Design , Synthesis , and Molecular Docking of 1-( 1-( 4-Chlorophenyl )-2-( phenylsulfonyl ) ethylidene )-2-phenylhydrazine as Potent Nonazole Anticandidal Agent

1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia 2Department of Clinical Microbiology and Immunology, College of Medicine, Mansoura University, Mansoura 35516, Egypt 3Department of Pharmaceutical Chemistry, College of Pharmacy, Egyptian Russian University, P.O. Box 11829, Badr City, Cairo, Egypt 4Stem Cell and Tissue Re-Engineering Program, Research Center, King Faisal Specialist Hospital and Research Center, P.O. Box 3354, MBC 03, Riyadh 11211, Saudi Arabia 5Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622, Egypt


Introduction
In the current medical era, human fungal infections have stood out as an important clinical threat, with serious associated morbidity and mortality [1].The human immunodeficiency virus (HIV) epidemic, stem cell transplantation, and modern progression in the fields of solid organ transplantation coupled with the advent of novel immunosuppressive drugs have collectively resulted in dramatic increase in the incidence and diversity of human fungal infections including those caused by Candida, Aspergillus, and Cryptococcus species [2].
In the last few decades, Candida has emerged as one of the most prevalent fungal pathogens, causing both mucosal candidiasis and invasive candidemia, although it is a part of the microbiota of gastrointestinal and urogenital tracts, skin, and mouth in healthy individuals [3].Many endogenous and exogenous predisposing factors such as immunological disorders, immoderate administration of antimicrobial agents, and prolonged use of invasive catheters may result in impaired Oxiconazole 6 Fluconazole 7 Zinoconazole 9 13 host defence against Candida spp.and, subsequently, Candida become pathogenic and disseminate into the bloodstream to infect different organs [4].
The azole antifungals are the mainstay of therapy usually used to treat different Candida infections such as oropharyngeal and esophageal candidiasis, vulvovaginal candidiasis, candidemia, and disseminated candidiasis [11].Azoles exert antifungal activity through inhibition of the fungal cytochrome P450-dependent 14-lanosterol demethylase through the binding of the N3-atom of the azole moiety to the sixth coordination of heme iron atom of the porphyrin in the substrate binding site of the enzyme [12].Due to the fungistatic action of azoles on Candida spp., cells repetitively exposed to these antifungals adapt to the drug pressure and eventually become azole resistant [13].
On the other hand, azole antifungal agents showed fatal hepatotoxicity [14][15][16].This side effect may be attributable to the imperfect specificity toward fungal enzyme where CYP51 is a member of the cytochrome P450 superfamily, which exists not only in fungi but in mammals [17,18].The limited success of azoles, due to severe resistance and fatal hepatotoxicity, has resulted in an urgent need to give much attention to update and modify drug leads from the point of view of medicinal chemistry and drug design to fulfill safe and more potent antifungals.
Concerning the SAR of azoles antifungals, the azole ring has been evidenced to be one of the most important pharmacophores for the activity; also, it is a key toxicophore for the hepatotoxicity of azole antifungal drugs due to the coordination binding of its nitrogen atom to the iron atom of heme [19,20].All of these findings inspired us to design and prepare novel nonazole lead compound with different structural pharmacophores hoping to obtain potent antifungal agent and separate the antifungal activity from toxicity.
Figure 1 summarized the main structural features of a series of azoles antifungals such as imidazoles 1-6 and triazole drugs 7 and 8 in addition to zinoconazole 9 (Figure 1).In the present study, the well-known azole antifungals [21,22] were chosen as lead compounds for development of new nonazole scaffold.Firstly, the heteroaryl rings were replaced with a phenyl ring.Since the anticandidal activity of hydrazones was extensively reported [23][24][25], we decided to retain the phenylhydrazine moiety of zinoconazole 9.
Finally, in the present work, utilization of sulfone group was performed depending on the reported antimicrobial activity of sulfones [26][27][28] and due to the incorporation of sulfur in some azole antifungals such as sulconazole 4 and butoconazole 5 in addition to the incorporation of oxygen in ether functions of econazole 1, miconazole 2, isoconazole 3, and hydroxyl group of fluconazole 7 and voriconazole 8.

Antifungal Activity.
In this study we used four reference strains Candida albicans ATCC 90029, Candida parapsilosis ATCC 22019, Candida krusei ATCC 14247, and Candida glabrata ATCC 15126.Antifungal activity of the synthesized compounds was determined by the agar well diffusion method [38].
A 1 × 10 6 CFUs/mL yeast suspension was prepared for each isolate in phosphate-buffered saline (PBS) and Mueller-Hinton agar plates were inoculated automatically with a Spiral plater (Autoplate 4000; Spiral Biotech, Inc., Bethesda, MD, USA).Subsequently, equidistant (1 cm diameter) holes were made using sterile cork borer in the agar.Holes were filled with 100 L of the tested compound at concentration (100 mol dissolved in 1 mL DMSO).25 g fluconazole and solvent were included as positive and negative controls, respectively.The plates were incubated for 24 h at 37 ∘ C. Afterwards, the antimicrobial activity of each newly synthesized compound was evaluated by measuring the inhibition zone diameters.The experiment was repeated on three separate occasions and the average zone of inhibition was calculated.

Minimal Inhibitory Concentration.
The microdilution method was performed as described by Irobi et al. [39], with 96-well, round-bottom microtiter plates using RPMI 1640 medium (Life Technologies, New York, NY, USA) and 2% glucose in MOPS buffered to pH 7. Dimethyl sulphoxide (DMSO) was used as solvent for the synthetic compounds, starting with 100 mol concentration of all compounds dissolved in 1 mL DMSO and then reduced by successive twofold dilutions of stock solution using a calibrated micropipette.The final solutions concentrations were 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, and 0.195 mol/mL.The stock solutions of the synthetic compounds were dispersed in the assay medium to obtain appropriate concentrations in wells 1-10 in each row; drug-free medium was dispensed in wells 11 and 12. Fluconazole is used as reference drug.Sabouraud dextrose agar was used to culture yeast strains.The inoculums of microorganisms were prepared from cultures and were adjusted to 0.5 McFarland standard; the suspensions were further diluted in RPMI 1640 medium to yield an inoculum concentration of approximately 10 4 CFU/mL.The inoculated plates were incubated for 48 h at 35 ∘ C. Triplicate tests were performed and the average was taken as final reading.The MIC endpoint was defined as the lowest drug concentration exhibiting 100% inhibition of growth compared with the control well [40].According to CLSI criteria 100% growth inhibition is defined as clear wells.Therefore, the readings at MIC value were similar to the negative control without Candida.The MIC of fluconazole was determined for each species in parallel as a control, and antibiotic-free solvent was included as a negative control.

Molecular Docking.
The crystal structure of cytochrome P450 14-sterol demethylase (Cyp51) from Mycobacterium tuberculosis in complex with fluconazole (PDB 1EA1) was provided from Brookhaven protein data bank (PDB; http://www .rcsb.org/pdb) and loaded to Molegro Virtual Docker (MVD 2013.6.0.0 [win32]) program, fully functional free trial version with time limiting license [41] The nonbonded oxygen atoms of water, present in the crystal structure, was removed.ChemBio3D Ultra 10 [42] was used to draw the 3D structures of different ligands.Ligands were further preoptimized using free version of Marvinsketch 4.1.13from Chemaxon Ltd [43] with MM force field and saved in Tripos mol2 file format.MolDock score functions were used with a 0.3 Å grid resolution.The binding sites were defined to any residues with 10 Å distant from the cocrystallized fluconazole in the complex crystal structure of the enzyme [44].epithelial cell line MCF10A by measurement of annexin-V binding by flow cytometry was performed according to the reported method with minor modification [45].

Minimum Inhibitory Concentration (MIC).
Thereafter, minimum inhibitory concentration (MIC) of the compound 13 is evaluated in vitro using the twofold serial dilution technique.The lowest concentration showing no growth was chosen as the MIC.The results of minimum inhibitory concentration were displayed in Table 2.
The antifungal activity of 13 was compared with that of fluconazole, a standard antifungal drug.Investigations of the antifungal activity against C. krusei and C. parapsilosis indicated that they were the most sensitive species to the influence of the compound 13 with MIC values of 0.195 and 0.39 mol/mL, respectively.Also, compound 13 almost was equipotent as fluconazole against C. glabrata (MIC = 1.56 mol/mL).On the other hand, compound 13 was 2-fold less active than fluconazole against C. albicans with MIC value of 0.39 mol/mL.

Structure Activity Relationship for Compound 13.
From the SAR point of view, the N-phenyl ring and hydrazone function of 13 is necessary for its activity.The activity of compound 13 may be due to the presence of three electron clouds of aryl groups with similar distribution with those of fluconazole (two triazole moieties and benzene ring).

Molecular Docking Study for Compound 13.
Docking study was performed for 13 in order to investigate the possible interactions with cytochrome P450 14-sterol demethylase from Mycobacterium tuberculosis (Mycobacterium P450 DM).The crystallographic structure of the complex between cytochrome P450 14-sterol demethylase from Mycobacterium tuberculosis (Mycobacterium P450 DM) and fluconazole (ID 1EA1) was used for the docking study [46].Figure 2(a) shows the docked reference drug fluconazole in the active site of the enzyme for validation of our docking protocol.Azole ring is positioned almost perpendicular to the porphyrin plane (cofactor) where the nitrogen of azole ring coordinated to the heme iron.The distance between nitrogen atom of azole ring in fluconazole and heme ring was 2.34 Å. Fluconazole revealed a MolDock score of −161.29.In case of compound 13, the oxygen of sulfone moiety is perpendicular to the porphyrin plane heme ion with distance 3.44 Å (Figure 2(b)).The phenyl ring of 13 occupied the hydrophobic region above the heme ring and showed good van der Waals interactions with heme and amino acids Tyr76, Phe78, Phe83, It was observed that compound 13 was oriented in the binding groove of enzyme in such fashion that favors the possibility of - interaction of its three benzene rings with the hydrophobic amino acid residues of the binding site of the enzyme (Figure 3).The latter binding interactions of 13 are the same as that of fluconazole with binding site of the enzyme (Figure 3).In addition, the sulfone moiety in 13 occupies the same position of the hydroxyl group in fluconazole as shown in Figure 3.

Apoptotic Activity Evaluation for Compound 13.
It has been shown that loss of phospholipid asymmetry of the plasma membrane is an early event of apoptosis.The annexin-V binds to negatively charged phospholipids, like phosphatidylserine.During apoptosis, the cells react to annexin-V once chromatin condenses but before the plasma membrane loses its ability to exclude DAPI.Hence, by staining cells with a combination of APC annexin-V and DAPI it is possible to detect nonapoptotic live cells, early apoptotic cells, and late apoptotic or necrotic cells.Apoptotic effect of compound 13 against noncancerous human breast epithelial cell line MCF10A showed no effect between the treated (10 M) and the untreated cells (Figure 4).

Conclusion
In conclusion, 1-(1-(4-chlorophenyl)-2-(phenylsulfonyl)ethylidene)-2-phenylhydrazine (13) was prepared by the reaction of -ketosulfone 12 with phenyl hydrazine and its structure was established under the basis of its spectral data.It showed excellent antifungal activities against Candida fungal species.The molecular modeling results showed a similar binding interaction of 13 and fluconazole in the active site of CYTP-450.The phenylhydrazone moiety plays an important role in the antifungal potentiality of 13.In addition, compound 13 showed no cytotoxicity against noncancerous cell line MCF10A.Annexin-V APC-A Annexin-V APC-A

Scheme 1 :
Scheme 1: Synthetic pathway of the title compound 13.

Figure 4 :
Figure 4: Measurement of annexin-V binding by flow cytometry.Apoptotic effects of compound 13 against noncancerous human breast epithelial cell line MCF10A showed no effect between the treated (10 M) and the untreated cells.