Synthesis, Characterization and Anti-Bacterial Activity of Isatin Schiff Base Derivatives

Initially, Isatin was treated with 4-amino acetanilide to form Isatin Schiff base, by the reaction of the free amino group on4-amino acetanilide with carbonyl group of Isatin in the presence of ethanol and glacial acetic acid with the elimination of water molecule. The obtained Isatin Schiff base was then treated with substituted aromatic aldehydes in the presence of potassium hydroxide and ethanol to form various Chalcone derivatives (C1-C7). The structures of the synthesized compounds (C1-C7) were examined and confirmed using IR, Mass spectroscopy, 1H-NMR and elemental analysis. By agar disc diffusion method, all given compounds were evaluated for their anti-bacterial activity against four bacteria at concentrations 25, 50 and 100 μg/ml. C4 and C6, were discovered to have possessed the most potent anti-bacterial activity.

Over the past years, potency of antibacterial therapy is somewhat in hesitant due to bacterial resistance to antibiotics. Quick development of drug resistant strains exerts a severe threat in present years. The lack of effective treatment is the main cause of this problem. The main challenge plunges into two parts: suitable target selection, mainly the requirement of pursuing molecular targets that are not susceptible to rapid resistance development and enhancement of chemical libraries to defeat the limitations of diversity particularly that which is essential to defeat barriers to bacterial species, especially, in gram negative organism. Even though there is advancement in expansion of antibacterial agents. The chemical reactivity and physical properties of Schiff bases are continued to be used to synthesize a large variety of various heterocyclic compounds and as a raw material for drug synthesis. Schiff bases are further known to possess wide spectrum of pharmacological activities such as antiviral, antimycobacterial, antibacterial, anti-inflammatory, cytotoxic, etc. Isatin shows a wide range of pharmacological activities reported in the literatures, including antiviral 12 , spermicidal 13 , anticorrosive 14 , analgesic 15 , anticonvulsant 16 , antioxidant 17 , antitubercular 18 , transthyretin fibrillogenesis inhibitory activity 19 , antidepressant 20 and antiepileptic 21 . The objective of the proposed study is to synthesize and evaluate the anti-bacterial activity of the compound with the aim to: • To Synthesize Schiff base from isatin derivative. • To incorporate the benzaldehyde derivatives to the synthesized Schiff base of isatin to form various chalcones ( Figure 1). • To characterize the new chalcone derivatives by IR, NMR, Mass spectroscopy and elemental analysis and to evaluate their anti-bacterial activity (in vitro)

Material and Methods
Melting point (mp) was determined in open capillary tubes and was uncorrected. The IR spectra were recorded in potassium bromide disks using IR Affinity-1 SHIMADZU spectrometer. The 1 H-NMR spectra were recorded on BRUKER ( 1 H NMR IN CDCl 3 ). The Chemical shifts were recorded in parts per million (ppm) relative to TMS as an internal reference. Mass spectra were recorded on a LCMS-2010A DATA REPORT SHIMADZU instrument using Fast Atom Bombardment (FAB Positive). The progress of all the reactions were monitored by readymade silica gel plates (Merck). Iodine and UV lamp were used as a developing agent. Spectral data (IR, 'HNMR and Mass spectra) were confirmed the structures of the synthesized compounds and the purity of these compounds were ascertained by microanalysis.

Synthesis of Isatin Schiff 's Bases
Equimolar quantity of isatin (1.47g, 0.01mol) and 4-amino acetanilide (0.01mol) was added into absolute ethanol (20ml) and refluxed for one hour in the presence of 2-3 drops of glacial acetic acid in round bottomed flask. The solvent was then distilled off and the product obtained was recrystallized using ethanol.

Synthesis of Chalcone Derivative. C1-C7
0.01 mol of isatin Schiff base (from step 1) and 0.01 mol of substituted benzaldehyde were mixed in 30 ml of ethanol in a RBF and placed in an ice bath. To this, 10 ml of 60% KOH solution was added with continuous stirring for 3 h at room temperature. The mixture was left still at room temperature overnight. Then, it was mixed with ice distilled water (40 mL), filtered and washed. Dried in air and recrystallized from rectified methanol to get the titled compound C1-C7 (Table 1). Figure 1. Synthetic scheme.

Antibacterial Activities
The standard strains of microorganisms were procured from the CSIR Instituted of Microbial Technology, Chandigarh, India. Agar disc diffusion method was used for the screening the anti-bacterial activities of all the synthesized compounds (C1-C7 Bacterial strains and yeast were cultured overnight at 37°C in Muller-Hinton broth and at 30°C in nutrient agar medium respectively. Amoxicillin was used as standard drugs for anti-bacterial 21 . In order to activate these cultures, subculture were freshly prepared, sterilized (autoclaved at 120°C for 30 min) and incubated at 37°C for 18 h to 24 h before use. A volume of 0.1 ml of each such culture was used as inoculums in all tests. The preparation of nutrient medium (broth), subculture, agar medium and peptone water was done following the standard procedure. The discs measurements were 6.25 millimeter in diameter and obtained from Whatman filter paper. The Stock of synthesized compounds (C1-C7) were diluted in 1% dimethyl sulphoxide to give 25 μg/ml, 50 μg/ml and 100 μg/ml as final concentration. A standard reference solution for gram negative and gram-positive bacteria were made by dissolving weighed amount of amoxicillin (50μg/ml) in sterile distilled water, separately. It was then incubated at 37°C for 24h. 0.1 mL of dimethyl sulfoxide was used to maintain the control and showed no inhibition. The zone of inhibition revealed by each compound was measured in millimeter.

Physical Data
The physical data and solubility data of all the title compounds are presented in the Table 2

Antibacterial Activity
All synthesized compounds showed a significant activity against all tested bacteria From the anti-bacterial study, it was discovered that in general the compounds with electron with drawing moiety showed better activity compared to compounds having electron releasing moieties.
Within the electron withdrawing moiety, compounds (C3 and C4) showed good activity among other tested compounds. The compounds (C3, C4 and C6) possessed activity towards entire tested microorganism. The zone of inhibition was observed and measured, and it is in the order of (C4>C3>C6).

Conclusion
The chemical structure and anti-bacterial activity of the synthesized compounds reveals that in general the compounds having electron withdrawing moiety exhibit better activity than compounds having releasing moieties.