Synthesis and Biological Activity of 3-Chloro-1-( 4-perimidine methylcarbonyl amino )-4-phenyl-azetidin-2one

Perimidine-1-acetic acid hydrazide (1) undergoes facile condensation with aromatic aldehydes to afford the corresponding azomethine (i.e. Schiff base) derivatives (2a-h) in good yield. Cyclocondensation of compounds (2a-h) with chloro acetyl chloride affords 3-chloro-1-(4-perimidine methylcarbonylamino)-4phenyl-azetidin-2-ones (3a-h). The structures of these compounds were established on the basis of analytical and spectral data. The newly synthesized compounds were evaluated for their antibacterial and antifungal activities.


Introduction
Perimidine derivatives are of wide interest because of their diverse biological activities and chemical application.Several classical synthetic methods have been reported for synthesis of perimidine derivatives.The most widely used method for preparation of perimidine is the cyclocondensation reaction of 1, 8-diaminonaphthalene with carboxylic acids under reflux condition microwave irradiation [1][2][3][4][5][6][7][8][9][10][11][12][13][14] .These heterocyclic systems find wide use in medicine, agriculture and industry.A large number of azetidinones containing β-lactam rings [17][18][19][20] are known to exhibit various biological activities like antibacterial, antifungal 21 and antibiotic 22 activities.More particularly and recently these types of compounds have been found in the treatment of T.B. and other chemotherapeutic diseases.Hence, it was thought of interest in merging of both azetidinone and perimidine-1-acetic acid hydrazide moieties may enhance the drug activity of compounds up to some extent or might posses some of the above mentioned biological activities.From this point of view, the objective of the present work is to prepare new derivatives of perimidine-1-acetic acid hydrazide containing an azetidinone moiety.Hence the present communication comprises the synthesis of 3-chloro-1-(4-perimidine methylcarbonylamino)-4-phenyl-azetidin-2-one.The research work is scanned in Scheme 1.

Experimental
Melting points were determined in open capillary tubes and are uncorrected.The IR spectra were recorded in KBr pellets on a Nicolet 400D spectrometer and 1 H NMR and 13 C NMR spectra were recorded in DMSO with TMS as internal standard on a Brucker spectrometer at 400 MHz and 100 MHz, respectively.LC-MS of selected samples taken on LC-MSD-Trap-SL_01046.

Materials
2-Methyl perimidine (1) was prepared by reported method 15 and other materials were used as AR grade.

Preparation of perimidine-1-acetic acid hydrazide
Equimolar solution of 2-methyl perimidine (0.1 mole) in the dry acetone (60 mL) and ethyl chloroacetate (0.1 mole) in the presence of anhydrous K 2 CO 3 (5 g) was refluxed for 8 h, cooled and the solid thus obtained was filtered, dried and crystallized from ethanol to furnish 1, yield is about 70%.m.p. 150 0 C. and this compound (0.05 mole) and hydrazine hydrate (0.05 mole) in 1,4-dioxane (35 mL) was refluxed on heating coil for 5 h. the excess of solvent was removed and the product crystallized from methanol to give 1, yield is about 80%, m.p. 122 0 C 16 .

Preparation of azomethyl derivatives (2a-h)
An equimolecular mixture of perimidine-1-acetic acid hydrazide (0.01 mole) and the aromatic aldehyde (a-h) in ethanol (15 mL) and conc.H 2 SO 4 (0.4 mL) was refluxed on a water bath for 1-2 h.The solid separated was collected by filtration, dried and recrystallized from ethanol.The yields, melting points and other characterization data of these compounds are given in Table 1.

Results and Discussion
It was observed that Perimidine-1-acetic acid hydrazide (1) on condensation with aromatic aldehydes to yield azomethyl derivatives (2a-h).The structures of (2a-h) were confirmed by elemental analysis and IR spectra showing absorption band at 1620-1640(C=N).The C,H,N analysis and 1 H NMR data of all compounds are presented in Table 1.
The cyclocondensation of (2a-h) with chloroacetyl chloride resulted in formation of 2azetidinones (3a-h).The structures assigned to (3a-h) were supported by the elemental analysis and IR spectra showing absorption bands at 1620-1640(C=N of-perimidine) and 1740-1750(C=O of monocyclic β-lactam).The C, H, N analysis and 1 H-NMR data of all compounds are presented in Table 2.
The examination of data reveals that the elemental contents are consistence with the predicted structure shown in scheme1.The IR data also direct for assignment of the predicted structure.The final structure of all compounds is confirmed by LC-MS data of selected samples.The LC-MS of samples 3c and 3f give the molecular ion peak (m/z) at 440 and 457 respectively.These values are corresponds to their molecular weight.

Biological screening Antibacterial activities
Antibacterial activities of all the compounds were studied against gram-positive bacteria (Bacillus subtilis and staphylococcus aureus) and gram-negative bacteria (E coli, Salmonella typhi and Klebsiella promioe) at a concentration of 50 µg/mL by agar cup plate method.Methanol system was used as control in this method.Under similar condition using tetracycline as a standard for comparison carried out control experiment.The area of inhibition of zone measured in cm.Compound 3d, 3f and 3g were found more active against the above microbes.Other compounds found to be less or moderate active than tetracycline (Table 3).

Antifungal activities
The fungicidal activity of all the compounds was studied in vitro at 1000 ppm concentration.Plant pathogenic organisms used were aspergillus, Nigrospora sp., Fusarium oxysporium, Botrydepladia thiobromine and Albicans.The antifungal activity of all the compounds (3a-h) was measured on each of these plant pathogenic strains on a potato dextrose agar (PDA) medium.Such a PDA medium contained potato 200 g, dextrose 20 g, agar 20 g and water one liter.Five days old cultures were employed.The compounds to be tested were suspended (1000 ppm) in a PDA medium and autoclaved at 120 o C for 15 min.at 15 atm.pressure.These medium were poured into sterile Petri plates and the organisms were inoculated after cooling the Petri plates.The percentage inhibition for fungi was calculated after five days using the formula given below: Percentage of inhibition = 100(X-Y) / X Where, X = Area of colony in control plate Y = Area of colony in test plate The fungicidal activity displayed by various compounds (3a-h) is shown in Table 4.