key: cord-1014850-3zynjxjx authors: Pogrebnoi, Serghei; Radul, Oleg; Stingaci, Eugenia; Lupascu, Lucian; Valica, Vladimir; Uncu, Livia; Smetanscaia, Anastasia; Petrou, Anthi; Ćirić, Ana; Glamočlija, Jasmina; Soković, Marina; Geronikaki, Athina; Macaev, Fliur Z. title: The Synthesis of Triazolium Salts as Antifungal Agents: A Biological and In Silico Evaluation date: 2022-04-27 journal: Antibiotics (Basel) DOI: 10.3390/antibiotics11050588 sha: 85fe0ecef43f981be813479390639f5143e76bcf doc_id: 1014850 cord_uid: 3zynjxjx The control of fungal pathogens is increasingly difficult due to the limited number of effective drugs available for antifungal therapy. In addition, both humans and fungi are eukaryotic organisms; antifungal drugs may have significant toxicity due to the inhibition of related human targets. Furthermore, another problem is increased incidents of fungal resistance to azoles, such as fluconazole, ketoconazole, voriconazole, etc. Thus, the interest in developing new azoles with an extended spectrum of activity still attracts the interest of the scientific community. Herein, we report the synthesis of a series of triazolium salts, an evaluation of their antifungal activity, and docking studies. Ketoconazole and bifonazole were used as reference drugs. All compounds showed good antifungal activity with MIC/MFC in the range of 0.0003 to 0.2/0.0006–0.4 mg/mL. Compound 19 exhibited the best activity among all tested with MIC/MFC in the range of 0.009 to 0.037 mg/mL and 0.0125–0.05 mg/mL, respectively. All compounds appeared to be more potent than both reference drugs. The docking studies are in accordance with experimental results. In recent years, the frequency and severity of life-threatening fungal infections have increased, mainly in patients with impaired or compromised immunity [1] , suggesting the urgent need for the development of new antifungal agents with different modes of action [2] . The majority of mycotic infections are caused by Candida and Aspergillus species. The main cases of nosocomial fungal infections are caused by Candida albicans, while Aspergillus fumigatus is most of the widespread airborne fungal pathogens [3] . The increased incidence of drug-resistant species of both yeasts and molds, which are causative pathogens of invasive, life-threatening infections, emphasizes the urgent need to develop more effective and safe innovative antifungal drugs [4] . Latest studies revealed that typical azole inhibitors fit the active site of CYP51, a member of the cytochrome P450 (CYP 450) family, through the hydrogen bonds formation, stacking, hydrophobic interactions, and heme coordination [5] . Many effective drugs presently used in clinical belong to a class of azoles (imidazole and triazoles). Nevertheless, the interest in developing new azoles and not only with an extended spectrum of activity still attracts the interest of scientists. On the other hand, there is a significant amount of information regarding improvements made by researchers in fighting bacteria and fungi drug resistance. Thus, there is a growing interest in phage therapy [6, 7] . Photodynamic therapy was used to fight against ESCAPE pathogens [8] . Combarros-Fuertes [9] reported that the combined use of honey and antibiotics reduces the concentrations of drugs necessary to achieve efficacy, thus limiting the likelihood of developing resistance and reverting the susceptibility of some resistant bacteria to antibiotics. There are some reviews regarding the antibiotics from sea microorganisms [10] [11] [12] [13] . Casertano et al. [14] , in their extensive review, reported the role of the ascidian s secondary metabolites as an extraordinary source of novel drug lead structures, describing 160 molecules, including sulfur-containing compounds, alkaloids, meroterpenes, peptides, furanones, and other aromatic derivatives with antimicrobial activity. The activity of some of the metabolites appears to be superior compared to reference drugs. Zalacain et al. [15] reported a novel specific metallo-β-lactamase inhibitor ANT2681, that restores the activity of meropenem to clinically effective levels against Enterobacterales. They showed that the addition of ANT2681 at 8 µg/mL decreased the MIC50/MIC90 from >32/>32 µg/mL to 0.25/8 µg/mL. Yang et al. [16] reported that based on the virtual screening approach, they found that carnosic acid exhibited an inhibitory effect on NDM-1. According to the MIC and timekilling assays, carnosic acid can restore the antibacterial activity of meropenem and inhibit NDM-1. Thus, the authors speculated that carnosic acid could improve the antibacterial potency of meropenem by inhibiting NDM-1. It was recently reported a new class of enzyme blockers, called indole carboxylates (InCs), which inhibit the activity of metallo-β-lactamase (MBL) resistance enzymes produced by bacteria. The results of Schofield et al. [17] revealed that InCs have a substantial potential for clinical development with β-lactam antibiotics. They are actively forwarding InCs toward clinical trials in humans, focusing on low-to-middle income countries in which NDM-mediated (New Delhi Metallo-β-lactamase) resistance is widespread. Five member heterocycles, with two of the three heteroatoms, are important units in approved drugs. Triazole derivatives are an attractive class of compounds with s wide spectrum of biological activities such as antibacterial [6] [7] [8] , antifungal [7, [9] [10] [11] , antitubercular [12, 13] , anti-inflammatory [14, 18] , analgesic [19] , antiviral [20, 21] , anti-HIV [22, 23] , antidiabetic [24] [25] [26] , anticancer [27] [28] [29] , anticonvulsive [26, 30, 31] , carbonic anhydrase inhibitory activity [32, 33] , antileishmanial [34] [35] [36] antimalarial [26, 37, 38] . and others [39] [40] [41] [42] . Furthermore, a triazole scaffold is present in the structure of many approved drugs, such as ribavirin, an antiviral drug for the treatment of Respiratory Syncytial Virus (RSV) infection, hepatitis C and some viral hemorrhagic fevers. In contrast, ketoconazole, fluconazole, and itraconazole are known antifungal drugs. Other drugs that include a triazole moiety are rizatriptan (for the treatment of migraine and headache), alprazolam (anxiolytic), and estazolam (tranquilizer) [39] (Figure 1 ). Following our research on the development of new antimicrobial agents [43, 44] , herein, we report the synthesis and evaluation of antifungal activity as well as docking studies of a series of triazolium salts. The three types of ammonium salts were synthesized by reacting 1,2,4-triazoles with a stoichiometric amount of the alkyl-, benzyl-, aryl-ethanone halides. Their chemical structures are illustrated in Scheme 1. The triazolylmethylketones 11-15 were synthesized by two methods. Method A: involves N1-alkylation of 1H-1,2,4-triazole by 1-aryl-2-bromoethanones 1-5 in the presence of a base. Method B is a reaction of 4-amino-1H-1,2,4-triazole and corresponding 1-aryl-2bromoethanones in acetonitrile following the removal of an amino group. According to method A, the reaction proceeds in one step, while method B consists of two, but the yield of the desired product is almost twice as high. For example, the yield of 1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl) ethanone 15 by method A is 47%, whereas by method B is 81%. Therefore, we chose method B for the synthesis of triazolylmethylketones 11-15 as the most practical. The obtained triazolylmethylketones 11-15 were quaternized with 1-aryl-2-bromoethanones, ethyl 2-bromoacetate, or ethyl iodide in acetonitrile at reflux. The final products 16-23 yield from 67% to 93%. Based on our experiences in preparing the quaternary ammonium salts 16-23, it was expected that 1-benzyl-1H-1,2,4-triazole also afford the triazolium salts 24-26. Therefore, a reaction sequence 1H-1,2,4-triazole→potassium 1,2,4-triazol-1-ide →1benzyl-1H-1,2,4-triazole →1-benzyl-substituted triazolium salts 24-26 was realized in which a benzyl fragment was introduced first at position 1 of 1H-1,2,4-triazole followed by the addition of benzyl-, phenacyl-and 2,4-dichlorophenacyl fragments of target substances (Scheme 2). Scheme 2. Synthesis of 1-Benzyl-substituted triazolium salts.Reagents and conditions: (1) benzyl chloride, K 2 CO 3 , acetone, rt, 24 h; (2) benzyl chloride, phenacyl bromide or 2,4-dichlorophenacyl bromide, acetone, reflux 8 h. The structure of the obtained compounds was supported by IR, 1 H, and 13 C NMR spectroscopic data and by elemental analysis. The IR spectrum of salts showed absorption bands in the range of 1652-1726 cm −1 for compounds 6-23, 25, and 26, which is characteristic of the stretching vibration of the carbonyl group. The presence of absorption bands in the range of 1504-1595 cm −1 for the compounds 6-26 cm −1 suggests the presence of a C=N bond. The examination of the 13 C NMR spectra of the discussed compounds further confirmed the formation of 1H-1,2,4-triazole functionalized salts. The peaks in the 13 C NMR spectra at 189.0-191.0 ppm are typical of the carbonyl nucleus (Supplementary Materials). In the 1 H-NMR spectra, the protons of the triazole ring appeared as broad singlets at 9.33-9.43 ppm and 10.17-10.61 ppm, while the methylene group's proton is environmental dependent, and their chemical shifts varied between 3.90 to 6.42 for most of the compounds. Other items analysed in the NMR spectra examined the partially decoupled spectra and appear in the experimental section. The purity of the triazolium salts was confirmed by HPLC with acetonitrile/water = 70:30, methanol/water = 70:30, and methanol as the mobile phase at a flow rate of 1.0 mL/min over the range of 2.5 mg/L, 50 mg/L, and 100 mg/L. The HPLC analyses were carried out at room temperature under isocratic conditions. The purity of salts with 1-phenylethanone moiety at the 1-arylethanone of triazole ring 6, 16, 25 was ≥ 98.70%. The HPLC chromatograms of the triazolium salts 6-10 and 16-26 appear as supporting information. All synthesized compounds were tested for their antifungal activity by the microdilution method against the panel of eight fungi. All compounds showed good antifungal activity with a MIC/MFC in the range of 0.0003-0.2/0.0006-0.4 mg/mL (0.0005-0.055/0.001-1, 116 mmol/mL) ( Compound 19 exhibited the best activity among all tested with MIC and MFC in the range of 0.009-0.037 mg/mL and 0.0125-0.05 mg/mL, respectively, while the lowest activity was displayed by compound 25 (MC/MFC in arrange of 0.025-0.20/0.05-0.40 mg/mL). Ketoconazole showed antifungal potential with MIC/MFC at 0.28-1.88/0.38-2.82 mg/mL, while MIC and MFC of bifonazole were in the range of 0.32-0.64/0.64-0.81 mg/mL. Thus, all compounds appeared to be more active than ketoconazole and bifonazole. Generally, compound 15 displayed up to ten times stronger antifungal potential than Ketoconazole and two to twelve times higher than Bifonazole. Compound 19 was a more powerful antifungal agent than the reference drugs. It possessed even up to 50 to 209 times stronger inhibitory and fungicidal potential against all tested micromycetes. Generally, all tested compounds displayed significantly higher antifungal activities than the reference compounds Ketoconazole and Bifonazole. Those compounds with the lowest activity were 24, 23, and 25 against A. fumigatus, which showed two-to-five times better antifungal potential compared to both Ketoconazole and Bifonazol. Differences in sensitivity were observed not only among different species, but also for each fungus. At the same time, almost all Aspergillus species except of A. versicolor were found to be sensitive towards compound 7, while Penicillium species mostly towards compound 8. Compounds 8, 19, 22 , and 17 (MIC /MFC at 0.009/0.0125 mg/mL) as well as compound 20 (MIC/MFC 0.0006/0.00125 mg/mL) displayed excellent activity against T. viride, while compound 20 showed the best activity among all tested compounds also against A. versicolor with MIC and MFC at 0.0003 mg/mL and 0.0006 mg/mL, respectively, followed by compound 16 (MIC/MFC at 0.0006/0.0125 mg/mL. Good activity against A. versicolor was also shown by compounds 8 and 19 with MIC /MFC at 0.0125/0.025 mg/mL. It should be mentioned that the same good activity was observed for compound 8 against P. funiculosum and P. ochraceus. The study of structure-activity relationships revealed the presence of (2,4-dichlorophenyl) ethanone at N-1 of a triazole moiety as a substituent as well as 1-phenylethanone at N-4 of triazole 19 is beneficial for antifungal activity. Replacement of (2,4-dichlorophenyl)ethanone by (2,4-dibromophenyl)ethanone and acetophenone by amino group led to a less potent compound 8. The replacement of 1-phenylethanone in compound 19 by 1-(4-methoxyphenyl)ethanone decreased more than activity 17. Finally, the presence of the benzyl group as a substituent at N-1 of a triazole ring and 1-phenylethanone at N-4 had a very negative effect on the antifungal activity, leading to the less active compound 25. Among the compounds with a 2,4-dichlorobenzene moiety at the 1-arylethanone of the triazole ring, the activity order can be presented as follows: 19 > 17 > 20 > 9 > 21 > 18 > 23. In general, it seems that these groups are the most active. Thus, the presence of 1-phenylethanone as a substituent at N-4 of triazole ring, as already mentioned, is favorable for the activity; the opposite was observed with 1-(4-nitrophenyl)ethanone 18 and ethyl group as substituents 23. On the other hand, the dibromo-substituted compound 8 is more favorable than bromo-substituted compound 7. In the case of an unsubstituted aromatic moiety at N-1 of triazole ring, the activity order is 16 > 24 > 6 > 25. This fact indicates that the presence of 1-(2,4-dichlorophenyl)ethanone at N-4 of triazole has a positive influence on the antifungal activity, whereas the presence of 1-phenylethanone in compound 25 has a negative one. Thus, the activity of compounds depends not only on their nature but also on their position on the triazole ring. In order to predict the possible mechanism of antifungal activity of the compounds, all the synthesized compounds and the reference drug ketoconazole were docked to lanosterol 14α-demethylase of C. albicans and DNA topoisomerase IV ( Table 2) . The docking results revealed that the most active compound 19 takes place inside the enzyme interacting with the heme group of the enzyme throughout its benzene ring, forming aromatic interactions. Additional positive ionizable interactions were detected between the N atom of the thiazole ring of the compound and the Fe atom of the heme group. Moreover, hydrophobic interactions between Phe233, Thr311, Leu376, Phe380, and Met508 and the benzene rings of the compound were detected (Figure 2 ). Ketoconazole also interacts, thus forming interactions with its hydrophobic benzene ring and aromatic interactions ( Figure 3) . Indeed, the superposition of compound 19 and ketoconazole showed that they bind to the enzyme in the same manner (Figure 4 ). This is probably the reason why compound 19 has a high inhibition profile. Regarding the second-most active compound 8 ( Figure 5 ), docking studies showed that it is placed inside the enzyme by the side of heme group, binding to the Fe (II) ion throughout its N atom of triazole ring. It is believed that this interaction increases its binding affinity and justifies its high antifungal activity. Furthermore, compound 2 forms one halogen bond between the Br substituent of the benzene ring and the residue of Met508. Hydrophobic interactions were also detected between residues Phe228, Thr311, Leu376, Met508, Val509, and the benzene ring of the compound 8 ( Figure 5B ). Molecular docking studies also were performed on 14-alpha demethylase (CYP51B) from the fungus Aspergillus fumigatus in a complex with voriconazole, because this yeast was used in the biological evaluation in order to have a clear picture of the binding mode of the compounds. Results of docking studies are presented in Table 3 and revealed that estimated free energies of binding of the compounds were in accordance with this experimental data for this yeast. Hydrophobic, Fe-binding The most active compounds 7 and 8 exhibited low estimated free energies of binding, which are reflected in their binding mode. In particular, compound 7 interacted with the heme group of enzymes, forming a hydrogen bond with its amino group substituent and several hydrophobic interactions with residues Phe130, Val135, Ala307, Ala303, and Leu304. All of these interactions contributed to the stabilization of the complex enzyme compound. Compound 8 also formed a hydrogen bond with the heme group of the enzyme through its amino substituent and another with residue Tyr122. Moreover, hydrophobic interactions with residues Ala307, Ala303, and Leu503 were detected ( Figure 6 ). It is important to note that the formation of a strong hydrogen bond with the heme group reflects the high inhibition profile of compounds 7 and 8 and likely explains it. Compounds 17 and 19, with high antifungal activity, also formed this interaction ( Table 3) . The superposition of most active compounds 7 (magenta), 7 (orange) with voriconazole (grey) in 14-alpha demethylase (CYP51B) from Aspergillus fumigatus (Figure 7) , revealed that they took place inside the enzyme along with the heme group and were bound to the enzyme in the same manner. The number of violations of various rules, viz. Lipinski, Ghose, Veber, Egan, and Muegge, along with the bioavailability and drug-likeness scores, are given in Table 4 . The results revealed that none of the compounds violated any rule, and their bioavailability score was approximately 0.55. All compounds exhibited moderate to good drug-likeness scores ranging from −1.15 to 0.17. Compound 26 appeared to be the best in terms of their in silico predictions, with a drug-likeness score of 0.16 without any rule violation. The chemicals used were of reagent grade and used as received. The removal of all solvents was carried out under reduced pressure. 1 H and 13 C NMR spectra were recorded for 2% solutions on a Bruker-Avance III spectrometer (400.13 and 100.61 MHz) (Karlsruhe, Germany). Chemical shifts δ are given in ppm, referring to the signal center using the deuterated solvent peaks for reference. The IR spectra were recorded on a Spectrum 100 FT-IR spectrophotometer (Perkin-Elmer, Waltham, MA, USA) using the universal ATR sampling accessory. All products were analyzed by CHN elemental analysis (Elementar Vario EL analyzer) (Santa Clara, CA, USA). Melting points (uncorrected) were determined on a Boetius apparatus (Dresden, Germany). The GC-MS analysis was performed using an Agilent Technologies 7890A gas chromatograph coupled with a 5975C Mass Selective Detector (MSD) equipped with a splitless injector (1 µL) (Santa Clara, CA, USA). The analysis was carried out on a fused silica capillary HP-5MS calibrated column (30 m × 0.25 mm i.d.; film thickness 0.25 µm). The injector and detector temperatures were kept at 250 • C. Helium was used as carrier gas at a flow rate of 1.1 mL/min; the oven temperature program was 70 • C/2 min, which was then programmed to 200 • C at the rate of 5 • C/min, and finally to 300 • C at the rate of 20 • C/min; the split ratio was 1:50. The MSD ionization energy of 70 eV, scan time of 1 s, acquisition mass range was from 30 to 450 amu, and a solvent delay of 3 min. For LC/MS analyses, a Shimadzu LC/MS 2020 single quadrupole mass spectrometer with an electrospray ion source (ESI) was used. A nitrogen gas generator N2LCMS (Nitrogen Generator, Claind) was employed throughout this study. The temperature of the desolvation line and heat block were set at 250 and 200 • C, respectively. The N 2 nebulizer gas flow was maintained at 1.5 L min −1, and the drying gas flow was set at 15 L min −1 , while the interface voltage was set at 4.5 kV in positive or negative mode. The sample injection volume was 5 µL in all cases. The carrier was a mixture of 0.1% aqueous formic acid/methanol, 50/50 v/v. The flow rate was set at 0.5 mL min −1 . MS scan: 100-600 m/z. Sample concentration: 500 µg mL −1 in MeOH. The HPLC analyses were performed using high-pressure liquid chromatography with a UV detector on a Shimadzu Prominence system coupled to a Shimadzu UV-SPD-20PD detector (Shimadzu, Kyoto, Japan) over the range of 2. Method (A). To a solution of 1H-1,2,4-triazole (0.76 g, 11 mmol) and triethylamine (1.39 mL, 11 mmol) in MeCN (20 mL) was added to a solution of 1-aryl-2-bromo-ethanone (2.33 g, 11 mmol) in MeCN (5 mL). The reaction mixture was stirred at 20 • C for 7 h (TLC control). The reaction mixture was poured into water, extracted with chloroform, and dried with anhydrous sodium sulfate. The usual work-up produced a crude residue, which was submitted to flash chromatography on a silica gel column. Elution with 3% EtOAc in petroleum ether gave pure (GC-MS analysis) target compounds. Method (B). In a three-necked flask equipped with a mechanical stirrer, cooler, and dropping funnel, a solution of 4-amino-1,2,4-triazole (0.92 g, 11 mmol) in MeCN (30 mL) was added a stirring solution of 1-aryl-2-bromo-ethanone (11 mmol) in MeCN (10 mL). The reaction mixture was boiled for 40 minutes. After cooling, the precipitated crystals were filtered, washed with cold acetonitrile, and dried. A solid substance was obtained. From the mother liquor, after distilling off the solvent, another amount of product was obtained. The diazotization of the obtained salt: In a three-necked flask equipped with a stirrer, a thermometer, and a dropping funnel, the substance (10 mmol) obtained in the previous experiment was suspended in water (50 mL). Then, the HCl (1.7 mL of 37% solution) was added and heated until the salt was completely dissolved. The reaction mixture was cooled to 0 • C, and a solution of sodium nitrite (0.83 g, 12 mmol) in water (10 mL) was added slowly with stirring for 1 hour, maintaining the temperature within + 5-0 • C. By the end of the addition of sodium nitrite, the reaction mixture thickened. Stirring at this temperature continued for another 1 hour, after which the temperature was raised to 20 • C. In this case, gas evolution is observed. The reaction mixture was kept at room temperature for 3 h, treated with NH 4 OH to pH = 8-9, and extracted with chloroform. The extract was washed with water, dried over anhydrous sodium sulfate, and then evaporated under reduced pressure. The resulting product was crystallized to give pure products (GC-MS analysis). A solution of 1-aryl-2-bromo-ethanones, ethyl 2-bromoacetate, or iodoethane (10.5 mmol) in MeCN (10 mL) was added to a solution of 1-substituted-2-(1H-1,2,4-triazol-1-yl)ethanone (10 mmol) in MeCN (30 mL). The reaction mixture was refluxed for 7 h. The white precipitate was filtered off, washed with acetonitrile, and dried in a vacuum. Sometimes it depends on the nature of the starting materials to see if a distillation of half of the solvent is necessary. 3.1.3. General Experimental Procedure for the Synthesis of 1-benzyl-4-substituted 1H-1,2,4-triazol-4-ium halides 24-26 To a solution of 1H-1,2,4-triazol (0.1 mmol) in acetone (10 mL) were added benzyl chloride (0.1 mmol) and K 2 CO 3 (0.1 mmol) in this order. The reaction mixture was stirred at room temperature for 24 h, after which acetone (10 mL) was added. The organic layer after filtration was evaporated under reduced pressure. The residue was dissolved in to the prepared enzyme for verification of the method (Figure 8 ) with an RMSD value of 0.855Å. Furthermore, the reference drug ketoconazole was also docked into the active site of 5V5Z structure. Drug-likeness is one of the qualitative ideas employed for predicting a drug-like property. It is designated as an intricate balance of diverse molecular and structural features, which plays a pivotal task in establishing if the specific drug candidate can be likened to the known drugs. The targeted molecules were appraised for predicting the Drug-likeness based on five separate filters, namely the Egan [51] , Ghose [52] , Veber [53] , Muegge [54] , and Lipinski [55] rules accompanying bioavailability, and the drug-likeness scores using the Molsoft software and SwissADME program(http://swissadme.ch, access on 11 January 2022) using the ChemAxon's Marvin JS structure drawing tool. Fifteen triazolium salts were synthesized and evaluated for their antifungal activity against a panel of eight fungi. All compounds showed good antifungal activity with MIC /MFC in the range of 0.0003-0.2 /0.0006-0.4 mg/mL. As reference compounds, ketoconazole and bifonazole were used. The most active compound (19) exhibited MIC/MFC in the range of 0.009-0.037 /0.0125-0.05 mg/mL, respectively. All compounds appeared to be more active than ketoconazole and bifonazole. Thus, compound 19 showed 209 times superior activity than ketoconazole and 53-fold more than bifonazole against T. viride, while compound 20 was 1267 times more potent than ketoconazole and 1067 than bifonazole against A. versicolor. Docking to DNA TopoIV and CYP51 of C. albicans revealed that the best estimated binding energy was shown by CYP51, indicating the probable involvement of this enzyme in the mechanism of antifungal activity. The docking results revealed that the most active compound 19 takes place inside the enzyme interacting with the heme group of the enzyme throughout its benzene ring, thus forming aromatic interactions in the same way with ketoconazole. This in silico prediction study indicated that none of the compounds violated any rule, and their bioavailability score was approximately 0.55. Solid products were obtained by filtration and drying at room temperature. 4-Amino-1-(2-oxo-2-phenylethyl)-1H-1,2,4-triazol-4-ium bromide (6) Yield 2.4 g, (85%), white crystals (MeCN), mp 185-187 • C. IR (ν/cm −1 ): 3150, 3122 2H, s), 6.30 (2H, s). 13 C NMR (DMSO-d6, 100 MHz): 190.9, 145.6, 144.6, 135.3, 133.9, 129.6, 128.8, 58.9. Anal. Calcd for C 10 H 11 BrN 4 O C 42.42; H 3.92; N 19.79. Br, 28.22%. Found C 42.34; H 3.95; N 19 25 (2H, br.s), 6.24 (2H, s). 13 C NMR (DMSO-d6 4-chlorophenyl)-2-oxoethyl)-1H-1,2,4-triazol-4-ium bromide (10) Yield 3.1 g, (98%), white crystals (ethanol) 4-triazol-1-yl)ethanone (11) Yield 1.85 g, (90%), white crystals (hexane), mp 107-108 • C 1-(4-Bromophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone (12) Method A: Yield 1.58 g, (60%), Method B: Yield 2.05g, (78%), white crystals (MeCN) 1-(2,4-Dibromophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone (13) Method A: Yield1 116-117 • C Anal. Calcd for C 10 H 7 Cl 2 N 3 O C 46.90; H 2.76; Cl 27.69; N 16.41%. Found C 46.99; H 2.59; Cl 27.47; N 16 Method A: Yield 1.03 g,(47%), Method B: Yield 2.05 g,(93%), white crystals 2-oxoethyl)-1-(2-oxo-2-phenylethyl)-1H-1,2,4-triazol-4-ium bromide (16) Yield 3.66 g (88%), white crystals (MeCN), mp 215-216 • C. IR (ν/cm −1 ): 3170, 3130 2-oxoethyl)-1H-1,2,4-triazol-4-ium bromide (17) Yield 3.2 g, (66%), white crystals (ethanol), mp 209-211 • C. IR (ν/cm −1 ): 3555, 3381 2-oxoethyl)-1H-1,2,4-triazol-4-ium bromide (18) Yield 4.5 g, (90%), white crystals (ethanol), mp 228-229 • C. IR (ν/cm −1 ): 3138, 3091 2H, s), 6.33 (2H, s). 13 C NMR (DMSO-d6 27%. MS: calcd for m/z 500.13, found: 419 (M-HBr) 4-triazol-4-ium bromide (19) Yield 4.23 g, (93%), white crystals (ethanol), mp 189-202 • C. IR (ν/cm −1 ): 3583, 3402 33 (1H, s), 8.14 (1H, d, J = 8.5 Hz) 39 (2H, s), 6.31 (2H, s). 13 C NMR (DMSO-d6 4-dichlorophenyl)-2-oxoethyl)-1H-1,2,4-triazol-4-ium bromide (20) Yield 3.51 g,(67%), white crystals (ethanol), mp 215-216 • C. IR (ν/cm −1 ): 3091, 3003, 2912 75 (2H, m), 6.39 (2H, s), 6.21 (2H, s). 13 C NMR (DMSO-d6 1H, s), 9.37 (1H, s), 8.12 (1H, d 4-dichlorophenyl)-2-oxoethyl)-1H-1,2,4-triazol-4-ium bromide (22) Yield 4.15 g, (85%), white crystals (ethanol), mp 227-229 • C. IR (ν/cm −1 ): 3555, 3380 H, 2.56; Br, 16 Anal. Calcd for C 12 H 12 Cl 2 IN 3 O C 34.98; H 2.94; Cl 17.21; N 10.20%. Found C 35.11; H 3.23; Cl 17.39; N 10 H NMR (DMSO-d6, 400 MHz): 10.09 (1H, s), 9.26 91H, s), 8.11 (3H, m), 7.93 (1H, d, J = 2.0 Hz), 7.80 (1H, t, J = 7.4 Hz), 7.76 (1H, dd, J = 2.0, 8.5 Hz), 7.67 (2H, t, J = 7.7 Hz) Aspergillus versicolor (ATCC 11730), Penicillium funiculosum (ATCC 36839), Trichoderma viride (IAM 5061), Penicillium verrucosum var. cyclopium (food isolate). The organisms were obtained from the Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research-Siniša Stankovic, Belgrade, Serbia. The antifungal activity of the synthesized compounds was determined by the modified microdilution method The inocula were stored at 4 • C until further use. The obtained results were presented as minimal inhibitory (MICs), and minimal fungicidal concentrations Molecular Docking Studies Docking studies were performed in an effort to predict the mechanism of action of the compounds To prepare the proteins, all water molecules were eliminated, and polar hydrogens were added, while for the preparation of the inhibitors, charges were added, and rotatable bonds were determined. Grid maps have been calculated utilizing the Autogrid algorithm and must contain the area to be connected. The Autogrid Box was computed by the X-, Y-, and Z-coordinates for each enzyme For the present system, the following settings were used: initial population: 300, 2,500,000 maximum energy ratings, and 27,000 as a maximum generation For each compound, 200 configurations were produced. The results from the Autodock calculations were grouped using a RMSD deviation value of 1.5 Å, while the lowest-energy configuration of the largest population group was chosen as the most likely tethering configuration. The discovery studio 2017 R2 silent and LigandScout The docking box was centered on the heme molecule, at the active center of the enzyme, with coordinates x = −47.731, y = −13.422, z = 22.982 with a target box of 50 × 50 × 50Å. As a first step of docking studies Susceptibility to five antifungals of Aspergillus fumigatus strains isolated from chronically colonised cystic fibrosis patients receiving azole therapy Design, synthesis & evaluation of condensed 2H-4-arylaminopyrimidines as novel antifungal agents Identification of virulence determinants of the human pathogenic fungi Aspergillus fumigatus and Candida albicans by proteomics Design, synthesis and antifungal activities of novel 1,2,4-triazole derivatives Design, synthesis and determination of antifungal activity of 5(6)-substituted benzotriazoles Antibacterial activity study of 1,2,4-triazole derivatives New vinyl-1,2,4-triazole derivatives as antimicrobial agents: Synthesis, biological evaluation and molecular docking studies Tetrazoloquinoline-1,2,3-Triazole Derivatives as Antimicrobial Agents: Synthesis, Biological Evaluation and Molecular Docking Study Synthesis, antifungal and antibacterial activity of novel 1,2,4-triazole derivatives 8-Hydroxyquinoline 1,2,3-triazole derivatives with promising and selective antifungal activity Biological exploration of a novel 1,2,4-triazole-indole hybrid molecule as antifungal agent Triazole derivatives and their anti-tubercular activity New Application of 1,2,4-Triazole Derivatives as Antitubercular Agents. Structure, In Vitro Screening and Docking Studies Novel Specific Metallo-β-Lactamase Inhibitor ANT2681 Restores Meropenem Activity to Clinically Effective Levels against NDM-Positive Enterobacterales Discovery of a Novel Natural Allosteric Inhibitor That Targets NDM-1 Against Escherichia coli Mechanistic Investigations of Metallo-βlactamase Inhibitors: StrongZinc Binding Is Not Required for Potent Enzyme Inhibition Synthesis of novel 2-mercapto benzothiazole and 1,2,3-triazole based bis-heterocycles: Their anti-inflammatory and anti-nociceptive activities Antiviral activity of 1,2,4-triazole derivatives (micro review) Novel 1,2,3-Triazole Derivatives as Potential Inhibitors against Covid-19 Main Protease: Synthesis, Characterization, Molecular Docking and DFT Studies Discovery of novel 1,4-disubstituted 1,2,3-triazole phenylalanine derivatives as HIV-1 capsid inhibitors 1,2,3-Triazole hybrids with anti-HIV-1 activity Design, synthesis, biological evaluation, and docking study of new acridine-9-carboxamide linked to 1,2,3-triazole derivatives as antidiabetic agents targeting α-glucosidase Synthesis of novel indole, 1,2,4-triazole derivatives as potential glucosidase inhibitors Triazole analogues as potential pharmacological agents: A brief review Al-Soud, Y.A. Design, synthesis and anticancer screening ofnovel benzothiazole-piperazine-1,2,3-triazole hybrids New 1,2,4-Triazole Scaffolds as Anticancer Agents: Synthesis, Biological Evaluation and Docking Studies Synthesis, biological evaluation and computational studies of fused acridine containing 1,2,4-triazole derivatives as anticancer agents Recent developments on triazole nucleus in anticonvulsant compounds: A review 1,2,4-Triazole-based anticonvulsant agents with additional ROS scavenging activity are effective in a model of pharmacoresistant epilepsy Synthesis of 1,2,4-triazole-5-on derivatives and determination of carbonic anhydrase II isoenzyme inhibition effects Carbonic anhydrase inhibition with a series ofnovel benzenesulfonamide-triazole conjugates Novel functionalized 1,2,3-triazole derivatives exhibit antileishmanial activity, increase in total and mitochondrial-ROS and depolarization of mitochondrial membrane potential of Leishmania amazonensis Synthesis, study of antileishmanial and antitrypanosomal activity of imidazo pyridine fused triazole analogues Antiprotozoal Activity of Triazole Derivatives of Dehydroabietic Acid and Oleanolic Acid Synthesis and Antiplasmodial Evaluation of Sulfoximine-triazole Hybrids as Potential Antimalarial Prototypes Molecular modeling and design of someβ-amino alcohol grafted1,4,5-trisubstituted 1,2,3-triazoles derivatives against chloroquine sensitive, 3D7 strain of Plasmodium falciparum Biological Evaluation of 4-(1H-triazol-1-yl)benzoic Acid Hybrids as Antioxidant Agents The synthesis and in silico antihypertensive activity prognosis of new mannich bases containing the 1, 2, 4-triazole moiety Biological effects of a new set 1,2,4-triazolo[1,5-a]quinazolines on heart rate and blood pressure Recent advances bioactive 1, 2, 4-triazole-3-thiones Chromenol Derivatives as Novel Antifungal Agents: Synthesis, In Silico and In Vitro Evaluation. Molecules Regiospecific synthesis of aryl-and alkyl(1,2,4-triazolyl-1-ylmethyl) ketones Efficient microwave-assisted synthesis of 1-(1H-indol-1-yl)-2-phenyl-3-(1H-1,2,4-triazol-1-yl)-propan-2-ols as antifungal agents Novel Hit Compounds as Putative Antifungals: The Case of Aspergillus fumigatus Pyrimethanil: Between efficient fungicide against Aspergillus rot on cherry tomato and cytotoxic agent on human cell lines Autodock4 and AutoDockTools4: Automated docking with selective receptor flexiblity AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading FAF-Drugs2: Free ADME/tox filtering tool to assist drug discovery and chemical biology projects A knowledge-based approach in designing combinatorial or medicinal chemistry libraries for drug discovery. A qualitative and quantitative characterization of known drug databases Molecular properties that influence the oral bioavailability of drug candidates Simple selection criteria for drug-like chemical matter Lead-and drug-like compounds: The rule-of-five revolution The authors declare no conflict of interest.