Molecular Sieves And Ultrasound-Assisted Synthesis Of Novel 1 , 3 , 4-Oxadiazole-2-Thiones Derivatives As Potential Antifungal Agents . ”

In the category of microorganism, fungi are considered as the special class of microbes responsible for opportunistic pathogenic infections in humans species. Due to the side effects of commercially available  antifungal drugs  and the emergence of new drug resistant fungal species in the past few years  has forced the researchers to search for  novel and efficient antifungal drug molecules. The 1,3,4-oxadiazoles scaffold is associated with diverse biological activities. The multipurpose use of the Mannich bases in pharmaceutical chemistry provoked us to prepare a new series of 1,3,4-oxadiazole Mannich bases derivatives, as antifungal agents. 
Herein we report Molecular sieves and Ultrasound assisted synthesis of novel series of Mannich bases of the 5-substituted 1,3,4-oxadiazole-2-thiones  by amino methylation with paraformaldehyde and substituted primary / secondary amines and their  evaluation for antifungal activity .The structures of the newly synthesized compounds were determined by IR, NMR and Mass spectral study. The synthesized compounds exhibited interesting moderate to excellent antifungal activity against Candida albicans (NCIM 3557),Candida albicans(NCIM3471), Candida glabrata(NCIM3237), Cryptococcus neoformans (NCIM 3542),Cryptococcus neoformans(NCIM 3378),Aspergillus fumigates(NCIM 902), Aspergillus niger( NCIM 628) using  Flucanazole as a standard reference drug. The synthesised compounds 6d, 6f ,6g, 6h and 6j exhibited promising antifungal activity as fungi static agents.


Introduction:
In the category of microorganism, fungi are considered as the special class of microbes responsible for opportunistic pathogenic infections in plant, animals and humans species.
Commercially available broad-spectrum antifungal drugs includes fluconazole, itraconazole, miconazole and voriconazole in which the mechanism of action is on target CYP51 which get inhibited and in turn switch off the biosynthesis of ergo sterols [1] .But the frequent use of these antifungal drugs in immune compromised patients who are undergoing the long term treatment of broad-spectrum antibiotics may be in cases of HIV infection, grafting surgery or anticancer therapy [2], has led to the development of resistance fungal strains.The emergence of new drug resistant fungal species in the past few years has forced the researchers around the world to search for novel and efficient antifungal drug molecules.[3,4,5].The 1,3,4-oxadiazoles scaffold is associated with diverse biological activities such as antifungal[6a-b] antibacterial [7], anti myco bacterial [8], anti HIV [9], anti-hepatitis B virus [10] anticancer [11] anticonvulsant [12], anti-inflammatory [13], anti malarial [14] analgesic [15] etc. 5-Substituted-1,3,4-oxadiazole-2thiones represent an important type of compound in the field of coordination chemistry because of their potential multifunctional donor sites, viz either exocyclic sulfur or endocyclic nitrogen [16,17] and possess CNS depressant [18]and tyrosinase inhibition [19] property.The multipurpose use of the Mannich bases in pharmaceutical chemistry [20,21] promote us to prepare a new series of 1,3,4-oxadiazole based amino methyl derivatives .
The use of ultrasound to endorse chemical reactions is called "Sonochemistry".Ultrasonicassisted organic synthesis is a green synthetic approach and it is a powerful technique towards the increase in reaction rate and yield [22,23].It offers the potential reaction in small time cycles, cheaper reagents and milder physical conditions [24,25].It can also be considered as important tool for conservation of energy and minimization of waste as compared to the conventional techniques [26].Herein, we introduced ultrasound-promoted Mannich reaction of the 5-substituted 1,3,4-oxadiazole-2-thiones with primary and secondary amines and its antifungal screening because of the structural resemblance with established antifungal molecule triazoles.As the majority of fungal infections are caused by Candida, Yeast, Cryptococcus Aspergillus species, [27,28].So we have screened the synthesized compound against the selected strains of these species.

Result and Discussion:
We herein report the synthesis and antifungal evaluation of some novel structural scaffolds (6a-o) as illustrated in Scheme 1.
The starting material methyl 4-(benzyloxy) benzoate (3) was synthesized by reaction of methyl4-hydroxybenzoate and chloromethyl benzene in K 2 CO 3 and DMF as solvent in ultrasonic processor up to 4hr.The compound Methyl 4-(benzyloxy) benzoate obtained with good yield in step I, is refluxed with NH 2 NH 2 to get 4-(benzyloxy) benzohydrazide (4).
The reaction of the above acid hydrazides (4) with carbon disulphide under basic conditions using KOH yielded 1, 3, 4-oxadiazol-2-thiones (5), which underwent N-amino methylation by the Mannich reaction in ultrasonic processor and various substituted primary and some secondary amines in presence of paraformaldehyde.
The structure of intermediate 1, 3, 4 oxadiazole-2-thiones (5) was confirmed by its spectroscopic analysis.The IR spectrum showed a weak SH stretching absorption at 2592 cm _1 .The absorption bands due to -C=N was observed at 1681 cm _1 .A broad downfield signal observed at d 11.8 in the 1 H NMR spectrum was assigned to NH/SH tautomeric proton.In 13 C NMR spectrum, -C=S carbon appeared at δ 177.13 in addition to other characteristic signals of 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 13 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 CH 2 group of 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.The 1 H NMR spectrum showed a sharp singlet at δ 5.02 for N-CH 2 -N protons.Eight protons of morpholine moiety resonated as two triplets at δ 2.75 and δ 3.62 with J ¼ 6.80 Hz.In 13 C NMR spectrum, the signals observed at δ 67.91 and 70.12 were assigned to C2, C6 and C3, C5 of morpholine ring respectively.The signal due to aryloxy methylene carbon appeared at δ 68.23 Experimental section: a)General procedure for the synthesis of 4-(benzyloxy)benzoate(3): For the synthesis of methyl-4-(benzyloxy) benzoate, methyl-4-hydroxybenzoate and chloromethyl benzene were taken in equal ratio (0.01mol). in N,N-dimethyl formamide (DMF) as solvent and reaction is carried out in K 2 CO 3 ultrasonic processor up to 4hr.After that, the solution was poured into ice-water.The precipitate was filtered and recrystallized from ethanol.
Colour: White M.P.105 0 C b) General procedure for the synthesis of 4-(benzyloxy) benzohydrazide (4): For the synthesis of substituted benzo hydrazines, a mixture of corresponding esters (20 mmol), 85% hydrazine hydrate (20 mmol) in ethanol (35 ml) was heated to reflux for 6 h.After that, the solution was poured into ice-water.The precipitate was filtered and recrystallized from ethanol.
Colour: White M.P.80 0 C c) General procedure for the synthesis of 5-phenyl- Equimolar quantities of the substituted benzo hydrazine (5 mmol) and potassium hydroxide were added with constant stirring.The resulting mixture was kept in ultrasonic processor for 2-4hrs.The precipitated solids were filtered, washed with water and recrystallized from methanol to yield the title compounds 6a-o (Table 1).
The physical characterization and spectral data of synthesized derivatives is as follows:

( 5
mmol) were dissolved in 20 mL of 95% ethanol.The mixture was allowed to stir for several minutes at room temperature and then carbon disulfide (15 mmol) was slowly added drop wise 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 values of the solution to 5-6.Then the precipitate was collected washed with water for several times and dried and recrystallized from ethanol.Colour: White M.P.160 0 C.d)General procedure for the synthesis of Mannich Base Derivatives of 1,3,4-Oxadiazole-2-thiones (6a-o): 1,3,4-Oxadiazole-2-thiones (10 mmol) was dissolved in methanol, para formaldehyde (15mmol) and primary/secondary amine (10 mmol) in methanol
(60.78)( 4.13)(12.86)BiologicalevaluationInvitroantifungalsusceptibility testing was performed by broth micro dilution method according to the Clinical and Laboratory Standards Institute (CLSI) to find out IC 50 concentration and minimum inhibitory concentration (gives excellent activity against the Aspergillus niger.(NCIM628),whereas6e(47µg/ml)give good activity against the same strain, standard drug fluconazole (46 µg/ml).Compound 6f (5.4 µg/ml) gives excellent activity against Candida glabrata(NCIM 3237) and 6e(24.5µg/ml)showsgoodactivity.Fluconazole is used as a standard drug (9.4 µg/ml).Other synthesized compounds have shown good to moderate activity for the remaining strain of fungi.