Chromenol Derivatives as Novel Antifungal Agents: Synthesis, In Silico and In Vitro Evaluation

Herein we report the synthesis of some new 1H-1,2,4-triazole functionalized chromenols (3a–3n) via tandem reactions of 1-(alkyl/aryl)-2-(1H-1,2,4-triazole-1-yl) with salicylic aldehydes and the evaluation of their antifungal activity. In silico prediction of biological activity with computer program PASS indicate that the compounds have a high novelty compared to the known antifungal agents. We did not find any close analog among the over 580,000 pharmaceutical agents in the Cortellis Drug Discovery Intelligence database at the similarity cutoff of 70%. The evaluation of antifungal activity in vitro revealed that the highest activity was exhibited by compound 3k, followed by 3n. Their MIC values for different fungi were 22.1–184.2 and 71.3–199.8 µM, respectively. Twelve from fourteen tested compounds were more active than the reference drugs ketoconazole and bifonazole. The most sensitive fungus appeared to be Trichoderma viride, while Aspergillus fumigatus was the most resistant one. It was found that the presence of the 2-(tert-butyl)-2H-chromen-2-ol substituent on the 4th position of the triazole ring is very beneficial for antifungal activity. Molecular docking studies on C. albicans sterol 14α-demethylase (CYP51) and DNA topoisomerase IV were used to predict the mechanism of antifungal activities. According to the docking results, the inhibition of CYP51 is a putative mechanism of antifungal activity of the novel chromenol derivatives. We also showed that most active compounds have a low cytotoxicity, which allows us to consider them promising antifungal agents for the subsequent testing activity in in vivo assays.


Introduction
Azoles constitute a significant class of antifungal drugs frequently used to treat infections caused by many pathogenic fungi. Azole antifungals are pretty inexpensive, have

Introduction
Azoles constitute a significant class of antifungal drugs frequently used to treat infections caused by many pathogenic fungi. Azole antifungals are pretty inexpensive, have similar chemical structures, and are effective against most fungal species. Azoles target a crucial enzyme in the ergosterol biosynthesis pathway, CYP51, whose inhibition leads to a reduced fungal growth [1,2]. Ketoconazole [3,4], itraconazole [3,5], bifonazole [3,6], ravuconazole [3,7], and voriconazole [3,8,9] (Figure 1) are examples of drugs from this class [3]. Since azoles are fungistatic instead of fungicidal, their prolonged use and abuse frequently results in resistance, which is a severe clinical problem in antifungal therapy. The gradual increase in systemic fungal infections over the last three decades has led to high morbidity and mortality rates due to the unavailability of effective medications. The development of resistant strains contributed markedly to this phenomenon. The most aggressive fungal infections occur due to immune suppression associated with several illnesses, such as AIDS, and several drugs which are applied for cancer chemotherapy, immunosuppressive therapy for organ transplantation, and corticosteroid therapy in inflammatory diseases. More than 90% of reported fungal-associated deaths result from species belonging to three genera: Cryptococcus, Candida, and Aspergillus [10]. Clinically, candidiasis and aspergillosis represent 80% to 90% of systemic fungal infections in immunocompromised patients. Extensive use and long-term therapy with azoles resulted in fungal resistance [11,12]. Most of the drugs used in antifungal treatment, except azoles and polyenes, are characterized by low potency, a narrow spectrum of activity, and many severe side effects. These precipitate an urgent need to develop novel effective antifungal azoles with a better clinical efficacy and low risk of side effects. One way to overcome this rapid development of drug resistance is to design new agents with chemical characteristics that are different from those of existing agents. Especially the mechanism of action should be ultimately the same, e.g., different binding sites or different targets are the best possibilities for avoiding cross-resistance to existing therapeutics [13].
Triazole derivatives have attracted considerable interest in the scientific community due to their vast range of biological activities. In addition to antifungal action [14][15][16][17][18][19], they were shown to possess other antimicrobial effects such as antibacterial, including antituberculous activity [20][21][22][23][24], antiparasitic [25][26][27] and anti-HIV effects [28] as well as anticholinesterase [29], antiangiogenic [30], anticancer [31,32], antidiabetic [33,34] and anticonvulsant activities [35]. Extensive use and long-term therapy with azoles resulted in fungal resistance [11,12]. Most of the drugs used in antifungal treatment, except azoles and polyenes, are characterized by low potency, a narrow spectrum of activity, and many severe side effects. These precipitate an urgent need to develop novel effective antifungal azoles with a better clinical efficacy and low risk of side effects. One way to overcome this rapid development of drug resistance is to design new agents with chemical characteristics that are different from those of existing agents. Especially the mechanism of action should be ultimately the same, e.g., different binding sites or different targets are the best possibilities for avoiding cross-resistance to existing therapeutics [13].
These findings focused particular interest on incorporating a triazole with a chromen ring in one frame to obtain compounds with improved antifungal activity. According to the current literature, more efficacious antibacterial compounds have been designed by joining two or more biologically active heterocyclic nuclei in a single molecular framework [50][51][52][53].
Taking all these into account, herein we report the design, synthesis, and evaluation of the antifungal activity of fourteen new chromenol-triazole hybrids.

Chemistry
The synthesis of new vinyl-1,2,4-triazole derivatives A (Figure 1) as antimicrobial agents was previously reported [54]. Recent attention has only been paid to the tandem reactions of salicylic aldehydes or salicylic imines with α,β-unsaturated compounds [55]. We considered that an applicable route to the synthesis of 1H-1,2,4-triazole-functionalized chromene B (Figure 2) would be possible through the coupling of substituted triazolyl ethanones with salicylic aldehydes.
These findings focused particular interest on incorporating a triazole with a chromen ring in one frame to obtain compounds with improved antifungal activity. According to the current literature, more efficacious antibacterial compounds have been designed by joining two or more biologically active heterocyclic nuclei in a single molecular framework [50][51][52][53].
Taking all these into account, herein we report the design, synthesis, and evaluation of the antifungal activity of fourteen new chromenol-triazole hybrids.
These findings focused particular interest on incorporating a triazole with a chromen ring in one frame to obtain compounds with improved antifungal activity. According to the current literature, more efficacious antibacterial compounds have been designed by joining two or more biologically active heterocyclic nuclei in a single molecular framework [50][51][52][53].
Taking all these into account, herein we report the design, synthesis, and evaluation of the antifungal activity of fourteen new chromenol-triazole hybrids.
The structure of the obtained derivatives was supported by IR, 1 H and 13 C NMR spectroscopic data and by elemental analysis (see Supplementary Materials).
The examination of the NMR spectra of the discussed compounds further confirmed the formation of 1H-1,2,4-triazole functionalized chromenols by the intramolecular addition of a hydroxy group to a ketonic carbonyl group. The peaks in the 13  , which is consistent with the structure assigned to it. The characterization data of compounds 3a-n are given in experimental part.

PASS Prediction and Similarity Search in CDDI Database
A PASS prediction of antifungal activity was performed for the whole set of designed molecules, from which fourteen were chosen for synthesis and biological testing. Antifungal activity for all selected compounds was predicted with the probability to be active Pa values ranging from 0.43 to 0.53 (Table 1). The mechanism of antifungal activity was predicted as well. The prediction revealed that lanosterol 14 alpha demethylase inhibition was estimated with Pa between 0.16 and 0.35. The calculated Pa values for most of the compounds were less than 0.5, indicating their relative novelty compared to the structures of the compounds from the PASS training set [56,57]. Such results lead to the conclusion that the studied compounds have some features different from those of well-known antifungal agents, which may indicate their innovative potential. To check this inference, we performed a chemical similarity search in the Cortellis Drug Discovery Intelligence (CDDI) database [58], one of the most comprehensive sources of current information about pharmacological agents. The CDDI contains data on more than 580 thousand pharmaceutical agents, 90% of which includes a chemical structure. Using the similarity search implemented in the CDDI, no close analogs of the compounds under study were identified with the Tanimoto coefficient (TC) exceeding 70%. Earlier, Yvonne Martin and co-authors [59] showed that even at the TC > 85%, there is only a 30% chance to find compounds with the same biological activity. Thus, the similarity search results in the CDDI database confirm that the designed compounds have a high novelty compared to the known antifungal agents.

PASS Prediction and Similarity Search in CDDI Database
A PASS prediction of antifungal activity was performed for the whole set of designed molecules, from which fourteen were chosen for synthesis and biological testing. Antifungal activity for all selected compounds was predicted with the probability to be active Pa values ranging from 0.43 to 0.53 (Table 1). The mechanism of antifungal activity was predicted as well. The prediction revealed that lanosterol 14 alpha demethylase inhibition was estimated with Pa between 0.16 and 0.35. The calculated Pa values for most of the compounds were less than 0.5, indicating their relative novelty compared to the structures of the compounds from the PASS training set [56,57]. Such results lead to the conclusion that the studied compounds have some features different from those of well-known antifungal agents, which may indicate their innovative potential. To check this inference, we performed a chemical similarity search in the Cortellis Drug Discovery Intelligence (CDDI) database [58], one of the most comprehensive sources of current information about pharmacological agents. The CDDI contains data on more than 580 thousand pharmaceutical agents, 90% of which includes a chemical structure. Using the similarity search implemented in the CDDI, no close analogs of the compounds under study were identified with the Tanimoto coefficient (TC) exceeding 70%. Earlier, Yvonne Martin and co-authors [59] showed that even at the TC > 85%, there is only a 30% chance to find compounds with the same biological activity. Thus, the similarity search results in the CDDI database confirm that the designed compounds have a high novelty compared to the known antifungal agents.

Biological Evaluation
Antifungal Activity Compounds 3a-3n were evaluated by a microdilution method against a panel of eight fungi using bifonazole and ketoconazole as reference drugs. All compounds showed antifungal activity, and the results are presented in Table 2. Table 2. Antifungal activity of the tested compounds (µM). The order of antifungal activity can be presented as follows: 3k > 3n > 3g > 3m > 3d > 3c > 3h > 3b > 3e > 3l > 3a > 3i > 3f > 3j. The best antifungal activity was achieved for compound 3k with MIC ranging 22.1-184.2 µM and MFC at 46.0-368.5 µM. The lowest antifungal activity was exhibited by compound 3j with MIC and MFC at 143.9-1151 µM and 575.6-1439 µM, respectively.
As far as reference drugs are concerned, ketoconazole showed an antifungal potential with MIC at 380-4750 µM and MFC at 950-5700 µM, whereas bifonazole showed MIC at 480-640 µM and MFC at 640-800 µM. Thus, all compounds were more effective antifungal agents than ketoconazole, and almost all (twelve from fourteen) exhibited a higher activity than bifonazole.
It is interesting to mention that the fungi showed a different sensitivity towards the compounds tested. Thus, the order of activity of tested compounds against the most sensitive fungi T. viride is 3k = 3n > 3b = 3g = 3m > 3c = 3d = 3e = 3h = 3i = 3l = 3j > 3a > 3f, whereas the sensitivity of the most resistant fungi, A. fumigatus, can be presented as 3d = 3e = 3k > 3g = 3h = 3m > 3b = 3n > 3c = 3l > 3j > 3a = 3i > 3f. Not only were there differences in the sensitivity among species, but also in each fungus were observed. At the same time, all species appeared to be sensitive to compound 3k and not sensitive to 3f and 3j.
Compound 3k showed perfect activity with MIC 22.1 µM against A. niger. It also exhibited good activity with MIC 33.1 µM against A. fumigatus, and T. viride and A. versicolor with MIC 92.1 µM. The same good activity was achieved against T. viride by compound 3n, against A. versicolor by 3d and 3e, and against P. ochraceus by 3b, 3c, 3d, and 3l.
The study of the structure-activity relationship revealed that the presence of the 2-(tert-butyl)-2H-chromen-2-ol substituent 3k on the 4th position of the triazole ring was very beneficial for antifungal activity. The introduction on 2-(tert-butyl)-2H-chromen-2-ol bromine at position six, resulted in a slightly less active compound 3n, while substitution on position two of the 2-(tert-butyl)-2H-chromen-2-ol by 2,4-dichlorophenyl group (in 3g) decreased the antifungal activity more. In series of 2-H chromen-2-ol derivatives with a tert-butyl substituent in position two of the chromen ring, the most beneficial was the unsubstituted benzene (3k) derivative followed by derivatives with a substituted benzene ring with electron withdrawal (EWG) substituents (Br, Cl, NO 2 ), while the electrondonating (EDG) substituent (Ph) (3j) was detrimental for antifungal activity of these derivatives. In the case of the 2,4-dichlorophenyl substituent in position two of the chromen-2-ol moiety, the favorable effect was observed for the derivative with bromine (3g) as a substituent in the benzene ring. The order of activity can be presented as Br > NO 2 > Ph > Cl. There is no strong correlation of activity with the EWG/EDG character of substituents in this case. From all mentioned above, it can be concluded that antifungal activity depends not only on the triazole ring, but also on its substituents.

Docking Studies
All the synthesized compounds and the reference drug ketoconazole were docked to lanosterol 14α-demethylase (known as well as cytochrome 51-CYP51) of C. albicans and DNA topoisomerase IV (Table 3). Results revealed that the values of free binding energy for DNA topoisomerase IV were higher than CYP51. Thus, the inhibition of this enzyme may be the putative mode of action of the analyzed novel chromenol derivatives.
Docking results revealed that the most active compound, 3k, bound the CYP51 enzyme of C. albicans in a way that allowed the interaction with the heme. In particular, the N atom of the piperazine ring of the compound interacted strongly with the Fe of the heme group. Moreover, hydrophobic interactions were detected with the residues Tyr118, Ile131, Tyr132, Phe126, Tyr122, Leu121, Thr311, Leu376 and Met508. Hydrophobic interactions were also observed between the methyl substituents and the heme group of the enzyme. The same interactions were detected in the binding of compound 3n ( Figure 5). Docking results revealed that the most active compound, 3k, bound the CY zyme of C. albicans in a way that allowed the interaction with the heme. In partic N atom of the piperazine ring of the compound interacted strongly with the Fe of group. Moreover, hydrophobic interactions were detected with the residues Tyr11 Tyr132, Phe126, Tyr122, Leu121, Thr311, Leu376 and Met508. Hydrophobic interacti also observed between the methyl substituents and the heme group of the enzyme. T interactions were detected in the binding of compound 3n ( Figure 5).
Interaction with the heme group was also observed with the benzene ring conazole, which formed a hydrophobic and aromatic interaction ( Figure 6). H compounds 3k and 3n had stronger interactions than ketoconazole and formed m ble complexes with the enzyme which is the likely reason for the better antifunga than ketoconazole.  Interaction with the heme group was also observed with the benzene ring of ketoconazole, which formed a hydrophobic and aromatic interaction ( Figure 6). However, compounds 3k and 3n had stronger interactions than ketoconazole and formed more stable complexes with the enzyme which is the likely reason for the better antifungal activity than ketoconazole.

In Silico Predictive Studies
Drug-likeness is an essential part of drug research and development that provides the base for the molecules to be powerful drug candidates. Various rules, such as, e.g., Lipinski, Ghose, Veber, Egan, and Muegge, were considered to measure the drug-likeness of the tested compounds to find out whether they can be bioactive drug candidates according to some critical criteria such as the molecular weight, LogP, number of hydrogen bond acceptors and donors. The number of violations to the rules, along with 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 around 0.55. All the tested molecules were suggested to pass the blood-brain barrier (BBB) except for in compounds 3h and 3l. All compounds exhibited moderate to good drug-likeness scores, ranging from −0.63 to 0.29.
Moreover, the bioavailability radar of some of the compounds is displayed in Figure 7. The most active compound, 3k, appeared to be the best in the in silico predictions, with a drug-likeness score of 0.29 without any rule violation. However, compounds 3k and 3n had stronger interactions than ketoconazole and formed more stable complexes with the enzyme which are the likely reasons for the better antifungal activity than ketoconazole.

Cytotoxicity Studies
To ensure the safety margin for the tested compounds, cytotoxicity was tested in both the cancerous breast cancer cell line MCF7/S0.5 and the non-cancerous cell renal epithelial line HK-2.
Initial screening at a high concentration was performed in the MCF7/S0.5 cell line. At a concentration of 100 µM, seven compounds (3c, d, j, k, l, m, and n) showed little toxicity. When tested at a, still high, concentration of 50 µM, several other compounds (3a and 3e) showed a higher survival compared to the 100 µM results (Figure 8a). Since cultured cancer cell lines are more sensitive to xenobiotic treatment, the same experiments were performed with the non-cancerous cell line HK-2 to further confirm the safety of the compounds.'. As shown in Figure 8b, general survival values were much higher for cells treated in the same way as in MCF7/S0.5 cells, as expected. These data indicate that nine out of fourteen synthesized compounds can be considered potential candidates for furthe drug development. When integrated with antifungal activity data, the most promising compounds seemed to be 3c, 3d, 3k, 3m and 3n.

Cytotoxicity Studies
To ensure the safety margin for the tested compounds, cytotoxicity was tested in both the cancerous breast cancer cell line MCF7/S0.5 and the non-cancerous cell renal epithelial line HK-2.
Initial screening at a high concentration was performed in the MCF7/S0.5 cell line. At a concentration of 100 µM, seven compounds (3c, d, j, k, l, m, and n) showed little toxicity. When tested at a, still high, concentration of 50 µM, several other compounds (3a and 3e) showed a higher survival compared to the 100 µM results (Figure 8a). Since cultured cancer cell lines are more sensitive to xenobiotic treatment, the same experiments were performed with the non-cancerous cell line HK-2 to further confirm the safety of the compounds.'. As shown in Figure 8b, general survival values were much higher for cells treated in the same way as in MCF7/S0.5 cells, as expected. These data indicate that nine out of fourteen synthesized compounds can be considered potential candidates for furthe drug development. When integrated with antifungal activity data, the most promising compounds seemed to be 3c, 3d, 3k, 3m and 3n.

General Information
The chemicals used were of reagent grade. Removal of all solvents was carried out under reduced pressure. The 1 H and 13 C NMR spectra were recorded in d6-DMSO 2% solutions on a "Bruker Avance III" (400.13 and 100.61 MHz) (Karlsruhe, Germany). Chemical shifts δ are given in ppm referring to the signal center using the solvent peaks for reference: d6-DMSO 2.50 ppm. IR spectra were recorded on a Spectrum 100 FT-IR spectrophotometer (PerkinElmer) using the universal ATR sampling accessory Agilent 5975C VL MSD (Waltham, Massachusetts, USA) with triple axis detector, performed using the method by Druta: temperature 60-320°C, run time: 32.333 min, flow 1.1062 mL/min, pressure 9.418 psi and method steroid 250, temperature 180-250°C, run time 29 (a) (b) Figure 8. Cytotoxicity activity of the tested compounds. The fourteen derivates were tested in (a) breast cancer cell line MCF7/S0.5 and (b) renal epithelia HK-2 cell line for 48 h in two high concentrations. Results are expressed as the mean ± s.d. of three independent experiments performed in triplicates, where 100% survival was set to the positive control wells (DMSO 0.1%). Statistical assay one-way ANOVA was performed to assess the significance of the results compared to positive control. * p < 0.001 vs. the negative control DMSO.

General Information
The chemicals used were of reagent grade. Removal of all solvents was carried out under reduced pressure. The 1 H and 13 C NMR spectra were recorded in d6-DMSO 2% solutions on a "Bruker Avance III" (400.13 and 100.61 MHz) (Karlsruhe, Germany). Chemical shifts δ are given in ppm referring to the signal center using the solvent peaks for reference: d6-DMSO 2.50 ppm. IR spectra were recorded on a Spectrum 100 FT-IR spectrophotometer (PerkinElmer) using the universal ATR sampling accessory Agilent 5975C VL MSD (Waltham, MA, USA) with triple axis detector, performed using the method by Druta: temperature 60-320 • C, run time: 32.333 min, flow 1.1062 mL/min, pressure 9.418 psi and method steroid 250, temperature 180-250 • C, run time 29 min, flow 1.1062 mL/ min, pressure 16.528 psi. 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). Thin-layer chromatography was carried out on Merck aluminum TLC plates, silica gel 60 coated with fluorescent indicator F254.

Synthesis
For the preparation of tested compounds, the following method was used: Onenecked flask equipped with a Dean-Stark receiver and reflux condenser were charged with triazolyl ketone (10 mmol), aldehyde (11 mmol), catalyst (1 mol%, mixture of acetic acid and piperidine 1:1) and benzene (100 mL). The resulting mixture was heated in an oil bath to maintain a gentle solvent reflux for 18 h. The reaction was monitored by TLC. After cooling to room temperature, the reaction mixture was washed with water (2 × 50 mL) in a separation funnel, dried with magnesium sulphate and distilled in vacuo.

Crystallographic Study
Diffraction measurements for single-crystal X-ray analysis of compound 3h were carried out on an Xcalibur E diffractometer (Cambridge, Great Britain) equipped with a CCD area detector and a graphite monochromator utilizing MoKα radiation at room temperature. All calculations to solve and refine the structure were carried out with the programs SHELXS-97 and SHELXL-2014 [60,61]. Non-hydrogen atoms were refined anisotropically. Positions of H atoms were calculated geometrically and refined isotropically using a rigid-body model. The 1,3-dichlorobenzene moiety of the 3h molecule was found to be disordered in the structure over two coplanar positions with probabilities of their occupancy 0.591(3):0.409(3). The structure was refined using 3079 (R(int) = 0.0512) independent reflections to R1 = 0.0630 and wR2 = 0.0727 for 1478 reflections with I > 2σ(I) and GOF = 1.003. The maximum and minimum residual electron densities in the difference synthesis were 0.196 and −0.277eÅ -3 . Crystallographic data were deposited with The Cambridge Crystallographic Data Centre, CCDC 2033740, and may be obtained free of charge from The Cambridge Crystallographic Data Centre.

PASS Predictions and Similarity Assessment
The online version of the computer program PASS (Prediction of Activity Spectra for Substances) [56,57] predicts about 4000 biological activities with an average accuracy of about 95%. Thousands of researchers widely use it from over 100 countries to identify the most promising directions of biological testing of the designed and synthesized compounds. Structural formulae of compounds presented as MOL or SDF files are used as input data. Predictions are presented as a list of probable activities with two probabilities that reflect the chance of compounds belonging to active (Pa) and inactive (Pi) classes. A high Pa value could be obtained for the compounds with close analogs in the PASS training set. If, for a particular activity, the Pa value is less than 0.5, the compound does not have close analogs with this activity in the PASS training set.
The Cortellis Drug Discovery Intelligence database [58] is one of the most comprehensive informational sources continually curated and updated by the skilled team manually annotated data about pharmaceutical research and development performed worldwide. Currently, the CDDI includes more than 500 thousand chemical structures of pharmaceutical agents under different biological testing stages. Chemical similarity search in the CDDI database allows identifying at different cutoff values (from 60 to 100%) the close analogs of the structure used as a query if such analogs are found.

Molecular Docking
The program AutoDock 4.2 ® software [63] was used for the docking stimulation. The free energy of binding (∆G) of E. coli DNA GyrB, thymidylate kinase, E. coli MurA, E. coli primase, E. coli MurB, DNA topoIV and CYP51 of C. albicans in a complex with the inhibitors was generated using this molecular docking program. The X-ray crystal structures data of all the enzymes used were obtained from the Protein Data Bank (PDB 1S16 and 5V5Z, respectively).
To prepare proteins, all water molecules were eliminated and polar hydrogens were added, while for preparation of the inhibitors, charges were added and the rotatable bonds determined. Grid maps were calculated utilizing the AutoGrid algorithm. Autogrid Box was computed by the X-, Y-and Z-coordinates for each enzyme. Three-dimensional structures of all compounds were constructed using ChemBio3D Ultra 12.0 software (Chemical Structure Drawing Standard; PerkinElmer Informatics, Waltham, MA, USA). For the present system, the Lamarckian genetic algorithm was applied for minimization, and the following settings were used: initial population 300, 2,500,000 maximum energy ratings and 27,000 as maximum generation. The pitch was 1.0 Å, while the quaternion and pivot angle were set to 5.0 degrees. For each compound, 200 configurations were produced. The results from the AutoDock calculations were grouped using an 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 Ligand-Scout were used to display the results and process the configurations with the highest tie rating [64]. Finally, the docking process methodology was first validated by redocking all the co-crystalized original ligands in the active sites of all enzymes with deviation (RMSD) values from 0.86 to 1.63 Å.

In Silico Predictive Studies
Drug-likeness is one of the qualitative ideas employed for predicting drug-like property. It is designated as an intricate balance of diverse molecular and structural features which plays a pivotal task in establishing whether the specific drug candidate is alike the known drugs or not. The targeted molecules were appraised for predicting the druglikeness based on 5 separate filters namely Egan [65], Ghose [66], Muegge [67], Veber [68] and Lipinski [69] rules, accompanying bioavailability and drug-likeness scores using the Molsoft software and SwissADME program (http://swissadme.ch, accessed on 7 July 2021) using the ChemAxon's Marvin JS structure drawing tool.

Cytotoxicity Experiments
CellTiter 96 ® Aqueous Non-Radioactive Cell Proliferation Assay (Promega Corporation, Madison, WI, USA) was performed to evaluate the in vitro effects of evaluated compounds in two cell lines. Cancerous MCF7/S0.5 cells, developed to grow in a low-sera environment, and non-cancerous HK-2 renal cells were used for these purposes. MCF7/S0.5 cells were cultivated as specified in our previous paper [70] and HK-2 cells were cultivated in high-glucose Dulbecco's Modified Eagle Medium (DMEM) complemented with 10% FBS and 2 mM L-glutamine. The assay is based in the reduction in a tetrazolium salt which happens only in the mitochondria of viable cells. Experiments were performed according to manufacturer indications as specified previously [66]. Briefly, cells were treated with the test compounds at concentrations of 100 and 50 µM, negative control (SDS 10%) or vehicle (DMSO 0.1%) for 48 h in 96-well plates. After incubation period, MTS reagent was added to each well and incubated for further 3 h. Absorbance was measured at 490 nm using a plate reader (Hidex Sense Beta Plus plate reader, Hidex Turku, Finland). Results are expressed as the relative cell viability, considering vehicle to have 100% viability.

Conclusions
We performed, for the first time, the synthesis of 1H-1,2,4-triazole-functionalized chromenols via tandem reactions of 1-(alkyl/aryl)-2-(1H-1,2,4-triazole-1-yl) with salicylic aldehydes and the evaluation of their antifungal activity. The antifungal activity evaluation was performed by a microdilution assay using, as reference, drugs bifonazole and ketoconazole. The best activity was achieved for compound 3k. The most sensitive fungal compounds tested were T. viride, whereas A. fumigatus was the most resistant one. It should be mentioned that almost all compounds, except for 3e, 3j and 3l, against P.v.c appeared to be more potent than ketoconazole against all fungi tested, while many compounds were even more active than bifonazole against all fungi tested. Compound 3k was found to be 32-fold more active than ketoconazole and 16 times more than bifonazole.
PASS predictions demonstrated that the compounds under study have a low structural similarity with the antivirals included in the training set. This conclusion was confirmed by the similarity search in the CDDI database: at the 70% similarity cutoff, no close analog was identified among the 580,000 pharmacological substances. Thus, the synthesized chromenol derivatives have a very high innovative potential in the pharmaceutical field.
According to molecular docking studies, it seems that the inhibition of 14α-demethylase of C. albicans (CYP51) was involved in the mechanism of antifungal activity of compounds tested. Biological experiments have shown that the compound 3k is not toxic to cells. Finally, we can state that the derivative 3k can be the lead compound for further discovery of more potent antifungal agents.