Synthesis and Anticandidal Activity of New Imidazole-Chalcones

In the present work, 15 new 1-(4-(1H-imidazol-1-yl)phenyl)-3-(4-substituedphenyl)prop-2-en-1-one derivatives (3a–3o) were synthesized to evaluate their antifungal activity. Structures of newly synthesized imidazole derivatives (3a–3o) were characterized by IR, 1H-NMR, 13C-NMR, and LCMSMS spectroscopic methods. The anticandidal activity of compounds (3a–3o) against C. albicans (ATCC 24433), C. krusei (ATCC 6258), C. parapsilosis (ATCC 22019), and C. glabrata (ATCC 90030) was elucidated according to the EUCAST definitive (EDef 7.1) method. Consistent with the activity studies, 3a–3d were found to be more potent derivatives with their MIC50 values (0.78 µg/mL–3.125 µg/mL) against Candida strains. Compound 3c indicated similar antifungal activity to ketoconazole against all Candida species and was evaluated as the most active derivative in the series. Effects of the most potent derivatives 3a–3d on ergosterol biosynthesis were observed by LC-MS-MS method, which is based on quantification of the ergosterol level in C. krusei. Moreover, these compounds were subjected to a cytotoxicity test for the preliminary toxicological profiles and were found as non-cytotoxic. Furthermore, docking studies for the most active derivative 3c were performed to evaluate its binding modes on lanosterol 14-α-demethylase. In addition to in vitro tests, docking studies also revealed that Compound 3c is a potential ergosterol biosynthesis inhibitor.


Introduction
During the last few years, there has been an increased awareness of morbidity and mortality related to invasive and systemic fungal disease due to resistant fungi and immunocompromised infections such as AIDS.Candidiasis, aspergillosis, and cryptococcosis represent the three most common invasive fungal infections and have the highest mortality rates [1][2][3].Cryptococcus, Trichosporon, Geotrichum, and Rhodotorula species also lead to diverse occurrence and restrict clinical therapy owing to the fungus variety.However, Candida species have been the primary reason of fungal infections [4,5].Candida spp.are presently the third-to-fourth leading reason of bloodstream infections in the USA [6].Candida is generally part of the normal flora of the mouth, vagina, skin, and intestinal tract and can exist in the oral cavity in 40-60% of the population without causing any problems [7,8].If the environment of normal flora changes in a way that encourages fungal growth, Candida can increase and cause infections.Currently, various antifungal drugs have been used clinically in an attempt to reduce effects of fungal infections.
The "azoles" are class of antifungal agents whose molecules are based on a pharmacophore that inhibits the activity of fungal cytochrome P45014DM (also known as lanosterol 14-α-demethylase, Erg11p, Cyp51p, and Erg16p).Azoles are administered against lanosterol 14-α-demethylase in the ergosterol pathway.Antifungal azoles bind with nitrogen atoms to iron atoms of the heme group in the target protein and block fungal membrane ergosterol biosynthesis by inhibiting the demethylation of lanosterol to ergosterol and altering the fungal membrane structure and function [9][10][11].The variability of azole nuclei is demonstrated in their present applications as pharmaceutical drugs used in the cure of bacterial, viral, and fungal infections, worm infestations, acid reflux, cancer, inflammations, and diabetes [12].Imidazoles, the first group to be developed in azole antifungals also block the accumulation of methylated sterols, and disrupt the ergosterol biosynthesis, which is an essential component of the fungal cell wall.Moreover, some imidazole drugs, at high concentrations, could display a direct inhibitory effect on membranes, without intervene in sterols and sterol esters [13][14][15].Ketoconazole, miconazole, and clotrimazole are some common drugs used for the treatment of patients affected by different Candida species [16][17][18].Chemical structures of these antifungal agents are summarized in Figure 1 along with compounds targeted in the present study.
In other respects, it is well known that chalcones (1,3-diaryl-2-propen-1-one) plays an important role for anticandidal activity [19][20][21][22].Their anticandidal action has been mainly related to the reactive enone moiety.As a Michael reaction acceptor, the enone unit binds thiol groups of certain proteins.Hence, most chalcones inhibit biosynthesis of the fungal cell wall and thus clarify their antifungal potential [23].
Knowledge of antifungal activities in both functional groups (azole and chalcone) has prompted curiosity about the antifungal effects of compounds containing these two groups.Therefore, within the scope of this study, a series of new imidazole-chalcone derivatives were synthesized and evaluated for their antifungal activities.
Consistent with the activity studies, Compounds 3a-3d, were found to be more potent derivatives with their MIC 50 values (0.78-3.125 µg/mL) against Candida strains.Compounds 3a-3c showed higher antifungal activity against C. krusei with an MIC 50 value of 0.78 µg/mL compared with standard drugs.Furthermore, MIC 50 value of 0.78 µg/mL was recorded for Compounds 3c and 3d against C. glabrata.Compound 3c indicated similar antifungal activity to ketoconazole and fluconazole against all Candida species and was evaluated as the most active derivative in the series.
Antimicrobial activity results clearly indicated that variable groups at the 4th position of the phenyl moiety have an essential impact on antifungal activity.It was observed that the presence of more liphophilic phenoxy and phenylthio groups in Compounds 3a-3d have significantly enhanced anticandidal activity when compared with the compounds that carry cyclic secondary amines.It is known that lipophilicity is a key property that influences the ability of a drug to reach the target by transmembrane diffusion and to have a major effect on the biological activity [26].Besides, augmented electron density may be another reason for better anticandidal activity due to the presence of electronically rich aromatic rings in Compounds 3a-3d.Thus, it may be suggested that increased lipophilic and/or electronic characters of Compounds 3a-3d caused an increase in anticandidal activity.

Quantification of the Ergosterol Level
Sterols are neutral lipids of eukaryotic cells, among which ergosterol is the main constituent of fungal membranes.Ergosterol has biological functions such as membrane fluidity, regulation, activity and distribution of integral membrane proteins, and control of the cell cycle [27,28].The critical role of sterols in maintenance of cell membranes make ergosterol and its biosynthetic pathway essential for fungal growth, and a primary target for antifungal drugs to treat fungal infections [6].From this point of view, we performed an LCMSMS (Shimadzu LCMS 8040, Kyoto, Japan) method for quantitative determination of ergosterol content of C. krusei as reported in our recent study [29].Compounds 3a-3d, displaying the best anticandidal activity and low cytotoxic effect, and reference agents ketoconazole and fluconazole were tested at concentrations of 0.78-3.12µg/mL.Ergosterol standard (Product No.: 45480, Sigma-Aldrich, Darmstadt, Germany) was used for the quantification of ergostrerol in samples, which were treated with reference agents and Compounds 3a-3d.Ergosterol quantity of the negative control was considered as 100%.Quadruplicate analyses were performed for all concentrations and the obtained data were expressed as mean ± standard deviation (SD) (Table 2).
According to obtained results, the decrease in ergosterol level after administration of Compounds 3a-3d are noticeable when compared to the reference agents.Compounds 3a-3d and reference agents significantly decreased the level of ergosterol at all tested concentrations.Therefore, it can be interpreted that Compounds 3a-3d play a role in the ergosterol biosynthesis pathway.

Cytotoxicity Test
The main cause of failure in all stages of the new drug development process is toxicity.Early identification of toxicities of drug candidates is very important in terms of drug development studies [30].Therefore, the MTT cell viability assay, which is suggested for cytotoxicity screening of drug candidates by ISO (10993-5, 2009) was performed [31].Cytotoxicity of selected compounds (3a-3d), displaying strong anticandidal activity and good predicted pharmacokinetics, was determined against NIH/3T3 mouse embryonic fibroblast cell lines (ATCC CRL1658).
The cytotoxicity results of the tested compounds are presented in Table 2. IC 50 of compounds against NIH/3T3 was much higher than their MIC 50 values (0.78-3.125 µg/mL) against Candida strains.This finding shows that the antifungal activity of Compounds 3a-3d 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 3a-3d as anticandidal drug candidates.

Molecular Docking Studies
Docking studies were performed in order to gain more insight into the binding mode of the most active compound 3c to lanosterol 14-α-demethylase.Lanosterol 14-α-demethylase from Mycobacterium tuberculosis has high homology compared with lanosterol 14-α-demethylase from Candida species.It has been reported that these two enzymes have a high degree of similarity between the hydrophobic cavities of the catalytic site [29,32].Therefore, docking studies were carried out using X-ray crystal structure of lanosterol 14-α-demethylase from Mycobacterium tuberculosis in complex with fluconazole (PDB ID: 1EA1) [33] obtained from the Protein Data Bank server (www.pdb.org).
As stated in the antifungal activity section, Compound 3c was found to be the most active derivative against C. albicans, C. glabrata, C. krusei, and C. parapsilosis with 1.56, 0.78, 0.78, and 0.78 µg/mL MIC values, respectively.Thus, the main purpose of docking studies was to investigate the possible interaction of this compound with lanosterol 14-α-demethylase enzyme.
The docking pose on lanesterol 14-α-sterol demethylase reveals that the interactions between Compound 3c and HEM450 are very important in terms of binding to the active site of the enzyme (Figure 2).Imidazole and its neighboring phenyl established two π-π interactions.The other two π-π interactions were formed by other phenyl rings of the structure.Phenyl of benzylidene was in interaction with Arg96, while phenyl substituted with methoxy group created a π-π interaction with the phenyl of Phe78.Additionally, the docking pose showed that there were three hydrogen bonds providing polar interactions.Carbonyl of Compound 3c established a hydrogen bond with the amino of Val395.Oxygen atoms of phenoxy created this interaction with the amino of Met79, whereas oxygen atoms of the methoxy group formed with the amino of Ile323.These interactions explain the stronger anticandidal activity of 3c.It could be that C-4 of phenyl is very important in terms of binding to the enzyme active site and anticandidal activity.As a result, this additional interaction could explain the greater binding capability and stronger activity of Compound 3c compared with other compounds.

Chemistry
All chemicals used in the syntheses were purchased either 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 measured by MP90 digital melting point apparatus (Mettler Toledo, Columbus, OH, USA) and were presented as uncorrected. 1H-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 of the compounds were recorded using an IRAffinity-1S Fourier transform IR (FTIR) spectrometer (Shimadzu, Tokyo, Japan).LC-MS-MS studies were performed on a Schimadzu, 8040 LCMSMS spectrophotometer (Shimadzu, Tokyo, Japan).The purities of compounds were checked by TLC on silica gel 60 F254 (Merck KGaA, Darmstadt, Germany).

Synthesis of Four Substituted Benzaldehydes (2a-2o)
A mixture of 4-fluoro benzaldehyde (0.259 mL, 0.002 mol), corresponding phenol, thiophenol, or amine (0.002 mol), and a catalytic quantity of potassium carbonate (K 2 CO 3 ) was refluxed in DMF (20 mL) for 36 h.After completion of the reaction, the mixture was poured into ice water (50 mL), and the precipitated product was filtered, washed with deionized water, dried, and recrystallized from ethanol.

Antifungal Activity
Microbiological studies were performed according to the EUCAST definitive (EDef 7.1) method [34] for C. albicans (ATCC 24433), C. krusei (ATCC 6258), C. parapsilosis (ATCC 22019), and C. glabrata (ATCC 90030).Fluconazole and ketoconazole were used as control drugs.Two MIC readings were carried out for each chemical agent.The yeasts were maintained in RPMI after overnight incubation at 37 • C. The inocula of test microorganisms was adjusted to match the turbidity of a MacFarland 0.5 standard tube as determined with a spectrophotometer, and the final inoculum size was 0.5-2.5 × 10 5 cfu/mL for the antifungal assay.Testing was carried out in RPMI at pH = 7, and the two-fold serial dilutions technique was applied.The last well on the microplates containing only inoculated broth was kept as a control, and the last well with no growth of microorganism was recorded to represent the MIC 50 expressed in µg/mL.For the antifungal assays, the compounds were

Figure 2 .
Figure 2. The interacting mode of Compound 3c in the active region of 14-alpha-sterol demethylase.The inhibitor is colored with purple and HEM with grey.