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

molecules Article Ultrasoundand 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 Urja D. Nimbalkar 1, Santosh G. Tupe 2, Julio A. Seijas Vazquez 3, Firoz A. Kalam Khan 4, Jaiprakash N. Sangshetti 4and Anna Pratima G. Nikalje 4,* 1Maulana Azad P. G. and Research Centre, Dr. Rafiq Zakaria Campus, Rauza Baug, Aurangabad 431001, India; [email protected] 2Biochemical Sciences Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pashan, Pune 411008, India; [email protected] 3Departamento de Química Orgánica, Facultad de Ciencias, Universidad of Santiago De Compostela, Alfonso X el Sabio, Lugo 27002, Spain; [email protected] 4Y. B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Rauza Baug, Aurangabad 431001, India; [email protected] (F.A.K.K.); [email protected] (J.N.S.) *Correspondence: [email protected]; Tel.: +91-916-892-9111 Academic Editor: Derek J. McPhee Received: 10 March 2016; Accepted: 1 April 2016; Published: 10 May 2016 Abstract: A novel series of 5-(4-(benzyloxy)substituted phenyl)-3-((phenyl amino)methyl)-1,3,4oxadiazole-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. Keywords: 1,3,4-oxadiazoles; ultrasound; Mannich reaction; molecular sieves; antifungal activity; molecular docking 1. 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 Molecules 2016,21, 484; doi:10.3390/molecules21050484 www.mdpi.com/journal/molecules Molecules 2016,21, 484 2 of 13 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]. Heterocycles containing nitrogen and oxygen atoms are considered an important class of compounds in medicinal chemistry because of their versatile biological applications. The 1,3,4-oxadiazole scaffold is associated with diverse biological activities such as antifungal [ 4 – 6 ] antibacterial [ 7 , 8 ], antimycobacterial [ 9 ], anti-HIV [ 10 ], anti-hepatitis B virus [ 11 ], anticancer [ 12 ], anticonvulsant [ 13 ], anti-inflammatory [ 14 ], anti-malarial [ 15 ] and analgesic effects [ 16 ]. 5-Substituted1,3,4-oxadiazole-2-thiones, which feature an exocylic sulphur at C-2 of a 1,3,4-oxadiazole skeleton represent an important type of compound class in the field of coordination chemistry because of their potential multifunctional donor sites, viz either exocyclic sulfur or endocyclic nitrogen [ 17 ] and known to possess CNS depressant [ 18 ] and tyrosinase inhibition [ 19 ] activity. 1,3,4-Oxadiazole-2-thiones belong to the class of five-membered aromatic N-heterocycles substrates with the N–H reactive center involved in tautomerism with S–H [ 20 ]. The three-component condensation of a compound containing an acidic hydrogen atom with substituted primary or secondary amines and an aldehyde is known as a Mannich reaction and the product of this reaction has reported to be of significance in the preparation of natural and synthetic molecules with a remarkable biological activity. The literature survey on Mannich reaction involving oxadiazole thiones has shown that these compounds undergo either N-aminomethylation or S-aminomethylation depending on the reaction conditions [ 21 ]. The multipurpose usage of the Mannich bases in pharmaceutical chemistry [ 22 , 23 ] prompted us to prepare a new series of 5-(4-(benzyloxy)substitutedphenyl)-3-((phenyl amino)methyl) -1,3,4-oxadiazole-2(3H)-thione based aminomethyl derivatives. 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 ultrasoundand 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. 2. Results and Discussion 2.1. 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 (Table 1). Characterization data of the synthesized derivatives is presented in Table S1 (Supplementary Materials). Molecules 2016,21, 484 3 of 13 Molecules 2016, 21, 484 3 of 13 R:6a ;6b ;6c ;6d ;6e ;6f ;6g ;6h ; 6i ;6j ;6k ;6l ;6m ;6n ;6o Scheme 1. Synthesis of Mannich bases 6a–o. Table 1. Comparison of reaction kinetics of conventional and ultrasonic irradiation methods for the synthesized compounds 6a–o. Entry Conventional Ultrasonic Irradiation Time (h) Yield (%) Time (h) Yield (%) 6a 15 68 1.5 80 6b 16 63 1.0 81 6c 15 62 1.5 78 6d 17 58 2.0 79 6e 18 45 1.0 81 6f 18 56 1.0 79 6g 19 57 2.0 81 6h 17 61 1.5 90 6i 18 53 1.0 82 6j 17 63 1.5 85 6k 15 58 1.5 80 6l 17 57 1.0 81 6m 15 61 2.0 79 6n 16 58 2.0 79 6o 15 45 2.0 78 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 Scheme 1. Synthesis of Mannich bases 6a–o. Table 1. Comparison of reaction kinetics of conventional and ultrasonic irradiation methods for the synthesized compounds 6a–o. Entry Conventional Ultrasonic Irradiation Time (h) Yield (%) Time (h) Yield (%) 6a 15 68 1.5 80 6b 16 63 1.0 81 6c 15 62 1.5 78 6d 17 58 2.0 79 6e 18 45 1.0 81 6f 18 56 1.0 79 6g 19 57 2.0 81 6h 17 61 1.5 90 6i 18 53 1.0 82 6j 17 63 1.5 85 6k 15 58 1.5 80 6l 17 57 1.0 81 6m 15 61 2.0 79 6n 16 58 2.0 79 6o 15 45 2.0 78 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 7.0–7.9 for 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 Molecules 2016,21, 484 4 of 13 carbon atoms. The HRMS mass spectrum showed (M + 1) molecular ion peak at m/z285, in agreement with its molecular formula, C15H12O2N2S. 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 7.06–8.02 for 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/z383, in agreement with the assigned molecular formula, C20H21N3O3S. 2.2. 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 µ g/mL), imidazole for 6d (6 µ g/mL), morpholine for 6j (8 µ g/mL), and triazole in case of 6c (11.2 µ g/mL). Compound 6f (5.4 µ g/mL) with ortho and para-disubstituted toluyl and 6e (24.5 µ g/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. Table 2. Antifungal activity of the compounds 6a – o (IC 50 * in µ g/mL) against different human pathogenic fungi. Compound Candida albicans ATCC 24433 Candida albicans ATCC 10231 Candida glabrata NCYC 388 Cryptococcus neoformans ATCC 34664 Cryptococcus neoformans PRL 518 Aspergillus fumigatus NCIM 902 Aspergillus niger ATCC 10578 6a 64 49.6 64 64 64 >256 27.7 6b 128 67.12 59.6 77.5 >128 24.2 24.1 6c 21.6 108 53.6 108.9 196.5 11.2 93 6d 68 159 65.6 128 >128 6130 6e 54.4 44.6 24.5 64 14.21 25.8 47 6f 16 84.9 5.4 >256 90 256 256 6g 62 107.6 256 76.8 94.7 4>256 6h 55.3 57.4 64 47.6 66.6 16 18.61 6i 23.3 80.6 40.3 256 93.5 24.6 >256 6j 38.4 31.5 56.6 192 39 8128 6k 150 53.3 36.2 64.2 146.6 35.1 22.9 6l 115 95.3 42.5 49.7 145.8 81 73.2 6m 91.5 49.7 78.6 62.5 54.6 87.4 84.8 6n 30.3 43 48.8 47.5 105.6 81.5 56.9 6o 95 28.1 50.7 42.7 156.5 76.1 70.5 Fluconazole 0.12 0.11 9.4 16 4 64 46 * IC50 is the concentration exhibiting 50% inhibition of the growth as compared to the growth of control. Molecules 2016,21, 484 5 of 13 2.3. Molecular Docking Study The azole class of antifungal drugs inhibits the cytochrome P450 lanosterol 14 α -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 14 α -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. Table 3. Results of the docking study of the compounds. Compound Binding Energy Hydrogen Bonds Hydrophobic Bonds 6a ´41.69 LEU412-O=C ALA343, GLY344, LEU412, MET415, PHE499, GLY508 and ALA512 6b ´49.33 LEU412-O=C ALA343, THR347, MET415, GLY500 and HIS504 6c ´57.85 CYS506-O-CH2 LEU412, SER414, MET415, CYC506, ILE507 and GLY508 6d ´45.23 CYS506-O-CH2 ALA343, LEU412, MET415, CYC506, ILE507 and GLY508 6e ´35.50 LEU412-O=C; GLY500-N of nitro and GLY500-O-N of NO2ALA343, LEU412, ILE507 and GLY508 6f ´65.25 CYS506-NH ALA343, THR347, MET415, PHE499, HIS504, CYC506, GLY508, GLU509, ALA512 and TYR513 6g ´52.37 LEU412-O=C THR347, LEU412, MET415, PHE499 and HIS504 6h ´47.18 VAL497 and GLY500-N of NO2ALA343, GLY344, PHE499, GLY500 and GLY508 6i ´61.03 TYR154-O-N of 2-nitro; TYR168O=C and MET415-N of 4-nitro LEU340 and ALA343 6j ´40.29 LEU412-O-CH2 ALA343, GLY344, LEU412, MET415, PHE499, GLY508 and ALA512 6k ´37.85 - ALA343, GLY344, THR347, PHE499, GLY500 and GLY508 6l ´42.91 - LEU186, LEU240, GLY344, THR347, MET415, PHE499, GLY500, CYS506, GLY508 and PHE511 6m ´36.83 - ALA343, LEU412 and PHE499 6n ´40.09 CYS506-O-CH2ALA343, VAL440, PRO442, VAL497, GLY500 and CYC506 6o ´38.97 TYR168-O=C LEU340, ALA343, ILE507 and GLY508 Fluconazole ´67.29 TYR168-F of phenyl GLY500 and HIS504 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 14 α -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 Molecules 2016,21, 484 6 of 13 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. Molecules 2016, 21, 484 6 of 13 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. 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. Molecules 2016,21, 484 7 of 13 3. Materials and Methods 3.1. 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 Hand 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). 3.2. 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) 3.3. 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) 3.4. 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. 3.5. 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). Molecules 2016,21, 484 8 of 13 5-(4-(Benzyloxy)phenyl)-1,3,4-oxadiazole-2(3H)-thione ( 5 ): Recrystallized from ethanol; yield: 90%; m. p.: 160 ˝ C. IR (KBr) νmax (cm ´1 ): 3235 (NH), 3100–3000 aromatic CH stretch,2870–2800 aliphatic CH stretch, 1621 (C=N), 1596 (C–C), 1250 (C=S), 1258 and 1093 (C–O–C), 1 H-NMR (CDCl 3 ), δ ppm: 3.8 (s, 1H, NH), 5.17 (s, 2H, C–CH 2 –O), 7.07.9 (m, 9H, two aromatic ring), 13 C-NMR (CDCl 3 , δ ppm): 70.8, 114.4, 116.3, 127.1, 127.6, 128.9, 130.2, 136.7, 157.0, 161.3, 189.9, m/z: 284.1 (100.0%), 285.1 (18.0%), 286.1 (6.5%), Molecular Formula: C 15 H 12 N 2 O 2 S. Elemental Analysis: Calculated: (C, H, N) 63.3, 4.25, 9.85 Found: 63.5, 4.22, 9.87. 5-(4-(Benzyloxy)phenyl)-3-(piperazine-1-ylmethyl)-1,3,4-oxadiazole-2(3H)-thione ( 6a ): Yield: 80%; m.p.: 205 ˝ C. IR (KBr) νmax (cm ´1 ): 3245 (NH), 3100–3000 aromatic CH stretch, 2870–2800 aliphatic CH stretch, 1623 (C=N), 1598 (C–C), 1252 (C=S), 1260 and 1095 (C–O–C), 1280 (C-N sec. amine piperazine), 1560 (N–H sec. amine piperazine), 1 H-NMR (CDCl 3 ), δ ppm: 1.91 (s, 1H, –NH piperazine), 2.37 (t, 4H, N–CH 2 –C piperazine), 2.65 (t, 4H, C–CH 2 –NH piperazine), 3.72 (s, 2H, N–CH 2 –N), 5.16 (s, 2H, C–CH 2 –O), 7.38–8.02 (m, 9H, aromatic ring), 13 C-NMR ( CDCl 3 , δ ppm): 45.91, 54.7, 70.8, 71.0, 114.4, 116.3, 127.1, 127.5, 128.9, 130.2, 136.7, 157.0, 163.3, 177.1., m/z382.15 (100.0%), 383.15 (22.8%), 384.14 (4.5%), Molecular Formula: C 20 H 22 N 4 O 2 S. Elemental Analysis: Calculated (C, H, N) 62.80, 5.80, 14.65, Found: 62.76, 5.74, 14.62. 5-(4-(Benzyloxy)phenyl)-3-(((4-chlorophenyl)amino)methyl)-1,3,4-oxadiazole-2(3H)-thione ( 6b ): Yield: 81%; m.p.: 180 ˝ C. IR (KBr) νmax (cm ´1 ):3230 (NH), 3100–3000 aromatic CH stretch, 2870–2800, aliphatic CH stretch, 1625 (C=N), 1592 (C–C), 1235 (C=S), 1261 and 1094 (C–O–C), 3470 (N–H primary amine) 744 (C–Cl), 1 H-NMR (CDCl 3 ), δ ppm: 4.0 (S, 1H, –NH), 4.42 (S, 2H, N–CH 2 –N–), 5.16 (S, 2H, –OCH 2 ), 6.54 and 8.02 (m,13H, aromatic rings), 13 C-NMR (CDCl 3 , δ ppm): 70.8, 114.4, 114.9, 116.3, 121.1, 127.1, 127.6, 128.9, 129.6, 130.2, 136.7, 145.7, 157.0, 161.3, 177.1., m/z423.08 (100.0%), 425.08 (36.9%), 424.08 (25.7%), Molecular Formula: C 22 H 18 ClN 3 O 2 S. Elemental Analysis: Calculated (C, H, N) 62.33, 4.28, 9.91, Found: 62.28, 4.22, 9.88. 3-((1H-1,2,4-Triazol-1-yl)methyl) 5-(4-(benzyloxy)phenyl)-1,3,4-oxadiazole-2(3H)-thione ( 6c ): Yield: 78%; m.p.: 235 ˝ C. IR (KBr) νmax (cm ´1 ): 3100–3000 aromatic CH stretch, 2870–2800, aliphatic CH stretch, 1621 (C=N), 1596 (C–C), 1425 (C=S), 1258 and 1093 (C–O–C), 1290 (C–N), 1622 (C=N triazole), 1 H-NMR (CDCl 3 ), δ ppm: 5.09 (s, 2H, N–CH 2 –N), 5.16 (S, 2H, –OCH 2 ),7.06–8.02 (m, 9H, aromatic rings), 8.05 and 8.68 (s, 1H, NH triazole ring), 13 C-NMR (CDCl 3 , δ ppm): 70.8, 73.6, 114.4, 116.3, 127.1, 127.6, 128.9, 130.2, 136.7, 143.8, 151.5, 157.0, 161.3, 177.1, m/z365.09 (100.0%), 366.10 (19.7%), 367.09 (4.5%), Molecular Formula: C 18 H 15 N 5 O 2 S; Elemental Analysis: Calculated (C, H, N) 59.16, 4.14, 19.17, Found: 59.11, 4.10, 19.11. 3-((1H-Imidazol-1-yl)methyl) 5-(4-(benzyloxyphenyl)-1,3,4-oxadiazole-2(3H)-thione ( 6d ): Yield: 79%; m.p.: 240 ˝ C. IR (KBr) νmax (cm ´1 ): 3100–3000 aromatic CH stretch, 2870–2800 aliphatic CH stretch, 1621 (C=N), 1596 (C–C), 1425 (C=S), 1258 and 1093 (C–O–C), 1290 (C–N), 1622 (C=N imidazole), 1625 (C=C imidazole ring), 1 H-NMR (CDCl 3 ), δ ppm: 5.09 (s, 2H, N–CH 2 –N), 5.16 (S, 2H, –OCH 2 ), 6.77, 7.13 and 7.83 (s, 1H each of imidazole ring), 7.06–8.02 (m, 9H, aromatic rings), 13 C-NMR (CDCl 3 , δ ppm): 70.8, 71.7, 114.4, 116.3, 120.6, 127.1, 127.6, 128.1, 128.9, 130.2, 136.7, 137.8, 157.0, 161.3, 177.1, m/z 364.10 (100.0%), 365.10 (22.9%), 366.10 (5.4%), Molecular Formula: C 19 H 16 N 4 O 2 . Elemental Analysis: Calculated (C, H, N) 62.62, 4.43, 15.37 Found: 62.58, 4.40, 15.34. 5-(4-(Benzyloxy)phenyl)-3-(((4-chloro-2-nitrophenyl)amino)methyl)-1,3,4-oxadiazole-2(3H)-thione ( 6e ): Yield: 81%; m.p.: 218 ˝ C. IR (KBr) νmax (cm ´1 ): 3549(NH), 3100–3000 aromatic CH stretch, 2870–2800 aliphatic CH stretch, 1625 (C=N), 1590 (C–C), 1526 and 1346 (NO 2 ), 1429 (C=S), 1263 and 1089 (C–O–C), 1490 (C–NO 2 ), 744 (C–Cl); 1 H-NMR (DMSO-d 6 ), δ ppm: 4.0 (S, 1H, –NH), 4.42 (S, 2H, N–CH 2 –N–), 5.16 (S, 2H, –OCH 2 ), 7.06–8.13 (m, 12H, aromatic rings). 13 C-NMR (CDCl 3 , δ ppm): 69.8, 70.8, 114.4, 115.8, 116.3, 122.0, 127.1, 127.6, 128.9, 130.2, 131.5, 135.7, 136.7, 144.8, 157.0, 161.3, 177.1, m/z468.07 (100.0%), 470.06 (36.5%), 469.07 (24.9%), Molecular Formula: C 22 H 17 ClN 4 O 4 S. Elemental Analysis: Calculated (C, H, N) 56.35, 3.65, 11.95, Found 56.30, 3.61, 11.92. Molecules 2016,21, 484 9 of 13 5-(4-(Benzyloxy)phenyl)-3-(((2,4-dimethylphenyl)amino)methyl)-1,3,4-oxadiazole-2(3H)-thione ( 6f ): Yield: 79%; mp: 230 ˝ C. IR (KBr) ν max (cm ´1 ): 3230 (NH), 3100–3000 aromatic CH stretch, 2870–2800 aliphatic CH stretch, 1616 (C=N), 1595 (C–C), 1427 (C=S), 1252 and 1151 (C–O–C), 1460 (Aromatic C–CH 3 ), 1 H-NMR (CDCl 3 ), δ ppm: 2.10 (s, 3H, –CH 3 ), 2.30 (s, 3H, –CH 3 ), 4.0 (S, 1H, –NH), 4.42 (S, 2H, N–CH 2 –N–), 5.16 (S, 2H, –OCH 2 ),6.36–8.02 (m, 12H, aromatic rings), 13 C-NMR (CDCl 3 , δ ppm): 17.9, 21.6, 70.8, 71.1, 113.3, 114.4, 116.3, 126.8, 127.1, 127.6, 128.9, 130.2, 131.7, 136.2, 136.7, 143.5, 157.0, 161.3, 177.1 m/z417.15 (100.0%), 418.15 (27.9%), 419.15 (5.0%) Molecular Formula: C 24 H 23 N 3 O 2 S. Elemental Analysis: Calculated (C, H, N) 69.04, 5.55, 10.06, Found: 69.00, 5.51, 10.02. 5-(4-(Benzyloxy)phenyl)-3-(((4-bromophenyl)amino)methyl)-1,3,4-oxadiazole-2(3H)-thione ( 6g ): Yield: 81%; m.p.: 228 ˝ C. IR (KBr) νmax (cm ´1 ): 3235 (NH), 3100–3000 aromatic CH stretch, 2870–2800 aliphatic CH stretch, 1621 (C=N), 1593 (C–C), 1424 (C=S), 1260 and 1123 (C–O–C), 690 (C–Br), 1 H-NMR (DMSO-d 6 ), δ ppm: 4.0 (S, 1H, –NH), 4.42 (S, 2H, N–CH 2 –N–), 5.16 (S, 2H, –OCH 2 ), 6.59–8.02 (m, 13H, aromatic rings), 13 C-NMR ( CDCl 3 , δ ppm): 70.8, 114.4, 114.5, 115.1, 116.3, 127.1, 127.6, 128.9, 130.2, 132.4, 136.7, 146.6, 157.0, 161.3, 177.1, m/z469 (100%), 467 (97.4%), 470 (25.7%), 471.2 (4.3%). Molecular Formula: C22H18BrN3O2S. Elemental Analysis: Calculated (C, H, N) 56.42, 3.87, 8.97, Found: 56.38, 3.84, 8.93. 5-(4-(Benzyloxy)phenyl)-3-(((2-nitrophenyl)amino)methyl)-1,3,4-oxadiazole-2(3H)-thione ( 6h ): Yield: 90%; m.p.: 220 ˝ C.IR (KBr) νmax (cm ´1 ): 3245 (NH), 3100–3000 aromatic CH stretch, 2870–2800 aliphatic CH stretch, 1622 (C=N), 1590 (C–C), 1522 and 1345 (NO 2 ), 1427 (C=S), 1261 and 1088 (C–O–C), 1401 (C–NO 2 ), 755; 1 H-NMR (DMSO-d 6 ), δ ppm: 4.0 (S, 1H, –NH), 4.42 (S, 2H, N–CH 2 –N–), 5.16 (S, 2H, –OCH 2 ), 7.06–8.04 (m, 13H, aromatic rings), 13 C-NMR (CDCl 3 , δ ppm): 69.8, 70.8, 114.4, 116.3, 118.0, 125.9, 127.1, 127.6, 128.9, 130.2, 131.7, 135.6, 136.7, 146.7, 157.0, 161.3, 177.1, m/z431.10 (100%), 435.11 (97.4%), 436.10 (4.5%), Molecular Formula: C 22 H 18 N 4 O 4 S. Elemental Analysis: Calculated (C, H, N) 60.82, 4.18, 12.90, Found: 60.78, 4.13, 12.86. 5-(4-(Benzyloxy)phenyl)-3-(((2,4-dinitrophenyl)amino)methyl)-1,3,4-oxadiazole-2(3H)-thione ( 6i ): Yield: 82%; m.p.: 145 ˝ C. IR (KBr) νmax (cm ´1 ): 3247 (NH), 3100–3000 aromatic CH stretch, 2870–2800 aliphatic CH stretch, 1621 (C=N), 1593 (C–C), 1524 and 1344 (NO 2 ), 1426 (C=S), 1262 and 1083 (C–O–C), 1405–1418 (C–NO 2 ), 1 H-NMR (DMSO-d 6 ), δ ppm: 4.0 (S, 1H, –NH), 4.42 (S, 2H, N–CH 2 –N–), 5.16 (S, 2H, –OCH 2 ), 7.06–8.88 (m, 12H, aromatic rings), 13 C-NMR (CDCl 3 , δ ppm): 69.8, 70.8, 114.4, 116.3, 120.8, 127.1, 127.6, 128.9, 130.2, 130.8, 135.2, 136.7, 137.2, 152.8, 157.0, 161.3, 177.1, m/z479.09 (100%), 480.09 (26.7%), 481.09 (6.4%), Molecular Formula: C 22 H 17 N 5 O 6 S. Elemental Analysis: Calculated (C, H, N) 55.11, 3.57, 14.61, Found: 55.08, 3.52, 14.57. 5-(4-(Benzyloxy)phenyl)-3-(morpholinomethyl)-1,3,4-oxadiazole-2(3H)-thione ( 6j ): Yield: 85%; m.p.: 200 ˝ C. IR (KBr) νmax (cm ´1 ): 3100–3000 aromatic CH stretch, 2870–2800 aliphatic CH stretch, 1623 (C–N), 1592 (aromatic C–C), 1437 (C=S), 1222 and 1036 (C–O–C), 2916/2850 (CH 2 of morpholine), 1 H-NMR (CDCl 3 ), δ ppm: 2.75 (t, 4H, N–CH 2 –C), 3.62 (t, 4H, O–CH 2 –C), 3.72 (S, 2H, N–CH 2 –N–), 5.16 (S, 2H, –OCH 2 ), 7.06–8.02 (m, 9H, aromatic rings), 13 C-NMR (CDCl 3 , δ ppm): 67.91, 68.23, 70.12, 115.47, 119.51, 125.68, 128.17, 128.43, 128.61, 129.00, 131.97, 135.64, 156.69,177.14 m/z383.13 (100%), 384.13 (23.7%), 385.13 (5.6%), Molecular Formula: C 20 H 21 N 3 O 3 S. Elemental Analysis: Calculated (C, H, N) 2.64, 5.52, 10.96 Found: 62.61, 5.48, 10.92. 5-(4-(Benzyloxy)phenyl)-3-((phenylamino)methyl)-1,3,4-oxadiazole-2(3H)-thione ( 6k ): Yield: 80%; m.p.: 80 ˝ C. IR (KBr) νmax (cm ´1 ): 3553 (NH), 3100–3000 aromatic CH stretch, 2870–2800 aliphatic CH stretch, 1612 (C=N), 1595 (C–C), 1428 (C=S), 1255 and 1151 (C–O–C), 1 H-NMR (CDCl 3 + DMSO-d 6 ), δ ppm: 4.0 (S, 1H, –NH), 4.42 (S, 2H, –N–CH– 2 N–), 5.16 (S, 2H, –OCH 2 ), 6.77–8.02 (m, 14H, aromatic rings), 13 C-NMR (CDCl 3 , δ ppm): 70.8 113.5, 114.4, 116.3, 120.8, 127.1, 127.6, 128.9, 129.5, 130.2, 147.6, 157.0, 161.3, 177.1, m/z383.13 (100%), 384.13 (23.7%), 385.13 (5.6%). Molecular Formula: C 22 H 19 N 3 O 2 S. Elemental Analysis: Calculated (C, H, N) 67.84, 4.92, 10.79 Found: 67.79, 4.88, 10.74. 5-(4-(Benzyloxy)phenyl)-3-(((4-methoxyphenyl)amino)methyl)-1,3,4-oxadiazole-2(3H)-thione ( 6l ): Yield: 81%; m.p.: 100 ˝ C IR (KBr) νmax (cm ´1 ): 3550–3235 (NH), 3100–3000 aromatic CH stretch, 2870–2800 aliphatic CH stretch, 1615 (C=N), 1593 (C–C), 1425 (C=S), 1250 and 1152 (C–O–C), 1450 (C–OCH 3 ),