COLISTE NA hOLLSCOILE CORCAIGH UNIVERSITY COLLEGE CORK

PF3002
Pharmaceutical Chemistry III

Practical Manual School of Pharmacy

Contents
Health Warning Introduction and general information Safety Practical reports Laboratory sessions 3 4 6 9

1. Synthesis of 5,5-diphenylhydantoin (Phenytoin)

10

2. Preparation of Nitrazepam

12

Chemistry Practical Health Warning Checklist

Read the manual before stepping inside the laboratory Read the safety precautions and emergency procedures before using any chemical Always wear protective measures appropriate to the risk situation Read data sheet before initiating any work and complete risk assessment Assess the situation in front of you before starting your procedure The laboratory is a shared facility be aware of your own and your neighbours safety Communicate, communicate, communicate Consult your demonstrator if unsure of anything Remember you will be marked on laboratory efficiency not speed

This list is intended to ensure your safety. It is not a comprehensive list of the safety aspects required for this series

Introduction
Pharmaceutical chemistry seeks to examine the synthetic methodology used in order to produce pure biologically active molecules to be formulated as medicines. An in depth understanding of organic chemistry is core to the study of this subject as it plays a key role in many of the processes utilised in the formation of medicines. PF3002 builds on the core knowledge attained in PF1001, PF2001 and PF2002 and applies this to the pharmaceutical process. Key to this is the manipulation and transformation of functional groups in a structured manner to produce a suitable end product. With this knowledge, known functional group chemistry will be applied to the formation of new medicines. An important factor in pharmaceutical chemistry is the adaptability of the chemistry in the initial stages of a project in order to achieve analogues and derivatives of a lead compound, and in the final stages the ability to take a laboratory scale process (medicinal chemistry route producing milligrams of product for biological testing) and reproduce the same compound on a plant scale (producing kilos of product for formulation and market). This course will give an insight into the problems faced by all pharmaceutical companies producing compounds for use as medicines and the industrialisation of chemistry. As will be seen the medicinal chemistry route to most products is usually adapted and changed completely as the project progresses. In this series of labs we will learn about the issues concerning pharmaceutical production from drug discovery to plant scale manufacture. This practical series on the design and synthesis of compounds for medical use requires familiarity with the practical aspects of organic chemistry and for this it is important you revise the practical issues learned in PF1001 and PF2001, at least for safety reasons if not for academic. It must also be noted that most of the compounds and chemicals used will be bio-active to some degree (as that is the purpose of the chemistry) and so must be handled with caution and care. There are two practical experiments to be completed over four weeks that are designed to enhance your chemical skills to the level required to manipulate organic compounds in the very first process of pharmaceutical production. The initial chemistry involved will be familiar from lectures however it is essential to read thoroughly the experimental instructions involved in each laboratory session. The following experiments will be carried out: Experiment 1: Synthesis of 5,5-diphenylhydantoin (Phenytoin) Experiment 2: Preparation of Nitrazepam

GENERAL INFORMATION
It is essential that you familiarise yourself with each practical before attending a laboratory session. The following MUST be brought to ALL pharmaceutical chemistry laboratory practicals: Laboratory coat and safety glasses Laboratory manual (this book) Small hardback notebook labelled with your name, course and student ID number. This will be used as your own record of the experiment Calculator J-Cloth or tissues to clean the bench

Each laboratory session begins with a brief talk from the laboratory instructor in which the experiment will be described and advice on the correct procedure given. You must be on time for all laboratory sessions. An attendance record is taken.

GENERAL RULES: 1. 2. 3. 4. 5. 6. 7. 8. Admission to the laboratory is not permitted without safety glasses or laboratory coat. Bags should not be brought into the laboratory or left in the corridor outside the labs. Use your locker. No food or drink is to be brought into the laboratory Always read all the safety notes for each experiment. Do not take off your safety spectacles or laboratory coat at any time. Keep your bench tidy and avoid spilling chemicals. Do not swallow or touch any chemicals. Consult your Demonstrator before attempting any technique which is unfamiliar.

Remember: There are many potential hazards in the laboratory. It is you and your colleagues that are at risk. Be careful and insist that your co-workers are just as careful.

Safety in Laboratory Classes

When in the laboratory it is essential that you observe all the regulations for your own safety and for that of your colleagues. SAFETY SPECTACLES MUST BE WORN AT ALL TIMES, FAILURE TO DO SO WILL RESULT IN YOUR REMOVAL FROM THE LABORATORY. EATING, DRINKING and SMOKING ARE ALL FORBIDDEN. You should only carry out practical work when a demonstrator is present. No bench work is permitted outside the set laboratory periods for safety reasons. Some of the products that you will be making are bio-active, and therefore MUST be handled with care to avoid contact either by inhalation, ingestion or through skin contact. Bio-active compounds will cause varying effects on people and it must be stressed that on no circumstances should any product or reagent come in contact with your person. Treat all compounds with respect, and ensure spillages are dealt with by either a technician or demonstrator. Most organic solvents are highly flammable (especially diethyl ether), and the use of naked flames (bunsen burners) nearby or in the same fume cupboard is forbidden. ALWAYS check what your neighbour is doing. All chemicals should be handled with care, but some are particularly hazardous. These should be handled in the fume cupboard and the appropriate glove/additional protective clothing should be worn. Avoid breathing vapours of any volatile organic compound. Check the data sheets before using any organic material and know the emergency procedures involved. The laboratory is a communal area and so benches must be left clean and tidy and apparatus such as balances and rotary evaporators must not be left in a dirty condition. Always clean up your own mess before you move on to the next stage of any procedure. It is not fair to expect someone else to clear up your mess. Not only is this good practice but it is an essential safety requirement. Before you start any practical you are required to carry out a risk assessment of ALL the compounds, reagents and solvents you will be using listing their hazards. This should be written at the start of each experiment (incorporated into the data sheets). A typical assessment is given below. Remember, that safety should always come first and if you are in any doubt, ASK a demonstrator. They are there to help you.

Sample Assessment: Chemical Approx. amount Acetylacetone 6g

Assessment standard

Hazards toxic corrosive toxic corrosive

Spillage wash with water wash with water

Sodium hydroxide

5M soln

standard

Toluene

20 ml

fume cupboard standard

flammable

see demonstrator see demonstrator

Petroleum

75 ml

flammable

IN THE EVENT OF AN ACCIDENT IN THE LABORATORY, NOTIFY THE DEMONSTRATORS, TECHNICIANS OR ACADEMIC STAFF IMMEDIATELY

Data Sheets, Notebook, Practical Reports and Seminars

This practical series contains 4 component marks: a laboratory efficiency mark, a data sheet mark, a practical report mark and a seminar mark. The hardback notebook you are required to have is for your own records. It is important that you keep a detailed laboratory notebook in which you should write down exactly what you have done and record any observations. This will be vital for future use and revision. It is also good laboratory practice and should be updated as the reaction proceeds, not completed when finished. All observations and results should be entered directly into your laboratory notebook. The efficiency mark is given for competency and cleanliness. Please note this mark is not time dependent faster does not mean better. The mark is based on the quality of your work. A well completed laboratory notebook is a good indicator of efficiency. A complete data sheet is required for each experiment. Details on the data sheet will include overall reaction equations, melting points, yields, relevant analysis, etc. This sheet must be signed by your demonstrator before you leave the laboratory. On completion of the practical, a practical report must be drawn up and handed to your demonstrator by the deadline given (usually 1 week after the practical has been completed). The report should consist of the following sections: Title of Experiment Date Introduction Detail briefly the objectives of the experiment (max half page A4) Safety Assessment Assessment of all reagents and solvents Experimental Procedure Insert reference to manual. Do not transcribe from the manual. Include details of any procedures that are different from those given in the laboratory manual only. Reaction details If not on data sheet: weights, equivalents and number of moles of starting materials and reaction equation. Results If not on data sheet, report the results clearly and in tabular form when appropriate. Weights and percentage yield(s) of product(s) along with their melting point and spectra required. No need to copy from data sheet. Discussion

Outline the significance of your results. Provide concluding remarks and answer all questions given in the manual (max 2 page A4).
Include mechanisms for the reactions in the experiments where appropriate, along with the interpretation of NMR/IR spectra and the answers to any questions that are required.

ALL your products should be handed in, in a clearly labelled sample tube (Your name, experiment number and structure).

Report should be submitted in a plastic A4 sheet including the signed data sheet. These reports will be returned for review and feedback after marking but will be recollected at end of practical series. Final practical mark will be determined by return of reports. If you are unsure about any technique or instruction given in the experimental script, ASK THE DEMONSTRATOR. A number of videos are available in the laboratory, which show the basic techniques you will need to use. Work should be presented for marking as soon as the experiment is completed. Seminar marks will be determined on presentation, content and delivery. Seminars will take place during laboratory time and topics will be assigned in the first week of practicals.

Experiment 1
Introduction

Synthesis of 5,5-diphenylhydantoin (Phenytoin)

Phenytoin has been widely prescribed for the control of epilepsy since its introduction as a pharmaceutical agent during 1950s, and although superseded by a number of newer drugs, it remains in use today in this role. The main structural challenge for the synthesis of this compound is the construction of the hydantoin ring. This hydantoin ring can be formed in a one-pot procedure starting from benzil. The procedure for this reaction is a base catalysed addition of urea to benzil that is an interesting example of a benzilic acid re-arrangement where the phenyl groups undergoes a 1,2-migration during formation of the hydantoin ring (Scheme 1).

KOH, NH2CONH2

O HN NH O

Ethanol

Scheme 1 Synthesis of phenytoin from benzil

Objectives Synthesis and purification of a pharmaceutical product and confirmation of purity by melting point, TLC and IR analysis. Materials Benzil Urea Potassium Hydroxide Hydrochloric Acid Ethanol Procedure Benzil (2.00 g, 10 mmol), potassium hydroxide (2.00 g, 17.82 mmol) and urea (780 mg, 13 mmol) were dissolved in dry ethanol (80 ml) and heated gently to reflux on a steam bath until TLC analysis [ethyl acetate:hexane (20:80)] indicated complete consumption of starting material. The reaction mixture is then poured onto an ice/water mixture (100 ml) and the solution filtered to remove a small quantity of 4,5-diphenyl-4.5-dihydroxy-2imidazolone. The remaining red solution is acidified by the drop wise addition of concentrated hydrochloric acid (CARE) to precipitate the product as a white solid, which was removed by filtration, washed with water and dried to give the product that is recrystallised from ethanol. You should calculate the yield of recrystallised material, and record the IR and melting point of your product.

2g 780 mg 2g 80 ml

Irritant Irritant Harmful / Corrosive Harmful / Corrosive Harmful

Questions
The most common method for the synthesis of phenytoin analogues is the Bergs synthesis which has been previously been used to prepare a large number of analogues for pharmacological screening studies (Scheme2). (i) Why do we not use this method today? (ii) Derive a mechanism for this reaction.
O

KCN, (NH4)2CO3

O HN NH O

EtOH,

Scheme 2 Bergs synthesis of phenytoin

Experiment 2
Introduction

Preparation of Nitrazepam

The discovery in 1957 by L. O. Randell of the tranquillising and sedative properties of chlorodiazepoxide (Librium) (1), prepared two years earlier by L. H. Sternbach at Hoffman La Roche laboratories, was the starting point for much pharmacological and chemical research leading to the important class of anxiolytic drugs known as benzodiazepines. Many substances of this group are used in anxiolytic, hypnotic and anticonvulsant therapy. The original Sternbach procedure is difficult to implement in teaching laboratories and we will use a modified procedure to prepare nitrazepam (2) a nitrated benzodiazepine derivative.
N Cl N O NHCH3 H N O2N N O

(1)

(2)

NOTE: This product is pharmacologically active and should be handled with extreme care. Objectives Synthesis and purification of Nitrazepam and analysis of the purity of intermediates and product by TLC, IR and melting point. Application of a three step synthetic process to form a pharmaceutical product. Procedure Step 1. Preparation of 2-bromoacetamido-5-nitrobenzophenone
O NH2 O2N O Toluene, Br Br O2N NH Br O O

Materials Bromoacetyl bromide 2-amino-5-nitrobenzophenone Toluene

3.23 g, 16 mmol 3 g, 12.4 mmol 20 ml

Irritant Irritant Irritant

Bromoacetyl bromide (3.23 g, 1.4 ml, 16 mmol) is added dropwise to a stirred solution of 2-amino-5-nitrobenzophenone (3 g, 12.4 mmol) in toluene (20 ml) at room temperature and then heated to reflux on a steam bath for 30 minutes. (NOTE: CARE hydrogen bromide gas is evolved. Carry out all procedures in a fume hood). On cooling, the product crystallises out from the reaction mixture and is isolated by filtration to yield a yellow solid. Record the yield of your product and its IR spectrum and melting point.

Step 2.

Preparation of 2-Azidoacetamido-5-nitrobenzophenone
O NH Br O2N O Ethanol, NaN3 O2N O NH N3 O

Materials 2-bromoacetamido-5-nitrobenzophenone Sodium azide Ethanol Acetone

2.0g, 5.5 mmol 0.45 g, 6.9 mmol 30 ml 20 ml

Irritant Toxic / Explosive Irritant Irritant

NOTE: Sodium azide is toxic and is potentially explosive. Pre-weighed samples will be provided for you, and this compound should not be handled directly by undergraduate student. 2-Bromoacetamido-5-nitrobenzophenone (2.0 g, 5.5 mmol) and sodium azide (0.45g, 6.9 mmol) in ethanol (30 ml) are refluxed for 30 minutes and then acetone (20 ml) is added carefully to redissolve the crude azide. The mixture is then filtered while hot to remove any residual sodium azide before concentrating to approximately 40 ml to precipitate the azide product that is collected by filtration to yield the product as a white solid used directly in the next reaction.

Step 3.

Preparation of Nitrazepam
O NH N3 O2N O Ph3P O2N Toluene, ( - N2) N H N O

Materials 2-azidoacetamido-5-nitrobenzophenone Triphenylphosphine Toluene

1.5 g, 4.6 mmol 1.82 g, 5 mmol 20 ml

Irritant Irritant Irritant

2-Azidoacetamido-5-nitrobenzophenone (1.5 g, 4.6 mmol) and triphenylphosphine (1.82 g, 5 mmol) in toluene (20 ml) are stirred at room temperature until no more nitrogen is evolved and all solids completely dissolve to give a yellow solution. The mixture is then refluxed for one hour with occasional stirring. The toluene is then removed on a rotary evaporator, the residue can be crystallised by the addition of ethanol (10 ml) to give the product as a yellow solid that is isolated by filtration. Recrystallisation from approximately 30-40 ml of ethanol gives the product as a light yellow solid. Questions

(i)

Describe the stereochemistry of the benzodiazepines.

SOME HAZARDS ASSOCIATED WITH MATERIALS USED ON THIS PRACTICAL COURSE

Acetone b.p 56 C Highly Flammable If contact with eyes flush with copious amounts of water for 15 minutes while keeping eyelids separated. Wash skin with soap. If inhaled remove to fresh air.

If contact with eyes flush with copious 2-Amino-5-nitrobenzophenone Irritating to eyes, skin and respiratory amounts of water for 15 minutes while keeping eyelids separated. Wash skin with system soap. If contact with eyes flush with copious Benzil Irritating to eyes, skin and respiratory amounts of water for 15 minutes while keeping eyelids separated. Wash skin with system soap. If contact with eyes flush with copious Bromoacetyl Bromide Causes burns. Irritating to eyes and amounts of water for 15 minutes while keeping eyelids separated. Wash skin with respiratory system. soap. If inhaled remove to fresh air. If contact with eyes flush with copious Ethanol b.p. 78 C Highly flammable. Irritating to eyes, amounts of water for 15 minutes while keeping eyelids separated. Wash skin with respiratory system and skin. soap. If inhaled remove to fresh air. Ethyl Acetate b.p. 77 C Highly flammable If contact with eyes flush with copious amounts of water for 15 minutes while keeping eyelids separated. Wash skin with soap. If inhaled remove to fresh air. If contact with eyes flush with copious amounts of water for 15 minutes while keeping eyelids separated. Wash skin with soap. If inhaled remove to fresh air.

Hexane b.p. 68 C Highly flammable. Possible serious damage to health by prolonged exposure through inhalation.

If contact with eyes flush with copious Hydrochloric Acid Causes burns. Irritating to eyes, skin and amounts of water for 15 minutes while keeping eyelids separated. Wash skin with respiratory system. soap.

If contact with eyes flush with copious Methanol b.p. 65 C Highly flammable. Toxic by inhalation and amounts of water for 15 minutes while if swallowed. keeping eyelids separated. Wash skin with soap. If inhaled remove to fresh air. Potassium Hydoxide Causes severe burns If contact with eyes flush with copious amounts of water for 15 minutes while keeping eyelids separated. Wash skin with soap. If contact with eyes flush with copious Contact with amounts of water for 15 minutes while keeping eyelids separated. Wash skin with soap. If contact with eyes flush with copious amounts of water for 15 minutes while keeping eyelids separated. Wash skin with soap. If inhaled remove to fresh air. If contact with eyes flush with copious amounts of water for 15 minutes while keeping eyelids separated. Wash skin with soap.

Sodium Azide Very toxic if swallowed. acids liberates toxic gas.

Toluene Highly flammable. Harmful by inhalation.

Triphenyl Phosphine Harmful if swallowed.

Urea Toxic by inhalation, by contact with skin If contact with eyes flush with copious and if swallowed. Irritating to eyes, skin amounts of water for 15 minutes while keeping eyelids separated. Wash skin with and respiratory system. soap.