Synthesis and Characterization of Benzocaine, DEET, and Lidocaine PDF

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synthesis and Characterization of Benzocaine, DEET, and Lidocaine...

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Preparation of Benzocaine, Lidocaine, and DEET through Fischer Esterification & Nucleophilic Acyl Substitution of Carboxylic Acids Abstract: The purpose of this lab procedure was to use the conversion of carboxylic acids to acyl derivatives in order to synthesize benzocaine, DEET, and lidocaine. The products that are created from the fischer esterification and nucleophilic acyl substitution are analyzed by infrared spectroscopy, proton nuclear magnetic resonance spectroscopy (HNMR), thin layer chromatography (TLC), and determination of melting point for the solid crystalline products created with lidocaine and benzocaine. Background: Benzocaine Benzocaine is used in the pharmaceutical industry as a topic pain reliever, it is most commonly known as the over the counter analgesic “Orajel”. Benzocaine is used as a pain reliever because of its ability to bind to the sodium gates which block the sodium channels, preventing the flow of sodium ions into the nerve endings which in turn block the pain signals to your brain. Benzocaine is generally non-toxic because it is not readily absorbed and it is poorly water soluble. The preparation of benzocaine is through the synthesis from esterification of PABA or p-aminobenzoic acid which is a material that is used by bacteria to produce folic acid. A recent discovery shows that p-aminobenzoic acid can be damaging to the structure of our DNA and has the potential to cause skin cancer. The mechanism of benzocaine is based on the esterification of PABA with the addition of absolute ethanol, concentrated sulfuric acid, and heat. The mechanism begins with the protonation of the carboxylic acid of p-aminobenzoic acid from the concentrated sulfuric acid. Then the addition of an alkyl hydroxy group from concentrated ethanol and leaving of the hydronium ion leaving group. This then forms the benzocaine final product. This mechanism is shown below in figure 1.

Figure 1:Fischer esterification of PABA to benzocaine Structure created on ChemDraw pro

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DEET DEET or N,N-diethyl-m-toluamide is the active ingredient in insect repellents. It is a colorless, oily liquid in the insect repellent that is intended to be used on the skin or clothing to prevent biting insects. DEET can be prepared by converting m-toluic acid to acyl chloride using SOCl2 and reacting with diethylamine. It binds to the anopheles gambiae odorant binding protein 1, which are receptors in insects that contribute to the transmission of the malaria infection. DEET does not allow insects to locate a host due to the fact that they can no longer sense human body odors once the product has been applied. To elaborate more on the synthesis of DEET, beginning with the m-toluic acid being converted into an acid chloride with a thionyl chloride. Then, it undergoes nucleophilic acyl substitution with a diethyl amine to create the final product of N,N-diethyl-m-toluamide. The mechanism begins with the carboxylic acid of m-toluic acid being treated with thionyl chloride which is followed by the addition of a chloride ion in a [1,2] addition. This adds to the carboxylic acid and then the chloride from the thionyl group leaves in a [1,2] elimination while another chloride ion deprotonates the acid chloride. This then leads to the formation of hydrochloric acid gas and acid chloride. Acid chloride is highly reactive so it is treated with diethyl amine which causes nucleophilic acyl substitution of the chloride with diethyl amine.

Figure 2: nucleophilic acyl substitution mechanism of DEET Mechanism created on ChemDraw Pro

Lidocaine Lidocaine is a local anesthetic and a drug commonly used to treat ventricular tachycardia. Lidocaine is a fast acting anesthesia that temporarily relieves pain and is most commonly administered as a nerve block or infiltration depending on the area being treated. A local anesthetic nerve block is a short term pain reliever involving the injection of the local anesthetic as close to the nerve as possible. This is a preferred treatment over general anesthesia due to the faster recovery time and less postoperative pain.

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Lidocaine works in a similar fashion as benzocaine does, blocking sodium channels and preventing sodium ions from flowing into the free nerve endings, thus blocking the pain signals to the brain. The structure of lidocaine contains an amino-amide which is why it is a longer lasting anesthetic and pain reliever than benzocaine. The synthesis of lidocaine begins with a one step synthesis of 2,6-dimethylaniline. 2-nitro-m-xylene is treated with tin chloride and a strong acid combined with alcohol. This is then neutralized with potassium hydroxide and water which yields the final reduction reaction of 2,6-dimethylaniline. The second step begins with treating 2,6-dimethylaniline with acetic acid, sodium acetate, and α-chloroacetylchloride. This causes an acylation reaction which produces α-chloro-2,6-dimethylacetanilide. The third step, also the final step, is a nucleophilic substitution that occurs after treating α-chloro-2,6-dimethylacetanilide with diethylamine and diethyl ether to reach the final product of lidocaine. Step 1:

Figure 3: Step 1 lidocaine synthesis Mechanism created on ChemDraw Pro

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Step 2:

Figure 4: Step 2 Lidocaine synthesis Mechanism created on ChemDraw Pro Step 3:

Figure 5: Step 3 Lidocaine synthesis Mechanism created on ChemDraw Pro

Characterization 4

After performing the synthesis of each of the materials, IR and HNMR spectroscopy as well as thin layer chromatography and melting point analysis were performed on the final products to determine if the synthesis reaction created a complete version of the product. Prior to running the final products through the tests, there were predictions of the peaks expected to be seen in IR and HNMR spectroscopy results. These are shown in tables 1 and 2 for benzocaine, tables 3 and 4 for Deet, and tables 5 and 6 for lidocaine.

Figure 6: Molecular structure of Benzocaine Structure drawn with ChemDraw Pro

The predicted melting point of benzocaine is 90 degrees celsius.

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Figure 7: Molecular structure of DEET Structure built using ChemDraw

Figure 8: Molecular structure of lidocaine Structure drawn by ChemDraw

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The melting point value of lidocaine is expected to be 68 degrees celsius.

With further analysis doing the calculation of each synthesis, we will be able to determine if each of the reactions reached completion if the HNMR and IR spectra peaks match the expected and predicted values. If values do not correspond, it is likely due to an error in the experimental procedure while performing the synthesis. Experimental procedure: Benzocaine Benzocaine synthesis begins with measuring 1 gram of p-aminobenzoic acid and adding it to a 25 mL round bottom flask that is equipped with a stir bar. Dissolve the p-aminobenzoic acid in 10 mL of 100% ethanol. Stir the solution with the magnetic stir bar until fully dissolved. Once dissolved, add 1 mL of sulfuric acid, drop wise. While the reaction is occuring in the flask, set up the reflux apparatus and allow the solution to reflux for 30 minutes. Begin the water flow and turn on the heating mantle. After the 30 minutes is complete, remove the heating mantle and allow the reaction to cool while still allowing the water to run so the solution cools without losing any solvent. Once cooled, pour the reaction over 30 mL of water. Neutralize the excess sulfuric acid with 10% sodium carbonate. Add an approximated amount and then take the pH to determine whether more sodium carbonate must be added. Keep the stir bar going while the sodium carbonate is being added. Once the pH tab is the same color as the litmus paper, the solution is no longer overly acidic. The solution made is mainly aqueous which leads the product to be floating on top. The next step is to vacuum filter the crude product. Add water to the filter paper in the funnel so the paper does not move. Turn on the water from the vacuum filtration apparatus and from there begin filtering the solution to isolate the crude product. If any crude product is remaining in the original beaker, use a spatula to scoop out any remainder. The rest can be rinsed with cold water and filtered. If the beaker gets full, simply take out the funnel and discard the contents. Then suction dry the crude product to ensure the removal of any excess water. The next part of the procedure is the process of recrystallizing the pure benzocaine product. Begin by transferring the product to an erlenmeyer flask. Be sure to remove as much of the product from the funnel as possible. Then dissolve the product in the minimum amount of hot methanol. Place the flask on a hot plate and make sure the stir bar is rotating. While on the hot plate, the solution should boil slightly to make sure that everything is dissolved. When the solution has fully dissolved, remove it from the hot plate and bring it down to room temperature. Once it is cooled and you can safely touch the bottom of the flask, place it into an ice bath. Once placed in the ice bath, if no crystals form after a minute or two, boil off 7

some of the solvent in the flask. Scratching the surface of the liquid can induce crystal formation. Using the same vacuum filtration method as earlier, empty the flask with the pure product into the funnel and vacuum filter the crystals to isolate the product. Let the product suction dry and then characterize the product. DEET Begin the procedure by flame drying all glassware and apparatus needed using a manifold. First put the flask under vacuum and then using a flame, dry the glassware. Close the vacuum and then refill it with nitrogen. Let the nitrogen circulate for a few seconds and then close it and reopen the vacuum. Repeat this process a few times. The last time you flame dry the glassware, leave the nitrogen tap open. Let the glass cool as it is extremely hot. Once the flask has cooled, measure and add 2.0 grams of m-Toluic acid. Immediately cap the flask so the reaction stays in a nitrogen filled atmosphere. Next add 2.2 mL Thionyl Chloride to the flask using a syringe. Make sure the syringe has no air bubbles. Add it slowly into the reaction. Allow the reaction to reflux for 15 minutes. After reflux is over, take away the heating mantle and allow the flask to cool. Put the whole reaction on ice. Then add 30 mL of diethyl ether to dilute the solution and cover the flask. Then add 5 mL of diethylamine in 10 mL of Ether slowly into the reaction flask. Then take the reaction mixture off the ice and keep it stirring for 15 minutes. After the stirring process is complete, add 15 mL of 2.5 M NaOH. Stir this mixture for another 15 minutes. Pour the reaction into a separatory funnel and from there allow the layers to settle. Let the layers settle and the ether layer is less dense than water because it contains hydroxide so the ether organic layer is on top. Remove the aqueous layer from the separatory funnel.Wash the organic layer with 3 M HCl to neutralize any excess base in the organic layer. Shake the funnel and ventilate to remove any excess pressure. Allow the layers to settle once again. Drain out the aqueous layer noce again and wash the organic layer with cold water. This will remove any excess salts or acids in the organic layer. Once again, let the layers settle and drain out the aqueous layer. Then, drain the organic layer into a flask and dry the organic layer with sodium sulfate to remove any excess water. If you see free grains of sodium sulfate moving at the bottom of the flask, this indicates there is no more water left in the solution. Additionally, if the solution is see through this also means that there is no more water in the flask. Next, decant the solution. Pour the solution into a round bottom flask and make sure not to get any of the sodium sulfate in. Then attach the flask to the rotovap to concentrate the solution. Turn on the vacuum, water, and hot water bath underneath. Attach the flask to the rotovap and turn off the vacuum and attach a keg clip to keep the flask attached. Slowly start spinning the flask and lower the flask into the hot water bath. This will remove all the solvent out of the flask and we are left with the crude reaction material. The next step is to purify the product using column chromatography. Start off with a column and add a small amount of sand to the bottom to make it even. Purify and make a slurry of the stationary phase by taking the mobile phase (Heptane) and add it to the alumina. Add this slurry into the column, rinse the flask with heptane to ensure all of the solvent has been added. Use compressed air to pack the alumina into the column. Ensure that the stationary phase in the column does not become dry. If the alumina dries out, you must start over. When the solvent is even with the stationary phase, add the sample directly onto the alumina. Add it slowly to make the alumina stays even. Push the solvent down to the alumina using compressed air. Add sand to protect the alumina so as to not disturb the level. Add the mobile phase on top of the sand.

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Now, start collecting the fractions into multiple test tubes. Use compressed air to push the mobile phase through the column. Keep collecting the fractions. Once all the fractions have been collected, TLC each fraction in order to determine which fraction the product is in. After TLC is done, place all the fractions into a round bottom flask. Concentrate the solution using a rotovap with the same process as before. After it is done rotovapping, we are able to calculate the percent yield and characterize the product. Lidocaine Synthesis of 2,6-dimethylaniline Begin the synthesis by dissolving 33.9 g of Tin (II) chloride in 40 mL HCl. Add it slowly and begin stirring using a magnetic stir bar as the solution is added. Then dissolve 5 grams of 2,6-dimethylnitrobenzene in 50 mL Acetic Acid and stir to dissolve with a stir bar. Once both solutions are completely dissolved, pour the tin (II) chloride solution into the nitrobenzene solution. Allow this mixture to be stirred for 15 minutes. After 15 minutes, the reaction will be warm and a precipitate will begin to form. Clamp the reaction mixture to the stand and place it in a cool water bath. Once the reaction mixture has cooled, transfer it to a buchner funnel. Wet the filter paper with the solvent of the reaction and turn on the water. Slowly pour the reaction mixture into the funnel to vacuum filtrate the solvent. Let the vacuum run to suction dry the liquid from the reaction mixture. Stop the water once you see no more liquid being collected. Transfer the precipitate along with the stir barto a secondary container using a weigh paper. Be sure to collect all the solids from the sides of the funnel. Scrape off any material stuck to the filter paper as well. Dissolve the solid in 25 mL of water as well as washing the spatula. Then add about 50 mL of 8M KOH to ensure that the solution is basic. Allow this mixture to stir with the stir bar for a moment. If solids are still present, add more potassium hydroxide and test the pH of the solution. T Transfer the solution to a separatory funnel. Add about 30 mL of diethyl ether to extract the organic layer. Shake and ventilate the separatory funnel. The organic layer is on the top and the aqueous layer will be on the bottom. Remove the aqueous layer into another flask. Then remove the organic layer into another flask as well. Transfer the aqueous layer back into the separatory funnel to once again wash it with diethyl ether. Wash the original container with organic solvent and transfer it back into the separatory funnel. Shake and ventilate the separatory funnel once again. Extract the organic and aqueous layers into separate funnels once again. The organic material that is extracted will be transferred back into the separatory funnel and washed with water to remove any excess base that is remaining. Add 25 mL of water, swirl and ventilate the separatory funnel. Collect the bottom aqueous layer once again into the flask used to collect it previously. Leave the organic layer in the separatory funnel and add the remaining amount of water. Once again, swirl and ventilate. Remove the aqueous layer and if emulsions form, swirl the flask again. Extract the organic layer and dry the excess water with sodium sulfate. Decant the solution into a round bottom flask without getting any of the solids into the new flask. Concentrate the solution using a rotovap. After the concentrate is done, you can now characterize the product and calculate the percent yield. Synthesis of α-chloro-2,6-dimethylacetanilide Once the synthesis of aniline derivative is done, transfer the pre weighed quantity into a clean erlenmeyer flask via a pipette. Dilute the oil in acetic acid and place the new solution on a stir plate. With 9

the solution of amine and acetic acid, add acyl fluoride drop wise. Let the reaction stir for 15 minutes. While it is stirring, dissolve sodium acetate in 80 mL of water. Swirl to make sure the solution is completely homogenous. Add this mixture to the reaction mixture. The flask will be slightly warm so transfer to an ice bath. Allow the solution to cool completely. Next, wet the filter paper in the buchner funnel, turn on the water and pour the heterogenous over the filter paper slowly to vacuum filter the solution. Rinse the solid with water until you stop smelling acetic acid. Rinse the original container first and then rinse the solid in the buchner funnel with water. Let the solid dry overnight and once completely dry, characterize the product and collect the percent yield. Lidocaine C Prior to beginning the final step of the synthesis, flame dry all parts of the apparatus and glassware by placing it under vacuum and heating it with a blow torch. Make sure the water is not running. Allow it to cool and close the vacuum and put it under nitrogen. Repeat this process a few times to ensure all the excess water is gone. Next, add 2.2 g of α-chloro-2,6-dimethylacetanilide into the round bottom flask. Carefully but quickly transfer the solids. Once all the material has been transferred, add 30 mL of Toluene to dilute the solids. Then, add 2.4 grams of diethylamine via syringe. Ensure no air bubbles are in the syringe. Add it slowly into the reaction mixture. Warm and reflux the reaction for 90 minutes while it's gently sitting on the heating mantle, also turning on the water prior to turning on the heat. After letting the reaction reflux, cool the reaction mixture to room temperature by removing the heating mantle. Further cool the reaction in an ice bath to crash out amine salts. Filter the precipitate in a vacuum filtration apparatus. Wash the precipitate with toluene and rinse the flask to ensure that all the material has been transferred. Once the drops of filtration have been more infrequent, stop the vacuum and turn off the water. Now, transfer the filtrate to a separatory funnel. Extract the product with 3M HCl, the amine will be protonated and brought into the aqueous phase. Swirl and ventilate the separatory funnel. Collect the aqueous layer on the bottom into a new flask. Then extract the remaining product with 3M HCl once again. Swirl and ventilate. Remove the aqueous layer into the flask and transfer the toluene into a separate flask. Now neutralize the acidic amine aqueous layer with 25 ml of 8M KOH. Gently pour in the solution to see the formation of the solvent. Then check the pH to make sure it is basic. Transfer it back into the separatory funnel and extract the organic layer, which is on top, with diethyl ether. Swirl and vent when the diethyl ether is added. Remove the aqueous layer in a flask. Collect the organic solvent in a separate flask. Transfer the aqueous layer back into the separatory funnel and extract again with ether. Rinse the aqueous container to ensure all the material gets accounted for. Now the SN2 product has been extracted, the mixture will separate. Dry the organic extracts with sodium sulfate which removes any residual water. Swirl and see that you have created a clear solution. Decant it into a ro...