A Multistep Synthesis of Sulfanilamide Background: An important area of research in the pharmaceutical industry is the discovery and development of new orally active antibiotics to treat bacterial infections. Commonly used antibiotics fall into several important classes including beta-­‐lactams, macrolides, the tetracyclines, the aminocyclitols, and the sulfa drugs. Each antibiotic has a different profile of biological activity, and all of them are used in modern medicine to treat various infections. The broad-­‐spectrum antibacterial activity of sulfanilamide (1) was first revealed in the mid-­‐
1930s by serendipity, as are many important discoveries in science. Over one thousand derivatives of sulfanilamide have been synthesized and tested as potential antibiotics, and some of these are still used today. The mode of action of the sulfa drugs is another interesting story because it provides some insights into strategies that might be generally exploited to design biologically active compounds. Early in the development of sulfa drugs as antibiotics, it was found that p-­aminobenzoic acid (PABA, 2), which is also used in sunscreens and sunblocks, inhibits the antibacterial action of sulfanilamide. Since p-­aminobenzoic acid and sulfanilamide are structurally similar, this discovery led to the speculation that the two compounds competed with each other in some biological process that was essential for bacterial growth. This speculation was eventually supported by experimentation. p-­Aminobenzoic acid is used by bacteria in the synthesis of the essential enzyme cofactor folic acid (3). When sulfanilamide is present, it successfully competes with p-­aminobenzoic acid for the active site in the enzyme that incorporates p-­aminobenzoic acid into folic acid. By functioning as a competitive inhibitor of this enzyme, sulfanilamide blocks the biosynthesis of folic acid, and without folic acid, the bacteria cannot grow. On the other hand, animal cells cannot synthesize folic acid, which is an essential vitamin, and it therefore must be part of the diet. Because only bacteria rely on the biosynthesis of folic acid from p-­aminobenzoic acid, the sulfa compounds are “ideal” drugs, as they kill only the bacteria and not the animal host. Of course, they are not truly ideal, since some people have allergic reactions to sulfa drugs and because bacteria develop resistance to them over time. Today and next week, we will be performing the reaction scheme shown below SAFETY NOTE: Chlorosulfonic acid is a strong acid and a strong irritant. It reacts violently with water, releasing HCl. Use ice to decompose any residual material. Concentrated ammonium hydroxide is caustic and may cause burns if it is allowed to come in contact with your skin. Wear latex gloves when handling or transferring this reagent, and use care when working with it. Should any of the liquid come in contact with your skin, immediately flood the affected area with cold water. Procedure: Step 1 (day 1). Chlorosulfonation of Acetanilide. Place 2.5g of acetanilide in a 125mL Erlenmeyer flask and, IN A FUME HOOD, heat the acetanilide on a hot plate attached to a ring stand until it is liquid, BUT DO NOT BOIL it. Allow the liquid to solidify, then, chill the flask in an ice bath. Add 6.5mL of cholorsulfonic acid (CAUTION: see safety note) and stopper the flask. While the chlorosulfonic acid is being added to the flask, clamp a water trap to a second ring stand. When the addition of 6.5ml of chlorosulfonic acid is complete, connect the reaction flask to a vacuum trap filled with ~30ml of water, keeping both flasks clamped to their respective ring stands. The glass tube inserted into the vacuum trap should be positioned ~1 inch above the surface of the water. Insert the rubber stopper at the end of the trap hose into the mouth of the reaction flask and place a new ice bath under the flask used as the trap. Continue stirring the reaction mixture and unclamp the reaction flask if necessary to swirl its contents. Let the flask stand at room temperature until the solid has dissolved (may need gentle swirling of flask). Heat the flask in a hot water bath for 10 minutes. Allow the flask to cool to room temperature and slowly pour the contents, with vigorous stirring, into a 250mL beaker containing 50mL of crushed ice. The hydrolysis is exothermic and a milky-­‐white precipitate is formed. Collect the solid by vacuum filtration, using two 5 mL portions of water to aid in the transfer. Press the solid with a spatula and using the vacuum, suck off as much water as possible. (NOTE-­‐this precipitate is fine and gelatinous. The dryer you can press the cholorsulfonate, the easier it will be to transfer into an Erlenmeyer flask for the next step). Transfer the wet sold to a tared 125mL Erlenmeyer flask. You should obtain about 4.25g of the wet solid. Step 2. Day 1 continued. Preparation of p-­‐
acetamidobenzenesulfonamide In a fume hood, add 25mL of 7.5M NH3 to the Erlenmeyer flask containing the product prepared in Step 1. Warm the flask on a hot plate in the hood for 10-­‐15 minutes (DO NOT BOIL the mixture). By the end of this period, some of the solid will have dissolved. Let the flask cool to room temperature, than chill it in an ice bath. Vacuum-­‐filter the solid, using 5 mL of water for washing. Air-­‐dry the product. We will allow this to dry for the week and use this material in step 3. Step 3. Day 2 Conversion to the Sulfanilamide. Place the crude p-­‐acetamidobenzenesulfonamide from step 2 in a 50 mL round bottom flask and add a solution of 2.5 mL of concentrated HCl in 5 mL of water. Equip the round-­‐
bottom flask with a condenser and heat the mixture at reflux for 20 minutes. At the end of the reflux period, cool the flask to room temperature and transfer the contentes to a 250 mL beaker, using 10 mL of water to wash out any residual material. Chill the beaker in an ice bath. Add small portions of a 10% NaHCO3 solution (~35 mL), stirring with a spatula and leeting the effervescence diminish between additions. Continue adding the NaHCO3 until the solution is alkaline to pH paper. Toward the end of the addition, the mixture will become a frothy paste. Vacuum-­‐filter the solid, using two 10 mL portions of cold water to wash the solid into the funnel. Air-­‐dry the solid overnight and determine the yield and melting point of the crude product. Purify the product by recrystallization (from hot water) if necessary??? Pre-­‐lab Please write up a brief procedural outline for the days experiment. Make sure you have a reaction scheme and a table of reagents in your lab notebook (including mmols and equivalents of the reagents used). Post-­‐lab questions: 1. What materials, organic or inorganic, may contaminate the crude sulfonyl chloride prepared in this reaction? Which of them are likely to react with the ammonia used in the next reaction step of the sequence? 2. Using curved arrows to symbolize the flow of electrons, write a stepwise mechanism for the conversion of acetanilide into 4-­‐acetamidobenzenesulfonic acid? 3. Why does the sulfonation of acetanilide only occur in the para position? 4. Following hydrolysis of 4-­‐acetamidobenzenesulfonamide with aqueous acid, the reaction mixture is homogeneous, whereas 4-­‐acetamidobenzenesulfonamide is insoluble in aqueous acid. Explain the change in solubility that occurs as a result of the hydrolysis. 5. What would be observed if 4-­‐acetamidobenzenesulfonamide were subjected to vigorous hydrolysis conditions, such as concentrated hydrochloric acid and heat for a long period of time? Write an equation for the reaction that might occur. 6. Calculate the overall yield of sulfanilamide obtained in the sequence of reactions that you performed. References: Techniques and Experiments for Organic Chemistry. Fessenden and Fessenden. 1983 A Small Scale Approach to Organic Laboratory Techniques, 3rd edition. Pavia, D., Lampman, G., et al. 2011 Cengage Learning