Synthesis and Antimicrobial Activity of Some Novel N-Mannich Bases of Imidazole Phenylazetidin-2-one

Azetidine-2-one (β-lactam) chemistry is of great importance because of the use of β-lactam derivatives as antibacterial agents. Since the discovery that the structure of penicillin contains a β-lactam function, a vast amount of effort has been devoted to producing other β-lactam antibiotics with a wider spectrum of activity and a greater resistance to enzymic cleavage by β-lactamases. The synthesis of β-lactam antibiotics has occupied an important place in the field of medicinal and research pharmaceutical. The antibiotic activity of Azetidine-2-ones (β-lactam) possessing antiviral, antifungal activities, antithrombotic and cholesterol inhibition [1-4].


Introduction
Azetidine-2-one (β-lactam) chemistry is of great importance because of the use of β-lactam derivatives as antibacterial agents. Since the discovery that the structure of penicillin contains a β-lactam function, a vast amount of effort has been devoted to producing other β-lactam antibiotics with a wider spectrum of activity and a greater resistance to enzymic cleavage by β-lactamases. The synthesis of β-lactam antibiotics has occupied an important place in the field of medicinal and research pharmaceutical. The antibiotic activity of Azetidine-2-ones (β-lactam) possessing antiviral, antifungal activities, antithrombotic and cholesterol inhibition [1][2][3][4].

Experimental Section Instrumentation and chemicals
All the chemicals used in the present investigation were purchased from Sigma-Aldrich Chemicals company, Inc. USA. And used without further purification. Thin Layer Chromatography was performed on aluminium sheet of silica gel 60F254, E-Merk, Germany using iodine as visualizing agent. Melting points were determined in open capillary tubes on Mel-Temp apparatus and are uncorrected. Column chromatography was performed on silica gel with different solvent systems as eluents to afford the pure compound. The IR Spectra were recorded as KBr pellets on Perkin-Elmer 1000 units, instruments. All 1 H and 13 C-NMR spectra were recorded on a Varian XL-300 spectrometer operating at 400MHz for 1 H -NMR and 75 MHz for Hawker's (Glucose 5 gr; KNO 3 3.5 g; KH 2 PO 4 1.75 g; MgSO 4 .7H 2 O 0.75 g and distilled water 1000 mL) media used for fungi growth. The media chemicals present study purchased from Merck. The standard bacterial and fungal strains were procured from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), and Chandigarh, India. The pure bacterial cultures were maintained on Nutrient Agar Media (NAM) for bacterial and fungal culture on potato dextrose agar (PDA).
The antimicrobial activity of these newly synthesized 3-chloro-4-(1-(morpholinomethyl) / ((4-methylpiperazin-1-yl) methyl)-1H-imidazol-4-yl)-1-(4-substituted phenyl) azetidin-2-one 7(a-h) performed according to Agar well diffusion method is preferred to be used in this study since it was found to be better than the disc diffusion method suggested by Parekh et al. [15] and also recommended by the National Committee for Clinical Laboratory. The synthesized compounds were used at the concentration of 2 mg/mL DMSO as a solvent [16]. A standardized 1 to 2 x 10 7 cfu/mL 0.5 MC Farland standard was introduced onto the surface of a sterile agar plate and evenly distributed inoculums by using a sterile glass spreader. Simultaneously, 6 mm wells were cut from the plate using a sterile cork borer. 50µl solution at a concentration of 2mg/mL of the compounds was introduced into well and incubated at 37ºC for 24 hrs, the inhibition zones were measured with a ruler and compared with the control well containing only 1mg/mL in DMSO of streptomycin as the standard. The antifungal assay of the compounds was carried out by agar well diffusion method as described by Magaldi et al. [17] 6 mm diameter open wells punched with a sterile cork borer on cultured plates with test organisms before incubated. The wells were filled with 50 µl solution at a concentration of 2 mg/mL of the compounds at 30°C. After 72 hours, the zones of inhibition were measured and compared with those of the control DMSO and the standard Fluconazole at a concentration of 1 mg/mL.

Reagents and Conditions:
( i ) Addition of alcohol , Glc CH 3 COOH and heated on steam bath for 4-5 hrs at 100 o C, after standin g for 24 hrs at room temparature.
To this solution, Farmaldehide (0.05 mol, 15 mL) and DMF (10ml) were added in ice cold condition and stirred for 2 hours in an ice bath and left over night at room temperature. The progress of the reaction was monitored by Thin Layer Chromatography using cyclohexane and ethylacetate (7:3) solvent mixture as a mobile phase. At the end of the reaction dichloromethane (30ml) was added to the mixture followed by neutralization with 50ml of 1N NaOH solution, after neutralization the mixture was extracted with CH 2 Cl 2 (3x25 mL). The combined extract was dried on anhydrous Na 2 SO 4 . After filtration, the solvent was removed with rota evaporator. The residue was purified by column chromatography, using 60-120 mesh silica and CHCl 3 solvent was used as an elutent. Finally the product 3-chloro-4-(1-((4-methylpiperazin-1-yl) methyl) -1H -imidazol -4 -yl) -1 -phenylazetidin-2-one (7e) was purified from aqueous dimethyl formamide. Yield 68% with 0.48g, m p 142-144 o C.
The structure of these newly synthesized compounds of 7(e-h) were established by IR, 1 H-NMR, 13 C-NMR, mass data and elemental analysis.