Synthesis and Antifungal Screening of 2-{[1-(5-Alkyl/arylalkylpyrazin-2-yl)ethylidene]hydrazono}-1,3-thiazolidin-4-ones †

Two novel thiosemicarbazones and eight novel 2-{[1-(5-alkyl/arylalkylpyrazin-2-yl)ethylidene]hydrazono}-1,3-thiazolidin-4-ones were prepared and tested against a panel of eight fungal strains–Candida albicans ATCC 44859, Candida tropicalis 156, Candida krusei E 28, Candida glabrata 20/I, Trichosporon asahii 1188, Aspergillus fumigatus 231, Lichtheimia corymbifera 272, and Trichophyton interdigitale 445. 1,3-Thiazolidin-4-ones exhibited activity against all strains, the most potent derivative was 2-{[1-(5-butylpyrazin-2-yl)ethylidene]hydrazono}e-1,3-thiazolidin-4-one. Susceptibility of C. glabrata to the studied 1,3-thiazolidin-4-ones (minimum inhibitory concentrations (MICs) were in the range 0.57 to 2.78 mg/L) is of great interest as this opportunistic pathogen is poorly susceptible to azoles and becomes resistant to echinocandins. Antifungal potency of thiosemicarbazones was slightly lower than that of 1,3-thiazolidin-4-ones.


Introduction
Fungal infections, especially invasive ones, represent a serious problem. Whilst topical fungal diseases are quite common and cause considerable morbidity, they are generally not life-threatening [1]. On the contrary, it has been estimated that invasive fungal infections are responsible for the deaths of 1.5 million people each year [2]. The increased incidence of life threatening systemic fungal infections is mainly due to the increasing numbers of immunocompromised people [2,3]. Besides, infections that were once uncommon emerge more frequently in the United States and Europe as a result of international travel, immigration from endemic areas, and changing climate conditions [4]. Fungal resistance also prevents successful treatment of mycoses [5][6][7][8]. Therefore, searching for new drugs and therapeutic options is of high importance [9][10][11].

Chemistry
Thiosemicarbazones 10g and 10h were prepared using the method reported in our previous paper [20]. Their spectral characteristics corresponded to the spectra of their previously reported congeners 10a-10f. Hence, it could be concluded that they are also E-isomers. Details concerning the determination of the configuration on the double bond can be found in ref. [20].
As can be seen in Table 1, MICs of fluconazole and voriconazole were uncommonly high for C. tropicalis. This indicates that the strain could have developed a resistance to azole antifungal agents during long-term passaging. However, as the most potent compounds (11a, 11c, and 11e) exhibited activity against the resistant strain, it can be presumed that their mechanism of action and/or resistance is different from that of azoles, and their antifungal activity is independent of the susceptibility of a given strain of C. tropicalis to azole derivatives. As it was already mentioned in the introduction, invasive fungal infections, especially those caused by resistant pathogens, represent a serious health problem [5][6][7][69][70][71]. In immunocompromised patients, they have high mortality rates (20%-40% for Candida albicans, 20%-70% for Cryptococus neoformans, and 50%-90% for Aspergillus fumigatus) [72]. Moreover, new infections due to opportunistic fungi, have emerged recently [73][74][75][76][77]. One of these difficult to treat pathogens is Candida glabrata. It exhibits some special features and is more similar to Saccharomyces cerevisiae than to Candida albicans [78,79]. C. glabrata belongs to the main fungal opportunistic pathogen in humans. It is poorly susceptible to azole antimycotic agents [78] and becomes resistant to echinocandins [79][80][81]. Strain resistance to amphotericin B was also reported [82]. 2-{[1-(5-Alkyl/arylalkylpyrazin-2-yl)ethylidene]hydrazono}-1,3-thiazolidin-4ones 11a-11h presented here showed promising activity against C. glabrata. The most potent derivative, 11c exhibited good activity against all studied fungal pathogens. These data make them prospective antifungal agents that deserve further studies.

Chemistry
Pyrazine-2-carbonitrile (Sigma-Aldrich, Prague, Czech Republic) was used as a starting compound. It was alkylated to yield intermediates 8a-8h, and these in turn were converted to the corresponding acetylpyrazines 9a-9h using methods reported previously [83,84]. Thiosemicarbazones 10a-10f were prepared and characterized in our previous paper [20]. Thiosemicarbazones 10g and 10h were prepared analogously using commercially available analytical grade thiosemicarbazide (Lachema, Brno, Czech Republic). Commercially available pure chloroacetic acid (Sigma-Aldrich, Prague, Czech Republic) and pure crystalline sodium acetate (Lachema, Brno, Czech Republic) were used for the cyclization of thiosemicarbazones. The purity of the products was checked by thin layer chromatography on TLC aluminium sheets, silica gel 60 F 254 (Merck, Darmstadt, Germany); mixtures of light petroleum and ethyl acetate 80:20 and 60:40 were used as mobile phases. Analytical samples were dried over anhydrous phosphorus pentoxide under reduced pressure at room temperature. Melting points were determined on a Boëtius BHMK 73/4615 apparatus (VEB Analytik, Dresden, Germany) and are uncorrected. Elemental analyses (EA) were performed on an EA 1110 CHNS instrument (CE Instruments, Milano, Italy). IR spectra were recorded by the attenuated total reflection (ATR-Ge) method on Nicolet Impact 400 spectrometer or Nicolet 6700 IR spectrophotometer (Nicolet-Thermo Scientific, Madison, WI, 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 or VNMR S500 (Varian) spectrometer operating at 500 MHz for 1 H-NMR and 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-d 6 ). Signal multiplicities are described as s, singlet; bs, broad singlet; m, multiplet; d, doublet and t, triplet.

General Procedure for the Preparation of Thiosemicarbazones 10a-10h
5-Alkylated acetylpyrazine (0.01 mol) and thiosemicarbazide (0.01 mol) were dissolved in methanol (10-15 mL). Three drops of concentrated acetic acid were added, and the mixture was heated at reflux for 5 h. Then it was cooled, the product was removed by filtration and crystallized from ethanol. Thiosemicarbazones 10a-10f (characterized in reference [20]) and two novel thiosemicarbazones 10g and 10h were prepared by this procedure. Thiosemicarbazone (7 mmol) and chloroacetic acid (0.99 g, 10.5 mmol) were dissolved in a minimum amount of anhydrous ethanol under stirring and heating to reflux. Then, 1.5% (w/w) ethanolic solution of sodium acetate (8 mL) was added, and the reaction mixture was heated under reflux for 10 h. After cooling, the precipitated crystals were sucked off, washed with water and 50 mL of water-ethanol mixture (1:1, v/v). Analytically pure products were obtained by crystallization from anhydrous ethanol. Using this procedure the following compounds were prepared:

Conclusions
The studied 2-{[1-(5-alkyl/arylalkylpyrazin-2-yl)ethylidene]hydrazono}-1,3-thiazolidin-4-ones showed promising activity against C. glabrata-an opportunistic pathogenic yeast that is often resistant to both azoles and echinocandins. The most potent derivative exhibited good activity against all eight fungal pathogens used in the susceptibility assay. In the light of the results obtained in the present study and the antifungal properties of 1,3-thiazolidin-4-one derivatives reported by other research groups, it can be concluded that substituted 1,3-thiazolidin-4-ones deserve additional studies as potential antifungal drugs.