Synthesis and Biological Activity of N-Substituted-3-chloro-2-azetidinones

2-Aminobenzothiazole-6-carboxylic acid (1), on condensation with chloroacetyl chloride yielded 2-(2-chloroacetylamino)benzothiazole-6-carboxylic acid (2), which on amination with hydrazine hydrate yielded in turn 2-(2-hydrazinoacetylamino)benzothiazole-6-carboxylic acid (3). Compound 3, on condensation with various aromatic aldehydes afforded a series of 2-{2-[N’-(arylidene)hydrazino]acetylamino}benzothiazole6-carboxylic acids 4a-h, which upon dehydrative annulation in the presence of chloroacetyl chloride and triethylamine yielded 2-{2-[3-chloro-2-(aryl)-4-oxoazetidin-1ylamino]-acetylamino}benzothiazole-6-carboxylic acids 5a-h. The synthesized compounds 4a-h and 5a-h were screened for their antibacterial activity against four microorganisms: Staphylococcus aureus (Gram positive), Bacillus subtilis (Gram positive), Psuedomonas aeruginosa (Gram negative) and Escherichia coli (Gram negative). They were found to exhibit good to moderate antibacterial activity. The antifungal activity of these compounds were also tested against three different fungal species. None of them were active against the species tested.


Introduction
The β-lactam heterocycles are still the most prescribed antibiotics used in medicine.They are considered as an important contribution of science to humanity [1].The most widely used antibiotics such as the penicillins, cephalosporins, carumonam, aztreonam, thienamycine and the nocardicins all contain β-lactam rings [2].The long-term use of β-lactam antibiotics exerts selective pressure on bacteria and permits the proliferation of resistant organisms [3].A comparative study of current antibiotics with those from previous decades shows an alarming increase in bacterial resistance to βlactam antibiotics [4].The development of several synthetic and semi-synthetic β-lactam antibiotics by the pharmaceutical industry was due to the growing resistance of bacteria towards the β-lactam antibiotics and the need for medicines with a more specific antibacterial activity [5].An interesting group of β-lactams are the monocyclic β-lactams, which are molecules that do not contain another ring fused to the β-lactam one.
Hence, with a view to further assess the pharmacological profile of this class of compounds, it was thought worthwhile to synthesize some new congeners of β-lactam heterocycles by incorporating the 2-aminobenzothiazole and azetidinone moieties in a single molecular framework.The present work deals with the synthesis of the title compounds starting from 2-aminobenzothiazole-6-carboxylic acid, followed by their antimicrobial screening.

Antibacterial activity
To determine the antibacterial activity of these agents, the cup plate method was used, with Ampicillin and Streptomycin as the reference antibiotics [22].The prepared compounds were examined against two strains each of gram positive and gram negative bacteria.The test results, presented in Table 2, suggest that compounds 5e, 5g, 5h and 4e, 4f, 4g, 5f, 5h are highly active against S. aureus and E. coli respectively.Compounds 4g and 4h are also highly active against P. aeruginosa.The rest of the compounds were found to be either moderately active, slightly active or inactive against the tested microorganisms.

Antifungal activity
The antifungal activities of the prepared compounds were tested against three different fungi such as C. tropicans, A. niger and F. heterosporium by filter paper disc technique [23].None of the compounds were found to be active against the fungi species tested.

General
Melting points were determined in open capillaries on Thomas Hoover apparatus and are uncorrected. 1H-NMR spectra were recorded on a Bruker AM400 (400 MHz) instrument using tetramethylsilane (TMS) as an internal standard and DMSO-d 6 as a solvent.Chemical shifts are given in parts per million (ppm).Mass spectra (MS) were recorded on Schimadzu GC-MS.Infrared spectra were recorded on Schimadzu-IR Prestige 21.Elemental analysis (C, H, N) was performed on Perkin Elmer 240 analyzer and all compounds are within ±0.4% of theory unless otherwise specified.All products were purified by recrystallisation.The reactions were followed up and the purity of products was carried out on pre-coated TLC plates (Silica gel 60 F 254 , Merck), visualizing the spots under ultraviolet light.Column chromatography was performed on Merck silicagel (60-120 mesh).The antimicrobial screening was carried out at Chemo Test Laboratory.2-Aminobenzothiazole-6carboxylic acid (1): was prepared by a reported method [21].

Antibacterial activity
The cup plate method using Hi-Media agar medium was employed to study the antibacterial activity of 4a-h and 5a-h against S. aureus (ATCC6538P), B. subtilis (ATCC6633), P. aeruginosa (ATCC9027) and E. coli (ATCC10536) [22].Preparation of nutrient broth, subculture, base layer medium, agar medium and peptone water was done as per the standard procedure.Each test compound (50 mg) was dissolved in dimethylformamide (50 mL, 1000 μg/mL), which was used as sample solution.Sample size for all the compounds was fixed at 0.1 mL.Using a sterilized cork borer cups were scooped out of agar medium contained in a petri dish which was previously inoculated with the microorganisms.The test compound solution (0.1 mL) was added in the cups and the petri dishes were subsequently incubated at 37 °C for 48 h.Ampicillin and Streptomycin were used as reference drugs and dimethylformamide as a negative control.Zones of inhibition produced by each compound was measured in mm, and the results are listed in Table 2.

Antifungal activity
The antifungal activities of compounds 4a-h and 5a-h were tested against three different fungi such as C. tropicans (ATCC9763), A. niger (ATCC16404) and F. heterosporium by the filter paper disc technique [23].

Table 2 .
Antibacterial activity of the compounds