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COLÁISTE NA hOLLSCOILE CORCAIGH UNIVERSITY COLLEGE CORK
PF3002
Pharmaceutical Chemistry III
Practical Manual
School of Pharmacy
Contents
Health Warning 3
Introduction and general information 4
Safety 6
Practical reports 9
Laboratory sessions
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. Admission to the laboratory is not permitted without safety glasses or laboratory
coat.
2. Bags should not be brought into the laboratory or left in the corridor outside the
labs. Use your locker.
3. No food or drink is to be brought into the laboratory
4. Always read all the safety notes for each experiment.
5. Do not take off your safety spectacles or laboratory coat at any time.
6. Keep your bench tidy and avoid spilling chemicals.
7. Do not swallow or touch any chemicals.
8. 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 Assessment Hazards Spillage
Acetylacetone
Sodium
hydroxide
Toluene
Petroleum
6g
5M soln
20 ml
75 ml
standard
standard
fume
cupboard
standard
toxic
corrosive
toxic
corrosive
flammable
flammable
wash with
water
wash with
water
see
demonstrator
see
demonstrator
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 Synthesis of 5,5-diphenylhydantoin (Phenytoin)
Introduction Phenytoin has been widely prescribed for the control of epilepsy since its introduction as
a pharmaceutical agent during 1950’s, 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).
O
O KOH, NH2CONH2
Ethanol HNNH
O
O 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 2g Irritant
Urea 780 mg Irritant
Potassium Hydroxide 2 g Harmful / Corrosive
Hydrochloric Acid Harmful / Corrosive
Ethanol 80 ml Harmful
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-2-
imidazolone. 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.
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.
OKCN, (NH4)2CO3
EtOH, ∆ HNNH
O
O Scheme 2 Bergs synthesis of phenytoin
Experiment 2 Preparation of Nitrazepam Introduction
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
N
ClO
NHCH3
N
HN
O2N
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
NH2
O2NO
NH
O2NO
O
Br
Toluene, ∆
Br Br
O
Materials Bromoacetyl bromide 3.23 g, 16 mmol Irritant
2-amino-5-nitrobenzophenone 3 g, 12.4 mmol Irritant
Toluene 20 ml 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
NH
O2NO
O
Br NaN3
Ethanol, ∆
NH
O2NO
O
N3
Materials 2-bromoacetamido-5-nitrobenzophenone 2.0g, 5.5 mmol Irritant
Sodium azide 0.45 g, 6.9 mmol Toxic / Explosive
Ethanol 30 ml Irritant
Acetone 20 ml 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
NH
O2NO
O
N3 Ph3P
Toluene, ∆
( - N2)
N
HN
O2N
O
Materials 2-azidoacetamido-5-nitrobenzophenone 1.5 g, 4.6 mmol Irritant
Triphenylphosphine 1.82 g, 5 mmol Irritant
Toluene 20 ml 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
2-Amino-5-nitrobenzophenone Irritating to eyes, skin and respiratory
system
Benzil Irritating to eyes, skin and respiratory
system
Bromoacetyl Bromide Causes burns. Irritating to eyes and
respiratory system.
Ethanol b.p. 78°C
Highly flammable. Irritating to eyes,
respiratory system and skin.
Ethyl Acetate b.p. 77°C
Highly flammable
Hexane b.p. 68°C
Highly flammable. Possible serious
damage to health by prolonged exposure
through inhalation.
Hydrochloric Acid Causes burns. Irritating to eyes, skin and
respiratory system.
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 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
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.
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.
If contact with eyes flush with copious
amounts of water for 15 minutes while
keeping eyelids separated. Wash skin with
soap.
Methanol b.p. 65°C
Highly flammable. Toxic by inhalation and
if swallowed.
Potassium Hydoxide Causes severe burns
Sodium Azide Very toxic if swallowed. Contact with
acids liberates toxic gas.
Toluene Highly flammable. Harmful by inhalation.
Triphenyl Phosphine Harmful if swallowed.
Urea Toxic by inhalation, by contact with skin
and if swallowed. Irritating to eyes, skin
and respiratory system.
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 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
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 contact with eyes flush with copious
amounts of water for 15 minutes while
keeping eyelids separated. Wash skin with
soap.