MSci ‘Chemistry with Biomedicine’ 4CCC0150 Integrated Chemistry Laboratory Experiment 19 Synthesis and analysis of Sulphanilamide Introduction In this experiment you will use a multi-‐step process to synthesise an antibacterial drug. You will then use reversed phase TLC to analyse the effect of different substituents on the lipophilicity of the drug. Learning Objectives •
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Multi step organic synthesis Antibacterial properties of sulphonamide drugs Principles of reversed phase TLC Before the laboratory workshop: Before arriving at the workshop for this laboratory you should do the following: • read and understand the following background information, safety information and instructions • write any pre-‐lab information you need in your laboratory notebook, including a table of reagents and their physical properties • complete the online quiz on KEATS • obtain a melting point of the product from the literature, complete with reference • write a balanced equation and a mechanism for each step in the reaction Background Theory Sulphanilamide was one of the first compounds developed for the clinical treatment of bacterial infections. It was discovered accidentally when biologists first noted that the biological stain known as Prontosil was toxic to bacteria. Further research then demonstrated that in vivo Prontosil decomposed to sulphanilamide and it was this compound that acted as an inhibitor of bacterial growth. Sulphanilamide and derivatives of it have been credited for saving the lifes of many wounded during World War II by preventing infection. Sulphanilamide can be synthesised from acetanilide in 3 steps following the scheme in fig 1. In the first step acetanilide undergoes chlorosulfonation which is followed by the reaction with ammonia to give the sulphonamide. Finally the acetyl amide group is hydrolysed to give sulphanilamide as the final product. Derivatives of sulfanilamide can be prepared by reacting the sulfonyl chloride formed in step 1 with different amines. In this experiment you will look at how different substituents affect the lipophilicity of the sulfonamide. MSci ‘Chemistry with Biomedicine’ 4CCC0150 Integrated Chemistry Laboratory Figure 1. Synthetic scheme for the synthesis of sulphanilamide The partition coefficient (P) of a drug can be an important factor in drug design as it describes the lipophilic character of the drug and therefore has implications for controlling whereabouts in the body drug action is most likely to occur. Hansch and coworkers derived an equation to calculate substituent constants for the contribution of individual atoms or groups to the partition coefficient. These are known as the lipophilicity constant, π, and are given by equation 1: !! = log
!!"
!!"
[1] Where, !!" and !!" are the partition coefficients, determined in a 1-‐octanol/water system, of substituted and unsubstituted compounds respectively. Thus the π constant indicates a change in the logarithm of the partition coefficient when a substituent of group X is introduced. A positive π constant means the substituent makes the drug more lipophilic compared to an H group and a negative substituent indicates that the drug is less lipophilic with the substituent in place. However, the direct measurement of partition coefficients can be tedious and is often difficult, therefore a simpler method, based on reversed phase thin layer chromatography, which uses the Rm value is often used. The Rm value is calculated from the Rf value for a substance using reversed phase chromatography. Equation 2 shows how to calculate the Rm value for neutral substances and equation 3 is used for acids and bases. !" = !"#
!" = !"#
!
!"
!
!"
− 1 [2] − 1 + log
!! ![! ! ]
[! ! ]
[3] Where !! , is the dissociation constant of the acid or base and [! ! ] is the hydrogen ion concentration of the mobile phase. The difference in the Rm value for the substituted and unsubstituted compounds is equal to the lipophilicity constant, π: !! = !"!" − !"!" [4] In this experiment you will synthesise the unsubstituted derivative and then use reversed phase chromatography to determine the lipophilicity constant for a series of substituents found in commercial sulphonamides. MSci ‘Chemistry with Biomedicine’ 4CCC0150 Integrated Chemistry Laboratory Safety Information Chemical Acetanilide Precautions Wear gloves, safety glasses and laboratory coat. If in contact with eyes or skin, rinse immediately with plenty of water. Chlorosulfonic acid Reacts violently with Wear gloves, safety glasses and laboratory Fumehood water. Causes severe coat. This must remain in the fume burns. Irritant. cupboard at all times. Add ice to any glassware which has contained this reagent prior to washing up. Conc. Aqueous ammonia Causes burns. Very Wear gloves, safety glasses and laboratory Fumehood toxic to aquatic systems coat. 6 M Hydrochloric acid Causes burns, irritating Wear gloves, safety glasses and laboratory to respiratory system coat. Saturated Sodium carbonate N/A Wear gloves, safety glasses and laboratory coat. If in contact with eyes or skin, rinse immediately with plenty of water. Ethanol Flammable Wear gloves, safety glasses and laboratory coat. Keep away from sources of ignition. Sulfamethazine Do not breathe dust. Keep away from sources of ignition. Wear Avoid contact with skin gloves, safety glasses and laboratory coat. and eyes If in contact with eyes or skin, rinse immediately with plenty of water. Sulfamethoxypyridazine Irritant. Risk of serious Wear gloves, safety glasses and laboratory damage to eyes coat. If in contact with eyes or skin, rinse immediately with plenty of water. Sulfathiazole Irritant. Wear gloves, safety glasses and laboratory coat. If in contact with eyes or skin, rinse immediately with plenty of water. Acetone Flammable. Irritant. Keep away from sources of ignition. Wear May cause drowsiness gloves, safety glasses and laboratory coat. If in contact with eyes or skin, rinse immediately with plenty of water. 1-‐octanol Irritant Wear gloves, safety glasses and laboratory coat. If in contact with eyes or skin, rinse immediately with plenty of water. Diethyl ether Extremely flammable. Keep away from sources of ignition. Wear May form explosive gloves, safety glasses and laboratory coat. peroxides. Harmful. If in contact with eyes or skin, rinse May cause drowsiness immediately with plenty of water. Phosphate buffer pH 7.4 N/A Wear gloves, safety glasses and laboratory coat. Dimethylaminobenzaldehyde Harmful. Toxic to Wear gloves, safety glasses and laboratory spray aquatic systems coat. If in contact with eyes or skin, rinse immediately with plenty of water. Use in the fumehood Instructions Hazards Harmful. Irritant. MSci ‘Chemistry with Biomedicine’ 4CCC0150 Integrated Chemistry Laboratory NOTE: This must be done the week before you are due to start this experiment. Prepare a RP TLC plate by marking a plate with your initials, a baseline and 4 crosses for each sample. Soak the plate in the 5 % v/v 1-‐octanol solution in diethyl ether. Leave the plate to dry until the last lab session. Set up the apparatus as shown in the picture on the left. Connect the rubber tubing to the arm on the receiver and place the pipette into a test tube that contains a small amount of water and a piece of damp cotton in the neck to trap hydrogen chloride vapors. Make sure all apparatus are secured with a clip or clamped. Add your purified acetanilide (18.5 mmol) to the flask below the dropping funnel and cool in a cold water bath. Add chlorosulfonic acid (6.25 mL) in one go using the funnel while stirring rapidly (NOTE: chlorosulfonic acid must be dispensed in a fumehood using the cylinder provided. Do not remove the cylinder from the fumehood. Place a stopper in your funnel and check for leaks before transporting the acid to your fumehood). After the reaction has subsided (5-‐10 min), remove the water bath and allow it to reach room temperature. Remove the trap and heat the mixture on a steam bath for 20-‐30 min to complete the reaction. Cool the flask to room temperature and add the contents dropwise, with stirring, to a 500 mL beaker containing ice (150 g). Stir the precipitated p-‐acetamidobenzenesulfonyl chloride until an even white suspension of granular solid is obtained, break up any large lumps with a glass rod. Collect the solid by vacuum filtration and wash with cold water until the filtrate is neutral to pH paper. Air dry and record the crude yield. Transfer your product to a conical flask, add conc. Aq NH3 (7.5 mL) and water (7.5 mL) to your product and heat on a steam bath until just below boiling, then continue to heat for 5 min. Cool the suspension in an ice bath and collect the amide using vacuum filtration. Record a crude yield for the product. Transfer this solid to a round bottom flask and add conc. 6 M HCl (10.0 mL) and water (5 mL), boil gently, while stirring for 30 mins. Cool to room temperature (if solids form then return to boiling for a further 15 mins). Once cool, neutralise the solution by slow addition of saturated sodium carbonate until it is slightly alkaline. Cool in an ice bath and collect the product by vacuum filtration. Recrystallise from boiling water and record the yield. Record the melting point for the product. Prepare solutions of your product and the 3 provided sulphonamides in acetone (10 mg in 2.5 mL). Spot each solution on your RP-‐TLC plate and place in a prepared TLC chamber containing a phosphate buffer pH 7.4 as the mobile phase. The mobile phase is prepared by combining 0.1 M potassium dihydrogen phosphate (50 mL) and 0.1 M NaOH (39.5 mL). Once developed, dry with hot air and visualize using the dimethylaminobenzaldehyde spray. Calculate the Rf, Rm and the lipophilicity constant (π) for each of the substituent groups using the pKas given in the table below: Sulphonamide pKa Sulphanilamide Sulphamethazine 10.4 7.4 MSci ‘Chemistry with Biomedicine’ Sulphamethoxypyridazine Sulphathiazole 4CCC0150 Integrated Chemistry Laboratory 7.2 7.1 Below is a proton NMR spectra for either N-‐(4-‐sulfamoylphenyl)-‐acetamide or for sulphanilamide. Identify which compound the spectrum belongs to and assign all the peaks with an explanation for each. Write-‐up If assigned this experiment the write up should be in the form of a full lab report including a title, aim, introduction, method section, results and discussion, conclusion and references (plus appendices if appropriate). Remember to show all results including any necessary calculations and to discuss your findings in full. All structures must be drawn using a chemical drawing program such as Accelrys Draw. Useful References: •
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Furniss, B.S; Hannaford, A.J; Smith, P.W.G and Tatchell, A.R. Vogel’s Textbook of Practical Organic Chemistry, 1989, Prentice Hall, pg 883 Fieser, L; Williamson, K. Organic experiments, 8th Ed, 1998, Houghton Mifflin, pg 426 Harwood, L.M. et al. Experimental organic chemistry, 2nd Ed, 1999, Blackwell science, pg 260 McMurry, J. Organic Chemistry, Intl Student Ed. 2004, Brooks/Cole, pg 916 Solomons, G; Fryhle, C. Organic chemistry, 7th Ed, , 2000, John Wiley & Sons, pg 975 Prado, M. J.Chem.Ed., 2001, 78, 533-‐534