CA2050216C

CA2050216C - Process for the preparation of peptides by solid-phase synthesis

Info

Publication number: CA2050216C
Application number: CA002050216A
Authority: CA
Inventors: Gerhard Breipohl; Jochen Knolle; Wolfgang Konig
Original assignee: Hoechst AG
Current assignee: Hoechst AG
Priority date: 1990-08-30
Filing date: 1991-08-29
Publication date: 2003-03-11
Anticipated expiration: 2011-08-29
Also published as: SK280319B6; HU208838B; FI914064L; IL99338A0; EP0475184A1; RU2036200C1; NZ239575A; TW295589B; NO913396L; PL291561A1; JP3177269B2; FI102380B1; CZ282881B6; HUT60749A; NO913396D0; HU912825D0; IL99338A; PT98813B; DK0475184T3; KR100203548B1

Abstract

A process for the preparation of peptides of the formula (X)n-A-NH2 in which X is a natural or unnatural amino acid, aza-amino acid or imino acid, n is an integer from 1 to 50, preferably 1 to 30, and A is an aza-amino acid, and the physiologically tolerated salts thereof is described. Said process comprises the steps of converting a spacer into a form which is capable of acylation, reacting the latter with a suitable formic acid derivative and subsequently with an appropriate amino hydrazide, where appropriate, converting the protective group into a protective group which is base-labile or labile to weak acids, coupling the spacer obtained in this way to a resin, synthesizing the required peptide stepwise from the C-terminal end, subsequently cleaving the peptide off the resin and, where appropriate, converting it into physiologically tolerated salts thereof.

Description

.o r 2.J
~.~1,(~r;.~6 HOECHST AKTIENGESELLSCHAFT HOE 90/F 257 Dr. WI/je Description A process for the preparation of peptides by solid-phase synthesis The invention relates to a process for the preparation of peptides with C-terminal aza-amino amide by solid-phase synthesis.
The object of the invention is to develop a low-racemiz ation process for the preparation of peptides with C
terminal aza-amino amides by solid-phase synthesis.
This object is achieved according to the invention by the process for the preparation of peptides of the formula I
(~~ - A - tdH~ y) in which 15~~ X is a natural or unnatural amino acid, aza-amino acid or imino acid, n is an integer from 1 to 50, preferably 1 to 30, and A is an aza-amino acid, and the physiologically tolerated salts thereof, which comprises converting a spacer into a form capable of acylation, reacting the latter with a suitable formic acid derivative and subsequently with an appropriate amino hydrazide, where appropriate converting the pro tective group into a protective group which is base labile or labile to weak acids, coupling the spacer obtained in this way to a resin, synthesizing the re quired peptide stepwise from the C-terminal end, subse quently cleaving the peptide off the resin and, where appropriate, converting it into physiologically tolerated salts thereof.
Natural or unnatural amino acids can, if chiral, be in the D or L form. «-Amino acids are preferred.

l4t~uq~?Bf a~_~) Examples which may be mentioned are: Aad, Abu, yAbu, AHz, 2ABz, eAca, Ach, Acp, Adpd, Ahb, Aib, ~Aib, Ala, ,BAla, ~Ala, Alg, All, Ama, Amt, Ape, Apm, Apr, Arg, Asn, Asp, Asu, Aze, Azi, Hai, Bph, Can, Cit, Cys, (Cys)2, Cyta, Daad, Dab, Dadd, Dap, Dapm, Dasu, Djen, Dpa, Dtc, Fel, Gln, Glu, GIy, Guv, hAla, hArg, hCys, hGln, hGlu, His, hIle, hLeu, hLys, hMet, hPhe, hero, hSer, hThr, hTrp, hTyr, Hyl, Hyp, 3Hyp, Ile, Ise, Iva, Ryn, Lant, Lcn, Leu, Lsg, Lys, pLys, nLys, Met, Mim, Min, nArg, Nle, Nva, Oly, Orn, Pan, Pec, Pen, Phe, Phg, Pic, Pro, Pro, Pse, Pya, Pyr, Pza, Qin, Ros, Sar, Sec, Sem, Ser, Thi, pThi, Thr, Thy, Thx, Tia, Tle, Tly, Trp, Trta, Tyr, Val, Nal, Tbg, Npg, Cha, Chg, Thia (cf., for example, Houben-Weyl, Methoden der organischen Chemie (Methods of organic chemistry), Volume XV/1 and 2, Stuttgart, 1974).
Aza-amino acids are derived from natural or unnatural amino acids with the central -CHR- or -CHZ- unit being replaced by -NR- or -NH- respectively. Examples which may be mentioned are azaglycine, azavaline, azaleucine, azaisoleucine and azaphenylalanine.
By an imino acid are meant in general natural or un-natural amino acids whose amino group is monosubstituted.
Particular mention may be made in this connection of compounds which are substituted by C1-C8-alkyl, which in turn is optionally mono- or diunsaturated and can be substituted by up to 3 identical or different radicals from the series comprising mercapto; hydroxyl; C1-C~-alkoxy; carbamoyl; C1-Cs-alkanoyloxy; carboxyl; C1-C~-alkoxycarbonyl; F; C1; Br; I; amino; amidino' which can optionally be substituted by one, two or three Cl-C8-alkyl radicals; guanidino which can optionally be substituted by one or two benzyloxycarbonyl radicals or by one, two, three or four C~-CB-alkyl radicals; C1-C~-alkylamino; di-Cl-C~-alkylamino; C1-C6-alkoxycarbonylamino; C~-C~5-aral-koxycarbonyl; C~-C15-aralkoxycarbonylamino; phenyl-C1-C,-alkoxy; 9-fluorenylmethoxyearbonylamino; C1-Cs-alkylsul-fonyl; C1-C6-alkylsulfinyl; C1-Cs-alkylthio; hydroxyamino;

3 ~:~~~r.Mx_~i hydrox;rimino; sulfataoyl.
Also suitable are heterocycles from the following group:
pyrrolidine-2-carboxylic acid; piperidine-2-carboxylic acid; 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;
decahydroisoquinoline-3-carboxylic acid;octahydroindole-2-carboxylic acid; decahydroquinoline-2-carboxylic acid;
octahydrocyclopenta[b]pyrrole-2-carboxylic acid; 2-aza-bicyclo[2.2.2]octane-3-carboxylic acid; 2-azabicyclo-[2.2.1]heptane-3-carboxylic acid; 2-azabicyclo[3.1.0]-hexane-3-carboxylic acid; 2-azaspiro[4.4]nonane-3-car-boxylic acid; 2-azaspiro[4.5]decane-3-carboxylic acid;
spiro[(bicyclo[2.2.1]heptane)-2,3-pyrrolidine-5-car-boxylic acid; spiro[(bicyclo[2.2.2]octane)-2,3-pyrroli-dine-5-carboxylic acid; 2-azatricyclo[4.3Ø16'9]decane-3-carboxylic acid; decahydrocyclohepta[b]pyrrole-2-carboxylic acid; octahydrocyclopenta[c]pyrrol-2-car-boxylic acid; octahydroisoindole-1-carboxylic acid;
2,3,3a,4,6a-hexahydrocyclopenta[b]pyrrole-2-carboxylic acid; 2,3,3a,4,5,7a-hexahydroindole-2-carboxylic acid;
tetrahydrothiazole-4-carboxylic acid; isoxazolidine-3-carboxylic acid; pyrazolidine-3-carboxylic acid; hydroxy-proline-2-carboxylic acid; all of which can optionally be substituted:

_ ø _ -co- ; - ;
ce ., 4 ~:i$~
co- co-: : ~co s ~co-I
~~co- ;
_i rv '~ CO- f ~ CO- ; ~ CO
I

,~ C O-~t-,~J- i NCO-' N
I I
~ CJ- ~CO- ; ~CrJ- :
V/~ N
N I
C
~ co- ; ' co- :
N NCO- ; \ N CO- ; ~CO-NN
NO
w _ _ O~NrCp ' N I~--CO ~ ~CO- :
o By salts of compounds of the formula I are meant, in particular, pharmaceutically utilizable or non-toxic salts . Particularly suitable are alkali metal or alkaline earth metal salts, salts with physiologically tolerated amines and salts with inorganic or organic acids, such as, for example, HC1, HBr, HZS04, H3P04, malefic acid, fumaric acid, citric acid, tartaric acid and acetic acid.

The process is advantageously carried out in such a way that a compound of the formula II
(II) in which Y1, YZ, Y3, Y' and YS are hydrogen, C1-C,-alkyl, Cl-C4-alkoxy or -O- ( CHZ ) n-COOH, - ( CHZ ) n-COOH or -r1H-co- ( cH2 ) A cooH, where the radicals can be identical or different, but at least one radical is -O-(CHZ)a-COON, - ( CH2 ) n-COON or -NH-CO- ( CHZ ) n-COON, n is an integer from 1 to 6, preferably 1 to 3, and R1 is hydrogen or C1-C6-alkoxy-C6-C12-aryl, preferably 4-methoxyphenyl, is reacted with a silylating reagent, for example tert.
butyldimethylsilyl chloride, tart. butylphenylsilyl chloride, trimethylchlorosilane, especially trimethylchlorosilane, in a solvent suitable for this purpose, such as, for example, THF, acetonitrile, methylene chloride, dimethylformamide or mixtures thereof, and subsequently the silylated compound is converted with a chloroformic acid derivative, especially the substituted ester derivatives, into compounds of the formula III
(III) ROC -in which R1, Y1, Y2, Y3, Y'' and YS are as defined above, and - s - ~:f)~~~w~
R2 is a C6-C~-aryl radical which is substituted by electron-attracting substituents, preferably nitro and halogen, for example F or C1, the compounds of the formula III obtained in this way are reacted with an amino hydrazide of the formula IV
R'-X-CO-NH-NH-R' (IV) in which X is a natural or unnatural amino acid or imino acid and is as defined above, R3 is a protective group which is base-labile or labile to weak acids or hydrogenation, such as, for ex-ample, a urethane protective group (see, for ex-ample, Hubbuch, Kontakte (Merck) 1979, No. 3, pages 14 to 23), and R' is C1-C8-alkyl, C3-Cg-cycloalkyl, Cs-C~-aryl, C6-Cla-aryl-C1-C8-alkyl, heteroaryl or heteroaryl-C1-Ca-alkyl or hydrogen, in a solvent in which the compounds of the formula III
and IV are soluble, such as, for example, DMF, to give 20~ the compounds of the formula V

(V) in which R1, R3, R' and y1, yz, y3, y4 and YS have the abovementioned meanings, if R3 is a protective group which is labile to hydrogenation, preferably benzyloxycarbonyl, this protective group is removed by hydrogenation on a Pd catalyst and, before the subsequent reaction, converted into a base-labile urethane protective group, preferably Fmoc, or a urethane protective group which is labile to - ~ - 2~3J~s ~:~~
weak acids, preferably Bpoc, subsequently the compound of the formula V in which R1, R°, Y1, Y2, Y3, Y° and YS have the abovementioned meanings, and R3 is a urethane protective group which is labile to bases or weak acids, is coupled with the coupling reagents customary in peptide chemistry via the -O- ( CH2 ) a-COON, - ( CHZ ) n-COOH or -NH-CO- ( CHZ ) E COOH group to a resin, the protective group R3 is eliminated, natural or unnatural amino, imino or aza-amino acids which have been temporarily protected by amino-protective groups which are base-labile or labile to weak acids and which are optionally in the form of their activated derivatives are coupled on stepwise, and, after synthesis is complete, the peptides of the formula I are liberated from the resin by treatment with a moderately strong acid, with elimination again, simultaneously or by suitable measures subsequent thereto, of temporarily introduced side-chain protective groups.
The compounds of the formula IV are prepared by reacting the natural or unnatural amino acids or imino acid with the appropriate hydrazines by coupling methods customary in peptide chemistry.
Alkyl can be straight-chain or branched. A corresponding statement applies to radicals derived therefrom, such as, for example, alkoxy, alkylthio, alkylamino, dialkylamino and alkanoyl. Alkyl is, in particular, C1-C~-alkyl.
Cycloalkyl also means alkyl-substituted radicals such as, for example, 4-methylcyclohexyl or 2,3-dimethylcyclo-pentyl.
Halogen is fluorine, chlorine, bromine or iodine, espe-cially fluorine or chlorine.
Cs-C1z-aryl is, for example, phenyl or naphthyl, prefer-ably phenyl. Heteroaryl is the radical of a 5- to 7-membered monocyclic or 8- to 10-membered bicyclic - 8 - ~:~~~E~?~~
aromatic ring system which can be benzo-fused and which can contain as hetero elements one, two, three or four different radicals from the group comprising N, 0, S, NO, SO and SOZ and which is substituted by 1 to 6 hydroxyl or by one, two or three identical or different radicals from the series comprising F, Cl, Br, I; hydroxyl, mono-, di-or trihyd,roxy-C~-C4-alkyl; trifluorcenethyl; forrnyl; carboxamido;
mono- or di-C~-C4-alkylaminocarbonyl; nitro; C~-C~ alkoxy: C~'~7-alkyl; C1-C~-alkoxycarbonyl; amino; C1-C~-alkylamino; di-C1-C~-alkylamino; carboxyl; carboxymethoxy; amino-Cl-C~-alkyl; C1-C~-alkyiamino-C1-C~-alkyl; di-C1-C~-alkylamino-C1-C~-alkyl; C1-C~-alkoxycarbonylmethoxy; carbamoyl;
sulfamoyl; C1-C~-alkoxysulfonyl; C,-CA-alkylsulfonyl;
sulfo-C~-C8 alkyl; guanidino-C~-C8 alkyl and C~-C6 alkc~xycarbonyl and/or mono-, di- or trisubstituted by oxo. Particular mention may be made of: furyl, thienyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrimidinyl, indolyl, isoindolyl, indazolyl, phthalazinyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, cinnolinyl, p-carbolinyl or a benzo-fused derivative of these radicals.
If necessary to prevent side reactions or for the syn-thesis of specific peptides, the functional groups in the side chain of amino, aza-amino and imino acids are additionally protected by suitable protective groups (see, for example, T.W. Greene, "Protective Groups in Organic Synthesis", New York, John Wiley & Sons, 1981;
Hubbuch, Kontakte (Merck) 1979, No. 3, pages 14-23;
Hiillesbach, Rontakte (Merck) 1980, No. 1, pages 23-35), employing primarily Arg(Tos), Arg(Mts), Arg(Mtr), Arg(Pmc), Asp(OBzl), Asp(OtBu), Cys(4-MeHzl), Cys(Acm), Cys(StBu), GIu(OBzI), Glu(OtBu), His(Tos), His{Fmoc), His(Dnp), His(Tzt), Lys{C1-2), Lys(Hoc), Met{0), Ser(Bzl), Ser(tBu), Thr(Bzl), Thr{tBu). It is also possible for the functional groups in the side chain to be glycosylated as described, for example, in EP-A 263 521 (HOE 86/F 253).

- 9 - ~~3~~i:'~~
The resins used as support material are commercially available or prepared by the user, such as, for example, alkoxybenzyl alcohol-resins, aminomethyl-resins or benzhydrylamino-resins. Aminomethyl-, benzhydrylamino-(BHA) and methylbenzhydrylamino-resins (MBHA) are pre-ferred. The loading is determined by amino-acid analysis and/or elemental analysis.
By suitable spacers are meant, for example, the spacers described in Atherton, Sheppard in Perspectives in Peptide Chemistry, pages 101-117 (Rarger, Basel 1981);
EP-A 264 802 (HOE 86/F 259), EP-A 287 882 (HOE 87/F 101) and EP-A 322 348 (HOE 87/F 386R), and derivatives derived therefrom, such as, for example, those whose protective group has been eliminated. 4-Carboxylatopropoxy-4'-methoxybenzhydrylamine and 5-carboxylatoethyl-2,4-di methoxy-4'-methoxybenzhydrylamine are preferred.
It is possible to use as coupling reagent for the spacer of the formula V and the other amino acid derivatives all the possible activating reagents used in peptide syn-thesis, see, for example, Houben-Weyl, Methoden der Organischen Chemie, Volume XV/2, Stuttgart 1974, but especially carbodiimides such as, for example, N,N'-dicyclohexylcarbodiimide,N,N'-diisopropylcarbodiimide or N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. This coupling can be carried out directly by addition of amino acid derivative with the activating reagent and, where appropriate, an additive suppressing racemization, such as, for example, 4-dimethylaminopyridine, 1-hydroxy-benzotriazole (HOBt) (W. Ronig, R. Geiger, Chem. Ber. 103 (1970) 788 - 798) or 3-hydroxy-4-oxo-3,4-dihydro-benzotriazine (HOObt) (W. RSnig, R. geiger, Chem.
Ber. 103 ( 1970 ) 2034 - 2040 ) to the resin, or else the preactivation of the amino acid derivative as symmetrical anhydride or HOBt or HOObt ester can take place separate-1y and the solution of the activated species in a suit-able solvent can be added to the peptide-resin capable of coupling.

Plw W .at ~v i :. (y_ ~) The coupling and activation of the spacer of the for-mula V and of the amino acid derivative with one of the abovementioned activating reagents can be carried out in dimethylformamide or methylene chloride or a mixture of the two. The activated amino acid derivative is normally employed in a 1.5- to 4-fold excess. In cases where incomplete coupling occurs, the coupling reaction is repeated without previously carrying out the deblocking of the a-amino group of the peptide-resin which is necessary for the coupling of the next amino acid in sequence.
The success of the coupling reaction can be checked using the ninhydrin reaction as described, for example, by E. Raiser et al. (Anal. Biochem. 34 (1970) 595). The synthesis can also be carried out automatically, for example with a model 430A peptide synthesizer from Applied Biosystems, it being possible to use either the synthesis programs provided by the equipment manufacturer or else those drawn up by the user himself. The latter are particularly employed when using amino acid deriv-atives protected by the Fmoc group.
The peptide amides are cleaved off the resin by treatment with moderately strong acids customarily used in peptide synthesis (for example trifluoroacetic acid), adding as cation traps substances such as phenol, cresol, thio-cresol, anisole, thioanisole, ethanedithiol, dimethyl sulfide, ethyl methyl sulfide or similar cation traps customary in solid-phase synthesis, singly or a mixture of two or more of these auxiliaries. In this connection the trifluoroacetic acid can also be used diluted by a suitable solvent such as, for example, methylene chlor-ide. When the spacer is cleaved off the resin there is simultaneous elimination of the side-chain protective groups.
The crude peptides obtained in this way are purified by chromatography on ~Sephadex, ion exchanger resins or by I

HPLC.
A process of solid-phase synthesis for the preparation of Ac-D-NaI(2)-p-C1-D-Phe-D-Trp-Ser-Tyr-D-Sera-L-Rha)-Leu Arg-Pro-Azagly-NHZ and pGlu-His-Trp-Ser-Tyr-D-Ser(t8u) Leu-Arg-Pro-Azagly-NH2 (ZoladexjMis preferred.
List of abbreviations used:

BSA bistrimethylsilylacetamide Cha cyclohexylalanine Chg cyclohexylglycine DCC dicyclohexylcarbodiimide DIC diisopropylcarbodifmide DMAP dimethylaminopyridine Fmoc 9-fluorenylmethoxycarbonyl HOBt 1-hydroxybenzotriazole HOObt 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine Nal naphthylalanine Npg neopentylglycine Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl Tbg tert.-butylglycine TIiF tetrahydrofuran Thia 2-thienylalanine The examples which follow serve to illustrate the present invention without intending to restrict it thereto.
Example I
a) 5-Carboxylatoethyl-2,4-dimethoxy-4'-methoxybenz-hydrylamine 17.5 g of 5-carboxylatoethyl-2,4-dimethoxy-4'-methoxy-benzophenone oxime were dissolved in 450 ml of a 1:1 mixture of ethanol and DID', and 2 ml of concentrated NH3 were added. After addition of the Pt/C catalyst, hydro-genation was carried out under atmospheric pressure for 5 days. After completion of the reaction, the catalyst was filtered off with suction, the filtrate was ~~J~
- 12 - ~~"~~) concentrated, and the product was precipitated with ether. It was employed as such without further purification.
1b) N-(p-Nitrophenyloxycarbonyl)-5-carboxylatoethyl-2,4-dimethoxy-4'-methoxybenzhydrylamine g of the title compound from Example la) were intro-duced into 100 ml of a 4:1 THF/DMF mixture and, at room temperature, 2.1 equiv. of bistrimethylsilylacet-amide (BSA) were added. The suspension became completely 10 clear in a short time, and the clear solution was then stirred for 2 hours. 3 g of nitrophenyl chloroformate wEre then added and the mixture was stirred for a further hour. After completion of the reaction, the solvent was removed under high vacuum. The residue was mixed with 300 ml of water, and the resulting oil was extracted with ethyl acetate. The ethyl acetate phase was washed with a 1 N RHS04 solution and water. The organic phase was dried over MgS04 and evaporated to dryness. The residue (12 g) is characterized by NMR, IR and MS.
N-(p-Nitrophenyloxycarbonyl)-5-carboxylatoethyl-2,4-dimethoxy-4'-methoxybenzhydrylamine was then reacted with amino hydrazides to give the suitable substituted anchors.
lc) Benzyloxycarbonyl-4-prolylazaglycine (5-carboxylato-ethyl-2,4-di.methoxy-4'-methoxybenzhydryl)amide.
3.27 g of benzyloxycarbonyl-prolyl hydrazide hydrochlor-ide and 6.94 g of the title compound from Example 1b) were dissolved in 40 ml of dimethylformamide (DMF), and 3 equiv. of N-ethylmorpholine and a catalytic amount of dimethylaminopyridine (DMAP) were added. Reaction was allowed to take place for 16 hours. After completion of the reaction, the mixture was evaporated to dryness. The residue was taken up in ethyl acetate/butanol, and the organic phase was washed with saturated NaHC03 solution, - 13 - ,. ~ ~, r.. , ,. , ~ , I~.o V;~.i:.~~~J
1 N RHSO, solution and water. The organic phase was dried over MgSO, and, after filtration, evaporated to dryness.
It was possible to recrystallize the residue from pure ethyl acetate. 6.6 g of the title compound were obtained.
FAB-MS: 641 (M+Li+) IR: CO 1695 cm1 1H-NMR (DMSO): s = 3.7 s (6H, ocH3) ppm 1d) 9-Fluorenylmethoxycarbonyl-L-prolylazaglycine (5 carboxylatoethyl-2,4-dimethoxy-4'methoxybenzhydryl) amide 26.5 g of the title compound from Example lc) were dissolved in 300 ml of methanol, and 2 g of Pd/C catalyst were added. The hydrogenation was complete after one hour. The catalyst was filtered off, and the filtrate was evaporated to dryness. The residue (17.5 g) was, without further purification, taken up in a mixture of 80 ml of water and 80 ml of dioxane and mixed with 8 g of sodium bicarbonate and 17 g of N-(9-fluorenylmethoxycarbonyl-oxy)succinimide (Fmoc-ONSu). Reaction was allowed to take 20. place for one day. After the reaction was complete, the mixture was filtered through a clarifying filter. The filtrate was adjusted to pH 6 with 2 N HZS04 and evapor-ated in vacuo to a volume of 80 ml. The mixture was diluted with 100 ml of water and extracted with a mixture of ethyl acetate and n-butanol (8.5:1.5). The organic phase was washed with 50 $ saturated NaCl solution and then evaporated to dryness. The residue was filtered through 500 g of silica gel with ethyl acetate. 20 g of the title compound were obtained.
FAB-MS: 729 (M+Li+) IR: CO 1695 cml 1e) Coupling of the title compound frem Example 1d) to a polystyrene resin 1.0 g of aminomethylpolystyrene resin (loading 1.07 mmol) and 1.2 g of the title compound from Example 1d) were - 14 - /Le~~a..7~~~: ~~~~.~) suspended in 10 ml of dimethylformamide, and 216 mg of 1-hydroxybenzotriazole (HOBt) and 0.75 ml of diisopropyl-carbodiimide (DIC) were added. Reaction was allowed to take place overnight until the ninhydrin test indicated complete reaction. The resin was filtered off and washed with dimethylformamide and methylene chloride and thor-oughly dried in vacuo. The loading of the resin with proline was 0.51 mmol/g.
1f) Ac-D-NaI(2)-p-C1-D-Phe-D-Trp-Ser-Tyr-D-Sera-L-Rha)-Leu-Arg-Pro-Azagly-NH2 The 9-fluorenylmethoxycarbonyl-Na-amino-protective group of the compound from Example 1e) was eliminated with a % strength piperidine/dimethylformamide solution (2x3 min, 2x8 ml). The resin was then washed with N-15 methylpyrrolidinone (5x10 ml) and the peptide was synthe-sized on the resin (785 mg of resin from Example lc)), carrying out the following steps in cycles:
- elimination of the Fmoc protective group with 20 %
piperidine in DMF
20 - washing of the resin with DMF/N-methylpyrrolidinone - coupling on of the Fmoc-amino acid with in situ activation as HOBt ester using diisopropyl carbodiimide as activating reagent (1.5 mmol of amino acid, 2.25 tnmol of HOBt, 1.6 mmol of diiso propylcarbodiimide) If the coupling was incomplete (Raiser test), the coupl-ing step was repeated. The last amino acid employed was Fmoc-D-Nal(2)-OH. The N-terminal acetyl group was intro-duced by reaction with acetic anhydride.
After completion of the solid-phase synthesis, the resin was washed (DMF, CH2Clz) and thoroughly dried. 1.35 g of substituted resin were obtained.

- 15 - ig.~~~ia~ f ~~i The dried resin was suspended at room temperature in 0.75 ml of ethanedithiol. After 15 minutes, 7.5 ml of trifluoroacetic acid were added and the suspension was stirred for 1.5 hours. After this time, the resin was filtered off and thoroughly washed with 80 $ strength trifluoroacetic acid. The filtrate was evaporated in vacuo and taken up in 30 ml of water. NaHC03 was added to adjust to pH 6-7, and the peptide was extracted by shaking with n-pentanol (4x30 ml). The n-pentanol phase was evaporated and taken up in 10 ml of methanol/H20 (9:1), and 0.5 g of RZC03 was added. The mixture was stirred for 30 minutes and filtered, and the filtrate was concentrated. The residue was taken up in 100 ml of n-pentanol, and the organic phase Was washed with water.
The organic phase was dried with MgS04 and filtered and then evaporated. 740 mg of crude product were obtained.
Chromatography on 'Sephadex G 25 (1 M acetic acid) and on silica gel resulted in 185 mg of the pure title compound.
FAB-MS: 1531 (M+H+)

Claims

1. A process for the preparation of peptides of the formula I
(X)n-A-NH2 (I) in which X is a natural or unnatural amino acid, aza-amino acid or imino acid, n is an integer from 1 to 50, and A is an aza-amino acid, and the physiologically tolerated salts thereof, which comprises converting a spacer into a form capable of acylation, reacting the latter with a suitable formic acid derivative and subsequently with an appropriate amino hydrazide, Where appropriate converting the pro-tective group into a protective group which is base-labile or labile to weak acids, coupling the spacer obtained in this way to a resin, synthesizing the re-quired peptide stepwise from the C-terminal end, subse-quently cleaving the peptide off the resin and, where appropriate, converting it into physiologically tolerated salts thereof.

2. The process as claimed in claim 1, wherein n is an integer from 1-30.

3. The process as claimed in claim 1, wherein a compound of the formula II
in which Y1, Y2, Y3, Y4 and Y5 are hydrogen, C1-C4-alkyl, C1-C4-alkoxy or -O-(CH2)n-COOH, -(CH2)n-COOH or -NH-CO-(CH2)n-COOH, where the radicals can be identical or different, but at least one radical is -O-(CH2)n-COOH, -(CH2)n-COOH or -NH-CO-(CH2)~-COOH, n is an integer from 1 to 6 , and R1 is hydrogen or C1-C6-alkoxy-C6-C12-aryl, is reacted with a silylating reagent in a solvent suitable for this purpose, and subsequently the silylated compound is converted with a chloroformic acid derivative into compounds of the formula III
in which R1, Y1, Y2, Y3, Y4 and Y5 are as defined above, and R2 is a C6-C12-aryl radical which is substituted by electron-attracting substituents, the compounds of the formula III obtained in this way are reacted with an amino hydrazide of the formula IV
R3 .cndot. X .cndot. CO .cndot. NH .cndot. NH .cndot. R4 (IV) in which X is a natural or unnatural amino acid or imino acid and is as defined above, R3 is a protective group which is base-labile or labile to weak acids or hydrogenation, and R4 is C1-C8-alkyl, C3-C9-cycloalkyl, C6-C12-aryl, C6-C12-aryl-C1-C8-alkyl, heteroaryl or heteroaryl-C1-C8-alkyl or hydrogen, in a suitable solvent to give the compounds of the formula V

in which R1, R3, R4 and y1, y2, y3, y4 and y5 have the abovementioned meanings, if R3 is a protective group labile to hydrogenation this protective group is removed by hydrogenation on a Pd catalyst and, before the subse-quent reaction, converted into a urethane protective group which is base-labile or labile to weak acids, subsequently the compound of the formula V in which R1, R4 y1, y2, y3, y4 and Y5 have the abovementioned meanings, and R3 is a urethane protective group which is labile to bases and weak acids, is coupled with the coupling reagents customary in peptide chemistry via the -O-(CH2)n-COON, -(CH2)n-COON or -NH-CO- (CH2) n-COOH group to a resin, the protective group R3 is eliminated, natural or unnatural amino, imino or aza-amino acids which have been tempor-arily protected by amino-protective groups which are base-labile or labile to weak acids and which are optionally in the form of their activated derivatives are coupled on stepwise, and, after the synthesis is complete, the peptides of the formula I are liberated from the resin by treatment with a moderately strong acid, with elimination again, simultaneously or by suitable measures subsequent thereto, of temporarily introduced side-chain protective groups.

4. The process as claimed in claim 3, wherein n is an integer from 1 to 3.

5. The process as claimed in claim 3 or 4, wherein R2 is a C6-C12-aryl radical which is substituted by electron-attracting substituents selected from nitro and halogen.

6. The process as claimed in any of claims 1 to 5 , wherein Ac-D-Nal(2)-p-C1-D-Phe-D-Trp-Ser-Tyr-D-Ser(.alpha.-L-Rha)-Leu-Arg-Pro-Azagly-NH2 is prepared.

7, The process as claimed in any of claims 1 to 5 , wherein pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-Azagly-NH2 is prepared.