Patent Application: US-26925794-A

Abstract:
novel antagonists of endothelin are described , as well as methods for the preparation and pharmaceutical compositions of the same , which are useful in treating elevated levels of endothelin , acute and chronic renal failure , hypertension , myocardial infarction , metabolic , endocrinological , neurological disorders especially cerebral vasospasm , stroke , and head injury , congestive heart failure , endotoxic shock , subarachnoid hemorrhage , arrhythmias , asthma , preeclampsia , atherosclerotic disorders including raynaud &# 39 ; s disease , restenosis , angina , cancer , pulmonary hypertension , ischemic disease , gastric mucosal damage , hemorrhagic shock , ischemic bowel disease , and diabetes .

Description:
in the compounds of formula i , the term &# 34 ; alkyl &# 34 ; means a straight or branched hydrocarbon radical having from 1 to 12 carbon atoms and includes , for example , methyl , ethyl , n - propyl , isopropyl , n - butyl , sec - butyl , isobutyl , tert - butyl , n - pentyl , n - hexyl , n - heptyl , n - octyl , n - nonyl , n - decyl , undecyl , dodecyl , and the like . the term &# 34 ; alkenyl &# 34 ; means a straight or branched unsaturated hydrocarbon radical having from 2 to 12 carbon atoms and includes , for example , ethenyl , 2 - propenyl , 1 - butenyl , 2 - butenyl , 1 - pentenyl , 2 - pentenyl , 3 - methyl - 3 - butenyl , 1 - hexenyl , 2 - hexenyl , 3 - hexenyl , 3 - heptenyl , 1 - octenyl , 1 - nonenyl , 1 - decenyl , 1 - undecenyl , 1 - dodecenyl , and the like . the term &# 34 ; alkynyl &# 34 ; means a straight or branched triple bonded unsaturated hydrocarbon radical having from 2 to 12 carbon atoms and includes , for example , ethynyl , 2 - propynyl , 1 - butynyl , 2 - butynyl , 3 - butynyl , 1 - pentynyl , 3opentynyl , 1 - hexynyl , 2 - hexynyl , 3 - hexynyl , 3 - heptenyl , 1 - octynyl , 2 - octynyl , 1 - nonynyl , 2 - nonynyl , 3 - nonynyl , 4 - nonynyl , 1 - decynyl , 2 - decynyl , 2 - undecynyl , 3 - undecynyl , 3 - dodecynyl , and the like . the term &# 34 ; cycloalkyl &# 34 ; means a saturated hydrocarbon ring which contains from 3 to 12 carbon atoms , for example , cyclopropyl , cyclobutyl , cyclopentyl , cyclohexyl , adamantyl , and the like . the term &# 34 ; cycloalkylalkyl &# 34 ; means a saturated hydrocarbon ring attached to an alkyl group wherein alkyl is as defined above . the saturated hydrocarbon ring contains from 3 to 12 carbon atoms . examples of such are cyclopropylmethyl , cyclopentylmethyl , cyclohexylmethyl , adamantylmethyl and the like . the terms &# 34 ; alkoxy &# 34 ; and &# 34 ; thioalkoxy &# 34 ; are o - alkyl or s - alkyl as defined above for alkyl . the term &# 34 ; aryl &# 34 ; means an aromatic radical which is a phenyl group , a benzyl group , a naphthyl group , a biphenyl group , a pyrenyl group , an anthracenyl group , 3 , 3 - diphenylalanyl , 10 , 11 - dihydro - 5h - dibenzo [ a , d ]-( cyclohepten - 5 - yl ) glycyl , or a fluorenyl group and the like , unsubstituted or substituted by 1 to 4 substituents selected from alkyl as defined above , alkoxy as defined above , thioalkoxy as defined above , hydroxy , thiol , nitro , halogen , amino , ## str30 ## wherein alkyl is as defined above , ## str31 ## wherein alkyl is as defined above , ## str32 ## wherein alkyl is as defined above , or aryl . the term &# 34 ; arylalkyl &# 34 ; means an aromatic radical attached to an alkyl radical wherein aryl and alkyl are as defined above for example benzyl , fluorenylmethyl and the like . the term &# 34 ; heteroaryl &# 34 ; means a heteroaromatic radical which is 2 - or 3 - thienyl , 2 - or 3 - furanyl , 2 - or 3 - pyrrolyl , 2 -, 4 -, or 5 - imidazolyl , 3 -, 4 -, or 5 - pyrazolyl , 2 -, 4 -, or 5 - thiazolyl , 3 -, 4 -, or 5 - isothiazolyl , 2 -, 4 -, or 5 - oxazolyl , 3 -, 4 -, or 5 - isoxazolyl , 3 - or 5 - 1 , 2 , 4 - triazolyl , 4 - or 5 - 1 , 2 , 3 - triazolyl , tetrazolyl , 2 -, 3 -, or 4 - pyridinyl , 3 -, 4 -, or 5 - pyridazinyl , 2 - pyrazinyl , 2 -, 4 -, or 5 - pyrimidinyl , 2 -, 3 -, 4 -, 5 -, 6 -, 7 -, or 8 - quinolinyl , 1 -, 3 -, 4 -, 5 -, 6 -, 7 -, or 8 - isoquinolinyl , 2 -, 3 -, 4 -, 5 -, 6 -, or 7 - indolyl , 2 -, 3 -, 4 -, 5 -, 6 -, or 7 - benzo [ b ] thienyl , or 2 -, 4 -, 5 -, 6 -, or 7 - benzoxazolyl , 2 -, 4 -, 5 -, 6 -, or 7 - benzimidazolyl , 2 -, 4 -, 5 -, 6 -, or 7 - benzothiazolyl , unsubstituted or substituted by 1 to 2 substituents selected from alkyl as defined above , aryl as defined above , alkoxy as defined above , thioalkoxy as defined above , hydroxy , thiol , nitro , halogen , formyl , amino , ## str33 ## wherein alkyl is as defined above , ## str34 ## wherein alkyl is as defined above , ## str35 ## wherein alkyl is as defined above or phenyl . the term &# 34 ; heterocycloalkyl &# 34 ; means 2 - or 3 - tetrahydrothieno , 2 - or 3 - tetrahydrofurano , 2 - or 3 - pyrrolidino , 2 -, 4 -, or 5 - thiazolidino , 2 -, 4 -, or 5 - oxazolidino , 2 -, 3 -, or 4 - piperidino , n - morpholinyl or n - thiamorpholinyl . the following table provides a list of abbreviations and definitions thereof used in the present invention . ______________________________________abbreviation * amino acid______________________________________ala alaninearg arginineasn asparagineasp aspartic acidcys cysteineglu glutamic acidgln glutaminegly glycinehis histidineile isoleucineleu leucinelys lysinemet methioninephe phenylalaninepro prolineser serinethr threoninetrp tryptophantyr tyrosineval valine______________________________________abbreviation * modified and unusual amino acid______________________________________bhg 10 , 11 - dihydro - 5h - dibenzo [ a , d ]- ( cyclohepten - s - yl ) glycine or α - amino - 10 , 11 - dihydro - 5h - dibenzo - [ a , d ] cycloheptene - 5 - acetic acidbip ( para - phenyl ) phenylalaninedip 3 , 3 - diphenylalanine3hyp 3 - hydroxyproline4hyp 4 - hydroxyprolinen - mephe n - methylphenylalaninen - measp n - methylaspartic acidn - meile n - methylisoleucinen - meval n - methylvalinenva norvalinenle norleucineorn ornithineabu 2 - aminobutyric acidalg 2 - amino - 4 - pentenoic acid ( allylglycine ) arg ( no . sub . 2 ) n . sup . g - nitroarginineatm 2 - amino - 3 -( 2 - amino - 5 - thiazole ) propanoic acidcpn 2 - amino - 3 - cyclopropanepropanoic acid ( cyclopropylalanine ) chx cyclohexylalanine ( hexahydrophenyl - alanine ) n - mechx n - methylcyclohexylalanine ( n - methylhexahydrophenylalanine ) emg 2 - amino - 4 , 5 ( rs )- epoxy - 4 - pentenoic acidhis ( dnp ) n . sup . im - 2 , 4 - dinitrophenylhistidinehomoglu 2 - aminoadipic acidhomophe 2 - amino - 5 - phenylpentanoic acid ( homophenylalanine ) met ( o ) methionine sulfoxidemet ( o . sub . 2 ) methionine sulfone1 - nal 3 -( 1 &# 39 ;- naphthyl ) alanine2 - nal 3 -( 2 &# 39 ;- naphthyl ) alaninenia 2 - amino - 3 - cyanopropanoic acid ( cyanoalanine ) pgl phenylglycinepgy 2 - aminopentanoic acid ( propylglycine ) pha 2 - amino - 6 -( 1 - pyrrolo )- hexanoic acidpyr 2 - amino - 3 -( 3 - pyridyl )- propanoic acid ( 3 - pyridylalanine ) tic 1 , 2 , 3 , 4 - tetrahydro - 3 - isoquinolinecarboxylic acidtza 2 - amino - 3 -( 4 - thiazolyl )- propanoic acidtyr ( ot - bu ) o - tertiary butyl - tyrosinetyr ( ome ) o - methyl - tyrosinetyr ( oet ) o - ethyl - tyrosinetrp ( for ) n . sup . in - formyl - tryptophanbheg 8h - dibenzo [ a , d ] cycloheptene glycinetxg 9h - thioxanthene glycineoxn 9h - xanthene glycine______________________________________abbreviation protecting group______________________________________ac acetylada 1 - adamantyl acetic acidadoc adamantyloxycarbonylbzl benzylmebzl 4 - methylbenzylz benzyloxycarbonyl2 - br - z ortho - bromobenzyloxycarbonyl2 - cl - z ortho - chlorobenzyloxycarbonylbom benzyloxymethylboc teriary butyloxycarbonyltbs tertiary butyldimethysilyldnp 2 , 4 - dinitrophenylfor formylfmoc 9 - fluorenylmethyloxycarbonylno . sub . 2 nitroltos 4 - toluenesulfonyl ( tosyl ) trt triphenylmethyl ( trityl ) ada 1 - adamantyl acetic acidbz benzylcarbonyltbu t - butylcarbonylcf . sub . 3 co trifluoroacetylcxl cyclohexylacetylcxl ( u ) cyclohexylureaet propionylpya 3 - pyridylacetylme ( u ) methylurea______________________________________abbreviation solvents and reagents______________________________________hoac acetic acidch . sub . 3 cn and acn acetonitriledcm dichloromethanedcc n , n &# 39 ;- dicyclohexylcarbodiimidediea n , n - diisopropylethylaminedmf dimethylformamidehcl hydrochloric acidhf hydrofluoric acidhobt 1 - hydroxybenzotriazolekoh potassium hydroxidetfa trifluoroacetic acidmbha resin methylbenzhydrylamine resinpam resin 4 -( oxymethyl )- phenylacetamidomethyl resin______________________________________ * if the optical activity of the amino acid is other than l ( s ), the amino acid or abbreviation is preceded by the appropriate configuration d ( r ), o dl ( rs ). the compounds of formula i are capable of further forming both pharmaceutically acceptable acid addition and / or base salts . all of these forms are within the scope of the present invention . pharmaceutically acceptable acid addition salts of the compounds of formula i include salts derived from nontoxic inorganic acids such as hydrochloric , nitric , phosphoric , sulfuric , hydrobromic , hydriodic , hydrofluoric , phosphorous , and the like , as well as the salts derived from nontoxic organic acids , such as aliphatic mono - and dicarboxylic acids , phenyl - substituted alkanoic acids , hydroxy alkanoic acids , alkanedioic acids , aromatic acids , aliphatic and aromatic sulfonic acids , etc . such salts thus include sulfate , pyrosulfate , bisulfate , sulfite , bisulfite , nitrate , phosphate , monohydrogenphosphate , dihydrogenphosphate , metaphosphate , pyrophosphate , chloride , bromide , iodide , acetate , trifluoroacetate , propionate , caprylate , isobutyrate , oxalate , malonate , succinate , suberate , sebacate , fumarate , maleate , mandelate , benzoate , chlorobenzoate , methylbenzoate , dinitrobenzoate , phthalate , benzenesulfonate , toluenesulfonate , phenylacetate , citrate , lactate , maleate , tartrate , methanesulfonate , and the like . also contemplated are salts of amino acids such as arginate and the like and gluconate , galacturonate ( see , for example , berge s . m ., et al ., &# 34 ; pharmaceutical salts ,&# 34 ; journal of pharmaceutical science , 66 : 1 - 19 ( 1977 )). the acid addition salts of said basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner . preferably a peptide of formula i can be converted to an acidic salt by treating with an aqueous solution of the desired acid , such that the resulting ph is less than 4 . the solution can be passed through a c18 cartridge to absorb the peptide , washed with copious amounts of water , the peptide eluted with a polar organic solvent such as , for example , methanol , acetonitrile , aqueous mixtures thereof , and the like , and isolated by concentrating under reduced pressure followed by lyophilization . the free base form may be regenerated by contacting the salt form with a base and isolating the free base in the conventional manner . the free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents , but otherwise the salts are equivalent to their respective free base for purposes of the present invention . pharmaceutically acceptable base addition salts are formed with metals or amines , such as alkali and alkaline earth metals or organic amines . examples of metals used as cations are sodium , potassium , magnesium , calcium , and the like . examples of suitable amines are n , n &# 39 ;- dibenzylethylenediamine , chloroprocaine , choline , diethanolamine , dicyclohexylamine , ethylenediamine , n - methylglucamine , and procaine ( see , for example , berge s . m ., et al ., &# 34 ; pharmaceutical salts ,&# 34 ; journal of pharmaceutical science , 66 : 1 - 19 ( 1977 )). the base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner . preferably , a peptide of formula i can be converted to a base salt by treating with an aqueous solution of the desired base , such that the resulting ph is greater than 9 . the solution can be passed through a c18 cartridge to absorb the peptide , washed with copious amounts of water , the peptide eluted with a polar organic solvent such as , for example , methanol , acetonitrile , aqueous mixtures thereof , and the like , and isolated by concentrating under reduced pressure followed by lyophilization . the free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner . the free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents , but otherwise the salts are equivalent to their respective free acid for purposes of the present invention . certain of the compounds of the present invention can exist in unsolvated forms as well as solvated forms , including hydrated forms . in general , the solvated forms , including hydrated forms , are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention . certain of the compounds of the present invention possess one or more chiral centers and each center may exist in the r ( d ) or s ( l ) configuration . the present invention includes all enantiomeric and epimeric forms as well as the appropriate mixtures thereof . aa 1 is ## str36 ## wherein r is ## str37 ## wherein r 2 and r 3 are each the same or different and each is hydrogen , fluorenylmethyl , ## str38 ## wherein r 2 and r 3 are as defined above , ## str39 ## wherein r 9 is f , cl , br , or i , ## str40 ## wherein r 3 is as defined above , or ## str41 ## wherein r 3 is as defined above excluding r 3 is hydrogen ; z is -- o --, -- s ( o ) m --, wherein m is zero or an integer of 1 or 2 , ## str42 ## wherein r 2 is as defined above , --( ch 2 ) n --, wherein n is zero or an integer of 1 , 2 , 3 , or 4 , --( ch 2 ) n -- ch ═ ch --( ch 2 ) n &# 39 ; --, wherein n and n &# 39 ; are each independently the same or different and each is as defined above for n , ## str43 ## wherein r 1 is hydrogen or alkyl , ## str44 ## wherein r 2 and r 3 are each the same or different and each is as defined above , and x and y are the same and substituted at the same position on the aromatic ring and each substituent is selected from the group consisting of heteroaryl , ## str46 ## wherein r 2b and r 3b are each the same or different and each is hydrogen , -- or 2b , wherein r 2b is as defined above , ## str47 ## wherein r 2b and r 3b are each the same or different and each is as defined above for r 2b and r 3b , ## str48 ## wherein r 2b is as defined above , ## str49 ## wherein r 2b is as defined above , or ## str50 ## wherein r 2b is as defined above , and r 1 and n are as defined above , or aa 2 is absent ; aa 3 is ## str51 ## wherein w is ## str52 ## r 5 is aryl , heteroaryl , ## str53 ## wherein r 2b and r 3b are each the same or different and each is as defined above , ## str54 ## wherein r 2b is as defined above , or ## str55 ## wherein r 2b is as defined above , and r 1 and n are as defined above , or aa 3 is absent ; aa 4 and aa 5 are each independently absent or each is independently ## str56 ## wherein r 6 is hydrogen alkyl , aa 6 is ## str57 ## wherein r 7 is aryl or heteroaryl , and r 1 and n are as defined above , or ## str58 ## wherein r 7 , r 1 , and n are as defined above ; stereochemistry at c * h in aa 1 , aa 2 , aa 3 , aa 4 , or aa 5 is d , l , or dl , and stereochemistry at c * h in aa 6 is l ; , or a pharmaceutically acceptable salt thereof . a more preferred compound of formula i is one wherein aa 1 is ## str59 ## wherein r is ## str60 ## wherein r 2 and r 3 are each the same or different and each is hydrogen , fluorenylmethyl , ## str61 ## wherein r 2 and r 3 are as defined above , ## str62 ## wherein r 9 is f , cl , br , or i , ## str63 ## wherein r 3 is as defined above , or ## str64 ## wherein r 10 is hydrogen , alkyl , aryl , arylalkyl , or fluorenylmethyl , excluding r 10 is hydrogen , --( ch 2 ) n , wherein n is zero or an integer of 1 , 2 , 3 , or 4 , or --( ch 2 ) n . spsp . a -- ch ═ ch --( ch 2 ) n . spsp . a - 1 --, wherein n a and n a - 1 are each independently the same or different and each is zero or an integer of 1 or 2 and x and y are each the same and substituted at the same position on the aromatic ring and each substituent is selected from the group consisting of aa 2 is ## str65 ## wherein r 1 &# 39 ; is hydrogen or methyl , r 4 is hydrogen , heteroaryl , ## str66 ## wherein r 2b and r 3b are each the same or different and each is hydrogen or alkyl , ## str67 ## wherein r 2b and r 3b are each the same or different and each is hydrogen or alkyl , ## str68 ## wherein r 2b is as defined above , or ## str69 ## wherein r 2b is as defined above , and n is zero or an integer of 1 , 2 , 3 , or 4 or aa 2 is absent ; aa 3 is ## str70 ## wherein w is ## str71 ## r 5 is aryl , heteroaryl , ## str72 ## wherein r 3b is hydrogen or alkyl , ## str73 ## wherein r 2b is hydrogen or alkyl , or ## str74 ## wherein r 2b is hydrogen or alkyl , and r 1 &# 39 ; and n are as defined above ; aa 4 and aa 5 are each independently ## str75 ## wherein r 6 is hydrogen , alkyl , aa 6 is ## str76 ## wherein r 7 is aryl or heteroaryl , and r 1 &# 39 ; and n are as defined above , or ## str77 ## wherein r 7 , r 1 , r 1 &# 39 ; , and n are as defined above ; stereochemistry at c * h in aa 1 , aa 2 , aa 3 , aa 4 , or aa 5 is d , l , or dl and stereochemistry at c * h in aa 6 is l ; the compounds of formula i are valuable antagonists of endothelin . the tests employed indicate that compounds of formula i possess endothelin antagonist activity . thus , the compounds of formula i were tested for their ability to inhibit [ 125 i ]- et - 1 ([ 125 i ]- endothelin - 1 ) binding in a receptor assay according to the following procedures : endothelin receptor binding assay - a ( erba - a ) intact cell binding of [ 125 i ]- et - 1 the cells used were rabbit renal artery vascular smooth muscle cells grown in a 48 - well dish ( 1 cm 2 ) ( confluent cells ). the growth media was dulbecco &# 39 ; s modified eagles / ham &# 39 ; s f12 which contained 10 % fetal bovine serum and antibiotics ( penicillin / streptomycin / fungizone ). the assay buffer was a medium 199 containing hanks salts and 25 mm hepes buffer ( gibco 380 - 2350aj ), supplemented with penicillin / streptomycin / fungizone ( 0 . 5 %) and bovine serum albumin ( 1 mg / ml ). amersham radioiodinated endothelin - 1 [ 125 i ]- et - 1 was used at final concentration of 20 , 000 cpm / 0 . 25 ml ( 25 pm ). first , add 0 . 5 ml warm assay buffer ( described above ) to the aspirated growth media and preincubate for 2 to 3 hours in a 37 ° c . water bath ( do not put back in the 5 % carbon dioxide ). second , remove the assay buffers , place the dish on ice , and add 150 μl of cold assay buffer described above to each well . third , add 50 ml each of cold [ 125 i ]- et - 1 and competing ligand to the solution ( at the same time if possible ). next , place dish in a 37 ° c . water bath for about 2 hours and gently agitate the dish every 15 minutes . discard the radioactive incubation mixture in the sink and wash wells 3 times with 1 ml of cold phosphate buffered saline . last , add 250 ml of 0 . 25 molar sodium hydroxide , agitate for 1 hour on a rotator , and then transfer the sodium hydroxide extract to gamma counting tubes and count the radioactivity . endothelin receptor binding assay - b ( erba - b ) [ 125 i ]- et - 1 binding in rat cerebellar membranes the tissue is made up of 20 mm tris ( hydroxymethyl ) aminomethane hydrochloride ( trizma ) buffer , 2 mm ethylenediaminetetra acetate , 100 μm phenylmethylsulfonyl fluoride . first , thaw one aliquot of frozen rat cerebellar membranes ( 2 mg protein in 0 . 5 ml ). next , add 0 . 5 ml membrane aliquot to 4 . 5 ml cold tissue buffer , polytron at 7 , 500 revolutions per minute for 10 seconds . finally , dilute tissue suspension 1 / 100 ( 0 . 1 ml suspension + 9 . 9 ml tissue buffer ), polytron again , and place ice . medium 199 with hank &# 39 ; s salts plus 25 mm hepes + 1 mg / ml bovine serum albumin . amersham [ 125 i ]- et - 1 ( aliquots of 2 × 10 6 cpm per 100 ml aliquot of [ 125 i ]- et - 1 with 5 . 2 ml dilution buffer , place on ice until use ( final concentration will be 20 , 000 cpm per tube , or 25 pm ). add 50 μl each of cold [ 125 ]- et - 1 and competing ligand to tubes on ice . mix in 150 μl of tissue to each tube , vortex briefly , then tap to force all liquids to bottom ( total assay volume = 250 μl ). then place the tubes in a 37 ° c . water bath for 2 hours . add 2 . 5 ml cold wash buffer ( 50 mm trizma buffer ) to each tube , filter , and then wash tube with additional 2 . 5 ml wash buffer and add to filter . finally , wash filters with an additional 2 . 5 ml of cold wash buffer . human endothelin receptor biding assay - b ( herba - b ) [ 125 i ]- et - 1 binding to human cloned receptor a human placenta cdna library was constructed in bacteriophage lambda gt11 and approximately 10 6 plaques were screened with a 32 p - labelled 1 . 3 kilobase hindiii / xbai restriction fragment of rat etbr cdna as a probe . plaque hybridization was carried out for 16 hours at 42 ° c . in a solution containing 100 μg / ml calf thymus dna , 1 × denhardt &# 39 ; s solution , 5 × sodium saline citrate ( ssc ), 50 mm sodium phosphate , and 0 . 1 % sodium lauryl sulfate ( sds ). the membranes were then washed twice for 30 minutes each in 2 × ssc and 0 . 1 % sds at 42 ° c . a final wash was carried out in 0 . 5 × ssc with 0 . 1 % sds at 55 ° c . the positive clones were purified and subcloned into a puc19 plasmid . dna sequencing was performed by the dideoxynucleotide chain termination method and human etbr ( h etbr ) was identified by reading both dna strands . the 1 . 35 kb hindiii / xbai restriction fragment of clone 12 of the hetbr was inserted into the eukaryotic expression vector prccmv ( prccmv - hetbr ). cho - k1 cells were transfected with 20 μg of prccmv - hetbr by electroporation at 300 v , 800 μf , low ohms for 1 second . cell populations expressing human etbr were selected with g418 ( 0 . 5 mg / ml ) and clonal cell lines were isolated from these selected cell populations by single cell cloning . expression levels of human etbr were determined by the receptor binding assay described below using [ 125 i ]- et - 3 as the radioligand . cho - k1 cells were grown in ham &# 39 ; s nutrient mixture f12 and dulbecco &# 39 ; s eagle medium ( 1 : 1 ) dme / f12 ( 1 : 1 ) supplemented with 10 % fetal bovine serum and g418 ( 0 . 5 mg / ml ). membranes were prepared from confluent transfected cho - k1 cells by lysing cells in cold lysis buffer ( 5 mm n -[ 2 - hydroxyethyl ] piperazine - n &# 39 ;-[ 2 - ethanesulfonic acid ] ( hepes ), 2 mm edta , ph 7 . 4 ), homogenizing with a dounce &# 34 ; a &# 34 ; homogenizer , and centrifuging the homogenate at 30 , 000 × g for 20 minutes at 4 ° c . cell pellets were resuspended in cold buffer ( 50 mm tris , 2 mm edta , 200 μm pefabloc , 10 μm phosphoramidon , 10 μm leupeptin , 1 μm pepstatin , ph 7 . 4 ) and frozen at - 80 ° c . until use . membranes were thawed and homogenized with a brinkmann polytron ( westbury , n . y . ), then diluted in binding buffer ( 20 mm tris , 2 mm edta , 100 μm pefabloc , 100 μm bacitracin , ph 7 . 4 ). radioligand and competing ligands were prepared in binding buffer containing 0 . 1 % bovine serum albumin ( bsa ). competition binding assays were initiated by combining membranes ( 0 . 7 μg human etbr , 3 μg rat etbr ), [ 125 i ]- et - 3 ( 30 , 000 cpm ), and competing ligand in a final volume of 250 μl and incubating 2 hours at 37 ° c . the assay was terminated by filtration over whatman gf / b filters which were presoaked with 50 mm tris , ph 7 . 4 , containing 0 . 2 % bsa and 100 μm bacitracin . nonspecific binding was defined as binding in the presence of 100 nm unlabelled et - 3 , and specific binding was defined as total binding minus nonspecific binding . specific binding was analyzed by nonlinear least squares curve fitting ( inplot , graphpad software , san diego , calif .). in vitro inhibition of et - 1 stimulated arachidonic acid release in cultured rabbit vascular smooth muscle cells ( aar - a ) or cho cells expressing rat recombinant et b receptor ( aar - b ) by compounds of formula i antagonist activity is measured by the ability of added compounds to reduce endothelin - stimulated arachidonic acid release in cultured vascular smooth muscle cells as arachidonic acid release ( aar ). [ 3 h ] arachidonic acid loading media ( lm ) is dme / f12 + 0 . 5 % fetal calf serum ( fcs )× 0 . 25 mci / ml [ 3 h ] arachidonic acid ( amersham ). confluent monolayers of cultured rabbit renal artery vascular smooth muscle cells ( aar - a ) or cho cells expressing rat recombinant et b receptor ( aar - b ) were incubated in 0 . 5 ml of the lm over 18 hours , at 37 ° c ., in 5 % co 2 . the lm was aspirated and the cells were washed once with the assay buffer ( hank &# 39 ; s balanced salt solution [ bss ]+ 10 mm hepes + fatty acid - free bsa ( 1 mg / ml )), and incubated for 5 minutes with 1 ml of the prewarmed assay buffer . this solution was aspirated , followed by an additional 1 ml of prewarmed assay buffer , and further incubated for another 5 minutes . a final 5 - minute incubation was carried out in a similar manner . the same procedure was repeated with the inclusion of 10 μl of the test compound ( 1 nm to 1 μm ) and 10 μl et - 1 ( 0 . 3 nm ) and the incubation was extended for 30 minutes . this solution was then collected , 10 μl of scintillation cocktail was added , and the amount of [ 3 h ] arachidonic acid was determined in a liquid scintillation counter . in vitro antagonism of et - 1 stimulated vasoconstriction in the rabbit femoral artery ( et a ) and sarafotoxin 6c stimulated vasoconstriction in the rabbit pulmonary artery ( et b ) male new zealand rabbits were killed by cervical dislocation and exsanguination . femoral and pulmonary arteries were isolated , cleaned of connective tissue , and cut into 4 - mm rings . the endothelium was denuded by placing the rings over hypodermic tubing ( 32 guage for femoral rings and 28 guage for pulmonary rings , small parts , inc , miami , fla .) and gently rolling them . denuded rings were mounted in 20 ml organ baths containing krebs - bicarbonate buffer ( composition in mm : nacl , 118 . 2 ; nahco 3 , 24 . 8 ; kcl , 4 . 6 ; mgso 4 7 . h 2 o , 1 . 2 ; kh 2 po 4 , 1 . 2 ; cacl 2 . 2h 2 o ; ca -- na 2 edta , 0 . 026 ; dextrose , 10 . 0 ), that was maintained at 37 ° c . and gassed continuously with 5 % co 2 in oxygen ( ph 7 . 4 ). resting tension was adjusted to 3 . 0 g for femoral and 4 . 0 g pulmonary arteries ; the rings were left for 90 minutes to equilibrate . vascular rings were tested for lack of functional endothelium ( i . e ., lack of an endothelium - dependent relaxation response to carbachol ( 1 . 0 μm ) in norepinephrine ( 0 . 03 μm ) contracted rings . agonist peptides , et - 1 ( femoral ), and s6c ( pulmonary ), were cumulatively added at 10 - minute intervals . the et antagonists were added 30 minutes prior to adding the agonist and pa 2 values were calculated ( table ii ). caco - 2 cell transport and stability in rat intestinal perfusate of endothelin antagonists caco - 2 cells ( human colon adenocarcinoma cell line ) were grown in corning t - 75 tissue culture flasks and passed when 50 % to 80 % confluent to new flasks ( 25 ml medium per flask ) using 1 ml of trypsin - edta and diluting with 10 ml media to stop trypsin activity . for experiments , the cells were counted , then diluted to 2 . 5 × 10 5 cells / ml for planting ( 400 μl per well ) onto snapwell culture membranes with 2 . 5 ml media in the lower chamber . media in the snapwells was changed every 2 to 3 days . cells used for this series of experiments were between passage 38 and 91 . 2 -[ n - morpholino ] ethanesulfonic acid ( mes )+ 25 mm glucose was used as incubation buffer for all the experiments and all incubation solutions were prepared to an osmotic pressure of 280 to 300 milliosmoles . the concentration of the endothelin antagonist being studied ( for all experiments ) was 100 μm and another endothelin antagonist with similar hplc characteristics ( retention time 2 - 3 minutes from the et antagonist being studied , and similar chemical characteristics ) was used at a concentration of 25 μm as an internal standard to compensate for any injection error . prior to the start of the experiment , the transepithelial electrical resistance ( teer ) was measured for each snapwell ( in media ) at three points of the membrane . any membrane with large variations in the three values was not used as this suggested compromises to the cell monolayer . the membranes were washed by swirling in 0 . 9 % normal saline and mounted in the pretreated diffusion chambers [ 0 . 1 mg / ml human serum albumin for 1 to 2 hours at 37 ° c . with o 2 bubbling followed by air drying at 37 ° c . overnight , then with 4 . 5 to 5 . 0 ml dosing solution ( mes + glucose + 14 c - peg - 4000 ( 5000 dpm / 50 μl )+ drug ) for 5 minutes and 4 . 5 to 5 . 0 ml mes + glucose rinse just before the experiment .] the apical ( donor ) side was filled with 4 . 5 to 5 . 0 ml dosing solution , the basolateral ( receiver ) side with mes + glucose . samples were removed from the donor and receiver compartments over the course of the experiment and analyzed for 14 c - peg 4000 ( cell membrane integrity ) and endothelin content . the amount of drug transferred as a function of time was used to calculate the apparent permeability , where v is the volume of the receiver chamber ( 4 . 5 or 5 . 0 ml ), a is the exposed surface of the cell monolayer ( 1 . 13 cm 2 ), c o is the donor drug concentration , and dc / dt is the change in receiver drug concentration over time . the starting concentration of each diffusion chamber was used for its individual c o value . the values in table ii suggest that examples 1 , 16 , and 19 may have 5 - 10 % absorption in the gut . for the stability experiments , male wistar rats ( 250 - 350 g ) were fasted overnight , then anesthetized with a cocktail of ketamine / xylasine ( prepared just before injection ) in the thigh muscle followed by pentobarbital injection in the alternate thigh muscle . after deep anesthesia , verified by loss of reflex reaction , a midline abdominal incision was made to open the peritoneal cavity . the ligament of treitz was located and the jejunum was cannulated approximately 5 cm distal as well as 15 cm further distal to isolate that segment of intestine for perfusion . the segment was perfused for 90 minutes with mes buffer at a flow rate of 30 ml / minute using a harvard apparatus perfusion pump in the infuse / refill mode . the perfusate was kept at 37 ° c . for each experiment . an oscillating 37 ° c . water bath set to 90 cycles / minute was used for all samples . the experimental time course for the compounds studied was 0 , 1 , 3 , 5 , 7 , 10 , 15 , 20 , 30 , 60 , 90 , 120 , and 180 minutes ( with some variations for individual compounds ). the reactions were run as follows : 90 μl of perfusate plus 10 μl of endothelin antagonist ( 250 μm stock in mes buffer for most compounds ) followed by brief vortexing and incubation for indicated time . the time zero was prepared by adding 100 μl acn and 90 μl perfusate , vortexing , then adding 10 μl stock drug , vortexing . all samples were centrifuged for 10 minutes at 14 , 000 rpm in an eppendorf centrifuge to pellet precipitated proteins . for hplc analysis 50 μl of the final supernatant was injected and loss of parent and appearance of metabolites was examined . half - life determinations were calculated based on loss of parent peak height using the calculation : the enzyme activity of leucine amino peptidase in the perfusate was determined as a convenient marker . the values in table i show the enhanced stability of example 19 . fifty microliter samples were collected from all time points and from each chamber , placed in 20 - ml scintillation vials , 10 ml of ready - gel scintillant was added . all samples were allowed to stabilize for at least 1 hour prior to counting . the samples were counted in the packard tricarb 4000 for 5 minutes each in the dpm mode for 3 cycles to assure that no chemiluminescence was present . typically the second and third counts were used in calculations . when membranes were analyzed for radiolabel uptake they were solubilized with 0 . 5 ml of soluene - 350 for at least 30 minutes then neutralized with 0 . 1 ml of a saturated solution of sodium pyruvate in methanol , glacial acetic acid , and methanol in the ratio of 4 : 3 : 1 by volume ( pgm ) followed by addition of scintillant ( 10 ml ) and counting as indicated above . for caco - 2 experiments 20 to 50 μl were removed from donor compartments , 150 to 200 μl from receiver compartments at each time point . internal standard ( solubilized in 95 - 99 % acn / h 2 o ) was measured to 1 . 5 ml eppendorf tubes in an equal volume to the receiver volume collected ( mes + glucose added to donor compartment tubes to equal receiver final volume ) within 30 minutes of sampling time point . the time point aliquots were added to the eppendorf tubes , mixed , then 125 μl was injected into the hplc . when membranes were analyzed for endothelin uptake the cells were lysed by adding 250 μl 0 . 1x triton vortexing briefly , sonicating 15 minutes , and rinsing the tube with an equal volume of acn / internal standard . a gradient hplc method was used for all samples ( caco - 2 cell experiments and stability experiments ): mobile phase a = 90 % h 2 o , 10 % acn , 0 . 1 % tfa , ph 3 . 5 ; mobile phase b = 24 % h 2 o , 76 % acn , 0 . 1 % tfa , ph 3 . 5 ; 100 % a to 100 % b over 20 minutes , 100 % b to 100 % a from 20 to 22 . 5 minutes , and 100 % a from 2 . 5 to 27 minutes . the ph of both mobile phases was adjusted after addition of all components with naoh . to 0 . 9 ml mes buffer ( blank ) and 0 . 9 ml perfusate 0 . 1 ml l - leucine - p - nitroanilide hydrochloride ( lpna ) solution ( 1 . 44 mg / ml ) was added . the solutions were mixed quickly and a 5 - minute kinetics program was run on the beckman du - 70 spectrophotometer ( 380 nm wavelength , 10 - second time interval ) to monitor the formation of p - nitroaniline . nonfasted rats ( 350 - 500 g ) were used and anesthetized with metofane ( methoxyflurane ) by inhalation . the rats were jugular cannulated ( pe50 ) for iv administration of mecamylamine - hc1 ( mec ), et - 1 and ac - d - bhg - leu - asp - ile - n - meile - trp ( example 19 ) seq id no : 20 , and carotid cannulated ( pe50 ) for arterial blood pressure measurements . rats were attached to a swivel for freedom of movement , and food and water were available ad lib . prior to the experiment , the animals were allowed to recover from anesthesia for 60 minutes . the rats were ganglionically blocked with mec 1 . 25 mg / kg iv 20 minutes prior to the et - 1 challenge . ac - d - bhg - leu - asp - ile - n - meile - trp ( example 19 ) seq id no : 20 was administered at a dose of 10 mg / kg iv . this compound inhibited et - 1 pressor activity by 81 % and 58 % at 5 and 30 minutes postdose , respectively ( n = 4 at each time point ). this illustrates the prolonged activity of ac - d - bhg - leu - asp - ile - n - meile - trp ( example 19 ) seq id no : 20 in vivo . male sprague - dawley rats ( 300 - 400 g ) were housed in metabolic cages for 2 days before and 7 days after renal injury ; urine output and plasma creatinine levels were monitored daily . on the day of renal injury , rats were anesthetized with sodium pentobarbital ( 50 mg / kg , ip ) heparinized ( 50 units , iv ), and instrumented with a tail vein canulae for drug or vehicle infusion . both kidneys were exposed via a flank incision and the right kidney was removed . the left renal artery was clamped for 60 minutes and released . example 1 was infused for 60 minutes prior to and following the ischemic period . renal injury was evident 1 and 2 days following ischemia from a ten - fold increase in plasma creatine levels and significant decrease in urine output . mortality occurred primarily between the second and third days post - injury . however , mortality was significantly less ( 52 %, n = 23 ) in rats treated with example 1 compared to vehicle rats ( 83 %, n = 23 ). in addition , urine output on the second day following renal injury was significantly greater in example 1 treated animals . creatine levels were not significantly different between treatment groups on either the first or second days post - injury ( haleen s ., et al ., faseb j ., april 1994 ). therefore , these data show that example 1 and related analogues in table i are effective in a model of ischemia - induced acute renal failure . the data in table ii below show the endothelin receptor binding , antagonist activity , caco - 2 cell transport , metabolic stability , and effectiveness in a model of ischemia - induced acute renal failure of representative compounds of formula i . table ii - biological activity of compounds of formula i stability rat erba - a erra - b herba - b aar - a aar - b intestinal ic . sub . 50 ic . sub . 50 ic . sub . 50 ic . sub . 50 ic . sub . 50 pa . sub . 2 perfusate caco - 2 example compound ( μm ) ( μm ) ( μm ) ( μm ) ( μm ) femoral pulmonary ( minutes ) ( cm / minute ) 1 ac -- d - bhg -- leu -- 0 . 0026 0 . 019 0 . 21 0 . 0049 0 . 02 6 . 8 7 . 1 10 . 6 ± 2 . 2 4 . 73 ± 0 . 86 seq id no : 2 asp -- ile -- ile -- trp 2 d - bhg -- leu -- asp -- 0 . 45 2 . 1 0 . 10 9 . 6 5 . 7 seq id no : 2 ile -- ile -- trp 3 l - bhg -- leu -- asp -- 2 . 5 3 . 0 2 . 36 seq id no : 2 ile -- ile -- trp 4 ac -- l - bhg -- leu -- 0 . 56 0 . 71 0 . 56 seq id no : 2 asp -- ile -- ile -- trp 5 ac -- d - txg -- leu -- 0 . 018 0 . 18 0 . 05 0 . 062 seq id no : 2 asp -- ile -- ile -- trp 6 ac -- d - bheg -- leu -- 0 . 005 0 . 019 0 . 003 0 . 017 6 . 8 7 . 4 seq id no : 2 asp -- ile -- ile -- trp 7 ac -- d - bhg -- orn -- 0 . 022 1 . 5 0 . 037 6 . 5 seq id no : 3 asp -- ile -- ile -- trp 8 ac -- d - bhg -- glu -- 0 . 0055 0 . 022 0 . 007 0 . 027 6 . 5 7 . 0 seq id no : 6 asp -- ile -- ile -- trp 9 ac -- l - oxn -- lys -- 0 . 05 0 . 93 seq id no : 4 asp -- ile -- ile -- trp 10 ac -- d - oxn -- lys -- 0 . 12 0 . 55 seq id no : 4 asp -- ile -- ile -- trp 11 l - txg -- leu -- asp -- 4 . 5 2 . 1 seq id no : 2 ile -- ile -- trp 12 ac -- l - txg -- leu -- 1 . 5 2 . 1 seq id no : 2 asp -- ile -- ile -- trp 13 d - txg -- leu -- asp -- 0 . 83 0 . 38 0 . 32 2 . 4 seq id no : 2 ile -- ile -- trp 14 ac -- d - bhg -- arg -- 0 . 0012 0 . 04 seq id no : 8 asp -- ile -- ile -- trp 15 ac -- d - bhg -- leu -- n -- 0 . 44 1 . 9 seq id no : 15 measp -- ile -- ile -- trp 16 ac -- d - bhg -- leu -- d - 0 . 14 0 . 4 37 . 4 ± 6 . 4 0 . 95 ± 0 . 19 seq id no : 2 asp -- ile -- ile -- trp 17 ac -- d - bhg -- leu -- 0 . 068 0 . 03 seq id no : 31 asp -- phe -- ile -- trp 18 ac -- d - bhg -- arg -- 0 . 079 4 . 9 seq id no : 64 asp -- ile -- ile -- trp ( for ) 19 ac -- d - bhg -- leu -- 0 . 0005 0 . 04 0 . 025 0 . 0043 7 . 3 6 . 6 538 ± 52 5 . 54 ± 2 . 24 seq id no : 20 asp -- ile -- n -- meile -- trp the compounds of formula i may be prepared by solid phase peptide synthesis on a peptide synthesizer , for example , an applied biosystems 430a peptide synthesizer using activated esters or anhydrides of n - alpha - boc protected amino acids , on pam or mbha resins . additionally , the compounds of formula i may also be prepared by conventional solution peptide synthesis . amino acid side chains are protected as follows : bzl ( asp , glu , ser ), 2 - cl - z ( lys ), 2 - br - z ( tyr ), bom ( his ), for ( trp ), and mebzl ( cys ). each peptide resin ( 1 . 0 g ) is cleaved with 9 ml of hf and 1 ml of anisole or p - cresol as a scavenger ( 60 minutes , 0 ° c .). the peptide resin is washed with cyclohexane , extracted with 30 % aqueous hoac , followed by glacial hoac , concentrated under reduced pressure , and lyophilized . ( a peptide containing for ( trp ) is dissolved in 0 ° c ., the ph is adjusted to 12 . 5 with 1n koh ( 2 minutes ), neutralized with glacial hoac , desalted on c 18 ( as described below ), and lyophilized . the crude peptide is purified by preparative reversed phase high performance liquid chromatography ( rp - hplc ) on a c 18 column ( 2 . 2 × 25 . 0 cm , 15 . 0 ml / min ) with a linear gradient of 0 . 1 % tfa in water to 0 . 1 % tfa in acetonitrile and lyophilized . the homogeneity and composition of the resulting peptide is verified by rp - hplc , capillary electrophoresis , thin layer chromatography ( tlc ), proton nuclear magnetic resonance spectrometry ( nmr ), and fast atom bombardment mass spectrometry ( fab - ms ). the compounds of the present invention can be prepared and administered in a wide variety of oral and parenteral dosage forms . thus , the compounds of the present invention can be administered by injection , that is , intravenously , intramuscularly , intracutaneously , subcutaneously , intraduodenally , or intraperitoneally . also , the compounds of the present invention can be administered by inhalation , for example , intranasally . additionally , the compounds of the present invention can be administered transdermally . it will be obvious to those skilled in the art that the following dosage forms may comprise as the active component , either a compound of formula i or a corresponding pharmaceutically acceptable salt of a compound of formula i . for preparing pharmaceutical compositions from the compounds of the present invention , pharmaceutically acceptable carriers can be either solid or liquid . solid form preparations include powders , tablets , pills , capsules , cachets , suppositories , and dispersible granules . a solid carrier can be one or more substances which may also act as diluents , flavoring agents , binders , preservatives , tablet disintegrating agents , or an encapsulating material . in powders , the carrier is a finely divided solid which is in a mixture with the finely divided active component . in tablets , the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired . the powders and tablets preferably contain from five or ten to about seventy percent of the active compound . suitable carriers are magnesium carbonate , magnesium stearate , talc , sugar , lactose , pectin , dextrin , starch , gelatin , tragacanth , methylcellulose , sodium carboxymethylcellulose , a low melting wax , cocoa butter , and the like . the term &# 34 ; preparation &# 34 ; is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers , is surrounded by a carrier , which is thus in association with it . similarly , cachets and lozenges are included . tablets , powders , capsules , pills , cachets , and lozenges can be used as solid dosage forms suitable for oral administration . for preparing suppositories , a low melting wax , such as a mixture of fatty acid glycerides or cocoa butter , is first melted and the active component is dispersed homogeneously therein , as by stirring . the molten homogenous mixture is then poured into convenient sized molds , allowed to cool , and thereby to solidify . liquid form preparations include solutions , suspensions , and emulsions , for example , water or water propylene glycol solutions . for parenteral injection liquid preparations can be formulated in solution in aqueous polyethylene glycol solution . aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants , flavors , stabilizing and thickening agents as desired . aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material , such as natural or synthetic gums , resins , methylcellulose , sodium carboxymethylcellulose , and other well - known suspending agents . also included are solid form preparations which are intended to be converted , shortly before use , to liquid form preparations for oral administration . such liquid forms include solutions , suspensions , and emulsions . these preparations may contain , in addition to the active component , colorants , flavors , stabilizers , buffers , artificial and natural sweeteners , dispersants , thickeners , solubilizing agents , and the like . the pharmaceutical preparation is preferably in unit dosage form . in such form the preparation is subdivided into unit doses containing appropriate quantities of the active component . the unit dosage form can be a packaged preparation , the package containing discrete quantities of preparation , such as packeted tablets , capsules , and powders in vials or ampoules . also , the unit dosage form can be a capsules , tablet , cachet , or lozenge itself , or it can be the appropriate number of any of these in packaged form . the quantity of active component in a unit dose preparation may be varied or adjusted from 0 . 1 mg to 100 mg preferably 0 . 5 mg to 100 mg according to the particular application and the potency of the active component . the composition can , if desired , also contain other compatible therapeutic agents . in therapeutic use as antagonist of endothelin , the compounds utilized in the pharmaceutical method of this invention are administered at the initial dosage of about 0 . 01 mg to about 20 mg per kilogram daily . a daily dose range of about 0 . 01 mg to about 10 mg per kilogram is preferred . the dosages , however , may be varied depending upon the requirements of the patient , the severity of the condition being treated , and the compound being employed . determination of the proper dosage for a particular situation is within the skill of the art . generally , treatment is initiated with smaller dosages which are less than the optimum dose of the compound . thereafter , the dosage is increased by small increments until the optimum effect under the circumstances is reached . for convenience , the total daily dosage may be divided and administered in portions during the day , if desired . the following nonlimiting examples illustrate the inventors &# 39 ; preferred methods for preparing the compounds of the invention . the linear hexapeptide is prepared by standard solid phase synthetic peptide methodology utilizing a boc / benzyl strategy ( stewart j . m . and young j . d ., solid phase peptide synthesis , pierce chemical co ., rockford , ill ., 1984 ). all protected amino acids and reagents are obtained from commercial sources with the exception of n - α - boc - dl - bhg and are not further purified . the protected peptide resin is prepared on an applied biosystems 430a peptide synthesizer , utilizing protocols supplied for a dicyclohexylcarbodiimide - mediated coupling scheme ( standard 1 . 0 , version 1 . 40 ). starting with 0 . 710 g of n - α - boc - trp - pam resin ( 0 . 70 meq / g , 0 . 497 meq of boc - trp ( for ) total ) the protected peptide is prepared by the stepwise coupling of the following amino acids ( in order of addition ): n - α - boc - ile . 0 . 5h 2 o , n - α - boc - ile . 0 . 5h 2 o , n - α - boc - asp ( bzl ), n - α - boc - leu · h 2 o , and n - α - boc - dl - bhg . a typical cycle for the coupling of an individual amino acid residue is illustrated below ( reproduced from the abi manual ): after the coupling of n - α - boc - dl - bhg , the boc group is removed with the end - nh 2 cycle ( 1 . 012 g ). the peptide is liberated from the solid support , and the carboxylate of aspartic acid deprotected by treatment with anhydrous hydrogen fluoride ( 9 . 0 ml ), anisole ( 0 . 5 ml ), and dimethyl sulfide ( 0 . 5 ml ) ( 60 minutes , 0 ° c .). after removing the hydrogen fluoride under a stream of nitrogen , the resin is washed with diethyl ether ( 3 × 30 ml ) and extracted with 20 % hoac in water ( 3 × 30 ml ) and glacial hoac ( 2 × 30 ml ). the aqueous extractions are combined , concentrated under reduced pressure , and lyophilized ( 360 mg ). the crude peptide is dissolved in 4 . 0 ml of 50 % tfa / h 2 o , filtered through a 0 . 4 l syringe filter , and chromatographed on a vydac 218tp 1022 column ( 2 . 2 × 25 . 0 cm , 15 . 0 ml / min , a : 0 . 1 % tfa / h 2 o , b : 0 . 1 % tfa / ch 3 cn , gradient ; 0 % b for 10 minutes , 10 % to 40 % b over 120 minutes ). two individual fractions are collected and combined based upon analysis by analytical hplc . the combined fractions are concentrated separately under reduced pressure ( 10 ml ), diluted with h 2 o ( 50 ml ), and lyophilized ( 40 . 0 mg / ea ). separation into the two diastereomers ( isomers a and b ) is effected under these conditions ( t r = isomer a 15 . 63 min ., isomer b 16 . 79 min .). the late running peak fractions ( isomer b ) are repurified under the same experimental conditions with a gradient of 30 % to 50 % b over 120 minutes at 15 ml / min to afford purified product . acetylation is carried out with 20 mg of isomer b in 90 % acetic acid followed by addition of acetic anhydride ( 5 ml ) and stirring overnight . after evaporation and drying the product ac - d - bhg - leu - asp - ile - ile - trp seq id no : 2 is 99 % pure by hplc . [ vydac 218 tp 1022 column ( 2 . 2 × 25 . 0 cm , 15 . 0 ml / min . a : 0 . 1 % tfa / ch 3 cn , gradient 20 % to 86 % b over 22 min .)] t r = 18 . 66 minutes . the homogeneity and structure of the resulting peptide is confirmed by analytical hplc . proton nuclear magnetic resonance spectroscopy ( h 1 - nmr ) and fast atom bombardment mass spectroscopy ( fab - ms ), m + na 972 . 0 , m + 2na + 995 . 9 . in a process analogous to example 1 using the appropriate amino acids , the corresponding compounds of formula i are prepared as follows : a saturated solution of sodium bicarbonate in water is prepared , diluted with water ( 1 : 10 ), chilled to 0 ° c ., and 10 ml of the solution is added to approximately 50 mg of ac - d - bhg - leu - asp - ile - ile - trp seq id no : 2 ( example 1 ) with stirring . the ph of the solution is greater than 9 . after 10 minutes , the solution is passed through a c18 cartridge , washed with water ( 100 ml ), and the absorbed peptide is eluted with methanol ( 50 ml ), concentrated under reduced pressure , resuspended in water ( 50 ml ), and lyophilized ( three times ) to give the title compound . ac - d - bhg - leu - asp - ile - ile - trp , disodium salt ; fab - ms , m + 1 950 . 4 , m + na 972 . 1 , m + 2na 994 . 3 . seq id no : 2 bhg . hcl ( 1 . 70 g , 5 . 43 mmol ) is suspended in 150 ml of p - dioxane : h 2 o ( 2 : 1 ) at room temperature . to the stirred solution is added 1 . 40 g ( 6 . 42 mmol ) of di - tert butyldicarbonate . the ph of the solution is adjusted to & gt ; 9 . 0 with 1n naoh and maintained at between ph 9 and 10 with aliquot additions of 1n naoh , until the ph is constant . the solution is concentrated under reduced pressure to approximately 75 ml , overlain with ethyl acetate ( 50 ml ), and acidified to approximately ph 2 . 5 with 10 % aqueous hcl . the organic layer is separated , washed successively with 10 % aqueous hcl ( 2 × 50 ml ), brine ( 2 × 50 ml ), h 2 o ( 3 × 50 ml ), and dried with mgso 4 . the solution is filtered , concentrated under reduced pressure , and the oil is recrystallized from ethyl acetate : heptane ( 1 . 82 g ). the white solid is characterized by proton nmr , fast atom bombardment mass spectrometry ( m + 1 = 368 ), and elemental analysis . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 64 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 1 : xaaxaaxaaxaaxaaxaa15 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 2 : xaaleuaspileiletrp15 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 3 : xaaxaaaspileiletrp15 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 4 : xaalysaspileiletrp15 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 5 : xaaaspaspileiletrp15 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 6 : xaagluaspileiletrp15 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 7 : xaapheaspileiletrp15 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 8 : xaaargaspileiletrp15 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 5 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 9 : xaaaspileiletrp15 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 10 : xaaleupheileiletrp15 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 11 : xaaleuasnileiletrp15 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 12 : xaaleugluileiletrp15 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 13 : xaaleuglnileiletrp15 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 14 : xaaleutyrileiletrp15 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 15 : xaaleuxaaileiletrp15 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 16 : xaaleutrpileiletrp15 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 17 : xaaleuaspvaliletrp15 ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 18 : xaaleuaspilevaltrp15 ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 19 : xaaleuaspxaailetrp15 ( 2 ) information for seq id no : 20 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 20 : xaaleuaspilexaatrp15 ( 2 ) information for seq id no : 21 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 21 : xaaargaspilexaatrp15 ( 2 ) information for seq id no : 22 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 22 : xaalysaspilexaatrp15 ( 2 ) information for seq id no : 23 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 23 : xaaxaaaspilexaatrp15 ( 2 ) information for seq id no : 24 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 24 : xaaaspaspilexaatrp15 ( 2 ) information for seq id no : 25 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 25 : xaagluaspilexaatrp15 ( 2 ) information for seq id no : 26 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 26 : xaaargaspxaailetrp15 ( 2 ) information for seq id no : 27 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 27 : xaalysaspxaailetrp15 ( 2 ) information for seq id no : 28 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 28 : xaaxaaaspxaailetrp15 ( 2 ) information for seq id no : 29 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 29 : xaaaspaspxaailetrp15 ( 2 ) information for seq id no : 30 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 30 : xaagluaspxaailetrp15 ( 2 ) information for seq id no : 31 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 31 : xaaleuasppheiletrp15 ( 2 ) information for seq id no : 32 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 32 : xaaxaaasppheiletrp15 ( 2 ) information for seq id no : 33 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 33 : xaalysasppheiletrp15 ( 2 ) information for seq id no : 34 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 34 : xaaaspasppheiletrp15 ( 2 ) information for seq id no : 35 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 35 : xaagluasppheiletrp15 ( 2 ) information for seq id no : 36 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 36 : xaapheasppheiletrp15 ( 2 ) information for seq id no : 37 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 37 : xaaargasppheiletrp15 ( 2 ) information for seq id no : 38 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 38 : xaaxaaaspilexaaxaa15 ( 2 ) information for seq id no : 39 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 39 : xaapheaspilexaatrp15 ( 2 ) information for seq id no : 40 :( i ) sequence characteristics :( a ) length : 5 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 40 : xaaaspilexaatrp15 ( 2 ) information for seq id no : 41 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 41 : xaaleupheilexaatrp15 ( 2 ) information for seq id no : 42 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 42 : xaaleugluilexaatrp15 ( 2 ) information for seq id no : 43 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 43 : xaaleuglnilexaatrp15 ( 2 ) information for seq id no : 44 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 44 : xaaleutyrilexaatrp15 ( 2 ) information for seq id no : 45 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 45 : xaaleuxaailexaatrp15 ( 2 ) information for seq id no : 46 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 46 : xaaleutrpilexaatrp15 ( 2 ) information for seq id no : 47 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 47 : xaaleuaspvalxaatrp15 ( 2 ) information for seq id no : 48 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 48 : xaaleuaspxaaxaatrp15 ( 2 ) information for seq id no : 49 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 49 : xaaleuaspxaaxaatrp15 ( 2 ) information for seq id no : 50 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 50 : xaaargaspxaaxaatrp15 ( 2 ) information for seq id no : 51 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 51 : xaalysaspxaaxaatrp15 ( 2 ) information for seq id no : 52 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 52 : xaaxaaaspxaaxaatrp15 ( 2 ) information for seq id no : 53 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 53 : xaaaspaspxaaxaatrp15 ( 2 ) information for seq id no : 54 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 54 : xaagluaspxaaxaatrp15 ( 2 ) information for seq id no : 55 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 55 : xaaleuasnilexaatrp15 ( 2 ) information for seq id no : 56 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 56 : xaaleuaspphexaatrp15 ( 2 ) information for seq id no : 57 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 57 : xaaxaaaspphexaatrp15 ( 2 ) information for seq id no : 58 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 58 : xaalysaspphexaatrp15 ( 2 ) information for seq id no : 59 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 59 : xaaaspaspphexaatrp15 ( 2 ) information for seq id no : 60 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 60 : xaagluaspphexaatrp15 ( 2 ) information for seq id no : 61 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 61 : xaapheaspphexaatrp15 ( 2 ) information for seq id no : 62 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 62 : xaaargaspphexaatrp15 ( 2 ) information for seq id no : 63 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 63 : xaaleuaspphexaatrp15 ( 2 ) information for seq id no : 64 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 64 : 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