Novel Pyrazine Analogs of Chalcones: Synthesis and Evaluation of Their Antifungal and Antimycobacterial Activity

Infectious diseases, such as tuberculosis and invasive mycoses, represent serious health problems. As a part of our long-term efforts to find new agents for the treatment of these diseases, a new series of pyrazine analogs of chalcones bearing an isopropyl group in position 5 of the pyrazine ring was prepared. The structures of the compounds were corroborated by IR and NMR spectroscopy and their purity confirmed by elemental analysis. The susceptibility of eight fungal strains to the studied compounds was tested. The results have been compared with the activity of some previously reported propyl derivatives. The only strain that was susceptible to the studied compounds was Trichophyton mentagrophytes. It was found that replacing a non-branched propyl with a branched isopropyl did not have a decisive and unequivocal influence on the in vitro antifungal activity against T. mentagrophytes. In vitro activity against Trichophyton mentagrophytes comparable with that of fluconazole was exhibited by nitro-substituted derivatives. Unfortunately, no compound exhibited efficacy comparable with that of terbinafine, which is the most widely used agent for treating mycoses caused by dermatophytes. Some of the prepared compounds were assayed for antimycobacterial activity against M. tuberculosis H37Rv. The highest potency was also displayed by nitro-substituted compounds. The results of the present study are in a good agreement with our previous findings and confirm the positive influence of electron-withdrawing groups on the B-ring of chalcones on the antifungal and antimycobacterial activity of these compounds.

Moreover, the differences in the potencies of propyl and isopropyl derivatives are sometimes very subtle. Therefore, it can only be concluded that replacing a non-branched propyl with a branched isopropyl does not have a decisive and unequivocal influence on the in vitro antifungal activity against T. mentagrophytes. Potency of (E)-1-(5-isopropylpyrazin-2-yl)-3-(2-nitrophenyl)-prop-2-en-1-one (4g), and (E)-1-(5-isopropyl-pyrazin-2-yl)-3-(4-nitrophenyl)prop-2-en-1-one (4i) was comparable to that of fluconazole, which is sometimes used for the treatment of mycoses caused by Trichophyton spp. [42,43], but lower than that of voriconazole and terbinafine. Terbinafine is most widely used agent to treat mycoses caused by dermatophytes and other fungi [42,44]. Voriconazole belongs to the highly effective systemic antifungal drugs with a favourable risk-benefit ratio, and with distinct in vitro activity against dermatophytes, yeasts and some molds [45] but clinically it is used to treat invasive aspergillosis [46,47]. The good potency of the nitro-substituted derivatives 4g and 4i is in a good agreement with our previous results [34], confirming the positive influence of a nitro group on the B-ring on the antifungal potency of chalcone-like derivatives.

Antimycobacterial Activity
Selected compounds were submitted to evaluation of antimycobacterial activity in the Tuberculosis Antimicrobial Acquisition and Coordination Facility (TAACF) through a research and development contract with the U.S. National Institute of Allergy and Infectious Diseases. The results are shown in Table 2. As expected according to our previous results [34], the best inhibition was displayed by 2-nitro (4g) and 4-nitro (4i) derivatives, but they were less potent than the previously reported (E)-1-(5-tertbutylpyrazin-2-yl)-3-(4-nitrophenyl)prop-2-en-1-one (MIC90 = 0.78 μg/mL). This confirms that tert-butyl is the best substituent for antimycobacterial potency. A lower efficacy was observed with 2-hydroxy-(4a) and 4-hydroxy-(4c) substituted compounds, which is also in agreement with our previous results [32,34]. Table 2. Antimycobacterial activity of compounds 4a-4j and 5a-5j compared to isoniazid and rifampicin.

Compd.
R % Inhibition at 6.25 μg/mL MIC 90 (μg/mL) CC 50  For moving compounds into in vivo testing MIC ≤ 6.25 µg/mL and an selectivity index (the ratio of the measured CC50 in VERO cells to the MIC) SI ≥ 10 are required. Unfortunately, the selectivity indexes of the two promising compounds were too low.

Materials and Methods
Pyrazine-2-carbonitrile (Sigma-Aldrich, Prague, Czech Republic) was used as a starting compound. 5-Isopropylpyrazine-2-carbonitrile and 1-(5-isopropylpyrazin-2-yl)ethan-1-one were prepared as described previously [40]. Commercially available substituted benzaldehydes (Sigma-Aldrich) were used as the starting materials. Silpearl (Kavalier, Votice, Czech Republic) was used for flash column chromatography. The purity of the products was checked by TLC on Silufol UV 254 plates (Kavalier). Mixtures of light petroleum and ethyl acetate were used for TLC. Analytical samples were dried over anhydrous phosphorus pentoxide under reduced pressure at room temperature. Melting points were determined on a Boëtius apparatus and are uncorrected. Elemental analyses were performed on an EA 1110 CHNS instrument (CE Instruments, Milano, Italy). Infrared spectra were recorded in KBr pellets on a Nicolet Impact 400 IR spectrophotometer (Thermo Scientific, Waltham, MA, USA). 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. 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 CDCl3). Coupling constants J are given in Hz.

Evaluation of in Vitro Antifungal Activity
The antifungal activity of all compounds was evaluated by the modified microdilution broth CSLI standards [50,51]. Fungal inocula were prepared by suspending yeasts, conidia, or sporangiospores in sterile 0.85% saline. The cell density was adjusted using a Bürker's chamber to yield a stock suspension of 1.0 ± 0.2 × 10 5 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-IC80), resp. 50% or greater (for molds-IC50) 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. mentagrophytes, the MICs were recorded after 72 and 120 h due to its slow growth rate. Fluconazole, voriconazole and terbinafine were used as reference antifungal drug.

Evaluation of in Vitro Antimycobacterial Activity
Primary screening of all compounds was conducted at 6.25 μg/mL against Mycobacterium tuberculosis H37Rv (ATCC 27294) in the BACTEC 12B medium using the Microplate Alamar Blue Assay (MABA). Compounds exhibiting fluorescence were tested in the BACTEC 460-radiometric system [52]. Compounds demonstrating at least 90% inhibition in the primary screen were re-tested at lower concentrations against M. tuberculosis H37Rv to determine the actual minimum inhibitory concentration (MIC) in the MABA. The MIC is defined as the lowest concentration effecting a reduction in fluorescence of 90% relative to controls.
The compounds that exhibited promising antimycobacterial activity were tested for cytotoxicity (CC50) in VERO cells at concentrations less than or equal to 10 times the MIC for M. tuberculosis H37Rv. After 72-h exposure, viability was assessed on the basis of cellular conversion of 1-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) into a formazan product using the Promega CellTiter 96 Non-radioactive Cell Proliferation Assay. The selectivity index was then calculated as the ratio of the measured CC50 in VERO cells to the MIC described above.

Conclusions
A series of (E)-1-(5-isopropylpyrazin-2-yl)-3-phenylprop-2-en-1-ones with various substituents on the phenyl ring (ring B) was prepared and tested for antifungal and antimycobacterial activity. Their in vitro antifungal potency was compared to previously reported propyl analogs. Only Trichophyton mentagrophytes was susceptible to the tested compounds, and it was found that replacing a non-branched propyl with a branched isopropyl has no decisive and unequivocal influence on the in vitro antifungal activity against T. mentagrophytes. Unfortunately, no compound exhibited efficacy comparable with that of terbinafine, which is most widely used agent for treating mycoses caused by dermatophytes. In both biological assays, the highest in vitro potency was displayed by nitro-substituted derivatives. This confirms our previous findings about the positive effect of electron-withdrawing groups on the B-ring of chalcones on their antimicrobial activity.

Author Contributions
Marta Kucerova-Chlupacova carried out synthesis, interpreted yields, elemental analysis, IR spectra, and antimycobacterial data; Jiri Kunes recorded and interpreted NMR data; Vladimir Buchta designed antifungal assays and interpreted their results; Marcela Vejsova carried out antifungals assays; Veronika Opletalova proposed the subject, designed the study, and wrote the paper. All the authors read and approved the final manuscript.