Synthesis , Spectral Characterization and Antibacterial activity of Functionalized Hydrazones

New hydrazones have been synthesized by condensing 2-acetylthiophene with acetic hydrazide and benzhydrazide. The synthesized hydrazones viz. 2-acetylthiophene acetoylhydrazone (ATAH), 2-acetylthiophene benzoylhydrazone (ATBH) are characterized in the light of physicochemical and analytical data. Structures of ATAH and ATBH are confirmed by FT-IR, 1H-NMR and Mass spectral data. The hydrazones are screened for their anti-bacterial activities against E coli, Bacillus cereus, Staphylococcus aureus and Pseudomonas aureoginos. Acetoyl hydrazones are found to show more antibacterial activity than the corresponding benzoyl hydrazones.


INTRODUCTION
Hydrazones having an azomethine (>C=N-NH-) aggregate establish an imperative class of ligands for new advancements in medication 1 .In this way, it is of interest to prepare new hydrazones and to evaluate their biological activities.Hydrazones possess diverse biological and pharmacological properties 2,3 such as antimicrobial 4 , anti-tubercular 5 anticonvulsant 6 , anti-inflammatory 7 , analgesic, antifungal, vasodilator 8 , antiviral, anticancer 9.10 , antiplatelet, antimalarial, cardio protective, antihelmintic, antiprotozoal, antitrypanosomal, cytotoxic 11 and antischistosomiasis activities.The chemical properties of hydrazones have been widely investigated due to their chelating capability 12 , pharmacological activity 13,14 and analytical applications 15,16 .Survey of literature 1,17,18 revealed that hydrazones possessing heterocyclic group show interesting biological and pharmacological properties.
Thiophenes are impor tant class of heterocyclic compounds.Thiophene derivatives are widely used as building blocks in many pharmaceuticals 19 .Thiophene derivatives are well known for their therapeutic applications.Thiophene nucleus is one of the most important heterocycles exhibiting remarkable pharmacological activities 20 .For example, 2-butylthiophene has been used as a raw material in the synthesis of anticancer agents.
In the light of the above literature and in continuation of our progressing research work, here in we report synthesis, spectral characterization and antibacterial activity of 2-acetylthiophene acetoylhydrazone(ATAH), 2-acetylthiophene benzoylhydrazone (ATBH).

EXPERIMENTAL
2-acetylthiophene (AR grade) was procured from Sigma-Aldrich Chemicals Pvt. Ltd.India.Acetic hydrazide and benzhydrazide were purchased from Merck chemicals.N,N-dimethyl formamide(DMF), ethanol were of AR quality and utilized as provided.Elemental data were obtained using a Perkin-Elmer 2400 CHNS/O analyzer.Mass spectra (in EI+ ionization mode)of compounds were recorded using JEOLGCMATEIIGC-Mass spectrometer.IR spectra of hydazones in solid state were recorded in 4000-400 cm -1 range on a Perkin-Elmer 100 FT-IR spectrophotometer. 1 H-NMR spectra were recorded using Avanc-400 Brucker, NMR spectrometer.

Preparation of 2-acetylthiophene acetoylhydrozone (ATAH)
A 3.0 g (0.03) of acetic hydrazide dissolved in 20 ml methanol was added to a hot methanolic solution (20 ml) of 2-acetylthiophene (0.03 mol, 5.03 ml)in a 100 ml round bottom flask.Glacial acetic acid (3-4drops) was added to the reaction mixture.The contents were refluxed over water bath for The antibacterial activity of hydrazones were screened against the Bacillus subtilis and Staphylococcus aureus which are gram positive and Pseudomonas aureoginosa and Escherichia coli which are gram negative organisms

Antibacterial Activity
Procedure for the Development Media: Bacillus subtilis and Staphylococcus aureus which are gram positive and Pseudomonas aureoginosa and Escherichia coli which are gram negative organisms were chosen in light of their clinical and pharmacological significance.The bacterial strains were got from Division of Microbiology, Osmania College, Hyderabad.Activities of present hydrazones are investigated on these microorganisms.The bacterial stock societies were hatched for one day at 37°C on supplement agar.The microscopic organisms were developed on Mueller-Hinton agar plates at 37°C.The stock cultures were kept up at 4°C for the development of organisms potato dextrose agar was utilized.Antibacterial activity of hydrazones was determined by using zone of hindrance technique

Preparation of Discs
Whatman No.1 channel paper plates of 5mm width were autoclaved by keeping in a spotless and dry Petri plate.The plates were absorbed compound answers for 5 h were taken as test material.Following 5 h the circles were dried in shade.The groupings of compound arrangements per plate are represented 0.1 g /1 ml.In this manner they were cautiously exchanged to spread on refined Petri plates.Channel paper plates inundated in ethanol are arranged and utilized as positive control and streptomycin as negative control.

Testing of antibacterial activity
LB agar medium was prepared and it was sanitized at 121°C for 30 minutes.The agar plates were prepared 21 by pouring about 10 ml of the medium into 10 cm Petri dishes under aseptic condition and left undisturbed for 2 h to harden the medium. 1 ml of inoculum (containing suspension) of microorganisms culture (Bacillus subtilis, Staphylococcus aureus, Pseudomonas aureoginosa and Escherichia coli) was poured on to the plates independently containing set agar media.The prepared sterile channel paper circles were impregnated with the sample solution and shaken completely and these test plates hatched for 2 days at 37 o C for the improvement of inhibitory zones and the average of two readings were recorded.

Measuring the diameter of inhibition zone
The inhibition zones were measured after 1 day at 37 o C. The diameter of the inhibition zone was measured and recorded with the aid of plastic ruler.Five paper discs placed in one Petri plate.

RESULTS AND DISCUSSION
The hydrazones are partially soluble in water, less soluble methanol more soluble in ethanol and readily soluble in acetonitrile (CH 3 CN), DMF and DMSO.The colours, formula weight, yields and elemental analysis of hydrazones are summarized in Table 1 The hydrazones are further characterized using (i) Fourier-Transform Infrared spectroscopy, (ii) NMR spectroscopy and (iii) Mass spectrometry (i) FT-IR spectroscopy: IR spectra of ATAH and ATBH are shown in Fig. 1 and 2 respectively.IR spectral data values and assignment of peaks are given in Table 2. (ii) 1 H-NMR spectroscopy 1 H-NMR spectra of ATAH and ATBH are shown in Fig. 3 and 4 respectively.Peak assignments are given in Table 3.

(iii) Mass spectra
Mass spectra of ATAH and ATBH are shown  Mass spectra of ATAH and ATBH show molecular ion peak at m/z values 182 and 244 respectively corresponding to their molecular weights.According to nitrogen rule even mass numbers of ATAH and ATBH indicate even number of nitrogen atoms.Fragmentation Scheme of ATAH is shown in Figure 7.  in Fig. 5 and 6 respectively.Fragmentation Schemes are given in Figs.7 and 8.
The diameters of the zones of complete inhibition were measured in millimeters(mm) and data are given in the Table 4.

Antibacterial assay
The antibacterial activity of hydrazones were screened against the Bacillus subtilis and gram negative bacterial species but at maximum concentrations the activity is not as expected due to less diffusion rate in agar medium.
In our present study we are investigating our compounds which are more antibacterial in nature and it makes platform for preparation of effective chemotherapeutics agents for therapy of human pathogenic diseases.

CONCLUSION
We have synthesized and characterized two closely related functionalized hydrazones bearing thiophenemoiety and evaluated their anti-bacterial activity.Acetoyl hydrazones are found to show more antibacterial activity than the corresponding benzoyl hydrazones.