DE1518288A1 - Process for the production of new polypeptides - Google Patents

Description

Dr. Expl. ^Jlr523

Terfahren max production of new Pt * peptides

The red-lying blight concerns the production of a new Kim ··· therapeutieofa w * rt-Toller polypeptides, eowle ton dwra g “solifltstftn and their salts. Ine "beeonder" ermWJfliolrt amm contemporary Verfahrea the Heretellun "bucket mum HUM tob polypeptides of the following ·" Straktart

aethioninaiiid (I) where Z a · der nmoheteheadea Qxupjem ! »» Eratoff, • in. ^ inoelnrr

and Ir-Alinim, or «la BsdiJnl« I * ·· ■ it Aotnahe »dee L-Aiptrtyl-l-Alaayl,» owl · geeobutBten Saliva * ·, w «lohe di · Aaiaocruppra» at ti · carboxyl group, which is not awe Hlm <d * r Piptltrerlcnüigung herejogesogen ein, in fMifseter fei ·· dura * · 1α ·

90983Ö / U72

BATH ORIGINAL

Sohwfcagruppe _s which can be removed by aoidolysis or ijydrolyee la known #eiae, have blooklert, e as well as their non-toxic, pharmaceutically acceptable acid alia. The components of the new class of polypeptides "have a high hypoteneiv" and vaaodllatatorieohe activity and are also used in the therapy of hypertension, fypiaohe meanings τοη Z and the following anlnoaacids and dipeptidesι H-Phe-H-iye-Ala

EhPTO- E-Gly-Ala-

fl-jlet B-Pyr-Ala-

H-Aap ( S & 2 ) - K-Aap (BB ₂ ) -iya- H-Ser = E-Iyr-Ser-

H-% r- H-Pyr-Ser-

H-Iry- a-iury-A * p (CE) -

Hiohtij dedto less can X auoh a-iciere amino acids and soft toü the obgenaccten aind aind,

jSe glbü many mögiXoxio üy in the he Ben sur Herateilung the new JECiaase from Polypepxidac di.e form the genus of the present invention ₀ tfana generally the juamoic acid or the poxypiytid η is brewed with another polyp ept id sur Umaetsung, whereby the lnino- ftfil dl · Carboocyl groups which are not involved in the formation of the i-eptidbinduaf

909830/1472. _{ßAD or} , g.nal

• Lad _f Ia feel «&# t« r tele · an Öruj> p «n done voduroh Md« r · feeebUtste PoLypej / tlde get miiwinrtgolf (lelon irk dts feeaeten amount dtr

Amino acid fol * »n. Zoa Bonu-fc «d« r frupp «fl kOttMS dl« To # yl- (p-Toluoleulfonjl),

fengrl- »trlflttor« ottyl- (2riipp · and AAder · »« le ei · übliohtrla Hr 7olyp # p ^ ideli «Bl · T« m «nd« tw «rd # n, iur an

Btlttts der Oaxboaqrlgrcippeii can use methyl, ithyl, t-butyl-, Beaeyl ·, y-liiro-'puiiqrl-erapp · !! and other dl ubliohtmtiee for diteea Zwtok elngoertst become, τβΓ- «• nd« t w «rd« n.

The condensation creeps the aoino group a · FolypeptldaolekÜls and the Carboxjlerappe are another - or amino acid molecules for the formation of the

can oh oh well follow the Ublion methods of the polyers, üeiepieireise through a suitable White activated acid derivative, such as the azide and the phosphoric acid derivative or by direct condensation between the free Aaioogruppe and the free carboxyl group in öegenWöirt a suitable condensingemlttela, such as a carbodiimide from the group dee [pizyiclohexyIoarbodiimids, l- ^ klohexyl-J-aorpholinyl-oarbodiimid, and others in known in literature. The condensation can be carried out in a suitable Lueungsaittel from the group of Ν, Ν-dialkyl-

909830 / U72

BATH

for «*« id «« * 4 tie * · ai * 4ri {#a aliphatic *! * «lit rile, before-SijMtr ^ ifornMde and aortic nitrile * Heektlon * «| fiaat Is #« r Ol Π a eaiaoben -6 wad

O ⁰ C uü «

tob> -70 iituodfro fe # «ae ^

au · tie »cfcoJ ^ t» t «nl« SE9> «ptid received« to JHietoJeraai ·, dler Seüut * -truypma in «c aioh fceJfanuter WtJ β» dee tutsprechönio tr «ie Foljr ^» »y <id or <5ao eniÄ> rooh «ßdt 3α1β der or ^ anitchea o ^ 1« r «öOjTßÄßlechen SUurtn j · canh dta ear Sntferniumf d * r Soba ^ fgruppea 7« r «« uöetoa Agtae. Tue *

b uu <3 kmrxn «la ·· d« r nccfcf ol ^ wafl ^ n ββί α ι in flüeei5 «3 AMieai · *« «ftteersioif Ια G-sonr ^ rt vou * ftlladlusfcatftlj »titorts,» a * o «arfrtt · H * ioe« tw »** Mretofi · Iq

«He ywp

i lti4 kaoa 4neh fe «l« «tteAf« ο «

"In

ο · γ B * o »a kind of v« reöe «rt« ii loacnhcrMa <ttr aiohaotid, Oeliforoi «USA ant« r Q% m AO 11A9 «riiAitefi be.

AUQα (Sie üelEklgung <S «r erfiüäaae« ge «ä tana naon bakaooten M «hod« n der warden, «la durob aegenatrünnrteilang and Gutoofttocrapbiβ on Alux.iQiuaoxyd or alneaionenauetauaoii

909830 / U72

BATH ORIGINAL

tar ·! »!« »!« Iwit— JeWrIIt · nc 90. eeUyiele # ei * e «iod • lsi «· HyoiMlieat fa * erfloittngtgeeiieea polypeptides arrived! TaeWaoedere "ground 41" oyntheeen of the following

(II)

(III) (IV) (T)

* tatfti * j »tl« (XI) kajui hexgtrttllt «« rden durofa «M« ti «&« la ·· # β · θ & α ** Ι · Α StriTct «d« Ieol «uoyl-glyoia-

• t German
(b «tt« fcri «b« fl la d ^^

«Lt wlMB g« i «küt * ttn derivative of jrhenylalanlna,

"-0" rbo - t-batoi3r-L-ph "a ^ lal * ßlii-p. -Nitro ptüoy 1- •" t "r UAd trglbt B4Mh der Terieiiuag dae geeohtttit" ^T ripeptid

«Eloh ··

by Koal «aiAtloA alt de · lUoe ^ tia L-Leuo ^ l-L-aebioflinaiiid

German /

(impressed in the · 4§ «μβ *« «« &. patent application d * e ent «prtoa« ad · geeoiiütite derivative of errt »peptide β (£ 1) results, au «» eloliee duroh JSllalnlerung dar eohüttendea druppea that free whole peptide is obtained. You Polyi> eptlö ·

aod (IT)
(III) / know are produced by condensation of the pentapeptlde (II) bit a * geaohütataa derivative of the phenylalanine, atapielawelae the J - Garbo-1-butoxy-

909830 / U72

BATH

anti »it niaetn

Aa ^ areglnyl -lyeio-dipefrtidö, beiealelmialae de »itOJty'-L-ae ^ arft ^ iQ ^ l-Ht-ctxbo-t-butoxy L-lyain u» d result 00 do «respectively * ge« iOhUtete l »olype * tld au a which by climatizing the ßonut "groups dl * ire leu Folypeptldio" men. Since a poly pt pt id (?) Can be produced by condensation dee Hexa ^ t ^ tlde H-AIa-Pfae-Ile-Gly-LoU-IBt-IH ₂ (toseltrieben and claimed in the patent * r. 629.243 of the ürftoderin) alt a derivative of the loreioa t, § -Dl-oerbo-t-butocry-L -

does de · protected polypeptide> u ergubea, eue «lelohea by allBinierun * the Orupyen d« »free l'oly ^ tide is obtained. ucblle011ch doe b can "xep" PTLD (TI) are hergeatellt by Kondeneatlon "5nea geeotiütsten Derivata the tripeptides Phenylftlanyllaoleuoylglyaln belopleloweie * L"phenylalanyl> L "iaoleuoylglyiin-ethyl" oterbromiijdr * t (beaonrieben in wäg ** · * Style Fatentaiuuoldun "WrE & rrS5 ) old a soaked derivative dea Serlae, for example N-Trityl-L-aerine, welohea oacb% drolyae dae geeohütate Tetraiiö ^ tld N-frityl-

Ir-iaoleuoyl ~ glycine yields which

durob Kondetittdtioa with the dipeytide L-leucyl-L-methioniamide in the · 4 ^ »· Λ = 4« »1. Patent application - * - "Λ—

dae

909830 / U72

BATH ORIGINAL

0a * eote ^ iweiMed «geeohfltite derivative dee Hexapeptlde (VI) results in μ »vtlofea * daroh Illvlnieruag d« r aobttt »end Groups de · free FMypeptld is obtained.

The present trfindaae can it fh # r * ple dtr yptrtool or for immediate treatment by Hy ^ ertoni eat, with v n • pftstiaolsau 8yadroa «ab« «oQd« ra in aunkular-outantn Ab- ■ otuiltt «B (Bur§ # r'ecb« disease, Balnaud ** ob · disease, Tr «i) O« «(leiti · oallttrana, hard · tfloara) "der Mttghautgaf" Mt (Imvon through 6paa "MJi der Betlnaientral" lrkanf), the ■ eolfealea e # fai3e _t (C "pnalta and Healorania duroh Vaaoapaaaen)

ler

uod / OoronargeflU "(Afg, naanfille).!> le connection of the present Äpfloduüf can on parent tr ale" way, nitalioh eubcutaneous, iateranttakulMr, intravenue (al or al · long · »·· Infoaion) or intraartarially verabreloht

You aa rent suitable loops bind iaeaer or physiological but non-alkaline sales solutions.

On »ubkutfcceii or intraauekulilreia away can auoh the connection i * ubatanüen old decelerator effect cugeaetst will. The proportion of active ingredient can vary depending on the naoh the particular pharaoh-eutical form and the customary

hypoteneiven

909830 / U72

BATH ORIGINAL

lqr * ottMlv * A tfiafct vary let jtfoeh Ib alli «MlDan

ML · AaMOdtaa; dtr WfIBdMaJNt-IKn polypeptide · Ttrareaont μ1μ * 1 · 1 iyaptoa «latr acute or

MoSUTolftaden BtliyltX »dltnto tor Ä« r ürUioic oifi · ei · Jodo ^ i üitrmaf tu to «fr« at # n V «rt · l« r tt ^ To — tofphl ^ oiua Analy ·· wit ei · ia n «brad« a «λ ·« · ι ^ · β · ΐ »· Α» in * «urd« n § «Ai d« r «« ι «iolMdk Mf lb» tM-Pn> l
η μΊιβοΊ ImI fit ι «ιμ ·· τ (4titi) · τ · 1 · ΐι. Pit! • rt · v «r4« a al · If ftufttfrtrttaltt. 01 · • ftad «r« BiSko «fflelaten« u «6« draokt in au · dtr b «k« Yurt «n egrakol ·» «in« eXslohftll · wlidff | «| it« B. BtI- · ρ1 · 1 · «· 1 ·· b« 4 «wfe« td · * Auedruok _ft , »

• lnM pH of 1 »9 <m unfaithful · Polypcptld eioh ■!% · 1α · τ βΜ0Μΐιιβ1« |; · ϋ τοη 0.909 faohM in bMu «« «f Ltooln al · Mault
B> lapi ·! 1

f ή

Una LOaong τοη 0.619 $ β dee

b "l" uoy 1 ~ L-Mtnion * anaaid onloraydret aaoh daa tigtnan b «lg. Patent 623,243) in 15 al

Dia »thylforaaaid

909830 / U72

ORIGINAL

Dimetnylioratmid and 0.14 al irietnylamine and 0.465 β ** »JI * -Dl-omrbo-t-batoxy ~ Ir-lyeia-p-nltropii * oyltet # r were opened during 1 wool / 1 * 1 ßauateapermtur. The Löeang was danaofc. Consolidated in Yakuua and diluted 150 al »» ■ • tr. De * aegeeo & iedene product was filtered, old deet, leaser fwwwiohea, am Tilter getrooiuMt and d «& aoh old Xthylttber gmmiQb * n * You got 0.58 g of crude product, which ·· in · 1α · Γ Hiaoluuig tojq methaaol ~ The "tbylforma" id e * ~ lört word "and then Sugab · τοα Ithylttther-Ithjlaottat was filed ·. Ϊ · eolileden Miuh 0.35 ^

Methloniaaald old a fp of 231-234 ⁰ O.:The product is shown in the earjatograpfeie on the plate and at the ÄleJrtropboreie ale honogen. Duroh Ujpdtrietallieleren aue MetoAnolwere 0.23 g of a product ait elnea Pp ▼ on Ü & 2W ^o fax ^ Uni \ jl l ^A - -27,? ° («« 1 in Dieetnyifor «« »ia) * 1, i? Ö ag dei geeohüt et en He ^ ta ^ eptid «treated ait äip. aqr solution of waeeerfreiea Chlorwaaeeretoffv in ÄlÄeaiiig (1 ^ 33'Jf) , resulted in 100 ing Ir ^ Lyeyl *> lr-alany 1-1-

dioWbrhydreit ': with a Tp of 24-4? 248 ⁰ C (under Zereetstuig) | y -29.6 ° (o «1 in 95 years of BSBlgäaure) j E«, _#g »0.78 Leu; - ■ '·' Γ example 2 ^;

909830 / U72

BATH ORIGINAL

Belapiel 2

-L-methionlnaa- id

Sine weight of 0.685 g Ii-Alaoyl-L-phenyl-alanfrl-L « isoleuoyl «gly» ^ 1-Ir-Irfmoy l-L-methioninaniid chlorohydrate, 0, H ml ! riethylamia and 0.30 if-carbo-t-butoxy-glyBin-p-nitrophenyl ester (produced according to Vol. Üaadria and B.A. Boleeonnaa: HeIv. Clilm. Aota ± 6, 1963 p. 1637) In 15 ml of dimethylformamide were during 1 fscthe at Haumteinheratürgelaaatn ·

Then the solution was concentrated in vacuo and old diluted 150 ffil water. Btr aioh abaoheidende Miedereohiagrfurde filtered off, cit deat. Water geviasoJaea »am Filter dried and then washed with Ä'thex. 0.70 g of the crude product with a P ^ of 260-268 ° 0 were obtained (Decomposition) 0.45 g of reinea if-Oarbo-t-Dulio ^ -glyByl-Ir-alanyl-L-phenylalanjrl-Ir-isoleuoyl-glyisyl-Ir-leuoyl-Ir-methionijiamide with an mp of 266-268 ° C (decomposition).

^ ⁵ --3O ° (0 »1 in dimethylformamide). 100 mg of the protected beptides, treated with 3.5 ml of a solution of anhydrous hydrogen chloride in glacial acetic acid (1.33 N), gave 95 mg of glycyl-I-alanyl-Lp.ienylalanyl-Ir-isoleucylglycyl-L-leucyl-L -methionlnaiiil6oQlorhydrat with an mp of

284-285 ⁰ C.

909830 / U72

BATH ORIGINAL

284-285 ⁰ C (Zereetzzu & g)! U] £! -44 ° (ο- 1 In 95 * igar Beeigeättra); 0.55

₁
Example 5

^ -L-alaavl-L-pheny IaJL any l ~ I «-iaolettOjl ~ glyoyl-l · -

~ 1euoy 1-L-methionlnamid

A solution of 0.68 g of L-Aienj1-L-phenylalany1-L-ieoleuoylglyayl ~ I-lanyl ~] ^ metmoninamide carbohydrate, 0.14 ml of triethylamine and 0.35 g of N-carbo-t-butoxy-L-pyroglutamate des p-Hitrophenyls in 15 ml of dimethylformamide were used for 3 days left to react at room temperature suspended in 150 ml water, since Produtt is filtered off, with least. #aeser used, dried on the plate and then bit Ethyl ether used.

0.63 g of the crude product β with a bottom point tob 270 ⁰ O (less than 2 · Γ · βteung) were obtained. Duron TJekrietallieieren from methanol obtained Q ₉ 41 g of pure I-Oarbo-tbutoxy-I ^ pyiOglutaeyl-L-alan / lL-phenjlalanyl-.L-isoleuojlglyayl-L-leuoyl-L-uethioninamide ait a ΐήτοη 277-278 ⁹ C (below zero)} Ca] | ⁰ «-28.8 ° (0« 0.5 in Dis * thjlforeaaid). 100 mg of the gosohatsten Heptapcptida treated alt 4 al a Lueung clay anhydrous Chlorwaeeeretoff in * ieeeeig (1.551), yielded ifO mg L-Pyroglutamyl-L-alanjl-L-phenylalanyl-L-ieoleuoyl-glyayl-L-leuoyl-L-Bethioninamid ait one 'ρ rom

263-265 ⁰ Q.

909830 / U72

BATH

263-265 ⁰ C (ent · iereetaunghta3 |? ² "-48 ° (ο - 1 in 95 * IGTR • eigtäure) * I ₁ Q!» 0 _f 46 Leu,
Belwlel 4

33 »7 β L-Ieoleuoyl-glyain-ttthylteterbroÄjire-t (produced geaäl der · 4 $ ώΜΑΙ« «1st patent anotldu & g and 43» 8 g I-OaLrbo-t-tjuloxy-L-ßhtnylal •• Itr (btrg «et« llt gtaftt Xd. Sandrin and HA Boleaovuu ItIv. Chia.Aot ·, ££, 1963 »S. 1637) were in 150 al I> lMth? lforaeeld gtlOet. Bar Luau & f were 15.8 al TriBthjlMdn UüA daaftoh 2-3 ^ ropfen ^ «Eiettur · eugeeetat, laoh 4 - ^^ | 1ι · β atehtn bold BftuBt« aptr «tur« tard «dl« Löeung la YftkuuB. At «itiör!« Nptra-lur not ^{concentrated above 45 0} O, dtr Bttoketand la 1000 al feee «r re-filtered, filtered, nlt d« it. fuiir gtiiaeoh · !!, aa filter dried and then old Ither gtnaeoben. Durob Uaücrietallieierea aue lUthanol-iLther became 44 gd · «I-Oarbo-t-butoxy-L-phenylalanyl - ^ itoleuoylglyiln-Ithyleeter · old a Tp τοη 160-162 ⁰ O. ⁰ UTOh further Uakrietalligation aue methanol-Ethylather received one "in" analysis sample which at 162.5 - 163 ⁰ O • ohoolii Oi ^ ¹ -57.2 ^C (0 «2 in methanol). 20 g of the IrI-peptldeetere were in a hollow τοη 45» 5 al more normal

3c

9098.30 / U72

■ Soda oil, 4.5 ml water and 50 ml ethanol glittst · laoh 80 min at tree temperature the resulting yellow

Solution with IOC ml Waener diluted felt by a ■ leiohten turbidity filtered off mi at ⁰ ° C with acetic acid. The dense precipitate was taken up bit Blewaeser, filtered, washed with SLewajfler and dried in a vacuum in the presence of phosphoric anhydride. Van obtained 17.40 g H-Carbo-t-butoiy-L-phenylalanyl-Ir-ieolettoylglyein with a Pp of 155-158 ⁰ C. By ümkrietalliaieren from methanol-ether to give an analytical sample · bit of a Sohmelspunkt of ie7-190 ° OrCa] ^ ⁰ ° -28.4 ° (o x 1 in ethanol). In a solution of 8.71 g of the N-Garbo-t-butoiy-L-phenjialanyl-L-isoleuoyl-glycine, 5 * 96 g of the L-Leuoyl-L-methionine amide chloride (prepared according to our own Italian Patantanaeldo 10.265 / 63) in 27 ml of dimethylformamide and 2.8 sheets

Triethylamine were stirred at O ⁰ C 4, ^ 5 g of H ,! ¹

■ ft
-Dizyklo-hoxyloarbodiimid Bugesetat ₉ Misohung was for 16 hours at O ⁰ C and danaah for 24 hours at room temperature, allowed to stand The gebildetβ'feste Maeie was suspended 500 al Wcisser _y filtered, washed with more water and dried in vacuo The crude product was dissolved in 150 ml of methyl formainide are cooked up and the undissolved

'■' - '■ Part,

909830 / U72

BATH ORIGINAL

Part, consisting of H _f N'-Dizyklohexy! Urea and I-Oarbot-'butoxy-L-phenylaXanyl-L-ieoleuayl-glyzyl-diayiaohexylur- • toff filtered off and discarded. & thylather ~ petrol & ther 4.78 g

Λ-

dee H-Carbo-t-butoxy-L-pfcenylelanyl-It-laoleuoyl-gljByl-L »· leuoyl-L-ethionineBid Bi-I a? p τοη 238-240 ⁰ O separated. With the recrystallization of dimethylforaamide-Kthyrather, an analysis sample was obtained with a Pp τοη 239-240 _t 5 ° 0) [α] Jp-21.5 ° (ο- 1 In DlmethxXfoTnanid).

2 g of dee geeohatiüen ^ entapeptide were during 20 »in

at 50 ⁰ C «it $ <) al a Löeuo« τοη waeee * £ retea Ohlomaeeeretoff in Biiceeifi, (1,331) twgeaetMt. Sae Lusungevtttel was evaporated under reduced pressure and the Rüoketand in

waeeer-free £ hbr suspended, filtered off ^ with waeeer-free hydrld

: '. '. Phoiphoric acid an /

Xther gewaeohea and la VeJcuüa in 'presence of Kaliuahydroxyd uad /

trooknet. 1.85 β was obtained

-Qhlorhydrs ^ e, welahee at · Brhit sen lmXabillarTohx «lon ^{begins to puff up at about 150 0} C» the Zeraetzuiig starts tei about 219 ⁰ C and.ee eohailat with charring at about 295 ⁰ O ¹ * ία ^ ,,. 2ο # 4 ^ ₀ ^ _{1 ln} ^ ₅ ^ i _gtr E _eB igeäure)) E _{1 Q} «Ö, 55 Leu.

At ept ti 5 .- »-'...

■ ethioninamide . ·

For a solution τοη 0.60 g L-ihenylalanyl-L-ieoleuoyl-

· '■. ·. ■' ■ glygyi - '. ■.

909830 / U72

in 16 ml ids * thy I-formamide 0.145 al triethylamine iugtt $ etst and aneohlieland 0.40 g of the N · -σaΓ ■ bo ··· i: · - ■ butc> 2y · -L-ρl ^ · Σl / l & laaia -i ,. _t t _t iίi · cpli · L ·> I ·· t · r «(made according to Bd, Sandria and S ₀ A ₀ Boiaaoa *« ,, £ 6, 1963, S »1637)» laoh 5 were at Baoateepeiraia * ward * dt · Lusung vacuum "at a temperature sees over 45 ⁰ O" ttf a gerilagea volume konaentritrt and danaoh μΙΊ 10 «1 Hetnaaol ▼ erdUnnt. ^ Uroa addition ron» afeer eohied eioh da * product from was danaoh filtered with DTET Faeeer Gewä * above, on Tilter giipgoknet and then grUndlloh old Ither durahcewaeohen.Mui received 0.48 g of dea H- ^ arbo-t-butoxy-L-phenylalanyl - ^ - Ptenjlalan L-i8ol * uoyl-gljayl-L-leuoyl -LB "ethioninaaide bit a" fp of 235-2 3β ° ο (Zereetiung). Duroh UekriataXlielei ^ a aoa MethMOl been 0.35 g dea peptides old a "fp ton 236-240 ° 0 (ttat" r S * - aeteong) get [α3 ^ 5 «-jo» («- 1 in SiaeUyUorMalA) ·

215 ag dee, geeohOtate »Basaptytli« · η · Λ · η Wl ■ it 6 as a solution τοη w «aaerfreleei OhlMemeiTitoff la eealff (1,331), 209 a «dea

i eoleuoy 1-f Iy «y 1-L ~ leaojl-» L- <ethAent naeri iohl orhjdrmf alt

fp from 2β7-289 ° 0 'llfreeteenf) ^ ·]! ³ - 1Tt5 ° (β · 1 4 »« 5 * «· τ Ifflgalure)} I ₁ ^ -0.47 Leu.

Example <

2a of a solution τοη 5 «5 I-Oarbo-t-butoaqr-L-proline

(herteeteUt

909830 / U72

-7.AD ORIGINAL

genii Sucre β V Other and Ami O, Mo. # · Α «· Οα # β * 8οο · Yes» ¹ 937 »8. 61fi0) and 4.2 g

in 90 nl Jtttyl & attat, «« Iahe on O ⁰ O gt-

kft & it "orden IMkTi was Btthru & g tia" soldering τοη 5 '2 g V ₉ 9 * -Dis7kio2i "i ^ lOarbodilBi4 Ii 10 BTI Ktheraaetet eye" • "did · Dft" l% & re & is wfthr "Ttae 90 BIB · toti O ⁰ O tmd d «B * oli

^ HiI 2 Sttm &# ii h% i ^ AUftteapesmtiar forteeaetst · Halt Mixture earn w & hs- ^ tti 1 Stuödt * ai O ⁰ O ₉ kuanen dl · to

4 ₉ 9β i ίΐββ! »M ^ SivylüLolitxylharBttofft ab-9a« Tiltr & t will bit tintr «tettttlfttii Um mn> Ifüiflrbußi tu ^ l iiünwh« lt tlatr

füi2 ff »atrality ftvotohtn, Saoh trook & ta (Sbti? Waiettrfrelt »ffatriu» eulfat is dft · ^ Daueetiiittti '^ i T * x- and «rtea Soruofc abfedaütpn. One hates 8 "7 i dtt" "^^ o-? * Tnitosjl" If-pr ^ lia "p.altropb" ijrl "tttre alt gelbta oil "Su" ia'T I £ tu & e foa 0.55 § "-O * rb" -t-botcaty "

Ä «ad 0.80 g dta l« lbtiyl.alafWl ~

la 10 el DI * ttbirlf """Bia" trdtii 0.19 al fritttlyrlaads uad tin dripping ta Jtealeaänrt sugeettst · dit Luatiag is "Thrond 6 Fagta hot fiauartstaptratur atthtagtlaatta, daaaoh daa itOta & etaltttl Ib vacuum bti tiatr ftaperatar Blobt About 50 ⁰ O afefoHaapft and the BUokataad in the methanol gelOat »DtU ¹ Oh SugaDt ton ffaeetr aoatldet eioti dae product

909830/1472

^BATH

from t It is filtered _f with de st. Waaaar gawaao & an, a «Tilter getrooknet and then« it lth «r gtttcobtn 0.47 g receives the H-

«It« in ·) « f $

from 232-235 ⁰ O weloh «e« loh at Ohroaatograpfei · Mf 4tr Platt · as a hoooger anraiit. SuroB. Uakrtatalllalcrtn 111 methanol Xthyljätn · * werdft 0.40 g 4 ·· ·· product "it a Pp τοπ 242-243 ⁰ C get! CeO ^ ⁵ --43 ° (β · 1 Il

Dimethylformamide) * X _{1 Q} = »0.09

11 $ mg treated alt 4 al ilwr LOavag tob • ot öhlornateeijetjpff in Blaaaalf _v Ci _f 3313 »·»! · & · »109 Sf

methioniimeid- <jh ^ orhydr * t · «it« in · «Ip Tea | 6O-H4 ° 0 (under Se * e« taWit) »t«] 2 ⁴ -47.9 ° (ο - 1 ta 99 ** «· * '

3 «lepi ·! 7th

Iu one on · 0 * 0 «» Whiten Mlfohaae ton 1Q ₉ CO f

t-butoiy ^ L-tte-bhionini ^ teetellt § «aal jjtooVf · W. Anderson and Aani 0. Md.Ore (place J. Aaph # aS« 4 · T9 ₉ 1997 · p. 6180) "η * 7.10 g p.Iitrophenol in 100 al were 8.77 B II

The B ** Jttione «leohttni warte vtfhr · * · 3 / i. "1"

'■■ ·

909830 / U72

BATH

diftaob w «iragi <2 2 hours at Iauataaparater etenaagalaeaea · loch AIkKaItKiS« a? d «fi 8 β K _v I * -4ttqrttotmjrlfearaatof * at»

2ae hiiitil becomes al * «liter & δ · ιαβ -of 5 jtleaa Batriva * kartooa * H * since SrtfSfftaie ob ^ fi »aaeto si-ft ilair Ißfetrlmaeiilerl * Me Aar

froeloMa Ot «? like "r2r" 19B Ätrloseulfat "irA te «Rfongeettttl% ·! wraiBteiitem Äruok a% fcdM9lt nad ^^ krietftUieisrt ate * Sta *** ·

· Ο 10.68 β Am

royat & jrlttter alt eiaea ffp tob 97-9area Hatqrletallltloreii becomes »In · Anmlyeenprob ·

| ¹ * 505 °

data Jfe tel 101-102 ⁰ O trb & lteni W | ¹ * -50 ₉ 5 ° (o - 1 la

₁ ^ ₉ IS Lou.
Ia * imr iOmm ^ by O ₉ 58 β ^ PhtiqrlalÄnjrl

oj3t-La «thioninaaidalilortiy <lrat In 10 al foraaald i» rd · »O ₉ 14 al Trl * ibyl« ain and 0.375 g! «Garbo» t-Twitosy «L-fcffch1 oaln-p, nltrophtnylaatar augaaatat» lach 5 Tagan bal Baaataaparatup is the solution la vacuum bal alB # r niaat !! bar 50 ⁰ O lying tea apparatus komaatrlart

Daa eo at «« aahladaa · Product is filtered, «lt datt · Waeaar faoaaehao, aa filter gatrooknat and danaoh alt ItHar gawaMhea. 0.40 g of I-Oarbo-t-butoxy-L-

attaloalaaald alt alaaa Ip τοη 255 ⁰ O (Saraatiung) Daa

9C9830 / U72 product

BATH ORIGINAL

Jfeoetekt enteist see at 02uroB & togr * pliie wf # «· fin« «boaeetti tmd result * through Vekritttllieltrti from 0, * 4 $ I tiae · ProdulrteB _v with a ··» ρ iron 265-2ST ¹¹ C MtPBBg) I £ «3 J ⁴ »· 51 ° <ο« 1 Ib ifeMttylforafJiiih

140 eg d ·· ge «GhttiBteii EexApeptld ·, treated ad ^ § nl tti & «F 3iösun« Ton wasterfr ·! «· Ohlorwaeivrctoff In Bj, ta ig, C1,3? B] results in "130 ag L-MethioByl-L-ptwnylalanyl-I" "

gljzyl ^ Ii ^ l ^ uoyl ^ L ^ MtliloeliifMldelilorlijrdrmt «It 9p tron 315-316 ⁰ C (outer femtsuiif) | £ »3 J ⁰ · ν-20» 1 * (ο- 1 la 95 Jtiger BetigeEex «) ^ ₁ ^ 0.44 Example 6

Sis «Misohung το» 163 mg
p.nitropfaenjleater (hergeatellt gea & S 14 · 3 * eixla WtA H.λ. Boieaonaaat I · It.Chim, lot »££, 1963, p. 165?) '« Si 273 eg Ton Ir-Ptienjlalaixyl-Ir-ieoleoayl ^ gljrtyl -L-leaejrl-Ip- • ethionina «L4 In 5 al OiMthylforaMld · β ft« · part at home temperature was atehengelaeetn.

The LöiuDgeelttel was la TvJraak at timer vijkt

50 ° C der BOekrlaaA im

Waattr euependiert, filtered, with dtrt · ** m — r gtwi »

Tilter getrookntt and daaaoh thr — l »ait ltbtr trtiitlt 248 a «dts

909830/1472

^ AD ORIGINAL

phenyXaXaXanyX ^ L-ieoXeuoyX-eXyeyl-I-XeuoyX-L-eethioninaaid with a Fp τοη 227-230 ° (Zereetsung)

The product erwie · aioh at Ghromf.tographlt On the PXatt · homogeneous and showed naoh Uekrirtallieieren jmm MtthaaoX ,,, 230 mg eineβ Products "Hit a gieionen SohneXipunkt. Ca] ^ ⁸ -29.5 ° (o - 1 in rieethylforsamid).

100 ag dee geeonUtaten Jieptapeptide benandeXt old 3 Bl of a solution τοη waaetrfreiea GnXorwaeeeretoff Ib 11 ·· » eaaig, (1,331), gave 87 ag tone L-AeparaginyX-L-phenyXeXanyX-

L-ieoleuoyl-glyiyl ^ L-leuoyl-I-eethioninamidonXorhydrat eit

an Fp τοη 240-245 ⁰ O (2ereets «ne) f Ca] J ⁷ .47.3 ° (· - 1 in 95 Jfiger Beeigeiur ·)! B ₁ ^ ₉ - 0.53 feaf H _f - 0.62

Example 9 n't

L-Agparfttf ^ ny H - ^ -! IiY IY? I ^ Ti- fffaf DTlil ftfly X — L * ^ boX <ttoy 1— * X y oy X—

Lüeung tob 4.06 g J! -OarDo-t-bttto "yL ^ ep * r" finp.nitrophenyieittr umd 2.99 g I * -Oareo-t-btttoxy-L-Xyein-ph # nyX-eeter (hergeeteXXt geaftf B. 8öh «TMr« b4 W. Bittelt ΗβΧτ · Ohim · ▲ ot «,, 1961, 8th 159) in 100 al ini ^ triWl« ■ tetÜd is »for 4 days with mood operator Mr Vmeetnng · braohtt JIe biXdet fled a krietoiXUer fiedereomXag · M «I ^ Jettngeaittel is fixed at Temindei'te · Draok and the BUoketand alt ithyXaeetat aofrraoween« b4. «AB tr» hilt «ο 3 * 94 g I-Oarbo-t-btitoiy-L-aepe ^ ginyX-J ^ Oar ^ ot-btttoijr-L-XyeiniwtÄyXteter old an« Ip tob 1β4-1ββ ^# 0 · [«] J ⁴ ^ -IJ * (t-1 MethanoX),

3.0% g de JDlpeptldeetere «orden la 100 al

909830 / U72

ßAD ORIGINAL

Methanol dissolved. To this on rauateaperatur abfekOkltea

The solution was 7.5 ml of a normal latrluakarboaetlOeuog and 50 oil Waeeer eugesttst "* aoa 1 stand" becomes dM

Methanol evaporated at reduced DruoJfc aad dl Ttr-

permanent Löoung with Smlewaacrer rerlcttant and 2 eel old

Ethyl acetate used. The wftSrlge Löeunf is responsible for this O ⁰ O old Chlorwaeeeretoffeiore Mi ta pH 1 anfeetaert ad

dae Product 4 nal old a "eeättigten I & Eung too latriia- etc.

Chloride extracted, for water-free «Batrluawolfat β ·» trooknet and b ^ l Termlnd «rt« a Druok drained. Otr old

Xtfcylaeetat-Ivtrölätlter aufeaaeM BOekataad is

filtered and gives 2.55 I ¥ -Qerb * -t-bate * jr-X-ljrtla alt elnea Ip ron 154-1? 5 ^{# (} 3

Dttroh Üakrletallielerem aM Mtaaael IHm> liii • ine Analjreenpro »· alt elaeei V» iw 19T-19efO Mka & wami «+ 4.85 ° (p * 1 la AttMAOl) ·

Ba one on O ⁰ O «tkaaltea Utaaag rm 995 m

ohlornydrat, produces «eat! Bele »lel 2» O ₉ 14 al ^ rllthjrlaain nad 465 Bf I ^ aveo-t-VftMar-L-Mfanfflafl-! • -carbo-t-btttojqr-L-ljfeln la 5 al J »iMiajlferMaU« erdta 207 ag Mtl ' -DiarkloAWcyloarbodlleid —eeetet, 9l · ReeJrtlonealeoBuai will wfihreod 7 StaaivB Wl O ⁰ O Md

909830 / U72

_BATH

then kept at construction temperature for 50 hours. The solid tfasee formed is suspended Ια 50 al of water and the product is then washed with ether on the filter. Vaoh Trooknen in 7akuum wei daa product Ib 25 B1 warm "dimethylformamide suspended" The suepene ion is ^{cooled to 0 0} O and kept at the temperature for several hours, then the 1, N ^r -Dieyklohexylharnetoff

with
filtered by ethyl acetate, and the filtrate

ι »

WaBOhfltttisigkditen are under twisted pressure on tin

small volume concentrates. The product is then precipitated, filtered off, dried and aue methanol recrystallized. Im gets 5 ^ 0 «f Ä-Carbo-tbut o3ty-L-aepÄraginyl-H ^c -oarbo-t-btttoxy-L-ljr ej lL-piieajlalaoy 1-L-ieoleuoyijlyayl-L-lettoyl-LHetthioninaeid bit a * p iron 244-245 ⁰ O.

Duroh Uak ^ etaXllelerva aoa athtanol «reveals an analytical test of an ip ton 240-251 ⁰ O (less than 2% test); [α] $ & 53 ° (ο - 1 in DlartB / lforMaid)

120 al dee geeohUtsitea heptapeptide treated old Q al a solution τοη "" eeerfreiea Ohlom "4Mrrloff in Sieeeeig, (1, 3H), result in 100 Bg of L-aeparafinyl-L-lyeyl-Ir-phenylalanjl-L-iaoltuoyl-flyegrl -L-leaoyl-L-Be'thionlnamid-diohlorhjdrat · alt a «fp τοη 295 ° (under iereeteung)! ^] ^. ^^ ( ₀ - 0.5 in 95 1 && · τ

e acid) i

909830 / U72

acid); E _{1 o} ~ 0.f; 3
Bgia.pl el, 10

aeihlpninami.i

To einsr eüf -6 ⁰ C cooled LÖeuag of 2.34 g HL-serine (prepared ^ eiaäß RA Boiieonnae, St. utoann ^G, P ₀ A ₀ Jacquenoud and JP, Waller: Ee It _£ Chi a, Aata ^ SLt ¹⁹⁵⁶ , a, 1421) and 3 g of L-phonylalanyl-li-ieoleuoyl-glyein-ethylea-fcarbroniäydrat, (according to the "* l + wi .."

■ QI022 / 6a ' ' -

Pai: ent: «. AuielduEg t0w« W5 »ö) in a mixture of 15 al

and, 15 ml Aoetc, aiiril were 0 ₍ 94 «1 ixnd 1.39 g NiH'-Biiyklohexyloarbodiimld« ugofUgto - ^ The action was taken from -6 ⁰ C for 2-3 levels and then during one Haoht to ^{0 0} C and Eohlleßllah for 24 hours the room temperature. The separated. S, N'-JH «yklofa« xjluratoff is filtered off and washed with ethyl acetate Tohaunige Huoketand was crystallized from a mixture of ethyl ether-xthyian acetate. This gave 2.33 g of H-trityl-L-eeryl-Lr-phenylalanyl-glyein-ethers old one _;

Mp of 204 ~ 208 ° ΰ {it was found in paper ear photography ale hoaogen. Duroh üafcrietallieieren aat ethyl

ethT> 909830 / U72

SAD ORiGiNAL

äthor ~ Ä.thylazei: at mode an Äsmlysenprobe with a

Pp at 207-270 ° C erb * Ilen · [α j £ ² "-46 _f J ° (o" 2 in methanol).

2.10 g of letropep-iideeters were suspended in a Miaotumg Yum 4 ml of a normal solution of potassium hydroxide in ethanol, 22 ml of oil and 1/2 ml of water. Ifaoh 60 min be :. Tree temperature, the resulting luaung was pre-thinned with 75 til Wa.esor, cooled down to ^{0 0} O and with jäaelgeuze a ^ igeeä'.ierto Sas old £ ea <sliiedene product, wclohθs aoh ^ he also filtered iei, is 4 eel with Ethylazetai : extrui: i rt and the JSxtralcte 2 »al bit more saturated

't

Natriuaobloilciiöeung and about waeaerfree " Sodium sulphate getrooknes'.

The L5e {mgami - !; tel is evaporated when the pressure is reduced and the RUoksstmd ii. ^ 'ajcuua la 0 * g * tmari tob Kailumhydroxyd uric Phceolioreiiureaiihydrid dried. Mail receives 1.9W .; robee n - ^. rl ^ jl-I ^ eeryl-Ii-phen ^^ onyl-I ^ iaoleuoyl-glyoin with an Ip of 212-216 ⁰ C. In finer ^{solution cooled to -6 0} C τοη 1.90 g I -irityl-L-eeryl-L-phenylalanyl "L.-iaolauoyl-glyöin and 0.85 f L-leueyl-L-methioninamide-ohiorlaydri ^ in 10 al ^ imtthylfonutmld are 0.397 nl triethyl" and 0 _f 59 g "" I ' - ^ ojolohexyl-oar'bodiimid eugeeetk ^ You reaction misolni & e will warm up

909830 / U72

S-feuntien "at -6 ⁰ O, about Νε-oht" at ^{0 0} O and aoüli »8iiciii kept for 2 toges at lauartempex-atur. The fled abaoheidenco SiJ ¹ -Dio ^ oloheoylhametoff is filtered off and first washed with a small amount of dimethylformamide ß'l ösnaoh with ethyl acetate. The filtrate and the combined Wasohn oils are dipped in under renainderfem aur Tioclcena. The huoketand is in Buepeüäiert, esa pllöer getroaloiet, again suspended in ethyl acetate and filtered off. 1 _f 30 g de a H-Trltyl ~ L-äfc; ryl ~ Ii-pheny lalany 1-L-iioleuoyl-gljroyl-Ir-

i - _Λ

leuoyl-L-methibninamlde with a W · tou 206-209 C »Da«

f.

Product turns out to be oil oh at the

Λ- I '

in the case of üromatoiraphie on the plate and% ·! Slektropaort ··

.V- '''ί · ^{: ί} «- ■■ ■' ■■■ ·. ■ -. ■■■ ■ · ·. ·

at pH 46 all homogeneous. Puroh tftpkriitalliile «tt tA · · ', r

Methanol ^ rden Q ₁ 97 g

227-230 ° ®Äersetaung) erüaiLtenf ^ el ^ —ie, ^. '. "(« .. V-Λ in

a ίββα ^. ^ τοη 0.75 ^ * ±% ν% * τ \ -Ίι- '''':.

methionine amide in 6 ml - ^ eelgaäure be etfgesetst 6 al distilled "water and the Usohung währeod Hinuten 40 to 100 ⁰ C. and danoo ndt 6 ml, d" etlllier1 »p faaeer diluted. By cooling to design temperature, a gelatinous Hezapeptld loosens next to the clock of the reaction

909830/1472

BATH ORIGINAL

formed crystalline Trlpfc.ei>ylaa:> * Mnol ab. Dl · 1 · -teere connection ν χ -d ivoash W .β up with Itfeer and decanting teilweie "tntfarnt.und dana & i c""r gelatiöee Bttoketand atoraali la üaüigellura geldit and dl · Ißeung under reduced Bi -Lot e ngedanpft · fixed the" Rliaketand becomes la Ethes. «. fiMp # m * iert ₅ abfiXtriert »nehrtnala with ether gewaaohön u * ti inJfbh Im Vaicuu» in öegdawart clay potassium hydroxide and Phoephorailureanh./drid dried. 0.53 β is obtained

ö,

. ■

with a 53 ?. 218-22O ⁰ C (Zereetaang)!

In U

1 1
Be «rtr ^ j-.efcii Lüeung Toa 0 _r 43H g lir« phergrl »lan / lI» -

0.097b! ftlllt ^ liiniii imd 0.112 g

153-154 ⁰ C,

(e * 0.3 la Mattüjafonaanid). J) i # VerMndaag will la The same t «ie # produced as with the 4-part Sjrntfc ··· tone I <-o «rhe« t ^ iit 0xy «lr-« eparagln * p. altro ^ enyleeter aagtveadert t Bd.Bandriß ua4 E. Boieaouaae, HelT.Ohlm.Aotß £ ^, 1637 (1963)]

In 5 flashes · »DiMthylform« thread 3 <· **** • at 3O ⁰ O etehen gelaeni danaeh wlnl die BeeJrtlon »-

909830/1472

^ß AD OFtlGt _NAL

niaoaong «it Waeeei» TordUnnt and 4a «« bfiltrierfc, nit Wan β er gewaaoae & _f «κ filter and then aehraala with lther gewaeaheiu I · wti * $ es 0.35 dta raw Hezapeetlde received, welofe · gel« et and duroh addition « tob Waeeer receives 0.25 g of H-

a? P * 233-235 ° C (below 3P Zereetitmf) ι [β] | ¹ · -19ι5 ° (ο = 1 in Dimethylformaaid) | X _{1 a} * 0 ₉ 12 Leu ·

0.1 θ g of the Η · χ * ρ · ρΊ1ύβ, contains4 * according to In Ublioher Weis · with $ al finer L0 «U9f τοη trookene» CJnlerwaeaerstoff in Eieeeaig <1.33 I) «give 0.12 g

Chlorohydrate ait tine «fP-248-250 ° 0 (« ter

^ ¹ -31.7 ° (9 W 1 la 99 jllf # r Ut *** »**). M ^ fQ, SO Sem

«FcX» Heethi otrt if ld

Ια-, a | ιΟ · «β§ of 1 $ 2 eg I ieiTig 1 .Wetlf-P

nltroplienjUeter (ierfeetellii enalM 4 «r SynKheae dee

chloAy4r »1 and Ο, Οβ« 1 TzUHlqrlaxin in

dettiilierkea i> i—

909830 / U72

BATH ORIGINAL

2 * -

acid auge3ai; at and daffoh the BeaJctlonanleohung during 5 days at tree tea time.

The resulting gelatine iteese "lrd In 50 tablespoons of water euspenäiart, filtered *, with deatilllertea Waeeer g * ~ waeoaen, put on the filter and then poured. old & * ^& ^^! * ^ * ^ο * γΐ * Λ · Ee «earth 350 mg N-oarbo-t-l5u-feoxy-2) -aaparaginyl-L-« laiiyl "-L · · / I-ißOleuoyl- glycyl-Ii-leaoyl-L-Mthioninwaid old tine «IT» 240-250 ⁰ O (2 # ratattng), which · nneh their aue methanol 25 ') ssg de · Heptaptptlds old a «· FIt 252-257 ⁰ C ( under Zerae-üBWigh 60 ag old a « ⁰

(under ^ ereatBurig) results in welohef «loh in the ohromatographle \ ΒΓ »1) and la der Bltktropoor ·« · at pH 1.9 (initial value) al Aoaogen ervele-fe "

The aue Dlaethylioraftald -Ither uakrletellielerte Analyeenprot · eohmllit he 262-265 ° 0 («ater Ser« etiac) i ,,

Ca] | ⁷ "-I7 | 1 ° (o - 1 in:> ia * toyUor""Bid).

100 mg de · geeohUt.atea heptnpeptide ·, treated in the usual feie · iiit 3 nl eiaev XSaang of. dry ·· Ghlorwaeeeretcff la Bieerjeig (1.35 I) yielded 80 ag te · products · which at 252-254 ⁰ O (under terirtsimg) «ohaeln I ⁹ -47.9 ° (ο« 1 la S ¹ S Jtlger BeeigeJlare) !!, _a --0.5C Ι · ε · Dl · la of the present Irf charge In the examples a

1-11

909830 / U72

BATH ORIGINAL

V ΊΟ teßc ^ irietianon uac marked as 3alß · Polypeptides generally germinate the corresponding free polypeptides by a signed · β baoieoh · «Af« η · vbA jJcM-U "b3rleiten About lorenauaiftueoheriiara ·,» .B. that new type of 'delayed' ion haria '1 · «le men Bin-Had laborauo ^ iee Kiohmond - Oaliforaia, Ü8A itttcr the base housing AC- τ 1 A 9 delivered «ground« transferred

909830 / U72

Claims (1)

J, Ar 523 30 a> θ H a α ι p F ι ο k ι Ancestors are »making a new QUH Polypeptides of the following · »8t * aktur · I-L-Paenj-lalanyl-I ^ ieoleuojl-fljoyl-L-leuoyl -Ii-methloninamla, where Z fateea-etoff "de" l # et an AalnoeHrre, aii AuwÄh * "from b-hjwi * " ad 3 # Ol "ai & · Am 1 · τΙ Dipeptide, Bit iaenaluM foa fr ^ parW-MUagrl eonie from the ceeohtttstea © ErlTatem, whose ta der The PeptldbiAdon "not tellaebaeodeii Aalno-Oarbo ^ l groups old geelgnetta ecbutifruppen blocks one that can easily through Aoidolyee vad ^ rdrogMaeSyaA i & * ·· known" elee split off "earth, oad ron their acidic" noxioble poisonous and pharmaceutical Saisen, dadnroh known »« is worthwhile, since dl · polypeptides der Above structure similar to that on the poljpeptideÄeeie knew method ufl produced 909830 / U72 BAD ORIG »NAL