EXPT 146 Synthesis Bioassay of Sulfanilamide th From K L Williamson R D Minard and K M Masters Macroscale and Microscale Organic Experiments 5 ed 2007 Houghton Mifflin Boston p 617 revised 10 18 06 PreLab Exercise What is the purpose of starting with the amide acetanilide in the beginning of the reaction scheme just to convert the amide to the primary amine sulfanilamide Introduction Paul Ehrlich the father of immunology and chemotherapy discovered Salvarsan an arsenical magic bullet a favorite phrase of his used to treat syphilis He hypothesized at the beginning of the 20th century that it might be possible to find a dye that would selectively stain or dye a bacterial cell and thus destroy it In 1932 I G Farbenindustrie patented a Prontosil a new azo dye that they put through routine testing for chemotherapeutic activity when it was noted that it had a particular affinity for protein fibers like silk H 2N N N SO2NH2 NH2 Prontosil Prontosil was found to be effective against streptococcal infections in mice but somewhat surprisingly it was ineffectual in vitro outside the living animal A number of other dyes were tested but only those having the group were effective French workers hypothesized that the antibacterial activity had nothing to do with the identity of the compounds as dyes but rather with the reduction of the dyes in the body to p aminobenzenesulfonamide known commonly as sulfanilamide On the basis of this hypothesis sulfanilamide was tested and found to be the active substance H2N SO2NHR Sulfanilamide R H Because sulfanilamide had been synthesized in 1908 its manufacture was not protected by patents so the new drug and thousands of its derivatives were rapidly synthesized and tested When the R group in sulfanilamide is replaced with a heterocyclic ring system for example pyridine thiazole diazine merazine and so on the sulfa drug so produced is often faster acting or less toxic than sulfanilamide Although they have been supplanted for the most part by antibiotics of microbial origin these drugs still find wide application in chemotherapy Unlike that of most drugs the mode of action of the sulfa drugs is now completely understood Bacteria must synthesize folic acid for growth Higher animals like humans do not synthesize folic acid and hence must acquire it in their food Sulfanilamide inhibits the formation of folic acid stopping the growth of bacteria and because the synthesis of folic acid does not occur in humans only bacteria are affected A closer look at these events reveals that bacteria synthesize folic acid using several enzymes including one called dihydropteroate synthetase which catalyzes the attachment of p aminobenzoic acid to a pteridine ring system When sulfanilamide is present it competes with the p aminobenzoic acid note the structural similarity for the active site on the enzyme O C OH H N H p Aminobenzoic acid H 2N N N N N OH O O H2 H2 O C N C C C OH H H H N OH Glutamic acid part Pteridine part p Aminobenzoic acid part Folic Acid This activity makes it a competitive inhibitor Once this site is occupied on the enzyme folic acid synthesis ceases and bacterial growth stops Folic acid can also be synthesized in the laboratory1 The Synthesis and Bioassay of Sulfanilamide This experiment is the synthesis and bioassay of sulfanilamide starting Acetanilide with reacts with chlorosulfonic acid in an electrophilic aromatic substitution The protecting amide group is removed from the p acetamidobenzenesulfonamide by acid hydrolysis The amide group is more easily hydrolyzed than the sulfonamide group O H N O O H CH3 N H CH3 p Acetaminobenzenesulfonyl chloride MW 233 68 NH2 CH3 NH3 HSO3Cl SO2Cl Acetanilide MW 135 16 mp 114 C N dil HCl SO2NH2 p Acetaminobenzenesulfonamide MW 214 25 SO2NH2 Sulfanilamide MW 172 20 mp 163 164 C IN THIS EXPERIMENT perfectly dry acetanilide is treated with chlorosulfonic acid a highly reactive reagent The hydrogen chloride evolved is trapped the reaction mixture is added carefully to water and the product p acetaminobenzenesulfonyl chloride is isolated by filtration This solid is added to aqueous ammonia to form p acetaminobenzenesulfonamide which is hydrolyzed with hot hydrochloric acid The resulting solution is neutralized with sodium bicarbonate to give sulfanilamide 1 L T Plante K L Williamson and E J Pastore in Methods in Enzymology eds D B MCCormick and L D Wright New York Academic Press 1980 66 533 aceta CAUTION Chlorosulfonic acid is a corrosive chemical and reacts violently with water Withdraw with a pipette and pipette Neutralize any spills and drips immediately The wearing of gloves and handling in the hood is required The chlorosulfonation of acetanilide in the preparation of sulfanilamide is conducted without solvent in a 25 mL Erlenmeyer flask Fit the Erlenmeyer flask with a septum connected to a short length of polyethylene tubing leading into a reaction tube that contains a small piece of damp cotton to trap hydrogen chloride vapors Figure 1 Place 0 25 g of acetanilide in the Erlenmeyer flask Add 0 625 mL of fresh chlorosulfonic acid pick up from the stockroom a few drops at a time to acetanilide using a Pasteur pipette not a syringe with metal needle Connect the flask to the gas trap between additions In 5 10 min the reaction subsides with only a few small pieces of acetanilide remaining undissolved Figure 1 Chlorosulfonation apparatus When this point has been reached heat the mixture on a sand bath for 10 min to complete the reaction cool the flask in ice and deliver the oily product by drops with a Pasteur pipette while stirring it into 3 5 mL of ice water contained in a 10 mL Erlenmeyer flask in the hood Should the material in the flask solidify it usually does not add 3 5 g of ice to the reaction flask Use extreme caution when adding the oil to ice water and when rinsing out any containers that have held chlorosulfonic acid Rinse the flask with cold water and stir the precipitated p acetaminobenzenesulfonyl chloride for a few minutes until an even suspension of granular white solid is obtained Collect and wash the solid with water on a Hirsch funnel After pressing and draining the solid transfer the solid to the rinsed reaction flask add 0 75 mL of concentrated aqueous ammonia solution ammonium hydroxide and 0 75 mL of water and in the hood heat the mixture over a hot sand bath to just below the boiling point with occasional swirling Heat the mixture in this manner for 5 min During this treatment a change can be