Synthesis of ( E )-2-( arylbenzylidene )-2-( ( 4-methoxyphenyl ) amino ) acetohydrazide derivatives and their antimicrobial activity

Article history: Received March 27, 2013 Received in Revised form July 27, 2013 Accepted 22 August 2013 Available online 24 August 2013 A series of some new (E)-2-(arylbenzylidene)-2-((4-methoxyphenyl)amino)acetohydrazides 4(a-j) have been conveniently synthesized by intermolecular oxidative cyclization of (E)-2(arylbenzylidene)-2-[(4-methoxyphenyl)amino]acetohydrazides promoted by iodobenzene diacetate as an oxidant. The structures of the synthesized compounds have been confirmed by H NMR, IR, MS and elemental analysis. All the synthesized compounds were tested for their inhibitory action against clinically isolated strains i.e., Bacillus subtilis, Staphylococcus aureus, Xanthomonas campestris, Escherichia coli and Fusarium oxysporum. Compounds 4f (21-28 mm, 73.8 %), 4i (20-24 mm, 72.4 %) and 4j (21-27 mm, 71.6 %) demonstrated good antimicrobial activity against all the tested bacterial and fungal strains. © 2013 Growing Science Ltd. All rights reserved.


Introduction
In the past decades, the problem of multidrug resistant micro-organism has reached on alarming level around the world, and the synthesis of new anti-infective compounds has become an urgent need for the treatment of microbial infections.There are various problems arising with the use of antimicrobials such as local tissue irritation, interference with wound healing process, hypersensitivity reactions, system toxicity, narrow antimicrobial spectrum, and emergence of resistance. 1,2Therefore, the increasing clinical importance of drug resistant microbial pathogens has additional urgency in microbiological and antifungal research.Compounds containing heterocyclic ring systems continue to attract considerable interest due to the wide range of biological activities they possess. 3Among them five membered rings gained importace because of their versatile biological properties. 3In particular, compounds bearing 1,3,4-oxadiazoles nucleus are known to have unique angioedema and anti-inflammatory activities. 4Substituted oxadiazoles molecules possess other interesting properties such as analgesic 5 , antimicrobial 6 , antiviral 7 , anticonvulsant 8 , antihypertensive 9 , anti-proliferative 10 , enzyme Inhibitors 11 , 5-HT-receptor antagonists 12 and inhibitors of muscle glycogen phosphorylase 13 .
Literature reveals that docking study of N-(hetarylmethyl)-aniline derivatives showed anticancer activity 14 and N-aryl-N-benzylamines were synthesized and evaluated for their antifungal activity, which was compared with their homoallylamine analogues that possessed an allyl group in the carbon next to the nitrogen atom.Results indicated that the absence of the allyl group caused an enhancement of the antifungal activity which could be correlated with the flexibility of the alkyl chain between both aromatic groups. 15This led to the preparation of N-substituted amines bearing a hetaryl fragments (N-(hetarylmethyl)-anilines).

Chemistry
The new compounds were prepared by using the synthetic strategy described in Scheme 1.The 2-(4-methoxyphenylamino)acetohydrazide 2 was synthesized by the reaction of ethyl-(4methoxyphenyl)glycinate 1 with hydrazine hydrate in ethanol as per the reported procedure 15 .

Scheme 1. Synthesis of compounds 4
The IR spectra of the compound 3a exhibited characteristic bands at absorption bands at 1631 cm -1 and 3150 cm -1 due to for carbonyl and NH group, respectively.The 1 H NMR spectra of 3a showed two singlet due at N=CH and NH at δ 8.58 and δ 9.34, respectively.The structure of all compounds 4a-j confirmed by their spectral (IR, LCMS, and 1 H NMR) and elemental analysis.The characterization of products 4a-j was based upon a careful comparison of their IR and 1 H NMR spectra with those of 3a-j.IR spectra of 4a were found to be transparent in the region of NH stretch and CO stretch.In 1 H NMR spectra of 4a the disappearance of their singlet due to N=CH around δ 8.4-8.6 and NH proton around δ 9.3-9.5, thus confirming the oxidation of 3a-j into 4a-j.The mass spectra of 4a showed molecular ion peak at m/z 342, which is in agreement with the molecular formula C 18 H 19 N 3 O 4 .3a-j (1 eq), IBD (1 eq), methanol, reflux.

Antimicrobial activity
Compounds (4a-j) were tested in vitro for their antibacterial activity against two gram positive and two gram negative bacterial strains.Commercial antibiotics such as bacteriomycin and gentamycin were used as standard drugs.The results were compared with standard drugs and depicted in Table 2. Compound 4f was found to be more potent against gram positive and gram negative bacterial strains with the zone of inhibition respectively 21-28 mm.Compounds 4i and 4j exhibited 20-24 mm and 21-27 mm, respectively against all the bacterial strains.Compounds 4a, 4b, 4d, 4e and 4g were showed moderate antibacterial activity and compound 4d was found to be slightly active than 4a, 4e, 4b and 4g.A compound 4h was weakly active against tested bacterial strains.
The in vitro antifungal activity of the new oxadiazole derivatives (4a-j) was studied against the fungal strain, Fusarium oxysporum.Nystatin was used as a standard drug and the results are given in Table 2. Compounds 4f, 4i and 4j showed 73.8 %, 72.4 % and 71.6 % inhibition, respectively when compared to standard drug (100 %).Compounds 4d (69.0 %), 4e (63.1 %), 4b (56.4 %), 4g (52.0 %) and 4a (53.8 %) exhibited moderate antifungal activity against tested fungal strain.On the other hand, the lowest antifungal effect was detected for compounds 4h (51.9 %) and 4c (50.2 %) against tested fungal strain.In the present study different groups attached to aryl ring as substituent linkage to 1,3,4oxadiazole ring.The close survey of antimicrobial efficacy indicated that the inhibition values of all the compounds exhibited a different range of activity against the tested strains.The biological results for compounds (4a-j) showed that the substitution pattern on phenyl ring appears to be vital for broad spectrum activity.Antibacterial activity was expressed in Zone of inhibition (mm), Antifungal activity was expressed in % inhibition (mm)

Conclusions
In conclusion, a series of new 2,5-disubstituted-1,3,4-oxadiazoles (4a-j) were synthesized in good yield, characterized by different spectral studies and their antimicrobial activities have been evaluated.Compounds 4f, 4i and 4j demonstrated good inhibition against microbial strains tested.The substituent on phenyl ring is responsible for the antimicrobial activity of these classes of agents.On the basis of their activity, these derivatives were identified as viable leads for further studies.

Materials and Methods
All solvents and reagents were purchased from Merck Chemicals, India.Melting point was determined by VMP III apparatus.An elemental analysis was recorded on Vario MICRO superuser V1.3.2Elementar.The IR spectra were recorded using KBr discs on IR Jasco 4100 infrared spectrophotometer. 1 H NMR spectra was recorded using d 6 -DMSO as solvent on Bruker DRX-500 spectrometer at 400 MHz.The mass spectrum was recorded using the instrument LC-MSD-Trap-XCT.

4.3.
General procedure for the synthesis of (

E)-2-(arylbenzylidene)-2-((4methoxyphenyl)amino)acetohydrazides (3a-j)
Compound 2 was refluxed with different aryl aldehydes in ethanolic solution (10 mL) for about 2 h.Reaction completion was confirmed by the thin layer chromatography (TLC).The reaction mass was cooled to 5 to 10 o C for 1 h.The reaction mass was filtered and washed with ethanol.The obtained solid was dried to get the pure product.

General procedure for the synthesis of 2,5-disubstituted-1,3,4-oxadiazoles (4a-j)
(E)-2-(arylbenzylidene)-2-((4-methoxyphenyl)amino)acetohydrazides (3a-j) was dissolved in dichloromethane and IBD were added to it.The contents were stirred for 2 h and the progress of the reaction was monitored by TLC.The solvent was removed and the residue was taken in petroleum ether and stirred for 30 min.The solid thus obtained was filtered, washed with petroleum ether and dried to afford (4a-j).

Antibacterial activity
Antibacterial activity of the newly synthesized compounds was determined in DMF by disc diffusion method on nutrient agar medium 18 by using Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and Gram-negative bacteria (Xanthomonas campestris and Escherichia coli).In each Petri plate the sterile media (Nutrient Agar Medium, 15 mL) was uniformly smeared with cultures of both bacteria.Sterile discs of 10 mm diameter (Hi-Media) was placed in the Petri plates, to which 50 µL (1 mg/ml i.e., 50 µg/disc) of the new compounds were added.This included 50 µL of DMF as negative, bacteriomycin and gentamycin as positive control.The plates were incubated at 37 ± 2 ºC for 24 h and the zone of inhibition was determined.

Antifungal activity
The synthesized compounds were screened for the antifungal activity in DMF by poisoned food technique 19 against Fusarium oxysporum.Prepare Potato Dextrose Agar (PDA) media and about 15 mL of PDA was poured into each Petri plate and allowed to solidify.5 mm disc of seven days old culture of the test fungi was placed at the center of the Petri plate and incubated at 26 °C for 7 days.After incubation the percentage inhibition was measured and three replicates were maintained for each treatment.Nystatin was used as standard.All the synthesized compounds were tested (at the dosage of 500 µL of the new compounds/petriplate, where concentration was 0.1 mg/mL) by poisoned food technique.