Patent Application: US-81283104-A

Abstract:
the present invention relates to novel anti - hypertensive molecules . the present invention also provides a process for the preparation of novel antihypertensive molecules . the present invention particularly relates to the preparation of novel angiotensin converting enzyme inhibitors with prolonged activity . ace inhibitors play an important role in renin - angiotensin - aldosteron system by inhibiting the activity of angiotensin converting enzyme and therefore are used to regulate blood pressure . ace inhibitors synthesized by the process of present invention have a peptide moiety and nonpeptide moiety . ace inhibitors , synthesized by this present invention , show enhanced bioavailability and fewer side effects .

Description:
for proper drug targeting there is need to synthesize the library of compounds i . e . peptidomimics . these peptidomimics are being designed according to structural activity relationship , synthesized by combinatorial chemistry and their inhibition is checked by in vitro studies . peptidomimic compounds that can act as ace inhibitor preferably must have following features ( fig3 ): ( 1 ) the peptidomimic compound should have aromatic , aliphatic amino acids like phe , trp , tyr pro ( cheung et al 1980 ), ( 2 ) a negatively charged coo − group should be present at c - terminal . ( 3 ) amino acids like ile , leu , lys and val are incorporated next to the c - terminal amino acid . ( 4 ) in peptidomimic compound the aromatic moieties of currently existing aceis are replaced with suitable heterocyclics or unusual amino acids . the process of present invention thus results in the synthesis of di and tripeptidomimics consisting of various combinations of aromatic amino acids ( phe , trp , tyr and pro ) and aliphatic amino acids ( ile , leu , lys and val ). the residues chosen at c - terminal will enhance hydrophobic interactions with subsites s1 ′, s2 ′. the negatively charged coo − group of these amino acids at c - terminal has hydrophobic interaction with positively charged arg residue in the enzyme &# 39 ; s active site . hydrophobic side chains of aliphatic amino acids , next to the c - terminal amino acid , establish interaction with ace hydrophobic subsite s1 ′. a carbonyl of amide bond of these amino acids forms h - bonds with hydrogen of ace active site . in new peptidomimics , n - terminal will be substituted with suitable hetrocyclic or unusual amino acids to enhance their bioavailability as well as their interaction with co - ordinated zn and hydrophobic subsite s1 present in enzyme &# 39 ; s active site . heterocyclic moieties are the molecules that have certain medicinally functional groups known as ‘ pharmacophores ’. these pharmacophores are supposed to be compatible with human system . these molecules are also expected to produce conformational constraints that help them to interact with active site in a better and effective way . the work as designed and described above involves synthesis of large number of peptidomimics . this can be best achieved by the combinatorial chemistry , which will be used in the present proposed work . this concept takes its origin with the aim of synthesizing 96 diverse peptides simultaneously ( furk et al , 1991 ). in this method , in each reaction vessel , peptides with different sequences are synthesized preferably by solid phase peptide synthesis . this technique is of two types : this method involves the synthesis of mixture of large number of peptides called as compound libraries of peptides . in this method , in each reaction vessel peptides with different sequences are simultaneously synthesized . this method , also called pool and split method , involves : ( a ) dividing or splitting of resin into different parts . ( b ) coupling of resin with amino acids . ( c ) mixing of coupled parts of resin and adding new amino acids to generate mixture of peptides . multiple synthesis of individual peptides involves synthesis of large number of individual peptides ( beck - sickinger and jung , 1996 ) one peptide sequence is synthesized in each reaction vessel . there is no splitting and mixing of reaction products like in previous case . this method requires coupling , washing and deprotection repeatedly . the library of peptidomimic compound was evaluated for their ace inhibition potencies by determining the ace activity by in - vitro enzyme assay using spectrophotometric method ( cheung et al , 1980 ). in this assay ace acts upon synthetic substrate hip - his - leu . the following assay components in a final volume of 0 . 25 ml are incubated for 20 minutes at 37 ° c . : 100 mm potassium phosphate buffer , ph 8 . 3 , 5 mm hip - his - leu , 300 mm nacl and angiotensin converting enzyme ( 12 miliunits / ml of assay volume ). the rate of hydrolysis of hip - his - leu is determined by measuring the absorbance of hippuric acid after extracting into ethyl acetate , evaporation of solvent at 120 ° c . and redissolution into water . extracted hippuric acid is then measured by reading absorbance at 228 nm . evaluation of ace inhibition of synthesized peptidomimics is done in hypertensive rats . wistar rats ( female , 225 - 250 gm ) were taken and hypertension was induced in these rats by administering the injection of methylprednisolone per week for two weeks ( elijovich and krakoff , 1980 ). after two weeks when rats become hypertensive , the efficacy of synthesized ace inhibitors is checked in in - vivo conditions by intra venous administration in pharmacologically accepted medium in doses ranging from 5 mg / kg of body weigh to 10 mg / kg of body weight . the following examples are given for the present invention and should be construed to limit the scope of the present invention . ( i ) unusual amino acids are coupled with dipeptide . for example for designing of l - abrine - ornithine - proline , proline was taken at ultimate position , ornithine was put at penultimate position and unusual amino acid l - abrine , present at antepenultimate position , was linked to dipeptide ornithine - proline . ( ii ) heterocyclic compounds are coupled with dipeptide . for example for designing of 3 -( 3 - thienyl )- l - alanine - ornithine - proline , proline was taken at ultimate position , ornithine was put at penultimate position and heterocyclic compound 3 -( 3 - thienyl )- l - alanine , present at antepenultimate position , was linked to dipeptide ornithine - proline . ( a ) 2 - chlorotrityl chloride resin ( substitution 1 . 5 mm / gm ) is taken as solid support and swelled in dichlorometane ( dcm ) ( b ) c - terminal of n - α - protected fmoc - pro - oh at is coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) to the solid support of 2 - chlorotrityl chloride resin . ( c ) deprotection of n - α - terminal protecting fmoc group of the anchored imino acid proline by 20 % piperidine in dimethyl formamide ( dmf ). ( d ) c - terminal of n - α fmoc protected ornithine is activated and coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) on to the deprotected α - imino group of the anchored imino acid proline . ( e ) deprotection of n - α - terminal protecting fmoc group of ornithine linked to anchored imino acid proline by 20 % piperidine in dimethyl formamide ( dmf ). ( f ) unusual amino acid fmoc - l - abrine ( n - methyltryptophan ) is coupled to deprotected n - α - terminal amino acid ornithine by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ). ( g ) deprotection of n - α - terminal protecting fmoc group of l - abrine linked to dipeptide ornithine - proline by 20 % piperidine in dimethyl formamide ( dmf ). ( h ) cleave the peptidomimic l - abrine - ornithine - proline from the solid support by known methods depending upon the side chain protecting groups of amino acids . ( i ) for amino acids with out boc and trt group , acetic acid : trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 : 8 . 10 to 20μ litre of ethyldiamine tetra acetate ( eta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( ii ) for boc containing amino acids trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 ( v / v ). 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( j ) cleaved product is purified in lh - 20 column using methanol as mobile phase ( a ) 2 - chlorotrityl chloride resin ( substitution 1 . 5 mm / gm ) is taken as solid support and swelled in dichlorometane ( dcm ) ( b ) c - terminal of n - α - protected fmoc - pro - oh at is coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) to the solid support of 2 - chlorotrityl - chloride resin . ( c ) deprotection of n - α - terminal protecting fmoc group of the anchored imino acid proline by 20 % piperidine in dimethyl formamide ( dmf ). ( d ) c - terminal of n - α fmoc protected ornithine is activated and coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) on to the deprotected α - imino group of the anchored imino acid proline . ( e ) deprotection of n - α - terminal protecting fmoc group of ornithine linked to anchored imino acid proline by 20 % piperidine in dimethyl formamide ( dmf ). ( f ) heterocyclic 3 -( 3 - thienyl )- l - alanine is coupled to deprotected n - α - terminal amino acid ornithine by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ). ( g ) deprotection of n - α - terminal protecting fmoc group of 3 -( 3 - thienyl )- l - alanine linked to dipeptide ornithine - proline by 20 % piperidine in dimethyl formamide ( dmf ). ( h ) cleaving the peptidomimic 3 -( 3 - thienyl )- l - alanine - ornithine - proline from the solid support by known methods depending upon the side chain protecting groups of amino acids . ( i ) for amino acids with out boc and trt soup , acetic acid : trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 : 8 . 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( ii ) for boc containing amino acids trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 ( v / v ). 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( i ) cleaved product is purified in lh - 20 column using methanol as mobile phase ( a ) 2 - chlorotrityl chloride resin ( substitution 1 . 5 mm / gm ) is taken as solid support and swelled in dichlorometane ( dcm ) ( b ) c - terminal of n - α - protected fmoc - trp - oh at is coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) to the solid support of 2 - chlorotrityl - chloride resin . ( c ) deprotection of n - α - terminal protecting fmoc group of the anchored amino acid tryptophan by 20 % piperidine in dimethyl formamide ( dmf ). ( d ) c - terminal of n - α fmoc protected lysine is activated and coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) on to the deprotected α - imino group of the anchored amino acid tryptophan . ( e ) deprotection of n - α - terminal protecting fmoc group of lysine linked to anchored imino acid proline by 20 % piperidine in dimethyl formamide ( dmw ). ( f ) heterocyclic 2 - oxo4 - phenyl - 3 - oxazolidine acetic acid is coupled to deprotected n - α - terminal amino acid lysine by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ). ( g ) deprotection of n - α - terminal protecting fmoc group of 2 - oxo - 4 - phenyl - 3 - oxazolidine acetic acid linked to dipeptide lysine - trytophan by 20 % piperidine in dimethyl formamide ( dmf ). ( h ) cleaving the peptidomimic 2 - oxo - 4 - phenyl - 3 - oxazolidine acetic acid - lys - trp from the solid support by known methods depending upon the side chain protecting groups of amino acids . ( i ) for amino acids with out boc and trt group , acetic acid : trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 : 8 . 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( ii ) for boc containing amino acids trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 ( v / v ). 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( i ) cleaved product is purified in lh - 20 column using methanol as mobile phase ( a ) 2 - chlorotrityl chloride resin ( substitution 1 . 5 mm / gm ) is taken as solid support and swelled in dichlorometane ( dcm ) ( b ) c - terminal of n - α - protected fmoc - trp - oh at is coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) to the solid support of 2 - chlorotrityl - chloride resin . ( c ) deprotection of n - α - terminal protecting fmoc group of the anchored amino acid tryptophan by 20 % piperidine in dimethyl formamide ( dmf ). ( d ) c - terminal of n - α fmoc protected ornithine is activated and coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) on to the deprotected α - amino group of the anchored amino acid tryptophan . ( e ) deprotection of n - α - terminal protecting fmoc group of ornithine linked to anchored amino acid tryptophan by 20 % piperidine in dimethyl formamide ( dmf ). ( f ) unusual amino acid fmoc 5 - hydroxytryptophan is coupled to deprotected n - α - terminal amino acid ornithine by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ). ( g ) deprotection of n - α - terminal protecting fmoc group of 5 - hydroxytryptophan linked to dipeptide ornithine - tryptophan by 20 % piperidine in dimethyl formamide ( dmf ). ( h ) cleaving the peptidomimic 5 - hydroxytryptophan - ornithine - tryptophan from the solid support by known methods depending upon the side chain protecting groups of amino acids . ( i ) for amino acids with out boc and trt group , acetic acid : trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 : 8 . 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( ii ) for boc containing amino acids trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 ( v / v ). 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( i ) cleaved product is purified in lh - 20 column using methanol as mobile phase ( a ) 2 - chlorotrityl chloride resin ( substitution 1 . 5 mm / gm ) is taken as solid support and swelled in dichlorometane ( dcm ) ( b ) c - terminal of n - αprotected fmoc - trp - oh at is coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) to the solid support of 2 - chlorotrityl - chloride resin . ( c ) deprotection of n - α - terminal protecting fmoc group of the anchored amino acid tryptophan by 20 % piperidine in dimethyl formamide ( dmf ). ( d ) c - terminal of n - α fmoc protected cyclohexylalanine is activated and coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) on to the deprotected α - amino group of the anchored amino acid tryptophan . ( e ) deprotection of n - α - terminal protecting fmoc group of cyclohexylalanine linked to anchored amino acid tryptophan by 20 % piperidine in dimethyl formamide ( dmf ). ( f ) heterocyclic benzimidazoleacetic acid is coupled to deprotected n - αterminal amino acid cyclohexylalanine by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ). ( g ) cleaving the peptidomimic benzimidazoleacetic acid - cyclohexylalanine - tryptophan from the solid support by known methods depending upon the side chain protecting groups of amino acids . ( i ) for amino acids with out boc and trt group , acetic acid : trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 : 8 . 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( ii ) for boc containing amino acids trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 ( v / v ). 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( h ) cleaved product is purified in lh - 20 column using methanol as mobile phase ( a ) 2 - chlorotrityl chloride resin ( substitution 1 . 5 mm / gm ) is taken as solid support and swelled in dichlorometane ( dcm ) ( b ) c - terminal of n - α - protected fmoc - phe - oh at is coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) to the solid support of 2 - chlorotrityl - chloride resin . ( c ) deprotection of n - α - terminal protecting fmoc group of the anchored amino acid phenylalanine by 20 % piperidine in dimethyl formamide ( dmf ). ( d ) c - terminal of n - α fmoc protected isoleucine is activated and coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) on to the deprotected α - amino group of the anchored amino acid phenylalanine . ( e ) deprotection of n - α - terminal protecting fmoc group of isoleucine linked to anchored amino acid phenylalanine by 20 % piperidine in dimethyl formamide ( dmf ). ( f ) unusual amino acid fmoc - l - abrine ( n - methyltrytophan ) is coupled to deprotected n - α - terminal amino acid isoleucine by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ). ( g ) deprotection of n - α - terminal protecting fmoc group of l - abrine linked to dipeptide isoleucine - phenylalanine by 20 % piperidine in dimethyl formamide ( dmf ). ( h ) cleaving the peptidomimic l - abrine - isoleucine - phenylalanine from the solid support by known methods depending upon the side chain protecting groups of amino acids . ( i ) for amino acids with out boc and trt group , acetic acid : trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 : 8 . 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( ii ) for boc containing amino acids trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 ( v / v ). 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( i ) cleaved product is purified in lh - 20 column using methanol as mobile phase ( a ) 2 - chlorotrityl chloride resin ( substitution 1 . 5 mm / gm ) is taken as solid support and swelled in dichlorometane ( dcm ) ( b ) c - terminal of n - α - protected fmoc - pro - oh at is coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) to the solid support of 2 - chlorotrityl - chloride resin . ( c ) deprotection of n - α - terminal protecting fmoc group of the anchored imino acid proline by 20 % piperidine in dimethyl formamide ( dmf ). ( d ) c - terminal of n - α fmoc protected pro is activated and coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) on to the deprotected α - imino group of the anchored amino acid pro . ( e ) deprotection of n - α - terminal protecting fmoc group of proline linked to anchored imino acid proline by 20 % piperidine in dimethyl formamide ( dmf ). ( f ) unusual ammo acid 1 , 2 , 3 , 4 - tetrahydroisoquinoline - 3 - carboxylic acid is coupled to deprotected n - α - terminal i imino acid proline by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ). ( g ) deprotection of n - α - terminal protecting fmoc group of 1 , 2 , 3 , 4 - tetrahydroisoquinoline - 3 - carboxylic acid linked to dipeptide pro - pro by 20 % piperidine in dimethyl formamide ( dmf ). ( h ) cleaving the peptidomimic 1 , 2 , 3 , 4 - tetrahydroisoquinoline - 3 - carboxylic acid - pro - pro from the solid support by known methods depending upon the side chain protecting groups of amino acids . ( i ) for amino acids with out boc and trt group , acetic acid : trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 : 8 . 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( ii ) for boc containing amino acids trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 ( v / v ). 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( i ) cleaved product is purified in lh - 20 column using methanol as mobile phase ( a ) 2 - chlorotrityl chloride resin ( substitution 1 . 5 mm / gm ) is taken as solid support and swelled in dichlorometane ( dcm ) ( b ) c - terminal of n - α - protected fmoc - pro - oh at is coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) to the solid support of 2 - chlorotrityl - chloride resin . ( c ) deprotection of n - α - terminal protecting fmoc group of the anchored imino acid proline by 20 % piperidine in dimethyl formamide ( dmw ). ( d ) c - terminal of n - α fmoc protected β - alanine is activated and coupled by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ) on to the deprotected α - imino group of the anchored amino acid pro . ( e ) deprotection of n - α - terminal protecting fmoc group of β - alanine linked to anchored imino acid proline by 20 % piperidine in dimethyl formamide ( dmf ). ( f ) heterocyclic 3 -( 2 - furyl )- l - alanine is coupled to deprotected n - α - terminal β - alanine by reactive ester formation method of 1 - hydroxybenzotriazole and diisopropylcarbodiimide ( dipcdi ). ( g ) deprotection of n - α - terminal protecting fmoc group of 3 -( 2 - furyl )- l - alanine linked to dipeptide β - alanine - pro by 20 % piperidine in dimethyl formamide ( dmf ). ( h ) cleaving the peptidomimic 3 -( 2 - furyl )- l - alanine - β - alanine - pro from the solid support by known methods depending upon the side chain protecting groups of amino acids . ( i ) for amino acids with out boc and trt group , acetic acid : trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 : 8 . 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( ii ) for boc containing amino acids trifluoroacetic acid : dichlorometane are taken in ratio of 1 : 1 ( v / v ). 10 to 20μ litre of ethyldiamine tetra acetate ( edta ) and a pinch of phenol are added in to the cleavage mixture . reaction is carried out for 30 minutes . ( i ) cleaved product is purified in lh - 20 column using methanol as mobile phase on the basis of the results of example 2 - 9 and according to same procedure as in example 2 - 9 all the peptidomimics , mentioned above , having general formula x — cx1 — nh - aa 1 - conh - aa 2 wherein x is a heterocyclic or unusual amino acid , x1 is o or h 2 and aa1 and aa2 are amino acids , are synthesized by using combinatorial chemistry . the peptidomimic compound l - abrine - ornithine - proline was evaluated for its ace inhibition potency by determining the ace activity assay using spectrophotometric method . in this assay ace acts upon synthetic substrate hip - his - leu . the following assay components in a final volume of 0 . 25 ml are incubated for 20 minutes at 37 ° c . : 100 mm potassium phosphate buffer , ph 8 . 3 , 5 mm hip - his - leu , 300 mm nacl and angiotensin converting enzyme ( 12 miliunits / ml of assay volume ). the rate of hydrolysis of hip - his - leu is determined by measuring the absorbance of hippuric acid after extracting into ethyl acetate , evaporation of solvent at 120 ° c . and redissolution into water . extracted hippuric acid is then measured by reading absorbance at 228 nm . l - abrine - ornithine - proline and 3 -( 3 - thienyl )- l - alanine - ornithine - proline were evaluated for their ace inhibition efficacy by this assay . 3 -( 3 - thienyl )- l - alanine - ornithine - proline showed its ic 50 to be 2 μ mole . the ic 50 of l - abrine - ornithine - proline was found to be 10 μ mole . inhibition kinetics of l - abrine - ornithine - proline is illustrated in fig4 . the control experiment contained no peptidomimic ace inhibitor . female wistar rats with an initial body weight of 225 - 250 gm were used in this experiment . three groups of animals were made : group i ( n = 3 ) of control animals ; group ii ( n = 3 ) of animals for reference inhibitor captopril and group iii ( n = 5 ) of animal for synthesized ace inhibitors measurement of body weight and tail systolic blood pressure were made in unanesthetsized animals . a long acting suspension of 20 mg / kg methylprednisolone ( depo - medrol , pharmacia ) was administered subcutaneously per week for two weeks in animals of group ii and iii . group i of control animals received only vehicle polyethylene glycol ( peg ). all animals had free access to tap water , were placed on regular rat chow and had their tail systolic pressure measured every week for two weeks . after two weeks of treatment , all the animal a became significantly hypertensive ( fig5 ). the rats were anaesthetized by intraperitoneal administration of urethane ( 1 gm / kg ). the trachea was cannulated with polyethylene tube . polyethylene cannulas were also placed into the rat femoral artery and vein . the arterial catheter was used for recording of blood pressure and the venous catheter was used for the administration of drugs / peptides . after surgery , animals were allowed 30 min for stabilization before starting the experiment . arterial blood pressure was measured with a pressure transducer ( wpi - model bplr ) with a strain gauge / bridge amplifier ( wpi - model tbm4 ) connected to four channel digital oscillograph tektronix ( model tds 420a ). permanent records were obtained by storing and copying on a floppy disk from the oscillograph . from the arterial blood pressure waveform , mean arterial pressure and diastolic blood pressure were measured . varying amounts of l - abrine - ornithine - proline ( 5 mg / kg , 8 mg / kg and 10 mg / kg ) were dissolved in normal saline . peptidomimics was injected i . v . in constant volume of 0 . 2 ml over a period of 10 seconds and the venous catheter was flushed with an additional 0 . 3 ml of saline . readings of mean arterial pressure ( map ) were made after each 10 minutes till blood pressure returned to base line . at 5mg / kg l - abrine - ornithine - proline showed its hypotensive effect which caused a fall of around 15 mm hg ( fig6 ). hypotensive effect started after almost five minutes and blood pressure returned to baseline within 30 minutes . a dose of 8 mg / kg of l - abrine - ornithine - proline began to show its hypotensive effect five minute after its administration . at this dose , blood pressure declined upto 46 mm hg . it nearly took 160 minutes for blood pressure to return to baseline . the highest dose used was 10 mg / kg . it showed strong hypotensive effect . the onset of hypotensive effect was again after five minutes of administration . a fall in blood pressure of almost 50 mmhg was reached within 70 minutes and blood pressure did not return to baseline even after 180 minutes reference inhibitor captopril was used at dose of 1 mg / kg . this dose of captopril caused fall of 60 mm hg and was quite effective in blocking angiotensin - converting enzyme even after 180 minutes and blood pressure returned to baseline after 240 minutes ( not shown ). the present invention adds following advantages to the ace inhibiting antihypertensive molecule as ( a ) it provides a process for the preparation of novel antihypertensive molecule . ( b ) it provides a process for the synthesis of peptidomimics that can act as ace inhibitors . ( c ) it provides a process to synthesize ace inhibitors that have better bioavailability in comparison to available inhibitors . ( d ) it provides a process to synthesize ace inhibitors that can strongly ligate the zn present in the active site in such a way that it can not compete with and carry out the hydrolysis of substrate . ( e ) it provides a process to make ace inhibitors having nonpeptide moiety or unusual amino acids ( pharmacophoric group ) that can make a nonpeptide bond resistant against proteolysis . ( f ) it provides a process to make ace inhibitors that can show selective inhibition of the two active sites i . e . n - domain & amp ; c - domain active site of the ace . 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