Ramipril is a prodrug belongs to
the group of angiotensin-converting enzyme (ACE) inhibitor class of
medications. It is converted to Ramiprilat in the liver by esterase enzymes1.
Ramipril competitively inhibits ACE and it prevents the formation of active octapeptide,
(angiotensin II) from inactive decapeptide, (angiotensin I).2 The inhibition
of angiotensin II leads to vasodilation and regulation of blood pressure which
is a key component of the renin angiotensin-aldosterone system (RAAS). RAAS is
a mechanism for regulating hemodynamic, water and electrolyte balance1.
When renal blood pressure is low, renin is secreted from juxtaglomerular kidney
cells in to the blood stream. Renin cleaves the circulating angiotensinogen to
ATI, which then gets converted to ATII by ACE. Therefore, increase in ATII
leads to vasoconstriction and increase in the blood pressure. Ramipril is used
in treatments of hypertension, it may also be used to reduce cardiovascular
deaths following myocardial infraction in hemodynamically stable patients.  Also used to reduce the rate of deaths in myocardial
infraction and stroke patients who are in high risk of developing
cardiovascular diseases.3  


1.2: Bioavailability and the effect of adding
methyl group on Ramipril.

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It has found that the
bioavalibilities of Ramipril and ramiprilat is 28% and 44%, respectively, when
oral administration is taken compared to intravenous administration1.
The bioavailability can be increased if changes are made to the Ramipril
structure. The way this can be carried forward is to look at the chemical
structure of Ramipril and the functional groups present. The two-important
active functional group of Ramipril is carboxylic acid and the aromatic ring.  The carboxylic
acid group fits into the enzymes pocket by exhibiting hydrophilic attraction
while the aromatic ring exhibits hydrophobic attraction with ACE at the region
S1′. 4,5

The addition of methyl group to the
aromatic ring on Ramipril by Friedel crafts alkylation will make the ring more
hydrophobic leading to stronger attraction between the enzymes pocket (S1′) and
the drug via Van der Waals forces. This will increase bioavailability because
it makes it difficult for the drug to disassociate from the enzyme and
therefore, harder for the drug to be broken before reached to the active site
in the body. The methyl group on aromatic ring will also increase the
therapeutic effect of Ramipril, as it provides extra binding with enzymes
pocket S1′. This in return can reduce possible side effects and adverse drug

1.3 Friedel crafts alkylation reaction.


The addition of methyl group is
carried by Friedel crafts alkylation, this is an organic reaction that is used
to substitute alkyl halide on to aromatic compound using a Lewis acid catalyst.
The reaction starts with Lewis acid attacking halide from the alkyl halide to
form an electrophilic intermittent and formation of tetrasubstituted aluminium
anion (AlCl4).  Then the
aromatic ring attacks the alkyl cation via electrophilic aromatic substitution
(SEAr) to a cationic product leading the aromaticity to be lost. The
loss of proton from the aromatic ring will result in final product and
regeneration of the Lewis acid catalyst. 6,7

Figure 1. The generation of carbocation using acid Lewis

Figure 1 shows a Lewis acid/base
complex. The complex has lead a partial positive charge on the chloride due to
the pulling of electrons from the R group making the dipole between R and
chloride bigger8.

 The ? electrons of the aromatic ring attacking
the electrophilic R+ group. This leads to aromaticity of the ring to be destroyed
forming a cation intermediate.

Figure 2. Formation of intermediate.

The last step is to detach the
proton on the ring to re-establish the aromaticity of the ring. This step also
involves the regeneration of the catalyst back to AlCl3.

Figure 3. Regeneration of aromatic ring by losing a proton.

The reaction shown above will be
performed on Ramipril on the aromatic ring. The position which methyl group
takes is extremely important, the drug will be most effective if it takes ortho
position however, the position of methyl group can either be para or ortho this
is because the aromatic ring is attached to CH2 group which is a
directing group and therefore determines the position of the methyl group.  The substituent present on the aromatic ring
(CH2) is also an activating group which means it is more electron
donating than hydrogen and enhances the rate of reaction due to increase in
electron density making the ring more reactive towards electrophiles9.



Structure of Ramipril:

Diagram 1: showing chemical
structure of Ramipril before Friedel Crafts alkylation.


Design of congener of Ramipril

Diagram 2: Structure of congener of
Ramipril at ortho position.


Diagram 3: structure of congener of
Ramipril at para position.

(Structures have been designed
using MolVeiw10)












2. Aims and objectives:


2.1: Aims


The aim is to produce a congener of
Ramipril to make the drug more effective in the treatment of hypertension and
cardiovascular associated diseases using Friedel crafts alkylation reaction to
increase the bioavailability of the drug.

2.2: Objectives


Objectives will be:

To create congener of Ramipril to increase
bioavailability by making aromatic ring more hydrophobic.

To increase overall therapeutic effect by adding
methyl group to the lead structure to probe for extra binding with the target

To test the final product synthesised using in vitro and in vivo testing.

To purify congener using different methods and












1.       The
first step is to weight out anhydrous aluminium chloride and transfer into 25 ml
round bottom flask containing a magnetic stir bar11.

2.       Fit
this with a claisen adaptor, dropping funnel and a
condenser. Ramipril is added to the flask, and chloromethane is added to the
dropping funnel.  An ice bath is also
prepared to cool the reaction if needed.

3.       While
Ramipril and aluminium chloride is being stirred, chloromethane is added slowly.  If the reaction becomes too vigorous the ice
bath is used to cool the reaction. After the addition of chloromethane is
complete continue stirring the mixture at room temperature for 10-15 minutes.

4.       Transfer
the mixture into 50ml beaker with ice in it. The mixture is stirred until
fuming has ended.

5.        The two phases: aqueous and organic mixture is
stirred while adding solid sodium chloride to the mixture to make the aqueous
phase saturated. Then the mixture is poured in to separate funnel. The organic
layer is separated and transferred into a conical flask and dried over
anhydrous Na2SO4.  Transfer the dried organic layer in to a clean
25ml round flask and filter the organic layer.12








3.1 Analysis of congener synthesised and
comparing with Ramipril.


needs to be performed on the congener synthases of Ramipril. Chromatography is a
process in which a chemical mixture is carried around the stationary phase and
mobile phase. Components of the mixture are separated because of differential
distribution of the solutes between carrier phase and the stationary phase. The
amount of time that each competent spends in each phase depends on their
affinity to the stationary phase or the mobile phase. The attraction depends on
the individuals physical and chemical structure13. High performance
liquid chromatography (HPLC) will be performed to compare the purity of
Ramipril to the product that has been produced. HPLC is a performed by
injecting the mixture in the mobile phase at high pressure through the column
(The stationary phase).14  (Appendix
1) shows that the retention time of Ramipril is 2.910 minutes, for the congener
synthesised this would be longer, due to addition of methyl group added on the
aromatic ring making the drug to become more hydrophobic which means the
congener will spend more time in the stationary phase and will take longer to
eluate form the column.

Infrared spectrometry (IR) is
another method that can be used to obtain information about the functional
groups of a molecule.  In IR spectrometry
substances are exposed to IR radiation this leads to vibrational energy to
occur within the molecule which then absorbs IR radiation at a specific
frequency leading to specific bands which can be used to determine the types of
bonds and therefore the functional group that are present within the molecule.15,16,18
By comparing the IR spectroscopy of Ramipril and the congener there is a
difference showing absence of methyl group on the aromatic ring in the
structure of Ramipril. (Appendix 2) shows the IR spectroscopy of Ramipril, showing
a band at 3032 wavelength/ cm-1 which a stretch caused by C-H in
aromatic ring. It is expected that congener synthesised will have another peak
which Ramipril will not have, this is due to C-H alkyl on the aromatic ring and
have a wavelength of 2925 cm-1 . The rest of the bands are expected
same because they have both have same function groups.

spectrometry (MS) can also be used to investigate the sample according to their
mass and electrical charge. This will produce a mass spectrum that would plot mass
to charge ratio of compounds in a mixture17. The molecular formula
of Ramipril is C23H32N2O5  and the molecular weight is 416.518 g/mol
(appendix 3).19 Comparing this to synthesised congener the molecular
formula is C24H35N2O5 and the molecular
weight is expected to be 431g/mol. The mass spectrometry of Ramipril has a
fragment of 91 which corresponds to the benzene ring and CH2  that is attached to the aromatic ring, this
fragment would be different for synthesised congener  due to addition of CH3 group. This
fragment would be 106 instead of 91. (appendix 3)

NMR is an
important technique that can be used to give essential information about
congener and allow the comparison to be made between Ramipril. There are two
types of NMR that can be used: proton NMR and Carbon-13 NMR. (Appendix 4) shows
proton NMR of Ramipril it has a chemical shift of 6.8-8 ppm which is
responsible for the aromatic ring on Ramipril, there are two resonance next to
one another (splitting) using the n+1 rule, 2-1 gives 1 which means there would
be 1 proton on the neighbouring atom. For the structure of congener, there will
be two peaks with chemical shit of 6.8-8 ppm, and 2.3 ppm. The 6.8-8 is
responsible for aromatic ring but the difference between the Ramipril and the
congener is resonance at 2.3 ppm a singlet and is responsible for the CH3
group20 which Ramipril does not have. C-13 NMR works in similar way
to proton NMR. (appendix 5)


4.Justification of method


The method chosen
is easy and simple to flow that can be carried by anyone. Previous studies that
have been conducted have used similar or same method which has led to
successful reaction with no complications that had been observed. However, the
limitation to this method is very specific to reaction being carried out. Polyalkylation
can be observed21 due to the substituent (CH2) being an
activating group which leads to many methyl groups to be attach to the aromatic
ring.  However, the use of simple alkyl
groups such as methyl or ethyl substituents allows high yield of monoalkyl
products to be obtained.22

















5.Ethical consideration


Once the congener
has been synthesised and purification techniques have all been performed on the
drug testing would need to be performed. Tests are performed to see the effect
of methyl group on the aromatic ring and whether it has led to increase in the
therapeutic effect of Ramipril and bioavailability. There are two main ways of
testing, in vitro and in vivo. Firstly, the drug would be tested
in vitro which is a technique of performing
an experiment in a controlled environment that is outside of a living organism.
Large number of experiments in cellular biology are performed outside of an
organism and cells23.  In vitro testing is done before in vivo to minimise possibility of harming
or causing damage to the living organism. Therefore, techniques such as
computer modelling is used   in
vitro to allow the researcher to assess the compatibility between the drug
and the enzymes.

Once the
synthesised congener of Ramipril has been proven to be effective in vitro
further testing would be performed in
vivo. In vivo testing is using
living organisms as opposed to partial or dead organism23.  Two most common examples of in vivo testing is animal testing and
clinical trials. In vivo experiment
would be preferred over in vitro this
is because Ramipril is a drug that is used in humans and testing would need to
be performed on living organisms to detect the effect of drug and how it
functions as if it is in human body.

Experiments which
are performed on animals would be undertaking in accordance of Animals
(scientific procedure) Act 1986, which will need personal licences and a
project license.24