Patent Application: US-92109206-A

Abstract:
this invention relates to a process for synthesising an amino acid derivative of a tripeptidomimetic , such as lisinopril . the invention also relates to a derivative of lisinopril , incorporating a amino acid moeity such as tryptophan at the p2 ′ position .

Description:
the synthesis of lisinopril incorporating a tryptophan moiety at the p 2 ′ position is described herein . ace is a complex two - domain enzyme , comprising an n and a c domain , each containing an active site with similar but distinct substrate specificities and chloride - activation requirements . the n - and c - domain sites of ace hydrolyze angiotensin 1 ( ang i ) and bradykinin ( bk ) at comparable rates in vitro , but in vivo it appears that the c - domain is primarily responsible for regulating blood pressure . this might suggest that a c - selective inhibitor would have a profile comparable to current mixed inhibitors , but this is not necessarily the case . first , while ang i is hydrolyzed predominantly by the c domain in vivo , bk is hydrolyzed by both domains and therefore selective inhibition of the c - domain site will allow some level of bk degradation to continue , catalyzed by the n - domain . this could be sufficient to prevent the excessive bk accumulation that has been observed during attacks of angioedema . second , bk potentiation by b 2 receptor resensitization is maximal when both the n - and c - domains are inhibited , suggesting that a pure c - selective inhibitor will have a lower propensity for excessive bk stimulation . third , the multiple ang and non - ang peptides known to be vaso - active are not hydrolyzed equally by the two domains , making it likely that the ratio of vasopressor to vasodilator peptides will differ between c - selective and mixed inhibitors . thus , a highly selective c - domain inhibitor has the potential for effective blood pressure control with reduced vasodilator - related side effects . in contrast to a c - selective inhibitor , an n - selective inhibitor may open up novel therapeutic areas . as discussed , the n - domain appears to play a minor role in blood pressure control in vivo . at least three physiologically important peptides are hydrolyzed preferentially or exclusively by the n domain : luteinizing hormone - releasing hormone ( lh - rh ), ang ( 1 - 7 ) , and acsdkp ( n - acetyl - seryl - aspartyl - lysyl - proline ). the contribution of ace to the metabolism of lh - rh and ang ( 1 - 7 ) in vivo is unclear , but there is increasing evidence that ace is the principal metabolizing enzyme for acsdkp , a natural hemoregulatory hormone . acsdkp has anti - proliferative and anti - fibrotic activities and may have utility in protecting hematopoietic stem cells against chemotherapy - induced injury and in limiting cardiac fibrosis . administration of ace inhibitors results in a 4 - 6 - fold elevation of acsdkp plasma levels . this may be the basis for the observed association between ace inhibitors and anemia , and the effective treatment of altitude polycythemia by the ace inhibitor enalaprilat . lisinopril , which is a commercially available ace inhibitor , is active with a nanomolar inhibition constant and has been shown to be relatively safe and effective for the treatment of patients with acute myocardial infarction , 32 hypertension , and heart - related diseases . 33 - 35 lisinopril is chemically described as ( s )- 1 -[ n 2 -( 1 - carboxy - 3 - phenylpropyl )- l - lysyl ]- l - proline dihydrate . its empirical formula is c 21 h 31 n 3 o 5 . 2h 2 o and its structural formula is : dive and co - workers 36 have recently reported that a phosphinic ace - inhibitor , rxp a380 , is ˜ 3000 - fold c - domain selective and these same researchers have further shown that one key feature of the c - domain selectivity is the tryptophan moiety at the p 2 ′ position . starting material 3 was prepared by the reductive amination of ethyl 2 - oxo - 4 - phenyl butyrate 1 and n - ε -( tert - butoxycarbonyl )- l - lysine 2 using an ethanolic solution of nabh 3 cn . 37 the ratio of the two diastereoisomers was observed as 60 : 40 from the 1 h nmr spectrum . peptide coupling of compound 3 with a l - tryptophan methyl ester 4 ( prepared earlier by methylation of tryptophan in the presence of thionyl chloride and methanol according to the method described by hvidt et al . 38 ) was effected using edc . hcl in the presence of hobt and diisopropyl ethyl amine as a base to afford the diastereomeric pseudopeptide 5 in 74 % yield . the characterisation of this diastereomeric mixture was achieved from the ei - ms and spectroscopic data . the ei - ms data indicated a molecular ion peak at 637 correponding to m + + h . the 1 h nmr spectrum showed two singlets at δ 3 . 71 and 3 . 73 ppm corresponding to the two methoxy groups of the diastereomeric mixture 5 . acid hydrolysis of the resulting diastereomeric mixture 5 produced the hydrochloride salt of the diastereomeric mixture 6 in a quantitative yield . evidence of the compound 6 was found from the disappearance of the boc , methyl and ethyl signals on the 1 h nmr spectrum . purification and separation of the diastereomeric mixture 6 was done by hplc . initial attempts to remove the boc group with concommitent hydrolysis of the ethyl and methyl esters under acid conditions afforded a mixture of four products separated by hplc . these four products p 1 , p 2 , p 3 and p 4 were generated as a result of incomplete hydrolysis of compound 5 , since acid hydrolysis of ester at room temperature is relatively slow . the ei - ms data revealed molecular ion peaks at 522 , 522 , 536 and 536 for p 1 , p 2 , p 3 and p 4 , respectively . however , the diastereomeric mixture 5 was stirred in 4n hcl at room temperature for 24 h , after which the solvent was evaporated and the mixture was then stirred with a solution of 0 . 5n lioh for a further 5 h to afford the desired product 6 . the characterisation of this diastereomeric mixture 6 was achieved from the ei - ms and spectroscopic data that gave a molecular ion peak at 495 corresponding to m + + h . purification and separation of the diastereomeric mixture 6 by hplc gave the required two diastereoisomers in a 60 : 40 ratio . ace inhibition assays were carried out on the incomplete hydrolysis products p 1 , p 2 , p 3 and p 4 . ( i ) ketone 1 ( 4 . 0 eq ), amino acid 2 ( 1 . 0 eq ), nabh 3 cn ( 2 . 0 eq ), 50 % etoh / h 2 o , rt , 12 h ; ( ii ) l - tryptophan methyl ester 4 , edc . hcl , hobt , ipr 2 net ( 1 . 0 equiv ), dry dmf , rt , 72 h ; ( iii ) ( a ) 4n hcl , etoac , rt , 24 h ; ( b )) 0 . 5n lioh , thf - meoh , rt , 5 h . it will be apparent to a person skilled in the art that it will also be possible to synthesize other compounds than those described above by the method of the invention . lisinopril is a carboxyalkyl dipeptide , essentially a phe - lys - pro analog . compound 6 is a tryptophan derivative of lisinopril — i . e . the pro in the p2 ′ position has been substituted by a trp . it should be possible to prepare numerous variations on this theme , with different trp - like groups in the p2 ′ position . also , the zn - binding carboxyl could be changed to a keto group or any one of the possibilities mentioned above . similarly , the functionalities in the p1 ′ and p1 position could be changed . in particular , the crystal structure indicates that the s1 ′ pocket can accommodate larger side chains , and the applicant is in the process of studying a lisinopril analog with an arginine ( or arginine - like ) residue replacing the lysine in the p1 ′ position . the invention will now be described in more detail with reference to the following non - limiting examples . all reactions were carried out under a nitrogen atmosphere , unless otherwise specified . reactions were monitored by tlc using merck 60 f 254 precoated silica gel plates . detection was effected by observation under a uv lamp ( wave - length of 254 nm ) and developed with i 2 . column chromatography was carried out on silica gel and the eluent mixture used is specified in each experiment . anhydrous solvents like dichloromethane were distilled from phosphorous pentoxide and stored over molecular sieves type 4 å . all other anhydrous solvents were obtained from aldrich or sigma or merck chemical co . all melting points were determined using a kofler hot plate apparatus and are uncorrected . specific rotations ([ α ] d ) were measured at 20 ° c ., unless otherwise specified , using a perkin - elmer 141 polarimeter and are recorded in units of 10 − 1 o cm 2 g − 1 . nmr spectra were obtained on a varian mercury 300 mhz or varian unity 400 mhz spectrometer . chemical shifts are reported in ppm relative to the residual signal of the solvent used . the coupling constants , where specified , are given in hertz ( hz ). mass spectra were obtained using electron impact ionization . a solution of n —[ n 6 - tert - butoxycarbonyl - n 2 —( r , s )-( 1 - ethoxycarbonyl - 3 - phenylpropyl )]- l - lysine 3 ( 200 mg , 0 . 459 mmol , 1 . 0 eq .) and l - tryptophan methyl ester 4 ( 100 mg , 0 . 459 mmol , 1 . 0 eq .) in dry dmf ( 10 ml ) was cooled at 0 ° c . 1 - hydroxybenzotriazole hydrate ( hobt ) ( 62 mg , 0 . 459 mmol , 1 . 0 eq . ), n - ethyl - n ′-( dimethylaminopropyl )- carbodiimide hydrochloride ( edc . hcl ) ( 88 mg , 0 . 459 mmol , 1 . 0 eq .) and ipr 2 net ( 0 . 079 ml , 0 . 459 mmol , 1 . 0 eq .) were added and the resulting mixture stirred at 0 ° c . for 2 h . the cooling bath was removed and the reaction mixture then stirred for a further 72 h at room temperature . the reaction mixture was diluted with h 2 o ( 30 ml ) and the extracted with etoac ( 3 × 30 ml ). the combined organic extract were washed sequentially with saturated aqueous nahco 3 ( 30 ml ), brine ( 30 ml ), and dried over anhydrous mgso 4 . evaporation of the solvent gave a crude residue , which was subjected to column chromatography on silica gel elution with 66 % etoac - hexane afforded the desired diastereomeric mixture n —[ n 5 - tert - butoxycarbonyl - l - lysyl - n 2 —( r , s )-( 1 - ethoxycarbonyl - 3 - phenylpropyl )]- l - tryptophan methyl ester 5 as a pale yellow oil ( 0 . 215 g , 74 %); r f 0 . 27 ( 66 % etoac - hexane ); ir ( liquid film ) ν 1718 ( co ), 3300 ( nh ) cm − 1 ; 1 h nmr ( 300 mhz , cdcl 3 ), duplication of the signals are due to the diastereomeric mixture , δ h 1 . 23 ( 3h , t , j = 7 . 1 hz , — och 2 ch 3 ), 1 . 25 ( 3h , t , j = 7 . 2 hz , — och 2 ch 3 ), 1 . 47 ( 9h , s , ( ch 3 ) 3 ), 1 . 48 ( 9h , s , ( ch 3 ) 3 ), 1 . 52 - 1 . 87 ( 12h , m , 6 ×- ch 2 ), 1 . 91 - 2 . 21 ( 4h , m , 2 ×- ch 2 ), 2 . 49 ( 2h , m , — ch 2 ph ), 2 . 58 ( 2h , t , j = 7 . 2 hz , — ch 2 ph ), 2 . 91 - 3 . 11 ( 4h , m ), 3 . 12 - 3 . 45 ( 2h , m ), 3 . 71 ( 3h , s , och 3 ), 3 . 73 ( 3h , s , och 3 ), 4 . 12 ( 2h , q , j = 7 . 2 hz , — och 2 ch 3 ), 4 . 13 ( 2h , q , j = 7 . 0 , — och 2 ch 3 ), 4 . 14 ( 2h , m , 2 ×- ch —), 4 . 58 ( 2h , br s , 2 ×— nh ), 4 . 89 ( 1h , m , — ch —), 4 . 95 ( 1h , m , — ch —), 6 . 97 - 7 . 48 ( 26h , m , ar — h ), 7 . 55 ( 1h , t , j = 8 . 4 hz ,), 7 . 56 ( 1h , t , j = 8 . 4 hz ,), 8 . 69 ( 1h , br s , — nh - pyrrole ) and 8 . 87 ( 1h , br s , — nh - pyrrole ); 13 c nmr ( 75 mhz , cdcl 3 ) δ c 14 . 2 , 21 . 0 , 22 . 2 , 22 . 5 , 27 . 6 , 27 . 7 , 28 . 5 , 30 . 0 , 31 . 7 , 32 . 0 , 32 . 7 , 33 . 6 , 35 . 5 , 40 . 3 , 51 . 8 , 52 . 3 , 58 . 8 , 60 . 2 , 60 . 4 , 61 . 0 , 62 . 0 , 111 . 3 , 111 . 4 , 118 . 5 , 119 . 4 , 119 . 5 , 122 . 0 , 122 . 1 , 122 . 9 , 126 . 0 , 126 . 1 , 127 . 6 , 128 . 3 , 128 . 4 , 128 . 5 , 136 . 2 , 136 . 3 , 141 . 0 , 141 . 6 , 156 . 2 , 156 . 3 , 162 . 53 , 171 . 1 , 172 . 4 , 173 . 6 and 174 . 96 ; ei - ms m / z 637 ( m + + h ). incomplete hydrolysis products p 1 , p 2 , p 3 and p 4 to a solution of the diastereomeric mixture n —[ n 6 - tert - butoxycarbonyl - l - lysyl - n 2 —( r , s )-( 1 - ethoxycarbon - yl - 3 - phenylpropyl )]- l - tryptophan methyl ester 5 ( 100 mg , 0 . 157 mmol ) in etoac ( 5 . 0 ml ) was added 4n hcl ( 10 ml ) and the resulting mixture was stirred at room temperature for 2 h . tlc showed the disappearance of the starting material . evaporation of the solvent after neutralization with 2n naoh gave incomplete hydrolysis products p 1 , p 2 , p 3 and p 4 as a cream white solid material in quantitative yield ; ir ( liquid film ) ν 1720 ( co ), 2980 ( oh ) and 3115 ( nh ) cm − 1 . the ei - ms data of indicated a molecular ion peaks at 522 , 522 , 536 and 536 for p 1 , p 2 , p 3 and p 4 respectively . to a solution of the diastereomeric mixture n —[ n 6 - tert - butoxycarbonyl - l - lysyl - n 2 —( r , s )-( 1 - ethoxycarbon - yl - 3 - phenylpropyl )]- l - tryptophan methyl ester 5 ( 50 mg , 0 . 0785 mmol ) in etoac ( 5 . 0 ml ) was added 4n hcl ( 10 ml ) and the resulting mixture was stirred at room temperature for 24 h . evaporation of the solvent and the residue was dissolved in 0 . 5n lioh ( 5 . 0 ml ) and stirred at room temperature for 5 h . tlc showed the disappearance of the starting material . evaporation of the solvent after neutralization with 1n hcl gave n 2 —( r , s )-( 1 - carbonyl - 3 - phenylpropyl )]- l - lysyl - l - tryptophan hydrochloride 6 as a cream white solid material in quantitative yield ; ir ( liquid film ) ν 1720 ( co ), 2980 ( oh ) and 3115 ( nh ) cm − 1 ; ir ( kbr ) ν 1715 ( co ) and 3250 ( broad , cooh / nh ) cm − 1 ; 1 h nmr ( 400 mhz , d 2 o ) δ h 1 . 09 - 1 . 36 ( 2h , m , — ch 2 ch 2 ch 2 nh 2 ), 1 . 56 - 1 . 80 ( 4h , m , — ch 2 ch 2 ch 2 ch 2 nh 2 ), 2 . 27 ( 2h , q , j = 7 . 3 hz , — ch 2 ch 2 ph ), 2 . 34 - 2 . 49 ( 4h , m , — ch 2 ph and — ch 2 nh 2 ), 2 . 83 ( 1h , dd , j = 6 . 2 and 13 . 0 hz , — ch α trp ), 3 . 05 ( 1h , dd , j = 6 . 3 and 13 . 2 hz , — ch β trp ), 3 . 31 ( 2h , m , 2 ×- ch —), 4 . 63 ( 1h , m , — ch —) and 7 . 01 - 7 . 70 ( 10h , m , ar — h ); 13 c nmr ( 100 mhz , d 2 o ) δ c 20 . 9 , 27 . 3 , 30 . 7 , 31 . 0 , 31 . 4 , 33 . 3 , 39 . 9 , 48 . 6 , 55 . 0 , 58 . 8 , 59 . 5 , 62 . 7 , 110 . 0 , 111 . 5 , 118 . 3 , 118 . 9 , 121 . 5 , 123 . 6 , 125 . 7 , 128 . 2 , 128 . 3 ( 2c ), 126 . 9 , 135 . 9 , 142 . 1 , 142 . 4 , 175 . 6 , 178 . 5 and 181 . 0 ( duplication of peaks due the two diastereoisomers ); ei - ms m / z calcd for c 29 h 34 n 4 o 5 ( m + ) 494 . 2429 , found 495 ( m + + h ). hplc purification and separation give the required two diastereoisomers in a 60 : 40 ratio . rp - hplc purification and separation of the incomplete hydrolysis products was performed using a jupiter 5 u c18 300a , size 250 × 4 . 60 mm column with gradient elution of t = 0 min ( 63 % a , 37 % b ) and t = 30 min ( 50 % a , 50 % b ); solvent a = 0 . 1 % tfa in h 2 o and solvent b = 0 . 1 % tfa and 75 % ch 3 cn in h 2 o ; a flow rate of 1 . 0 ml / min and a 215 and 280 nm uv wavelength detection . t r = 12 . 77 min for p 1 , t r = 14 . 5 min for p 2 , t r = 17 . 1 min for p 3 and t r = 21 . 1 min for p 4 . rp - hplc purification and separation of the diastereomeric mixture 4 was performed using a jupiter 5 u c18 300a , size 250 × 4 . 60 mm column with gradient elution of t = 0 min ( 50 % a , 50 % b ) and t = 50 min ( 40 % a , 60 % b ); solvent a = 0 . 1 % tfa in h 2 o and solvent b = 0 . 1 % tfa and 75 % ch 3 cn in h 2 o ; a flow rate of 1 . 0 ml / min and a 215 and 280 nm uv wavelength detection . t r = 32 . 1 min for p 1 and t r = 41 . 0 min for p 2 . the ace inhibitory activity ( as tabulated in table 1 ) was measured by the fluorometric determination of pthaldialdehyde - derivatised histidylleucine , a product of the enzyme reaction according to the method of almquist et al ., 12 with some modifications . for tacedelta36nj and n - domain inhibition using z - phe - his - leu as substrate : — 10 μl of 0 . 05 mg / ml enzyme + 120 μl inhibitor at ambient temperature for 3 hours . inhibitor concentrations ranged from 2 . 0 μm to 500 μm . a 3 . 0 μl aliquot of this was assayed for enzyme activity using 30 μl of 1 . 0 mm hip - his - leu . this was incubated ( in triplicate ) for 30 mins at 37 ° c . and stopped with 180 μl solution of 0 . 28n naoh . to this alkalized mixture 12 μl of o - pthaldialdehyde ( 150 mm ) was added and the mixture incubated for another 10 mins at room temperature . reactions were stopped by adding 26 μl solution of 3n hcl . fluorescence was measured at ex = 360 nm ; em = 486 nm , 5 × 5 slit width along with a his - leu standard calibration curve fluorometer : varian cary eclipse plate reader . all assays included enzyme incubated with buffer in the absence of inhibitor ( i . e . 0 μm inhibitor concentration ). lisinopril was also used as a positive control . the 50 % inhibition ( ic 50 ) of ace activity was calculated as the concentrations of samples that inhibited 50 % of ace activity under these conditions . compounds 6 and 6a are two diastereoisomers of a lisinopril derivative incorporating a trp at the p2 ′ position . their ace inhibitory activities are shown in table 1 . although they displayed ki values in the low micromolar range , the introduction of a tryptophan moiety at the p2 ′ position resulted in a marked increase in c domain - selectivity (& gt ; 19 -& gt ; 72 fold ) as compared with lisinopril ( 2 . 6 - fold ). neither compound inhibited the n domain up to a concentration of 500 μm ( table 1 ). compound 6a , with an s - configuration at the stereogenic centre bearing the zinc - binding carboxylate group , was more c - selective than its r - diastereoisomer ( compound 6 ). 1 skeggs , l . t . ; 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( 1998 ) angiotensin - converting enzyme inhibitors , circulation , 97 ( 14 ), 1411 - 1420 . 36 georgíadis , d . ; cuniasse , p . ; cotton , j . ; yiotakis , a . and dive , v . ( 2004 ) structural determinants of rxpa380 , a potent and highly selective inhibitor of the angiotensin - converting enzyme c - domain , biochemistry , 43 , 8048 - 8054 . 37 schnorrenberg , g . ; roos , o . ; losel , w . ; weidemann , i . ; gaida , w . ; hoefke , w . ; arndts , d . and streller , i . ( 1989 ) amino acid derivatives and their pharmaceutical use . in uspto patent full text and image database , boehringer ingelheim gmbh ; us , pp . 1 - 18 . 38 ( a ) hvidt , t . and szarek , w . a . ( 1988 ) synthesis of enantiomerically pure β - amino - α - methylene - γ - butyrolactones by way of ozonolysis of aromatic α - amino acids , can . j . chem ., 66 ( 4 ), 779 - 782 ; ( b ) peng , s , and winterfeldt , e . 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