of 2a-r are shown in Table  1.
The studied compounds inhibited photosynthetic electron transport in spinach chloroplasts, which was reflected in the inhibition of oxygen evolution rate. The photosynthesis inhibitory activity of the compounds has been expressed as IC 50 values (see Table 1). The IC 50 values varied in the range from 0.026 (2m) to 1.072 mmol·dm -3 (2a). In general, the photosynthesis-inhibiting activity of the studied compounds depended on their lipophilicity showing a quasi-parabolic trend. However, the studied compounds could be divided into two groups. The compounds with 2-CH 3 substituents on the phenyl ring (2a, 2b, 2c, 2p, 2q and 2r, squares in Figure 1) had lower biological activity than the other investigated compounds with comparable log P values. Consequently, we assume that the methyl substituent in ortho position of the benzene ring is disadvantageous from the viewpoint of interactions with the photosynhetic apparatus. On the other hand, compound 2m exhibited higher inhibitory activity than expected. Additionally some inhibition of chlorophyll production in green algae Chlorella vulgaris was studied at the compounds 2f, 2l, 2m, 2n, 2o and 2p. Results of their antialgal activity are given in Table 2. The antialgal activity of these six studied compounds showed a quasi-parabolic dependence upon log P with maximum activity for compounds having log P in the range from 3.18 to 5.16 (see Figure 2). With the further increasing of the lipophilicity a dramatic decrease of antialgal activity was observed.

Figure 2.
Quasi-parabolic dependence between antialgal activity and log P of studied amides 2f, 2l, 2m, 2n, 2o and 2p.  Melting points were determined on a Kofler block, and are uncorrected. Elemental analyses were obtained using an EA 1110 CHNS-O CE apparatus (Fisons Instruments S.p.A., Milan). The IR spectra were recorded on a Nicolet Impact 400 spectrometer in KBr pellets. The 1 H and 13 C NMR spectra were measured for CDCl 3 solutions with a Varian Mercury -Vx BB 300 spectrometer operating at 300 MHz. Chemical shifts were recorded as δ values in parts per million (ppm), and were indirectly referenced to tetramethylsilane via the solvent signal (7.26 for 1 H and 77.0 for 13 C). Multiplicities are given together with the coupling constants (in Hz). Log P values were computed using a program ACD/Log P ver. 1.0 (Advanced Chemistry Development Inc., Toronto).

Synthesis of amides 2a-r
A mixture of acid (i.e. 6-chloropyrazine-2-carboxylic [19], 5-tert-butylpyrazine-2-carboxylic [17] or 5-tert-butyl-6-chloropyrazine-2-carboxylic [17] acid, 0.05 mol) and thionyl chloride (5.5 mL, 75 mmol) in dry benzene (20 mL) was refluxed for about 1 h. Excess thionyl chloride was removed by repeated evaporation with dry benzene in vacuo. The crude acyl chloride dissolved in dry acetone (50 mL) was added dropwise to a stirred solution of the corresponding substituted aniline (50 mmol) in dry pyridine (50 mL) kept at room temperature. After the addition was complete, stirring was continued for another 30 min. The reaction mixture was then poured into cold water (200 mL) and the crude amide was collected and recrystallized from aqueous ethanol.

Antimycobacterial Assay
Antimycobacterial evaluation was carried out in Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF), Southern Research Institute, Birmingham, Alabama, USA, which is a part of National Institutes of Health (NIH). Primary screening of all compounds were conducted at 6.5 or 12.5 µg·ml -1 against Mycobacterium tuberculosis H 37 Rv in BACTEC 12B medium using the BACTEC 460 radiometric system [20]. The MIC was defined as the lowest concentration effecting a reduction in fluorescence of 99% relative to controls. For the results see Table 1.

In vitro antifungal susceptibility testing
Broth microdilution test [21,22] was used for the assessment of in vitro antifungal activity of ketoconazole (standard) and the synthetized compounds against Candida albicans ATCC 44859, Candida tropicalis 156, Candida krusei E28, Candida glabrata 20/I, Trichosporon beigelii 1188, Trichophyton mentagrophytes 445, Aspergillus fumigatus 231, and Absidia corymbifera 272. The procedure was performed with twofold compound dilutions in RPMI 1640 buffered to pH 7.0 with 0.165 mol morpholinopropanesulfonic acid. The final concentrations of the compounds ranged from 1000 to 0.975 µmol/L. Drug free controls were included. The MICs were determined after 24 and 48 h of static incubation at 35 o C. In the case of Trichophyton mentagrophytes the MICs were determined after 72 and 120 h of incubation.

Study of inhibition of oxygen evolution rate in spinach chloroplasts
The oxygen evolution rate in spinach chloroplasts was investigated spectrophotometrically (Specord UV VIS, Zeiss, Jena) in the presence of an electron acceptor 2,6-dichlorophenol-indophenol, by method described in Ref. [23]. The compounds were dissolved in dimethyl sulfoxide (DMSO) because of their low water solubility. The DMSO volume fractions used (up to 5 vol. %) did not affect the oxygen evolution. The inhibitory efficiency of the studied compounds has been expressed by IC 50 values, i.e. by molar concentration of the compounds causing 50 % decrease in the oxygen evolution relative to the untreated control. IC 50 value for the standard, a selective herbicide 3-(3,4dichlorophenyl)-1,1-dimethylurea, DCMU (DIURON) was measured about 1.9 µmol dm -3 . For the results see Table 1.

Study of inhibition of chlorophyll production in green algae Chlorella vulgaris
The algae Chlorella vulgaris were cultivated statically at room temperature according to Sidóová et al. [24] (photoperiod 16 h light/8 h dark; illumination 4000 lx; pH = 7.2). The effect of compounds 2f, 2l, 2m, 2n, 2o and 2p on algal chlorophyll (Chl) content was determined after 4-day cultivation in the presence of the tested compounds, expressing the response as percentage of the corresponding values obtained for control. The Chl content in the algal suspension was determined spectrophotometrically (Specord UV VIS, Zeiss Jena, Germany) after extraction into N,Ndimethylformamide according to Inskeep and Bloom [25]. The Chl content in the suspensions at the beginning of cultivation was 0.5 mg dm -3 . Because of their low water solubility, the tested compounds were dissolved in DMSO. DMSO concentration in the algal suspensions did not exceed 0.25 v/v % and the control samples contained the same DMSO amount as the suspensions treated with the tested compounds. IC 50 value for the standard, a selective herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DCMU (DIURON) was measured about 7.3 µmol dm -3 . For the results see Table 2.