SYNTHESIS AND ANTIBACTERIAL ACTIVITY OF SOME HETEROCYCLIC DERIVATIVES OF SULFANILAMIDE

Considering the promising antimicrobial potential of carbonic anhydrase inhibitors and heterocyclic compounds some heterocyclic derivative s of sulfanilamide ( 2a-e) were synthesized. The diazotisation of sulfanilamide followed by substitu tion with ethylacetoacetate and further condensatio n y elded compounds 2a-c. Schiff base of sulfanilamide with salicylaldehyde on reaction with thioglycollic acid and chloroacetyl chloride resulted in compound 2 -e. The susceptibility of Staphylococcus aureus , Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa to the title compounds (300 μg/disc) was investigated and compared to that of nitrofurantoin (300 μg/disc) and ciprofloxacin (25 μg/disc). The title compounds showed good antimicrobial activity.


INTRODUCTION
The field of research on development of carbonic anhydrase inhibitor based antimicrobials has shown promising results due to presence of carbonic anhydrases in a multitude of bacteria and protozoa [1][2][3].Sulfanilamide, a prototype of carbonic anhydrase inhibitor has good potential for antibacterial action [4][5].Conjugations of heterocyclic groups reportedly enhance antibacterial action of the original compound [6].Besides derivatives of azetidinone, thiazolidinone [7], oxazoles [8] and imidazoles [9] are widely reported with antibacterial action.It is thus envisageable that conjugation of heterocyclic compounds with sulfanilamide will be able to enhance the antibacterial action of sulfanilamide leading to novel types of pharmacological agents useful in the fight against infections.Keeping this in view, we have attempted synthesis of heterocyclic derivatives of sulfanilamide, and investigated the in vitro susceptibility of two gram-positive bacteria (S. aureus, E. faecalis) and two gram-negative bacteria (E. coli and P. aeruginosa) to them.

EXPERIMENTAL
The synthesized compounds 2a-e (Scheme 1) were purified with repeated washing and recrystallisation from different solvents.Purity of compounds was checked by TLC using chloroform: methanol: DMF (100+10+05 v/v) as developing solvents and iodine as visualizing agent.Melting points were determined in open capillary tubes (Sisco) and were uncorrected.Infrared spectra were recorded on Shimadzu-8400S spectrophotometer using KBr powder. 1 H NMR spectra were recorded in CDCl 3 on a Bruker DRX-300 NMR spectrophotometer (300 MHz) using TMS as internal standard.The CHN elemental analysis was carried out using EURO-EA elemental analyzer.a. sodium nitrite, b. ethylacetoacetate, c. phenyl hydrazine, d. hydrazine hydrate, e. hydroxylamine hydrochloride, f.Salycylaldehye, g. chloroacetyl chloride, h.thioglycollic acid.
Scheme 1. Synthesis of heterocyclic derivatives of sulfanilamide.
Synthesis of compound 1 and 2a-e were done as per the reported method [7][8] with little modification.(1).Sulfanilamide (0.01 mol) was taken as the starting material in a mixture of HCl (8 mL) and water (6 mL).It was cooled to 0 o C in an ice bath and a cold aqueous solution of sodium nitrite (0.03 mol) was added.The diazonium salt solution was filtered directly into a cold solution of ethylacetoacetate (0.01 mol) and sodium acetate (0.122 mol) in ethanol (50 mL).The resulting solid was washed with water and recrystallised from alcohol to yield ethyl-3-oxo-2-(2-(4-sulfamoylphenyl) hydrazono) butanoate (1).

RESULTS AND DISCUSSION
The physico-chemical data were used to characterize the compounds.The synthesized compounds exhibited characteristic IR (KBr, ν cm -1 ) peaks in the region of 3319.For the in vitro screening pure strains were obtained from Post Graduate Department of Microbiology, Orissa University of Agricultural Technology, Bhubaneswar, India.The organisms were identified [7] and screened using disc diffusion method [11][12].The compounds were dissolved in dimethyl formamide (6%), which was previously tested for antibacterial activity against all test bacteria and found to have no antibacterial activity.A solution of concentration 30 mg/mL was made for each test compounds and finally sterilized by filtration using 0.45 µm millipore filters.The sterile discs (Hi-media, 6mm) were impregnated with 10 µL of the test solutions (300 µg/disc) and placed in inoculated agar.The density of the bacterial suspension was standardized by using McFarland standard method [8][9].Nitrofurantoin (300 µg/disc) and ciprofloxacin (25 µg/disc) were used as standard drugs.The control was prepared using dimethyl fomamide.The inoculated plates were incubated at 37 o C for 24 h.The antibacterial activity of test compounds against the bacterial strains is given in Table 1 as zone of inhibition (mm).
The control did not show any zone of inhibition.Compounds 2a-e exhibited significant (p < 0.001) antimicrobial action compared to control.Compound 2d showed highest zones of inhibition against E. coli and P. aureginosa.Activity was better for 2b, 2c and 2d, against S. aureus.Compared to nitrofurantoin, most of the compounds exhibited comparable or better antimicrobial activity against all the strains (Table 1).The zone of inhibition against E. faecalis was highest for compound 2c.All the compounds exhibited better zones of inhibition than that of sulfanilamide against all microbial strains.Analysis of structural features reveals that substitution with heterocyclic group has increased the antimicrobial potential of sulfanilamide and the increment was more pronounced for the thiazolidinone derivative.