Medicinal Chemistry Practical Manual 2023/2024 (Hashemite University)

Hashemite University Faculty of Pharmaceutical Sciences Department of Pharmaceutical Chemistry Practical Medicinal chemistry Manual (131703425) Student Name: _______________________ Student number: _____________________ Edited by:...

Hashemite University Faculty of Pharmaceutical Sciences Department of Pharmaceutical Chemistry Practical Medicinal chemistry Manual (131703425) Student Name: _______________________ Student number: _____________________ Edited by: Farah Hudaib 2023/2024 -i-  Course Description Medicinal Chemistry is the study of how new drugs are developed and tested. In this course, you learn the basics of drug's synthesis, drug design and development. Medicinal chemistry requires an understanding of how chemistry, biology, mathematics and computing interact with each other to allow the scientist to effectively create new pharmaceuticals that will prevent or stop one or more disease conditions. This practical course in medicinal chemistry concerned with multistep synthesis of selected medicinal compounds. The successful medicinal chemist is an expert organic chemist who has, or can acquire, sufficient knowledge in other disciplines to apply that knowledge to drug synthesis and design. We shall have opportunities to illustrate the dependence of medicinal chemistry on knowledge from other disciplines as we progress through this course. So our course will build on the experience gained from organic chemistry lab through the synthesis and characterization of complex molecules, the acquisition and interpretation of physical data and the investigation of chemical systems through computational techniques gained in analytical and instrumental labs. It consists of a series of laboratory-based experiments aimed at developing skills in the synthesis, safe handling and analysis of chemical substances of a range of different classes of compounds; an understanding of modern characterization techniques (e.g. chromatography, atomic and molecular spectroscopy); and the operation of instrumentation for the acquisition of kinetic, structural and thermodynamic data. In addition to increased proficiency in standard techniques, this course provides an introduction into research-based chemistry through integrated and themed experiments. It will provide skill development in a range of techniques utilized in the modern chemistry laboratory. The subject provides experience across multiple traditional chemical disciplines whilst highlighting the importance of these disciplines in diverse 'real world' applications such as materials science and medicinal chemistry. This Lab class is divided into two parts: the first part will focus on three synthesis cycles; multi – step Synthesis of Sulfanilamide, Benzocaine and Phenytoin, using techniques of organic compounds that an organic chemist uses daily; including crystallization, distillation, and extraction and will be will be run in groups. The second part focuses on Molecular modeling using computer software’s for drug design. Course Objectives 1. Familiarize students with techniques commonly used in the medicinal chemistry laboratory. 2. Learn how synthesis a drug in multisteps and how get it in good yield and in pure form. 3. Demonstrate the effect of the different synthetic methodology. 4. Clarify theoretical concepts of chemical synthesis of drug molecules. 5. Working in the laboratory will give the students experience in handling and proper usage of laboratory glassware, equipment, and chemicals. 6. The students should learn how to keep an accurate and readable record of all experimental work and how to write a scientific report. 7. Students are expected at the completion of this course to master a variety of synthetic techniques including purification methods and should gain the ability to design a synthetic scheme for a proposed drug molecule. 8. Learn how to use different computer sofwares to draw & design drugs and learn how drug interact with its target. 9. Equip students with both oral and written communication skills, through your practical report and your written assignments and oral tasks, and through the communications that will be engaged in with lecturers, demonstrators and classmates, especially in the group laboratory work. -ii-  Intended Learning Outcomes (ILOs) Successful completion of the course should lead to the following outcomes: A. Knowledge and Understanding: 1. To understand. 2. To know the structures of different drugs. 3. To know the general laboratory safety and basic techniques. 4. To understand the principle of drug synthesis. B. Intellectual skills (cognitive and analytical): 1. To realize any mistakes done during the assay and try to avoid it. 2. To be able to create a chemical assay for identification the quality of drug. 3. The student is expected to develop the ability to suggest suitable techniques to synthesis different drug molecules. 4. The student is expected to interpret scientific data and make sound scientific conclusions. C. Subject specific skills The student is expected to learn how to conduct chemical reactions within medicinal chemistry context this includes: 1. How to set up chemical instruments and tools in an experiment. 2. How to mix reactants, solvents and reagents within experimental context. 3. How to isolate and purify reaction products through (not limited to) chromatography, crystallization, distillation. 4. Identification and characterization of the final products through standard chemical procedures such as melting point, NMR, etc. D. Transferable Skills 1. Team work. 2. Use oral communication to effectively transmit ideas and conclusions to a scientific audience. 3. Develop of problem solving and critical thinking skills. Reading List / References: Supplementary Textbook(s) Wilson and Gisvold's Textbook of Organic, Medicinal and Pharmaceutical Chemistry, 1 12th Edition, 2011, Lea & Febiger. Foye's Principles of Medicinal Chemistry, David A Williams, William O Foye and 2 Thomas L Lemke, 6th Edition, 2008, Lippincott Williams & Wilkins. Organic Chemistry: A Short Course. By Harold Hart, Leslie E. Craine, David J. Hart. 3 Publisher: Houghton Mifflin College; 10th edition (January 1999) ISBN. 0395902258 -iii-  Course Contents Credit Week ILOs Topics Teaching Procedure Assessment methods Hours Lecture+ discussion A1, A3, General instruction and safety rules And 1 1 Video presentations & - Class participation C1, D1, laboratory apparatus Animation A1, A2, - Class participation A3, A4, Brief discussion + - Laboratory Report B1, B2, 1 3 Benzocaine synthesis Video for laboratory - Quizzes C1, C3, work + Brain storming - Lab work evaluation C4, B3, - Assignment D3, D1 A1, A2, - Class participation A3, A4, Multi – step Synthesis of Phenytoin: Brief discussion + - Laboratory Report B1, B2, 2 3 Video for laboratory - Quizzes C1, C3, Benzoin condensation work + Brain storming - Lab work evaluation C4, B3, - Assignment D3, D1 A1, A2, A3, A4, - Class participation Brief discussion + B1, B2, Multi – step Synthesis of Phenytoin: - Laboratory Report 3 3 Video for laboratory C1, C3, Oxidation of Benzoin to Benzil Quizzes work + Brain storming C4, B3, - Lab work evaluation D3, D1 A1, A2, A3, A4, Multi – step Synthesis of Phenytoin: - Class participation Brief discussion + B1, B2, condensation of Benzil with urea to form - Laboratory Report 4 3 Video for laboratory C1, C3, dilantin (Phenytoin). - Quizzes work + Brain storming C4, B3, - Lab work evaluation D3, D1 6 Mid-term examination - Class participation A1, A2, Brief discussion + - Laboratory Report Multi-step Synthesis of Sulfanilamide: 7 3 A4, B2, Video for laboratory - Quizzes B3, D1 Synthesis of Acetanilide work + Brain storming - Lab work evaluation - Assignment A1, A2, - Class participation A3, A4, Multi-step Synthesis of Sulfanilamide: Brief discussion + - Laboratory Report B1, B2, 8 3 Synthesis of p- Video for laboratory - Quizzes C1, C3, Acetamidobenzenesulfonyl chloride work + Brain storming - Lab work evaluation C4, B3, - Assignment D3, D1 A1, A2, - Class participation A3, A4, Multi-step Synthesis of Sulfanilamide: Brief discussion + - Laboratory Report B1, B2, 9 3 Synthesis of p- Video for laboratory - Quizzes C1, C3, Acetamidobenzenesulfonamide work + Brain storming - Lab work evaluation C4, B3, - Assignment D3, D1 A1, A2, Multi-step Synthesis of Sulfanilamide: - Class participation A3, A4, Synthesis of p- Brief discussion + - Laboratory Report B1, B2, 10 3 Video for laboratory - Quizzes C1, C3, Aminobenzenesulfonamide work + Brain storming - Lab work evaluation C4, B3, (Sulfanilamide) - Assignment D3, D1 Lecture+ discussion - Class participation A1, B4, Molecular modeling: In silico prediction 11 3 Video for laboratory - Laboratory Report D2, D3 of Ionization Constants of Drugs work - Quizzes - Class participation Lecture+ discussion A1, B4, Molecular modeling: SAR analysis using - Laboratory Report 12 3 Video for laboratory D2, D3 accelrys software - Quizzes work - Lab work evaluation 14 Final examination week -iv-  Grade Distribution Assessment Grade Date 1. Quiz 10% weekly 2. Midterm exam 30% To be arranged 3. Report 10% weekly 4. Evaluation 10% weekly 5. Final Examination 40% To be arranged Important regulations On average, students need to spend 3 hrs of study and preparation weekly. Excellent attendence is expected. According to the university policy, students who miss more than 15% of the lecture hours with or without excuse will be dismissed from the course At the beginning of the lab, be on time and don't leave before the end of the lab session without an accepted excuse If you missed a lab session, it is your responsibility to find out about any announcements or assignments you have missed For any clarification, please communicate your instructor at his posted office hours or by appointment Switch off your mobile or keep it silent throughout the lecture Listen well to the lab disscution and avoid side discussions, if you have a question, ask your instructor and not your collegue If you have any information, document your reference, if you didn't, then you broke the intellectual property rights law and the law will be applied Exams are scheduled to be given two times throughout the semester, your are expected to attend all. If not, make-up exams will be offered for valid reasons. It may be different from regular exams in content and format. Cheating, academic diconduct, fabrication and plagiarism will not be tolerated, and the university policy will be applied Each student is expected to familirize himself with laboratory rules and safty precution. -v-  Index Exp. Experiment Page no. No. 00 General Instructions and Laboratory Safety Rules vii Cycle 1: Multistep Synthesis of Benzocaine 1 1 Part 4: Benzocaine synthesis 3 Cycle 2: Multistep Synthesis of Phenytoin 8 2 Part1: Benzoin condensation 10 3 Part 2: Oxidation of Benzoin to Benzil 15 Part 3: Condensation of Benzil with urea to form dilantin 4 20 (Phenytoin). Cycle 3: Multistep Synthesis of Sulfanilamide 26 5 Part 1: Synthesis of Acetanilide 30 6 Part 2: Synthesis of p-Acetamidobenzenesulfonyl chloride 36 7 Part 3: Synthesis of p-Acetamidobenzenesulfonamide 40 Part 4: Synthesis of p-Aminobenzenesulfonamide 8 42 (Sulfanilamide) Molecular modeling Molecular modeling: In silico prediction of Ionization 9 53 Constants of Drugs 10 Molecular modeling: SAR analysis using Accelrys software 80 Appendix I Percent Yields Calculations 90 II Melting Point Measurement 93 III Recrystallization and filtration techniques 97 IV Reflux and the reflux condenser 102 V Boiling chips 103 VI Other suggested mechanism for Step1 in cycle 1 104 VII Extraction 105 VIII Benzoin condensation using cyanide 109 IX Oxidation of Benzoin to Benzil using nitric acid 110 -vi-  General Instructions A. Lab Safety Precautions Gloves and Eye protection Goggles must be worn at all time in the lab, regardless of what is being done. Prescription glasses (not sunglasses) are acceptable. Contact lenses provide no protection. Familiarize yourself with the location of fire extinguishers , safety showers, fire blankets and eye wash fountains and know how and when to use these devices. If chemical are splashed in or near the eye, wash immediately with clean and cold running water for 10 -15 min (remove contact lenses in case you have been wearing them). Consult a physician afterwards. When inserting glass tubing or thermometer into rubber stoppers, always lubricate both the glass and the whole with glycerin and protect hands with a towel. Never taste any compound in the laboratory. To determine the odor of a compound, bring the stopper of the bottle cautiously toward the nose. Do not inhale. Avoid any contact of chemicals with the skin, especially the face. Wash your hands as soon as possible after making transfers or other manipulations. When heating a test tube or carrying out a reaction in one, never point the tube toward yourself or your neighbor. Never heat an organic liquid directly over a flame except under a condenser. When refluxing a liquid, be sure that the condenser is tightly fitted. If a temperature below 95oC is sufficient, use a steam bath rather than a burner. Before lighting a flame check to see that volatile liquids are not being poured or evaporated in your vicinity. Always turn a burner off as soon as you finish using it. As a general practice, and particularly if a burner is in use, avoid loose- fitting long sleeves and cuffs. Long hair should be tied back during laboratory work. -vii- Any experiment involving the use or production of objectionable (i.e. poisonous or irritating) liquids or gases, must be performed in the hoods. -viii-  B. Laboratory rules Each student is expected to attend each lab session and to be in the laboratory on time. Those students, who come early, should wait inside the lab but never gather in the corridors. Each student must wear a clean and buttoned up lab coat with his\here tag name before enter your lab. Skirt, short clothe, sandal (open shoes) and head caps forbidden in lab. Smoking, Drinking, Eating or Chewing gum is prohibited in the lab. Each student is responsible for keeping the laboratory clean and in good order. Coats, book and personal belongings should be kept in your lockers. Do not bring them with you to the lab. All working areas and balances must be kept clean. Powdered drugs, greasy or waxy materials or any insoluble waste materials will block the sink if thrown in to it. Thus, dispose them properly in a waste basket. Water immiscible organic solvents and other liquids should be discarded in a designated waste – solvent can but never poured in to a sink. Chemicals that react vigorously with water, such as acid chlorides or alkali- metals should be decomposed in a hood in a suitable way. If students are assigned to work as groups, each group is expected to work quietly and independently. Do not carry reagent bottles to your desk. Never return excess materials to reagent bottles. Each student must bring: ✓ Two hand towels ✓ Marker ✓ Sponge for disk cleaning ✓ Pair of gloves (nitrile gloves is ✓ Detergent preferred) ✓ Matches ✓ Calculator to each lab You Will Be Held Responsible For Each Rule Mentioned Above, Any Violation Will Expose You To Either Being Dismissed From The Lab Or Losing Evaluation Marks Of That Particular Experiment Without Prior Notification -ix- Common problems / easy solutions: 1- NH4OH: Serious eye damage if contacts with eye. Very destructive to mucous membranes. Corrosive – severe burns 2- Chlorinated solvents such as CHCl3 and DCM. Dispose in designated waste- solvent bottles found inside the hood. 3- Chlorosulfonic acid : Corrosive acid that may cause severe skin mburns and eye damage, vapor extremely irritating to lungs and mucous membranes and is fatal if inhaled. 4- Alkali metals: must always be kept covered with dry organic inert solvent such as Kerosene or paraffin oil. Remaining metal pieces, tools, knifes and spatulas must be placed in a beaker, then treated with absolute dry ethanol and leave it bubble (H2 gas) for minutes. 5- Mercury waste from a broken thermometer must be treated with solid sulfur powder bit never thrown in the waste basket. 6- Ether is very volatile and highly explosive. Keep in cold place. Dry solvent bottles on the shelves can be easily oxidized upon evaporation and leave explosive compounds. Please inform the technician. 7- You must report any broken glass. Don not remove yourself. 8- Do not draw any liquid with your mouth or smell any chemical. 9- Concentrated mineral acids and bases should always be kept at the hood and you should wear gloves when using. 10- Maintain labels on chemical containers received from manufacturers and label secondary containers. Replace old and deteriorated labels. 11- Smelly chemicals such as acetic anhydride, glacial acetic acid, pyridine, and benzaldehyde should be kept always in the hood and disposed in waste closed bottles. 12- Radioactive material, biological and chemical hazards should be handled and disposed in collaboration with faculty administration and local civil defense agency following special protocols. -i- Labs common glassware -ii-  Cycle 1 Multistep Synthesis of Benzocaine Target Product Benzocaine is a local anesthetic from the ester family. The drug benzocaine is used in multiple forms including lotion, gel, liquid, lozenges, and sprays as a topical pain reliever. When Benzocaine is applied in any form, it temporarily numbs or blocks the nerve endings by inhibiting the voltage dependent Na channels on the neuron membrane, which leads to decrease in the amount of pain. -1-  General scheme of synthesis Acetylation Oxidation Acid Hydrolysis Esterification Four-step synthesis is used to create benzocaine: Part 1: Synthesis of p-Acetotoluidide Part 2: Synthesis of p-Acetamidobenzoic acid Part 3: Synthesis of p-Amino benzoic acid (PABA) Part 4: Synthesis of Benzocaine You will see the mechanism only for the forth steps. -2-  Experiment 1 Multistep Synthesis of Benzocaine Part 4: Benzocaine synthesis HO O O O H + HO + H2 O Ethanol NH2 NH2 p- aminobenzoic acid Ethyl p-aminobenzoate PABA (Benzocaine) Benzocaine is synthesized through the Fischer esterification of p- aminobenzoic acid (PABA) and ethanol, using sulfuric acid as a catalyst. PABA: is amphoteric that has weak acidic and weak basic properties. 5 Assignment 5.1 Find a commercial synthesis pathway of PABA. -3-  Mechanism This reaction is called Fischer esterification reaction: a type of condensation reaction (both way reaction- limited yield) It is acid catalyzed (it not consume in the reaction). It proceeds very slowly in absence of strong acids as HCl or H2SO4. OH HO O HO OH HO O H H HO NH2 NH3 NH3 p-aminobenzoic acid Proton transfer OH2 O O O O O O H Hydrolysis - H3O Na2CO3 Neutralization Acid not of acid to ppt consume product (It is catalyst) NH2 NH3 NH3 To increase the ester yield : - Use excess from the reactants - Remove water from reaction mixture as it is formed -4-  Procedure 1. In a 100 ml round-bottomed flask place 1.32 g of PABA, 10 ml of ethanol, and 1 ml of sulfuric acid (add cautiously). Add a couple of boiling chips* then attach a reflux condenser and heat under reflux for 1 hour. 2. Cool the solution to room temperature, neutralize with 10% sodium carbonate (foaming), and extract⁂ with two 10-ml portions of dichloromethane (DCM) [use separatory funnel in extraction]. Then dry the combined organic layers over anhydrous magnesium sulfate [drying agent].(1) 3. Remove the dichloromethane by distillation using a steam bath as a heat source. 4. Then recrystallize the residue from methanol-water [Mixed solvent recrystallization‡].(2) (1) Add 3-4 gm of anhydrous magnesium sulfate, swirl the mixture for about 5 minutes, and then remove it by gravity filtration. (2) Add 5 ml portions of methanol (with heating) until all the amount is dissolved, then add water drop wise until the solution becomes turbid, after that cool it in an ice bath to complete crystallization. Weight (gm) M.P Benzocaine 2 Brain storming Question 2.1 Discus the reason behind doing extraction in DCM, and define the component for each layer. 2.2 Discus the reason behind adding drying agent for the organic layer. * Refer to appendix V: Boiling chips ‡ Refer to appendix III: Recrystallization-Mixed solvent recrystallization. ⁂ Refer to appendix VII: Extraction. -5-  pre-lab. Report sheet (1) Synthesis of Benzocaine Student name: Objectives : MW Solubility Compound Hazards (g/mol) in water p- aminobenzoic acid HCl Benzocaine Find out : 1. Mwt of Benzocaine…………… 2. The melting point of Benzocaine …………. Draw a simple flowchart for the procedure of the exp. Or write an outline steps in your own words? -6-  Post-lab. Report sheet (1) Synthesis of Benzocaine Student name: 1. data and results: The actual yield of Benzocaine: The theoretical yield of Benzocaine(show detailed calculation): The yield%: 2.chemical equations: Write down the balanced chemical equations that represent the preparation of Benzocaine: -7-  Cycle 2 Multistep synthesis of Phenytoin Phenytoin, sold under the brand name Dilantin among others. It is an anti-seizure medication. It is useful for the prevention of tonic-clonic seizures and partial seizures. Phenytoin blocks the spread of seizure activity in the brain by causing voltage dependent block of the voltage gated Na channels. It may also be used for certain heart arrhythmias or neuropathic pain. It doesn’t have sedative hypnotic activity It can be taken intravenously or by mouth -8-  General scheme of synthesis Condensation Oxidation Condensation Phenytoin can synthesize from benzaldehyde in three synthetic steps: Part 1: Benzoin condensation. Part 2: Oxidation of benzoin to benzil. Part 3: Condensation of benzil with urea to form phenytoin. You will see the mechanism for each steps. -9-  Experiment 2 Multistep synthesis of Phenytoin Part1: Benzoin Condensation O O OH Thiamine HCL NaOH 2 Ethanol Benzaldehyde Benzoin Benzoin is produced by dimerization of 2 molecules of benzaldehyde, The benzoin condensation§ is in fact a dimerization and not a condensation because a small molecule like water is not released in this reaction This reaction doesn’t occur spontaneously. It needs a catalyst to render the aldehydic carbon acidic so that it can be deprotonated and function as a nucleophile to attack the second benzaldehyde carbon. Cyanide* [𝐾 + 𝐶 ≡ 𝑁 − ] can use as a catalytic reagent use at 75°C (give faster rate) but they are extremely poisonous. Thiamine (contains a thiazole unit) is the catalyst was used here at Room temp, (give very slow rate). - Thiamine (Vit B1) is non-toxic (edible) material - Thiamine is heat sensitive and may decompose if heated vigorously - Instead of running the reaction at elevated temperature. We will allow it to proceed closer to room temperature for at least 24 hours § Condensation reaction: a reaction in which two molecules combine to form a larger molecule, producing a small molecule such as H2O as a by-product. * Refer to appendix VIII: Benzoin condensation using cyanide. -10-  Mechanism First: Formation of the catalyst (Ylide*): Acidic proton Second: The condensation reaction start with umpolung‡ to the carbonyl center of benzaldehyde: Acidic proton e-rich center e-difficult center Other molecule e-difficult center Anti- elimination * Yalide: is a species with opposite formal charges on adjacent atoms (both on same bond, no distance between them like in zwitter ion). zwitter ion: a molecule or ion having separate positively and negatively charged groups. ‡ Umpolung or polarity inversion in organic chemistry: is the chemical modification of a functional group with the aim of the reversal of polarity of that group. This modification allows secondary reactions of this functional group that would otherwise not be possible. -11-  Procedure 1. In a stoppered E. Flask, prepare a solution of 1.04 g of thiamine hydrochloride in 3 ml of water‡. 2. When all the thiamine hydrochloride has dissolved, add 8 ml of 95% ethanol with mixing. 3. Cool the solution for a few minutes in an ice bath [because thiamine is heat sensitive]. 4. Very carefully and slowly add 3 ml of 10% NaOH with mixing “making sure that the temperature of the solution never rises above 20 C” [because NaOH with water exothermic]. 5. Add to the mixture 7 ml of pure Benzaldehyde with mixing. 6. Stopper the flask and allow it to stand at room temp at least overnight (longer period do no harm). At the end of the reaction period, the benzoin should have separated as fine crystals. 7. Cool the reaction mixture in an ice bath to complete the crystallization. 8. Collect the product by vacuum filtration and wash them thoroughly with two 7 ml portions of cold 50% ethanol and several portions of water§ [color change from yellow to white]. 9. Drain well and leave your product to dry (no need for recrystallization*). Weight (gm) M.P Benzoin 3 Brain storming Question 3.1 Discus the chirality of the product, does it optically active? Explain your answer. ‡ Water is required in this reaction BUT in specific amount: - Too much water will force benzaldehyde out of the solution preventing an efficient reaction. - Too little water prevents thiamine-HCl from dissolving. * Re-crystallizion an done using 95% ethanol. § Ethanol to remove excess benzaldehyde and water to get rid of thiamine. -12-  Pre-lab. Report sheet (2) Benzoin Condensation Student name: Objectives : MW Solubility Compound Hazards (g/mol) in water Thiamine hydrochloride NaOH Benzaldehyde Benzoin Draw a simple flowchart for the procedure of the exp. Or write an outline steps in your own words? -13-  Post-lab. Report sheet (2) Benzoin Condensation Student name: 1. data and results: The actual yield of Benzoin: The theoretical yield of Benzoin (show detailed calculation): The yield%: 2.chemical equations: Write down the balanced chemical equations that represent the preparation of Benzoin: -14-  Experiment 3 Multistep synthesis of Phenytoin Part2: Oxidation of Benzoin to Benzil Preparation of benzil can be done by Mild oxidation of benzoin using catalytic oxidation reaction, nitric acid or any other mild oxidizing agent, where the OH group is converted to a ketone group. α-OH ketone Diketone Benzoin can be oxidized to the diketone benzyl using a Cu2+ salt and ammonium nitrate. Only catalytic amounts of copper(II) acetate are necessary because the Cu2+ is continuously recycled. Cupric acetate (Cu+2) is used in a catalytic amount (less than 1% of stoichiometric requirement) and is continuously re-oxidized from the reduced (cuprous state Cu+) by ammonium nitrate (NO3-) which is present in excess and is reduced to ammonium nitrite (NO2-) which decompose in the reaction mixture into nitrogen gas. Refer to appendix IX: Oxidation of Benzoin to Benzil using nitric acid. -15-  Mechanism The reaction is Coupled Oxidation; using Cu+2 as the catalytic transfer oxidant. Blue color Pale yellow color White color Yellow color In the first redox cycle, benzoin donates an electron to Cu2+, forming Cu+ and benzoin redical cation A. The benzoin radical cation loses a proton to acetate ion (AcO-), forming acetic acid(AcOH) and a resonance stabilized radical, depicted by structure B and C. Another redox cycle between Cu2+ and the radical takes place, forming a second Cu+ ion and cation D, which loses a proton to another acetate ion to form benzil. The Cu+ ions formed in these redox cycles are re-oxidized to Cu2+ by ammonium nitrate, which is present in excess. Forms ammonium nitrite (NH4NO2), which decomposes to nitrogen and water under the reaction conditions. 7 Assignment 7.1 Find other Mild oxidizing agents. -16-  Procedure 1. In a round bottomed flask place 1.75 g of unrecrystallized benzoin, 5 ml of glacial acetic acid, 0.8 g of pulverized (reduced to fine particle) ammonium nitrate, and 1 ml of a 2% solution of cupric acetate. 2. Add 1-2 boiling chips, attach a reflux condenser and bring the solution to a gentle boil. As the reactants dissolve, evolution of nitrogen begins. 3. Boil the blue solution for 1.5 hr to complete the reaction. 4. Cool the solution to 50-60°C and pour it into 10 ml of ice-water mixture in a beaker [to ppt product, since benzil insoluble in water], while stirring it. Then Benzil separates out as yellow oil, which immediately solidifies. 5. After crystallization of benzil is complete, collect the crystals on suction filtration and wash them thoroughly with water. 6. Press the product as dry as possible on the filter. 7. If desired, it may be purified by re-crystallize from methanol or 75% ethanol. (After dissolving the product in hot ethanol, add water dropwise to reach the cloud point and allow it to crystallize). Weight (gm) M.P Benzil 4 Brain storming Question 4.1 Discus the chirality of the product, does it optically active? Explain your answer. 4.2 What structural features make benzil yellow and benzoin colorless (white)? 4.3 Would you have obtained the same results for the oxidation of benzoin if the label on the copper acetate bottle had read “cuprous acetate? -17-  pre-lab. Report sheet (3) Oxidation of Benzoin to Benzil Student name: Objectives : MW Compound Hazards (g/mol) Benzoin Ammonium nitrate Cupric acetate Benzil Find out the melting point of Benzil ? Draw a simple flowchart for the procedure of the exp. Or write an outline steps in your own words? -18-  Post-lab. Report sheet (3) Oxidation of Benzoin to Benzil Student name: 1. data and results: The actual yield of Benzil: The theoretical yield of Benzil(show detailed calculation): The yield%: 2.chemical equations: Write down the balanced chemical equations that represent the preparation of Benzil: -19-  Experiment 4 Multistep synthesis of Phenytoin Part3: condensation of Benzil with urea to form Phenytoin (Dilantin ®) The preparartion of phentytoin carried out by the condensation of urea with benzil in basic media (catalyst). The reaction involves a skeletal re-arrangement step (Benzylic acid re-arrangement), both phenyls ending up on the same carbon atom. The rearrangement may occur due to the stability of imide (CO-NH- CO) group that drives it or due to formation of benzyl cation (very reactive- very high-energy intermediate). Acidification step is required to precipitate phenytoin Possible intermediates: 4,5-diphenyl-4,5-dihydroxyl imidazolone Migration reaction: is slow reactions need heat (thermodynamic reaction root) Give a very stable product due to very high transition stat energy for the intermediate. -20-  Mechanism The mechanism of this reaction begins with the nucleophilic attack of a urea nitrogen atom on one of the carbonyls of benzil, then itermoleculare cyclization and end up with Benzylic acid re-arrangement (Migration reaction). Suggested Mechanism (1) Imid proton is subtracted rather than amid proton. -21- Suggested mechanism (2): This reaction is proceeding via intermolecular cyclization to form an intermediate heterocyclic pinacol (4,5-diphenyl-4,5-dihydroxyl imidazolone), which on acidification yield hydantoin (phenytoin) as a result of 1,2-diphenyl shift in pinacol rearrangement reaction. Benzylic acid re-arrangement (general mechanism) Rearrangement intermediate -Slow moving- -22-  Procedure Because Dilantin® has significant biological activity, it is very important that protective gloves and safety goggles be worn when handling the product of this reaction! 1. In a small round bottomed flask place 400 mg of unrecrystallized benzil, 200 mg of urea, 6.0 ml of ethanol, and 1.2 ml of 30% NaOH. 2. Attach an upright condenser, add a boiling chip, and boil the mixture gently for at least one hour. 3. Cool the reaction mixture, add 10 ml of water, and filter the solution to remove a sparingly soluble side product [pinacol side product] that sometimes forms. 4. Acidify the filtrate with 3-4 ml of HCL (20%) to pH 2-3. Then collect the product on a suction filter, and wash it thoroughly with water. Weight (gm) M.P Phenytoin -23-  Pre-lab. Report sheet (4) Synthesis of Phenytoin Student name: Objectives : MW Solubility Compound Hazards (g/mol) in water Urea NaOH HCl Phenytoin 4,5-diphenyl-4,5- dihydroxyl imidazolone Find out the melting point of Phenytoin ? Draw a simple flowchart for the procedure of the exp. Or write an outline steps in your own words? -24-  Post-lab. Report sheet (4) Synthesis of Phenytoin Student name: 1. data and results: The actual yield of Phenytoin: The theoretical yield of Phenytoin(show detailed calculation): The yield%: 2.chemical equations: Write down the balanced chemical equations that represent the preparation of Phenytoin: -25-  Cycle 3 Multistep Synthesis of Sulfanilamide Target Product Sulfa drugs were discovered in the early 1900’s and found to be active anti- bacterial agents. Sulfanilamide inhibits the formation of folic acid in bacteria, thus preventing its further growth. Sulfanilamides are sulfonamide synthetic anti-bacterials. Were the 1st successful selectively toxic antibacterial drugs. Used for treatment of acute uncomplicated UTI caused by E- coli , malarial infections, vaginal yeast infection and ocular infection Toxicity: Kidney damage (stones formation), hypersensitivity reactions. 1 Assignment 1.1 Find the story for discovery of sulfonamide as anti-bacterial drug. -26-  General scheme of synthesis Nitration Reduction Acetylation Chloro-sulfonation Acid Hydrolysis -27-  Step1: Nitration of benzene. Mechanism: 1- Activation of nitric acid ( protonation of nitric acid to form nitronium ion) 2- Electrophile aromatic substitution mechanism. Nitronium ion -28-  Step2: Chemical reduction of Nitrobenzene to Aniline 1. 2. NaOH Reflux 30 min Neutralization of excess HCL Sulfanilamide is easily synthesized from aniline in four steps. You will see mechanism for each steps. -29-  Experiment 5 Multistep Synthesis of Sulfanilamide Part 1: synthesis of acetanilide Acetylation Reaction + CH3COOH Acetic acid NH2 is an electron donating group that directs the electrophilic aromatic substitution on ortho-para position (it is electron reach so good nucleophile). Aniline resonance Acetanilide resonance -30-  Acetylation of NH2- [ convert amine group to amide group] It was for: ✓ Protection ✓ Blocking ortho and para director: Acetyl group (Bulk) makes steric hindrance and blocks the ortho position so sulfonyl chloride group will be added to para position in the next step Figure 2. Sulfonation of Acetanilide Figure 1. Sulfonation of aniline all positions (o,p, Directed only to the para position amine) are possible option Acetyl group will also protect from polymerization that may occur between sulfonyl chloride from one molecule and the amine group from another molecule and give polymeric material containing sulfonamide linkages. Figure 3. Polymerization The free amine, under the strong acid conditions of chlorosulfonation, would protonate or react with strong Lewis acids present (such as SO3), resulting in deactivation of the ring toward chlorosulfonation and sulfonation of the free amine. Figure 4. Protonation of aniline in acidic condition 1 Assignment 1.2 Find other amine protective groups. -31-  Mechanism This reaction is a nucleophilic addition-elimination reaction Acetic anhydride is used instead of acetic acid because its carbonyl carbon is more electrophilic than that of acetic acid so the amide formation will be faster and easier otherwise it may need reflux for 3-4 hours. 1 Brain storming Question 1.1 Explains in your words the following side reaction (di-acetylation of aniline) and comments on its rate? Is it faster or slower than the previous reaction? How can minimize this side reaction? 1.2 Mention another side reaction suggested to be happen during this experiment. How can minimize this side reaction? -32-  Procedure 1. Place 16 ml of aniline, measured using a 25ml-graduated cylinder, in to a 500 ml Erlenmeyer flask. 2. Add 120 ml of water to the flask and then while swirling the flask add 20 ml of acetic anhydride [measure it using a dry cylinder] in several small portions. 3. Add 200 ml of water and boiling stones (chips)⁂, and then heat until the entire solid and oil have dissolved. 4. Add about 1 gm charcoal slowly to the main hot solution. Swirl the mixture and boil gently for 5 min. 5. Filter through hot gravity filtration‡ into a 500 ml Erlenmeyer flask. Have available 100 ml of boiling water for washing. 6. Cool the filtrate in an ice water bath for 15 min to complete the crystallization. 7. Filter [cold suction filtration‡], then dry the crystals and determine the m.p*. Weight (gm) M.P Acetanilide 1 Brain storming Question 1.3 Justify the following: A. Water used as solvent instead of acetic anhydride in this reaction? B. Addition of water to aniline with swirling (shaking)? Addition of acetic anhydride in several small portion? C. Hot filtration. * Refer to appendix II: Melting point. To measure M.P. ‡ Refer to appendix III, Part II: Filtration techniques. ⁂ Refer to appendix V: Boiling chips. -33-  Pre-lab. Report sheet (5) Synthesis of acetanilide Student name: Objectives: Miscibility MW Compound with solvent Hazards (g/mol) (water) Aniline Acetic anhydride Charcoal Acetanilide Find out: 1. The density of Aniline ……….. 2. The density of Acetic anhydride 3. The melting point of Acetanilide …………. Draw a simple flowchart for the procedure of the exp. Or write an outline steps in your own words? -34-  Post-lab. Report sheet (5) Synthesis of acetanilide Student name: 1. Data and results: The actual weight (yield) of acetanilide: The theoretical weight (yield) of acetanilide (show detailed calculation): The yield%§: 2. Chemical equations: Write down the balanced chemical equations that represent the preparation of acetanilide: § Refer to appendix I: Percent Yields Calculations. To calculate % yield. -35-  Experiment 6 Multistep Synthesis of Sulfanilamide Part 2: synthesis of p-acetamidobenzenesulfonyl chloride Two equivalent 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 reaction is conducted without solvent Water serves to hydrolyze the excess chlorosulfonic acid and to stop the reaction [ +∆] CAUTION: Chlorosulfonic acid is a corrosive chemical and reacts violently with water. Withdraw with a pipette. Neutralize any spills and drips immediately. The wearing of gloves and handling in the hood is required. 2 Brain storming Question 4.4 Discus the effect of water on the product (p-acetaminobenzenesulfonyl chloride) -36-  Mechanism This reaction is more complicated than it looks at first inspection. It is happen in two steps. Step 1: Exothermic reaction (spontaneous). The electrophile that initially adds to the ring is probably SO3, forming the sulfonic acid (Ar-SO3H). The initially formed substitution product is the sulfonic acid. ✓ Substitution is essentially, all para due to combined electronic and steric effects. 𝒇𝒊𝒓𝒔𝒕 ∶ 𝑪𝒍𝑺𝑶𝟑 𝑯 ↔ 𝑺𝑶𝟑 (𝒈) + 𝑯𝑪𝑳 (𝒈) ∆ +∆ Step 2: Endothermic reaction (nonspontaneous). The sulfonic acid is then converted to 4- acetamidobenzenesulfonyl chloride (Ar-SO2Cl) by reaction with excess chlorosulfonic acid, generating sulfuric acid as the co-product. ∆ This reaction is: Typical electrophilic aromatic substitution Byproducts of this reaction : HCl (g), H2SO4 (g) , SO3 (g) gas and heat (Since the overall reaction is exothermic) Other suggested mechanism summarized in appendix VI. -37-  Procedure 1. To a dry 100 ml Erlenmeyer flask, add 40 ml of Chlorosulfonic acid (ClSO3H) carefully while the flask in an ice bath. [corrosive and reacts violently with water] 2. Add 15 g of dry finely powdered [↑SA for rxn] Acetanilide in small portions and with good mixing. [because it is vigorous reaction] 3. Allow the mixture to warm at room temperature and then heat the mixture on a steam bath for 30 min. (Adjust the temperature to 50 - 60oC). [to complete the reaction –step 2] 4. Cool to room temperature (using an ice bath). Place 400g of crushed ice and 65 ml of D.W [water for ease stirring and and to get read of excess ClS 𝑂3 𝐻] in a large beaker and pour the reaction mixture slowly and carefully with stirring [to prevent caking – keep product suspended] onto the ice. 5. Rinse the flask with a little of cold water and transfer this to the beaker. [Fill the flask with water and leave it under fume hood to the end to get read of all gases out: HCL ,SO3.] 6. Collect the crude material by suction filtration [Cold Suction Filtration] and wash the precipitate with a small amount of cold water. 7. Recrystallize‡ the product from 200ml Chloroform (1). The purified product should be dried in air to be used in the next lab. [the chloroform used in recrystallization bcz the product soluble in it at high temp and ppt at low temp] (1) Dissolve the crude material in hot chloroform, and while the solution is still hot, put it in a preheated separatory funnel and take the lower organic layer. [To remove residual water bcz the product sensitive to it] Evaporate part of the chloroform until reach 100ml [keep your eyes on it will heating] and cool the solution to R.T then in an ice bath, then collect the crystals and wash them with cold chloroform. [Cold Suction Filtration] Step 1-5 under fume hood. ‡ Refer to appendix III: Recrystallization. -38-  Weight (gm) M.P p-Acetamidobenzenesulfonyl chloride 2 Brain storming Question 4.5 Discus cooling step (4) and use of both water and ice. 4.6 Discus is it possible to use drying agent like (MgSO4) instead of using extraction and separation step in recrystallization. 4.7 In recrystallization you used separatory funnel for sepration describe component for each layer. 4.8 In recrystallization‘Evaporate part of the chloroform until reach 100ml’. Why? -39-  Pre-lab. Report sheet (6) Synthesis of p-acetamidobenzenesulfonyl chloride Student name: Objectives: MW Stability Compound Hazards (g/mol) in water Chlorosulfonic acid Acetanilide Chloroform p- acetamidobenzenesulfonyl chloride Find out the melting point of p-acetamidobenzenesulfonyl chloride? Draw a simple flowchart for the procedure of the exp. Or write an outline steps in your own words? -40-  Post-lab. Report sheet (6) Synthesis of p-acetamidobenzenesulfonyl chloride Student name: 1. Data and results: The actual weight (yield) of p-acetamidobenzenesulfonyl chloride: The theoretical weight (yield) of p-acetamidobenzenesulfonyl chloride (show detailed calculation): The yield%: 2. Chemical equations: Write down the balanced chemical equations that represent the preparation of p-acetamidobenzenesulfonyl chloride: -41-  Experiment 7 Multistep Synthesis of Sulfanilamide Part 3: synthesis of p-acetamidobenzene- sulfonamide A whole family of sulfa drug is possible at this stage if any other amine RNH2 is used instead of ammonia. Examples: -42-  Mechanism The reaction is a nucleophilic acyl substitution reaction. The amide is produced by treatment of the sulfonyl chloride with an excess of aqueous ammonia. NH3 is a good nucleophile Excess ammonia will neutralize the produced HCl by forming a salt water soluble (NH4+CL-) [acid base side reaction]. Sulfuric acid is used to neutralize the excess ammonium hydroxide and to decrease the solubility of our product and precipitate it. -43-  Procedure 1. Transfer the product obtained in experiment 2 (~10g); p- Acetamidobenzenesulfonyl chloride to a 250 ml Erlenmyer flask. 2. Add 60 ml of conc. Ammonium hydroxide solution (28 %) and heat the mixture on an 80 oC steam bath for 30 min. [a homogenous thick mixture produced like past due to amide intermolecular H-bonding, and heating bcz the reaction is endothermic] 3. Cool the mixture in an ice bath and add of sulfuric acid (6 M) dropwise until it become acidic (pH 3-5). [add 2ml then drop by drop] 4. Cool the mixture again in an ice bath and collect the product by suction filtration. [Cold Suction Filtration] 5. Wash the crystals with a small amount of cold water and dry them. 6. If necessary, recrystallize from a small amount of hot water. Weight (gm) M.P p-acetamidobenzene-sulfonamide 5 Brain storming Question 5.1 Discus the use of diluted ammonia instead of concentrated liquid. 3.2 Suggest other side reaction that could happen. -44-  Pre-lab. Report sheet (7) Synthesis of p_acetamidobenzene-sulfonamide Student name: Objectives: Solidity MW Compound in Hazards (g/mol) water p- Acetamidobenzenesulfonyl chloride Ammonium hydroxide sulfuric acid p_acetamidobenzene- sulfonamide Find out the melting point of p_acetamidobenzene-sulfonamide? Draw a simple flowchart for the procedure of the exp. Or write an outline steps in your own words? -45-  Post-lab. Report sheet (7) Synthesis of p_acetamidobenzene-sulfonamide Student name: 1. Data and results: The actual weight (yield) of p_acetamidobenzene-sulfonamide: The theoretical weight (yield) of p_acetamidobenzene-sulfonamide (show detailed calculation): The yield%: 2. Chemical equations: Write down the balanced chemical equations that represent the preparation of p_acetamidobenzene-sulfonamide: -46-  Experiment 8 Multistep Synthesis of Sulfanilamide Part 4: Synthesis of sulfanilamide Sulfonamide group is slowly hydrolyzed and more stable in diluted acid, due to: I. steric hindrance to the approach of water molecules on sulfur. II. Resonance stabilization, it is more resonance stabilized than aceta-amide. Carboxylic amide (aceta-amide) group hydrolyzes easily in very diluted acid. Amide and esters are easily hydrolyzed in both diluted acid or diluted base condition with reflux. Neutralization with sodium carbonate (pH 6-7) in order to get the free base and precipitate sulfanilamide. -47- ❖ Sulfanilamide is amphoteric that has weak acidic and weak basic properties. -48-  Mechanism The reaction is Hydrolysis reaction in diluted acid condition. The mechanism involve proton transfer. Sulfanilamide in acidic condition will be protonated form of amine group, so neutralization step to deprotonate it and get it free of charge to ppt. + 𝑪𝑶−𝟐 𝟑 → + 𝑯𝑪𝑶− 𝟑 𝑯𝑪𝑶− + 𝟑 + 𝑯𝟑 𝑶 → 𝑪𝑶𝟐(𝒈) ↑ +𝑯𝟐 𝑶 -49-  Procedure 1. Weigh previous product (~5g) of p-Acetamidobenzenesulfonamide and transfer it to a 100 ml round-bottomed flask. 2. Prepare a solution of dilute HCl by mixing equal volumes of conc. acid and water. 3. Add to the amide an amount of dilute acid solution twice [in excess] the weight of the amide. [excess to prevent drying during reflux] 4. Attach a reflux* condenser to the flask and heat at a gentle reflux for 30 minutes. 5. To the reaction mixture add equal quantity of water (equal to dil.acid added before) and transfer the new mixture to a 600 ml beaker. [added water to dilute the acid] 6. Neutralize the excess acid by the addition of small quantities of solid sodium bicarbonate (Na2CO3) until the solution is just alkaline to litmus paper (pH 6-7) and stope foaming. [added carbonate slowly to ↓ foaming and prevent loss of our product and avoid extra addition] 7. Cool the mixture in an ice bath and collect the crystals by suction filtration. Wash the crystals with a small amount of cold water. 8. Recrystallize from small amount of hot water, decolorize the solution with charcoal (if necessary) then collect the pure product and allow it to dry in air. Weight (gm) M.P Sulfanilamide 6 Brain storming Question 6.1 Discus the reason behind using reflux. 6.2 Foaming will be resulted in this reaction, why? 6.3 In step 5, discus the effect of excess carbonate (Na2CO3) addition. * Please refer to appendix IV: Reflux and the reflux condenser. For more information. -50-  Pre-lab. Report sheet (8) Synthesis of sulfanilamide Student name: Objectives: MW Compound Hazards (g/mol) p- Acetamidobenzene sulfonamide HCl sodium bicarbonate sulfanilamide Find out the melting point of sulfanilamide? Draw a simple flowchart for the procedure of the exp. Or write an outline steps in your own words? -51-  Post-lab. Report sheet (8) Synthesis of sulfanilamide Student name: 1. Data and results: The actual weight (yield) of sulfanilamide: The theoretical weight of sulfanilamide (show detailed calculation): The yield%: 2. Chemical equations: Write down the balanced chemical equations that represent the preparation of sulfanilamide: -52-  Experiment 9 Molecular modeling In silico prediction of Ionization Constants of Drugs One of the basic tenets of medicinal chemistry is that biological activity is dependent on the three-dimensional placement of specific functional groups (the pharmacophore). Over the past few years, advances in the development of new mathematical models which describe chemical phenomena and development of more intuitive program interfaces coupled with the availability of faster, smaller and affordable computer hardware have provided experimental scientists with a new set of computational tools. These tools are being successfully used, in conjunction with traditional research techniques, to examine the structural properties of existing compounds, develop and quantify a hypothesis which relates these properties to observed activity and utilize these "rules" to predict properties and activities for new chemical entities. The development of molecular modeling programs and their application in pharmaceutical research has been formalized as a field of study known as computer assisted drug design (CADD) or computer assisted molecular design (CAMD). Molecular modeling allows scientists to use computers to visualize molecules, to discover new lead compounds for drugs, or to refine existing drugs in silico. "Molecular modeling" is a term for which the definition has evolved along with the capabilities of computer hard-ware and algorithms. The term referred to software capable of displaying and manipulating simple structures of molecules. As computer became faster and algorithms more -53- accurate, the term grew to include algorithms for calculating the structures of small molecules Modern molecular modeling software is used to study small molecules, proteins, lipids, DNA, and non biological work. One goal of molecular modeling is to develop a sufficiently accurate model of the system so that the physical experiment may not be necessary. Most pharmacologically active molecules contain one or more ionizing groups, and it's well-known that knowledge of the ionization state of a drug, indicated by the pKa value, is critical for understanding many properties important to the drug discovery and development process. The ionization state of a compound directly influences such important pharmaceutical characteristics as aqueous solubility, permeability, crystal structure, etc. Tremendous advances have been made in the field of experimental determination of pKa, in terms of both quantity / speed and quality / accuracy. However, there still remains a need for accurate in-silico predictions of pKa both to estimate this parameter for virtual compounds and to focus screening efforts of real compounds. Methods of molecular modeling: 1. Quantum mechanics. 2. Molecular mechanics. This experiment aims to teach you How to convert 2-D structure in to 3-D structure? How to find the most stable conformer for a given molecule? -54-  Conformation of cyclohexane The chair conformer is the most stable cyclohexane conformer consists of 6 tetrahedral carbons bonded in a ring. Bond angels are 109.5o with no angle strain. No eclipsing C-H bonds and no torsional strain (all staggered). The axial bonds looks like they eclipse, but the axial bonds are too far away to contain torsional strain. When all hydrogen atoms bonded (Cyclohexane), there is no steric strain in the chair conformer. Two chair forms in equilibrium Boat , intermediate between chairs -55-  Software Marvin software Marvin is an advanced; Java based chemical editor for drawing chemical structures, queries and reactions. It has pre-loaded structure templates. It is capable of : 2-D to 3-D structural conversion ; 2-D cleaning and conformer generation ; drawing and formatting shapes , arrows and text boxes ; structure-based calculations ( e.g. charg log P, etc ). The default layout of the MarvinSketch user interface is shown in the following picture. -56- Opening and Saving a Molecule-File To Open an Existing Molecule File You can open existing molecule files (from supported file formats) by choosing File > Open on the menu bar. It will load the content of the molecule file into Marvin and discard any unsaved changes. To Save a Molecule File You can save the molecule to any of the supported file formats. This will allow you to open and work with this molecule later. The default behavior of the Save button is to save the molecule to the same file it was opened from, in the same format. If you want to change the file name or format, choose Save As. If you are working with a new molecule, Save will function the same as Save As. To Save 'As Image' The Save As Image choice in the File menu allows you to save an image of the molecule in the sketcher. General Toolbar Rectangle Allows selection in rectangle mode on mouse drag. Selection Lasso Allows selection in lasso mode on mouse drag. Selection Allows selection in structure selection mode on Structure mouse drag. With this selection mode only whole Selection fragments can be selected. Erase Removes all structures upon selection. Reverses the last command or the last entry you Undo typed. Redo Reverses the action of the last Undo command. Cut Removes and copies the selection to the clipboard. Copy Copies the selection to the clipboard. Inserts the contents of the clipboard at the location Paste of the cursor, without replacing selection. Checks and corrects chemical structures. Check See Structure Checker in MarvinSketch for more Structure details. Zoom In Increases the canvas's magnification. Zoom Out Decreases the canvas's magnification. -57-  Changes the canvas's magnification to a specific Zoom Tool value. It can also do autoscale using named values: All, Selection, Scaffold, R-groups. Help Contents Shows MarvinSketch User's Guide. Tools Toolbar Insert Bond Places various bond types on the canvas. Places a carbon chain on the canvas. The number of carbon atoms can be increased or decreased by Insert Chain dragging the mouse. Selection of straight or curved chain drawing is available. Thickens the selected bond. See details on bold Bold Tool tool function. Hashed Bond Makes the selected bond hashed. It only retains single Tool original bond type. Places a Text object on the canvas. Allows changing Insert Text text properties on the appearing toolbar. Insert Reaction Places various reaction arrow objects on the canvas. Arrow Create Group Creates a custom abbreviation group. Places brackets, parentheses, chevrons or braces on the Insert Brackets canvas. Insert Graphics Places various graphical objects on the canvas. Increases the charge of the selected atom. The number of implicit hydrogens will be adjusted if possible to Increase Charge accommodate the new charge. Valence errors will be highlighted in red. Decreases the charge of the selected atom. The number Decrease of implicit hydrogens will be adjusted if possible to Charge accommodate the new charge. Valence errors will be highlighted in red. -58- Atoms Toolbar Shows periodic system and query/atom property drawing Periodic System window. Insert Hydrogen Places Hydrogen atom on the canvas. Insert Carbon Places Carbon atom on the canvas. Insert Nitrogen Places Nitrogen atom on the canvas. Insert Oxygen Places Oxygen atom on the canvas. Insert Sulfur Places Sulfur atom on the canvas. Insert Fluorine Places Fluorine atom on the canvas. Insert Places Phosphorus atom on the canvas. Phosphorus Insert Chlorine Places Chlorine atom on the canvas. Insert Bromine Places Bromine atom on the canvas. Insert Iodine Places Iodine atom on the canvas. Chemical Toolbar This toolbar contains chemical functions and it is not visible by default. To make it visible, choose View > Toolbars > Chemical. Clean 2D Calculates new 2D coordinates for the molecule. Calculates new 3D coordinates for the molecule. Clean3D builds up conformers of fragments from which the best, i.e. the lowest Clean 3D energy conformer is given back. The quality of the structures is measured by a simple energy function (Dreiding type molecular mechanics). Convert to Transforms the molecule to aromatic representation using the Aromatic transformation method set. Form Convert to Kekulé Transforms the molecule to non-aromatic representation. Form -59-  Simple Templates Toolbar If you only wish to use the 6 generic template structures without additional functions, you can use the Simple Templates Toolbar. This toolbar is not visible by default. To make it visible, choose View > Toolbars > Simple Templates. Cyclopentane (house) Pyrrole Cyclopentane Cyclohexane Benzene Naphthalene Advanced Templates Toolbar This toolbar contains special buttons holding structure templates. Additional functions of this toolbar: 1. The toolbar can show different template groups. General and My Templates: Crown Ethers and Bridged Polycyclics: To control which template sets are displayed on the toolbar, use the Properties panel in the Template Library from insert menu: -60- Checking the 'Use molecules as templates at 2D cleaning' checkbox will effect the structures containing that template during cleaning of the structure: the default cleaning form is overwritten by the template structure. This way, you can cutomize your drawings: add or draw a set of templates and check this option. Any structure can be added to the My Templates group. Using the Pop-up menu Set the name of the new template. Right-click on the template icon on the template toolbar and select Properties. -61-  Set the name and/or the abbreviation of the template in the Template Properties box. After that the template is identified with its name and/or abbreviation. Templates without a name If the template does not have a name, hovering the cursor over its icon on the template toolbar magnifies the image on the icon. This improves the visibility of the template icon, especially for big structures. The template can be removed from the toolbar. Right-click on the template icon and select Remove to remove the template from the toolbar and from the My Templates list. Status Bar of MarvinSketch The Status Bar appears at the bottom of the main frame, and unlike toolbars, it cannot be customized or moved. The Status Bar consists of 3 parts: 1. Dimension Button Switches between 2D and 3D modes. If the current structure is represented in 3D, then switching to 2D mode performs a 2D cleaning upon confirmation. 2. File Status Indicator This sign appears dynamically if there are unsaved modifications on the current structure, and disappears upon a Save command. -62-  3. Structure Checker Status By default it is disabled as seen on the first image. To enable manual checking double-click on it. Right-click enables automatic checking. The status bar displays different images when there is no problem, if checking is in progress or if problems were found. 4. Navigation Buttons The Navigation Buttons appearing on the Status Bar dynamically using multipage molecular documents provide a quick way to navigate between pages. Elemental Analysis Basic molecular values related to the elemental composition of the molecule are calculated by the Elemental Analysis. In the Elemental Analysis Options panel you can check different properties: Type Mass: average molecular mass calculated from the standard atomic weights 1. Exact mass: monoisotopic mass calculated from the weights 2 of the most abundant natural isotopes of the elements. Formula: chemical formula of the molecule according to the Hill system 3: the number of carbon atoms is indicated first, the number of hydrogen atoms next, and then the number of all other chemical elements subsequently, in alphabetical order. Isotopes (like Deuterium and Tritium) are not listed separately but counted together (e.g., deuterium and tritium atoms are counted as hydrogens). When the formula contains no carbon, all the elements, including hydrogen, are listed alphabetically. If the molecule contains an SRU or Repeating Unit S-group, it will be taken into account and Polymer Formula will be generated. Note: For polymer structures, mass, composition, and atom count calculations are not available and will return NaN, N/A, and -1, respectively. Isotope formula: chemical formula of the molecule listing isotopes separately according to the Hill system. -63-  Dot-disconnected formula: chemical formula of the molecule(s) separating fragment formulas by dots (e.g. salts, counterions, solvent molecules etc. are present). Dot-disconnected isotope formula: chemical formula of the molecule separating fragment formulas by dots and listing isotopes separately. Composition: elemental composition given in weight percentage (w/w %) calculated from the atomic masses. Isotope composition: elemental composition listing isotopes separately (w/w %). Atom count: number of all atoms in the molecule. Use D/T symbols for deuterium/Tritium: if unchecked (default), isotopes of hydrogen are displayed in formulas as 2H and 3H, if checked, D and T symbols are used. Single fragment mode: if unchecked (default), the calculation handles unlinked molecules together (e.g. salt molecules), summing up the masses of each component, if checked, the results are displayed in a scroll window. Name generator -64- Log P/ log D -65- PKa -66-  Decimal places: setting the number of decimal places with which the result value is given. Mode: micro, macro: micro and macro acidic dissociation constants. Acid/base prefix: o static: submitted ionic forms are converted to their neutral forms (adding or removing protons) and their pKa is calculated. o dynamic: the pKa of ionic forms are calculated, not their conjugated acids or bases. Min basic pKa: widens the calculation range because weak bases will have lower pKa values than the default -10. Max acidic pKa: widens the calculation range because weak acids will have higher pKa values than the default 20. Temperature: setting the temperature in Kelvin. Results are shown in a separate window -67- 2D and 3D Viewer Windows Choosing View >Open 2D Viewer or Open 3D Viewer launches a MarvinView window containing the current molecule of MarvinSketch -68- Isomers and their energies -69- Conformers Ex. Cyclopentane -70- You can choose the option calculate the lowest energy conformer and just the lowest energy conformer will be displayed -71- Stereoisomers(cis/trans) Ex. 2-pentene -72- Ionization of compounds at different PH values and isoelectric point: Ex. Oxazole -73- -74- Major microspecies at specific PH value: Oxazole not ionized at physiological Ph (7.4) -75- Working with Multipage Molecular Documents How to create a multipage molecular document Multipage molecular documents help to work with large drawings by dividing them into pages. You can create a multipage molecular document by choosing File > Document Settings..., then checking in the Multipage document checkbox. You can set the number of horizontal and vertical pages in the Document Grid part, and you can also define the title, the page size and the margins in the corresponding sections of this dialog window. After pushing the OK button, the following controls become automatically available: The items in the View > Pages menu are enabled A navigation status bar appears on the bottom of the window The frame of the pages appear on the canvas, while the title, the margins and the page numbers are displayed on each page -76- Explicit/implicit hydrogens -77- structure with explicit hydrogens implicit hydrogen options implicit hydrogens on all -78-  Post-lab. Report sheet (9) Marvin software Student name: 1. Draw the chemical structure of both cis- and trans-2butene, minimize them and report their energies. Which of these isomers has the lowest energy? Can you explain why? 2. Draw the structure of sulfamethoxazole. 3. Find the IUPAC name. 4. Calculate Log P value. 5. Do you think that the drug at physiological pH will be ionized or not (study the degree of ionization of the drug at different pH's). 6. Draw the chemical structure of cyclohexane (determine the shape and energy of the least conformer). Use the different visualization models. 7. Draw the chemical structure of cis- 1,2dimethylcyclohexane and trans- 1,2dimethylcyclohexane (compare between the energy of the two isomers). 8. Draw the chemical structure of the compounds provided by the instructor. -79-  Experiment 10 Molecular modeling SAR analysis using accelrys software Once the structure of a lead compound is known, the medicinal chemist moves on to study its structure activity relationships (SAR). The aim here is to discover which parts of the molecule are important to biological activity and which are not. If it's possible to crystallize the target with the drug bound to the binding site, the crystal structure of the complex could be solved by x- ray crystallography, and then studied with molecular modeling soft ware to identify important binding interactions. However, this may not be possible, either because the target structure cannot be crystallized or because the target structure has not been identified. If that is the case, it will be necessary to revert to the traditional method of synthesizing a selected number of compounds that vary slightly from the original structure, then studying what effect that has on the biological activity. In this lab, we will use the computer soft ware accelrys to study a protein binding pocket and decide the most important forces that stabilize drug protein complex. Student must know 1. How to find binding pocket from the crystallographic image for protein. 2. How to measure the distance between two atoms. 3. Requirements for each binding force to occur between enzyme and inhibitor. -80- The default layout of the accelrys DS visualizer user interface is shown in the following picture. Atom Display toolbar The Atom Display toolbar contains buttons that allow you easy access to the commands on the Atom tab of the Graphics View Display Style dialog. Toolbar Action Effect button Atom Display Blanks selected atoms so they are not displayed on the Off screen. Displays atoms using a traditional wire frame display. Line Nonbonded atoms are displayed as "jacks". Displays bonds using solid cylinders. Nonbonded Stick atoms are displayed as "jacks" as in Line style. Depicts bonds using cylinders for bonds and balls for Ball and Stick atoms. Scaled Ball Depicts bonds using cylinders and atoms using balls and Stick scaled to the van der Waals (VDW) radii. Displays spheres sized to the van der Waals (VDW) CPK radii. -81- Sketching toolbar The Sketching toolbar allows you to sketch structures directly in the 3D Window by giving you access to tools to rotate torsions, to sketch atoms, chains, and rings, as well as to add text to your structures. View toolbar The View toolbar provides tools that manipulate the 3D Window or edit molecules in the document. Select: Use the Select tool to select one or more objects using the mouse. Multiple objects can be selected by clicking with the SHIFT key down on additional objects. Rotate: The Rotate tool is used to change the angle from which a molecule is viewed. Translate: The Translate tool is used to move a molecule in the plane of the computer screen. Zoom: The Zoom tool zooms the view in or out from a molecule. Home: Undoes all rotations, returning the structure to the original orientation it was in when it was imported or created. Note that centering (Center Structure or Fit To Screen) actions are not undone. Fit To Screen: Works in the same way as View | Fit To Screen. Center Structure: Carries out the same operation as as View | Center. Display Style: Accesses the same functionality as View | Display Style.... Measure: The Measure tool measures the distance, angle, or torsion between selected objects. To measure the distance between two atoms, deselect all objects, select two atoms or one bond, and click the Measure tool. To measure the angle between three atoms, deselect all objects, select three atoms or two connected bonds, and click the Measure tool. To measure the torsion angle between four atoms, deselect all objects, select four atoms, two disconnected bonds, or three connected bonds, and click the Measure tool. Display: The Display tool makes the object you click in either the 3D Structure View or the Hierarchy View visible, and everything else invisible. -82- Chemistry toolbar The Chemistry toolbar provides shortcuts to commands in the Chemistry menu that assist you in building molecules. Toolbar button Action Effect Add Hydrogens Chemistry | Hydrogens | Add Hide Hydrogens Chemistry | Hydrogens | Hide Single Bond Chemistry | Bond | Single Double Bond Chemistry | Bond | Double Aromatic Bond Chemistry | Bond | Aromatic Triple Bond Chemistry | Bond | Triple Periodic Table Chemistry | Element | Table... Clean Geometry Structure | Clean Geometry Ex. 1. Open the DS visualizer. 2. From the toolbar, go to File-Open-protein data bank file-Captopril-ACE complex. -83- 3. To change the color of the background From the toolbar, under edit open preferences then select 3D window from the left side (double click on background color and choose the color you want). 4. To identify the binding pocket, from the cell window (on the left side): Open the second A (click on + ) then select captopril (code X8Z1615). 5. From the toolbar, under Structure open Show by radius and enter the No. 10 then ok. 6. To differentiate between the amino acids in the binding pocket and the drug go to View-Display Style and select stick model. 7. Try to identify the Zn atom and identify how the drug is strongly attached to the metal atom in the binding pocket. 8. To find hydrogen bonding interactions between drug and the enzyme: select captopril (as shown above) then go to Structure Monitor –intermolecular H-Bond ). -84- 9. To identify the amino acids in the binding pocket that have interactions with the ligand ,select the amino acid then right click and choose label Choose amino acid from object and name from attribute then apply and ok and the abbreviated name of the amino acid will appear. Or from the cell window open H bond monitor 1(click on +) and all h-bond details will appear. -85- 10. Fill the drug enzyme interaction tables listed below: Drug Functional Type of No Amino acid Group interaction -86-  Pre-lab. Report sheet (10) SAR analysis using accelrys software Student name: Draw the structures of the 20 standard amino acids and classify them? Draw the structure of captopril then find out the IUPAC name? Draw the structure of levothyroxine then find out the IUPAC name? -87-  Post-lab. Report sheet (10) SAR analysis using accelrys software Student name: Procedure 1. Open the DS visualizer. 2. From the toolbar, go to File-Open-protein data bank file-thyroxine thyroid hormone receptor interactions. 3. To identify the binding pocket, from the cell window (on the left side): Open the second X (click on + ) then select thyroxine (code T44500). 4. From the toolbar, under Structure open Show by radius and enter the No. 10 then ok. 5. To differentiate between the amino acids in the binding pocket and the drug go to View-Display Style and select stick model. 6. To find hydrogen bonding interactions between drug and the receptor: select thyroxine (as shown above) then go to Structure Monitor –intermolecular H-Bond ). 7. Fill the drug enzyme interaction tables listed below. Drug Functional type of No Amino acid Group interaction -88-  Appendix I Percent Yields Calculations Many synthesis require more than one reaction, with each reaction yielding an isolated compound before the final product is reached. Each individual step in the sequence has a percent yield, and the total synthesis has an overall yield calculated from the steps yields Limiting reagent : the reagent that is present in the smallest equivalent stoichiometric amount and limits how much product will be produced. It determines the maximum amount of product(theoretical yield) that will be formed. 𝒂𝒄𝒕𝒖𝒂𝒍 𝒎𝒂𝒔𝒔 𝒚𝒊𝒆𝒍𝒅 Percent yield = * 100% 𝒕𝒉𝒆𝒐𝒓𝒆𝒕𝒊𝒄𝒂𝒍 𝒚𝒊𝒆𝒍𝒅 % 𝒚𝒊𝒆𝒍𝒅 𝒇𝒐𝒓 𝒔𝒕𝒆𝒑 𝟏 % 𝒚𝒊𝒆𝒍𝒅 𝒇𝒐𝒓 𝒔𝒕𝒆𝒑 𝟐 % 𝒚𝒊𝒆𝒍𝒅 𝒇𝒐𝒓 𝒔𝒕𝒆𝒑 𝟑 Overall yield = * ∗ 𝟏𝟎𝟎 𝟏𝟎𝟎 𝟏𝟎𝟎 Each individual step has a relatively high efficiency, but the overall efficiency is low. Each subsequent reaction further reduces the actual amount of product that is formed from the initial starting material. -89- Example (1) from cycle 1: Excess Excess Excess Acetanilide p-acetaminobenzene p-acetaminobenzene sulfanilamide sulfonylchloride sulfonamide 1. You have 3 steps for this reaction, so you have to find percent yield for each step then multiply them to find the overall yield for the synthesis. 2. In this cycle the other reactants were used in excess so your main reactant will be the rate limiting reactant. For Step 1: Acetanilide + chlorosulfonic acid > p acetaminobenzene sulfony