Ramipril is a prodrug belongs tothe group of angiotensin-converting enzyme (ACE) inhibitor class ofmedications. 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 inhibitionof angiotensin II leads to vasodilation and regulation of blood pressure whichis a key component of the renin angiotensin-aldosterone system (RAAS). RAAS isa mechanism for regulating hemodynamic, water and electrolyte balance1.When renal blood pressure is low, renin is secreted from juxtaglomerular kidneycells in to the blood stream. Renin cleaves the circulating angiotensinogen toATI, which then gets converted to ATII by ACE.

Therefore, increase in ATIIleads to vasoconstriction and increase in the blood pressure. Ramipril is usedin treatments of hypertension, it may also be used to reduce cardiovasculardeaths following myocardial infraction in hemodynamically stable patients.  Also used to reduce the rate of deaths in myocardialinfraction and stroke patients who are in high risk of developingcardiovascular diseases.

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3   1.2: Bioavailability and the effect of addingmethyl group on Ramipril.  It has found that thebioavalibilities of Ramipril and ramiprilat is 28% and 44%, respectively, whenoral administration is taken compared to intravenous administration1.The bioavailability can be increased if changes are made to the Ramiprilstructure. The way this can be carried forward is to look at the chemicalstructure of Ramipril and the functional groups present. The two-importantactive functional group of Ramipril is carboxylic acid and the aromatic ring.

 The carboxylicacid group fits into the enzymes pocket by exhibiting hydrophilic attractionwhile the aromatic ring exhibits hydrophobic attraction with ACE at the regionS1′. 4,5The addition of methyl group to thearomatic ring on Ramipril by Friedel crafts alkylation will make the ring morehydrophobic leading to stronger attraction between the enzymes pocket (S1′) andthe drug via Van der Waals forces. This will increase bioavailability becauseit makes it difficult for the drug to disassociate from the enzyme andtherefore, harder for the drug to be broken before reached to the active sitein the body. The methyl group on aromatic ring will also increase thetherapeutic effect of Ramipril, as it provides extra binding with enzymespocket S1′. This in return can reduce possible side effects and adverse drugreaction 1.3 Friedel crafts alkylation reaction.

 The addition of methyl group iscarried by Friedel crafts alkylation, this is an organic reaction that is usedto substitute alkyl halide on to aromatic compound using a Lewis acid catalyst.The reaction starts with Lewis acid attacking halide from the alkyl halide toform an electrophilic intermittent and formation of tetrasubstituted aluminiumanion (AlCl4).  Then thearomatic ring attacks the alkyl cation via electrophilic aromatic substitution(SEAr) to a cationic product leading the aromaticity to be lost. Theloss of proton from the aromatic ring will result in final product andregeneration of the Lewis acid catalyst. 6,7Figure 1. The generation of carbocation using acid Lewiscatalyst. Figure 1 shows a Lewis acid/basecomplex.

The complex has lead a partial positive charge on the chloride due tothe pulling of electrons from the R group making the dipole between R andchloride bigger8.  The ? electrons of the aromatic ring attackingthe electrophilic R+ group. This leads to aromaticity of the ring to be destroyedforming a cation intermediate. Figure 2. Formation of intermediate.

The last step is to detach theproton on the ring to re-establish the aromaticity of the ring. This step alsoinvolves the regeneration of the catalyst back to AlCl3.Figure 3. Regeneration of aromatic ring by losing a proton. The reaction shown above will beperformed on Ramipril on the aromatic ring. The position which methyl grouptakes is extremely important, the drug will be most effective if it takes orthoposition however, the position of methyl group can either be para or ortho thisis because the aromatic ring is attached to CH2 group which is adirecting 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 electrondonating than hydrogen and enhances the rate of reaction due to increase inelectron density making the ring more reactive towards electrophiles9.  Structure of Ramipril: Diagram 1: showing chemicalstructure of Ramipril before Friedel Crafts alkylation.  Design of congener of Ramipril Diagram 2: Structure of congener ofRamipril at ortho position.  Diagram 3: structure of congener ofRamipril at para position. (Structures have been designedusing MolVeiw10)           2. Aims and objectives: 2.1: Aims The aim is to produce a congener ofRamipril to make the drug more effective in the treatment of hypertension andcardiovascular associated diseases using Friedel crafts alkylation reaction toincrease the bioavailability of the drug.

2.2: Objectives Objectives will be: ·        To create congener of Ramipril to increasebioavailability by making aromatic ring more hydrophobic.·        To increase overall therapeutic effect by addingmethyl group to the lead structure to probe for extra binding with the targetenzymes.  ·        To test the final product synthesised using in vitro and in vivo testing.

·        To purify congener using different methods andtechniques.           3.Methodology1.       Thefirst step is to weight out anhydrous aluminium chloride and transfer into 25 mlround bottom flask containing a magnetic stir bar11. 2.       Fitthis with a claisen adaptor, dropping funnel and acondenser. Ramipril is added to the flask, and chloromethane is added to thedropping funnel.

 An ice bath is alsoprepared to cool the reaction if needed. 3.       WhileRamipril and aluminium chloride is being stirred, chloromethane is added slowly.  If the reaction becomes too vigorous the icebath is used to cool the reaction. After the addition of chloromethane iscomplete continue stirring the mixture at room temperature for 10-15 minutes. 4.

       Transferthe mixture into 50ml beaker with ice in it. The mixture is stirred untilfuming has ended.5.        The two phases: aqueous and organic mixture isstirred while adding solid sodium chloride to the mixture to make the aqueousphase saturated. Then the mixture is poured in to separate funnel. The organiclayer is separated and transferred into a conical flask and dried overanhydrous Na2SO4.  Transfer the dried organic layer in to a clean25ml round flask and filter the organic layer.12        3.

1 Analysis of congener synthesised andcomparing with Ramipril.  Purificationneeds to be performed on the congener synthases of Ramipril. Chromatography is aprocess in which a chemical mixture is carried around the stationary phase andmobile phase. Components of the mixture are separated because of differentialdistribution of the solutes between carrier phase and the stationary phase. Theamount of time that each competent spends in each phase depends on theiraffinity to the stationary phase or the mobile phase.

The attraction depends onthe individuals physical and chemical structure13. High performanceliquid chromatography (HPLC) will be performed to compare the purity ofRamipril to the product that has been produced. HPLC is a performed byinjecting the mixture in the mobile phase at high pressure through the column(The stationary phase).14  (Appendix1) shows that the retention time of Ramipril is 2.910 minutes, for the congenersynthesised this would be longer, due to addition of methyl group added on thearomatic ring making the drug to become more hydrophobic which means thecongener will spend more time in the stationary phase and will take longer toeluate form the column.Infrared spectrometry (IR) isanother method that can be used to obtain information about the functionalgroups of a molecule.

  In IR spectrometrysubstances are exposed to IR radiation this leads to vibrational energy tooccur within the molecule which then absorbs IR radiation at a specificfrequency leading to specific bands which can be used to determine the types ofbonds and therefore the functional group that are present within the molecule.15,16,18By comparing the IR spectroscopy of Ramipril and the congener there is adifference showing absence of methyl group on the aromatic ring in thestructure of Ramipril. (Appendix 2) shows the IR spectroscopy of Ramipril, showinga band at 3032 wavelength/ cm-1 which a stretch caused by C-H inaromatic ring. It is expected that congener synthesised will have another peakwhich Ramipril will not have, this is due to C-H alkyl on the aromatic ring andhave a wavelength of 2925 cm-1 . The rest of the bands are expectedsame because they have both have same function groups. Massspectrometry (MS) can also be used to investigate the sample according to theirmass and electrical charge. This will produce a mass spectrum that would plot massto charge ratio of compounds in a mixture17. The molecular formulaof Ramipril is C23H32N2O5  and the molecular weight is 416.

518 g/mol(appendix 3).19 Comparing this to synthesised congener the molecularformula is C24H35N2O5 and the molecularweight is expected to be 431g/mol. The mass spectrometry of Ramipril has afragment of 91 which corresponds to the benzene ring and CH2  that is attached to the aromatic ring, thisfragment would be different for synthesised congener  due to addition of CH3 group. Thisfragment would be 106 instead of 91. (appendix 3)NMR is animportant technique that can be used to give essential information aboutcongener and allow the comparison to be made between Ramipril. There are twotypes of NMR that can be used: proton NMR and Carbon-13 NMR. (Appendix 4) showsproton NMR of Ramipril it has a chemical shift of 6.8-8 ppm which isresponsible for the aromatic ring on Ramipril, there are two resonance next toone another (splitting) using the n+1 rule, 2-1 gives 1 which means there wouldbe 1 proton on the neighbouring atom.

For the structure of congener, there willbe two peaks with chemical shit of 6.8-8 ppm, and 2.3 ppm. The 6.8-8 isresponsible for aromatic ring but the difference between the Ramipril and thecongener is resonance at 2.3 ppm a singlet and is responsible for the CH3group20 which Ramipril does not have.

C-13 NMR works in similar wayto proton NMR. (appendix 5)


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