Ultrasound- and Molecular Sieves-Assisted Synthesis, Molecular Docking and Antifungal Evaluation of 5-(4-(Benzyloxy)-substituted phenyl)-3-((phenylamino)methyl)-1,3,4-oxadiazole-2(3H)-thiones

A novel series of 5-(4-(benzyloxy)substituted phenyl)-3-((phenyl amino)methyl)-1,3,4-oxadiazole-2(3H)-thione Mannich bases 6a–o were synthesized in good yield from the key compound 5-(4-(benzyloxy)phenyl)-1,3,4-oxadiazole-2(3H)-thione by aminomethylation with paraformaldehyde and substituted amines using molecular sieves and sonication as green chemistry tools. The antifungal activity of the new products was evaluated against seven human pathogenic fungal strains, namely, Candida albicans ATCC 24433, Candida albicans ATCC 10231, Candida glabrata NCYC 388, Cryptococcus neoformans ATCC 34664, Cryptococcus neoformans PRL 518, Aspergillus fumigatus NCIM 902 and Aspergillus niger ATCC 10578. The synthesized compounds 6d, 6f, 6g, 6h and 6j exhibited promising antifungal activity against the tested fungal pathogens. In molecular docking studies, derivatives 6c, 6f and 6i showed good binding at the active site of C. albicans cytochrome P450 enzyme lanosterol 14 α-demethylase. The in vitro antifungal activity results and docking studies indicated that the synthesized compounds have potential antifungal activity and can be further optimized as privileged scaffolds to design and develop potent antifungal drugs.


Introduction
Mortality due to secondary fungal infections in immunocompromised patients such as patients with AIDS, autoimmune diseases, burns, radiotherapy or chemotherapy is becoming a serious problem worldwide, with 1.5 to 2 million deaths every year [1].Most of the mortality is caused by species belonging to four genera, namely Candida, Aspergillus, Cryptococcus and Pneumocystis.Emerging fungal pathogens and development of resistance in pathogenic fungi to currently used antifungal drugs has further aggravated the situation [2,3].Currently, a limited repertoire of drugs from five classes, namely polyenes, azoles, echinocandins, allylamines and fluoropyrimidines are used for the treatment of fungal infections.Apart from drug resistance in pathogens, drawbacks of existing drugs such as acute and chronic side-effects, less clinical efficiency, and effects on non-targeted cells has forced researchers around the world to search for novel and efficient antifungal drugs [2].
Ultrasound-assisted organic synthesis is a green synthetic approach and a powerful technique for the enhancement of reaction rates and yields [24,25].Ultrasonic irradiation is enhanced due to the formation of high energy intermediates.It offers the possibility of accomplishing potential reactions in short time cycles with cheaper reagents and less extreme physical conditions [26,27].It can also be considered as an important tool for conservation of energy and minimization of waste as compared to the conventional techniques [28].Microporous 3Å molecular sieves are aluminosilicate minerals with chemical composition of 2/3K 2 O¨1/3Na 2 O¨Al 2 O 3 ¨2SiO 2 ¨9/2H 2 O. Since the 1990s, these molecular sieves have attracted considerable attention due to their potential use in catalysis [29].Mannich reactions by conventional methods take 15 to 20 h for the synthesis of the same derivatives, whereas using ultrasound the reaction time is reduced to 1 to 2 h.In the present study we report the use of ultrasound-and molecular sieves-assisted Mannich reactions for the synthesis of 5-substituted 1,3,4-oxadiazole-2-thiones with primary and secondary amines.Molecular docking of the synthesized compounds and screening for antifungal potential against different yeast and filamentous fungal pathogens is also reported.

Chemistry
All the final compounds 6a-o were synthesized following the procedure depicted in Scheme 1.The starting material methyl 4-(benzyloxy) benzoate (3) was synthesized by the reaction of methyl 4-hydroxybenzoate (2) and benzyl chloride(1) in K 2 CO 3 and DMF as solvent in an ultrasonic processor for up to 4 h.Synthesis by a conventional method like stirring at room temperature required 20-28 h and by reflux nearly 8-10 h.The compound 3 obtained in good yield in step I is next refluxed with NH 2 NH 2 to get 4-(benzyloxy) benzohydrazide (4).The reaction of the acid hydrazide 4 with carbon disulphide under basic conditions using KOH yielded 5-(4-(benzyloxy)phenyl)-1,3,4-oxadiazole-2(3H)-thione 5 [30].N-Amino-methylation of (5) with various substituted primary amines and secondary amines and paraformaldehyde in presence of activated molecular sieves in ultrasonic processor under the Mannich reaction gave 6a-o with good yields within 1-2 h (      The structure of intermediate 5-(4-(benzyloxy)phenyl)-1,3,4-oxadiazole-2(3H)-thione (5) was confirmed by spectroscopic analysis.The IR spectrum showed bands at 3235 (NH), 1621 (C=N), 1596 (C-C), 1425 (C=S), 1258 and 1093 cm ´1 (C-O-C).The 1 H-NMR spectrum displayed a singlet at 3.8 ppm, assigned to the NH/SH tautomeric proton, a singlet at 5.17 ppm, integrating to 2H and assigned to the C-CH 2 -O group and a 9H multiplet at 7.0-7.9for the two aromatic rings.In the 13 C-NMR the -C=S carbon appeared at δ 189.9, along with the characteristic signals of the remaining carbon atoms.The HRMS mass spectrum showed (M + 1) molecular ion peak at m/z 285, in agreement with its molecular formula, C 15 H 12 O 2 N 2 S.
In the IR spectrum of compound 6j, the aromatic C-H stretching vibration was observed at 3047 cm ´1.The absorption band due to the CH 2 groups of the morpholine moiety was seen at 2916/2850 cm ´1.The C=N and C=S moieties showed their characteristic absorption bands at 1681 and 1356 cm ´1, respectively.In 1 H-NMR spectrum, the eight protons of the morpholine moiety resonated as two triplets at δ 2.75 and δ 3.62, while two singlet were seen at δ 5.02 and δ 5.16 for the N-CH 2 -N and -OCH 2 protons, along with a multiplet at δ 7.06-8.02for the nine aromatic ring protons.In the 13 C-NMR spectrum, the signals observed at δ 67.91 and 70.12 were assigned to C2, C6 and C3, C5 of the morpholine ring, respectively.The signal due to the aryloxy methylene carbon appeared at δ 68.23.The aryloxymethine carbon atom resonated at δ 156.69, and the C=S carbon appeared at 177.14, while other aromatic carbon atoms appeared at δ 115.47, 119.51, 125.68, 128.17, 128.43, 128.61, 129.00, 131.97 and 135.64.Further, the structure of 6j was also confirmed by recording its mass spectrum, which showed a molecular ion peak at m/z 383, in agreement with the assigned molecular formula, C 20 H 21 N 3 O 3 S.

In Vitro Antifungal Activity
The newly synthesized compounds 6a-o were screened for in vitro antifungal activity against different yeast and filamentous fungal pathogens and were found to be fungistatic.All the compounds exhibited moderate antifungal activity against the tested yeast pathogens, viz.Candida and Cryptococcus strains, as compared to the standard fluconazole, the results are exhibited in Table 2. Derivatives 6i and 6e exhibited better antifungal activity than fluconazole against the filamentous Aspergillus fumigatus and Aspergillus niger, respectively.The antifungal activity exhibited by these compounds may be mainly due to the presence of various electron withdrawing groups such as bromine at the phenyl para-position for 6g (4 µg/mL), imidazole for 6d (6 µg/mL), morpholine for 6j (8 µg/mL), and triazole in case of 6c (11.2 µg/mL).Compound 6f (5.4 µg/mL) with ortho and para-disubstituted toluyl and 6e (24.5 µg/mL) showed good activity against Candida glabrata NCYC 388.The antifungal susceptibility testing results indicated that 5-(4-(benzyloxy) substituted phenyl)-3-((phenylamino)methyl)-1,3,4-oxadiazole-2(3H)-thione derivatives can act as a good scaffold to develop lead molecules with potent antifungal activity.The final derivatives possessing electron withdrawing groups on the phenyl ring at position 3 of the nitrogen of the 1,3,4-oxadiazole-2-thione ring acted as more potent compounds and exhibited excellent antifungal activity.

Molecular Docking Study
The azole class of antifungal drugs inhibits the cytochrome P450 lanosterol 14α-demethylase enzyme in the ergosterol synthesis pathway.As ergosterol is the major sterol component of the fungal cell membrane, inhibition of this enzyme and subsequent ergosterol depletion by the compounds leads to loss in fungal cell integrity and function [29].Therefore, we carried out molecular docking of our oxadiazole thiones 6a-o and the standard drug fluconazole into the active site of cytochrome P450 lanosterol 14α-demethylase of Candida albicans, using the VLife MDS 4.3 software, to understand the binding interactions.The binding energy and hydrogen bond interactions are presented in Table 3.The most active synthesized compounds 6c, 6f and 6i showed the lowest interaction energy, i.e., ´57.85, ´65-25 and ´61.03 kcal/mol, respectively.The standard drug fluconazole also showed good interaction energy (´67.29 kcal/mol).The docking results indicated that compounds were held in the active pocket by combination of various hydrogen and hydrophobic interactions with cytochrome P450 lanosterol 14α-demethylase.Diverse hydrophobic interactions occurred between the synthesized compounds and the active site chains of LEU186, LEU240, LEU340, ALA343, GLY344, THR347, LEU412, MET415, VAL440, PRO442, VAL497, PHE499, GLY500, HIS504, CYS506, ILE507, GLY508, GLU509, PHE511, ALA512 and TYR513.The amino acid residues such as TYR154, TYR168, LEU412, MET415, VAL497, GLY500 and CYS506 formed hydrogen bonds with the synthesized compounds.The docking interactions of the most active compounds 6c, 6f and 6i are shown in Figure 1.The amino acid CYS506 formed hydrogen bonding (1.95 Å) with the -O-CH 2 -oxygen of the synthesized compound 6c.The 1,2,4-triazole ring of compound 6c was held in the active site by forming Vander Waal's interactions with amino acid residues like PRO410, LEU412, PRO442, VAL497 and PRO498.The amino acid residue CYS506 (2.40 Å) formed a hydrogen bond with the -NH-nitrogen of compound 6f.The amino acids TYR154 (2.19 Å), TYR168 (2.52 Å) and MET415 (2.82 Å) all formed hydrogen bonds with the 2-NO 2 oxygen, the carbonyl oxygen and the 4-NO 2 nitrogen, respectively, of compound 6i.On the basis of the antifungal activity and docking results, it was found that compounds 6c, 6f and 6i had potential to inhibit cytochrome P450 lanosterol 14α-demethylase of C. albicans.

General Information
All the chemicals used for synthesis were procured from Merck (Mumbai, Maharashtra, India), Sigma (Mumbai), HiMedia (Mumbai) or Qualigens (Mumbai) and used without further purification.The progress of each reaction was monitored by ascending thin layer chromatography (TLC) using pre-coated silica gel F254 aluminum TLC sheets (Merck) and the spots were visualized by UV light and iodine vapors.Elemental analyses (C, H, and N) were done with a FLASHEA 112 Shimadzu' analyzer (Mumbai) and all analyses were consistent (within 0.4%) with theoretical values.Infrared (IR) spectra were recorded on a PS 4000 FTIR (JASCO, Tokyo, Japan) using KBr pellets. 1 H-and 13 C-NMR (200 MHz) spectra were recorded on a ACF 200 spectrometer (Bruker, Billerica, MA, USA) fitted with an Aspect 3000 computer and all the chemical shifts (ppm) were referred to internal TMS for 1H and chloroform-d for 13 C-NMR. 1 H-NMR data are reported in the order of chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; br, broad; br s, broad singlet; m, multiplet and/ or multiple resonance), number of protons.A Micro TOF-Q-II (Bruker Daltonics, Billerica, MA, USA with electron spray ionization (ESI) was used to obtain the HRMS data.For ultrasound irradiation Vibra cell VCX-500 with solid probe was used (Sonics, Newtown, CT, USA).

Synthesis of Methyl-4-(Benzyloxy)benzoate (3)
For the synthesis of methyl-4-(benzyloxy)benzoate, the reaction between equal 0.01 molamounts of methyl-4-hydroxybenzoate and chloromethylbenzene in N,N-dimethylformamide (DMF) as solvent usingK 2 CO 3 as mild base was carried with under an ultrasonic processor at room temperature for up to 4 h.The solution was then poured into ice-water.The solid obtained was filtered and recrystallized from ethanol.Colour: white; m.p. 105 ˝C (100 ˝C reported in chemspider)

Synthesis of 4-(Benzyloxy)benzohydrazide (4)
For the synthesis of the substituted benzohydrazide, a mixture of the corresponding ester (20 mmol), 85% hydrazine hydrate (20 mmol) in ethanol (35 mL) was refluxed for 6 h.After that, the solution was poured into ice-water.The solid was filtered and recrystallized from ethanol.Colour: white; m.p.138 ˝C (140 ˝C reported in chemspider)

Synthesis of 5-(4-(Benzyloxy)phenyl)-1,3,4-oxadiazole-2(3H)-thione
Equimolar quantities of the substituted benzohydrazide (5 mmol) and potassium hydroxide (5 mmol) were dissolved in 95% ethanol (20 mL).The mixture was allowed to stir for several minutes at room temperature and then carbon disulfide (15 mmol) was slowly added dropwise to the reaction system and the mixture was heated to reflux.The residue obtained was dissolved in water (50 mL) and diluted hydrochloric acid was added to adjust the pH value of the solution to 5-6.The precipitate was collected washed with water for several times and dried and recrystallized from ethanol.Colour: white; m.p. 160 ˝C.

General Procedure for the Synthesis of 5-(4-(Benzyloxy)substituted phenyl)-3-((phenylamino)methyl)-1,3,4-oxadiazole-2(3H)-thiones 6a-o
To a methanolic solution of 5-(4-(benzyloxy) phenyl)-1,3,4-oxadiazole 2(3H) thione ( 5), (10 mmol), paraformaldehyde (15 mmol) and a substituted primary/secondary amine (10 mmol) in methanol 25 mL were added with constant stirring.Preactivated molecular sieves (3Å) were also added to the reaction mixture to absorb the water formed.The resulting mixture was subjected to ultrasonic irradiation for 1-2 h at room temperature.The precipitated solids were filtered, washed with ice water.The progress of the reaction was monitored by TLC using 1:4 ethyl acetate-n-hexane as a solvent system.The reaction was quenched with crushed ice and the solid obtained was recrystallized from methanol to yield the title compounds 6(a-o) mentioned in Table 1 (see also Table S1).[31][32][33][34].Briefly, appropriate amount of compounds were dissolved in dimethyl sulfoxide to get 100ˆfinal strength.The stock was then diluted 1:50 in RPMI 1640 medium and 200 µL of this solution was added to the first row of a 96-well microtitre plate.The compounds were serially diluted two-fold in successive wells to get a range of 2-256 µg/mL.Spores of the filamentous fungi (~2 ˆ10 4 spores/mL) and yeast cells freshly grown in YPG broth in logarithmic phase (~2 ˆ10 3 cfu/mL) were suspended in the RPMI 1640 medium and 100 µL from these were inoculated in the wells of the plate.The micro titer plates were incubated for 24-48 h.Growth was checked by visual observation and measuring absorbance at 600 nm using micro titer plate reader.The IC 50 was defined as the concentration exhibiting 50% inhibition of the growth as compared to the growth of control.

Molecular Docking Data
The 3D model structure of cytochrome P450 lanosterol 14α-demethylase of C. albicans was built using homology modeling [3,33].The amino acid sequence of the enzyme was obtained from the Universal Protein Resource (http://www.uniprot.org/,Accession Code: P10613) and sequence homologs obtained from Protein Data Bank (PDB) using Blast search.Based on the Blast search result, we used the crystal structure of human lanosterol 14α-demethylase (CYP51) with azole as a template for homology modeling (PDB ID: 3LD6).The VLifeMDS 4.3 ProModel was used for modeling of the 3D structure of protein based on the amino acid sequences of a close homologue.Alignment of amino acid sequence of CA-CYP51 (P10613) and human CYP51 (3LD6_B) is shown in Figure S1.The Blosum-62 matrix was used with a gap penalty of 1.The model was then energy minimized using the MMFF94 force field [35].Manual inspection was made to ensure the conserved motifs and loops were correctly aligned.The quality of generated C. albicans lanosterol 14α-demethylase model was assessed by using a well validated program like PROCHECK [36].The structural validation was performed by using Ramachandran plot and is shown in Figure 2. The further structural super imposition was performed to know the structural coordinate of target protein and RMSD value was found within standard range of 0.997607 Å.The molecular docking study of the synthesized compounds 6a-o and the standard drugs fluconazole and miconazole were performed against homology built cytochrome P450 lanosterol 14α-demethylase of C. albicans to understand the binding interactions using VLife MDS 4.3 package following standard procedures [37].

Molecular Docking Data
The 3D model structure of cytochrome P450 lanosterol 14α-demethylase of C. albicans was built using homology modeling [3,33].The amino acid sequence of the enzyme was obtained from the Universal Protein Resource (http://www.uniprot.org/,Accession Code: P10613) and sequence homologs were obtained from Protein Data Bank (PDB) using Blast search.Based on the Blast search result, we used the crystal structure of human lanosterol 14α-demethylase (CYP51) with azole as a template for homology modeling (PDB ID: 3LD6).The VLifeMDS 4.3 ProModel was used for modeling of the 3D structure of protein based on the amino acid sequences of a close homologue.Alignment of amino acid sequence of CA-CYP51 (P10613) and human CYP51 (3LD6_B) is shown in Figure S1.The Blosum-62 matrix was used with a gap penalty of 1.The model was then energy minimized using the MMFF94 force field [35].Manual inspection was made to ensure the conserved motifs and loops were correctly aligned.The quality of generated C. albicans lanosterol 14α-demethylase model was assessed by using a well validated program like PROCHECK [36].The structural validation was performed by using Ramachandran plot and is shown in Figure 2. The further structural super imposition was performed to know the structural coordinate of target protein and RMSD value was found within standard range of 0.997607 Å.The molecular docking study of the synthesized compounds 6a-o and the standard drugs fluconazole and miconazole were performed against homology built cytochrome P450 lanosterol 14α-demethylase of C. albicans to understand the binding interactions using VLife MDS 4.3 package following standard procedures [37].

Molecules 2016, 21 , 484 6 of 13 Figure 1 .
Figure 1.Docking of compounds 6c, 6f, 6i and standard fluconazole.Ligands are shown in red color.Hydrogen bonds are shown in green color.Hydrophobic bonds are shown in sky blue color.

Figure 1 .
Figure 1.Docking of compounds 6c, 6f, 6i and standard fluconazole.Ligands are shown in red color.Hydrogen bonds are shown in green color.Hydrophobic bonds are shown in sky blue color.

Table
). Characterization data of the synthesized derivatives is presented in TableS1(Supplementary Materials).

6a-o. Entry Conventional Ultrasonic Irradiation Time (h) Yield (%) Time (h) Yield (%) 6a
. Comparison of reaction kinetics of conventional and ultrasonic irradiation methods for the synthesized compounds
* IC 50 is the concentration exhibiting 50% inhibition of the growth as compared to the growth of control.

Table 3 .
Results of the docking study of the compounds.