AU595390B2

AU595390B2 - The synthesis of peptide aminoalkylamides and peptide hydrazides by the solid-phase method

Info

Publication number: AU595390B2
Application number: AU79935/87A
Authority: AU
Inventors: Gerhard Breipohl; Jochen Knolle
Original assignee: Hoechst AG
Current assignee: Hoechst AG
Priority date: 1986-10-21
Filing date: 1987-10-20
Publication date: 1990-03-29
Anticipated expiration: 2007-10-20
Also published as: HU197719B; FI88031B; ZA877862B; IE60864B1; DK175126B1; ES2052535T3; PT85952B; EP0264802B1; AU7993587A; NO172895B; IE872821L; JPS63104951A; NO172895C; DE3783009D1; FI88031C; IL84195A; KR960013073B1; CA1340421C; ATE83244T1; EP0264802A3

Abstract

Compounds of the formula I <IMAGE> in which A is hydrogen or an amino protective group, B is an amino acid residue, X is alkylene or aralkylene, Y<1>, Y<2>, Y<3> and Y<4> are identical or different and are hydrogen, methyl, methoxy or nitro, V is hydrogen or a carboxyl protective group, W is -[CH2]n- or -O-[CH2]n-, m is 0 or 1, n is 0 to 6 and p is 0 to 5, are prepared as described and used in the solid-phase synthesis.

Description

Form COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION Class I t. Class Application Number: Lodged; 595390 t~Complete Specification Lodged: Accepted: Published: Priority: This document contains the amiendmenits niade Linder Section 49 and is correct for printing.,3 i* V' Related Art Name of Applicant: Address of Applicant HOECHST AKTIENGESELLSCHAFT 45 Bruningstrasse, D-623Q Frankfurt/Main 80, Federal Republic of Germany Actual Inventor: GERHARD BREIPOHL and JOCHEN KNOLLE E(OWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Address for Service, Complete Specification for the invention entitled: THE SYNTHESIS OF PEPTIDE AMINOALKYLAMIDES AND PEPTIDE HYDRAZIDES BY THE SOLID-PHASE METHOD The following statement Is a full description of this Invention, Including the best method of performing It known to us HOECHST AKTIENGESELLSCHAFT HOE 86/F 259 Dr.WI/mu Specification: The synthesis of peptide aminoalkylamides and peptide hydrazides by the solid-phase method The introduction of an aminoalkyLamide into the C-terminal end of a biologicaLLy active peptide has in some cases had beneficial effects on the metabolic stability and activity (EP-A 179 332). The preparation of the peptides modified in this way has made use of the classical coupling of fragments in solution.

In the solid-phase synthesis of peptides (see Patchornik, Cohen in Perspectives in Peptide Chemistry, pages 118 128 (Karger, Basle 1981)) the reactive chains are often not S grafted directly onto the synthetic resin material, but Sare bonded to the carrier material by what are called spacers Scc or links. The literature (for example Atherton, Sheppard in Perspectives in Peptide Chemistry, pages 101 117 (Karger, Basle 1981)) discloses, for example, reagents for introducing such spacers (called "Linkage agents") which have the formulae VI, VII and VIII.

£t HOCHZ- CH2-CH 2

-CO

2 H HOCH 2

OCH

2

-CO

2 H HOCH 2

CO

2

H

(VI) (VII) (VIIIl) New Linkage agents which allow direct construction, by solid phase synthesis, of peptides modified by C-terminal aminoalkylamide or hydrazide have been found.

Thus the present invention relates to compounds of the formuLa I yl y 2 (A-[B]p-NH-[X]n-NH-CO-0-CH2 j W-CO2-V y 3 y 4 A L r_ il- i I i i( -Y~ -2in which A denotes hydrogen or an amino protective group which is labile to bases or labile to weak acids, B represents identical or different amino acid residues, X denotes (C1-C 1 2)-alkylene or (C 6 -C10)-aryL-(Cl-C 1 2)alkylene, 1 2 3 4 Y Y Y and 4 are identical or different and denote hydrogen, methyl, methoxy or nitro, at least one of these radicals denoting hydrogen, V denotes hydrogen or a carboxyl protective group, W denotes -CCH2]n- or -O-CCH21nm is 0 or 1, n is an integer from 0 to 6, and p is an integer from 0 to ti.

t Preferred compounds of the formula I are those in which p is 0, 1 or 2, in particular 0, and/or in which m is 1.

X is preferably -CCH2]q-, it being possible for q to be 1 12, preferably 1 8.

Preferably at least 2, in particular at least 3, of the radicats Y Y Y 3 and Y 4 denote hydrogen.

Protective groups which are Labile to bases or labile to weak acids are, in particular, urethane protective groups, such as Fmoc, Ddz, Bpoc, Msc, Peoc, Pse and Tse, preferably Fmoc (see, for example, Hubbuch, Kontakte (Merck) 1979, No.

3, pages 14 23).

B represents the residue of an amino acid, preferably of an a-amino acid, which, if chiral, can be present in the D or L f-rm. Preference is given to residues of naturaLLy occurring amino acids, their enantiomors, homologs, derivatives and simple metabolites (see, for example, WUnsch et al., Houben-Weyl 15/1 and 2, Stuttgart, Thieme 1974). Thus, for example, the following are suitable: i -3- Aad, Abu, YAbu, ABz, 2ABz, E Aca, Ach, Acp, Adpd, Ahb, Aib, B Aib, Ala, ALa, A Ala, ALg, All, Ama, Amt, Ape, Apm, Apr, Arg, Asn, Asp, Asu, Aze, Azi, Bai, Bph, Can, Cit, Cys, Cyta, Daad, Dab, Dadd, Dap, Dapm, Dasu, Djen, Dpa, Dtc, Fel, Gin, Glu, Gly, Guv, hCys, His, hSer, Hyl, Hyp, 3Hyp, ILe, Ise, Iva, Kyn, Lant, Lcn, Leu, Lsg, Lys, Lys, A Lys, Met, Mim, Min, nArg, NIe, Nva, OLy, Orn, Pan, Pec, Pen, Phe, Phg, Pic, Pro, A Pro, Pse, Pya, Pyr, Pza, Qin, Ros, Sar, Sec, Sem, Ser, Thi, 6 Thi, Thr, Thy, Thx, Tia, TLe, Ty, Trp, Trta, Tyr, Vat and the residues of the corresponding enantiomeric D-amino acids.

Functional groups in the side chains of the said amino acid residues can be in protected form. Suitable protective groups are described in Hubbuch, Kontakte (Merck) 1979, No.

3, pages 14 23, and in Bullesbach, Kontakte (Merck) 1980, No. 1, pages 23 35. The preferred groups are those which xIt ~*th s w ic ttttk' are stable to bases and weak acids and can be eliminated using strong acids.

AlkyLene can be straight-chain or branched. Examples of definitions of (C6-Co10)-aryl are phenyL, tolyl or naphthyl; phenyl is preferred.

A carboxy protective group V is, for example, (C 1

-C

6 )-akyl or (C 7

-C

11 )-aralky; preference is given to methyl, ethyl, tert.butyl, benzyl and modified benzyl, such as p-chloro-i p-bromo-, p-nitro- and P-methoxybenzy and the nitrogen anarttct Log picoLyL. In the wider sense, such protective groups incude activated ester groups such as ONSu, OBt, O6bt or p- I nitrophenoxy.

The invention also relates to a process for the preparation of the compounds of the formula 1, which comprises a) reaction of a compound of the formula II 1 2 R CO- 02 W-C02,V y 3 y 4 jj -4in which R represents a leaving group which can be detached nucleophiLically, V represents a carboxyl protective group, and 1 2 3 4 W, Y Y, Y and Y are as defined in claim 1, with a compound of the formula III A- [B]p-NH- [Xm-NH 2 (III) in which A represents an amino protective group which is labile to bases or Labile to weak acids, and B, X, p and m are as defined in claim 1, and elimination of, where appropriate, one or both of the protective groups A and/or V in the resulting protected compound of the formula I, with the formation of the free NH 2 and/or CO 2 H group(s), the preferred processes being those in which V is selectively eliminated, for exam- Spie by reductive cleavage with Zn/glacial acetic acid, or b) reaction of a compound of the formula I in which A denotes hydrogen, and B, X, Y Y 2

Y

3 Y 4 V, W, m, n and p are as defined in claim 1, with a compound of the formula IV

(IV

in which A, B and p are as defined above, but A does not denote hydrogen, or its active ester, halide or azide, and, if V is not hydrogen, where appropriate Selimination of a carboxyl protective group V with the formation of the carboxyl group.

A leaving group R which can be detached nucleophilically is, for example, halogen, such as chlorine, bromine and iodine, or activated aryloxy, such as p-nitrophenoxy.

The reaction of a compound of the formula II with a compound of the formula IIl is preferably carried out in an 5 aprotic solvent such as, for example, THF, DMF, CHCL 3 or

CH

2

CI

2 advantageously in the presence of a base such as, for example, a tertiary amine, for example ethyl triisopropylamine, triethylamine or pyridine, the addition of an acylation catalyst such as, for example, DMAP, HOObt or HOBt having an advantageous effect, at a temperature between 0 0 C and the boiling point of the reaction mixture, preferably between 0° C and 400C.

Compounds of the formula I (A hydrogen) are reacted with compounds of the formula IV, their active ester, halide or azide preferably in an organic solvent, such as DMF, advantageously in the presence of a base such as, for example, a tert.amine, at a temperature between and the boiling point of the reaction mixture, preferably at room temperature. Examples of suitable active esters o are the ONSu, OBt, OObt and p-nitrophenoxy compounds.

Preferred halogen derivatives are the chlorides. Pyridinium perchlorate can be added to improve the solubility.

Compounds of the formula II are prepared by, for example, reacting esters of the formula IX yl y 2 HO-CHZ-^ (IX) 25 HO-CH 2 W-CO2-V (IX y 3 y 4 1 2 3 4 in which Y Y 2 Y 3

Y

4 W and V are as defined above, but V does not denote hydrogen, with phosgene or phosgene derivatives such as, for example, nitrophenyl chloroform- I* ate in an aprotic polar solvent, for example THF or DMF, mixed with a tert. base, for example a tert. amine such as pyridine, preferably in the ratio 1 1, at a temperature between -40 0 C and room temperature, preferably between -20°C and OOC.

The invention also relates to the use of a compound of the formula I, in which V denotes hydrogen and A does not 9 bt 99 9 @99 9 09 9* 9Q 0 9949 o1 94 *9 9 9*9 99 9rJ 9 .9 99 990 99 9 9P 9 9 9 r~S~ .999 6 denote hydrogen, in the solid-phase synthesis of compounds of the formula V P-NH-[X]m-NH 2

(V)

in which P represents a peptide residue comprising q S p+1 a-amino acids, and X, m and p are as defined above, and to a process for the preparation of a peptide of the formula V, in which P, X, m and p are as defined above, by solid-phase synthesis, which comprises coupling a compound of the formula I, in which A does not denote hydrogen, and V represents hydrogen, to a resin, eliminating the protective group A, stepwise coupling on q-p a-amino acids which are, where appropriate, in the form of their activated derivatives and which have been temporarily protected by amino protective groups which are labile to bases or labile to weak acids and, after construction is complete, liberating the peptide from the resin by 20 treatment with a moderately strong to strong acid, the temporarily introduced side-chain protective groups being eliminated again at the same time or, by suitable measures, subsequent thereto.

25 If necessary to prevent side reactions or for the synthesis of specific peptides, the functional groups in the side chain of amino acids are additionally protected by suitable protective groups (see, for example, T.W. Greene, "Protective Groups in Organic Syntheses", New York, John 30 Wiley Sons, 1981), those primarily used being Arg(Tos), Arg(Mts), Arg(Mtr), Asp(OBzl), Asp(OBut), Cys(4-MeBzL), Cys(Acm), Cys(SBut), Glu(OBzl), GLu(OBut), His(Tos), His(Fmoc), His(Dnp), His(Trt), Lys(CI-2), Lys(Boc), Met(O), Ser(Bzl), Ser(But), hr(Bzl), Thr(But).

The resins used as carrier material are commercially available. BHA and MBHA resins are preferred.

The peptide of the formula V is then cleaved off by -7treatment with the moderately strong to strong acids customarily used in peptide synthesis (for example trifluoracetic acid and HF), there being cleavage of the urethane protective group contained in the spacer.

It is possible to use as coupling reagent for the compound of the formula I (V H) and the other amino acid derivatives all possible activating reagents used in peptide synthesis, see, for example, Houben-Weyl, Methoden der organischen Chemie (Methods of organic chemistry), volume 15/2, but in particular 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 the amino acid derivative with the activating reagent and, where appropriate, an additive which suppresses racemization, such as, for example, 1-hydroxybenzotriazole (HOBt) Konig, R. Geiger, Chem. Ber. 102, 708 S,(1970)) or 3-hydroxy-4-oxo-3,4-dihydroxybenzotrlazine 44 (HOOBt) Konig, R. Geiger, Chem. Ber. 103, 2054 (1970)) l to the resin, or the preactivation of the amino acid derivative can be carried out separately as the symmetric anhydride or HOBt or HOObt ester, and the solution of the o activated species in a suitable solvent can be added to 4 #0 25 the peptide-resin which is ready for coupling.

The coupling and activation of the compound of the formula I (V H) and of the amino acid derivatives with one of the abovementioned activating reagents can be carried out S 30 in dimethylformamide or methylene chloride or a mixture Sof the two. The activated amino acid derivative is nort 6 mally used 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 coupling the next amino acid in the sequence.

Successful completion of the coupling reactio., can be checked using the ninhydrin reaction as described, for _111 -8 example, by E. Kaiser et al. Anal. Biochem. 34, 595 (1970).

The synthesis can also be carried out automatically, for example using an Applied Biosystems model 430A peptide synthesizer, it being possible to use either the synthesis programs provided by the apparatus manufacturer or those constructed by the user himself. The latter are particuLarly employed when amino acid derivatives protected with the Fmoc group are used.

When the peptide amides are cleaved off the resin with hydrogen fluoride and trifluoroacetic acid, it is customary to add substances as cation traps, such as phenol, cresol, thiocresol, thioanisole, anisole, ethanedithiol, dimethyl sulfide, ethyl methyl sulfide or a mixture of two or more of these auxiliaries. In this connection, the trifluoroacetic acid can also be used diluted by suitable solvents such as, for example, methylene chloride.

t Abbreviations used: Fmoc 9-fluorenylmethy oxycarbonyL Ddz Bpoc 2-E4-biphenyly]-2-propyloxycarbonyl Msc Methylsulfonylethyloxycarbonyl Peoc pyridylethyloxycarbonyl Pse phenylsulfonylethyloxycarbonyl Tse tolylsulfonylethyloxycarbonyl HONSu N-hydroxysuccinimide HOBt 1-hydroxybenzotriazole HOObt 3-hydroxy-4-oxo-3,4-dihydrobenzotriazine THF tetrahydrofuran DMF dimethylformamide DMAP dimethylaminopyridine The examples which follow serve to illustrate the present iijvention without intending to confine it to them.

-9- Example 1: MethyL 4- hydroxyethylphenoxyacetate 18.2 g of 4-hydroxymethyLphenoxyacetic acid are dissolved together with 17.1 ml of N,N-diisopropyLethylamine in 50 ml of DMF, and then 6.1 ml of methyl iodide are added to the stirred solution. The mixture warms slightly during this. The reaction is complete after 3 h. The solvent is removed in vacuo. The residue is taken up in ether, and the solution is extracted once with 0.5 N hydrochloric acid. The aqueous phase is then extracted three times with ether, and the combined ether phases are washed with aqueous sodium bicarbonate iaolution and concentrated. The residue is dissolved in ethyl acetate and filtered through a short silica gel column. The pale yellowish oil which is obtained after concentration crystallizes on being left to stand.

I

NMR and mass spectrum are consistent with the indicated structure.

Example 2: Fmoc-NH- (C 2 4 NH--0-CHZ r

O-CH

2

-COOCH

9.8 g of methyl 4-hydroxymetr.ylphenoxyacetate are dissolved in 200 ml of dry CH 2

CL

2 and then 10.1 g of p-nitrophenyl chloroformate and 7 ml of triethylamine are added.

The mixture is boiled under reflux for about 6 h, until the precursor has completely reacted. Then a suspension of 15.5 g of Fmoc-NH-(CH2)4-NH2 (prepared by reaction of Boc-NH-(CH 2 )4-NH 2 with Fmoc-ONSu foLLowed by elimin- 30 ation of Boc) in 100 ml of dry CH 2 C1 2 and a further 7 ml of triethylamine are added, and he mixture is boiled under reflux. After the reaction is complete, the solvent is removed in vacuo, and the residue is digested with ether and filtered off with suction. The residue on the filter is washed with aqueous I N Na 2 C03 solution and then with hot water, and is dried under high vacuum in a desiccator.

Melting point 122-124 0 NMR and mass spectrum are 8 ~8 8 888 8 #8 68 I 6

I

66 10 consistent with the indicated structure.

Example 3: H 2

N-(CH

2 4 -NH-CO-O-CH2 0 d1H 2

C"OOH

5.2 g of the ester obtained as in ExampLe 2 are suspended in 100 ml of methanol, and 6 equivalents of an aqueous 1 N NaOH soLution are added. After the reaction is ccniplete, the pH is adjusted to 3 with aqueous 1 N HC L, and the methanol is removed in vacuo. The precipitate is filtered off with suction, washed with a little H120, and then digested in ether and again filtered with suction.

Melting point starts at 1960C (decomposition), NMR and m3ss spectrum are consistent with the indicated structure.

ExampLe 4: Fmoc-Phe-NH-(CH 2 4 -NH-CO--CH2.(ij- O-CH 2

-COOH

1.5 g of the product obtained as in Example 3 are suspended in 50 mL of dry DMF. Then, successiveLy, 0.9 g of pyridlinium perchLorate (to improve the solubility) and 2.6 g of Fmoc-Phe-OObt and 0.5 mL of triethyLamine are.

added. The mixture is sti rred at room temperature. Af ter the reaction is complete, the solvent is removed in vacuo, and the residue is partit ioned between ethyL acetate and 25 H 2 0. The aqueous phase is extracted once more with ethy( acetite, and the combined organic phases are dried anr.i concentrated. The residue is digested with a little CH~l andc is filtered off with suction. The res idue on the f116, ter is washed with a little ether and is dried.

30 Melting point starts at 140Q 0 C (decomposition),, NMR and mass spectr um are cons istent with, the indicated structure.

Example 5: FDIoc-Phe-N-(CH 2 4 -NH..CO-0.OCH 2 J_\V -HC- (4-*ethyLbenzhydrykaqine resin) 1.4 g of the Fmoc-phenyLaLanlne spacer acid obtaino-~d ki in Example 4 are dlissoLved together with 350 "19 In 40 rL dry DMF, and the solution is added 4-me~hyLbenzhydryLamine resin (Novta Biochem,, I I 8* 88 8 *8* 8 4* AS 8 A *8 *888 *5*8 8 888*86 8 6 48

I

Ii *6

A

8

I

4 44- 4*44 4 *4 4 444 4 44 444 11 4 11 0.4 mmol/g). Then 0.6 mL of diisopropylcarbodiimide is added, and the reaction is allowed to go to completion, mixing continuously. After the reaction is complete, the product is filtered off with suction, washed with DMF, isopropanol, CH 2

CI

2 and tert.-butyl methyl ether and is dried under high vacuum. Loading according to elemental analysis (nitrogen determination): 0.3 mmol/g.

Example 6: Synthesis of [des-Tyr 2 4 des-Arg 23 3-ratriopeptinIII-(4-amino)butylamide The peptide synthesis is carried out on 1 g of the abovementioned resin using OOBt esters of Fmoc-amino acids with an Applied Biosystems model 430A automatic peptide synthesizer and synthesis programs modified by ourselves.

For this, 1 mmol of each of the appropriate amino acid derivatives is weighed into the cartridges supplied by the manufacturer, Fmoc-Arg(Mtr)-OH, Fmoc-Asn-OH and Fmoc- 20 Gin-OH are weighed together with 1.5 mmoL of HOBt into the cartridges. These amino acids are preactivated directly in the cartridges by dissolving in 4 ml of DMF and adding 2 ml of a 0.55 M solution of diisopropylcarbodiimide in DMF. The HOObt esters are dissolved in 6 ml of DMF and then pumped, in the same way as the afino acids arginine, asparagine and glutamine which are preactivated in situ, onto the resin which has previously been deblocked with 20% piperidine in DMF. The amino acids which are activated in situ are coupled twice.

After the synthesis is complete, the peptide butylamide is cleaved off the resin, simuLtaneously removing the side-chain protective groups with trifluoroacetic acid which contains thioanisole and m-cresol as cation traps.

The residue obtained after removal of the trifluoroacetic acid in vacuo is subjected to digestion with ethyl acetate and centrifugation several times. The remaining crude peptide is treated with tributylphosphine in trifluoroethanol to remove the cysteine protective group.

l 4lll I i 12 After the solvent has been removed, the residue is again digested with ethyl acetate and centrifuged. The reduced crude peptide is immediately oxidized with iodine in strength aqueous acetic acid, the excess 12 is removed with ascorbic acid, and the reaction mixture is concentrated to a small volume and then salt is removed on Sephadex with aqueous 1 N acetic acid. The fractions containing the pure peptide are combined and freeze-dried.

According to amino acid analysis, the amino acid composition of the peptide corresponds to the indicated formula.

Example 7: Phenacyl 4-hydroxymethylphenoxyacetate 182 g of 4-hydroxymethylphenoxyacetic acid and 199 g of a-bromoacetophenone are dissolved in 600 ml of dry DMF, and then, at 00 C, 138 ml of triethylamine are rapidly added dropwise. The mixture is allowed to reach room temperature, and is stirred overnight. The DMF solution is poured into 3.5 l of water, and the aqueous phase is extracted with ethyl acetate. 'he organic phase is washed with water, dried over sodium sulfate and concentrated.

The product precipitates out on evaporation. It is filtered off with suction, washed with ethyl acetate/n-hexane S 25 1:1 and dried under high vacuum.

Melting point: 94-950C, NMR is consistent with the indicated structure.

Example 8: 0 2

O-CO-O-CH

2

O-CH

2 .C02-CH 2

-CO

30 g of phenacyl 4-hydroxymethylphenoxyacetate are dissolved, under protective gas, in 500 ml of a 1:1 mixture of THF and pyridine, and the solution is cooled to -20 0

C.

Then 21 g of p-nitrophenyl chloroformate dissolved in 100 ml of THF are added dropwise. After the mixture has been stirred at this temperature for 30 minutes, it is allowed to warm to 0OC and stirred into 1 I of a halfsaturated aqueous NaCL solution at 0°C, and the mixture is then stirred for 30 minutes. The precipitate is filtered i 13 off with suction, washed with ice-water and, after drying, triturated with n-hexane.

Melting point: 142-145 0 C, NMR is consistent with the indicated structure.

Example 9: Fmoc-Phe-NH-(CH 2 8-H-CO-O-CH2-j O-C 2

-CO

2

-CH

2

-CO-

9.3 of the compound prepared in Example 8, 12.25 g of Fmoc-Phe-NH-(CH 2 8

-NH

2 trifluoroacetate and 3.26 g of HOObt are placed as the solid substances in a flask, and then a mixture of 2.58 g of ethyl diisopropylamine in 100 mL of dry DMF is poured over. The mixture is then stirred at 400 C for 3.5 hours and then stirred into 500 mL of halfsaturated aqueous NaCL solution. The precipitate which separates out is filtered off with suction, washed with ice-water and, after drying, triturated with ether/ethyl acetate.

Melting point: 147-1500oC, NMR and MS are consistent with the indicated formula.

The following compounds (ExampLes 10 to 14) are prepared in analogy to Example 9: Example t fC Fmoc Phe-NH-(cH 2 )4 NK-CO-O-CH2 co-C C 2 -CH eC tt tt Melting point 144-147 0 C, NMR and Ms correspond to the indicated formula.

Example 11: 3 Fmoc-Ala-NH- (CH2)8-H-cO-o-c2 0-CH2-CH-c 2-ca-o Melting point 179-181 0 C, NMR and MS correspond to the indicated formula.

-14- ExmpleNH (CH 2 8 M- CO 0- C H CH 2 C2 MeLt ing point 144-145 0 C, NMR and MS correspond to the indli- {i cated formuLa.

FormuLa 13: Fmoc-NH- (CH 2 6 .NH-CO- 0-C2 C0 2 -CH2-C MeL ting point 172-175 0 C, NMR corresponds to the indicated formul a.

Example 14: Fmoc- NH- (CH 2 NH- CO- 0- CH 2

CH

2 C0 2

CH

2

CO

MeLting point 165"166 0 C, NMR corresponds to the indicated formuLa.

Exam!,te

(CH

2 8 NH-CO-0-CH 2

O-CH

2

-CO

2

H

8.4 g Fmoc-Plie-NH- (CM 2 )8-HCOOc are suspended in a mixture of 150 mL of glaciaL acetic tot,, 0 aidand 50 ml of dlichLoromethane, and 12 g of zinc pow- "404 der which has previously been activated by washing with 1 N HCL and dry ethanol are added in portions. After a few minutes, the suspension becomes more viscous and difficult to stir, while there is sLight evoLution of heat.

Hence a further 80 ml of glacial acet ic acid and 5Q ml of dichioromethane are added, and stirring is continued overn ig h t The mixture is then filtered with suction through a filter with a clarifying Layer, washing with glacial acetic acid and dichLoronethale. The filtrate is I, i ii;i~ 15 concentrated, and the oil which remains as residue is taken up in a Little dichLoromethane and stirred with ethyl acetate and ether. The precipitated product is filtered off with suction and dried under high vacuum.

Melting point: decomposition above 1600C, NMR and MS are consistent with the indicated formula.

In addition, the compounds of Examples 16 to 18 are prepared by the method described in Example Example 16: Fmoc- Phe-NH- (H 2 4-NH- CO- 0- CH2 O- CH2-CO 2

H

Melting point: decomposition above 1500C, NMR and MS are consistent with the indicated formula.

Example 17: Fmoc-Phe-NH-(CH 2 8 -NH-CO-0-CH2 -0-CH 2

-CO

2

H

S0 4 t °Melting point: decomposition above 1600C, NMR and MS are consistent with the indicated formula.

a ab 61 25 Example 18: S. Fmoc- NH- CH2)8-NH-CO-0-CH2- Q-CHCOC02H 4 4 Melting point: decomposition above 154 0 C, NMR and MS are 30 consistent with the indicated formula.

Example 19: Fmoc-NH-(CH 2 6 -NH-CO-0-CH 2 -j O-CH 2

-CO

2

CH

3 The synthesis is carried out in analogy to Example 2.

Melting point: 115-118 0 C, NMR and MS are consistent with the indicated formula.

16 Example

NH

2 -(CH2) 6 -NH-CO-0-CH 2

KJ--CH

2

.CO

2

H

was prepared by the method described in Example 3.

Melting point: 184-187 OC decomposition, NMR and MS are consistent with the indicated formula.

ExampLe 21: Fm'oc- Phe- NH- (CH 2 6 -NlCO-O- CH2-J/ CH 2

CO

2

H

The synthesis is carried out in analogy to Example 4.

Melting point: decomposition above 120 0 C, NMR and MS are consistent with the indicated formula.

4 f4a

Claims

1. A compound of the formula I yl v2 A-[B]p-NH-[X]m-NH-CO-0-CH 2 W-CO 2 -V y 3 y 4 in which A denotes hydrogen or an amino protective group which is Labile to bases or labile to weak acids, B represents identical or different amino acid resi- dues, X denotes (C1-C 12 )-alkylene or (C 6 -C 10 )-aryL-(C 1 -C 12 alkylene, y1 y3 4 Y 1 2 Y and Y are identical or different and denote hydrogen, methyl, methoxy or nitro, at least one of these radicals denoting hydrogen, V denotes hydrogen or a carboxyl protective group, W denotes -[CH23 n or -0-[CH23n-, m is 0 or 1, n is an integer from 0 to 6, and p is an integer from 0 to

2. A compound of the formula I as claimed in claim 1, in which p is 0, 1 or 2.

3. A compound of the formula I as claimed in claim 1 or 2, in which m is 1.

4. A compound of the formula I as claimed in one of claims C. I c. 2. Q \ky 0 1 3, in which X denotes.C -d l dnots into A compound of the formula I as claimed in one of claims 1 4, in which at Least 2 of the radicals Y 1 Y 2 Y and u Y 4 denote hydrogen. 18 18

6. A process for the preparation of a compound as claimed in one of claims 1 5, which comprises a) reaction of a compound of the formula II Sl y 2 R-CO-0-CH 2 WC0 2 -V Y3 y4 in which R represents a leaving group which can be detached nucLeophilically, V represents a carboxyl protective group, and W, Y Y y 3 and Y4 are as defined in claim 1, with a com- pound of the formula III A- [B]p-NH- [X]m-NH 2 in which A represents an amino protective group which is Labile to bases or labile to weak acids, and B, X, p and m are as defined in claim 1, and eLimination of, where appropriate, one or both of the protective groups A and/or V in the resulting protected compound of the formula I, with the formation of the free NH 2 and/or CO 2 H group(s) or b) reaction of a compound of the formula I in which A denotes hydrogen, and B, X, Y1, Y 2 Y 3 Y 4 V, W, m, n and p are as defined in claim 1, with a compound of the formula IV A- [B15-p-OH (IV) in which A, B and p are as defined above, but A does not denote hydrogen, or its active ester, halide or azide, and, if V is not hydrogen, where appropriate elimination of a carboxyl protective group V with the formation of the carboxyl group. -19-

7. The use of a compound of the formula I as claimed in one of claims 1 5, in which V denotes hydrogen, and A does not denote hydrogen, in the solid-phase synthesis of com- pounds of the formula VI P- NH- [Xm-Nm2 (VI) in which P represents a peptide residue comprising q p+1 a-amino acids, and X, m and p are defined as in claim 1.

8. A process for the preparation of a peptide of the formula V, in wh'ch P, X, m and p are de'fined as in claim 7, by solid-phase synthesis, which comprises coupling a com- pound of the formula I, as claimed in one of claims 1 in which A does not denote hydrogen, and V represents hydrogen, to a resin, eliminating the protective group ft A, stepwise coupling on of q-p t-amino acids which are, where appropriate, in the form of their activated deriva- Stives and which have been temporarily protected by amino protective groups which are labile to bases or labile to weak acids and, after construction is complete, liberating the peptide of the formula V from the resin by treatment with a moderately strong to strong acid, the temporarily tI introduced side-chain protective groups being eliminated again at the same time or, by suitable measures, subsequent thereto. DATED this 19th day of October 1987. HOECHST AKTIENGESELLSCHAFT EDWD. WATERS SONS PATENT ATTORNEYS QUEEN STREET MELBOURNE. VIC. 3000.