DK150618B - ANALOGUE PROCEDURE FOR PREPARING SOMATOSTATIN ANALOGUE OR PHARMACEUTICAL ACCEPTABLE SALTS THEREOF - Google Patents

Description

150518150518

Den foreliggende opfindelse angår en analogifremgangsmåde til . fremstilling af hidtil ukendte somatostatinanaloge eller farmaceutisk acceptable salte deraf.The present invention relates to an analogous method for. preparation of novel somatostatin analogs or pharmaceutically acceptable salts thereof.

Det er blevet påvist, jf. Brazeau et al., Science, 179, 77 (1973), at den cycliske somatotropin-frigørelsesinhiberingsfaktor (SRIF), kendt som somatostatin, har nedenstående strukturIt has been demonstrated, cf. Brazeau et al., Science, 179, 77 (1973), that the cyclic somatotropin release inhibition factor (SRIF), known as somatostatin, has the following structure

H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH S--—SH-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH S - - S

(A) hvor alle aminosyrerne har "naturlig" eller L-konfiguration.· 2 150518(A) wherein all amino acids have "natural" or L-configuration. · 2 150518

Adskillige metoder til fremstilling af somatostatin er beskrevet i litteraturen indbefattende faststoffasemetoden ifølge Rivier, J. Am. Chem. Soc., 96_, 2986 (1974),, og opløsningsmetoderne ifølge Sarantakis et al., Biochemical Biophysical Research Communications, _54, 234, (1973) og Immer et al-, Helv. Chim. Acta., Sl_, 730 (1974), og der forskes meget inden for peptidområdet med henblik på at forbedre soma-tostatins farmakologiske virkning ved syntetisk modifikation af dets struktur.Several methods for preparing somatostatin are described in the literature including the solid phase method of Rivier, J. Am. Chem. Soc., 96, 2986 (1974), and the dissolution methods of Sarantakis et al., Biochemical Biophysical Research Communications, 544, 234 (1973) and Immer et al., Helv. Chim. Acta., SL, 730 (1974), and much research is conducted in the peptide field to improve the pharmacological action of soma-tostatin by synthetic modification of its structure.

Den reducerede form (åben kædeform) af somatostatin (RS) er det lineære tetradecapeptid med formlenThe reduced (open chain) form of somatostatin (RS) is the linear tetradecapeptide of the formula

SH SHSH SH

I {In {

H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OHH-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH

(B)(B)

Den reducerede form (B) er blevet fremstillet ved totalsyntese, jf. Rivier et al., C.R. Acad. Sci. Ser. p. Sci. Natur (Paris), 276, 2737 (1973) og Sarantakis and McKinley, Biochem. and Biophys. Res. Communications, 54, 234 (1973), og denne form (B) kan omdannes til somatostatin (A) ved oxidation, hvorved der dannes en tværbinding imellem de to sulfhydrylgrupper i de to cysteinylaminosyrerester i tetradecapeptidet.The reduced form (B) has been prepared by total synthesis, cf. Rivier et al., C.R. Acad. Sci. Ser. p. Sci. Nature (Paris), 276, 2737 (1973) and Sarantakis and McKinley, Biochem. and Biophys. Res. Communications, 54, 234 (1973), and this form (B) can be converted to somatostatin (A) by oxidation, thereby forming a cross-link between the two sulfhydryl groups in the two cysteinylamino acid residues of the tetradecapeptide.

Mange polypeptider, som kan anses for at være strukturelle modifikationer af somatostatin, kan fremstilles syntetisk og er beskrevet i den kemiske litteratur. Sådanne polypeptider har visse strukturelle træk til fælles med somatostatin og adskiller sig fra somatostatin ved, at specifikke aminosyrerester eller funktionelle grupper, som oprindeligt var til stede i somatostatinmolekylet, enten mangler eller er erstattet af andre aminosyrerester eller funktionelle grupper. .Many polypeptides, which may be considered to be structural modifications of somatostatin, can be synthetically prepared and described in the chemical literature. Such polypeptides have certain structural features in common with somatostatin and differ from somatostatin in that specific amino acid residues or functional groups originally present in the somatostatin molecule are either missing or replaced by other amino acid residues or functional groups. .

Således angår dansk patentansøgning nr. 3515/76 såvel som en tilsvarende artikel i Biochemical and Biophysical Res. Commun. bind 65,Thus, Danish Patent Application No. 3515/76, as well as a corresponding article in Biochemical and Biophysical Res. Commun. Volume 65,

OISLAND

nr. 2 (1975), side 746-751, D-Trp -Somatostatin og dens virkningsforøgelse i forhold til somatostatin under forskellige afprøvningsbetingelser, navnlig i henseende til modstandsdygtighed mod enzymatisk nedbrydning.No. 2 (1975), pages 746-751, D-Trp -Somatostatin and its effect increase over somatostatin under various assay conditions, in particular with respect to resistance to enzymatic degradation.

Dansk patentansøgning nr. 885/76 angår en bred mangfoldighed af N-terminerede og C-terminerede modifikationer af somatostatin og giver ikke udtryk for videre kritiske forhold, for såvidt angår strukturen i disse endestillinger.Danish Patent Application No. 885/76 relates to a wide variety of N-terminated and C-terminated modifications of somatostatin and does not indicate further critical conditions as far as the structure of these end positions is concerned.

3 1506183 150618

Det har dog efterhånden vist sig, at mange polypeptider, som er endog særdeles nærbeslægtede med somatostatin i strukturel henseende, kan være fuldstændig blottet for somatostatin--agtig aktivitet, se således Rivier et al., Peptides 1976, hvor dette punkt specielt illustreres i tabel III på side 439--440, hvoraf det klart fremgår, at selv meget små ændringer i struktur hyppigt resulterer i udtalte forskelle med hensyn til 5 aktivitet. Dette gælder f.eks. for en forbindelse ([Asp ]-SS), hvori den hydrofobe amidgruppe Asn i somatostatin-stilling er ændret til den hydrofile frie carboxylsyre Asp. Det ses, at denne ændring resulterer i et næsten fuldstændigt tab af aktivitet.However, it has gradually been found that many polypeptides, which are even very closely related to somatostatin in structural terms, may be completely devoid of somatostatin-like activity, see thus Rivier et al., Peptides 1976, where this point is specifically illustrated in Table III on pages 439--440, which clearly states that even very small changes in structure frequently result in pronounced differences in activity. This applies for example. for a compound ([Asp] -SS) wherein the hydrophobic amide group Asn in somatostatin position is changed to the hydrophilic free carboxylic acid Asp. It is seen that this change results in an almost complete loss of activity.

Dansk patentansøgning nr. 1404/75 beskriver eventuelle modifikationer af somatostatin med ornithin i 4- og/eller 9-stillingen og med alanin i 5-stillingen. Ud fra det, som er anført i beskrivelsen til sidstnævnte danske patentansøgning, må det konkluderes, at en hvilken som helst ændring i aktivitet set i forhold til somatostatin må hidrøre fra modifikationen ved molekylets N-termi-nåle stilling, fordi aminosyrerne i 4-, 5- og 9-stillingerne kan være de samme som i somatostatin. I hvert fald anføres det på side 4 i det tilsvarende tyske patentskrift, at peptiderne har nogenlunde samme aktivitetsspektrum som naturligt somatostatin, omend de er kraftigere virkende og har den fordel, at virkningen holder sig længere, idet kun 20-60 vægtprocent er nødvendigt for at opnå samme biologiske aktivitet som med det naturlige peptid.Danish Patent Application No. 1404/75 discloses any modifications of somatostatin with ornithine in the 4- and / or 9-position and with alanine in the 5-position. From what is stated in the description of the latter Danish patent application, it must be concluded that any change in activity in relation to somatostatin must result from the modification at the N-terminal position of the molecule, because the amino acids in 4- The 5 and 9 positions may be the same as in somatostatin. At least on page 4 of the corresponding German patent it is stated that the peptides have roughly the same activity spectrum as natural somatostatin, although they are more potent and have the advantage that the effect lasts longer, with only 20-60% by weight needed to achieve the same biological activity as with the natural peptide.

Hvorledes kraften står i forbindelse med virkningens varighed, fremgår dog ikke tydeligt.However, how the force relates to the duration of the effect is not clear.

Den foreliggende opfindelse bygger nu på, at visse hidtil u-kendte, syntetiske polypeptider, som kan anses for at være en strukturel modifikation af somatostatin, men afviger fra somatostatin i følgende henseender: 1 2 (a) Ala -Gly -segmentet er fraværende, 4 (b) Lys -resten er erstattet af Arg eller His, 5 (c) Asn -resten er erstattet af His eller Glu og 8 (d) Trp -resten er erstattet af D-trp, har vist sig at være biologisk aktive på en helt særegen måde ved selektivt at sænke niveauet af såvel væksthormon som glucagon i blodet uden at påvirke dets insulinniveau i væsentlig grady når de anvendes i afpassede doser. Hertil kommer, at disse nye somato-statinanaloge udviser overrasksende lang virkningsvarighed.The present invention is now based on certain novel synthetic polypeptides which may be considered a structural modification of somatostatin but differ from somatostatin in the following respects: (a) the Ala-Gly segment is absent; 4 (b) The Lys residue is replaced by Arg or His, 5 (c) The Asn residue is replaced by His or Glu and 8 (d) The Trp residue is replaced by D-trp, has been found to be biologically active on a very peculiar way of selectively lowering the level of both growth hormone and glucagon in the blood without significantly affecting its insulin level when used at appropriate doses. In addition, these new somato-statin analogs exhibit surprisingly long duration of action.

150618 4 I forhold til somatostatins struktur er det aminosyrerester-ne i 4- og 5-stillingerne, der her er ændret.Relative to the structure of the somatostatin, the amino acid residues at the 4 and 5 positions have changed here.

Alle de optisk aktive aminosyrerester og aminosyrer har L-kon-figuration, medmindre andet er angivet.All optically active amino acid residues and amino acids have L configuration unless otherwise indicated.

I overensstemmelse hermed tilvejebringer opfindelsen en analogifremgangsmåde til fremstilling af somatostatinanaloge med den almene formel H-Cys-X1-X2-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-X4OH iAccordingly, the invention provides an analogous method for preparing somatostatin analogs of the general formula H-Cys-X1-X2-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-X4OH in

i Ii

SA ASSA AS

hvori A er hydrogen, eller begge A'er tilsammen repræsenterer en S-S- 12 4wherein A is hydrogen or both A's together represent an S-S-12 4

-binding, X betyder His eller Arg, X betyder His eller Glu og Xbond, X means His or Arg, X means His or Glu and X

betyder Cys eller D-Cys, eller farmaceutisk acceptable salte deraf.means Cys or D-Cys, or pharmaceutically acceptable salts thereof.

Symbolerne, som identificerer aminosyrerne og aminosyre-resterne i de her beskrevne polypeptider, er vedtaget af the IUPAC--IVB Committee on Biochemical Nomenclature Recommendation (1971) og beskrevet i the Archives of Biochemistry and Biophysics, 150, 1-8 (1972).The symbols identifying the amino acids and amino acid residues of the polypeptides described herein were adopted by the IUPAC - IVB Committee on Biochemical Nomenclature Recommendation (1971) and described in the Archives of Biochemistry and Biophysics, 150, 1-8 (1972).

De fremstillede forbindelser er hvide til svagt gulbrune faste stoffer, som praktisk taget er uopløselige i chloroform, benzen o.lign., men som er opløselige i vand og vandige syreopløsninger, som f.eks. saltsyre og eddikesyre. Smeltepunkterne for de omhandlede stoffer er ikke klart erkendelige, og stofferne kan f.eks. renses ved kromatografiske fremgangsmåder. Ved hydrolyse af de omhandlede stoffer i f.eks. 4-N-methansulfonsyre kan deres ami-nosyreindhold bestemmes, hvilket er i overensstemmelse med de o-venfor angivne strukturer.The compounds prepared are white to slightly yellowish brown solids which are practically insoluble in chloroform, benzene and the like, but which are soluble in water and aqueous acid solutions such as e.g. hydrochloric and acetic acid. The melting points of the substances in question are not clearly recognizable, and the substances may e.g. purified by chromatographic procedures. By hydrolysis of the subject substances in e.g. 4-N-methanesulfonic acid, their amino acid content can be determined, which is in accordance with the o-friend mentioned structures.

De omhandlede forbindelser har farmaceutisk virkning, især ved anvendelsen, som karakteriseres ved inhibering af frigørelsen af et eller flere af hormonerne somatotropin, glucagon og insulin, som påvist ved farmakologiske standardtests. Ved dosisregulering muliggør en virkningsselektivitet inhibering af somatotropin- og glucagonfri-gørelse, uden at endogene insulinsekretionsniveauer påvirkes væsentligt.The compounds of the invention have a pharmaceutical effect, especially in the use characterized by inhibition of the release of one or more of the hormones somatotropin, glucagon and insulin, as demonstrated by standard pharmacological tests. In dose control, efficacy selectivity allows inhibition of somatotropin and glucagon release without significantly affecting endogenous insulin secretion levels.

De omhandlede forbindelser kan endvidere anvendes i blanding med insulin til behandling af varmblodede dyr, som lider af diabetes melli-tus.The present compounds can further be used in admixture with insulin for the treatment of warm-blooded animals suffering from diabetes mellitus.

Den Jiu påviste selektive aktivitet samtidig med meget lang vxrkningsvarighed er bemærkelsesværdig, når man betænker, at aktivitetsvarigheden for somatostatin indgivet intravenøst er på ca.The Jiu detected selective activity at the same time as very long waking duration is noteworthy considering that the activity duration of somatostatin administered intravenously is approx.

150618 5 15 minutter (idet dets biologiske halvlevetid er under 4 minutter). Derfor må det regnes for en helt overraskende erkendelse når de ovenfor definerede somatostatinanaloge med formlen I har vist sig at være aktive i 2 timer og under visse omstændigheder endda længere. Tilvejebringelsen af sådanne forbindelser er ganske bemærkelsesværdig og frembyder bl.a. den tekniske fordel, at det ikke er nødvendigt at foretage intravenøse infusioner, således som man hidtil har været henvist til at benytte som almen indgivelsesvej med somatostatin for at udnytte dettes kendte aktivitet.15 minutes (with its biological half-life being less than 4 minutes). Therefore, it must be considered quite surprising when the somatostatin analogs of formula I defined above have been found to be active for 2 hours and in some circumstances even longer. The provision of such compounds is quite remarkable. the technical advantage that it is not necessary to make intravenous infusions, as has hitherto been referred to as the general route of administration of somatostatin to utilize its known activity.

Analogifremgangsmåden ifølge opfindelsen er ejendommelig ved, at forbindelser med formlen l' 21 4The analogous process of the invention is characterized in that compounds of formula I '21 4

X-Cys-X -X -Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-X -ORX-Cys-X -X -Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-X -OR

1 I C · 7 I O I Q I1 I C · 7 I O I Q I

Sa Rb r' R Ry S3 (II) hvori R betyder en carboxybeskyttelsesgruppe eller en polysty- renharpiksbærer bundet via en methylengruppe, X er H eller en a- 1 ' aminobeskyttelsesgruppe, X betyderSa Rb r 'R Ry S3 (II) wherein R means a carboxy protecting group or a polystyrene resin support bonded via a methylene group, X is H or an α-1' amino protecting group, X means

His Arg i2 eller R1 2 3 hvori R betyder en beskyttelsesgruppe for sidekædenitrogenato- 2 merne i argxnin, og R betyder hydrogen eller en beskyttelses- 2' gruppe for imidazolnitrogenatornet i histidin; X betyderHis Arg i2 or R1 23 wherein R represents a protecting group for the side chain nitrogen atoms in arginine and R means hydrogen or a protecting 2 'group for the imidazole nitrogen atom in histidine; X means

His ' 2His' 2

RR

2* 2 hvori R har den ovenfor angivne betydning, eller X betyder2 * 2 wherein R is as defined above or X is

Glu R3 4 5 6 3 4 hvor R betyder en beskyttelsesgruppe for sidekædecarboxygruppen 5 i glutaminsyre; R^ betyder en beskyttelsesgruppe for sidekædeami- 6 8 9 nogruppen i lysin, R , R og R hver især betyder beskyttelses- 4 grupper for hydroxylgrupperne i threonin og serin, X er som o-venfor anført, og α og 3 betyder hydrogenatomer, sulfhydrylbe-skyttelsesgrupper eller tilsammen udgør en direkte binding imellem svovlatomerne, befries for alle beskyttelsesgrupper og fjernes fra den eventuelle polystyrenharpiksbærer, om nødvendigt oxideres til dannelse af en direkte S-S-binding, og om nødvendigt produktet isoleres som den frie base eller et farmaceutisk acceptabelt salt deraf.Glu R3 4 5 6 3 4 wherein R represents a protecting group for the side chain carboxy group 5 in glutamic acid; R 2 represents a protecting group for the side chain amino group of 6 8 9 the lysine group, R, R and R each represent protective 4 groups for the hydroxyl groups of threonine and serine, X is as o-friend listed, and α and 3 are hydrogen atoms, sulfhydrylbe protecting groups or together form a direct bond between the sulfur atoms, are freed of all protecting groups and removed from the optional polystyrene resin support, oxidized if necessary to form a direct SS bond, and if necessary isolated the product as the free base or pharmaceutically acceptable salt thereof.

150618 6 12 1150618 6 12 1

Fortrinsvis betyder X og X His, eller X betyder Arg og X2 betyder Glu.Preferably, X and X mean His or X means Arg and X 2 means Glu.

Som eksempler på særligt foretrukne forbindelser, som fremstilles ved fremgangsmåden ifølge opfindelsen, kan nævnes: H-Cys-Arg-His-Phe-Phe-D-Trp-Lys-Examples of particularly preferred compounds prepared by the process of the invention are: H-Cys-Arg-His-Phe-Phe-D-Trp-Lys

S-SS-S

-Thr-Phe-Thr-Ser-Cys-OH-Thr-Phe-Thr-Ser-Cys-OH

H-Cys-His-His-Phe-Phe-D-Trp-Lys- å—......... fH-Cys-His-His-Phe-Phe-D-Trp-Lys-å —......... f

-Thr-Phe-Thr-Ser-Cys-OH-Thr-Phe-Thr-Ser-Cys-OH

H-Cys-His-His-Phe-Phe-D-Trp-Lys—H-Cys-His-His-Phe-Phe-D-Trp-Lys-

S--—SS --- S

iin

-Thr-Phe-Thr-Ser-D-Cys-OH-Thr-Phe-Thr-Ser-D-Cys-OH

. H-Cys-Arg-Glu-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH ί *. H-Cys-Arg-Glu-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH

S________________________________ SS________________________________ S

H-Cys-His-Glu-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OHH-Cys-Glu-His-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH

k.—..........—-----Sk. S-..........------

De ved fremgangsmåden ifølge opfindelsen anvendte udgangsforbindelser for forbindelserne med formlen (I) er hidtil ukendte. Disse forbindelser har formlen 1* 9' Δ X-Cys-X -X —Phe—Phe—D-Trp-Lys—Thr—Phe—Thr—Ser—X -OR n 8a P P P P h hvor R betyder en carboxylbeskyttelsesgruppe (f.eks. alkyl eller aral-alkyl) eller en ved hjælp af en methylengruppe bundet polystyren-The starting compounds used in the process of the invention for the compounds of formula (I) are novel. These compounds have the formula 1 * 9 'Δ X-Cys-X -X -Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-X -OR n 8a PPPP h where R represents a carboxyl protecting group (f. (alkyl or aralalkyl) or a polystyrene bonded by a methylene group.

fpolystyren-Jfpolystyren-J

harpiksbærer, (dvs. R betyder en -C^- i harpiks- i,) X betyder nydro- ! bærer i’ 1 i gen eller en α-aminobeskyttelsesgruppe, χ betyder 150518 7 2 3resin carrier, (i.e. R means a -C 2 - in resin- i,) X means nehydro-! carries in '1 in gene or an α-amino protecting group, χ means 150518 7 2 3

R i i RR i i R

I eller |I or |

His Arg 3 hvor R betyder en beskyttelsesgruppe for sidekædenitrogenatomerne 2 i arginin, og R betyder hydrogen eller en beskyttelsesgruppe for 2' imidazolnitrogenatomet i histidin, X betyderHis Arg 3 wherein R represents a protecting group for the side chain nitrogen atoms 2 in arginine and R represents hydrogen or a protecting group for the 2 'imidazole nitrogen atom in histidine, X means

His , 12 4 R R4 2 2’ ’ hvori R er som ovenfor anført, eller X betyder Glu.His, 12 4 R R 4 2 2 '' wherein R is as above or X is Glu.

4 hvor R betyder en beskyttelsesgruppe for sidekædecarboxygruppen g i glutaminsyre, R betyder en beskyttelsesgruppe for sidekæde- . 7 8 9 ammogruppen i lysxn, R , R og R hver især betyder beskyttelsesgrupper for hydroxylgrupperne i threonin og serin, X^ som ovenfor anført, og α og β betyder hydrogenatomer, sulfhydrylbeskyttelses-grupper eller tilsammen udgør en direkte binding imellem svovlatomerne.4 where R represents a protecting group for the side chain carboxy group g in glutamic acid, R means a protecting group for the side chain. 7 8 9 the ammo group of lysxn, R, R and R each means protecting groups for the hydroxyl groups of threonine and serine, X 1 as indicated above, and α and β represent hydrogen atoms, sulfhydryl protecting groups or together form a direct bond between the sulfur atoms.

Som eksempler på α-aminobeskyttelsesgruppen X kan nævnes (1) acylbeskyttelsesgrupper, som f.eks. formyl, trifluoracetyl, phthalyl, p-toluensulfonyl (tosyl), nitrophenylsulfe-nyl med flere, (2) aromatiske urethanbeskyttelsesgrupper, som f.eks. benzyloxycarbonyl, og substitueret benzyloxycarbonyl, som f.eks. p--chlorbenzyloxycarbonyl og p-nitrobenzyloxycarbonyl, (3) aliphatiske urethanbeskyttelsesgrupper, som f.eks. tert.butyloxycarbonyl, diiso-propylmethoxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl, 2,2,2--trichlorethoxycarbonyl og amyloxycarbonyl, (4) cycloalkylurethan-beskyttelsesgrupper, som f.eks. cyclopentyloxycarbonyl, adamantyl-carbonyl og cyclohexyloxycarbonyl, (5) thiourethanbeskyttelsesgrupper, som f.eks. phenylthiocarbonyl, (6) alkylbeskyttelsesgrupper, som f.eks. triphenylmethyl (trityl), og (7) trialkylsilangrupper, som f.eks. trimethylsilan. Som α-aminobeskyttelsesgruppe foretrækkes tert.butyloxycarbonyl.As examples of the α-amino protecting group X may be mentioned (1) acyl protecting groups, such as e.g. formyl, trifluoroacetyl, phthalyl, p-toluenesulfonyl (tosyl), nitrophenylsulfonyl and several, (2) aromatic urethane protecting groups such as e.g. benzyloxycarbonyl, and substituted benzyloxycarbonyl, e.g. p - chlorobenzyloxycarbonyl and p-nitrobenzyloxycarbonyl, (3) aliphatic urethane protecting groups such as e.g. tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl and amyloxycarbonyl, (4) cycloalkylurethane protecting groups such as e.g. cyclopentyloxycarbonyl, adamantylcarbonyl and cyclohexyloxycarbonyl, (5) thiourethane protecting groups such as e.g. phenylthiocarbonyl, (6) alkyl protecting groups such as e.g. triphenylmethyl (trityl), and (7) trialkylsilane groups such as e.g. trimethylsilane. As α-amino protecting group, tert-butyloxycarbonyl is preferred.

33

Som eksempler på R kan nævnes nitro, tosyl, benzyloxycarbonyl, 3 adamantyloxycarbonyl eller tert.butyloxycarbonyl. R betyder fortrinsvis tosyl.Examples of R include nitro, tosyl, benzyloxycarbonyl, 3 adamantyloxycarbonyl or tert-butyloxycarbonyl. Preferably R is tosyl.

Nitro- eller tosylgruppen beskytter enten N**'- eller N^-nitrogen-atomerne i argininet, medens oxycarbonylbeskyttelsesgrupperne beskytter N^- og enten N4*'- eller N^-nitrogenatomerne.The nitro or tosyl group protects either the N ** or N 2 nitrogen atoms in the arginine, while the oxycarbonyl protecting groups protect the N 2 and either N 4 * or N 2 nitrogen atoms.

22

Som eksempler på R kan nævnes tosyl, benzyloxycarbonyl, adaman- 2 8 150818 tyloxycarbonyl og tert.butyloxycarbonyl. R betyder fortrinsvis tosyl.Examples of R include tosyl, benzyloxycarbonyl, adam-amyloxycarbonyl and tert-butyloxycarbonyl. Preferably R is tosyl.

gg

Sidekædeaminogruppen i lysin beskyttes ved hjælp af R , som f.eks. kan være tosyl, tert.amyloxycarbonyl, tert.butyloxycarbonyl, diisopropyloxycarbonyl, benzyloxycarbonyl, halogenbenzyloxycarbonyl, nitrobenzyloxycarbonyl o.lign., fortrinsvis 2-chlorbenzyloxycarbonyl.The side chain amino group of lysine is protected by R, e.g. may be tosyl, tert.amyloxycarbonyl, tert.butyloxycarbonyl, diisopropyloxycarbonyl, benzyloxycarbonyl, halogenobenzyloxycarbonyl, nitrobenzyloxycarbonyl and the like, preferably 2-chlorobenzyloxycarbonyl.

Som eksempler på beskyttelsesgrupper R , R og R for hydroxyl-gruppen i threonin og serin kan nævnes acetyl, benzoyl, tert.butyl og fortrinsvis benzyl.Examples of protecting groups R, R and R for the hydroxyl group in threonine and serine include acetyl, benzoyl, tert-butyl and preferably benzyl.

44

Som eksempler på R kan nævnes benzyl og tert.butyl.Examples of R include benzyl and tert-butyl.

Som eksempler på a- og β-beskyttelsesgrupper for sulfhydryl-grupperne i cysteinylaminosyreresterne kan nævnes benzyl, substitueret benzyl, hvor substituenten er mindst en af grupperne methyl, methoxy, nitro eller halogen (f.eks. 3,4-dimethylbenzyl, p-methoxybenzyl, p-chlorbenzyl, p-nitrobenzyl med flere), trityl, benzyloxycarbonyl, benzhydryl, p-methoxybenzyloxycarbonyl, benzylthiomethyl, ethylcarba-moyl, thioethyl, tetrahydropyranyl, acetamidomethyl, benzoyl, s-sulfonsalt med flere. p-Methoxybenzylgruppen foretrækkes.Examples of α and β protecting groups for the sulfhydryl groups in the cysteinyl amino acid residues may be mentioned benzyl, substituted benzyl, wherein the substituent is at least one of the groups methyl, methoxy, nitro or halogen (eg 3,4-dimethylbenzyl, p-methoxybenzyl , p-chlorobenzyl, p-nitrobenzyl, etc.), trityl, benzyloxycarbonyl, benzhydryl, p-methoxybenzyloxycarbonyl, benzylthiomethyl, ethylcarbamoyl, thioethyl, tetrahydropyranyl, acetamidomethyl, benzoyl, s-sulfonic salt, and more. The p-methoxybenzyl group is preferred.

Forbindelserne med formlen (I) fremstilles således ved fjernelse af alle beskyttelsesgrupperne og polystyrenharpiksbæreren, når den er til stede, fra en forbindelse med formlen (II) med den ovenfor angivne definition. Produktet oxideres om nødvendigt til dannelse af en direkte S-S-binding, så man får den cycliske kædeform, og isoleres om nødvendigt som den frie base eller som et farmakologisk acceptabelt salt.Thus, the compounds of formula (I) are prepared by removing all the protecting groups and polystyrene resin support, when present, from a compound of formula (II) with the above definition. If necessary, the product is oxidized to form a direct S-S bond to give the cyclic chain form and, if necessary, isolated as the free base or as a pharmacologically acceptable salt.

Fjernelsen af beskyttelsesgrupperne kan gennemføres på de for de respektive beskyttelsesgrupper kendte måder. Fortrinsvis fjernes beskyttelsesgrupperne i ét enkelt trin, f.eks. ved anvendelse af hydrogenfluorid i nærværelse af anisol.The removal of the protecting groups can be carried out in ways known to the respective protecting groups. Preferably, the protecting groups are removed in a single step, e.g. using hydrogen fluoride in the presence of anisole.

Polystyrenharpiksbæreren kan fraspaltes samtidigt med fjernelsen af beskyttelsesgrupperne, f.eks. ved anvendelse af hydrogenfluorid i nærværelse af anisol, eller den kan fraspaltes ved transesterifikation, hvorved der fås et carboxylbeskyttet mellemprodukt med formlen (II), hvor R er en esterfunktion. F.eks. tilvejebringes ved methanolyse et methylestermellemprodukt med formlen (II), hvor R betyder methyl.The polystyrene resin support can be cleaved simultaneously with the removal of the protecting groups, e.g. using hydrogen fluoride in the presence of anisole or it may be cleaved by transesterification to give a carboxyl protected intermediate of formula (II) wherein R is an ester function. Eg. For example, methanolysis provides a methyl ester intermediate of formula (II) wherein R is methyl.

Ved hydrolyse af sådanne estere tilvejebringes den nødvendige C-ende-stillede carboxylfunktion.Hydrolysis of such esters provides the necessary C-terminal carboxyl function.

Ved passende valg af beskyttelsesgrupper kan a- og β-beskyttel-sesgrupperne fjernes selektivt fra forbindelserne med formlen (II), hvorved fås de frie disulfhydrylderivater med formlen (II), hvor α og β 9 150518 betyder hydrogen. Når a og β betyder trityl eller acetamido, kan de f.eks. fjernes selektivt ved indvirkning af et merkuri- eller sølvsalt, hvorved fås det tilsvarende disulfhydrylderivat i form af dets tilsvarende merkuri- eller disølvsalt. Sidstnævnte salt kan underkastes virkningen af hydrogensulfid til dannelse af det tilsvarende frie disulfhydrylderivat med formlen (II).By appropriate selection of protecting groups, the α and β protecting groups can be selectively removed from the compounds of formula (II) to give the free disulfhydryl derivatives of formula (II) where α and β are hydrogen. When α and β mean trityl or acetamido, they may e.g. is selectively removed by the action of a mercury or silver salt to give the corresponding disulfhydryl derivative in the form of its corresponding mercury or dis silver salt. The latter salt may be subjected to the action of hydrogen sulfide to form the corresponding free disulfhydryl derivative of formula (II).

Det frie disulfhydrylderivat med formlen (II) kan især cy-cliseres ved oxidation ved anvendelse af et oxidationsmiddel, som f.eks. iod, oxygen (f.eks. luft), 1,2-diiodoethan eller natriumeller kaliumferricyanid, til dannelse af dentilsvarende forbindelse med formlen (I), hvori A-A er en direkte S-S-binding.In particular, the free disulfhydryl derivative of formula (II) can be cyclized by oxidation using an oxidizing agent such as e.g. iodine, oxygen (e.g., air), 1,2-diiodoethane or sodium or potassium ferricyanide, to form a dentate-like compound of formula (I) wherein A-A is a direct S-S bond.

De nødvendige mellemprodukter kan fremstilles ved den kendte faststoffasemetode, jf. f.eks. Merrifield, J. Am. Chem. Soc. 8j5, 2149 (1963), eller ved klassisk syntese. Til brug ved fremgangsmåden ifølge opfindelsen tilvejebringes således forbindelser med formlen (II) , hvor α og β er beskyttelsesgrupper, og R betyder en polystyrenharpiksbærer, hvorved de nødvendige, passende beskyttede og/eller aktiverede aminosyrer kobles fortløbende under faststoffasesyntesebetingelser til en chlormethyleret eller hydroxymethylharpiksbærer. Alternativt kan forbindelserne med formlen (II), hvor α og β er beskyttelsesgrupper, og R er en carboxylbeskyttelsesgruppe, fremstilles ved kobling af de nødvendige passende beskyttede og/eller aktiverede aminosyrer eller peptiddele på de til dannelse af peptidbindinger kendte måder til dannelse af den tilsigtede sekvens af amionsyrer. α- og β-beskyt-telsesgrupperne kan derpå fjernes selektivt, og produktet kan oxideres, hvorved fås det cycliske disulfid med formlen (II), hvor α og β danner en direkte binding.The necessary intermediates can be prepared by the known solid phase method, cf. Merrifield, J. Am. Chem. Soc. 8j5, 2149 (1963), or by classical synthesis. Thus, for use in the process of the invention, compounds of formula (II) wherein α and β are protecting groups are provided, and R is a polystyrene resin support, whereby the necessary, suitably protected and / or activated amino acids are continuously coupled under solid phase synthesis conditions to a chloromethylated or hydroxymethyl resin carrier. Alternatively, the compounds of formula (II), wherein α and β are protecting groups and R is a carboxyl protecting group, can be prepared by coupling the necessary suitably protected and / or activated amino acids or peptide moieties in the known peptide bonding methods to form the intended sequence of amino acids. The α and β protecting groups can then be selectively removed and the product can be oxidized to give the cyclic disulfide of formula (II) where α and β form a direct bond.

Ved valg af en bestemt sidekædebeskyttelsesgruppe, som skal anvendes til fremstilling af de omhandlede peptider, bør følgende retningslinjer overholdes: (a) sidekædebeskyttelsesgruppen skal være stabil over for reagenset og under reaktionsbetingelserne valgt til fjernelse af α-aminobeskyttelsesgruppen i hvert fremstillingstrin, (b) beskyttelsesgruppen skal bibeholde dens beskyttende egenskaber (dvs. ikke fraspaltes under koblingsbetingelser), og (c) sidekædebeskyttelsesgruppen skal kunne fjernes ved syntesens afslutning under reaktionsbetingelser, som ikke forandrer peptidkæden.In selecting a particular side chain protecting group to be used for the preparation of the peptides, the following guidelines should be adhered to: (a) the side chain protecting group must be stable to the reagent and under the reaction conditions selected to remove the α-amino protecting group in each preparation step, (b) the protecting group must retain its protective properties (i.e., not cleave under coupling conditions), and (c) the side chain protecting group must be removable at the end of synthesis under reaction conditions which do not alter the peptide chain.

Udgangsforbindelserne kan fremstilles efter i og for sig kendte metoder, hvorved man 150618 ίο oparbejder det tilsigtede peptid ved kondensation af aminosyrer eller peptiddele, som om nødvendigt beskyttes. Kondensationsreaktionerne kan gennemføres ved anvendelse af de inden for peptid-og penicillinkemien alment kendte metoder til dannelse af peptidbindinger. Med henblik på at fremme kondensationen af aminosyrerne anvendes fortrinsvis et kondensationsmiddel. Som eksempler på kondensationsmidler kan nævnes carbodiimider, som f.eks. N,N'-dicyclohexylcarbodi-imid (DCC) og N,N'-diisopropylcarbodiimid. Alternativt kan kondensationen gennemføres ved aktivering af én eller begge endestillede grupper. Som eksempler på en aktiveret form af endestillet carboxyl kan nævnes syrechloridet, anhydridet, azidet eller den aktiverede ester. Det vil være indlysende for en fagmand, at den foreslåede metode til gennemførelse af kondensationsreaktionerne bør være forenelig med beskyttelsesgrupperne i aminosyrerne.The starting compounds can be prepared by methods known per se, thereby working up the intended peptide by condensation of amino acids or peptide moieties, which are protected if necessary. The condensation reactions can be carried out using the peptide and penicillin chemistry commonly known methods for forming peptide bonds. In order to promote the condensation of the amino acids, a condensing agent is preferably used. Examples of condensing agents include carbodiimides, e.g. N, N'-dicyclohexylcarbodiimide (DCC) and N, N'-diisopropylcarbodiimide. Alternatively, the condensation can be accomplished by activating one or both terminated groups. Examples of an activated form of terminal carboxyl include the acid chloride, anhydride, azide or activated ester. It will be obvious to one skilled in the art that the proposed method of conducting the condensation reactions should be compatible with the protecting groups in the amino acids.

Ovennævnte kendte metode· til oparbejdning af molekylet kan i princippet belyses ved kondensering af fortrinsvis et beskyttet peptid med formlenThe above-mentioned known method for working up the molecule can in principle be elucidated by condensing preferably a protected peptide of the formula

X-Cys-X1-X2-Phe-OHX-Cys-X1-X2-Phe-OH

Sa (Via) med et beskyttet peptid med formlen 4Sa (Via) with a protected peptide of formula 4

H-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-X -ORH-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-X -OR

IC in i o IQ IIC in i o IQ I

Rb R Ra Ry S3 hvor R betyder en carboxybeskyttelsesgruppe (fortrinsvis benzyl), X, R6, R2, R8, r9, X1, X2 og X4 har de ovennævnte betydninger, og α og β betyder beskyttelsesgrupper, fortrinsvis p-methoxybenzyl, hvorved fås en forbindelse med formlen (II), som derpå befries for beskyttelse og om ønsket cycliseres ved fremgangsmåden ifølge opfindelsen.Rb R Ra Ry S3 wherein R is a carboxy protecting group (preferably benzyl), X, R6, R2, R8, r9, X1, X2 and X4 have the above meanings, and α and β are protecting groups, preferably p-methoxybenzyl, whereby a compound of formula (II) which is then freed for protection and, if desired, cyclized by the process of the invention.

Når faststoffasemetoden anvendes på de omhandlede forbindelser kobles α-amino- og sulfhydryl-beskyttet cystein først til en chlor-methyleret polystyrenharpiks, hvorpå α-aminobeskyttelsesgruppen fjernes med trifluoreddikesyre i methylenchlorid, trifluoreddikesyre alene eller HC1 i dioxan. Fjernelsen af α-aminobeskyttelsesgruppen gennemføres ved en temperatur på mellem 0°C og stuetemperatur. Andre standard-fraspaltningsreagenser og betingelser til fjernelse af specifikke a-aminobeskyttelsesgrupper kan anvendes, jf. Schroder and Lubke, "The Peptides", 1, side 72-75 (Academic Press, 1965). Når a-amino-beskyttelsesgruppen er fjernet, kobles de næste tilsigtede beskyttede 150618 11 aminosyrer individuelt til den harpiksbårne sekvens i rækkefølge. Alternativt kan der fremstilles små peptidfragmenter, f.eks. ved opløsningsmetoden, som kan sættes til en faststoffase-reaktor i den ønskede rækkefølge. Den individuelle beskyttede aminosyre eller aminosyresekvens sættes til faststoffase-reaktoren i et omtrentligt firedobbelt overskud. Koblingen gennemføres i dimethylformamid, methylenchlorid eller en blanding af de to opløsningsmidler. Udfaldet af hver koblingsreaktion i hvert trin i syntesen bestemmes ved hjælp af ninhydrinreaktionen, jf. E. Kaiser et al.,When the solid phase method is applied to the subject compounds, α-amino- and sulfhydryl-protected cysteine is first coupled to a chloro-methylated polystyrene resin, whereupon the α-amino protecting group is removed with trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or HCl in dioxane. The removal of the α-amino protecting group is carried out at a temperature between 0 ° C and room temperature. Other standard cleavage reagents and conditions for the removal of specific α-amino protecting groups may be used, see Schroder and Lubke, "The Peptides", 1, pages 72-75 (Academic Press, 1965). Once the α-amino protecting group is removed, the next intended protected amino acids are individually coupled to the resin-borne sequence in sequence. Alternatively, small peptide fragments may be prepared, e.g. by the dissolution method which can be added to a solid phase reactor in the desired order. The individual protected amino acid or amino acid sequence is added to the solid phase reactor in an approximately quadruple excess. The coupling is carried out in dimethylformamide, methylene chloride or a mixture of the two solvents. The outcome of each coupling reaction at each step of the synthesis is determined by the ninhydrin reaction, cf. E. Kaiser et al.

Analyt. Biochem., 2Λ, 595 (1970). I tilfælde af ufuldstændig kobling, gentages reaktionen, før α-aminobeskyttelsesgruppen fjernes til indføring af den næste aminosyre eller aminosyresekvens.Analyte. Biochem., 2Λ, 595 (1970). In the case of incomplete coupling, the reaction is repeated before removing the α-amino protecting group to introduce the next amino acid or amino acid sequence.

De foretrukne koblingsreagenser er 1-hydroxybenzotriazol og diisopropylcarbodiimid. Andre sådanne reagenser vil være kendte for en fagmand.The preferred coupling reagents are 1-hydroxybenzotriazole and diisopropylcarbodiimide. Other such reagents will be known to those skilled in the art.

Når den tilsigtede aminosyresekvens er fremstillet, fjernes polypeptidet fra harpiksbæreren ved behandling med f.eks. hydrogenfluorid og anisol, hvorved fås det ikke længere på nogen måde beskyttede lineære polypeptid. Det cycliske disulfid kan om ønsket fremstilles, f.eks. ved luftoxidation eller ved oxidation med K^FeiCNjg.When the intended amino acid sequence is prepared, the polypeptide is removed from the resin support by treatment with e.g. hydrogen fluoride and anisole, thereby no longer protecting the linear polypeptide in any way. The cyclic disulfide can be prepared if desired, e.g. by air oxidation or by oxidation with K ^ FeiCNjg.

Ikke giftige additionssalte af de lineære og cycliske polypep-tider fremstilles ved de inden for teknikken kendte metoder ud fra saltsyre, hydrogenbromidsyre, svovlsyre, phosphorsyre, polyphosphor-syre, maleinsyre, eddikesyre, citronsyre, benzoesyre, ravsyre, malon-syre eller ascorbinsyre o.lign. Eddikesyresaltet foretrækkes.Non-toxic addition salts of the linear and cyclic polypeptides are prepared by the methods known in the art from hydrochloric, hydrobromic, sulfuric, phosphoric, polyphosphoric, maleic, acetic, citric, benzoic, succinic, malonic or ascorbic acids. like. The acetic acid salt is preferred.

De under hele faststoffasefremstillingen anvendte beskyttelsesgrupper er kendte inden for teknikken. a-Aminobeskyttelsesgrupperne anvendt med hver aminosyre indført i sekvensen af det endelige polypeptid er (1) acylbeskyttelsesgrupper, som f.eks. formyl, trifluor-acetyl, phthalyl, p-toluensulfonyl (tosyl), nitrophenylsulfenyl o.lign., (2) aromatiske urethanbeskyttelsesgrupper, som f.eks. benzyl-oxycarbonyl, og substitueret benzyloxycarbonyl, som f.eks. p-chlor-benzyloxycarbonyl og p-nitrobenzyloxycarbonyl, (3) aliphatiske urethanbeskyttelsesgrupper, som f.eks. tert.butyloxycarbony1, diisopropyl-methoxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl, 2,2,2-tri-chlorethoxycarbonyl og amyloxycarbonyl, (4) cycloalkylurethanbeskyt-telsesgrupper, som f.eks. cyclopentyloxycarbonyl, adamantyloxycarbo-nyl og cyclohexyloxycarbonyl, (5) thiourethanbeskyttelsesgrupper, som f.eks. phenylthiocarbonyl, (6) alkylbeskyttelsesgrupper, som f.eks. triphenylmethyl (trityl) og (7) trialkylsilangrupper, som f.eks. tri- 12 150613 methylsilan. Den foretrukne α-atoinobeskyttelsesgruppe er tert.butyl-oxycarbonyl.The protecting groups used throughout the solid phase preparation are known in the art. The α-amino protecting groups used with each amino acid introduced into the sequence of the final polypeptide are (1) acyl protecting groups such as e.g. formyl, trifluoroacetyl, phthalyl, p-toluenesulfonyl (tosyl), nitrophenylsulphenyl and the like; (2) aromatic urethane protecting groups such as e.g. benzyl oxycarbonyl, and substituted benzyloxycarbonyl, e.g. p-chlorobenzyloxycarbonyl and p-nitrobenzyloxycarbonyl, (3) aliphatic urethane protecting groups such as e.g. tert-butyloxycarbonyl, diisopropyl-methoxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl and amyloxycarbonyl, (4) cycloalkylurethane protecting groups such as e.g. cyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl, (5) thiourethane protecting groups such as e.g. phenylthiocarbonyl, (6) alkyl protecting groups such as e.g. triphenylmethyl (trityl) and (7) trialkylsilane groups such as e.g. tri-methylsilane. The preferred α-atoin protecting group is tert-butyl-oxycarbonyl.

De fremstillede forbindelser kan forekomme i den monomere cycliske form (også betegnet den "oxiderede" form), dvs. A-for-men hvor der er en enkeltbinding imellem svovlatomerne på de toThe compounds prepared may exist in the monomeric cyclic form (also referred to as the "oxidized" form), ie. A-form but there is a single bond between the sulfur atoms of the two

Cys-aminosyreester, eller de kan forekomme i den lineære B-form, hvori A betegner hydrogenatomer, dvs. den reducerede form.Cys amino acid esters, or they may exist in the linear B form wherein A represents hydrogen atoms, i. the reduced form.

På grund af de fremstillede somatostatinanaloges farmakologiske virkning, kan de anvendes ved behandlingen af akromegali og diabetes på samme måde som det er tilfældet for somatostatin selv. Peptiderne kan indgives på den for somatostatin og beslægtede polypeptider gængse måde under vejledning af en læge i en mængde, som retter sig efter dysfunktionens omfang konstateret af lægen. Forbindelserne kan indgives alene eller sammen med konventionelle farmaceutisk acceptabel bærere og hjælpestoffer i enhedsdosisform.Due to the pharmacological action of the somatostatin analogues, they can be used in the treatment of acromegaly and diabetes in the same way as for somatostatin itself. The peptides may be administered in the usual manner for somatostatin and related polypeptides under the guidance of a physician in an amount which is directed to the extent of dysfunction determined by the physician. The compounds may be administered alone or together with conventional pharmaceutically acceptable carriers and excipients in unit dosage form.

Som tidligere anført er de fremstillede forbindelser også anvendelige i blanding med insulin til behandling af varmblodede dyr, som lider af diabetes mellitus, jf. f.eks. beskrivelsen til US patent nr. 3.912.807, hvori er beskrevet anvendelsen af en effektiv mængde af et præparat indeholdende somatostatin blandet med insulin, til behandling af et varmblodet dyr, som lider af diabetes mellitus.As previously stated, the compounds prepared are also useful in admixture with insulin for the treatment of warm-blooded animals suffering from diabetes mellitus, cf. U.S. Patent No. 3,912,807, which discloses the use of an effective amount of a composition containing somatostatin mixed with insulin, for the treatment of a warm-blooded animal suffering from diabetes mellitus.

Når forbindelserne anvendes terapeutisk som midler til behandling af akromegali, juvenil diabetes og diabetes mellitus, påbegyndes behandlingen med små doser, som er mindre end den optimale dosis af forbindelsen. Derefter forøges dosis langsomt, indtil den under omstændighederne optimale virkning er opnået. Generelt indgives de fremstillede somatostatinanaloge i sådanne mængder, som sædvanligvis vil tilvejebringe effektive resultater u-den at forårsage skadelige eller ødelæggende bivirkninger. Dosis kan imidlertid varieres alt afhængigt af patientens behov og den anvendte forbindelse. For nemheds skyld kan den samlede dagdosis eventuelt deles og indgives portionsvis i løbet af dagen.When the compounds are used therapeutically as agents for the treatment of acromegaly, juvenile diabetes and diabetes mellitus, treatment is started with small doses that are less than the optimal dose of the compound. Thereafter, the dose is increased slowly until the optimal effect is obtained under the circumstances. Generally, the prepared somatostatin analogs are administered in such amounts that will usually provide effective results without causing harmful or destructive side effects. However, the dose can be varied depending on the patient's needs and the compound used. For convenience, the total daily dose may be split and administered in portions throughout the day.

I overensstemmelse hermed bringes et sådant farmaceutisk præparat bestående af en forbindelse med formlen (I) eller et farmakologisk acceptabelt salt deraf i kombination med en farmakologisk acceptabel bærer fortrinsvis på enhedsdosisform.Accordingly, such a pharmaceutical composition consisting of a compound of formula (I) or a pharmacologically acceptable salt thereof is preferably combined with a pharmacologically acceptable carrier preferably in unit dosage form.

Analogifremgangsmåden ifølge opfindelsen illustreres nær- 150618 13 mere i nedenstående eksempler, hvori benyttes følgende forkortelser for beskyttelsesgrupper, opløsningsmidler m.v.The analogous method of the invention is further illustrated in the examples below, which use the following abbreviations for protecting groups, solvents, etc.

Boc = tert.butyloxycarbonyl C^Bzl = dichlorbenzyl C1Z = 2-chlorbenzyloxycarbonyl Tos = tosyl(p-toluensulfonyl) TFA = trifluoreddikesyre EDT = 1,2-ethandithiol DMF = dimethylformamid TEA = triethylaminBoc = tert-butyloxycarbonyl C ^Bzl = dichlorobenzyl C1Z = 2-chlorobenzyloxycarbonyl Tos = tosyl (p-toluenesulfonyl) TFA = trifluoroacetic acid EDT = 1,2-ethanedithiol DMF = dimethylformamide TEA = triethylamine

AcOH = eddikesyreAcOH = acetic acid

EtOAc = ethylacetat n-BuOH = n-butanol NEt^ = triethylamin DIC = diisopropylcarbondiimid HF = hydrogenfluorid NH^OAc = ammoniumacetat 14 150618EtOAc = ethyl acetate n-BuOH = n-butanol NEt ^ = triethylamine DIC = diisopropylcarbondiimide HF = hydrogen fluoride NH4 OAc = ammonium acetate 14 150618

Eksempel 1Example 1

Tert.-butyloxycarbonyl-S-p-methoxybenzyl-L-cysteinyl-Nim-tosyl-L--histidyl-Nim-tosyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D-tryp-tophyl-Nf-2-chlorbenzyloxycarbonyl-L-lysyl-0-benzyl-L-threonyl-L--phenylalanyl-O-benzyl-L—threonyl-0-benzyl-L-seryl-S“p-methoxybenzyl- 2L2cγsteinγl·2ίiZÉE22Σ2®ίίϊΣlE°iΣ£^Σ££2__T-butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-Nim-tosyl-L - histidyl-Nim-tosyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D-Tryp-tophyl-N-2-chlorbenzyloxycarbonyl- L-lysyl-0-benzyl-L-threonyl-L - phenylalanyl-O-benzyl-L-threonyl-0-benzyl-L-seryl-S “p-methoxybenzyl-2L2cγsteinγl · 2ίiZÉE22Σ2® £ 2__

Chlormethyleret polystyrenharpiks (Lab Systems, Inc.), der er 1% tværbundet med divinylbenzen, esterificeres med BOC-Cys(SMBzl)OH i overensstemmelse med fremgangsmåden ifølge Gisin, Helv. Chim. Acta., 56, 1976 (1973). Polystyrenharpiksesteren behandles ifølge nedenstående forskrift A til inkorporering af BOC-Ser(Bzl)OH, BOC-Thr(Bzl)OH, BOC-Phe-OH, BOC-Thr(Bzl)OH, BOC-Lys(Clz)OH, BOC-D-Trp-OH, BOC-Phe-OH, BOC-Phe-OH, BOC-His(Tos)OH, BOC-His(Tos)OH og BOC-Cys(SMBzl)OH, hvorved fås den ønskede peptidoharpiks.Chloromethylated polystyrene resin (Lab Systems, Inc.), which is 1% cross-linked with divinylbenzene, is esterified with BOC-Cys (SMBzl) OH according to the method of Gisin, Helv. Chim. Acta., 56, 1976 (1973). The polystyrene resin ester is treated according to the following specification A for incorporation of BOC-Ser (Bzl) OH, BOC-Thr (Bzl) OH, BOC-Phe-OH, BOC-Thr (Bzl) OH, BOC-Lys (Clz) OH, BOC-D -Trp-OH, BOC-Phe-OH, BOC-Phe-OH, BOC-His (Tos) OH, BOC-His (Tos) OH and BOC-Cys (SMBzl) OH to give the desired peptide resin.

Forskrift ARegulation A

1. Vaskning med 3 x CH2C12.1. Wash with 3 x CH2 Cl2.

2. Behandling med TFA:CH2C12:EDT (1:1:5% , efter rumfang/rumfang) i 5 minutter. Å 3. Behandling som i trin 2 i 25 minutter.2. Treatment with TFA: CH2 Cl2: EDT (1: 1: 5%, by volume / volume) for 5 minutes. Å 3. Treatment as in step 2 for 25 minutes.

4. Vaskning med 3 x CH2C12.4. Washing with 3 x CH2 Cl2.

5. Vaskning med DMF.5. Washing with DMF.

6. Behandling med 12%' s TEA i DMF to gange i 3 minutter.6. Treatment with 12% TEA in DMF twice for 3 minutes.

7. Vaskning med DMF.7. Washing with DMF.

8. Vaskning med 3 x CH2C12.8. Washing with 3 x CH2 Cl2.

9. Behandling med 4 ækvivalenter af det tilsvarende aminosyrederivat i CH2C12:DMF og omrøring i 5 minutter.9. Treatment with 4 equivalents of the corresponding amino acid derivative in CH 2 Cl 2: DMF and stirring for 5 minutes.

10. Tilsætning i to portioner af 5 ækvivalenter DIC opløst i CH2C12 i løbet af 30 minutter. Reaktionstid = 6 timer.10. Addition in two portions of 5 equivalents of DIC dissolved in CH 2 Cl 2 over 30 minutes. Reaction time = 6 hours.

11. Vaskning med 3 x DMF.11. Wash with 3 x DMF.

12. Vaskning med 3 x CH2C12- 13. Gennemførelse af ninhydrinreaktionen i overensstemmelse med fremgangsmåden ifølge Kaiser et al., Annal. Biochem., 34 595 (1970).12. Washing with 3 x CH 2 Cl 2 - 13. Performing the ninhydrin reaction according to the method of Kaiser et al., Annal. Biochem., 34,595 (1970).

I tilfælde af ufuldstændig reaktion gentages ovennævnte trin 9-13.In case of incomplete reaction, steps 9-13 above are repeated.

Eksempel 2 15 150618 L-Cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D--tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L--cysteln-cyclisk (1-12) disulfid_ 9 g af peptidoharpiksen fremstillet ved den i eksempel 1 angivne fremgangsmåde blandes med 18 mg anisol og behandles med 180 ml flydende HF i 45 minutter i isbad. Overskydende HF fjernes i vakuum så hurtigt som muligt (ca. 1 time), og remanensen ekstraheres med afluftet 2M iseddike, hvorpå den filtreres. Filtratet vaskes med ether, og det vandige lag oxideres med K^FeiCNjg ved en pH-værdi på 7. Overskuddet af oxidationsmidlet fjernes ved hjælp af en "Bio Rad'^*AG-3-ionbytter-harpiks, hvorpå peptidet absorberes på en "Bio Rex "^-70-ionby tterharpiks og elueres med en pyridinpuffer med en pH-værdi på 7, og efter lyofili- sation fås 1,5 g materiale. Dette materiale sættes til en ,,Sephadex,,vsy G-15-søjle (2,5 x 160 cm) og elueres med 15%'s vandig eddikesyre. Fraktionerne (hver 5,2 ml) i rør 88-90 hældes sammen og lyofiliseres, hvorved fås den ønskede forbindelse i en mængde på 174 mg.Example 2 L-Cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D - tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L - Cysteine-cyclic (1-12) disulfide-9 g of the peptido resin prepared by the procedure of Example 1 are mixed with 18 mg of anisole and treated with 180 ml of liquid HF for 45 minutes in an ice bath. Excess HF is removed in vacuo as soon as possible (about 1 hour) and the residue is extracted with de-aerated 2M glacial acetic acid and filtered. The filtrate is washed with ether and the aqueous layer is oxidized with K 2 Fe 2 CN 2 at a pH of 7. The excess oxidant is removed by a "Bio Rad" * AG-3 ion exchange resin and the peptide is absorbed onto a " Bio Rex® 70-Ion exchange resin and eluted with a pyridine buffer of pH 7, and after lyophilization 1.5 g of material is obtained. This material is added to a "Sephadex" vsy G-15 column. (2.5 x 160 cm) and eluted with 15% aqueous acetic acid The fractions (each 5.2 ml) in tubes 88-90 are combined and lyophilized to give the desired compound in an amount of 174 mg.

Rf (BWA, 4:1:1) 0,45Rf (BWA, 4: 1: 1) 0.45

Rf (BWAP, 30:24:6:20) 0,64Rf (BWAP, 30: 24: 6: 20) 0.64

Aminosyreanalyse: Thr(2) 1,87, Ser(l) 0,89, Cys(2) 1,16, Phe(3) 3,Amino acid analysis: Thr (2) 1.87, Ser (1) 0.89, Cys (2) 1.16, Phe (3) 3,

Lys(1) 1,05, His(2) 1,76, Trp ej bestemt.Lys (1) 1.05, His (2) 1.76, Trp not determined.

Eksempel 3Example 3

Den farmakologiske virkning in vivo for dodecapeptidet fremstillet ved den i eksempel 2 angivne fremgangsmåde påvises som beskrevet nedenfor med de anførte resultater.The in vivo pharmacological effect of the dodecapeptide prepared by the method of Example 2 is demonstrated as described below with the results given.

Undertrykkelse af væksthormonSuppression of growth hormone

En subcutan (s.c.) injektion af peptid opløseliggjort eller suspenderet i fysiologisk saltopløsning gives til "Charles River" CD hanrotter, som ikke har fastet. Rotter, som tilsvarende har fået en s.c. injektion af en kontrolsaltopløsning, tjener som kontroldyr, således at hver forsøgsrotte sammenlignes med en kontrolrotte. Rotterne holdes i separate bure, og 20 minutter før forsøgsperiodens ophør giver man dem en intraperitoneal (i.p.) injektion "Nembutal"®i en dosis på 50 mg/-kcr. Blodprøver fås ved cardialounktur, οσ olasmaet isoleres til radio- 150618 16 på 2 og 4 timer efter injektion anvendes til at undersøge varigheden af peptidets evne til at undertrykke cirkulerende perifere VH-niveauer. VH-Værdier for kontrol og forsøg sammenlignes hver gang og bedømmes ifølge the Student "t"-test, og den statistiske signifikans (p) ved niveauet 0,05 eller derunder anvendes som virkningsindeks.A subcutaneous (s.c.) injection of peptide solubilized or suspended in physiological saline solution is given to non-fasted "Charles River" CD rats. Rats that have similarly received a s.c. injection of a control salt solution serves as a control animal such that each test rat is compared to a control rat. The rats are kept in separate cages and 20 minutes before the end of the experimental period they are given an intraperitoneal (i.p.) injection "Nembutal" ® at a dose of 50 mg / kcr. Blood samples are obtained by cardiac reconstruction, οσ the olasma isolated to radio at 2 and 4 hours after injection used to examine the duration of the peptide's ability to suppress circulating peripheral VH levels. VH values for control and trials are compared each time and rated according to the Student "t" test, and the statistical significance (p) at the level of 0.05 or below is used as the effect index.

Forbindelse (dosis) 2 Timer 4 TimerCompound (dose) 2 Hours 4 Hours

Kontrol 118 + 20 115 + 23Control 118 + 20 115 + 23

Forbindelse iflg. eksmpl. 2 32+6 62+13 (1 mg/kg) p = <0,01 p =^0,01Connection according to eksmpl. 2 32 + 6 62 + 13 (1 mg / kg) p = <0.01 p = ^ 0.01

Undertrykkelse af væksthormon, glucagon og insulin.Suppression of growth hormone, glucagon and insulin.

Albino-hanrotter fordeles i tre grupper (9 rotter/gruppe) og injiceres i.p. med 50 mg/kg "Nembutal" Efter 15 minutter injiceres de s.c. afhængigt af deres gruppe med (a) testforbindelsen, typisk 10-2000 pg/kg, (b) SRIF 200 }ig/kg eller (c) fysiologisk saltopløsning. Efter 10 minutter injiceres 0,5 ml arginin (300 mg/ml, pH = 7,2) ind i hjertet på dyrene. Fem minutter efter arginininjektionen skæres hovederne af rotterne, og blodet opsamles i trasylol-EDTA. Passende alikvo-ter undersøges derpå for væksthormon, glucagon og insulin. En aktiv forbindelse er en forbindelse, som forandrer plasmaniveauet betydeligt for et vilkårligt af disse hormoner i forhold til plasmaniveauet for saltopløsningskontrollerne. Sammenligninger imellem kontrol-og forsøgsværdier foretages ved statistisk bedømmelse ved variansanalyse, og den statistiske signifikans (p) ved 0,05 eller derunder anvendes som virkningsindeks.Male albino rats are divided into three groups (9 rats / group) and injected i.p. with 50 mg / kg "Nembutal" After 15 minutes they are injected s.c. depending on their group with (a) the test compound, typically 10-2000 µg / kg, (b) SRIF 200 µg / kg, or (c) physiological saline. After 10 minutes, 0.5 ml of arginine (300 mg / ml, pH = 7.2) is injected into the heart of the animals. Five minutes after the arginine injection, the heads are cut by the rats and the blood is collected in trasylol-EDTA. Appropriate aliquots are then examined for growth hormone, glucagon and insulin. An active compound is a compound that significantly changes the plasma level of any of these hormones relative to the plasma level of the saline controls. Comparisons of control and experimental values are made by statistical evaluation by analysis of variance, and the statistical significance (p) at 0.05 or below is used as the index of effect.

Forbindelse Insulin Glucagon (dosis ytg/kq) VH (ng/ml) (pl/ml) pg/mlCompound Insulin Glucagon (dose yg / kq) VH (ng / ml) (pl / ml) pg / ml

Kontrol (fysiologisk saltvand) 163 + 29 278 +32 69 + 13Control (physiological saline) 163 + 29 278 +32 69 + 13

Forbindelse iflg.Connection according to

éksmpl. 2 (100^ug/kg) 20 + 8 202 + 49 20 + 8 p=<0,01 p = <0,05 p=<0,01éksmpl. 2 (100 µg / kg) 20 + 8 202 + 49 20 + 8 p = <0.01 p = <0.05 p = <0.01

Eksempel 4 17 150618Example 4 17 150618

Tert.-butyloxycarbonyl-S-p-methoxybenzyl-L-cysteinyl-N^-tosyl-L--arginyl-Nim-tosyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D-tryp-tophyl-N^-2-chlorbenzyloxycarbonyl-L-lysyl-O-benzyl-L-threonyl-L--phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-S-p-methoxybenzyl--L-cysteinyl-hydroxymethyl-polystyrenester_T-butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-N ^ -tosyl-L - arginyl-Nim-tosyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D-Tryp-tophyl-N ^ -2- chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-threonyl-L - phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-Sp-methoxybenzyl - L-cysteinyl-hydroxymethyl-polystyrenester_

Chlormethyleret polystyrenharpiks (Lab Systems, Inc.), der er 1% tværbundet med divinylbenzen, esterificeres med Boc-Cys(SMBzl)--OH i overensstemmelse med fremgangsmåden ifølge Gisin, Helv. Chim. Acta., 56, 1976 (1973). Polystyrenharpiksesteren behandles ifølge forskrift A til inkorporering af Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Thr(Bzl)-OH, Boc-Lys(C1Z)-OH, Boc-D-Trp-OH, Boc-Phe--OH, Boc-Phe-OH, Boc-His(Tos)-OH, Boc-Arg(Tos)-OH og Boc-Cys(SMBzl)--OH, hvorved fås den ønskede peptidoharpiks.Chloromethylated polystyrene resin (Lab Systems, Inc.), which is 1% cross-linked with divinylbenzene, is esterified with Boc-Cys (SMBzl) - OH according to the method of Gisin, Helv. Chim. Acta., 56, 1976 (1973). The polystyrene resin ester is treated according to Regulation A for incorporation of Boc-Ser (Bzl) -OH, Boc-Thr (Bzl) -OH, Boc-Phe-OH, Boc-Thr (Bzl) -OH, Boc-Lys (C1Z) -OH, Boc-D-Trp-OH, Boc-Phe - OH, Boc-Phe-OH, Boc-His (Tos) -OH, Boc-Arg (Tos) -OH and Boc-Cys (SMBzl) - OH, whereby the desired peptide resin is obtained.

Forskrift ARegulation A

1. Vaskning med 3 x CH2CI2· 2. Behandling med TFA: : EDT (1:1:5%, r/r) i 5 minutter.1. Wash with 3 x CH 2 Cl 2 · 2. Treatment with TFA:: EDT (1: 1: 5%, r / r) for 5 minutes.

3. Behandling som i trin 2 i 25 minutter.3. Treatment as in step 2 for 25 minutes.

4. Vaskning med 3 x C^C^.4. Washing with 3 x C ^ C ^.

5. Vaskning med DMF.5. Washing with DMF.

6. Behandling med 12%'s TEA i DMF to gange i 3 minutter.6. Treatment with 12% TEA in DMF twice for 3 minutes.

7. Vaskning med DMF.7. Washing with DMF.

8. Vaskning med 3 x C^C^· 9. Behandling med 4 ækvivalenter af det tilsvarende aminosyrederivat i C^C^iDMF og omrøring i 5 minutter.8. Washing with 3 x C ^ C ^ 9. Treatment with 4 equivalents of the corresponding amino acid derivative in C ^ C ^ iDMF and stirring for 5 minutes.

10. Tilsætning i to portioner af 5 ækvivalenter DIC opløst i i løbet af 30 minutter. Reaktionstid = 6 timer.10. Add in two portions of 5 equivalents of DIC dissolved in 30 minutes. Reaction time = 6 hours.

11. Vaskning med 3 x DMF.11. Wash with 3 x DMF.

12. Vaskning med 3 x 13. Gennemførelse af ninhydrinreaktionen i overensstemmelse med fremgangsmåden ifølge Kaiser et al., Anal. Biochem., 34, 595 (1970). I tilfælde af ufuldstændig reaktion gentages trinene 9-13.12. 3x Washing. Performing the ninhydrin reaction according to the method of Kaiser et al., Anal. Biochem., 34, 595 (1970). In case of incomplete reaction, steps 9-13 are repeated.

18 15061818 150618

Eksempel 5 L-Cysteinyl-L-arginyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L—threonyl-L-seryl-L--cystein-cyclisk (1-12) disulfid_ 8,5 g af peptidoharpiksen fremstillet yed den i eksempel 4 angivne fremgangsmåde blandes med 16 ml anisol og behandles med 100 ml flydende HF i 45 minutter. Overskydende HF fjernes i vakuum så hurtigt som muligt, og remanensen optages i 25%'s vandig eddikesyre. Polymerbæreren frafiltreres, og filtratet vaskes med ether. Det vandige lag hældes i 3,5 liter vand, pH-værdien indstilles med NH^OH på 7, hvorpå sulfhydrylforbindelsen oxideres med K^Fe (CN)g. pH-Værdien indstilles på 5 med iseddike, og det uorganiske oxidationsmiddel fjernes med "Bio Rad" AG 3. Peptidmaterialet absorberes på "Bio Rex"® 70 og elueres med en pyridinpuffer med en pH-værdi på 7, hvorved fås 2 g af den rå forbindelse. Dette materiale ledes igennem en "Sephadex"-G-15-søjle (2,5 x 160 cm) og elueres med 15%'s vandig eddikesyre. 886 mg' af dette materiale i fraktioner 47 til 93 (hver fraktion = 5,2 ml) sættes til en CM-"Sephadex" ®G-25-søjle og elueres med en trinvis NH4OAc-gradient (0,1 til 0,3 molært NH^OAc), hvorved fås den ønskede forbindelse. Dette materiale sættes til en "Sephadex"®,LH-20-søjle (2,5 x 92 cm) og elueres med 10%*s vandig eddikesyre. Den rene ønskede forbindelse fremkommer i fraktioner 55 til 63 (hver fraktion = 4,1 ml) i en mængde på 159 mg.Example 5 L-Cysteinyl-L-arginyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L - cysteine -cyclic (1-12) disulfide 8.5 g of the peptido resin prepared by the procedure of Example 4 are mixed with 16 ml of anisole and treated with 100 ml of liquid HF for 45 minutes. Excess HF is removed in vacuo as soon as possible and the residue is taken up in 25% aqueous acetic acid. The polymer support is filtered off and the filtrate is washed with ether. The aqueous layer is poured into 3.5 liters of water, the pH is adjusted with NH 2 OH of 7, and the sulfhydryl compound is oxidized with K 2 Fe (CN) g. The pH is adjusted to 5 with glacial acetic acid and the inorganic oxidant is removed with "Bio Rad" AG 3. The peptide material is absorbed on "Bio Rex" ® 70 and eluted with a pyridine buffer of pH 7 to give 2 g of the raw compound. This material is passed through a "Sephadex" G-15 column (2.5 x 160 cm) and eluted with 15% aqueous acetic acid. 886 mg 'of this material in fractions 47 to 93 (each fraction = 5.2 ml) is added to a CM "Sephadex" ® G-25 column and eluted with a stepwise NH4OAc gradient (0.1 to 0.3 molar NH ^OAc) to give the desired compound. This material is added to a "Sephadex" ®, LH-20 column (2.5 x 92 cm) and eluted with 10% * aqueous acetic acid. The pure desired compound appears in fractions 55 to 63 (each fraction = 4.1 ml) in an amount of 159 mg.

Rf (BWA, 4:1:1) 0,46Rf (BWA, 4: 1: 1) 0.46

Rf (BWAP, 30:24:6:20) 0,65Rf (BWAP, 30: 24: 6: 20) 0.65

Aminosyreanalyse: Thr (2) 1,94, Ser (1) 0,91, Cys (2) 1,79,Amino acid analysis: Thr (2) 1.94, Ser (1) 0.91, Cys (2) 1.79,

Phe (3) 3, His (1) 1,02, Lys (1) 0,85,Phe (3) 3, His (1) 1.02, Lys (1) 0.85,

Arg (1) 0,95, Trp ej bestemt.Arg (1) 0.95, Trp not determined.

Eksempel 6Example 6

Den farmakologiske virkning in vivo for den ønskede forbindelse fremstillet ved den i eksempel 5 angivne fremgangsmåde påvises som beskrevet nedenfor med de anførte resultater:The in vivo pharmacological effect of the desired compound prepared by the procedure of Example 5 is demonstrated as described below with the results given:

Undertrykkelse af væksthormon, glucagon og insulin.Suppression of growth hormone, glucagon and insulin.

Albino-hanrotter fordeles i to grupper (9 rotter/gruppe) og injiceres i.p. med 50 mg/kg "Nembutal"®. Efter 15 minutter injiceres 150618 19 rotterne s.c. afhængigt af deres gruppe med testforbindelsen eller fysiologisk saltopløsning. Efter 10 minutter injiceres 0,5 ml arginin (300 mg/ml, pH = 7,2) ind i hjertet. Rotternes hoveder skæres af 5 minutter efter arginininjektionen, og blodet opsamles i trasylol-EDTA. Passende alikvoter undersøges derpå for væksthormon (VII) , glucagon (GLUN) og insulin (INS). En aktiv forbindelse er en forbindelse, som forandrer plasmaniveauet betydeligt for et vilkårligt af disse hormoner i forhold til plasmaniveauet for saltopløsningskontrollerne. Sammenligninger imellem kontrol- og forsøgsværdier foretages ved statistisk bedømmelse ved var.iansanalyse, og den statistiske signifikans (p) ved 0,05 eller derunder anvendes som virkningsindeks.Male albino rats are divided into two groups (9 rats / group) and injected i.p. with 50 mg / kg "Nembutal" ®. After 15 minutes, the rats are injected s.c. depending on their group with the test compound or physiological saline. After 10 minutes, 0.5 ml of arginine (300 mg / ml, pH = 7.2) is injected into the heart. The heads of the rats are cut 5 minutes after the arginine injection and the blood is collected in trasylol EDTA. Appropriate aliquots are then examined for growth hormone (VII), glucagon (GLUN) and insulin (INS). An active compound is a compound that significantly changes the plasma level of any of these hormones relative to the plasma level of the saline controls. Comparisons of control and experimental values are made by statistical evaluation by variance analysis, and the statistical significance (p) at 0.05 or below is used as the effect index.

Resultaterresults

Dosis VH INS GLUNDose VH INS GLUN

Forsøg pg/kg ng/ml μI/ml pg/mlTest pg / kg ng / ml μI / ml pg / ml

Kontrol --- 257 + 49 177 +19 61+9 (saltvand) ^ 6]_ ± 5* 52 ± 16* 0 + 0* 2 (saltvand) 131 ± 43 262 ± 26 42+6Control --- 257 + 49 177 +19 61 + 9 (saline) ^ 6] _ ± 5 * 52 ± 16 * 0 + 0 * 2 (saline) 131 ± 43 262 ± 26 42 + 6

10 71 + 21* 238+72 4 + 3X10 71 + 21 * 238 + 72 4 + 3X

x p =<0,01x p = <0.01

Eksempel 7Example 7

Tert.-butyloxy-carbony1-S-p-methoxybenzy1-L-cysteinyl-Nim-tosyl-L--histidyl-N^-tosyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D-tryp-tophyl-£-2-chlorbenzyloxycarbonyl-L-lysyl-0-benzyl-L-threonyl-L--phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-S-p-methoxybenzyl--D-cysteinyl-hydroxymethyl-polystyrenester_Tert-butyloxy-carbony1-Sp-methoxybenzy1-L-cysteinyl-Nim-tosyl-L - histidyl-N ^ -tosyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D-Tryp-tophyl- £ -2 -chlorbenzyloxycarbonyl-L-lysyl-0-benzyl-L-threonyl-L - phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-Sp-methoxybenzyl - D-cysteinyl-hydroxymethyl-polystyrenester_

Chlormethyleret polystyrenharpiks (Lab Systems, Inc.), der er 1% tværbundet med divinylbenzen, esterificeres med Boc-D-Cys(SMBzl)OH i overensstemmelse med fremgangsmåden ifølge Gisin, Helv. Chim. Acta, 56 1976 (1973). Polystyrenharpiksen behandles i overensstemmelse med forskrift A, jfr. eks. 1 til inkorporering af Boc-Ser(Bzl)OH, Boc-Thr(Bzl)- 150618 20 OH, Boc-Phe-OH, Boc-Thr (Bzlj OH, Boc-Lys(C1Z)OH, Boc-D-Trp-OH, Boc-Phe--0H, Boc-Phe-OH, Boc-His(Tos)OH, Boc-His(Tos)OH og Boc-Cys(SmBzl)OH, hvorved fås den ønskede peptidoharpiks.Chloromethylated polystyrene resin (Lab Systems, Inc.), which is 1% cross-linked with divinylbenzene, is esterified with Boc-D-Cys (SMBzl) OH according to the method of Gisin, Helv. Chim. Acta, 56 1976 (1973). The polystyrene resin is treated in accordance with Regulation A, cf. Example 1 for incorporating Boc-Ser (Bzl) OH, Boc-Thr (Bzl) - Boc-Phe-OH, Boc-Thr (Bzlj OH, Boc-Lys (C1Z) OH, Boc-D- Trp-OH, Boc-Phe-OH, Boc-Phe-OH, Boc-His (Tos) OH, Boc-His (Tos) OH, and Boc-Cys (SmBzl) OH to give the desired peptide resin.

Eksempel 8 L-Cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D--tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-D--cystein-cyclisk (1-12) disulfid-diacetatsalt_ 10 g af peptidoharpiksen fremstillet ved den i eksempel 7 angivne fremgangsmåde blandes med 20 ml anisol og behandles med 150 ml flydende HF i isbad i 45 minutter under udelukkelse af luft. Overskydende flydende HF fjernes under vakuum, og remanensen optages i 50%'s vandig eddikesyre. Blandingen filtreres, og filtratet fortyndes med vand indtil 3,5 liter. pH-Værdien indstilles på 7 med fortyndet NH^OH, og disulfhydrylpeptidet oxideres med K^FtCNjg til cyclisk disulfid. Blandingens pH-værdi indstilles på 5 med iseddike og be-handles med "Bio-Rad"®AG 3. Peptidmaterialet absorberes på "Bio Rex"^ 70 (H -form) og elueres med en pyridinpuffer (Py-^O-eddikesyre, 30:66:4, r/r). Fraktionerne indeholdende peptidmaterialet hældes sammen og lyofiliseres, hvorved fås 1,6 g råmateriale. Dette råprodukt sættes til en "Sephadex"®-LH-20-søjle (2,5 x 90 cm) og elueres med 10%'s vandig eddikesyre (hver fraktion = 5,8 ml). Materialet, som fremkommer i fraktioner 97-101 hældes sammen og lyofiliseres, hvorved fås det ønskede dodecapeptid i en mængde på 171 mg.Example 8 L-Cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D - tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-D cysteine-cyclic (1-12) disulfide diacetate salt-10 g of the peptido resin prepared by the procedure of Example 7 is mixed with 20 ml of anisole and treated with 150 ml of liquid HF in an ice bath for 45 minutes, excluding air. Excess liquid HF is removed under vacuum and the residue is taken up in 50% aqueous acetic acid. The mixture is filtered and the filtrate is diluted with water to 3.5 liters. The pH is adjusted to 7 with dilute NH 2 OH and the disulfhydryl peptide is oxidized with K 2 FtCNjg to cyclic disulfide. The pH of the mixture is adjusted to 5 with glacial acetic acid and treated with "Bio-Rad" ®AG 3. The peptide material is absorbed on "Bio Rex" 70 (H-form) and eluted with a pyridine buffer (Py-O-acetic acid, 30: 66: 4, r / r). The fractions containing the peptide material are combined and lyophilized to give 1.6 g of crude material. This crude product is added to a "Sephadex" ® LH-20 column (2.5 x 90 cm) and eluted with 10% aqueous acetic acid (each fraction = 5.8 ml). The material obtained in fractions 97-101 is combined and lyophilized to give the desired dodecapeptide in an amount of 171 mg.

Tyndtlagskromatografi: forovertrukne glasplader ("Avicel",Thin-layer chromatography: pre-coated glass sheets ("Avicel",

Rf (BWA, 4:1:1, r/r) 0,32, Rf (BWAP, 30:24:6:20, r/r) 0,56.Rf (BWA, 4: 1: 1, r / r) 0.32, Rf (BWAP, 30: 24: 6: 20, r / r) 0.56.

Aminosyreanalyse: Thr(2) 1,85, Ser(l) 0,84, Cys(2) 1,29, Phe(3) 3, Lys(1) 1,05, His(2) 1,41, Trp(l) 0,75.Amino acid analysis: Thr (2) 1.85, Ser (1) 0.84, Cys (2) 1.29, Phe (3) 3, Lys (1) 1.05, His (2) 1.41, Trp ( l) 0.75.

Den farmakologiske virkning in vivo for dodecapeptidet fremstillet ved den i eksempel 8 angivne fremgangsmåde påvises som beskrevet i eksempel 13. De fremkomne data er anført nedenfor: 150618 21The in vivo pharmacological effect of the dodecapeptide prepared by the procedure of Example 8 is demonstrated as described in Example 13. The data obtained are set forth below:

Undertrykkelse af væksthormonSuppression of growth hormone

Forbindelse (dosis) 2 Timer 4 TimerCompound (dose) 2 Hours 4 Hours

Kontrol 127 +22 59 + 20Control 127 +22 59 + 20

Forbindelse ifølge eksempel 8 (1 mg/kg) 20 + 3XX 107 + 34 xx p = <.0,001Compound of Example 8 (1 mg / kg) 20 + 3XX 107 + 34 xx p = <0.001

Undertrykkelse af væksthormon, glucagon og insulin.Suppression of growth hormone, glucagon and insulin.

Forbindelse Insulin Glucagon (dosis pg/kg) VH (ng/ml) jil/ml pg/mlCompound Insulin Glucagon (dose pg / kg) VH (ng / ml) µl / ml pg / ml

Kontrol (saltvand) 279 + 53 323 + 30 42 +6Control (saline) 279 + 53 323 + 30 42 +6

Forbindelse ifølge eksempel 8 (100) 64 + 20x 165 + 21* 5 + 2*Compound of Example 8 (100) 64 + 20x 165 + 21 * 5 + 2 *

Undertrykkelse af væksthormon, glucagon og insulin, (fortsat)Suppression of growth hormone, glucagon and insulin, (continued)

Forbindelse T n . _______Compound T n. _______

Insulin Glucagon (dosis (pg/kg) VH (ng/ml) (μΐ/itil) (pg/ml)Insulin Glucagon (dose (pg / kg) VH (ng / ml) (μΐ / itil) (pg / ml)

Kontrol (saltvand) 201 + 35 397 + 36 117 + 11Control (saline) 201 + 35 397 + 36 117 + 11

Forb. iflg.Conn. acc.

eksmpl. 8 (100) 110 + 22 318 + 54 70 + 7 x p «ί.0,01 + p ·<0,05eksmpl. 8 (100) 110 + 22 318 + 54 70 + 7 x p «ί.0.01 + p · <0.05

Af det ovenfor anførte fremgår det, at peptidet ifølge Eksempel 8 er karakteriseret ved undertrykkelsen af glucagon og væksthormon, uden at det påvirker insulinsekretionen væsentligt.From the foregoing, it appears that the peptide of Example 8 is characterized by the suppression of glucagon and growth hormone without significantly affecting insulin secretion.

Eksempel 9 22 150618Example 9 22 150618

Tert.-butyloxycarbonyl-S-p-methoxybenzyl-L-cysteinyl-N^n-tosyl-L--arginyl-y-benzyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D-tryp-tophyl-Ni-2-chlor-benzyloxycarbonyl-L-lysyl-0-benzyl-L-threonyl-L--phenylal^iyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-S-p-methoxybenzyl--L-cystein-hydroxymethyl-polystyrenester_T-butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-N ^ N-tosyl-L - arginyl-y-benzyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D-Tryp-tophyl-Ni-2- chloro-benzyloxycarbonyl-L-lysyl-0-benzyl-L-threonyl-L - phenylal ^ iyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-Sp-methoxybenzyl - L-cysteine-hydroxymethyl- polystyrenester_

Chlormethyleret polystyrenharpiks esterificeres med Boc-Cys-* (SMBzl)-OH i overensstemmelse med fremgangsmåden ifølge Gisin, Helv. Chim. Acta., 56_, 1976 (1973). Aminosyreharpiksesteren behandles derpå ifølge nedenstående forskrift A, hvor der som den beskyttede aminosyre i trin 9 anvendes Boc-Ser(Bzl)-OH. Forskrift A gentages derpå med henblik på at inkorporere nedenstående aminosyrer fortløbende i peptidoharpiksen:Chloromethylated polystyrene resin is esterified with Boc-Cys- (SMBzl) -OH according to the method of Gisin, Helv. Chim. Acta., 56, 1976 (1973). The amino acid resin ester is then treated according to the following specification A, where Boc-Ser (Bzl) -OH is used as the protected amino acid in step 9. Regulation A is then repeated to incorporate the following amino acids sequentially into the peptido resin:

Boc-Thr(Bzl)-OH Boc-Phe-OH Boc-Thr(Bzl)-OH Boc-Lys(C1Z)-OH Boc-D-Trp-OH Boc-Phe-OH Boc-Phe-OH Boc-Glu(OBzl)-OHBoc-Thr (Bzl) -OH Boc-Phe-OH Boc-Thr (Bzl) -OH Boc-Lys (C1Z) -OH Boc-D-Trp-OH Boc-Phe-OH Boc-Phe-OH Boc-Glu ( OBzl) -OH

Boc-Arg(Tos)-OH Boc-Cys(SMBzl)-OHBoc-Arg (Tos) -OH Boc-Cys (SMBzl) -OH

Forskrift A: (til behandling af harpiksesteren) 1. Vaskning med 3 x methylenchlorid (C^C^) · 2. Behandling med trifluoreddikesyresmethylenchlorid (1:1, r/r) indeholdende 5%‘s 1,2-ethandithiol i 5 minutter 3. Gentagelse af trin 2 i 25 minutter.Regulation A: (for the treatment of the resin ester) 1. Washing with 3 x methylene chloride (C ^C ^) · 2. Treatment with trifluoroacetic acid methylene chloride (1: 1, r / r) containing 5% 1,2-ethanedithiol for 5 minutes 3. Repeat step 2 for 25 minutes.

4. Vaskning med 3 x CH^^· 5. Vaskning med dimethylformamid (DMF).4. Washing with 3 x CH 2 · 5. Washing with dimethylformamide (DMF).

6. Behandling med 12%'s triethylamin i DMF i 3 minutter.6. Treatment with 12% triethylamine in DMF for 3 minutes.

7. Vaskning med DMF.7. Washing with DMF.

8. Vaskning med 3 x C^C^· 23 150618 9. Behandling med 4 ækvivalenter af den passende beskyttede aminosyre i C^C^tDMF og omrøring i 5 minutter.8. Washing with 3 x C ^ C ^ 23 Treatment with 4 equivalents of the appropriately protected amino acid in C ^ C ^ tDMF and stirring for 5 minutes.

10. Tilsætning i to portioner af 5 ækvivalenter diisopropylcarbodiimid opløst i CH2CI2 i løbet af 30 minutter. Reaktionstid = 6 timer.10. Add in two portions of 5 equivalents of diisopropylcarbodiimide dissolved in CH 2 Cl 2 over 30 minutes. Reaction time = 6 hours.

11. Vaskning med 3 x DMF.11. Wash with 3 x DMF.

12. Vaskning med 3 x CH2C12< 13. Gennemførelse af ninhydrinreaktionen i overensstemmelse med fremgangsmåden ifølge Kaiser et al., Annal. Biochem., M 595 (1970).12. Washing with 3 x CH 2 Cl 2 <13. Performing the ninhydrin reaction according to the method of Kaiser et al., Annal. Biochem., M 595 (1970).

I tilfælde af ufuldstændig reaktion gentagelse af trin 9-13.In the case of incomplete reaction, repeat steps 9-13.

Eksempel 10 L-Cysteinyl-L-arginyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D--tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L--cystein-cyclisk (1-12) disulfid__ 8,5 g af peptidoharpiksen fremstillet ved den i eksempel 9 angivne fremgangsmåde suspenderes i 16 ml anisol og behandles med vandfrit flydende hydrogenfluorid (HF) i 45 minutter i isbad, hvorpå overskydende HF fjer.^a under vakuum, og remanensen ekstraheres med 10%’s vandig eddikesyre. Filtratet vaskes med ether, og det vandige lag hældes i 5 liter vand, hvorpå pH-værdien bringes på 7 med fortyndet ammoniumhydroxid. Blandingen omrøres i fri luft i 48 timer, hvorefter pH-værdien indstilles på 5 med iseddike, og peptidet absorberes på "Amberlite" ® CG-50 (H+-form) . Peptidmateriale elueres med 50%'s vandig eddikesyre og lyofiliseres, hvorved fås 850 mg af et fast materiale.Example 10 L-Cysteinyl-L-arginyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D - tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-- cysteine cyclic (1-12) disulfide 8.5 g of the peptido resin prepared by the procedure of Example 9 is suspended in 16 ml of anisole and treated with anhydrous liquid hydrogen fluoride (HF) for 45 minutes in an ice bath, whereupon excess HF feathers. under vacuum and the residue is extracted with 10% aqueous acetic acid. The filtrate is washed with ether and the aqueous layer is poured into 5 liters of water, then the pH is brought to 7 with dilute ammonium hydroxide. The mixture is stirred in free air for 48 hours, then the pH is adjusted to 5 with glacial acetic acid and the peptide is absorbed on "Amberlite" ® CG-50 (H + form). Peptide material is eluted with 50% aqueous acetic acid and lyophilized to give 850 mg of a solid.

Råproduktet sættes til en "Sephadex" ® LH-20-søjle (2,5 x 150 cm) og elueres med 10%'s eddikesyre. Fraktionerne 94-130 hældes sammen og lyofiliseres, hvorved fås 483 mg af materialet. Dette materiale sættes til en "Sephadex" ® 62 5-søj le (1,5 x 115 cm) og elueres med 10%'s vandig eddikesyre. Fraktionerne 43-53 hældes sammen og lyofiliseres, hvorved fås det ønskede peptid i en mængde på 286 mg.The crude product is added to a "Sephadex" ® LH-20 column (2.5 x 150 cm) and eluted with 10% acetic acid. Fractions 94-130 are combined and lyophilized to give 483 mg of the material. This material is added to a "Sephadex" ® 62 5 column (1.5 x 115 cm) and eluted with 10% aqueous acetic acid. Fractions 43-53 are pooled and lyophilized to give the desired peptide in an amount of 286 mg.

Tyndtlagskromatografi: forovertrukne glasplader (Avicel),Thin-layer chromatography: pre-coated glass sheets (Avicel),

Rf (BWA, 4:1:1, r/r) 0,40, Rf (BWAP, 30:24:6:20, r/r) 0,65.Rf (BWA, 4: 1: 1, r / r) 0.40, Rf (BWAP, 30: 24: 6: 20, r / r) 0.65.

Aminosyreanalyse: Thr(2) 1,92, Ser(l) 0,89, Glu(l) 1,03, Cys(2) 1,49, Phe(3) 3, Lys(l) 1,05, Trp(l) 0,81,Amino acid analysis: Thr (2) 1.92, Ser (l) 0.89, Glu (l) 1.03, Cys (2) 1.49, Phe (3) 3, Lys (l) 1.05, Trp ( l) 0.81,

Eksempel 11 24 150618Example 11 24 150618

Tert.-butyloxycarbonyl-S-p-methoxybenzyl-L-cysteinyl-Nim-tosyl-L--histidyHT-benzyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D-tryp-tophyl-N£-2-chlorbenzyloxycarbonyl-L-lysyl“0-benzyl-L-threonyl-L--phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-S-p-methoxybenzyl--Ii-cysteln-hydroxyme thyl-polys tyrenes ter _T-butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-Nim-tosyl-L - histidyHT-benzyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D-Tryp-tophyl-N £ -2-chlorobenzyloxycarbonyl-L -lysyl "O-benzyl-L-threonyl-L - phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-Sp-methoxybenzyl - 1-cysteine hydroxyme thyl-polysyrenes ter

Chlormethyleret polystyrenharpiks esterificeres med Boc-Cys-(SMBzl)-OH i overensstemmelse med fremgangsmåden ifølge Gisin, Helv. Chim. Acta., i>6, 1976 (1973). Aminosyreharpiksen behandles derpå i-følge forskrift A (jf. eksempel 9), idet der som den beskyttede aminosyre i trin 9 anvendes Boc-Ser(Bzl)-OH. Forskrift A gentages derpå med henblik på at inkorporere nedenstående aminosyrer fortløbende i pep-tidoharpiksen:Chloromethylated polystyrene resin is esterified with Boc-Cys- (SMBzl) -OH according to the method of Gisin, Helv. Chim. Acta., I> 6, 1976 (1973). The amino acid resin is then treated in accordance with Regulation A (cf. Example 9), using as the protected amino acid in Step 9, Boc-Ser (Bzl) -OH. Specification A is then repeated to incorporate the following amino acids consecutively into the peptide resin:

Boc-Thr(Bzl)-OH Boc-Phe-OH Boc-Thr(Bzl)-OH Boc-Lys(C1Z)-OH Boc-D-Trp-OH Boc-Phe-OH Boc-Phe-OH Boc-Glu(OBzl)-OH Boc-His(Tos)-OH Boc-Cys(SMBzl)-OHBoc-Thr (Bzl) -OH Boc-Phe-OH Boc-Thr (Bzl) -OH Boc-Lys (C1Z) -OH Boc-D-Trp-OH Boc-Phe-OH Boc-Phe-OH Boc-Glu ( OBzl) -OH Boc-His (Tos) -OH Boc-Cys (SMBzl) -OH

Eksempel 12 L-Cysteinyl-L-histidyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D--tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl--L-cystein-cyclisk (1-12) disulfid_ 8 g af peptidoharpiksen fremstillet ved den i eksempel 11 angivne fremgangsmåde behandles med flydende vandfrit HF som beskrevet i eksempel 10, og det lineære disulfhydryldodecapeptid cycliseres ved oxidation med K^FeiCNjg ved en pH-værdi på 7,3 og i kraftig fortynding. pH-Værdien indstilles på 5 med iseddike, og blandingen behandles med en "Bio-Rad" ® -AG3-X4-ionbytterharpiks (Cl-form), hvorpå den absorberes på "Amberlite” ® CG 50 (H+-form). Peptidmaterialet elueres 150618 25 med 30%'s vandig eddikesyre og lyofiliseres, hvorved fås 500 mg af et råmateriale. Dette materiale kromatograferes igennem en "Sephadex"-LH-20-søjle, hvorved fås det ønskede dodecapeptid.Example 12 L-Cysteinyl-L-histidyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D - tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl - L cysteine cyclic (1-12) disulfide-8 g of the peptido resin prepared by the procedure of Example 11 is treated with liquid anhydrous HF as described in Example 10, and the linear disulfhydryl dodecapeptide is cyclized by oxidation with K 2 FeiCNjg at a pH of 7 , 3 and in heavy dilution. The pH is adjusted to 5 with glacial acetic acid and the mixture is treated with a "Bio-Rad" ® -AG3-X4 ion exchange resin (C1 form) and then absorbed onto the “Amberlite” ® CG 50 (H + form). With 30% aqueous acetic acid and lyophilized to give 500 mg of a crude material which is chromatographed through a "Sephadex" LH-20 column to give the desired dodecapeptide.

Tyndtlagskromatografi: forovertrukne glasplader (Avicel), R^ (BWA, 4:1:1, r/r) 0,43, Rf (tert.-AmOH-Py-W, 7:7:6, r/r) 0,74.Thin-layer chromatography: pre-coated glass sheets (Avicel), R 2 (BWA, 4: 1: 1, r / r) 0.43, Rf (tert-AmOH-Py-W, 7: 7: 6, r / r) 0, 74th

Aminosyreanalyse: Thr(2) 1,92, Ser(l) 0,93, Glu(l) 0,94, Cys(2) 1,59, Phe(3) 3, Lys(l) 1,02, His(l) 0,91,Amino acid analysis: Thr (2) 1.92, Ser (l) 0.93, Glu (l) 0.94, Cys (2) 1.59, Phe (3) 3, Lys (l) 1.02, His ( l) 0.91,

Trp(l) 0,81.Trp (l) 0.81.

Eksempel 13Example 13

Den biologiske virkning for peptiderne fremstillet ved de i eksemplerne 10 og 12 angivne fremgangsmåder påvises som følger.The biological effect of the peptides prepared by the methods set forth in Examples 10 and 12 is demonstrated as follows.

Albino-hanrotter indgives i.p. 50 mg/kg "Nembutal"^· Efter 15 minutter injiceres rotterne s.c. med testforbindelsen af fysiologisk saltopløsning. Efter 10 minutter injiceres 0,5 ml arginin (300 mg/ml, pH = 7,2) ind i hjertet på dyrene. 5 Minutter senere skæres rotternes hoveder af, og blodet opsamles i trasylol-EDTA. En passende alikvote undersøges for væksthormon (VH), insulin og glucagon ved radioimmuno-analyse. Resultaterne er anført nedenfor:Male albino rats are administered i.p. 50 mg / kg "Nembutal" ^ · After 15 minutes, the rats are injected s.c. with the test compound of physiological saline. After 10 minutes, 0.5 ml of arginine (300 mg / ml, pH = 7.2) is injected into the heart of the animals. 5 minutes later, the heads of the rats are cut off and the blood is collected in trasylol-EDTA. A suitable aliquot is examined for growth hormone (VH), insulin and glucagon by radioimmunoassay. The results are listed below:

Dosis VH Insulin Glucagon AntalDose VH Insulin Glucagon Qty

Forbindelse pg/kg ng/kg_p. I/ml_ pg/ml dyr t iCompound pg / kg ng / kg_p. I / ml_ pg / ml animal t i

Kontrol ' — 277 + 69 301 +27 46+8 10Controls' - 277 + 69 301 +27 46 + 8 10

Forb. iflg. „ „ w eksmpl. 10 200 42 + 6 115 + 16x 1,5 + lx 10Conn. acc. "" W ex. 10 200 42 + 6 115 + 16x 1.5 + lx 10

Kontrol ' -- 216 + 35 283 + 28 55 + 5 j 10Checks' - 216 + 35 283 + 28 55 + 5 j 10

Forb. iflg. ' eksmpl. 10 40 28 + 5X 158 +57 12 + 2 10Conn. acc. 'exmpl. 10 40 28 + 5X 158 +57 12 + 2 10

Kontrol — 454 + 106 269 +37 60 + 10 10Control - 454 + 106 269 +37 60 + 10 10

Forb. iflg.Conn. acc.

eksmpl. 12 200 107 + 29x 205 +36 4 + 2X 10eksmpl. 12 200 107 + 29x 205 +36 4 + 2X 10

Kontrol — 233 + 35 342 + 45 j 44 + 4 10 ]Control - 233 + 35 342 + 45 j 44 + 4 10]

Forb. iflg.Conn. acc.

eksmpl. 12 50 84 + 17x 322 +40 10 + 4X 10 26 150618eksmpl. 12 50 84 + 17x 322 +40 10 + 4X 10 26 150618

Disse data indicerer, at peptiderne ifølge eksempel 10 og 12, er effektive midler til mindskelse af væksthormon og glucagon, uden at de væsentligt påvirker insulinniveauer ved en dosis på henholdsvis 40 mg/kg og 50 mg/kg. Peptiderne ifølge eksempel 10 og 12 testes også for varigheden af deres virkning på VH-sekrétion hos rotter behandlet med "Nembutal" ®. Peptidet ifølge eksempel 10 udviser en betydelig inhibering af VH i mindst 4 timer ved en s.c. dosis på 1000 mg/kg. Peptidet ifølge eksempel 12 udviser ingen betydelig inhibering af VH ved 2 og 4 timer.These data indicate that the peptides of Examples 10 and 12 are effective agents for decreasing growth hormone and glucagon without significantly affecting insulin levels at a dose of 40 mg / kg and 50 mg / kg, respectively. The peptides of Examples 10 and 12 are also tested for the duration of their effect on VH secretion in rats treated with "Nembutal" ®. The peptide of Example 10 exhibits significant inhibition of VH for at least 4 hours at a s.c. dose of 1000 mg / kg. The peptide of Example 12 shows no significant inhibition of VH at 2 and 4 hours.

De omhandlede forbindelser kan indgives til varmblodede pattedyr enten intravenøst, subcutant, intramuskulært eller oralt til regulering af serumglucose ved behandlingen af diabetes. Dosismængden vil variere alt afhængigt af hvilken tilstand, der skal behandles, graden af tilstanden og behandlingens varighed.The present compounds may be administered to warm-blooded mammals either intravenously, subcutaneously, intramuscularly or orally to control serum glucose in the treatment of diabetes. The amount of dose will vary depending on the condition to be treated, the degree of the condition and the duration of treatment.

De omhandlede aktive forbindelser kan indgives alene eller i kombination med farmaceutisk acceptable bærere eller strækkemidler. Egnede farmaceutiske præparater vil være indlysende for en fagmand.The present active compounds can be administered alone or in combination with pharmaceutically acceptable carriers or excipients. Suitable pharmaceutical compositions will be apparent to those skilled in the art.

Eksempel 14Example 14

Klassisk syntese af L-cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl--L-phenylalanyl-D-tryptophyl-L-lysyl-L-treonyl-L-phenylalanyl-L-treo-nyl-D-seryl-L-cystein-cyclisk (1—»12) disulfid (a) N-benzyloxycarbonyl-im-benzyloxycarbonyl-L-histidyl-L-phenylalanyl--methylester (I)Classic synthesis of L-cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl - L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-D-seryl L-cysteine cyclic (1- 12) disulfide (a) N-benzyloxycarbonyl-im-benzyloxycarbonyl-L-histidyl-L-phenylalanyl - methyl ester (I)

En suspension af 42,3 g (0,1 mol) Z-His(Z)-OH og 21,8 g (0,1 mol) Phe-OMe-HCl (98%'s) i 1000 ml afkøles til 0°C, hvorpå der til sættes 22 g (0,12 mol) DCC og 10,1 g (0,10 mol) triethylamin. Blandingen henstilles ved stuetemperatur natten over.A suspension of 42.3 g (0.1 mole) of Z-His (Z) -OH and 21.8 g (0.1 mole) of Phe-OMe-HCl (98% s) in 1000 ml is cooled to 0 ° C, to which are added 22 g (0.12 mole) of DCC and 10.1 g (0.10 mole) of triethylamine. The mixture is left at room temperature overnight.

Det udskilte dicyclohexylurinstof frafiltreres, og filtratet koncentreres til et gummiagtigt materiale. Materialet opløses i ethylacetat, og uopløst materiale frafiltreres. Ethylacetatopløsningen vaskes med 5%'s citronsyre, I^O, iskold 5%'s natriumhydrogencarbonat-opløsning og ^0. Det organiske lag tørres over natriumsulfat og koncentreres til 1/3 af dets oprindelige volumen, der tilsættes 500 ml ether, indtil opløsningen bliver uklar, og opløsningen henstilles koldt natten over. Det fremkomne hvide faste stof isoleres og tørres. Udbytte: 32 g. Smp. 131-133°C.The separated dicyclohexylurea is filtered off and the filtrate is concentrated to a gummy material. The material is dissolved in ethyl acetate and the undissolved material is filtered off. The ethyl acetate solution is washed with 5% citric acid, 10 ° C, ice cold 5% sodium bicarbonate solution and 50 ° C. The organic layer is dried over sodium sulfate and concentrated to 1/3 of its original volume, added with 500 ml of ether until the solution becomes cloudy and the solution is left to cool overnight. The resulting white solid is isolated and dried. Yield: 32 g. 131-133 ° C.

Tyndtlagskromatografi: silicagel F-254, CH^C^iMeOH (9:1),Thin-layer chromatography: silica gel F-254, CH ^C ^ iMeOH (9: 1),

Rf = 0,8.Rf = 0.8.

2? 150613 (b) Im-benzyloxycarbonyl-L-histidyl-L-phenylalanylmethylester-dihydro-bromidsalt (II) 30 g (0,5 mol) Z-His(Z)-Phe-OMe (I) opløses i 300 ml 32%'s HBr-HOAc (opløsningen bliver uklar efter 2 minutter), og blandingen henstilles ved stuetemperatur i 30 minutter. Der tilsættes ether med med henblik på udfældning. Det bløde faste stof filtreres og tørres natten over. Udbytte: 32 g fast stof, smp. 135-137°C.2? (B) Im-benzyloxycarbonyl-L-histidyl-L-phenylalanyl methyl ester dihydrobromide salt (II) dissolve 30 g (0.5 mole) of Z-His (Z) -Phe-OMe (I) in 300 ml of 32% s HBr-HOAc (the solution becomes cloudy after 2 minutes) and the mixture is left at room temperature for 30 minutes. Ether is added for precipitation. The soft solid is filtered and dried overnight. Yield: 32 g of solid, m.p. 135-137 ° C.

Tyndtlagskromatografi: ingen bevægelse.Thin-layer chromatography: no movement.

(c) N-Benzyloxycarbonyl-im-benzyloxycarbony1-L-histidyl-im-benzyloxy-carbony1-L-histidy1-L-phenylalanyl-methylester (III)(c) N-Benzyloxycarbonyl-im-benzyloxycarbonyl-L-histidyl-im-benzyloxy-carbonyl-L-histidyl-L-phenylalanyl methyl ester (III)

En suspension af 30,5 g (0,05 mol) His(Z)-Phe-OMe-2HBr (II) og 21,7 g (0,05 mol) Z-His(Z)-OH i 500 ml CH2C12 afkøles til 0°C. Derpå tilsættes 11 g DCC og 10,1 g (0,10 mol) triethylamin, og blandingen henstilles ved stuetemperatur natten over.A suspension of 30.5 g (0.05 mole) of His (Z) -Phe-OMe-2HBr (II) and 21.7 g (0.05 mole) of Z-His (Z) -OH in 500 ml of CH 2 Cl 2 is cooled to 0 ° C. Then 11 g of DCC and 10.1 g (0.10 mol) of triethylamine are added and the mixture is allowed to stand at room temperature overnight.

Det dannede dicyclohexylurinstof frafiltreres, og filtratet koncentreres til et glasagtigt materiale. Remanensen opløses i ethyl-acetat, og ethylacetatopløsningen vaskes med 10%'s citronsyre, H20, iskold 5%'s natriumhydrogencarbonatopløsning og H20. Ethylacetatopløsningen tørres over natriumsulfat og koncentreres til ca. 1/3 af dets volumen, der tilsættes ether, indtil opløsningen bliver uklar, og opløsningen henstilles koldt. Der isoleres et hvidt fast stof. Udbytte: 29,5 g.The dicyclohexylurea formed is filtered off and the filtrate is concentrated to a glassy material. The residue is dissolved in ethyl acetate and the ethyl acetate solution is washed with 10% citric acid, H2 O, ice cold 5% sodium hydrogen carbonate solution and H2 O. The ethyl acetate solution is dried over sodium sulfate and concentrated to ca. 1/3 of its volume is added to ether until the solution becomes cloudy and the solution is left to cool. A white solid is isolated. Yield: 29.5 g.

(d) L-Histidy1-L-histidy1-L-phenylalanyl-methylester-trihydrochloridsalt (IV) Z Z(d) L-Histidyl-L-histidyl-L-phenylalanyl methyl ester trihydrochloride salt (IV) Z Z

En blanding af 29 g Z-Åis-His-Phe-OMe (IV), 10,8 "1 koncentreret HC1, 180 ml MeOH og to spatelfulde 10%'s Pd-C hydrogeneres i 5 timer under anvendelse af et Parr-hydrogeneringsapparatur.A mixture of 29 g of Z-Åis-His-Phe-OMe (IV), 10.8 "1 concentrated HCl, 180 ml of MeOH and two spatulate 10% Pd-C is hydrogenated for 5 hours using a Parr hydrogenation apparatus. .

Efter filtrering koncentreres methanolet til en olie, og der tilsættes ether til dannelse af et fast stof. 18 g råmateriale anvendes til den næste reaktion uden yderligere rensning.After filtration, the methanol is concentrated to an oil and ether is added to give a solid. 18 g of raw material are used for the next reaction without further purification.

(e) Tert.-butyloxycarbonyl-S-p-methoxybenzyl-L-cysteiny1-L-histidy1--L-histidyl-L-phenylalanyl-methylester (V) 13 g (0,038 mol) Boc-Cys(MBzl)-OH, 5,4 g (0,040 mol) hydroxy--benztriazol og 8,3 g (0,040 mol) dicyclohexylcarbodiimid omrøres i 1 liter iskoldt methylenchlorid i 30 minutter. Der tilsættes 19,5 g (0,0345 mol) His-His-Phe-OMe-3HCl og 10,5 ml (0,096 mol) N-methyl- 28 150618 morpholin, og blandingen omrøres natten over, efterhånden som den får stuetemperatur. Suspensionen filtreres til fjernelse af dicyclohexyl-urinstoffet, og filtratet vaskes successivt med vand, 10%'s I^CO^ og H20 og tørres over Na2S04· Efter filtrering fjernes opløsningsmidlet i vakuum, hvorved der bliver 21,0 g råprodukt tilbage. Dette materiale omrøres i 100 ml acetonitril i 30 minutter ved stuetemperatur og filtreres. Bundfaldet vaskes med acetonitril og derpå med ether og tørres. Udbytte: 11 g.(e) Tert-butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl methyl ester (V) 13 g (0.038 mol) of Boc-Cys (MBzl) -OH, 5, 4 g (0.040 mol) of hydroxy-benztriazole and 8.3 g (0.040 mol) of dicyclohexylcarbodiimide are stirred in 1 liter of ice-cold methylene chloride for 30 minutes. 19.5 g (0.0345 mole) of His-His-Phe-OMe-3HCl and 10.5 ml (0.096 mole) of N-methyl-morpholine are added and the mixture is stirred overnight as it reaches room temperature. The suspension is filtered to remove the dicyclohexyl urea and the filtrate is washed successively with water, 10% I 2 CO 2 and H2 O and dried over Na 2 SO 4 · After filtration, the solvent is removed in vacuo to leave 21.0 g of crude product. This material is stirred in 100 ml of acetonitrile for 30 minutes at room temperature and filtered. The precipitate is washed with acetonitrile and then with ether and dried. Yield: 11 g.

Tyndtlagskromatografi: Rf 0,12,silicagel 60, CHC13:CH3:OH-HAc, 9:1:0,5, r/r.Thin layer chromatography: Rf 0.12, silica gel 60, CHCl3: CH3: OH-HAc, 9: 1: 0.5, r / r.

Filtratet fra acetonitrilekstraktionen indeholder produkt og urenheder og kan anvendes til ekstraktion af andre råcharger.The filtrate from the acetonitrile extract contains product and impurities and can be used for extraction of other crude charges.

(f) Tert.-butyloxycarbonyl-S-p-methoxybenzyl-L-cysteinyl-L-histidyl--L-histidyl-L-phenylalanin (VI) 11,0 g (0,0142 mol) Boc-Cys(MBzl)-His-His-Phe-OMe opløses i 200 ml methanol, og der tilsættes 30 ml IN kaliumhydroxid. Opløsningen omrøres ved stuetemperatur i 2 timer, og methanolet fjernes i vakuum. Remanensen fortyndes med 200 ml vand og filtreres. Filtratet indstilles på en pH-værdi på 5 med 10%‘s citronsyre, og det gummi-agtige bundfald henstilles, efterhånden som det krystalliserer. Produktet filtreres, vaskes med vand og tørres i vakuum over P20j-. Udbytte: 7,3 g (67%).(f) Tert-Butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-L-histidyl - L-histidyl-L-phenylalanine (VI) 11.0 g (0.0142 mol) Boc-Cys (MBzl) -His His-Phe-OMe is dissolved in 200 ml of methanol and 30 ml of 1 N potassium hydroxide is added. The solution is stirred at room temperature for 2 hours and the methanol is removed in vacuo. The residue is diluted with 200 ml of water and filtered. The filtrate is adjusted to a pH of 5 with 10% citric acid and the gummy precipitate is allowed to crystallize. The product is filtered, washed with water and dried in vacuo over P 2 O 2. Yield: 7.3 g (67%).

Tyndtlagskromatografi: Rf 0,52, silicagel 60, n-butanol:ethyl-acetat:eddikesyre:H20, 1:1:1:1, r/r, Rf 0,43, silicagel 60, tert.--amylalkohol:pyridin:H20, 7:6:7, r/r.Thin layer chromatography: Rf 0.52, silica gel 60, n-butanol: ethyl acetate: acetic acid: H 2 O, 1: 1: 1: 1, r / r, Rf 0.43, silica gel 60, tert-amyl alcohol: pyridine: H20, 7: 6: 7, r / r.

(g) N-Tert.-butyloxycarbonyl-O-benzyl-L-threonyl-L-phenylalanin--methylester (VII)(g) N-Tert-butyloxycarbonyl-O-benzyl-L-threonyl-L-phenylalanine - methyl ester (VII)

En opløsning af 61,8 g (0,2 mol) Boc-L-Thr(Bzl)-OH og 22,4 ml (0,2 mol) N-methylmorpholin i THF afkøles til -15°C. Der tilsættes portionsvis 26,2 ml (0,2 mol) isobutylchlorformiat, idet temperaturen holdes på -15 - -10°C. Efter omrøring ved -15°C i 15 minutter tilsættes portionsvis en kold blanding af 43,1 g (0,2 mol) L-Phe-OMe-HCl og 22,4 ml (0,2 mol) N-methylmorpholin i DMF, idet temperaturen holdes på -10 - -5°C. Blandingen omrøres ved 0°C i 2 timer og derpå ved stuetemperatur natten over. Den filtrerede reaktionsblanding koncentreres i vakuum, og remanensen optages i ethylacetat. Ethylacetat-opløsningen vaskes fortløbende med 5%'s KHSO^, 5%'s KHC03 og salt- ?9 150618 opløsning og tørres over Na^SO^. Efter koncentration i vakuum fås en olie, som krystalliserer ved henstand. Det faste stof omkrystalliseres fra isopropylether:hexan. Udbytte: 74,9 g (80%). Smp. 78-81°C, 24 [<x]d + 10,75 (c 1,023, MeOH) , Rf (CHCI3) 0,35.A solution of 61.8 g (0.2 mole) of Boc-L-Thr (Bzl) -OH and 22.4 ml (0.2 mole) of N-methylmorpholine in THF is cooled to -15 ° C. 26.2 ml (0.2 mole) of isobutyl chloroformate are added portionwise keeping the temperature at -15 - -10 ° C. After stirring at -15 ° C for 15 minutes, a cold mixture of 43.1 g (0.2 mole) of L-Phe-OMe-HCl and 22.4 ml (0.2 mole) of N-methylmorpholine in DMF is added portionwise. keeping the temperature at -10 - -5 ° C. The mixture is stirred at 0 ° C for 2 hours and then at room temperature overnight. The filtered reaction mixture is concentrated in vacuo and the residue is taken up in ethyl acetate. The ethyl acetate solution is washed continuously with 5% KHSO 3, 5% KHCO 3 and brine solution and dried over Na 2 SO 4. After concentration in vacuo, an oil is obtained which crystallizes on standing. The solid is recrystallized from isopropyl ether: hexane. Yield: 74.9 g (80%). Mp. 78-81 ° C, 24 [≤ x] d + 10.75 (c 1.023, MeOH), R f (CHCl 3) 0.35.

Analyse for C26H34N2°6: (4?0/55) C% H% N%Analysis for C26H34N2 ° 6: (4? 0/55) C%

Beregnet: 66,36 7,28 5,95Calcd: 66.36 7.28 5.95

Fundet: 66,72 7,32 5,85 (h) N-Tert.-butyloxycarbonyl-O-benzyl-L-threonin-L-phenylalanin (VIII) 23,5 g (0,05 mol) Boc-Thr(Bzl)-Phe-OMe opløses i en blanding af 100 ml MeOH:dioxan (1:1) og behandles med 55 ml IN NaoH i 3 timer, indtil udgangsmaterialet ikke kan påvises ved tyndtlagskromatografi.Found: 66.72 7.32 5.85 (h) N-Tert.-Butyloxycarbonyl-O-benzyl-L-threonine-L-phenylalanine (VIII) 23.5 g (0.05 mole) Boc-Thr (Bzl) ) -Phe-OMe is dissolved in a mixture of 100 ml of MeOH: dioxane (1: 1) and treated with 55 ml of 1N NaoH for 3 hours until the starting material cannot be detected by thin layer chromatography.

Det meste af opløsningsmidlet inddampes i vakuum, og remanensen fortyndes med vand, hvorpå den syrnes med 5%'s citronsyre. Det udskilte gummiagtige stof optages i EtOAc og vaskes med vand (saltopløsning), hvorpå det inddampes til tørhed. Remanensen krystalliseres fra Et20:hexan. Udbytte 19,8 g (87%). Smp. 118-119°C.Most of the solvent is evaporated in vacuo and the residue is diluted with water and then acidified with 5% citric acid. The separated rubbery substance is taken up in EtOAc and washed with water (brine) and then evaporated to dryness. The residue is crystallized from Et 2 O: hexane. Yield 19.8 g (87%). Mp. 118-119 ° C.

(i) N-Tert.-butyloxycarbonyl-O-benzyl-L-threonyl-O-benzyl-L-serin--methylester (IX) 30,9 g (0,1 mol) N-tert.-butyloxycarbonyl-O-benzyl-L-threonin opløses i 200 ml tørt tetrahydrofuran, afkøles ved -20°C og behandles med 11 ml N-methylmorpholin og derpå med 13,4 ml isobutylchlorformiat. Den kolde reaktionsblanding omrøres i 5 minutter ved -20°C, hvorpå den behandles med en opløsning af O-benzyl-L-serin—.methylester og 25 g (ca. 0,1 mol) hydrogenchlorid indeholdende 11 ml N-methylmorpholin i DME, og blandingen henstilles natten over for at få stuetemperatur .(i) N-Tert.-butyloxycarbonyl-O-benzyl-L-threonyl-O-benzyl-L-serine methyl ester (IX) 30.9 g (0.1 mole) of N-tert.-butyloxycarbonyl-O-benzyl benzyl-L-threonine is dissolved in 200 ml of dry tetrahydrofuran, cooled at -20 ° C and treated with 11 ml of N-methylmorpholine and then with 13.4 ml of isobutyl chloroformate. The cold reaction mixture is stirred for 5 minutes at -20 ° C, then treated with a solution of O-benzyl-L-serine methyl ester and 25 g (about 0.1 mole) of hydrogen chloride containing 11 ml of N-methylmorpholine in DME. , and the mixture is left to stand overnight to get room temperature.

Opløsningsmidlet fjernes i vakuum, og remanensen fordeles imellem vand og ethylacetat. Den organiske fase vaskes med 5%'s citronsyre, vand, vandigt KHCO^ og vand og tørres over MgSO^, hvorpå den inddampes til tørhed, hvorved fås en olieformig remanens, som krystalliserer fra diethylether:hexan til et geléagtigt fast stof. Udbytte: 29 g.The solvent is removed in vacuo and the residue partitioned between water and ethyl acetate. The organic phase is washed with 5% citric acid, water, aqueous KHCO 3 and water and dried over MgSO 4, then evaporated to dryness to give an oily residue which crystallizes from diethyl ether: hexane to a jelly solid. Yield: 29 g.

Rf (CHCl3:MeOH, 25:1) 0,85, Rf (EtOAc:hexan, 1:1) 0,65.Rf (CHCl 3: MeOH, 25: 1) 0.85, Rf (EtOAc: hexane, 1: 1) 0.65.

1 b O 618 (j) N-Tert.-butyloxycarbonyl-O-benzyl-L-threonyl-O-benzyl-L-serin (X) 28.2 g (0,0563 mol) Boc-Thr(Bzl)-Ser(Bzl)-OMe opløses i ca. 50 ml methanol og behandles med 75 ml IN natriumhydroxid i 1,5 timer ved stuetemperatur. Den alkaliske opløsning neutraliseres til en pH-værdi på 7 med 10%'s citronsyre, og størstedelen af methanolet fjernes i vakuum. Remanensen fortyndes med noget vand og syrnes med 5%'s vandigt KHSO^, hvorpå den ekstraheres med EtOAc. Den organiske fase vaskes med H20, tørres over Na2S04 og inddampes til tørhed til en olie. Udbytte: kvantitativt.1 b O 618 (j) N-Tert-butyloxycarbonyl-O-benzyl-L-threonyl-O-benzyl-L-serine (X) 28.2 g (0.0563 mol) Boc-Thr (Bzl) -Ser (Bzl ) -To dissolve in approx. 50 ml of methanol and treated with 75 ml of 1N sodium hydroxide for 1.5 hours at room temperature. The alkaline solution is neutralized to a pH of 7 with 10% citric acid and the majority of the methanol is removed in vacuo. The residue is diluted with some water and acidified with 5% aqueous KHSO 4, then extracted with EtOAc. The organic phase is washed with H 2 O, dried over Na 2 SO 4 and evaporated to dryness to an oil. Yield: quantitative.

(EtOAc:hexan, 1:1) 0,15 (lang plet).(EtOAc: hexane, 1: 1) 0.15 (long stain).

(k) N-Tert.-butyloxycarbonyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-S--p-methoxybenzyl-L-cystein-benzylester (XI) 24.3 g (50 mmol) Boc-Thr(Bzl)-Ser(Bzl)-OH opløses i 250 ml ace-tonitril og dichlormethan (2:3) og blandes med 25 g (50 mmol) HCys-(SMBzl)-Bzl-TosOH, derpå med 6,8 ml triethylamin og 6,8 g N-hydroxy-benzotriazol, og blandingen afkøles i isbad.(k) N-Tert-butyloxycarbonyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-S - p-methoxybenzyl-L-cysteine-benzyl ester (XI) 24.3 g (50 mmol) Boc-Thr (Bzl) -Ser (Bzl) -OH is dissolved in 250 ml of acetonitrile and dichloromethane (2: 3) and mixed with 25 g (50 mmol) of HCys- (SMBzl) -Bzl-TosOH, then with 6.8 ml of triethylamine and 6.8 g of N-hydroxy-benzotriazole and the mixture is cooled in an ice bath.

Der tilsættes en opløsning af 11 g DCC (53 mmol) i 50 ml ace-tonitril, og reaktionsblandingen omrøres koldt i 2 timer og derpå i 2 dage ved stuetemperatur. Det udskilte DCU frafiltreres, og filtratet inddampes til tørhed. Den olieformige remanens fordeles imellem ethylacetat og vand, og den organiske fase vaskes med 10%'s citronsyre, vand, 5%'s KHC03 og saltopløsning og tørres over Na2S04· Opløsningsmidlet inddampes, og den olieformige remanens krystalliseres fra Et20:hexan, hvorved fås et hvidt fast stof i en mængde på 24,5 g, smp. 87-90°C.A solution of 11 g DCC (53 mmol) in 50 ml acetonitrile is added and the reaction mixture is stirred cold for 2 hours and then for 2 days at room temperature. The separated DCU is filtered off and the filtrate is evaporated to dryness. The oily residue is partitioned between ethyl acetate and water, and the organic phase is washed with 10% citric acid, water, 5% KHCO3 and brine and dried over Na 2 SO 4 · The solvent is evaporated and the oily residue is crystallized from Et 2 O: hexane to give a white solid in an amount of 24.5 g, m.p. 87-90 ° C.

(chloroform:methanol, 25:1) 0,54, spor ved 0,4 og 0,3 25 (heptan:EtOAc, 1:1) 0,64, spor ved 0,25, I2 positiv [a]D -14,7 (C 0,98, DMF).(chloroform: methanol, 25: 1) 0.54, trace at 0.4 and 0.3 (heptane: EtOAc, 1: 1) 0.64, trace at 0.25, I2 positive [a] D -14 7 (C 0.98, DMF).

Elementæranalyse for ^^H^N^SO^: (798,9) C% H% N%Elemental Analysis for ^^ H ^ N ^ SO ^: (798.9) C% H% N%

Beregnet: 66,15 6,56 5,26Calculated: 66.15 6.56 5.26

Fundet: 66,22 6,95 5,29 51 150818 (l) O-Benzyl-L-threonyl-O-benzyl-L-seryl-S-p-methoxybenzyl-L-cystein--benzylester-trifluoracetat (XII) 8 g (10 mmol) Boc-Thr(Bzl)-Ser(Bzl)-Cys(SMBzl)-OBzl blandes med 100 mmol anisol og behandles med 100 ml TFA i 45 minutter. Opløsningsmidlet inddampes i vakuum, og remanensen opløses i tørt Et£0, hvorpå den inddampes til tørhed i højvakuum, hvorved fås en olieformig forbindelse i en mængde på 7,2 g (90%).Found: 66.22 6.95 5.29 51 150818 (1) O-Benzyl-L-threonyl-O-benzyl-L-seryl-Sp-methoxybenzyl-L-cysteine - benzyl ester trifluoroacetate (XII) 8 g ( 10 mmol) Boc-Thr (Bzl) -Ser (Bzl) -Cys (SMBzl) -OBzl are mixed with 100 mmol of anisole and treated with 100 ml of TFA for 45 minutes. The solvent is evaporated in vacuo and the residue is dissolved in dry Et 2 O, then evaporated to dryness in high vacuum to give an oily compound in 7.2 g (90%).

(n-butanol:vand:iseddike, 4:1:1) 0,9, (heptan:EtOAc) 0,0-0,1 lang plet, I2 positiv og nihydrin positiv.(n-butanol: water: glacial acetic acid, 4: 1: 1) 0.9, (heptane: EtOAc) 0.0-0.1 long stain, I2 positive and nihydrine positive.

(m) N-Tert.-butyloxycarbonyl-O-benzyl-L-threonyl-L-phenylalanyl-O--benzyl-L-threonyl-O-benzyl-L-seryl-S-p-methoxy-benzyl-L-cystein--benzylester (XIII) 9,2 g (20 mmol) Boc-Thr(Bzl)-Phe-OH fremstillet ved den i eksempel (c) angivne fremgangsmåde opløses i 100 ml DFM og blandes med 3,4 g N-hydroxysuccinimid og en opløsning af Thr(Bzl)Ser(Bzl)--Cys(SMBzl)OBzl-TFA-salt (20 mmol) i 20 ml DMF neutraliseret med triethylamin til en pH-værdi på 7. Blandingen afkøles i isbad og behandles med 4,5 g DCC i 2 timer under kulde og derpå i 2 dage ved stuetemperatur. DCU filtreres, og filtratet inddampes til tørhed. Remanensen finsønderdeles med vand til dannelse af et bundfald, som optages i EtOAc, vaskes med 5%'s citronsyre, vand, 5%'s Na2C03 og vand, tørres over Na2SO^ og inddampes til tørhed. Remanensen bringes til at størkne fra Et20:hexan og kan krystalliseres fra MeOH (letopløselig i CHCl^). Udbytte: 17,3 g (70%). Smp. 105-107°C.(m) N-Tert-butyloxycarbonyl-O-benzyl-L-threonyl-L-phenylalanyl-O - benzyl-L-threonyl-O-benzyl-L-seryl-Sp-methoxy-benzyl-L-cysteine benzyl ester (XIII) 9.2 g (20 mmol) of Boc-Thr (Bzl) -Phe-OH prepared by the procedure of Example (c) are dissolved in 100 ml of DFM and mixed with 3.4 g of N-hydroxysuccinimide and a solution of Thr (Bzl) Ser (Bzl) - Cys (SMBzl) OBzl-TFA salt (20 mmol) in 20 ml of DMF neutralized with triethylamine to a pH of 7. The mixture is cooled in an ice bath and treated with 4.5 g DCC for 2 hours under cold and then for 2 days at room temperature. The DCU is filtered and the filtrate is evaporated to dryness. The residue is triturated with water to give a precipitate which is taken up in EtOAc, washed with 5% citric acid, water, 5% Na 2 CO 3 and water, dried over Na 2 SO 4 and evaporated to dryness. The residue is solidified from Et 2 O: hexane and can be crystallized from MeOH (readily soluble in CHCl3). Yield: 17.3 g (70%). Mp. 105-107 ° C.

Rf (CHCl3:MeOH, 10:1) 0,90, [α]§5-3,6 (C 1, DMF).Rf (CHCl3: MeOH, 10: 1) 0.90, [α] §5-3.6 (C1, DMF).

Analyse for (1156,2) C% H% N%Analysis for (1156.2) C% H% N%

Beregnet: 66,46 6,71 6,05Calculated: 66.46 6.71 6.05

Fundet: 66,68 6,59 6,20 32 150618 (n) Tert.-butyloxycarbonyl-D-tryptophyl-N^-benzyloxycarbonyl-L-lysyl--methylester (XIV) 30,4 g (0,1 mol) Boc-D-Trp-OH, 33 g (0,1 mol) Lys(Z)-OMe-HCl og 13,6 ml triethylamin opløses i 400 ml DMF og afkøles i isbad, derpå tilsættes 22 g dicyclohexylcarbodiimid, og blandingen henstilles natten over ved stuetemperatur. Det udskilte dicyclohexylurinstof filtreres, filtratet koncentreres til et lille volumen, og derpå tilsættes rigeligt med vand til dannelse af et gummiagtigt materiale. Dette materiale optages i ethylacetat og vaskes med 10%'s vandig citronsyre, H20, mættet NaHC03 og H20, tørres over Na2S04 og inddampes til tørhed. Remanensen finsønderdeles med ether og derpå med hexan, hvorved fås et krystallinsk fast stof i en mængde på 28 g.Found: 66.68 6.59 6.20 32 150618 (n) Tert.-Butyloxycarbonyl-D-tryptophyl-N + -benzyloxycarbonyl-L-lysyl methyl ester (XIV) 30.4 g (0.1 mole) Boc -D-Trp-OH, 33 g (0.1 mol) of Lys (Z) -OMe-HCl and 13.6 ml of triethylamine are dissolved in 400 ml of DMF and cooled in an ice bath, then 22 g of dicyclohexylcarbodiimide is added and the mixture is allowed to stand overnight. at room temperature. The separated dicyclohexylurea is filtered, the filtrate is concentrated to a small volume, and then plenty of water is added to form a gummy material. This material is taken up in ethyl acetate and washed with 10% aqueous citric acid, H 2 O, saturated NaHCO 3 and H 2 O, dried over Na 2 SO 4 and evaporated to dryness. The residue is triturated with ether and then with hexane to give a crystalline solid in an amount of 28 g.

Tyndtlagskromatografi: silicagel G, hexan:EtOAc, 1:1, r/r,Thin layer chromatography: silica gel G, hexane: EtOAc, 1: 1, r / r,

Rf 0,3.Rf 0.3.

(o) Tert.-butyloxycarbonyl-L-phenylalanyl-D-tryptophyl-N^-benzyloxy-carbonyl-L-lysin (XV) A. 22 g (44 mmol) Boc-D-Trp-Lys(Z)-OMe sættes til en i forvejen afkølet blanding af 220 ml TEA og 25 ml anisol, og blandingen ararøres i isbad i 30 minutter. TFA inddampes så hurtigt som muligt ved 27°C på en rotationsfordamper, og den resterende olie finsønderdeles flere gange med en opløsning af hexan:ether (2:1), dekanteres og pumpes sluttelig til et gummiagtigt stof. Udbytte 21,3 g (36 mmol).(o) Tert-Butyloxycarbonyl-L-phenylalanyl-D-tryptophyl-N + -benzyloxy-carbonyl-L-lysine (XV) A. 22 g (44 mmol) of Boc-D-Trp-Lys (Z) -Ome are added to a pre-cooled mixture of 220 ml of TEA and 25 ml of anisole and the mixture is stirred in an ice bath for 30 minutes. The TFA is evaporated as quickly as possible at 27 ° C on a rotary evaporator and the residual oil is finely decomposed several times with a solution of hexane: ether (2: 1), finally decanted and pumped to a rubbery substance. Yield 21.3 g (36 mmol).

B. 9,6 g (36 mmol) Boc-Phe-OH, 5,3 g (39 mmol) HOBT og 8 g (39 mmol) DCC omrøres i 500 ml koldt dichlormethan i 20 minutter.B. 9.6 g (36 mmol) of Boc-Phe-OH, 5.3 g (39 mmol) of HOBT and 8 g (39 mmol) of DCC are stirred in 500 ml of cold dichloromethane for 20 minutes.

Der tilsættes 21,3 g (36 mmol) D-Trp-Lys(Z)-OMe-TFA-salt i 2000 ml dichlormethan og derpå 4,25 ml (39 mmol) N-methylmorpholin. Reaktionsblandingen omrøres natten over, efterhånden som den får stuetemperatur. Blandingen filtreres til fjernelse af DCC, og filtratet vaskes med 2 x H20, 2 x 10%'s citronsyre, 2 x mættet NaHCO^ og 2 x vand og tørres over Na2S04· Efter filtrering og inddampning fås remanensen i en mængde på 21,4 g (82%), der kan anvendes uden yderligere rensning.21.3 g (36 mmol) of D-Trp-Lys (Z) -OMe-TFA salt are added in 2000 ml of dichloromethane and then 4.25 ml (39 mmol) of N-methylmorpholine. The reaction mixture is stirred overnight as it gets to room temperature. The mixture is filtered to remove DCC and the filtrate is washed with 2 x H 2 O, 2 x 10% citric acid, 2 x saturated NaHCO 3 and 2 x water and dried over Na 2 SO 4 · After filtration and evaporation, the residue is obtained in an amount of 21.4 g (82%) which can be used without further purification.

Tyndtlagskromatografi: silicagel F-254/CHCl2 9:CHgOH 1: HAc 0,5, Rf 0,76 samt spor af mindre urenheder.Thin layer chromatography: silica gel F-254 / CHCl2 9: CHgOH 1: HAc 0.5, Rf 0.76 and traces of minor impurities.

„ 150618 C. 22,9 g (31,4 mmol) Boc-Phe-D-Trp-Lys(Z)-OMe opløses i 450 ml p-dioxan og 100 ml methanol, der tilsættes 39 ml vandigt IN KOH, og blandingen omrøres natten over ved stuetemperatur. Opløsningen inddampes på en rotationsfordamper. Remanensen opløses i 500 ml varmt vand, syrnes til en pH-værdi på 3 med 10%'s citronsyre, omrøres i 30 minutter og filtreres. Efter tørring i vakuum natten over fås et udbytte på 21,6 g (96%). Smp. 85-90°C. [α]ϋ6= +7,09 c, 0,987 MeOH.Dissolve 22.9 g (31.4 mmol) of Boc-Phe-D-Trp-Lys (Z) -OMe in 450 ml of p-dioxane and 100 ml of methanol, add 39 ml of aqueous 1N KOH and the mixture Stir overnight at room temperature. The solution is evaporated on a rotary evaporator. The residue is dissolved in 500 ml of warm water, acidified to a pH of 3 with 10% citric acid, stirred for 30 minutes and filtered. After drying in vacuo overnight, a yield of 21.6 g (96%) is obtained. Mp. 85-90 ° C. [α] ϋ6 = +7.09 c, 0.987 MeOH.

0,63 CHCl^, 9:CH3OH, l=HAc, 0,5, silicagel F-254.0.63 CHCl3, 9: CH3OH, l = HAc, 0.5, silica gel F-254.

(p) Tert.-butyloxycarbony1-L-phenylalany1-D-tryptophy1-N^-benzyloxy-carbonyl-L-lysyl-O-benzyl-L-threonyl-L-phenylalanyl-Q-benzyl-L-threo-nyl-O-benzyl-L-seryl-S-p-methoxybenzyl-L-cysteinyl-benzylester (XVI) A. 40,5 g (35 mmol) Boc-Thr(Bzl)-Phe-Thr(Bzl)-Ser(Bzl)-Cys(SMBzl)--OBzl (XIII) sættes til en kold opløsning af 400 ml TFA og 40 ml ani-sol, og blandingen omrøres i kold tilstand i 30 minutter. TFA inddampes på en rotationsfordamper, og remanensen finsønderdeles med 1 liter ether:hexan (1:1) til et hvidt pulver. Efter filtrering, vaskning med etherrhexan og tørring i vakuum over °9 NaOH-kugler fås et udbytte på 34,6 g (82%). Smp. 136-139°C, blødgøres ved 120°C. [cx]q6= -9,93 c, 1,04 MeOH. Rf 0,63 silicagel CHC13, 9:CH3OH, l:HAc, 0,05 lille mere polær urenhed. R^ 0,53.(p) Tert-Butyloxycarbonyl-L-phenylalanyl-D-tryptophyl-N + -benzyloxy-carbonyl-L-lysyl-O-benzyl-L-threonyl-L-phenylalanyl-Q-benzyl-L-threonyl-O -benzyl-L-seryl-Sp-methoxybenzyl-L-cysteinyl-benzyl ester (XVI) A. 40.5 g (35 mmol) of Boc-Thr (Bzl) -Phe-Thr (Bzl) -Ser (Bzl) -Cys ( SMBzl) - OBzl (XIII) is added to a cold solution of 400 ml of TFA and 40 ml of anisole and the mixture is stirred in a cold state for 30 minutes. The TFA is evaporated on a rotary evaporator and the residue is triturated with 1 liter ether: hexane (1: 1) to a white powder. After filtration, washing with ether hexane and drying in vacuo over 9 NaOH beads, a yield of 34.6 g (82%) is obtained. Mp. 136-139 ° C, softened at 120 ° C. [cx] q6 = -9.93 c, 1.04 MeOH. Rf 0.63 silica gel CHCl 3, 9: CH 3 OH, 1: HAc, 0.05 slightly more polar impurity. R f 0.53.

B. 20,0 g (27,9 mmol) Boc-Phe-D-Trp-Lys(Z)-OH, 4,15 g (30,8 mmol) HOBT og 6,3 g (30,8 mmol) DCC omrøres i 2,0 liter koldt dichlormethan 1 isbad i 30 minutter. Der tilsættes 32,6 g (27,9 mol) H-Thr(Bzl)--Phe-Thr(Bzl)-Ser(Bzl)-Cys(SMBzl)-OBzl-TFA-salt og derp* 3,35 ml (30,8 mmol) N-methylmorpholin, og omrøringen fortsættes natten over, efterhånden som blandingen opvarmes til stuetemperatur. Tyndtlags-kromatografi viser, at reaktionen praktisk taget er afsluttet efter 2 timer.B. 20.0 g (27.9 mmol) of Boc-Phe-D-Trp-Lys (Z) -OH, 4.15 g (30.8 mmol) of HOBT and 6.3 g (30.8 mmol) of DCC Stir in 2.0 liters of cold dichloromethane 1 ice bath for 30 minutes. 32.6 g (27.9 mol) of H-Thr (Bzl) - Phe-Thr (Bzl) -Ser (Bzl) -Cys (SMBzl) -OBzl-TFA salt are added and therep3.35 ml ( 30.8 mmol) of N-methylmorpholine and stirring is continued overnight as the mixture is warmed to room temperature. Thin layer chromatography shows that the reaction is practically complete after 2 hours.

Blandingen filtreres, og filtratet vaskes med vand, 10%'s citronsyre, vand, mættet NaHCO^ og vand og tørres. Efter filtrering og inddampning viser tyndtlagskromatografi produkt og en mere polær urenhed, som fjernes ved, at råproduktet opløses i 200 ml varmt DMF, og der tilsættes 1 liter 2%'s vandigt NaHCO^. Produktet filtreres, vaskes med fortyndet NaHCO^ og vand og tørres i vakuum over ‘ Udbytte: 36,5 g (75%). Smp. 182-184°C (svinder ved 150°C) . [a]j*°= 0 c, 0,988 150618 34 CHCl^· &£ (enkelt plet) 0,76, silicagel CHClg, QtCH^OH, l:HAc, 0,05.The mixture is filtered and the filtrate is washed with water, 10% citric acid, water, saturated NaHCO 3 and water and dried. After filtration and evaporation, thin layer chromatography shows product and a more polar impurity which is removed by dissolving the crude product in 200 ml of hot DMF and adding 1 liter of 2% aqueous NaHCO 3. The product is filtered, washed with dilute NaHCO 3 and water and dried in vacuo over Yield: 36.5 g (75%). Mp. 182-184 ° C (decays at 150 ° C). [.alpha.] D @ 20 = 0 c, 0.988 CHCl3 (single spot) 0.76, silica gel CHCl3, QtCH2 OH, l: HAc, 0.05.

(q) H-Phe-D-Trp-Lys(Z)-Thr(Bzl)-Phe-Thr(Bzl)-Ser(Bzl)-Cys(SMBzl)-OBzl--trifluoracetatsalt (XVII) 32 g Boc-Phe-D-Trp-Lys(Z)-Thr(Bzl)-Phe-Thr(Bzl)-Ser(Bzl)-Cys-(SMBzl)-OBzl og 33 ml anisol opløses i 50 ml dichlormethan, der tilsættes 100 ml trifluoreddikesyre, og blandingen omrøres i 30 minutter ved stuetemperatur. Opløsningen inddampes på en rotationsfordamper ved 30°C. Remanensen finsønderdeles med vandfrit ether til et pulver, som filtreres, vaskes med ether og tørres i vakuum over ?2°5 °9 natriumhydroxidperler.(q) H-Phe-D-Trp-Lys (Z) -Thr (Bzl) -Phe-Thr (Bzl) -Ser (Bzl) -Cys (SMBzl) -OBzl - trifluoroacetate salt (XVII) 32 g Boc-Phe -D-Trp-Lys (Z) -Thr (Bzl) -Phe-Thr (Bzl) -Ser (Bzl) -Cys- (SMBzl) -OBzl and 33 ml of anisole are dissolved in 50 ml of dichloromethane, which is added 100 ml of trifluoroacetic acid, and the mixture is stirred for 30 minutes at room temperature. The solution is evaporated on a rotary evaporator at 30 ° C. The residue is triturated with anhydrous ether to a powder which is filtered, washed with ether and dried in vacuo over 2 ° 5 ° 9 sodium hydroxide beads.

Tyndtlagskromatografi på silicagel 60 i CHCl^iCH^OHiHOAC (9:1:0:5) viser en enkelt plet. R_p = 0,65. Kernemagnetresonansspektroskopi (HNMR) viser ingen top for tert.butyl.Thin layer chromatography on silica gel 60 in CHCl3 / CH2 OHiHOAC (9: 1: 0: 5) shows a single stain. R_p = 0.65. Nuclear Magnetic Resonance Spectroscopy (HNMR) shows no peak for tert-butyl.

(r) Tert.-butyloxycarbonyl-S-p-methoxybenzyl-L-cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-N^-benzyloxycar-bonyl-L-lysyl-O-benzyl-L-threonyl-L-phenylalanyl-O-benzyl-L-threonyl--O-benzyl-L-seryl-S-p-methoxybenzyl-L-cystein-benzylester (XVIII) 5,5 g (7,2 mmol) Boc-Cys(SMBzl)-His-His-PheOH, 1,07 g (7,9 mmol) 1-hydroxy-benztriazol-monohydrat og 1,65 g (7,9 mmol) dicyclohexyl-carbodiimid omrøres i 100 ml tørt dimethylformamid i isbad i 30 minutter. Der tilsættes 12,5 g (7,2 mmol) H-Phe-D-Trp-Lys(z)-Thr(Bzl)-Phe--Thr(Bzl)-Ser(Bzl)-Cys(SMBzl)OBzl-trifluoracetatsalt og 0,855 g (7,65 mmol) N-methylmorpholin, og blandingen henstilles til opnåelse af stuetemperatur natten over med omrøring. Suspensionen filtreres til fjernelse af dicyclohexylurinstof, og filtratet inddampes til 50 ml og fortyndes med 500 ml mættet vandig natriumhydrogencarbonatopløsning. Det fremkomne faste stof filtreres, vaskes med vand og opløses på ny i 200 ml DMF. Opløsningen fortyndes med 1 liter 5%'s vandig citronsyre og filtreres. Det faste stof vaskes med vand, opløses i 200 ml DMF og udfældes med 1 liter mættet natriumhydrogencarbonatopløsning, filtreres, vaskes med vand og tørres i vakuum over P2®5 ve^ stuetemperatur. Udbytte: 15 g.(r) Tert-Butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-N N-benzyloxycarbonyl-L-lysyl-O-benzyl -L-threonyl-L-phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-Sp-methoxybenzyl-L-cysteine-benzyl ester (XVIII) 5.5 g (7.2 mmol) Cys (SMBzl) -His-His-PheOH, 1.07 g (7.9 mmol) of 1-hydroxybenztriazole monohydrate and 1.65 g (7.9 mmol) of dicyclohexylcarbodiimide are stirred in 100 ml of dry dimethylformamide in an ice bath for 30 minutes. 12.5 g (7.2 mmol) of H-Phe-D-Trp-Lys (z) -Thr (Bzl) -Phe - Thr (Bzl) -Ser (Bzl) -Cys (SMBzl) OBzl trifluoroacetate salt are added. and 0.855 g (7.65 mmol) of N-methylmorpholine, and the mixture is allowed to reach room temperature overnight with stirring. The suspension is filtered to remove dicyclohexylurea and the filtrate is evaporated to 50 ml and diluted with 500 ml of saturated aqueous sodium bicarbonate solution. The resulting solid is filtered, washed with water and redissolved in 200 ml of DMF. The solution is diluted with 1 liter of 5% aqueous citric acid and filtered. The solid is washed with water, dissolved in 200 ml of DMF and precipitated with 1 liter of saturated sodium hydrogen carbonate solution, filtered, washed with water and dried in vacuo over P2® 5 room temperature. Yield: 15 g.

Aminosyreanalyse: His 1,66(2,0), Lys 1,05(1,0), Phe (3,0),Amino acid analysis: His 1.66 (2.0), Lys 1.05 (1.0), Phe (3.0),

Ser 1,02(1,0), Thr 2,2 (2,0).Ser 1.02 (1.0), Thr 2.2 (2.0).

55 150618 (s) L-Cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl-L-phenylalanyl--D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-Ii-threonyl-L-seryl--L-cystein-cyclisk (1-12) disulfid (XIX) 5 g Boc-Cys(SMBzl)-His-His-Phe-Phe-D-Trp-Lys(z)-Thr(Bzl)-Phe--Thr(Bzl)-Ser(Bzl)-Cys(SMBzl)OBzl blandes med 10 ml anisol og behandles med 100 ml flydende HF under udelukkelse af luft og i isbad i 60 minutter. Overskydende HF fjernes i vakuum, og remanensen optages i 10%‘s vandig eddikesyre (200 ml) og hældes i 7 liter afluftet vand. pH-Værdien indstilles på 7 med fortyndet NH^OH og omrøres i fri luft i 24 timer, pH-værdien indstilles på 5 med iseddike, og opløsningerne ledes igennem en "Amberlite"®-CG-50-søjle (H^-form). Peptidmaterialet, som absorberes på ionbytterharpiksen, fortyndes med 50%'s vandig eddikesyre, og fraktionerne indeholdende peptidmaterialet hældes sammen og lyofiliseres, hvorved fås 2 g af råmaterialet. Dette rådodecapeptid renses ved søjlekromatografi ved hjælp af "Sephadex"® G 25 og elueringer med 10%'s vandig eddikesyre og fordelingskromatografi ved hjælp af "Sephadex" G 25 med tofasesysternet N-BuOH:vand:-iseddike, 4:5:1, r/r.(S) L-Cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl-L-phenylalanyl - D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-1-threonyl-L-seryl -L-cysteine cyclic (1-12) disulfide (XIX) 5 g Boc-Cys (SMBzl) -His-His-Phe-Phe-D-Trp-Lys (z) -Thr (Bzl) -Phe - Thr (Bzl) -Ser (Bzl) -Cys (SMBzl) OBzl is mixed with 10 ml of anisole and treated with 100 ml of liquid HF under exclusion of air and in an ice bath for 60 minutes. Excess HF is removed in vacuo and the residue is taken up in 10% aqueous acetic acid (200 ml) and poured into 7 liters of de-aerated water. The pH is adjusted to 7 with dilute NH 2 OH and stirred in free air for 24 hours, the pH is adjusted to 5 with glacial acetic acid and the solutions are passed through an "Amberlite" ® CG-50 column (H . The peptide material which is absorbed on the ion exchange resin is diluted with 50% aqueous acetic acid and the fractions containing the peptide material are combined and lyophilized to give 2 g of the crude material. This crude decapapeptide is purified by column chromatography using "Sephadex" ® G 25 and elution with 10% aqueous acetic acid and partition chromatography using "Sephadex" G 25 with two-phase N-BuOH: water: -acetic acid, 4: 5: 1, r / s.

Tyndtlagskromatografi: silicagel (A.G. Merck), chlorpeptidspray, jf. D.E. Nitecki et al. Biochem. i), 665 (1966).Thin-layer chromatography: silica gel (A.G. Merck), chloride peptide spray, cf. D.E. Nitecki et al. Biochem. i), 665 (1966).

Rf (BWA, 4:1:1) 0,45, Rf (BWAP, 30:24:6:20) 0,64.Rf (BWA, 4: 1: 1) 0.45, Rf (BWAP, 30: 24: 6: 20) 0.64.

Aminosyreanalyse: Thr(2) 1,85, Ser(l) 0,79, Phe(3) 3, Lys(l) 1,02, His(2) 1,75, Cys, Trp ND.Amino acid analysis: Thr (2) 1.85, Ser (l) 0.79, Phe (3) 3, Lys (l) 1.02, His (2) 1.75, Cys, Trp ND.

Claims (3)

150818 PATENTKRÅV.150818 PATENT REQUIREMENTS. 1. Analogifremgangsmåde til fremstilling af somatosta-tinanaloge med den almene formel H-Cys-X1-X2-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-X4-OH (I) SA AS hvori A er hydrogen, eller begge A'er tilsammen repræsenterer 1 2 en S-S-binding, X betyder His eller Arg, X betyder His eller 4 Glu, og X betyder Cys eller D-Cys, eller farmaceutisk acceptable salte deraf/ kendetegnet ved, at forbindelser med formlen X-Cys-X1-X2-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-X4-OR (II)An analogous method for preparing somatostatin analogs of the general formula H-Cys-X1-X2-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-X4-OH (I) SA AS wherein A is hydrogen, or both A's together represent 1 2 an SS bond, X means His or Arg, X means His or 4 Glu, and X means Cys or D-Cys, or pharmaceutically acceptable salts thereof / characterized in that compounds having formula X-Cys-X1-X2-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-X4-OR (II) 1. C 1 7 f Q I Q I Sa R6 R7 R8 R9 S3 hvori R betyder en carboxybeskyttelsesgruppe eller en polystyren-harpiksbærer bundet via en methylengruppe, X er H eller en a-amino- I* beskyttelsesgruppe, X betyder His eller Arg ' 2 ’3 R R-3 3 hvori R betyder en beskyttelsesgruppe for sidekædenitrogenato- 2 merne i arginin, og R betyder hydrogen eller en beskyttelsesgrup- 9/ pe for imidazolnitrogenatomet i histidin; X betyder His R2 2 2/ hvori R har den ovenfor angivne betydning, eller X betyder Glu έ4 4 hvor R betyder en beskyttelsesgruppe for sidekædecarboxygruppen i glutaminsyre; R8 betyder en beskyttelsesgruppe for sidekædeamino-gruppen i lysin, R , R og R hver især betyder beskyttelsesgrup- 4 per for hydroxylgrupperne i threonin og serin, X er som ovenfor ‘ anført, og α og β betyder hydrogenatomer, sulfhydrylbeskyttelses-grupper eller tilsammen udgør en direkte binding imellem svovlatomerne, befries for alle beskyttelsesgrupper og fjernes fra den eventuelle polystyrenharpiksbærer, og om nødvendigt oxideres til dannelse af en direkte S-S-binding, og om nødvendigt produktet isoleres som den frie base eller et farmaceutisk acceptabelt salt1. C 17 f QIQI Sa R6 R7 R8 R9 S3 wherein R means a carboxy protecting group or a polystyrene resin support bonded via a methylene group, X is H or an α-amino-1 * protecting group, X means His or Arg '2' 3 R is R 3-3 wherein R is a protecting group for the side chain nitrogen atoms in arginine and R is hydrogen or a protecting group 9 / pe for the imidazole nitrogen atom in histidine; X represents His R2 2 2 / wherein R is as defined above, or X is Glu έ 4 4 where R represents a protecting group for the side chain carboxy group in glutamic acid; R8 means a protecting group for the side chain amino group of lysine, R, R and R each mean protecting groups 4 for the hydroxyl groups of threonine and serine, X is as indicated above and α and β represent hydrogen atoms, sulfhydryl protecting groups or together form a direct bond between the sulfur atoms, is freed of all protecting groups and removed from the optional polystyrene resin support and oxidized, if necessary, to form a direct SS bond, and if necessary the product is isolated as the free base or pharmaceutically acceptable salt