New Benzimidazole-1,2,4-Triazole Hybrid Compounds: Synthesis, Anticandidal Activity and Cytotoxicity Evaluation

Owing to the growing need for antifungal agents, we synthesized a new series 2-((5-(4-(5-substituted-1H-benzimidazol-2-yl)phenyl)-4-substituted-4H-1,2,4-triazol-3-yl)thio)-1-(substitutedphenyl)ethan-1-one derivatives, which were tested against Candida species. The synthesized compounds were characterized and elucidated by FT-IR, 1H-NMR, 13C-NMR and HR-MS spectroscopies. The synthesized compounds were screened in vitro anticandidal activity against Candida species by broth microdiluation methods. In vitro cytotoxic effects of the final compounds were determined by MTT assay. Microbiological studies revealed that compounds 5m, 5o, 5r, 5t, 5y, 5ab, and 5ad possess a good antifungal profile. Compounds 5w was the most active derivative and showed comparable antifungal activity to those of reference drugs ketoconazole and fluconazole. Cytotoxicity evaluation of compounds 5m, 5o, 5r, 5w, 5y, 5ab and 5ad showed that compounds 5w and 5ad were the least cytotoxic agents. Effects of these two compounds against ergosterol biosynthesis were observed by LC-MS-MS method, which is based on quantification of ergosterol level in C. albicans. Compounds 5w and 5d inhibited ergosterol biosynthesis concentration dependently. A fluorescence microscopy study was performed to visualize effect of compound 5w against C. albicans at cellular level. It was determined that compound 5w has a membrane damaging effect, which may be related with inhibition of biosynthesis of ergosterol.


Introduction
The Candida species are definitely the most important opportunistic fungal pathogens for individuals [1]. Candidiasis can range from non-life threatening mucocutaneous infections to incursive progressions [2]. In the past two decades, a dramatic rise has been observed in the incidence of fungal infections due to increasing number of immunocompromised hosts [3]. The most common reasons of this challenge contain treatment with broad spectrum antibiotics, use of central venous catheters and implantable prosthetic devices, parenteral nutrition, prolonged intensive care unit stay, hemodialysis, immunosuppression, organ transplantation, HIV infection, neutropenia, and use of glucocorticosteroids, chemotherapeutic agents, and immunomodulators [4][5][6][7][8][9]. As a result, antifungal therapy still seems to be one of the most problematic medication issue.
For the therapeutic management of fungal infections, only four important classes of antifungal agents are available in clinical use. These are azoles such as fluconazole, itraconazole, and ketoconazole; polyene macrolides such as Amphotericin B and nystatin; 5-flucytosine; and echinocandins such as caspofungin and micafungin [10]. Among them, azoles are the most widely used antifungal agents on account of their high therapeutic index, broad spectrum of activity and more favorable safety profile [11]. Azole type antifungal drugs have been divided into two groups: triazoles and imidazoles [12]. The most frequently used triazoles are fluconazole and itraconazole that display a wide spectrum of antifungal activity [13]. Several novel triazole antifungal drugs, such as voriconazole, posaconazole, ravuconazole and albaconazole, are available in the market or are at the late stages of clinical trials [14]. These drugs act by competitive inhibition of the lanosterol 14α-demethylase (CYP51A1), which is the key enzyme in sterol biosynthesis of fungi. Selective inhibition of CYP51A1 would cause depletion of ergosterol, a major component of the fungal cell membrane, and accumulation of lanosterol and other 14-methyl sterols resulting in the growth inhibition of fungal cells [14,15].
Benzimidazole compounds have always been important pharmacophores in studies on new antimicrobial agent development. The reason for this special interest can be explained by the structural similarity between purine and benzimidazole [16,17]. Furthermore, the benzimidazole scaffold has an ability to form hydrogen bonds with biological enzymes and receptors and participates in π-π and hydrophobic interactions, which may be related to its mechanism of action [18,19]. Because of these features, several pharmacological and biochemical research have been performed, and many of them have supported the finding that benzimidazole derivatives are potent against various microorganism strains [20][21][22][23][24][25][26][27][28][29][30][31][32][33]. Benzimidazoles can be classified as one the most important group of fungicides with systemic activity and are well-known for their pronounced ability to control a large number of fungal diseases. Thiabendazole, benomyl, carbendazim, chlorfenazole, cypendazole, debacarb, fuberidazole, mecarbinzid, and rabenzazole, which include the benzimidazole moiety, are the main examples of this fungicide class [34,35].

Chemistry
The synthesis of target compounds 5a-5ad was performed as outlined in Scheme 1. Initially, methyl 4-(5(6)-substituted-1H-benzimidazol-2-yl)benzoate (1a-1c) derivatives were synthesized by the reaction of methyl 4-formylbenzoate and corresponding o-phenylenediamine in the presence of Na 2 S 2 O 5 . In this step, we applied one of the most used approaches, involving the condensation of o-diaminoaromatic compounds with aromatic aldehydes in a two-step procedure, which includes an oxidative cyclodehydrogenation of the corresponding aniline Shiff bases intermediates. In the previous studies, several reagents, such as nitrobenzene [43], 1,4-benzoquinone [44], MnO 2 [45], benzofuroxan [46], tetracyanoethylene [47], oxone [48], Pb(OAc) 4 [49], NaHSO 3 [50], and Na 2 S 2 O 5 [51] have been used for this purpose. Among these reagents, we preferred Na 2 S 2 O 5 because it has been reported that high yield reactions could be achieved using Na 2 S 2 O 5 [52]. In the second reaction step, 4-(5(6)-substituted-1H-benzimidazol-2-yl)benzoate (1a-1c) derivatives were treated with excess of hydrazine hydrate to obtain 4-(5(6)-substituted-1H-benzimidazol-2-yl)benzoic acid hydrazides (2a-2c). In the first and second steps, due to microwave irradiation, the benzimidazole products (1a-1c and 2a-2c) were obtained in good yields (81%-89%) with a short reaction time (10 min), while in previously reported classical methods [53,54] the similar products were obtained in lower yields with longer reaction times. In the third step, benzimidazole-hydrazides (2a-2c) were treated with alkylisothiocyanates to afford compounds 3a-3f, which were converted to 5-substituted-1,2,4-triazole-3-thiols (4a-4f) by the effect of NaOH. In the final step, substitution reaction between 2-bromoacetophenones and compounds 4a-4f gave the target compounds (5a-5ad). Some characteristics of compounds 5a-5ad are presented in Table 1. The IR, 1 H-NMR, 13 C-NMR, and HRMS spectral data (Supplementary materials) were in agreement with the expected structures of compounds 5a-5ad. Structural elucidation of final compounds was performed by spectral analyses. In the IR spectra, N-H and C=O stretching bands were observed between 3472-3069 cm −1 and 1686-1661 cm −1 , respectively. It is known that benzimidazoles, which contain a hydrogen atom attached to N-1 position readily tautomerize. However, benzimidazoles present a rapid tautomerism which does not allow to observe separate peaks for each tautomers in the NMR spectra [55]. Thus, single signals were assigned for all protons in the NMR analysis. Protons of methylene between the sulfur atom and carbonyl group were recorded as a singlet between 4.58 and 5.03 ppm, and carbon of methylene was observed between 41.08 and 46.21 ppm. Benzimidazole proton at N-1 position gave a singlet between 13.03 and 13.32 ppm. Carbonyl carbons were recorded over 190 ppm as a singlet peak except for difluorophenyl derivatives, which were split into two having 3.0-4.0 Hz coupling constant values. The other hydrogens and carbons were recorded at expected regions. Fluorinated derivatives had coherent carbon-fluorine coupling constants. In the HRMS spectra, all measured mass and isotope ratios were compatible with theoretical values. Scheme 1. Synthesis route to target compounds 5a-5ad.
The differences in the chemical structures and the antibacterial activity profiles of compounds directed us to discuss structure activity relationships (SARs) (Figure 1). The structural variations of compounds can be classified in three regions. The first one is benzimidazole ring, carrying chloro or fluoro substituents at C-5 position or it is not substituted. The second region is triazole ring in which there is a methyl or ethyl substituents at N-4 position. The last region is phenyl ring of 1-phenyl-1ethan-1-one substructure that carries chloro or fluoro at C-4 position and dichloro or difluoro substituents at C-2 and C-4 positions. Looking at the chemical structure of the compounds (5m-5o, 5r-5t, 5w-5y, and 5ab-5ad) that showed stronger anticandidal activity, they commonly bear fluoro substituent at C-4 position of phenyl. Hence, it can be declared that C-4 of phenyl is very important position in terms of anticandidal activity. Fluoro substituents at this position significantly enhance the biological activity. However, compounds 5c, 5e, 5h, and 5j, which also contain fluoro substituent at C-4 position of phenyl, could not indicate strong antifungal activity. The main structural difference between initial and later compounds is the substituents at C-5 position of benzimidazole. At this position, compounds 5m-5o, 5r-5t, 5w-5y, and 5ab-5ad carry fluoro or chloro substituents, while compounds 5c, 5e, 5h, and 5j do not include any substituents. Thus, it can be suggested that Scheme 1. Synthesis route to target compounds 5a-5ad.
The differences in the chemical structures and the antibacterial activity profiles of compounds directed us to discuss structure activity relationships (SARs) (Figure 1). The structural variations of compounds can be classified in three regions. The first one is benzimidazole ring, carrying chloro or fluoro substituents at C-5 position or it is not substituted. The second region is triazole ring in which there is a methyl or ethyl substituents at N-4 position. The last region is phenyl ring of 1-phenyl-1-ethan-1-one substructure that carries chloro or fluoro at C-4 position and dichloro or difluoro substituents at C-2 and C-4 positions. Looking at the chemical structure of the compounds (5m-5o, 5r-5t, 5w-5y, and 5ab-5ad) that showed stronger anticandidal activity, they commonly bear fluoro substituent at C-4 position of phenyl. Hence, it can be declared that C-4 of phenyl is very important position in terms of anticandidal activity. Fluoro substituents at this position significantly enhance the biological activity. However, compounds 5c, 5e, 5h, and 5j, which also contain fluoro substituent at C-4 position of phenyl, could not indicate strong antifungal activity. The main structural difference between initial and later compounds is the substituents at C-5 position of benzimidazole.
At this position, compounds 5m-5o, 5r-5t, 5w-5y, and 5ab-5ad carry fluoro or chloro substituents, while compounds 5c, 5e, 5h, and 5j do not include any substituents. Thus, it can be suggested that C-5 position of benzimidazole is essential and fluoro or chloro substitution of this position significantly increases the antifungal activity. The methyl or ethyl substituents at N-4 position of triazole did not cause a meaningful difference on biological activity. Similarly, second fluoro substituent at C-2 position of phenyl in compounds 5e, 5j, 5o, 5t, 5y, and 5ad did not alter the antifungal activity, when compared with monofluoro substitution at C-4 position of phenyl in compounds 5c, 5h, 5m, 5r, 5w, and 5ab. C-5 position of benzimidazole is essential and fluoro or chloro substitution of this position significantly increases the antifungal activity. The methyl or ethyl substituents at N-4 position of triazole did not cause a meaningful difference on biological activity. Similarly, second fluoro substituent at C-2 position of phenyl in compounds 5e, 5j, 5o, 5t, 5y, and 5ad did not alter the antifungal activity, when compared with monofluoro substitution at C-4 position of phenyl in compounds 5c, 5h, 5m, 5r, 5w, and 5ab.

Prediction of ADME Parameters
Regrettably, significant pharmacological activity is not sufficient for a compound to become drug candidate. An appropriate profile of pharmacokinetics and lowest possible toxicity are also very essential for the new drug candidates and they should be assessed as early as possible in the drug development process. In recent years, an important increase in combinatorial chemistry has arisen and hence preliminary information needed on absorption, distribution, metabolism and excretion (ADME) can be easily provided [58]. Owing to importance of pharmacokinetics, in the present study ADME properties of compounds 5m-5o, 5r-5t, 5w-5y, and 5ab-5ad, which showed strong antifungal activity, were calculated by online Molinspiration property program [59]. This program provides the data of Lipinski s rule, which evaluates the ADME properties of new compounds and is imperative for the optimization of a biologically active compound. The rule highlights that an orally active drug should not have more than one violation [60].
The theoretical calculations of ADME parameters (molecular weight (MW), log P, topological polar surface are (tPSA), number of hydrogen donors (nON) and acceptors (nOHNH) and volume) and DLS are presented in Table 3 along with the violations of Lipinski's rule. According to these data, compounds 5m, 5o, 5r, 5w, 5y, 5ab, and 5ad comply with the rule of Lipinski by causing one violation. However, compounds 5n, 5s, 5t, 5x, and 5ac possess two violations and thus they may not display an ideal pharmacokinetics. As a result, it can be declared that compounds 5m, 5o, 5r, 5w, 5y, 5ab, and 5ad may have good pharmacokinetics profile, which improves their biological importance.

Cytotoxicity Evaluation
Toxicity is a key cause of failure at all steps of the new drug development process. The most part of safety-related problems occur at preclinical phases to predict preclinical safety liabilities earlier in the new drug development process. This approach allows to design and/or selection of better drug candidates that have more potentials to be marketed drugs [61]. Therefore, the MTT cell viability assay, which is suggested for cytotoxicity screening of drug candidates by ISO (10993-5, 2009) was performed [62]. Cytotoxicities of selected compounds (5m, 5o, 5r, 5w, 5y, 5ab, and 5ad), displaying strong anticandidal activity and good predicted pharmacokinetics, were determined against NIH/3T3 mouse embryonic fibroblast cell lines (ATCC CRL1658).
The cytotoxicity results of the tested compounds are presented in Table 4. IC 50 of compounds 5w (65.28 µg/mL) and 5ad (119.55 µg/mL) against NIH/3T3 was very higher than their MIC 50 values (0.78-1.56 µg/mL) against Candida strains. These findings show that the antifungal activity of the compounds 5w and 5ad is not due to general toxicity, but can be ascribed to their selective action against Candida species. Thus, cytotoxicity test findings enhanced the importance of compounds 5w and 5ad as anticandidal drug candidates.

Inhibition of Ergosterol Biosynthesis
The most of therapies, planned to treat fungal infections, target in the ergosterol biosynthesis pathway or its end product, ergosterol, a membrane sterol that is unique to fungi. It is the main sterol, and thus is essential for growth and normal membrane function of fungal cell. Besides serving as a bioregulator of membrane fluidity, asymmetry and integrity, it contributes to proper function of membrane-bound enzymes [63]. Hence, we applied an LC-MS-MS (Shimadzu LCMS 8040, Kyoto, Japan) method for quantitative determination of ergosterol content of C. albicans. Total intracellular sterols were extracted as reported by Breivik and Owades [64]. Exhibiting stronger anticandidal activities, lower cytotoxicities and better predicted profiles of pharmacokinetics, compounds 5w and 5ad, reference agents fluconazole and ketoconazole were used in the assay at MI 0.78 µg/mL, 1.56 µg/mL, and 3.12 µg/mL concentrations, respectively. Ergosterol standard (Product No. 45480, Sigma-Aldrich, Darmstadt, Germany) was used for quantification of ergosterol in both inhibitor-free (negative control) and inhibitor including samples. Ergosterol quantity in negative control samples was regarded as 100%. All concentrations were analyzed in quadruplicate, and the results were expressed as mean ± standard deviation (SD) (Figure 2).
Results showed that the decline in ergosterol levels after treatment with compounds 5w and 5ad was noticeable compared to the reference agents. Compounds 5w, 5ad and reference agents significantly decreased the level of ergosterol at all tested concentrations. Thus, it can be clearly demonstrated that compounds 5w and 5ad have a role in ergosterol biosynthesis pathway. (0.78-1.56 µg/mL) against Candida strains. These findings show that the antifungal activity of the compounds 5w and 5ad is not due to general toxicity, but can be ascribed to their selective action against Candida species. Thus, cytotoxicity test findings enhanced the importance of compounds 5w and 5ad as anticandidal drug candidates. 11.14 ± 0.57 5ab 43.13 ± 2.88 5ad 119.55 ± 4.39

Inhibition of Ergosterol Biosynthesis
The most of therapies, planned to treat fungal infections, target in the ergosterol biosynthesis pathway or its end product, ergosterol, a membrane sterol that is unique to fungi. It is the main sterol, and thus is essential for growth and normal membrane function of fungal cell. Besides serving as a bioregulator of membrane fluidity, asymmetry and integrity, it contributes to proper function of membrane-bound enzymes [63]. Hence, we applied an LC-MS-MS (Shimadzu LCMS 8040, Kyoto, Japan) method for quantitative determination of ergosterol content of C. albicans. Total intracellular sterols were extracted as reported by Breivik and Owades [64]. Exhibiting stronger anticandidal activities, lower cytotoxicities and better predicted profiles of pharmacokinetics, compounds 5w and 5ad, reference agents fluconazole and ketoconazole were used in the assay at MI 0.78 µg/mL, 1.56 µg/mL, and 3.12 µg/mL concentrations, respectively. Ergosterol standard (Product No. 45480, Sigma-Aldrich, Darmstadt, Germany) was used for quantification of ergosterol in both inhibitor-free (negative control) and inhibitor including samples. Ergosterol quantity in negative control samples was regarded as 100%. All concentrations were analyzed in quadruplicate, and the results were expressed as mean ± standard deviation (SD) (Figure 2).
Results showed that the decline in ergosterol levels after treatment with compounds 5w and 5ad was noticeable compared to the reference agents. Compounds 5w, 5ad and reference agents significantly decreased the level of ergosterol at all tested concentrations. Thus, it can be clearly demonstrated that compounds 5w and 5ad have a role in ergosterol biosynthesis pathway.

Fluorescence Microscopy
Ergosterol is the major fungal membrane sterol that regulates membrane fluidity, plasma membrane biogenesis and functions [65]. Hence, the inhibition of ergosterol biosynthesis causes a damage in cell membrane of fungi. To visualize effect of compound 5w, which possesses the strong anticandidal activity, a low cytotoxicity and a good predicted pharmacokinetics profile, against C. albicans at cellular level, a fluorescence microscopy study was performed. Figure 3 shows the cells treated with 5w and not treated with a compound. Green cells in the photomicrograph represent untreated cells, whereas orange cells depict treated ones with 3.12 µg/mL (4 × MIC 50 ) concentration of 5w. It is known that propidium iodide (PI) enters only cells with damaged membranes, while SYTO-9 marks cells with both damaged and unaffected membranes [66]. Thus, Figure 3 reveals membrane damaging effect of 5w due to penetration of PI through cell membrane. Consequently, fluorescence microscopic experiments supported the findings observed in inhibition of biosynthesis of ergosterol, which is a vital sterol regulating membrane functions.

Fluorescence Microscopy
Ergosterol is the major fungal membrane sterol that regulates membrane fluidity, plasma membrane biogenesis and functions [65]. Hence, the inhibition of ergosterol biosynthesis causes a damage in cell membrane of fungi. To visualize effect of compound 5w, which possesses the strong anticandidal activity, a low cytotoxicity and a good predicted pharmacokinetics profile, against C. albicans at cellular level, a fluorescence microscopy study was performed. Figure 3 shows the cells treated with 5w and not treated with a compound. Green cells in the photomicrograph represent untreated cells, whereas orange cells depict treated ones with 3.12 µg/mL (4 × MIC50) concentration of 5w. It is known that propidium iodide (PI) enters only cells with damaged membranes, while SYTO-9 marks cells with both damaged and unaffected membranes [66]. Thus, Figure 3 reveals membrane damaging effect of 5w due to penetration of PI through cell membrane. Consequently, fluorescence microscopic experiments supported the findings observed in inhibition of biosynthesis of ergosterol, which is a vital sterol regulating membrane functions.

Chemistry
Entire chemicals used in the syntheses were purchased from Sigma-Aldrich Chemicals Methyl-4-formyl benzoate (4.8 g, 0.030 mol), sodium disulfite (5.70 g, 0.030 mol) and DMF (5 mL) were added into a vial (30 mL) of microwave synthesis reactor (Anton-Paar, Monowave 300, Graz, Austria). The reaction mixture, was heated under conditions of 240 °C and 10 bar for 5 min. The mixture was cooled down, 5-substituted-1,2-phenylenediamine (0.028 mol) was added and then reaction mixture was kept under the same reaction conditions in microwave reactor. After cooling, the mixture was poured into iced-water, precipitated product was washed with water, dried and recrystallized from ethanol [53,54].

Chemistry
Entire chemicals used in the syntheses were purchased from Sigma-Aldrich Chemicals (Sigma-Aldrich Corp., St. Louis, MO, USA) or Merck Chemicals (Merck KGaA, Darmstadt, Germany). Melting points of the synthesized compounds were determined by MP90 digital melting point apparatus (Mettler Toledo, OH, USA) and were uncorrected. 1 H-NMR and 13 C-NMR spectra were recorded by a Bruker 300 MHz and 75 MHz digital FT-NMR spectrometer (Bruker Bioscience, Billerica, MA, USA) in DMSO-d 6 , respectively. In the NMR spectra splitting patterns were designated as follows: s: singlet; d: doublet; t: triplet; m: multiplet. Coupling constants (J) were reported as Hertz. The IR spectra were obtained on a Shimadzu, IR Prestige-21 (Shimadzu, Tokyo, Japan). LC-MS-MS studies were performed on a Shimadzu, 8040 LC-MS-MS spectrophotometer (Shimadzu, Tokyo, Japan). The purities of compounds were checked by TLC on silica gel 60 F254 (Merck KGaA, Darmstadt, Germany).

Cytotoxicity Assay
Cytotoxicity was tested using the NIH/3T3 mouse embryonic fibroblast cell line (ATCC ® CRL-1658™, London, UK). NIH/3T3 cells were incubated according to the supplier's recommendations and they were seeded at 1 × 10 4 cells into each well of 96-well plates. MTT assay was performed as previously described [68,69]. The compounds were tested in a concentration range of 800 µg/mL and 0.78 µg/mL concentrations. Percent inhibition was calculated for each concentration according to the formula below, and IC 50 values were determined by plotting a concentration-response curve of inhibition percent versus compound concentrations tested [70].

Quantification of Ergosterol Level
Total intracellular sterols were extracted as reported by Breivik and Owades [64] with slight modifications. Briefly, a single C. albicans colony from an overnight Sabouraud dextrose agar plate culture was used to inoculate 50 mL of Sabouraud dextrose broth (Difco) containing 0, 0.78, 1.56, and 3.12 µg/mL of compounds 5w, 5ad, fluconazole, and ketoconazole. The cultures were incubated for 16 h with shaking at 35 • C. The stationary-phase cells were harvested by centrifugation at 2700 rpm (Hettich, Rotina 380 R, Tuttlingen, Germany) for 5 min and washed once with sterile distilled water. Three milliliters of 25% alcoholic potassium hydroxide solution was added to each pellet and vortex mixed for 1 min. Cell suspensions were transferred to sterile borosilicate glass screw-cap tubes and were incubated in an 85 • C water bath for 1 h. Following incubation, tubes were allowed to cool to room temperature. Sterols were then extracted by addition of a mixture of 1 mL of sterile distilled water and 3 mL of chloroform followed by vigorous vortex mixing for 3 min. The chloroform layer was transferred to a clean borosilicate glass screw-cap tube and 1 µL of sterol extract was injected to LCMSMS system (Shimadzu LCMS 8040, Kyoto, Japan). The mass spectrometric analysis were achieved by employing the Nexera XR UFLC system coupled to an LCMS-8040 tandem quadrupole mass spectrometer (Shimadzu, Kyoto, Japan). Labsolutions LCMS software (Version 5.86, Shimadzu) was used to control the instruments and process the data. The Nexera UFLC system used in the analysis consisted of two pumps (LC-20ADxr), an autosampler (SIL-20ADxr), a column heater (CTO-10ASvp), and a degasser (DGU-20A5R). This instrument was equipped with ESI sources. Chromatographic separation was performed using a Shimadzu Shimpack FC-ODS C18 column (150 mm × 2.0 mm, 3 µm) at a flow rate of 0.25 mL/min in ESI source. The isocratic mobile phase consisted of acetonitrile-water 0.1% formic acid (50:50, v/v). The mass spectrometer operating parameters were optimized as follows: nebulizer gas flow, 3 L/min; drying gas flow, 15 L/min; desolvation line (DL) temperature, 250 • C; heat block temperature, 400 • C in ESI source. Other parameters were tuned automatically. MRM method was optimized using ergosterol standard stock solution with concentration of 20 µg/mL. Ergosterol quantity in negative control samples was regarded as 100%. All concentrations were analyzed in quadruplicate, and the results were expressed as mean ± standard deviation (SD).

Fluorescence Microscopy
The Live/Dead BacLight viability kit from Molecular Probes, Inc. (Eugene, OR, USA) was used as reported by Nobman et al. [65]. In this assay, the SYTO-9 and PI stains are in competition to bind to fungal nucleic acid. PI enters only cells with damaged membranes, while SYTO-9 marks cells with both damaged and unaffected membranes. A culture of C. albicans was grownup to mid-log phase in 20 mL of SDB. Twenty milliliters of the yeast culture was divided into two equal parts. Initial culture were incubated for 2 h with 3.12 µg/mL (4 × MIC 50 ) of compound 5w. The second culture was incubated under same conditions without a compound. Both cultures were centrifuged at 3000 rpm for 10 min. and then cell pellets were incubated with PI and SYTO9 at 40 • C for 30 min. The stained yeast cultures were observed under a fluorescence microscope (Carl-Zeis, Axio Scope.A1, Göttingen, Germany) using 100× oil-immersion objective [71].

Conclusions
In summary, preliminary evaluation of new 2-((5-(4-(5-substituted-1H-benzimidazol-2-yl)phenyl)-4-substituted-4H-1,2,4-triazol-3-yl)thio)-1-(substitutedphenyl)ethan-1-one derivatives as antifungal agents result in promising findings. Compounds 5w and 5ad exerted a good antifungal profile. Furthermore, toxicological and ADME studies indicated the relative potency of compounds 5w and 5ad. Results of ergosterol level quantification assay and fluorescence microscopy studies revealed that the mechanism of action of compounds is associated with the inhibition of ergosterol biosynthesis which may subsequently results in altered membrane fluidity, plasma membrane biogenesis and functions of fungi. Consequently, all these data may pave the way for researchers to synthesize similar compounds possessing enhanced antimicrobial profile.
Supplementary Materials: Supplementary materials are available online. The spectra of compounds 5w and 5ad are available online.