NO300216B1 - Process for the preparation of peptides by solid phase synthesis - Google Patents

Description

The present invention relates to a method for the production of peptides with C-terminal azaamino acid amide by means of solid phase synthesis.

The invention therefore relates to a method for the production of peptides of general formula I:

there

X means a natural or unnatural amino acid,

azaamlic acid or imino acid,

n means an integer from 1 to 50, preferably 1 to

30, and

A" means an aza amino acid,

as well as physiologically tolerable salts, characterized in that a compound of formula II is converted:

there

Y1, Y2, Y<3>, Y4 and Y<5> mean hydrogen, C1-C4-alkyl, C1-C4-alkoxy or -O-(CH2)n-C00H, -(CH2)n-C00E or -NH -CO-(CHgln-COOH, where the residues can be the same or different, where at least one residue means -0-(CH2 )n-C00H, -(CH2)n-C00H or -NH-C0-(CH2 )n- C00H, n is an integer from 1 to 6, R<1 >is hydrogen or C1-C5-Alkoxy-C2-C2-aryl > with a silylation reagent in a suitable solvent, and finally transfers the silylated compound with a chloroformic acid derivative to compounds of formula III:

there

R1, Y1, Y2, Y3, Y<4> and Y<5> are as defined above, and

R<2> means a C<6>_Ci2-aryl residue" which is substituted with electro-withdrawing substituents, preferably nitro and halogen, react the thus obtained compounds of formula III with an amino acid hydrazide of formula IV,

there

X means a natural or unnatural amino acid or imino acid and is as defined above, R<3> means a base labile or towards weak acids or hydrogenation-labile protecting group, and R<4> means C1-C8-alkyl, C3-C8-cycloalkyl, C6-C12<->aryl-C1-C8-alkyl, heteroaryl or heteroaryl-C1-C8-alkyl or hydrogen, in a suitable solvent, for compounds of formula V:

where R1, R3, R4 and Y<1>, Y<2>, Y<3>, Y<4> and Y<5> have the meanings given above, if R<3> means a protection group labile against hydration, removes this protection group by

hydrogenation over a Pd catalyst, and before further reaction transfers in a base labile or a weak acid labile urethane protecting group, then couples the compound of formula V, where R<1>, R<4>, Y<1>, Y2, Y3, Y4 and Y<5> have the meanings stated above, and R<3> means a counter base or weak acid labile urethane protecting group, with the coupling reagents common in peptide chemistry over the -0-(CE2)n-COOH,-(C<H>2)n-C00H or -NH-C0-(CH2)n-COOH grouping on a resin , cleaves off the protection group R<3>, step by step with base-labile or against weak acids labile amino protection groups temporarily protected natural or unnatural amino, imino or aza amino acids, possibly in the form of their activated derivatives and after completion of the construction of the peptides of formula I through treatment with a medium-strong acid from the resin, while at the same time or with the help of suitable steps temporarily introduced side chain protecting groups are cleaved off again.

Natural or unnatural amino acids can, if they are chiral, exist in the D or L form. Preferred are α-amino acids. For example, the following can be mentioned: AAd, Abu, yAbu, ABz, 2ABz, cAca, Ach, Acp, Adpd, Ahb, Aib, <p>Aib, Ala, PAla, AAla, Alg, All, Ama, Amt, Ape, Apm, Apr, Arg, Asn, Asp, Asu, Aze, Azi, Bai, Bph, Can, Cit, Cys, (Cys)2, Cyta, Daad, Dab, Dadd, Dap, Dapm, Dasu, Djen, Dpa, Dtc, Fel , Gin, Glu, Gly, Guv, hAla, hArg, hCys, hGln, hGlu, His, hile, hLeu, hLys, hMet, hPhe, hPro, hSer, hThr, hTrp, hTyr, Hyl, Hyp, 3Hyp, lie, Ise , I va, Kyn, Lant, Len, Leu, Lsg, Lys, eLys, ALys, Met, Mim, Min, nArg, Nie, Nva, Oly, Orn, Pan, Pec, Pen, Phe, Phg, Pic, Pro, APro, Pse, Pya, Pyr, Pza, Qin, Ros, , Sar, Sec, Sem, Ser, Thi, pThi , Thr, Thy, Thx, Tia, Tie, Tly,Trp, Trta, Tyr, Val, Nal, Tbg , Npg, Cha, Chg, Thia (cf. e.g. Houben-Weyl, Methoden der organischen Chemie, volumes XV/1 and 2, Stuttgart, 1974).

Azaamino acids are derived from natural or unnatural amino acids, as the central building block -CHR- or -CH2- is replaced by -NR- or -NH-. For example, the following can be mentioned: azaglycine, azavaline, azaleucine, azaisoleucine and azaphenylalanine.

An imino acid is understood to mean general natural or unnatural amino acids where their amino group is monosubstituted. Compounds that can be mentioned in this context are substituted with C 1 -C 8 alkyl, which in turn is possibly singly or doubly unsaturated and which may be substituted with up to 3 identical or different residues from the series mercapto;

hydroxy; C 1 -C 6 -Alkoxy, carbamoyl; C 1 -C 8 alkanoyl; carboxy;

C 1 -C 8 -C 6 -Alkoxycarbonyl; F; Cl; Bro; IN; amino; amidino, which may optionally be substituted with one, two or three C 1 -C 8 alkyl radicals; guanidino, which may optionally be substituted with one or two benzyloxycarbonyl residues or with one, two, three or four C 1 -C 8 alkyl residues; C 1 -C y alkylamino; di-C 1 -C 7 -alkylamino; C 3 -C 4 -Alkoxycarbonylamino; C 1 -C 4 -aralkoxycarbonyl; C 7 -C 15 -aralkoxycarbonylamino; phenyl-C 1 -C 6 alkoxy, 9-fluoronylmethoxycarbonylamino; C 1 -C 4 -alkylsulfonyl; C 1 -C 4 -alkylsulfinyl; C 1 -C 4 -alkylthio; hydroxamino; hydroxyimino;

sulfamoyl.

Furthermore, heterocycles from the following group come into consideration: pyrrolidone-2-carboxylic acid; piperidine-2-carboxylic acid;

1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; decahydroiso-quinoline-3-carboxylic acid; octahydroindole-2-carboxylic acid;

decahydroquinoline-2-carboxylic acid; octahydrocyclopenta[b]-2-carboxylic acid; 2-aza-bicyclo[2.2.2]octane-3-carboxylic acid; 2-azabicyclo[2.2.1]heptane-3-carboxylic acid; 2-azabicyclo[3.1.9]-hexane-3-carboxylic acid; 2-azaspiro[4.4.]nonane-3-carboxylic acid; 2-azaspiro[4.5]decane-3-carboxylic acid; spiro[(bicyclo-[2.2.1]neptane]-2,3-pyrrolidine-5-carboxylic acid; spiro[(bicyclo-[2.2.2]octane)-2,3-pyrrolidine-5-carboxylic acid; 2-azatricyclo- [4.3.0.<6>»<9>]decane-3-carboxylic acid; decahydrocyclohepta[b]-pyrrole-2-carboxylic acid; octahydrocyclopenta[c]pyrrole-2-

carboxylic acid; octahydroisoindole-1-carboxylic acid; 2,3,3a,4,6a-hex ahydrocyclopent a[b]pyr roi-2-carboxylic acid; 2,3,3a,4,5,7a-hexahydroindole-2-carboxylic acid; tetrahydro-thiazole-4-carboxylic acid; isoxazolidine-3-carboxylic acid; pyrazolidine-3-carboxylic acid; hydroxyproline-2-carboxylic acid; all of which may optionally be substituted:

By salts of compounds of formula I are meant particularly pharmaceutically usable or non-toxic salts. Particular mention may be made here of alkali or alkaline earth salts, salts with physiologically tolerable amines and salts with inorganic or organic acids such as e.g. HC1, HBr, H2SO4, H3PO4, maleic acid, fumaric acid, citric acid, tartaric acid, acetic acid.

The compounds with formula IV are produced through conversion of natural or unnatural amino acids or imino acids with corresponding hydrazides according to known coupling methods in peptide chemistry.

Alkyl can be linear or branched. The same applies to each of the derived residues, such as e.g. alkoxy, alkylthio, alkylamino, dialkylamino and alkanoyl. In particular, alkyl stands for C1-C4 alkyl.

Cycloalkyl also means alkyl-substituted residues, such as e.g. 4-methylcyclohexyl or 2,3-dimethylcyclopentyl.

Halogen stands for fluorine, chlorine, bromine or iodine, especially for fluorine or chlorine.

c6~c12_arvl is, for example, phenyl or naphthyl, preferably phenyl. Heteroaryl means the residue of a 5- to 7-membered monocyclic or 8- to 10-membered bicyclic aromatic ring system which may be benzannilated which may contain as heteroelements one, two, three or four different residues from the group N, 0, S, NO, SO, SO2, which can be mono-, di- or tri-substituted with 1 to 6 hydroxy or with one, two or three identical or different residues from the series F, Cl, Br, I; hydroxy; mono, di- or trihydroxy-C 1 -C 4 alkyl; trifluoromethyl; formyl; carboxamido; mono- or di-C 1 -C 4 -alkylaminocarbonyl; nitro; C 1 -Cyalkoxy; C 1 -C 6 alkyl; C 1 -C 7 -Alkoxycarbonyl, amino, C 1 -C 7 -Alkylamino; di-C 1 -C 7 -alkylamino; carboxy; carboxymethoxy; amino-Ci-Cy-alkyl; C 1 -C 7 -alkylamino-C 1 -C 7 -alkyl; di-C 1 -C 7 -alkylamino-C 1 -C 7 -alkyl; ci~c7~

alkoxycarbonylmethoxy; carbamoyl; sulfamoyl; C 1 -C 7 -Alkoxy-sulfonyl; C 1 -C 8 alkylsulfonyl; sulfo-C 1 -C 8 alkyl; guanidino-C 1 -C 8 -alkyl and C 1 -C 8 -alkoxycarbonyl and/or with oxo. In particular, the following may be mentioned: furyl, thienyl, imidazolyl, pyrazolyl, oxazolyl, isoazolyl, thiazolyl, isothiazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrimidinyl, indolyl, iso-indolyl, indazolyl, phthalazinyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, quinolinyl, g <->carbolinyl or a benzannelated derivative of this residue.

For the prevention of side reactions or for the synthesis of special peptides, the functional groups in the side chain of amino-, azaamino- or imino acids further protected with suitable protecting groups (see, e.g., T.W. Greene, "Protectice Groups in Organic Synthesis", New York, John Wiley & Sons, 1981; Hubbuch, Kontakte (Merck) 1979, No. 3, pages 14-23; Bullesbach, Kontakte (Merck) 1980 no. 1, pages 23-35), whereby arg (Tos), Arg (Mtr), Arg (Pmc), Asp (OBzl), Asp (OtBu), Cys ( 4-MeBz), Cys (Acm), Cys (StBu), Glu (OBzl), Glu (OtBu), His (Tos), His (Fmoc), His (Dnp), His (Trt), Lys (Cl-2 ), Lys (Boe), Met (0), Ser (Bzl), Ser (tBu), Thr (Bzl), Thr (tBu). Furthermore, the functional groups can also be glycosylated in the side chain, e.g. described in EP-A 263,521 (HOE 86/F 253).

The resins used as carrier material are commercially available or can be prepared yourself, such as e.g. alkoxy-benzyl alcohol resins, aminomethyl resins or benzhydrylamino resins. Preferred are aminomethyl, benhydrylamino (BHA) and methylbenzhydrylamine (MBHA) resins. The load is determined through amino acid analysis and/or elemental analysis.

Suitable spacers include, for example, those in Atherton, Sheppard in Perspectives in Peptide Chemistry, pages 101-117 (Karger, Basel 1981); EP-A- 264.8023 (HOE 86/F 259), EP-A 287.882 (HOE 87/F 101) and EP-A 322.348 (HOE 87/F 386K) describing spacers as well as derivatives derived from them, such as those where their protecting group is split off. Preferred are 4-carboxylatopropoxy-4'-methoxy-benzhydrylamine and 5-carboxyatoethyl-2,4-dimethoxy-4'-methoxy-benzhydrylamine.

All possible activation reagents used in peptide synthesis can be used as coupling reagents for the spacer with formula V and the further amino acid derivatives, see e.g. Houben-Weyl, Methoden der Organischen Chemie, Band XV/2, Stuttgart 1974, be used, especially carbodiimides such as e.g. N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide or N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. The coupling can, on the other hand, be carried out directly through the addition of an amino acid derivative with the activation reagent and possibly an addition that suppresses the racemization such as e.g. 4-dimethylaminopyridine, 1-hydroxybenzotriazole (HOBt) (W. Konig, R. Geiger, Chem. Ber. 103 (1970) 788-798) or 3-hydroxy-4-oxo-3,4-dihydrobenzotriazine (EOObt) (W K5nig, R. Geiger, Chem. Ber. 103 (1970) 2034-2040) to the resin or can the pre-activation of the amino acid derivative take place separately as symmetrical anhydride or HOBt or HOObt ester, and the solution of the activated species can be added in a suitable solvent for coupling capable peptide resin.

Coupling or activation of the spacers of formula V and the amino acid derivatives with one of the above activation reagents can be carried out in dimethylformamide or methylene chloride or a mixture of both. The activated amino acid derivative is usually added in a 1.4- to 4-fold excess. In those cases where an incomplete coupling occurs, the coupling reaction is repeated without the end-blocking of the cx-amino group of the peptide resin necessary in advance for the coupling of the subsequent amino acid.

The progress of the coupling reaction can be tested using the ninhydrin reaction, which e.g. described by E-Kaiser et al. (Anal. Biochem. 34 (1970) 595. The synthesis can also be carried out automatically, e.g. with a peptide synthesizer model 430A Fa. Applied Biosystems, where either the synthesis program from the device manufacturer or also self-determined by the user can be used. The latter is particularly used when used with Fmoc group-protected amino acid derivatives.

Cleavage of the peptide amides from the resin takes place through treatment with the medium-strong acids usually used in peptide synthesis (e.g. trifluoroacetic acid), whereas compounds such as phenol, cresol, thiocresol, anisole, thioanisole, ethanedithiol, dimethyl sulphide, ethyl methyl sulphide or common in the phosphase synthesis common cation traps individually or a mixture of two or more of these aids. Trifluoroacetic acid can thus be used diluted with a suitable solvent, such as e.g. methylene chloride.

When splitting off the spacer from resin, side chain protecting groups are simultaneously split off.

Purification of the crude peptide obtained in this way takes place by means of chromatography on Sephadex, ion exchange resin or

EPLC.

Preferred is a process in phosphase synthesis for the production of Ac-D-Nal(2)-p-Cl-D-Phe-D-Trp-Ser-Tyr-D-Ser(a-L-Rha)-Leu-Arg-Proazagly-NE2 and pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-azagly-NH2 (Zoladex).

List of abbreviations used:

The examples below shall explain the present invention without limiting it.

Example 1

a) 5-carboxyl ethyl-2,4-dimethocy-4'-methoxybenz-hydrylamine

17.5 g of 5-carboxylatoethyl-2,4-dimethoxy-4'-dimethoxybenz-phenoxime were dissolved in 450 ml of a 1:1 mixture of ethanol and DMF and reacted with 2 ml of conc. NE3. After addition of the Pt/C catalyst, it was hydrated at normal pressure for 5 days. After completion of the reaction, the catalyst was sucked off, the filtrate reduced and the product precipitated with ether. Further purification was thus applied.

lb) N-(p-n i trophenyloxycarbonyl)-5-carboxylatoethyl-2,4-dimethoxy-4'-methoxybenzhydrylamine

10 g of the title compound from example la) was added to 100 ml of a THF/DMF 4:1 mixture and reacted at room temperature with 2.1 eq. bistrimethylsilylacetamide (BSA). The suspension became clear in a short time, and the clear solution was then stirred for 2 hours. Then 3 g of chloroformate nitrophenyl ester were added, and this was stirred for a further hour. After completion of the reaction, the solvent was removed under high vacuum. The residue was reacted with 300 ml of water and the oil obtained was extracted with acetic acid. The acetate phase was washed with an IN KHSO 4 solution and water. The organic phase was dried over MgSO 4 and evaporated to dryness. The remainder (12 g) was characterized by NMR, IR and MS.

N-(p-n i trof enyloxycarbonyl)-5-car bok sy la toet yl-2 ,4-di-methoxy-4'-methoxy-benzhydrylamine was finally reacted with amino acid hydrazides to the appropriate substituted anchor.

lc ) Benzyl oxycarbonyl 1-4-propyl-azaglycyl-(5-carboxy-latoethyl-2,4-dimethoxy-4'-methoxybenzylhydryl)amide

3.27 g of benzyloxycarbonyl-propylhydrazide hydrochloride and 6.94 g of the title compound according to example 1b) were dissolved in 40 ml of dimethylformamide (DMF) and reacted with 3 eq. N-ethylmorpholine and a catalytic amount of dimethylaminopyridine (DMÅP). This is traded in 16 hours. After completion of the reaction, it was evaporated to dryness. The residue was taken up in ethyl acetate/butanol, and the organic phase was washed with saturated NaHCO 3 ~ solution, 1 N KESO 4 solution and water. The organic phase was dried over MgSO 4 and evaporated to dryness after filtration. The remainder can be recrystallized from pure vinegar. 6.6 g of the title compound is obtained.

FAB-MS: 641 (M+L1<+>)

IR: CO 1695 cm-<1>

% NMR (DMSO): A = 3.7 s (6E, 0CH 2 ) ppm

ld) 9-fluorenylmethoxycarbonyl-L-propyl-azaglycyl-(5-carboxylatoethyl)-2,4-dimethoxy-4'-methoxy-benzhydryl)amide

26.5 g of the title compound according to example lc) was dissolved in 300 ml of methanol and reacted with 2 g of Pd/catalyst. After one hour, the hydration was terminated. The catalyst was titrated out, and the filtrate reduced to dryness. Without further purification, the residue (17.5 g) was taken up in a mixture of 80 ml of water and 80 ml of dioxane and reacted with 8 g of sodium bicarbonate and 17 g of 9-fluorenylmethoxycarbonyl-N-hydroxysuccinimide (Fmoc-ONSu). This will be responded to in one day. After the end of the reaction, it was filtered over a clear filter. The filtrate was adjusted to pH 6 with 2 N H2SO4 and evaporated in vacuo to a volume of 80 ml. This is diluted with 100 ml of water and extracted with a mixture of ethyl acetate/n-butanol (8.5 : 1.5). The organic phase is washed with half-saturated NaCl solution and then evaporated to dryness. The residue was filtered over 500 g of silica gel with acetic acid. 20 g of the title compound is obtained.

FAB-MS: 729 (M+Li<+>)

IR: CO 1695 cm-<1>

le) Connecting the title compound from example Id) on 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 suspended in 10 ml of dimethylformamide and reacted with 216 mg of 1-hydroxy-benzo-triazole (HOBt) and 0.75 ml of diisopropylcarbodiimide (DIC). This is reacted overnight until the ninhydrin test shows a complete reaction. The resin was filtered off and washed with dimethylformamide and methylene chloride and thoroughly dried in vacuo. The loading of the resin with proline amounted to 0.51 mmol/g.

lf) Ac-D-Na(2 )-p-ClD-Phe-D-Trp-Ser-Tyr-D-Ser(a-L-Rha)-Leu-Arg-Pro-azagly-NH2

The 9-fluorenylmethoxycarbonyl-Na-amino protecting group of the compound from example le) was cleaved with a 2056 piperidine/dimethylformamide solution (2x3 min, 2x8 ml). Finally, the resin was washed with N-methylpyrrolidinone (5x10 ml), and the peptide was built on the resin (785 mg of resin from example 1c), the following steps being carried out cyclically: removal of the Fmoc protecting group by 20%

piperidine in DMF

washing the resin with DMF/N-methylpyrrolidinone

attachment of Fmoc amino acid during in situ activation of HOBt ester using diisopropylcarbodiimide as activation reagent (1.5 mmol amino acid, 2.25 mmol HOBt, 1.6 mmol diisopropylcarbodiimide)

If the coupling was incomplete (Kaiser test), the coupling step was repeated. As the last amino acid, Fmoc-D-Nal(2)-OH was added. The N-terminal acetyl group was carried out by reaction with acetic anhydride.

After completion of phosphase synthesis, the resin was washed (DMF, CH 2 Cl 2 ) and thoroughly dried. 1.35 g of substituted resin is obtained.

The dried resin was suspended with 0.75 ml of ethanol dithiol at room temperature. After 15 min. 7.5 mml of trifluoroacetic acid was added and the suspension was stirred for 1.5 hours. After this time, the resin was filtered off and thoroughly washed with 805é trifluoroacetic acid. The filtrate was evaporated in vacuo and taken up in 30 ml of water. Through the addition of NaHCO3, pH 6-7 was adjusted and the peptide shaken with n-pentanol (4x30 ml), the n-pentanol phase was evaporated and taken up in 10 ml methanol/H2O (9:1) and reacted with 0.5 g H2CO3. This was stirred for 30 min, filtered and the filtrate evaporated. The residue was taken up in 100 ml of n-pentanol, and the organic phase was washed with water. After drying with MgSO 4 and filtration, the organic phase was evaporated. 740 mg of crude product is obtained. After chromatography on Sephadex G 25 (IM acetic acid) and silica gel, 185 g of the title compound are obtained.

FAB-MS: 1531 (M+H<+>)

Claims (3)

1. Procedure for the preparation of peptides of general formula: there X means a natural or unnatural amino acid, azaamino acid or imino acid, n means an integer of 1 to 50, preferably 1 to 30, and A means an aza-amino acid, as well as physiologically tolerable salts, characterized by converting a compound of formula II: there Y1, Y2, Y3, Y<4> and Y<5> mean hydrogen, C1-C4-alkyl, C1-C4-alkoxy or -O-(CH2)n-<C>00H, -(CH2)n-C00H or -NH-CO-(CH2)n-C00H, where the residues may be the same or different, where at least one residue means -0-(CE2)n-C00H, -(CH2)n-C00H or -NH-C0- (CE2 )n-C00H, n is an integer from 1 to 6, R<1 >means hydrogen or C1-C4)-Alkoxy-C6)-C12-aryl, with a silylation reagent in a suitable solvent, and finally transfers the silylated compound with a chloroformic acid derivative to compounds of formula III: where R1, Y1, Y2, Y3, Y<4> and Y<5> are as defined above, and R<2> means a c<6>_Ci2-aryl residue, which is substituted with electro-withdrawing substituents, preferably nitro and halogen, reacts the thus obtained compounds of formula III with an amino acid hydrazide of formula IV, there X means a natural or unnatural amino acid or imino acid and is as defined above, R<3> means a base labile or towards weak acids or hydrogenation-labile protecting group, and R<4> means C 1 -C 8 -alkyl, C 3 -C 8 -cycloalkyl, C 6 -C 12<->aryl-C 1 -C 8 -alkyl, heteroaryl or heteroaryl-C 1 -C 8 -alkyl or hydrogen, in a suitable solvent, for compounds of formula V: where R1, R3, R<4> and Y1, Y2, Y3, Y<4> and Y<5> have the meanings given above, if R<3> means a protection group labile against hydrogenation, this protection group is removed by hydrogenation over a Pd catalyst, and before further reaction transfers in a base labile or a weak acid labile urethane protecting group, then couples the compound of formula V, where R<1>, R<4>, Y<1>, Y2, Y3, Y4 and Y <5> has the meanings stated above, and R<3> means a counter base or weak acid labile urethane protecting group, with the coupling reagents common in peptide chemistry over the -0-(CH2)n-C00H,-(CH2)n-C00H or -NH-CO-(C<E>2)n-COOH group on a resin , cleaves off the protection group R<3>, step by step with base-labile or against weak acids labile amino protection groups temporarily protected natural or unnatural amino, imino or aza amino acids, possibly in the form of their activated derivatives and after completion of the construction of the peptides of formula I through treatment with a medium-strong acid from the resin, while at the same time or with the help of suitable steps temporarily introduced side chain protecting groups are cleaved off again. 2. Method according to claim 1, characterized in that one prepares Ac-D-Nal(2)-p-Cl-D-Phe-D-Trp-Ser-Tyr-D-Ser(a-L-Rha)-Leu-Arg- Proazagly-NH2. 3. Method according to one of claims 1 to 2, characterized in that one prepares pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-azagly-NH2•