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Background • First synthesized in 1908 • Used primarily for Streptococcal species • Domagk received Nobel Prize for derivatives in 1940 • Large influx of derivatives in 1940s • P-aminobenzoic acid (PABA) is a substrate that binds with the enzyme dihydropteroate synthase to produce folic acid which is necessary for cell replication • Sulfanilamide is analogous structurally and chemically to PABA – inhibiting the production of folic acid • Antibacterial resistance on the rise and one way is to combat with new drugs Fig 1. Sulfanilamide (left) Fig 2. Structure of Folic Acid and P-aminobenzoic acid (right) Combating Antibacterial Resistance: Synthesis of Sulfanilamide Derivatives and their Efficacy Mariecris Madayag, Department of Biology, Derivatives Space filling model of Sulfanilamide http://www.chm.bris.ac.uk/motm/ sulfanilamide/sulfa1.gif Discussion •According to IR and ¹³C APT NMR spectra, the overall syntheses of derivatives were successful, except Diisopropylamine. • Dihydroxypyridazine significantly inhibited several organisms possibly due to high reactivity of hydroxyl groups. •Dimethylsulfamide also inhibited saccharomyces, a eukaryotic cell, successfully which Sulfanilamide did not. •Tetracholoroaniline and Thiabendazole were not soluble to be fully effective, a problem that arose in Woods (1940). Future Work Methods I HN O S O O OH Cl HN O S O O Cl NH 2 Cl Cl Cl Cl HN O S O O NH Cl Cl Cl Cl HCl H 2 O NH 3 S O O NH Cl Cl Cl Cl Cl N aH C O 3 S NH Cl Cl Cl Cl O O NH 2 4-amino- N -(2,3,5,6- tetrachlorophenyl)benzen esulfonamide Hypothesis/Objectives H0 : There is a difference between the efficacy of sulfanilamide derivatives and sulfanilamide (control) against microorganisms. • Successfully synthesize derivatives and confirm with instrumentation • Test derivative efficacy by running minimum inhibition S NH Cl Cl Cl Cl O O NH 2 4-amino- N -(2,3,5,6- tetrachlorophenyl)benzenesulfonamide NH 2 S O O NH S O O N 4-amino- N -( N , N - dimethylsulfamoyl)benzenesulfonamide NH 2 S O O N 4-amino- N , N - diisopropylbenzenesulfonamide Figure 3. Proper safety for handling chlorosulfonic acid My Sulfanilamide Derivatives time and guidance she has given me, even during the summer and weekends. I would also take Dr. Singleton for his understanding, guidance, and also his weekends! Thank you to everyone who have helped me with this project both from the Chemistry and Biology departments. Literature • Woods, D.D. 1940. The relation of para-aminobenzoic acid to the mode of action of sulfanilamide. British Journal of Experimental Pathology 21:74-90. • Wood, W.B. 1941. Studies on the antibacterial action of Sulfonamide Drugs. JEM 369-381. • Badr, E. E. 2008. Novel Sulfanilamide as potent surfactants and antibacterial agents. Journal of Dispersion Science and Technology 29:1143-1149 • Williamson, K.L., Minard, R.D., and Masters, K.M. 2007. Macroscale and Microscale Organic Experiments . Houghton Mifflin: Boston, MA. Results •Dimethylsulfamide (n=64) •Inhibited only saccharomyces at 0.1M •Tetracholoroaniline and Thiabendazole (n=64) did not yield in inhibition. •Diisoproprylamine was not synthesized successfully. Figure 9. ¹³C APT NMR spectrum of 2,3,5,6- Tetrachloroaniline Derivative Methods II •Bacteria diluted to 0.5 MacFarlin then to 1:1,000 •Column 1 – 100ul antibiotic, 50ul nutrient broth •Columns 2-10 – 50ul broth, 50ul culture, 50 ul transferred •Column 11- Control #1 – Bacteria and broth only •Column 12 – Control #2 – Drug and broth only NH 2 NH 2 S C O O NH 2 O HO H N H 2 C N N N N H 2 N OH O H N CH O OH CH 2 CH 2 O OH Figure 4. Reflux Set-Up (right) Figure 5. Recrystal lized products NH 2 S O O N SH N NH NH 2 S O O NH N N HO OH Thiabendazole Dihydroxypyridazi ne S. Aureus E. Coli B. cereus K. pneumonae P. aerugino sa S. pyogenes Saccharom yces 0.0 0.1 0.2 0.3 0.4 Sulfanilamide Dihydroxypyridazine * * * * M inim um Inhibition Concentration (M ) Figure 10. Comparison of minimum inhibition concentration (M) in various organisms between Sulfanilamide (control) and Dihydroxypyridazine. Bars represent means (n=64) and standard error of means with error bars. Data analysis conducted results in significant difference(*) p=0.0003, p=0.0014, p=0.0029, respectively. Figure 6. 0.5 MacFarlin dilution Figure 7. Sample 90-well plate Figure 8. Stack of sample well plates
