AU626128B2 - Peptides having vasorelaxant, natriuretic and diuretic actions, a process for their preparation, agents containing them, and their use - Google Patents

Description

run;uwrsrrr~*aacn=nsm~~ 1MU-CIIII IF 6 2 6 1 8 Form COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class Application Number: Lodged: Int. Class So Complete Specification Lodged: 0 o o 4 o o a o a °Priority V 4 i o Related Art: Accepted: Published: 4 44 440 0 00 o *4 Name of Applicant: 4 0 o o a a Address of Applicant HOECHST AKTIENGESELLSCHAFT 45 Bruningstrasse, D6230 Frankfurt/Main 80, Federal Republic of Germany GERHARD BREIPOHL, JOCHEN KNOLLE, WOLFGANG KONIG and MAX HROPOT 44*400 Actual Inventor: Address for Service EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the invention entitled: PEPTIDES HAVING VASORELAXANT, NATRIURETIC AND DIURETIC ACTIONS, A PROCESS FOR THEIR PREPARATION, AGENTS CONTAINING THEM, AND THEIR USE The following statement is a full description of this invention, including the best method of performing it known to us p: HOECHST AKTIENGESELLSCHAFT Dr.WJ./gm HOE 87/F 204 Peptidles having vasoreLaxant, natriuretic and diuretic actions, a process for their preparation, agents containing them, and their use EP-A2 140731, EP-A2 142487 and EP-A2 152333 discLose peptides whose sequence is a part-sequence of naturaL atriaL natriuretic factor (ANF) of humans or rats. ANF peptidles aLe aLso described in EP-A 231,752 (ZA 87/0 272).

The invention relates to new pept ides of the formuLa I 0:::in wh ic h X denotes hydrogen, (Cl-C 5 )-aLkoxycarbonyL, (C 6 -Cl 2 aryLoxycarbonyL, (C 7

-C

13 )-araLkyLoxycarbonyL, (Cl-C 6 aLkanoyL, (C 7 -Cl 3 )-aroyL, arginyL, LysyL, E-aminocaproyl, 000 arginyL-arginyl, arginyL-LysyL, LysyL-arginyL or LysyL- LysyL; (Cl-Cl 2 )-aLkyLcarbonyL or (C -C)-ccoklar bonyl which are op-Eionally branched in the d- position and ontiona11y rnonosubstituted in the w position byj amino or guanidino; or represents Ser, Thr, Ser(Y), Thr(Y), Q or Leu, each in their L or D configuration, or Ser-Ser, Thr-Thr, Ser-Thr, Q-Ser, Q-Thr, Thr-Q, Ser-Q, SerCY)-Ser or Ser-SerCY), it being possibLe for each amino acid to be present in its L or D configuration, and the N-terminal amino group of the amino acid or of the dipeptidle residue being in the free form (N-terminal radlical= H) or acyLated by (C 1

C

5 )-aLkoxycarbonyL, (C 6 -Cl 2 )-aryLoxycarbonyL, (C 7 -C 13) araLkyLoxycarbonyL, (C 1

-C

6 )-aLkanoyL, (C 7 -C 13 )-aroyL, arginyL, LysyL, c-aminocaproyL, arginyL-arginyL, arginyL-Lysyl, LysyL-arginyL cr LysyL-LysyL, or it being possible for X to be absent; V denotes Cys or, if X is absent, w-thio-CC 2

-C

8 )-aLkyLcarbonyL such as, for example, mercaptopropionic acid, -2 mercaptoacetic acid or mercaptobutyric acid; A denotes Phe, Tyr(Me), Tyr(Bu 4-haLogenophenyLaLanine, Trp or an L-2-thienyLaLanine residue; A 1denotes GLy, Ala or D-ALa; 9 9 99 9 04 9999 0990 o 9 9 999999 o oo 9 0 4 94 9.

9 990 A 2 denotes GLy, ALa or D-ALa; or A 1 and A 2 together denote an optionaLLy branched w-amino- (C 3 C 8 )-aL k yL c ar bo xyL i c a c idc; 8 denotes Arg, Lys, Orn or an L-homoarginire residue; 15 C denotes Lie,, Met, Phe, Trp, Leu,, Ser, Thr, VaL, His, Pro, Asn, Ser(Bu cycLohexyLaLanine, tert.-butyLgLycine, neopentyiglycine or an L-2-thienyLaLanine re s i due; 20 D denotes Asp, GLu, Gin, Asn, Phe, Leu, ILe, Trp, Pro, Tyr, Ala, Asp(OBu Asp(OBzL), GLuCO8u GLu(OBzL), a 2-thienyiaLanine residue, Aad, Tyr(Bu t) or Tyr(Me),, it being possible for each of the amino acids to be present in its L or D configuration; E denotes Gin, Thr or Pro; F denotes case in Ser, Thr, Pro, Ala, Ser(But or Thr(Bu t in each its L or D configuration; G denotes GLy, Ala or 0-ALa; Q denotes a radlicaL of the formula IV 1 2 R R 0 1 1 11 N CH C

(IV)

in which 3 R and R together with the atoms bearing these radicaLs, form a heterocycLic mono-, bi- or tricycLic system having 3 to 15 carbon atoms; Y denotes tert.-butyl or an optionally protected or partiaLLy protected gLycosyl radical; and Z represents a radical of the formula II -H-I-J-K-L-M (II) in which H denotes Asn, Ser, Q or Thr, in each case in its L or D form, or GLy; 1 I denotes Ser, Thr, ALa, Ser(Bu Thr(Bu t Soo or Pro, in each case in its L or D form; S 15 J denotes Phe, Trp, D-Phe, D-Trp, Tyr(Me), cycLohexyLalanyl, or a 2-thienylaLa nine 440a a residue; K denotes Arg, Lys, Orn or a bond; L denotes Q, Gly, Tyr, Tyr(But), Tyr(Me) or a bond; M denotes Arg-OH, Arg-NH 2 OH, OR, NH 2

NHR',

GLy-Lys-Arg-OH, Gly-Lys-Arg-NH2 or L-argininol; Q is as defined above; R denotes unbranched (C 1

-C

6 )-aLkyL, and R denotes -CCH 2 3n-NH 2 or -CCH2]n-NH-

C(NH)NH

2 n being an integer and representing 3-8; and their physiologically tolerated salts, excepting the peptides which correspond to the sequence of natural ANF and are of the formula III (III) X-Cys-Phe-Gly-Gly-Arg-C-Asp-Arg-Ile-Gly-Ala-Gln- -Ser-Gly-Leu-Gly-Cys-Z t n '.in wh ich -4 X denotes Ser, Ser-Ser, Arg-Ser-Ser or A rg-A rg-Se r-Se r, C denotes Lie or Met, Z denotes Asn-Ser-Phe-K-L-M, K denotes Arg or a bond, L denotes Tyr or a bond, and M denotes OH or NH 2 and their saLts compounds described in EP-A 231,752 (ZA 87/0 272; 0 9 9J) and the HOE 86/F rat-CMac 10 5 ]IANF( 101-126), rat-[Mpr 10 5 5 ANF (101-126), rat-[Mbu 10 5 ]IANF( 101-126) and rat-CMpr 105 D-Ala 107] ANF (105-126).

04 0 0 0 0 o .4 04 0004 o 0 0000 0 0 044004 0 0 00 0 *0 0 0 0 00 15 P ar t icu La r Ly s u ita b Le a s t he ra d ic aL o f a h et er o cy c Lic ring system of the formula IV are radicals of heterocycl es from tfle following group: PyrroLidine piperidine tetrahydroisoquino- 20 Line decahydroisoquinoLine octahydroindoLe octahydrocycLopentalblpyrroLe 2-azabicycLo- 12.2.2.]octane 2-azabicycLoC2.2.1]heptane 2- 2-azaspiroE4.41nonane J) spiro[(bicycLo[2.2.l]heptane)-2,3-pyrroL idine] spirc[(bicycLoL2.2.2]octane)-2,3-pyrroLidineI 2-azatricycLoE4.3.O.1 6 9 Jdecane decahydrocycLohepta~b]pyrroLe octahydroisoindoLe octahydrocycLopenta~clpyrroLe 2,3,3a,4,5,7a-hexahydroindoLe tetrahydrothiazoLe 2-azabicycLo[3.1.O]hexane all of which can optionally be substituted.

A

H

p 5 CoCO- H N H C6- I z

I.

N

K

"N

I

os 0 S6 0 9 0D 0 0 0* 0 a 00 00 o 0 00 0 000 D 0 1 cc-

L

M3- C

N

N

R

The heterocycles from which the abovementioned radicals are derived are disclosed in, for example, US Patent 4,344,949, US Patent 4,374,847, US Patent 4,350,704, EP-A 50,800, EP-A 31,741, EP-A 51,020, EP-A 49,658, EP-A 49,605, EP-A 29,488, EP-A 46,953, EP-A 52,870, EP-A 72,022, EP-A 84,164, EP-A 89,637, EP-A 90,341, EP-A 90,362, EP-A 105,102, EP-A 109,020, EP-A 111,873 and EP-A 113,880 Y is tert.-butyl or a glycosyl radical which is protected or partially protected by protective groups customary in carbohydrate chemistry, or is unprotected, and which is derived from a glycopyranose, glycofuranose or an oligosaccharide.

'f" -6- Protected glycosyl radicals are preferred. Both a- and BgLycosidic Linkage of the glycosyl radicals to the serine residue is possible.

Possible examples of Y are a glycofuranosy or a glycopyranosyl radical which is derived from naturally occurring aLdotetroses, aldopentoses, aldohexoses, ketopentoses, deoxyaLdoses, aminoaldoses and oLigosaccharides, such as di- and trisaccharides, and their stereoisomers.

The glycosyl radicals Y are derived, in particular, from natural D- or L-monosaccharides which occur in microorganisms, plants, animals or humans, such as ribose (Rib), 00. arabinose (Ara), xylose (Xyl), lyxose (Lyx), allose (ALL), 15 altrose (Alt), glucose (GLc), mannose (Man), gulose (GuI), co* idose (Ido), galactose (Gal), talose (Tal), erythrose (Ery), threose (Thr), psicose (Psi), fructose (Fru), sorbose (Sor), tagatose (Tag), xylulose (Xyu), fucose (Fuc) rhamnose (Rha), olivose (OLi), oliose (Olo), mycarose 20 (Myc), rhodosamine N-acetylglucosamine (GLcNAc), NacetyLgalactosamine (GaINAc) or N-acetyLmannosainine (ManNAc), or disaccharides such as maltose (Mal), lactose (Lac), cellobiose (CeL), gentiobiose (Gen), N-acetyllactosamine (LacNAc), chitobiose (Chit), B-galactopyranosyl- (1-3)-N-acetygaactosamine and B-galactopyranosyl-(1-3)or -(1-4)-N-acetygucosamine, as well as their synthetic derivatives such as 2-deoxy, 2-amino, 2-acetamido or 2halogeno, preferably bromo and iodo, sugars.

The protective groups customary in carbohydrate chemistry are to be understood to be, for example, the (C 1

-C

10 )-acyL protective groups such as (C1-C6)-alkanoy (for example acetyl, trichloroacetyl, trifluoroacetyl), benzyl or pnitrobenzoyl, as well as optionally modified methyl, methyloxymethyl, benzyl, tetrahydropyranyl, benzylidene, isopropylidene or trityl group, preference being given to the acyl protective groups, in particular the acetyl (Ac) group, in this connection.

-7- Preferred peptides of the formuLa I are those in which X denotes hydrogen, (Cl-C 5 )-aLkoxycarbonyL, (C 6 -Cl 2 aryloxycarbonyL, (C 7

-C

13 )-araLkyLoxycarbonyL, (C 1

C

6 aLkanoyL, (C 7

-C

13 )-aroyL, arginyL, LysyL or arginytarginyl; or denotes (Cl-C 12 )-aLkyLcarbonyL or (C 3 -C 8)cvcloalkylcarbonyl which are optionally branched in the o position and optionally ronosubstituted in the a position by amino or guanidino-or represents Ser, Thr or Q, each in its L or D configuration, or Ser-Ser, Thr-Thr, Ser-Thr, Pro-Ser, Pro-Thr, Thr-Pro or Ser-Pro, preferably Ser-Ser, it being possibLe for each 00 0 o amino acid to be present in its L or D configuration, 00 and the N-terminaL amino group of the amino acid or of the dipeptide residue being in the free form or acyLated by (C 1

-C

5 )-aLkoxycarbonyL, C 6

-C

12 )-aryLoxycarbonyL, 00 (C 7 -C 13 )-araLkyLoxycarbonyL, (C 1

-C

6 )-aLkanoyL, (C 7 -C 13)- 0 aryl arinl 0yy.r00iyLagnyo i en 0r yL ar i y L s L o r i yL a g n r i e n possible for X to be absent; 0 00V denotes Cys or, if X is absent, mercaptopropionic acid, I 90 mercaptoacetic acid or mercaptobutyric acid; 0 A denotes Phe, Tyr(Me), Tyr(Bu 4-haLogenophenyLaLanine or a L-2-thienyLaLanine residue, preferabLy 0: P he; 0:000:1 A 1 dntsGy l r Aa

A

2 denotes GLy, Ala or D-ALa; o A 1 and A2 together denote an optionally branched w-amino-

(C

3 -CS)-aLkyLcarboxyL ic acid; B3 denotes Arg or Lys; C denotes Lie, cycLohexyLaLanine, tert.-butyLgLycine, neopentyLgLcyine, Phe, Leu, VaL or an L-2-thienyLaLanine residue;

I

1. 8 D denotes Asp, Glu, GLn, Asn, Leu, ILe, Trp, Asp(OBu GLu(OBu GLu(OBzL) or a 2-thienylalanine residue, preferably Asp, Glu or Leu, it being possible for each of the amino acids to be present in its L or D configuration; E denotes GLn, Thr or Pro; F denotes Ser, Thr or ALa, preferably Ser or ALa, in each case in its L or D configuration; G denotes GLy, Ala or D-Ala, a a a a 00 a~ a o a.

a, a o a a 00 a a a a, and 4 tI

I

Z represents a radical of the formula II in which H denotes Asn, Ser, Pro or Thr, preferably Pro, Thr or Asn, in each case in its L or D form, or Gly; I denotes Ser, Thr, Ala or Ser(Bu preferably Ser, in each case in its L or D form; J denotes Phe, Tyr(Me), cyclohexylalanyl or an L-2-thienyLaLanine residue, preferably Phe; K denotes Arg, Lys, Orn or a bond; L denotes Tyr, Q, GLy, Tyr(Me) or a bond; M denotes Arg-OH, Arg-NH 2 OH, OR, NH 2

NHR',

Gly-Lys-Arg-OH or GLy-Lys-Arg-NH 2 R denotes unbranched (C 1

-C

6 )-akyL, and R' denotes -[CH23n-NH 2 or -CCH 2 ]n-NH-C(NH)NH 2 n being an integer and representing 3-8, as well as their physiologically tolerated salts, excepting peptides of the formula III, the compounds described in EP-A 231,752 and their salts, rat-EMac 105 ANF(101-126), rat-CMpr 10 5

]ANF

(101-126), rat-EMbu 105 ANF (101-126) and rat-EMpr 10 5 D-ALa 1 0 7 ANF (105-126).

Particularly preferred peptides of the formula I are those in which 71q 00 *0.

0 I *4 0444 4 t

I

9 X denotes hydrogen, arginyl or arginyL-arginyl, Ser, Q or Ser-Ser, it being possible for each of Q and Ser to be present in the L or D configuration, and the N-terminal amino group of the amino acid or dipeptide residue being present in free form or acylated by arginyl or arginylarginyl, or it being possible for X to be absent; V denctes Cys or, if X is absent, mercaptopropionic acid; A denotes Phe; B denotes Arg or Lys: C denotes ILe, Leu, Vat or an L-2-thienyLalanine residue; D denotes Asp, GLn, Leu, ILe, Asp(OBu GLu(OBut), Tyr(But) or Tyr(Me); E denotes GLn, Thr or Pro; F denotes Ser or ALa, in each case in its L or D configuration; G denotes Gly, ALa or D-Ala, and Z represents a radical of the formula II in which H denotes Asn, Pro or Thr, in each case in 20 its L or D form, or GLy; I denotes Ser, Thr or ALa, in each case in its L or D form; J denotes Phe, Tyr(Me), cyclohexyLaLanyl or a 2-thienylalanine residu.; K denotes Arg, Lys or a bond; L denotes Tyr, Tyr(Bu or a bond; M denotes OH, NH 2 NHR', GLy-Lys-Arg-OH or GLy-Lys-Arg-NH 2 and R' denotes -CCH 2 2

-NH

2 or -CCH2]n-NH-C(NH)NH 2 n being an integer and representing 3-8; as weLL as their physiologically tolerated salts, excepting peptides of the formula III, the compounds described in EP-A 231,752 and their salts, rat-CMac105 3ANF (101-126), rat- EMpr 10 5 ANF (101-126), rat-[Mbu 10 5 ANF (101- 126) and rat-EMpr 10 5 D-Ala 107 ANF (105-126).

IS

r 4 t

F

0 ii~~ Unless otherwise indicated in the individual case, aLkyL may be straight-chain or branched. A corresponding statement applies to radicals derived therefrom, such as alkoxy, aralkyl or alkanoyl.

(C6-C 12 )-Aryl preferably denotes phenyl, naphthyl or biphenylyl. Radicals derived therefrom, such as aryloxy, aralkyl or aroyl, are to be formulated correspondingly.

Unless indicated otherwise, the abbreviation of an amino acid residue without a stereodescriptor represents the residue in the L form (cf. Schrbder, L'bke, The Peptides, Volume I, New York 1965, pages xiii-xxix).

Particularly suitable salts are alkali metal or alkaline S 15 earth metal salts, salts with physiologically tolerated O" amines and salts with inorganic or organic acids such as, Sfor example, HCI, HBr, H 2 S0 4 maleic acid and fumaric acid.

f The invention also relates to a process for the preparation of peptides of the formula I, which comprises reaction of a fragrent having a free C-terminal carboxyl group, or one activated derivative, with an appropri- S 'r ate fragment having a free N-terminal amino group, or b) stepwise synthesis of the peptide, elimination, where appropriate, of one or more protective groups which have been temporarily introduced to protect other groups in the compound obtained as in or formation of a disulfide bridge between the two Cys residues, it also being possible for the two latter measures to be carried out in the reverse sequence, and, where appropriate, conversion of the resulting compound of the formula I into its physiologically tolerated salt.

The peptides of the present invention have been prepared by generally known methods of peptide chemistry, see, for example, Houben-Weyl, Methoden der organischen Chemie __~-aasn n;aa~ 11 (Methods of Organic Chemistry) Volume 15/2, preferably by solid-phase synthesis as described, for example, by B.

Merrifield, J.Am.Chem.Soc. 85, 2149 (1963) or R.C.

Sheppard, Int.J.Peptide Protein Res. 21, 118 (1983), or equivalent known methods. The a-amino protective groups used are urethane protective groups such as, for example, the tert.-butyloxycarbonyl(Boc) or fluorenylmethyLoxycarbonyL(Fmoc) protective group. If necessary to prevent side-reactions or for the synthesis of specific peptides, S 10 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 Synthesis"), use being made primarily of Arg(Tos), o o Arg(Mts), Arg(Mtr), Asp(OBzl), Asp(OBu Cys(4-MeBzl), 15 Cys(Acm), Cys(SBut), Glu(OBzl), Glu(OBut), His(Tos), His(Fmoc), His(Dnp), His(Trt), Lys(CL-2), Lys (Boc), Met(O), Ser(Bzl), Ser(But), Thr(Bzl), Thr(But), Trp(Mts), Trp(CHO), Tyr(Br-Z), Tyr(Bzl) or Tyr(Bu The solid-phase o|o synthesis starts at the C-terminal end of the peptide with o o the coupling of a protected amino acid onto an appropriate o resin. Starting materials of this type can be obtained by linking a protected amino acid via an ester or amide bond to a polystyrene or polyacrylamide resin which has been oo modified with a chloromethyl, hydroxymethyl, benzhydryl- 25 amino(BHA) or methylbenzhydrylamino(MBHA) group. The resins which are used as carrier materials are commercially available. E:A- and MBHA-resins are usually employed if the synthesized peptide is to contain a free amide group at the C-terminal end. If the peptide is to contain a secondary amide group at the C-terminal end, a chloromethyl- or hydroxymethyl-resin is used, and the elimination is carried out with the appropriate amines. If it is desired to obtain, for example, the ethylamide, it is possible to cleave the peptide off the resin with ethylamine, in which case the elimination of the side-chain protective groups is subsequently carried out with other suitable reagents. If the peptide is to retain the tert.-butyl protective groups on the amino acid side chain, the synthesis is carried out using the Fmoc protective group for ii -IWYI IP qX~I~rr 12 the temporary blocking of the a-amino group of the amino acid, using the methods described, for example, in R.C.

Sheppard, J.Chem.Soc., Chem.Comm. 1982, 587, the guanidino group of the arginine being protected by protonation with pyridinium perchlorate, and the other amino acids functionalized in the side chain being protected by benzyl protective groups which can be eliminated by catalytic transfer hydrogenation Felix et al. J. Org. Chem. 13, 4194 (1978)) or by sodium in liquid ammonia Roberts, 10 J.Am.Chem.Soc. 76, 6203 (1954)).

"After the amino protective group on the amino acid which S is coupled to the resin has been eliminated using a suit- S able reagent such as, for -ple, trifluoroacetic acid S 15 in methylene chloride in t' case of the 8oc protective group, or a 20% strength solution of piperidine in dimethylformamide in the case of the Fmoc protective group, o' the subsequent protected amino acids are successively coupled on in the desired sequence. The N-terminal protected peptide-resins which are produced as intermediates i are deblocked by the reagents described above before linkage to the subsequent amino acid derivative.

S It is possible to use as coupling reagents all the pos- 25 sible activating reagents used in peptide synthesis, see, for example, Houben-Weyl, Methoden der organischen Chemie, 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 amino acid derivative with the activating reagent and, where appropriate, an additive which suppresses racemization, such as, for example, 1-hydroxybenzotriazole (HOBt) K'nig, R. Geiger, Chem. Ber. 103, 708 (1970)) or 3 -hydroxy-4-oxo-3,4-dihydrobenzotriazine (HOObt) Konig, R. Geiger, Chem. Ber. 103, 2054 (1970)) to the resin, or the preliminary activation of the amino acid derivative as a symmetric anhydride or HOBt or HOObt ester can be carried out separately, and the solution of the __i 13 activated species in a suitable solvent added to the peptide-resin which is suitable for coupling.

The coupling and activation of the amino acid derivatives using 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 used in a 1.5- to 4-fold excess.

In cases where incomplete coupling occurs, the coupling 10 reaction is repeated without previously carrying out the V" deblocking of the a-amino group of the peptide-resin which is necessary for the coupling of the next amino acid (c .in sequence. Successful completion of the coupling reo °action can be checked using the ninhydrin reaction as S 15 described, for example, by E. Kaiser et al., Anal. Bio- 4 chem. 34 595 (1970). The synthesis can also be carried out automatically, for example using an Applied Biosystems 4.

Smodel 430A peptide synthesizer, it being possible to use synthesis programs either provided by the manufacturer of the instrument or drawn up by the user himself. The o latter are employed especially when amino acid derivatives protected with the Fmoc group are used.

1 After synthesis of the peptides in the manner described above, the peptide can be cleaved off the resin using reagents such as, for example, liquid hydrogen fluoride (preferred for the peptides prepared by the Boc method) or trifluoroacetic acid (preferred for the peptides synthesized by the Fmoc method). These reagents cleave not only the peptide from the resin but also the other sidechain protective groups on the amino acid derivatives.

Except when BHA- and MBHA-resins are used, the peptide obtained in this manner is in the form of the free acid.

In the case of BHA- and MBHA-resins, the cleavage with hydrogen fluoride or trifluoromethanesulfonic acid results in the peptide as amide. Further processes for the preparation of peptide amides are described in German Patent Applications P 37 11 866.8 and P 37 43 620.1. In this case, the peptide amides are cleaved off the resin by treatment

~II

14 with the moderately strong acids customarily used in peptide synthesis (for example trifluoroacetic acid), with there being added as cation traps substances such as phenol, cresol, thiocresol, anisole, thioanisole, ethanediol, 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. The trifluoroacetic acid can also be used for this diluted by suitable solvents such as, for example, methylene chloride.

On cleaving off with hydrogen fluoride, trifLuoromethaneo0 4 sulfonic acid and trifluoroacetic acid, it is usual to °oI a add, as cation traps, substances such as phenol, cresol, Sthiocresol, thioanisole, dimethyl sulfide, ethyl methyl Sa.. 15 sulfide or a mixture of two or more of these auxiliaries.

The trifluoroacetic acid can in this case also be employed diluted by suitable solvents such as, for example, r.methylene chloride.

If the tert.-butyl or benzyl side-chain protective groups on the peptides are to be retained, the peptide which has been synthesized on a specially modified carrier resin is cleaved off using 1% trifluoroacetic acid in methylene S chloride as described, for example, in R.C. Sheppard .r 25 J.Chem.Soc., Chem.Comm. 1982, 587. If individual tert.butyl or benzyl side chain protective groups are to be retained, then a suitable combination of these methods of synthesis and cleavage off is used.

The modified carrier resin described by Sheppard is likewise used for the synthesis of peptides having a C-terminal amide group or an w-amino- or w-guanidinoalkyl group. After the synthesis, the peptide which is fully protected in the side chain is cleaved off the resin and then reacted in a classical solution synthesis with the appropriate amine or w-aminoalkylamine or w-guanidinoalkylamine, it being possible, where appropriate, for further functional groups which are present to be temporarily protected in a known manner.

15 Another process for the preparation of peptides having an w-aminoalkyl group is described in German Patent Application P 36 35 670.0.

The peptides of the present invention were preferably synthesized using the solid phase technique and two general protective group tactics: The synthesis was carried out with an Applied Biosystems 10 model 4300 automatic peptide synthesizer using Boc and e6 Fmoc protective groups for the temporary blocking of the o° a-amino group.

9 1 Where Boc was used as the protective group, the synthesis i 15 cycles preprogrammed by the manufacturer of the apparatus Swere used for the synthesis.

The peptides having a free carboxyl group on the C-ter- 0 *90 minal end were synthesized on a 4-(hydroxymethyl)phenyl- I 20 acetamidomethylpolystyrene resin Merrifield, J.Org.

o .Chem. 43, 2845 (1978)) obtained from Applied Biosystems and functionalized with the appropriate Boc-amino acid.

An MBHA-resin from the same company was used to prepare the peptide amides. N,N'-Dicyclohexylcarbodiimide or 25 N,N'-diisopropylcarbodiimide were used as activating reagents. The activation was carried out as symmetric anhydride or as HOBt ester in CH 2 CL2 or CH 2

CI

2 /DMF mixtures or DMF. 2-4 equivalents of activated amino acid derivative were used for the coupling. In cases where the coupling did not go to completion, the reaction was repeated.

The linkage of the tw, Cys residues by a disulfide bridge is preferably carried out by one of the methods described in "Perspectives in Peptide Chemistry", Karger Basel 1981, pages 31-44 or Schr'o'der, L'ubke, "The Peptides", Volume I, Academic Press, New York, London 1965, pages 235-239.

Oxidation with air or 12 is preferred.

When the Fmoc protective group was used for the temporary L a

U

-16protection of the a-amino group, our own s/nthesis programs were entered for the synthesis using the Applied Biosystems model 430A automatic peptide synthesizer. The synthesis was carried out on a (p-benzyloxybenzyl alcohol)-resin Wang, J.Am.Chem.Soc. 95, 1328 (1973)) which was obtained from Bachem and was esterified with the appropriate amino acid by a known method Atherton et aL., J.C.S.Chem.Comm. 1981, 336). .The amino acid derivatives were activated as HOBt esters directly in the 10 amino acid cartridges provided by the manufacturer of the S" apparatus, by adding a solution of diisopropylcarbodiimide o in DMF to the mixture of amino acid derivative and HOBt .o a *or HOOBt which had previously been weighed in. It is a a* likewise possibLe to use Fmoc-amino acid OObt esters pre- .o.I 15 pared in the free state, as have been described in European Patent Application 87107634.5. The Fmoc protective group was eliminated using a 20% strength solution of piperidine o in DMF in the reaction vessel. The excess of reactive amino acid derivative used was 1.5 to 2.5 equivalents.

Where the coupling was incomplete, it was repeated as in j the Boc method.

In order to introduce the glycosyl radicals into serine I* or threonine, it is necessary for the amino group and the 25 carboxyl group to be suitably protected beforehand. Particularly favorable protective groups for this purpose have proved to be those which can be eliminated by catalytic hydrogenation or by secondary amines. In the former case, these protective groups are of the benzyl type such as, for example, the benzyloxycarbonyl(Z) or p-nitroben- A p zyloxycarbonyl radical as amino protective groups, and the benzyl(OBzl) or p-nitrobenzyl ester for the carboxyl group.

The 9-fLuorenylmethyloxycarbony (Fmoc) radical can be eliminated using secondary amines. It has proved particularly favorable to use Fmoc-L- or Fmoc-D-Ser-OBzl because,

*I

17 after glycosylation, it was possible selectively to eliminate the benzyl radicals in the corresponding Fmoc-L- or Fmoc-D-Ser(R 1 )-OBzl by catalytic hydrogenation while retaining the Fmoc. This is particularly surprising because there have been many reports recently that the Fmoc radical is eliminated by catalytic hydrogenation [for example by R.

Geiger and W. K'nig in E. Gross and J. Meienhofer (Eds): The Peptides, Vol. 3, p. 24, Academic Press, 19813.

t 10 The synthesis of the 0-glycosyl-amino acid units requires two polyfunctional reactants (carbohydrate and serine or a n threonine) to be linked. Selective blocking and unblocking must be possible for both. It must be possible to lib- O* erate and functionalize the anomeric center in the glyco- 15 syl component, and only the hydroxyl group needed for the linkage must be unblocked in the amino acid component. Depending on the type of glycosidic bond which is required (1,2-cis- or 1,2-trans-glycosides), it is necessary to introduce suitable protective groups for blocking the hydroxyl or amino groups in the glycosyl component, and to work out reaction conditiro s for the linkage step which leads stereoselectively to only one of the two possible anomers.

0 0 To prepare the compounds of the formula I, according to the 25 invention, containing an 0-glycosyl radical, use is made both of the 0-glycosylamino acid units which are known in the literature and most of which are natural, such as, for example, those described by K. Dill et al. [Carbohydr.

Res. 123 (1983) 137-144], H. Kunz [Nach. Chem. Tech. Lab.

32 (1984) 11] and H. Paulsen [Chem. Soc. Res. 13 (1984) 25-45], and of the artificial 0-glycosylserine and glycosylthreonine derivatives which are prepared by the glycosylation processes customary in carbohydrate chemistry, such as those described by A.F. Bochkov and G.E. Zaikov [Chemistry of the 0-glycosidic bond, Pergamon Press 157 (1979)], H. Paulsen CAngew. Chem. 94 (1982) 184-201] and R.R. Schmidt CAngew. Chem. 98 (1986) 213-2363 or modifications thereof. The glycosylated amino acid units are introduced into the cyclized peptide.

18 The peptides according to the invention are analogs of ANF (atrial natriuretic factor), a peptide which is formed in the atrium of mammalian hearts and has natriuretic, diuretic and vasoactive effects (Currie et al. Science 223, 67 1984; Kangawa, Matsuo, Biochem.

Biophys. Res. Commun. 118, 131 1984). The rat-ANF (103-126) sequence utilized in the instant invention is disclosed in D.M. Geller et a (1984) Biochem. Biophys. Res.

Commun. 120:332. It is possible by the modifications according to the invention to enhance or change specifically the effects of ANF. Thus, for example, the duration of action and profile of action of the natriuretic properties can be beneficially affected, or the vasoactive effect can be diminished, or even the opposite effect can be achieved.

1 0 Furthermore, the peptides according to the invention have immunomodulaing properties which have not previously been described for peptides isolated from the atria of mammalian hearts.

The receptor-binding assay described hereinafter is used to investigate the binding power Snecessary for the biological effect of the peptides according to the invention.

Preparations of membranes from the bovine adrenal cortex.

4 t Fresh bovine adrenals were obtained from a slaughterhouse and transported on ice. After the adrenal medulla had been removed, the cortex was cut into small pieces with scissors in ice-cold buffer A (5 mmol/l Tris, 1 mmol/l MgCl 2 250 mmol/l sucrose, pH 7.4, at The tissue was homogenized in buffer A in a Waring blender. Larger particles were removed by subsequent filtration through a gauze, and the filtrate was rehomogenized in a Potter homogenizer with 20 strokes. Thereafter, the cell particles of higher density were removed in a 10-minute centrifugation at 3000 x g and 4°C and S were discarded. The plasma membranes in the supernatant were then removed at 39,000 x g for 10 minutes and resuspended in buffer B (75 mmol/l Tris, 35 mmol/l MgCI 2 pH 7.4 at After this centrifugation step had been repeated twice, the plasma membranes were taken up in buffer B containing 250 mmol/l sucrose and were frozen in portions corresponding -pC o rF^ s

J

I_ I; 19 to 1 g of original cortex tissue/mL in liquid nitrogen.

On subsequent storage at -70 0 C, no decrease in the binding activity was found over several months.

Receptor binding For the binding experiments, the membranes were thawed, centrifuged at 39,000 x g for 10 minutes and suspended in I buffer C (100 mmol/l NaCL, 0.1 mmol/l EDTA-Na 2 50 mmol/l 4 HEPES, pH 7.4, and 0.2% bovine serum albumin to reduce Sadsorption to the walls). 40 pmol/L aprotinin, a peptid- S° ase inhibitor, were added to the plasma membrane suspeno sion to prevent breakdown of the radiolabeled Ligand o +B i (radioligand) during the incubation time. The binding experiment was carried out in micro titer plates at 25 0

C

V with 60 minutes to equilibrium. Binding was started by addition of the membrane suspension. The final volume v of each sample was 250 pL. These contained about 14,000 Scpm of the radio igand 12 5 I-human atrial natriuretic peptidel- 2 8 from Amersham Buchler, FRG) with a speci- S fic activity of 74 TBq/mmol and a quantity of the membrane protein which had bound about 50% of the added radioligand. Free and bound radioligand were separated by rapid filtration through a Whatman GF/C glass fiber filter using a R Skatron cell harvester. In order to reduce the non-specific binding, the filters had previously been placed for 1 hour in freshly prepared 3% strength polyethyleneimine at pH 10. After the filtration, the amount of the radioligand retained on the filters was measured in a gamma scintillation counter. The non-specific binding was defined as the binding in the presence of 0.5 pmol/L atriopeptin III (H-Ser-Ser-Cys-Phe-Gly-GLy Arg-Ile-Asp-Arg-ILe-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser- Phe-Arg-Tyr-OH), an amount which suffices completely to displace the radioligand from the receptors.

Calculation of the data The IC 5 0 values represent the concentration of the test

V

I~*cll-sl na~- 20 substance which is necessary to inhibit 50% of the binding of the radioligand. The values were calculated from the measured values using the median effect equation of Chen (1976), J. Theor. Biol. 59, 253-276, with a microcomputer.

Binding data (atriopeptin III displacement) iI ft I

I

Example No.

8 9 11 12 13 14 15 17 20 21 22

IC

5 0 [nm] substance 0.49 1.40 0.05 0.07 0.06 0.79 1.10 2.40 3.20 1.40 0.48

IC

50 Cnm3 atriopeptin III 7.40 7.40 3.40 8.30 8.30 3.50 8.60 8.50 5.40 3.50 5.40 ft 41 ft *~l 4( 4 *4 ~9 i ,:1 :i ii, ft 4.

ftI t 444.

ft 1 The vasorelaxant effect of the peptides according to the invention was tested in vitro on strips of guinea pig 25 aorta which had previously been contracted with 25 mM KCL.

The diuretic and natriuretic effect was demonstrated in vivo by i.v. administration to anesthetized rats. Atriopeptin III was used for comparison.

Hence the invention also relates to the use of the pep- Stides of the formula I as medicines and to pharmaceutical compositions which contain these peptides. Use in humans is preferred.

The new peptides according to the invention have, individually or in combination, diuretic, saluretic, vasorelaxant and immunomodulating effects, or can be used in cases of chronic or/and acute renal insufficiency and for protection of the kidney from nephrotoxic substances, for 61 21 example cyclosporin, especially in cases of renal transplantation, in a dose of 10 to 20,000 picomole/kg (25 ng 50 For this purpose the peptides can be administered parenterally i.m. or also by infusion into the arteria renalis) in a physiologically tolerated medium.

For parental administration, the active compounds or their physiologically tolerated salts are converted into solutions, suspensions or emulsions, if desired using the substances customary for this purpose, such as solubilizers, emulsifiers or other auxiliaries.

Examples of suitable solvents for the new active compounds and the corresponding physiologically tolerated salts are: water, physiological saline solutions or alcohols, for examples, ethanol, propanediol or glycerol, as well as sugar solutions such as glucose or mannitol solutions, or a mixture of the various solvents mentioned.

It is likewise possible to administer the active substances by implants, for example i,'5 composed of polylactide, polyglycolide, copolymers of lactic and glycolic acid, or poly-3- ,ot hydroxybutyric acid, or by intranasal compositions. On intranasal administration it is o necessary, because of the poorer adsorption, for the dose to be multiplied by ten. It is furthermore possible to administer the peptides in the form of their physiologically S tolerated salts or metal complexes.

The invention also relates to the use of compounds of the formula I for the treatment of glaucoma and/or for reducing the intraocular pressure in mammals, preferably in humans, by topical or systemic administration thereof. The invention also relates to the use of the compounds of formula I for treatment of renal insufficiency.

For the ophthalmological use of the compounds according to the invention, the latter are j 25 expediently incorporated in pharmaceutical products in a customary manner. They can be converted into the customary administration forms, such as solutions, ointments, emulsions, as well as in 7r+

C

-I

22 depot form. The active substance can also, where appropriate, be in microencapsulated form. The products can contain tolerated organic or inorganic additional substances, for example suspending agents, solvents, antibacterial agents, wetting agents and preservatives. Topical administration forms are preferred, It is also possible to use parenteral compositions. In the case of systemic administration forms, about 25 ng 5 pg/kg are administered once to three times a day. Solutions, ointe- 10 ments or ophthalmic inserts (solid inserts) are preferably used for topical administration. Topical administration forms can contain 0.001 5 percent by weight of the active substance. Higher or lower dosages can also be 0 o administered, provided they lower the intraocular pres- 15 sure. Preferably, 0.01 mg 1 mg of the active substance is administered to the human eye. For the preferred topical administration to the eye, the compounds of the formula I are administered in combination with physiologi- 0"1 cally tolerated vehicles such as, for example, aqueous 20 methylcellulose. The combination may be in the form of 0* a suspension, solution, ointment, emulsion or ocusert.

A preferred combination is one which facilitates the penetration of the active substances into the eye.

Another preferred topical administration form is the com- 25 bination of the compounds of the formula I with compounds such as, for example, benzalkonium chloride which facilitates the penetration of the active substance into the eye. Compounds of the formula I can also be used in combination with other antiglaucoma compounds for the treatment of glaucomatous disease.

The topical vehicles can be organic or inorganic compounds. Typical vehicles which are used pharmaceutically are aqueous solutions which are, for example, buffer systems or isotonic mixtures of water and water-miscible solvents such as, for example, alcohols or aryl alcohols, oils, polyalkylene glycols, ethylcellulose, carboxymethylcellulose, polyvin ,pyrrolidone or isopropyl myristate.

Examples of suitable buffer substances are sodium chloride, 23 sodium borate, sodium phosphate, sodium acetate or gluconate buffer. The topical administration form can also contain non-toxical auxiliai es, suli as, for example, emulsifying preservatives, wetting agents such as, for example, polyethylene glycols, antibacterial compounds such as, for example, quaternary ammonium compounds, benzalkonium chloride, phenylmercury salts, benzyl alcohoL, phenylethanol, triethanolamine oleate, thiosorbitol and other simi'ar substances which are used in topical oph- 10 thatmic compositions. The topical administration form can also be in the form of an ophthalmic insert (solid insert). For this purpose, it is possible to use, for *example, a solid water-soluble polymer as carrier for the active substance. Every water-soluble, non-toxic polymer 15 can be used as the poLymer, such as, for example, cellu- Lose derivatives such as, for example, methylcellulose, sodium carboxymethylcellulose, (C 1

-C

6 )-hydroxyakyceLLu- 9 04t lose such as, for example, hydroxyethylcelluose, hydroxypropylcellulose and hydroxypropylmethylcellulose, acrylic acid derivatives such as polyacrylic acid salts, ethyL acrylates and polyacrylamides. It is furthermore possible to use, for example, gelatin, starch derivatives, alginates, pectins, polyvinyl ?lcohos, polyvinyLpyrrolidones, polyvinyl methyl ethers, polyethylenz oxides or mixtures 25 of the various polymers. Solid inserts, which are suit- 0 t able for topical administration, are described in British Patent Application 1524405.

List of abbreviations: The abbreviations used for the amino acids correspond to the three-letter code which is customary in peptide chemistry, and described in, for example, Europ. J. Biochem.

138, 9 (1984). Other abbreviations used are listed below.

Acm Acetamidonmethy c-Ahx c-AminohexanoyL Aoc cis, endo- 2 -Azabicyclo[3.3.0octane-3-S-carbonyL Boc tert.-Butyoxycarbony But 8z L C L- Z D MF Dnp F m o c Ma c Mbu M e 10 4-M e bz L M p r M tr Mt s T FA 15 Tc s Tr t 24 tert ButyL Benzy I 4-ChLorobenzyLoxycarbonyL DimethyLformamidle 2,4-DinitrophenyL 9-FL uorenyLmethy LoxycarbonyL Mercaptoacetic acid Mercaptobutyr ic acid Methyl 4-MethyLbenzyL Mercaptopropionic acid 4-Methoxy-2,3,6-trimethyLphenyLsuLfonyL MesityLene-2-suLfoniyL Trifluoroacetic acid 4-MethyiphenyLsuLfonyL T r ty L ~e 8 8 8 *9 8 88 0 8 *0 *9*4 0

CO*S

010* o 0 0 *9 0 *98048 8 8 8# 9 8 88 8 88 8 8 *0 4;, o 8 088 I 1<1 I I t.

III I CI! III The examples which foLLow are intended to explain the preferred methods for the solid-phase synthesis of the 20 peptidles according to the invention without intending to confine the invention to them.

Reference example 1: 25 The synthesis of the ANF derivative which has natriuretic, diuretic and vasoactive effects and has the formula 13 H- Ser- Ser- Cys- Ph.- Giy-Giy-Arg-I ie-Giu- Arg-Ile- Gly-Ala- 14 1 -24 Gin- 5r- Gly- Leu- Gly- Cys- Asi- Ser- Ph.- Arg- Tyr- OH was carl-ied out using an Applied Biosystems modlel 430A automatic peptidle synthesizer and using the Boc method on a 4-(hydroxymethyL )phenylacetamido-methyLpoLystyrene resin substituted with Boc-Tyr(Bz-Z)-OH, which was obtained from AppLied 6iosystems and contained 0.5 mmoL of tyrosine. The foLlowing amino acid derivatives, which were Likewise supplied by Applied Biosystems, were used for the synthesis, 2 mmol of each already being weighed out into cartridges for use in the peptide synthesizer: 61,

-'T

4.

88 8 8 88a 9C 8 04 88 8 8~L 8O 8 *B 8 I 9 8 8 98 *r S 89 112 t 25 Boc-Arg(Tos)-OH, Boc-Phe-OH, Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Cys(4-MeBzl)-OH, Boc-Gly-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Ala-OH, Boc-ILe-OH, Boc-GLu(OBzL)-OH. The programs provided by the manufacturer of the apparatus were used for the synthesis, an example being listed hereinafter.

1) 65% trifLuoroacetic acid/CH 2

CL

2 5 ml, 2 min 2) 65% trifluoroacetic acid/CH 2

CL

2 5 mL, 15 min 3) Washing with CH 2

CL

2 (3 x 10 mL) 4) Washing with 10% diisopropyLethyLamine in DMF (2 x 10 ml) 10 5) Washing with DMF (3 x 10 mL) 6) Coupling of the amino acid derivative which had previously been activated as symmetric anhydride or HOBt ester (15 30 min), possibly repeating where coupling was incomplete 15 7) Washing with CH 2

CL

2 (6 x 10 mL) After completion of the synthesis, the peptide-resin was dried over molecular sieves in vacuo. The peptide was cleaved off the resin by weighing 1 g of peptide-resin 20 together with 0.5 g of p-cresol and 0.5 g of thiocresol into the reaction vessel of a HF apparatus obtained from Protein Research Foundation, Minowa, Osaka, Japan and, after evacuation of the apparatus, distilling in about ml of liquid HF at dry-ice temperature. The mixture is then left to react at 0°C for 1 h, and thereafter the excess HF is removed by cautious distillation in vacuo.

The remaining yellowish red resin/peptide mixture is washed several times with ethyl acetate and dry ether in order to remove the cation traps. After drying, the peptide is extracted from the resin using 30% strength aqueous acetic acid, and the resulting crude peptide solution is freeze-dried. The freeze-dried crude peptide is separated from any cation trap which is still present on Sephadex( G25 superfine using 1 N acetic acid, and the peptide-containing fractions are worked up further. This entails the crude peptide being dissolved in trifluoroethanol and reduced overnight with a 5-fold excess of trin-butylphosphine. After the solvent has been removed in vacuo, the residue is extracted several times with ethyl

.I

j rrr* 26 acetate, and the resulting precipitate of crude peptide is briefly dried and immediately oxidized with 12.

mmol of peptide is dissolved in 50 ml of 90% strength acetic acid, and this solution is rapidly added dropwise to a vigorously stirred solution of 0.5 mmol of 12 and 1 mmol of sodium acetate in 450 ml of 80% strength aqueous acetic acid. After the solution has been stirred for min it is decolorized by addition of 0.1 N aqueous ascorbic acid, and is concentrated to about 10 ml, and 10 salts are immediately removed on Sephadex G25 superfine e o r o using 1 N aqueous acetic acid as eluent. The fractions 9 AO containing the peptide are combined and freeze-dried. The see*

(R)

s purity is checked by HPLC on Vydac C 18 or by Vydac C 4 4a columns using a gradient of acetonitrile in 0.1% strength *e o 15 aqueous trifluoroacetic acid. For final purification, a preparative HPLC separation is carried out on the same packing in the same solvent mixture.

JO

Sco Reference example 2: 4,1 The peptide which has natriuretic, diuretic and vasorelaxant effects and has the formula 1 14 25 H-Ser-Ser-Cys-Phe-Gly-Gly-Lys-Ile-Asp-Arg-Ile-Gly-Ala-Gln- I 24 -Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH was synthesized stepwise using an Applied Biosystems model 430A peptide synthesizer and using the Fmoc method on a (p-benzyloxybenzyl alcohol)-resin esterified with Fmoc-Tyr(Bu )-OH obtained from Bachem (loading 0.40 mmol/g of resin). 1 g of this resin was used, and the synthesis was carried out using synthesis programs modified for the Fmoc method.

The following amino acid derivatives were used: Fmoc- Arg(Mtr)-OH, Fmoc-Phe-OH, Fmoc-Ser(But)-OH, Fmoc-Asn-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gln-OH, 27- Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Asp(OBut)-OH and Fmoc-Lys (Boc)-OH. 1 mmol of each amino acid derivative was weighed, together with 1.5 2.5 equivalents of HOBt, into the cartridges of the synthesizer. The amino acids were activated directly in the cartridges by dissolving in 4 ml of DMF and having a 0.55 molar solution of N,N'-diisopropylcarbodiimide in DMF (2 ml). A typical synthesis cycle is listed below: 1) elimination of the Fmoc group using 20% piperidine in 10 DMF (twice 8 ml for 10 min each time) .o 2) washing with DMF (6 times with 8 ml of DMF each time) 3) coupling of the amino acid derivative which has preo° viously been activated in the cartridge as the HOBt ester, possibly repeating if coupling is incomplete S 15 4) washing with DMF (6 times with 8 ml of DMF each time).

After the synthesis was complete, first the Fmoc protective group was eliminated from the peptide-resin by treatment with 20% piperidine in DMF, and the resin, which had been thoroughly washed with DMF, was treated several times with isopropanol and methyl tert.-butyl ether to

O*

shrink it. After drying under high vacuum, the peptide was cleaved off the resin by stirring with a mixture of trifluoroacetic acid/CH 2 Cl2/phenol (70:30:5) at room temperature for 4 h. Any remaining protective groups are eliminated by subsequent treatment with trifluoroacetic acid/phenol/dimethyl sulfide The resin is then removed by filtration and washed with elimination solution, and the filtrate is concentrated in vacuo. The cation traps were removed by stirring several times with i. ethyl acetate. After the crude peptide had been dried under high vacuum, any cation traps and traces of trifluoroacetic acid which were still present were removed on Sephadex G25 superfine using 1 N aqueous acetic acid. The peptide-containing fragments were freeze-dried and then worked up further. The oxidative elimination of the Acm protective groups on cysteine was carried out by the method of Kamber et al., Helv. Chim. Acta, 63, 899 (1980) in 90% strength aqueous acetic acid with a I II- s~ nayc- O 4 09.

to..

0 4 0 0i 0 P It to' 28 excess of 12 and a reaction time of 15 min. Salts were removed from the resulting crude peptide, and it was finally purified as described in Example 1.

Reference example 3: Synthesis of Cdes-Tyr 2 4 des-Arg23 -r-atriopeptin III-(4amino)butylamide 10 The peptide is synthesized on 1 g of the resin described in German patent application P 36 35 670.0 using Fmoc-amino acid OObt esters and an Applied Biosystems model 430A automatic peptide synthesizer and synthesis programs modified by ourselves.

This entails 1 mmol each of the appropriate amino acid derivatives being weighed into cartridges supplied by the manufacturer; Fmoc-Arg(Mtr)-OH, Fmoc-Asn-OH and Fmoc-GLn- 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 amino acids argenine, 25 asparagine and glutamine which are preactivated in situ, onto the resin which has previously been unblocked with 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 sidechain protective groups, using trifluoroacetic acid which contains thioanisole and m-cresol as cation traps. The residue obtained after removal of the trifluoroacetic acid in vacuo is digested with ethyl acetate and centrifuged several times. The remaining crude peptide is treated with tributylphosphine in trifluoroethanol to remove the cysteine protective group. After removal of the solvent, the residue is again digested with ethyl acetate and centrifuged. The 29 reduced crude peptide is immediately oxidized with iodine in strength aqueous acetic acid, the 12 excess is removed with ascorbic acid, and the reaction mixture is concentrated to a small volume, and then salts are removed on Sephadex G25 using aqueous 1 N acetic acid. The fractions containing pure peptide are combined and freeze-dried.

Reference Example 4: Synthesis of SA" H-Aoc-Cys-Phe-D-Ala-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln- Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Aoc-NH 2 The peptide synthesis is carried out on 1 g of the resin 15 described in German Patent Application P 37 43 620.1 using Fmoc-amino acid OObt esters and an applied Biosystems Model 430A automatic peptide synthesizer and synthesis programs modified by ourselves.

This entails 1 mmol each of the appropriate amino acid derivatives being weighed into cartridges sup-plied by the manufacturer; Fmoc-Arg(Mtr)-OH, Fmoc-Asn-OH and Fmoc-GLn- OH are weighed together with 1.5 mmol of HOBt into the cartridges. These amino acids are preactivated directly 25 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 amino acids argenine, asparagine and glutamine which are preactivated in situ, onto the resin which has previously been unblocked with piperidine in DMF. The amino acids which are activated in situ are coupled twice.

After the synthesis is complete, the peptide butylami.' is cleaved off the resin, simultaneously removing the sio.

chain protective groups, using trifluoroacetic acid which contains thioanisole and m-cresol as cation traps. The residue obtained after removal of the trifluoroacetic acid in vacuo is digested with ethyl acetate and centrifuged several 4 times. The remaining crude peptidle is treated with tributyiphosphine in trifluoroethanol to remove the cysteine protective group. After removal of the solvent, the residue is again digested with ethyl acetate and centrifuged. The reduced crude peptidle is immediately oxidized with iodine in strength aqueous acetic acid, the 12 excess is removed with ascorbic acid, and the reaction mixture is concentrated to a smalL volume, and then saLts are removed on Q- ephadex using aqueous 1 N acetic acid. The fractions containing pure peptidle are combined and freeze-dried.

following compounds are prepared in analogy to reference example 2 1. H-Ser-Ser-Cys-Phe-Giy-Giy-Arg-Iie-Asp-Arg-Ile.Gly.Ala.

Gin-Ser-Giy-Leu-Giy-Cys-Asn-Ser-Tyr(Me)-Arg-Tyr-OH 2. H-Ser-Ser-Cys-Tyr(Me)-Gly-Gly-Arg-Iie-Asp-Arg-Iie- Gly- Ala- Gin- Ser- Gly- r6Jeu- Gly- Cys-Asn- Ser- Phe-Arg-Tyr- OH 3. H-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asn-Arg-Iie-Gly-Ala- Gin- Ser- Gly- Leu- Gly- Cys-Asn- Ser- Phe-Arg-Tyr- OH 4. H- Ser- Ser- Cys-Phe- Gly- Gly- Arg-Ile-Asp- Arg- Pro- Gly-Ala- Gin- Ser- Gly- Leu- Gly- Cys- Aan- Ser- Phe- Arg- Tyr- OH H- Ser- Ser- Cys- Phe- Giy-Giy-Arg- Ile-Giu-Arg-Ile- Giy-Ala- Pro- Ser- Gly- Lei- Gly- Cys- Thr- Ser- Phe- Arg= Tyr- OH 6. H-Cys-Phe-D-Ala-Gly-Arg-Iie-Asp-Arg-Cyciohexylalanyl.

Giy- Al a- Gin-Ser- Giy- Leu- Gly- Cys- Asn- Ser- Ph.- Arg- Tyr- OH 7. H-Ser-Ser-Cys-Phe-NH- (CH 2 5 -CO-Arg-Ile-Asp-Arg-Ile-Gly- Al a- Gin-Ser- Gly- Leu- Gly- Cys- Asn- 5r- Ph.- Arg- Tyr- OH4 0 0 #0 6400 0000 0 0 0* 0 000.0.

0 00 0 00 00 0 *0 00 a, *50 a I C Lit C 31 The following compounds are prepared as in reference example 2, with the first amino acid being coupled onto a MBHA-resin, and the cleavage off being carried out with hydrogen fluoride or trifLouromethanesuLfonic acid as described in reference example 1.

8. H- Aoc-Cys-Phe-D-Aia..oiyArg-.Iie-Asp-Arg- Ile-Giy-AJla- Gin- Ser- Gly- Leu G-Cs- Asn- Ser- Phe- Lys-NH 2 9. H-D-Aoc-Cys-Phe-D-Aia-Giy-Arg- Ile-Asp-Arg-Ile-Gly-Ala- Gin- cr- Gly- Leu- Gly- Cys- Asn- Ser- Phe- Lys- NH 2 10. H-Aoc-Cys-Phe-D-Aa-Gy-Arg-Iie-Asp-Arg-Ile-Giy-Aia- Gin- Ser- Giy- Leu- Giy- Cys- Asn- Ser- Phe- Arg- Aoc- NH 2 11. H-Aoc-Cys-Phe-D-Aa-ly-Arg-Ie-Asp-Arg-tert.Butylglycyi- Giy- Aia-Gin- Ser- Giy- Leu- Gly- Cys- Asn- Ser- Phe- Arg- Ni 2 12. H-Aoc-Cys-Phe-D-Aa-Gy-Arg-Ile-Asp-Arg-Neopentyiglycyi- Gly- Ala- Gin-Ser-Gly- Leu- iy- Cys- Asn- cr- Ph.-Arg- NH 2 13. H- Aoc- Cys- Ph.- iy- Giy-Arg- Iie-Asp-Arg- Ile- Gly- Ala- Gin- Ser- Gly- Leu- Gly- Cys- Asn- 5cr- Cyci ohexyl aianyl -Arg- NH 2 The folLowing compounds (ExampLes 14-22) are prepared as described in reference example 3.

14. H-Cys-Phe-D-Ala-Gly-Arg- Ile-Glu-Arg- Ile-Gly-Ala-Thr- Ser-Gly- Leu- Gly- Cys- Pro- Ser- Phe-NH- (CH 2 4-H2' -32 H-Aoc- Ser- Cys-Plhe-Gly-Gly-Arg- Iie-Asp-Arg-Ile- Gly-Ala-

U-

Gin-Ser-Giy-Leu-Giy-Cys-Asn-Ser-Phe-NH-

(CH

2 8 -N1 2 16. H-Cys-Phe-Giy-Giy-Arg-Iie-Asp-Arg-Iie-Giy-Aia-Gin-Ser- V ~Giy-Leu-Giy-Cys-Asn-Ser-Phe-NH- (CH 2

)-H

17. H-Cys-Phe-D-Aia-Giy-Arg-Ile-Asp- Arg- Iie-Giy-Aa-Gl-- 19 H-Arg-Cys-Phe-D)-Aia-Giy-Arg- Iie-Asp-Arg- Iie-Giy-Ala- Gin- Ser-Giy-Leu-Giy-Cys-Asn-Ser-PheNH-(CH 2 6

-NH

2 204-r-r-y-h-0l-GyAgIeApAgIeGy Aia- Gin-Ser- Giy- Leu- Giy-Cys- Asn- Ser- Phe-NH- (CH 2 6~ 21. H- Aoc- Cys- Phe-D-Aia-Giy- Arg- Iie-Giu-Arg-Ile- Gly-Ala- Gin-Ser-Gly-Leu-Gly-Cys-Giy-D-Ala-Phe-NH-(CH 2

)-H

22. H-Aoc-Cys-Phe-D-Ala-Giy-Arg- Iie-Giu-Arg- Iie-Aia-Aia- Thr- Ser- Gly- Leu- Gly- Cys- Pro- Ser- Phe- NH- (CH 2

NIH

2 The peptidles were characterized by amino acid analysis.

The peptidles were hydrolyzed using 6 N hydlrochloric acid at 1200C for 12 hours. In this, cysteine resulted in a mixture of cysteine and cysteic acid and was present in all the peptidles.

2 33 Example 1 2 3 4 5 6 7 8 9 ASP 2.00 1.97 1.98 2.00 1.98 1.95 1.98 1.97 2.09 THR 0.88 SER 3.46 3.46 3.52 3.67 3.24 1.62 3.51 1.83 1.95 1.70 GLU 1.08 1.05 1.07 1.05 1.07 1.00 1.05 1.08 1.06 1.14 PRO 1.11 1.05 BLY 5.00 5.03 5.11 5.11 5.00 3.98 3.01 4.07 4.01 3.98 ALA 1.01 1.06 1.16 1.09 1.02 2.06 1.04 1.98 2.17 2.02 ILE 1.85 1.81 1.93 0.94 1.92 0.99 1.89 1.75 1.79 2.09 LEU 0.95 0.94 1.03 1.00 1.02 0.97 1.05 1.01 1.03 1.10 TYR 1.62 1.37 0.57 0.78 0.92 0.71 0.82 PHE 1.04 1.07 1.99 1.99 2.16 1.98 1.95 2.04 2.00 2.08 LYS 1.05 1.04 ARG 2.88 2.8 2.91 2.97 3.06 2.94 2.97 2.04 1.97 3.00 0 4 4*40 0 0 4 00 4 4 0 04 4.

0 4' 00* 04 404 T~I 'I Example

ASP

THR

DER

BLU

m~o

SLY

ALA

ILE

LEU

T'YR

PHE

LYS

11 12 13 14 15 16 17 18 19 20 21 22 2.05 2.11 2.00 1.93 1.95 1.97 2.00 1.96 0.92 0.98 1.06 1.69 1.82 1.83 1.83 2.39 1.68 1.86 1.33 1.75 1.68 1.03 2.29 1.18 1.29 1.16 1.00 1.10 1.05 1.03 1.04 1.07 1.06 2.05 1.00 0.98 1.13 3.39 4.00 4.93 4.05 5.00 5.00 4.03 5.01 4.06 4.05 4.90 2.98 2.36 2.41 2.17 2.11 1.05 1.09 1.98 1.98 1.96 2.01 2.84 2.85 0.95 0.87 1.83 1.98 1.79 1.86 1.83 1.97 1.91 1.97 1.87 1.93 0.92 1.13 1.16 0.97 0.99 1.07 1.03 1.00 1.00 1.00 1.04 2.12 2.04 1.00 2.00 1.99 1.97 1.99 2.00 1.39 1.97 1.83 1.83 3. 0 .9 12 95 1. 3 .0 164 2 0 5 .6 396 .9 119 3.00 2.90 2.95 l." 2.00 1.64 2.00 i I" 2.17 3.98 1.90 1.81

Claims (13)

1. A peptide of the formuLa I j 119 X-V -A-A-A 2 -B-C-D-B-C-ly-Ala-E-F-G-Leu-Cly-Cys-Z (I) i n wh ic h X denotes hydrogen, (C 1 -C 5 )-alkoxycarbonyl, (C 6 C 12 aryloxycarbonyl, (C C )-aralkyloxycarbonyl, (C C)- aikanoyl, (C 7 C 13 )-aroyl, arginyl, lysyl, S£-amnino- caproyl, arginyl-arginyl, arginyl-lysyl, lysyl-arainyl or lysyl-lysyl; (C 1 C 12 )-alkylcarbonyl or (C 3- C 8 cycloylkylcarbonyl which are optionally branched in the d- position and optionally monosubstituted in the o position by amin~o or guanidino; or represents Ser, Thr, SerCY), ThrCY), Q or Leu, each in their L or D configuration, or Ser-Ser, Thr-Thr, Ser-Thr, Q-Ser, Q-Thr, Thr-Q, Ser-Q, S e r( Y) Ser o r S er S er( i t b e ing p os s ibL e fo r each amino acid to be present in its L or D con- figuration, and the N-terminaL amino group of the amino acid or of the dipeptide residue being in the f r ee f o rm (N -t er m in aL ra d ic aL H o r ac y La t ed b y (C 1-C5)-aLkox>'carbonyL, (C6-C12)-ary'LoxycarbonyL, (C 7 -C 13 )-araLkyLoxycarbonyL, (Cl-C 6 )-aLkanoyL, (C 7 C13)-aroyL, arginyL, LysyL, E-aminocaproyL, arginyL- arginyL, arginyL-LysyL, LysyL-arginyL or Lysyi- LysyL, or it being possibLe for X to be absent; V denotes Cys or, if X is absent, w-thio-(C 2 -C 8 )-aLkyL- carbonyL such as, for exampLe, mercaptopropionic, acid, mecaptoacetic acid or mercaptobutyric acid; A denotes Phe, Tyr(Me), Tyr(But 4-haLogenophenyL- aLanine, Trp or an L-2-thienvLaLanine residue; A 1 denotes GLy, Ala or 0-Ala; A 2 denotes GLy, Ala or D-ALa; or ILF A 1 and A 2 together denote an optionally branched w-amino- C 3 -C 8 )a Lk y Lc ar bo x yL i c ac id; 8 denotes Arg, Lys, Orn or an L-homoargini ne residue; C denotes lie, Met, Phe, Trp, Leu, Ser, Thr, VaL, His, P r o, A sn S e r (Bu c yc Lo he xy La La n in e, t er t but y L- gLycine, neopentyLgLycine or an L-2-thienyLaLanine r es id ue; 0090 D denotes Asp, GLu, G~n, Asn, Phe, Leu, ILe, Trp, Pro, 0t 00 -eta* 0Tyr, Ala, AspCOBu Asp(OBzl), GLu(OBu GLu(oBzL), a

0002-thienyiaLanine residue, Aad, Tyr(Bu t) or Tyr(Me), it 0being possibLe for each of the amino acids to be pre- 0: e sn t i n i ts L o r D c o n f igura t ion; E denotes G~n, Thr or Pro; 000 F de n o tes S er T h r, P r o, A La S er 6u o r T h rCBu in e ac h c a se i n i ts L o r D c on fig u ra t ion; G denotes GLy, ALa or 0-Ala; :0:Q denotes a radical of the formula IV 01 2 R R 0 N H I I i n wh ic h R 1 n together with the atoms bearing these radi- cals, form a heterocycLic mono-, bi- or tricycLic system having 3 to 15 carbon atoms; Y denotes tert,--butyL or an optionally protected 'or par- tiaLLy protected gLycosyL radical; and -36- Z represents a radicaL of the formuLa II -H-l-J-K-L-M (I in which H denotes Asn, Ser, Q or Thr, in each case in its L or D form, or GLy; I denotes Ser, Thr, Ala, Se(B hr(Bu or Pro, in each case in its L or D form; J denotes Phe, Trp, D-Phe, D-Trp, Tyr(Me), cycLohexyLaLanyL or a 2-thienyLaLanine r es id ue K denotes Arg, Lys, Orn or a bond; L denotes Q, GLy, Tyr, Tyr(But Tyr(Me) or a b ond; M denotes Arg-OH, Arg-NH 2 OH, OR, NH 2 NHR', GLy-Lys-Arg-OH, GLy-Lys-Arg-NH 2 or L-argini- n oL; Q is as defined above; R denotes unbranched (Cl-C 6 )-aLkyL, an d R denotes -ECH 2 ]n-NH 2 or -ECH23n-NH- CC( N H) N H 2 n being an integer and representing 3-8; and its physioLogically tolerated salts, excepting the peptides which correspond to the sequence of natural ANF and are of the formula III (III) X- Cys-Phe-Gly-Gly-Arg-C- Asp- Arg-Ile- Gly-Ala-Gin- Ser- Gly- Leu- Gly- Cys- Z in which X denotes Ser, Ser-Ser, Arg-Ser-Ser or Arg-Arg-Ser--Ser, C denotes Le or Met, Z denotes Asn-Ser-Phe-K-L-M, K denotes Arg or a aond, L denotes Tyr or a bond, and M denotes OH or NH 2 and their salts; and the peptides of the formula V X-Cys-A-Gly-Gly-E,-C-D-B-C-Gly-A~ia-E-F-G-Leu-Gl y-t..ys-Z MV in which X denotes .9er or Ser-Ser, it being possible for each Ser to be in the L- or D-configuration, and the N-termiibi amino group of the amino acid or dipeptide residue being free or being acylated by arginyl-arginyl, c-aminohexanoyl or cis-endo-r Azabicyclo[3.3.Ojoctane-3-S- carbonyl, or represents cis-endo-2-Azabicyclof3 .3,OJoctane-3-S-cerbonyl, A denotes Phe; 8 denotes Arg, D-Arg or Lys; o 8 denotes Arg, D-Arg, ys; tu 8 *C denotes Ilie; C denotes Ilie or Pro; denotes Glu, Asp or D-Leu: :E denotes Gin, Pro or Thr; F denotes Saror D-Ala; G denotes Gly or D-Ala and Z represents a residue of the formula VI j (VD) in which H denotes As,,R or Thr; I denotes Gar in its L- or D-form; J denotes Phe, K denotes Arg, Lys or a bond; L denotes Tyr or a bond; M denote3 OH, NH 2 NHR' or Gly-Lyl-Arg-OH-; R' denotes -ICH 2 ],-NH 2 n being Sri integer end representing 4-8; and the peptldes of the formulas C) C C. W 36b H-Ser-Sar-Cys-Ph-Gly-Gly-Arg-Ile-Asp-Arg-lie- ro- 14 -Gin-Ser-Gly-Leu-Gly-Cys-Asn-Se -Arg-Tyr-OH 1 13 H-Ser-'er-C -P a- ly-Gly-Arg-Ile-Asp-Arg-Ie-Pro-Ala- 6c l C l 'i n6rP Arg Tyr-O-H and 3 H-Cys-Phe-D-Ala-Gly-Arg-le-Asp-Arg-1s-Gly-Ala-Gin-Ser- 16 -Gly-Leu-Gly-Cys-Asri-Sr-Phe-NH-(CH 2 4 -NH 2 and I C CCCIII C I I a K Cs-' CC: 37 rat-[Maclo5]ANF(105-126), rat-[MpriO5]ANF(105-126), rat=[Mbulos]ANF(105-126) and rat-[MprlO5, D-Alalo7] ANF(105-12) 2. A peptide of the formula I as claimed in claim 1, in which Q denotes Pro, and its physiologically tolerated salts.

3. A peptide of the formula I as claimed in claim 1 or 2, in which Y denotes tert.- butyl, and its physiologically tolerated salts.

4. A peptide of the formula I as claimed in one of claims 1 to 3, Sin which d X denotes hydrogen, (Ci -Cs)-alkoxycarbonyl, (C6-C12)- aryloxycarbonyl, (C 7 -C 13 )-aralkyloxycarbonyl, (Ci alkanoyl, (C7-C1 3 )-aroyl, arginyl, -u lysyl or arginyl-arginyl; or denotes (C 1 -C 12 )alkylcarbonyl or (C3-08)- cycloalkylcarbonyl which are optionally branched in the a position and optionally monosubstituted in the co position by amino or guanidino; or represents Ser, Thr or Q, each in its L or D configuration, or Ser-Ser, Thr-Thr, Pro-Ser, Pro-Thr, Thr-Pro or Ser-Pro, preferably Ser-Ser, it being possible for each amino acid to be present in its L or D configuration, and the N-terminal amino group of the amino acid or of the dipeptide residue being in the free form or acylated by (Ci -Cs 5 )-alkoxycarbonyl, (C6-C1 2 )-aryloxycarbonyl, (C7-Cl 3) tt~ aralkyloxycarbonyl, (C1 -C 6 )-alkanoyl, (C 7 -C 1 3 )-aroyl, arginyl, lysyl or arginyl-arginyl, or it being possible for X to be absent; V denotes Cys or, if X is absent, mercaptopropionic acid, mercaptoatetic acid or mercaptobutyric acid; usp- 2 n~ -1 I a. II~- YII~DUI~ III~IfP~7 PIP I~U- I.' 38 A denotes Phe, Tyr(Me), Tyr(Bu 4-halogenophenyl- aLanine or a L-2-thienyLaLanine residue, preferably Phe; A 1 denotes GLy, ALa or D-ALa; 2 A denotes GLy, ALa or D-ALa; or A and A 2 together denote an optionally branched w-amino- (C3-C 8 )-alkylcarboxy ic acid; B denotes Arg or Lys; C denotes ILe, cyclohexylalanine, tert.-butylglycine, neopentyLgLcyine, Phe, Leu, VaL or an L-2-thienylala- nine residue; D denotes Asp, GLu, GLn, Asn, Leu, ILe, Trp, Asp(OBut), GLu(OBut), GLu(OBzl) or a 2-thienylalanine residue, preferably Asp, Glu or Leu, it being possible for each of the amino acids to be present in its L or D con- figuration; E denotes GLn, Thr or Pro; F denotes Ser, Thr or ALa, preferabLy Ser or Ala, in each case in its L or D configuration; G denotes GLy, ALa or D-ALa, and Z represents a radical of the formuLa II in which H denotes Asn, Ser, Pro or Thr, preferabLy Pro, Thr or Asn, in each case in its L or D form, or Gly; I denotes Ser, Thr, ALa or Ser(But), pref- erably Ser, in each case in its L or D form; J denotes Phe, Tyr(Me), cycLohexylalanyl or -1 39 an L-2-thienyalanine residue, preferably Phe; K denotes Arg, Lys, Orn or a bond; L denotes Tyr, Q, Gly, Tye(Me) or a bond; M denotes Arg-OH, Arg-NH 2 OH, OR, NH 2 NHR', Gly-Lys-Arg-OH or GLy-Lys-Arg-NH 2 R denotes unbranched (C 1 -C 6 )-aLkyL, and R' denotes -[CH2]n-NH 2 or -CCH2]n-NH-C(NH)NH 2 n being an integer and represent ing 3-8, as well as its physiologically tolerated salts, excepting peptides of the formula III, the compounds described in EP-A 231,752 and their salts, rat [Mac 10 5 JANF (101-126), rat-[Mpr 10 5]ANF(101-126), rat-[Mbu105]ANF(101-126) and rat-CMpr 1 D-Ala 10 7 ]ANF(105-126). 04 0 4 '444 40 'I 4 44 04 4 44 44 4 44

5. A peptide of the formula I as claimed in one of claims 1 to 4, in which X denotes hydrogen, arginyl or arginyl-arginyl, Ser, Q or Ser-Ser, it being possible for each of Q and Ser to be present in the L or D configuration, and the N-terminal amino group of the amino acid or di- peptide residue being present in free form or acyl- ated by arginyl or arginyl-arginyl, or it being possible for X to be absent; V denotes Cys or, if X is absent, mercaptopropionic acid; A denotes Phe; B denotes Arg or Lys: C denotes lie, Leu, Va! or a L-2-thienylalanine residue; D denotes Asp, GLn, Leu, ILe, Asp(OBut), Glu(OBut), Tyr(Bu t or Tyr(Me); E denotes Gin, Thr or Pro; F denotes Ser or Ala, in each case in its L dr D con- figuration; G denotes Gly, ALa or D-Ala, and Im. ll~a* -ar~ -4 04 o so so o d 00041: 4I II I I I 444 40 Z represents a radical of the formula II in which H denotes Asn, Pro or Thr, in each case in its L or D form, or Gly; I denotes Ser, Thr or Ala, in each case in its L or D form; J denotes Phe, Tyr(Me), cyclohexylaLanyL or a 2-thienylalanine residue; K denotes Arg, Lys or a bond; L denotes Tyr, Tyr(Bu t or a bond; M denotes OH, NH 2 NHR', Gly-Lys-Arg-OH or Gly-Lys-Arg-NH 2 and R' denotes -CCH 2 2 -NH 2 or -CCH2]n-NH- C(NH)NH 2 n being an integer and representing 3-8; as well as its physiologically tolerated salts, excepting peptides of the formula III, the compounds described in EP-A 231,752 and their salts, rat-EMac 105 ANF (101-126), rat-CMpr 10 5 ]ANF (101-126), 105 105 rat-CMbu 10 5 ANF(101-126) and rat- Mpr 0 D-ALa 10 7 ]ANF(105-126).

6. A process for the preparation of a peptide of the formula I as claimed in one of claims 1 to 5, which comprises a) reaction of a fragment having a free C-terminal car- boxyl group, or one activated derivative, with an appropriate fragment having a free N-terminal amino group, or b) stepwise synthesis of the peptide, elimination, where appropriate, of one or more protective groups which have been temporarily introduced to protect other groups in the compound obtained as in or formation of a disulfide bridge between the two Cys residues, it also being possible for the two latter measures to be carried out in the reverse sequence, and, where appropriate, conversion of d, rA 'I B 0 00 0n 0 8 0 41 the resulting compound of the formula I into its physiologically tolerated salt.

7. The process as claimed in claim 6, wherein one fragment is covalently bonded to a solid body during the synthetic procedure.

8. The use of a compound as claimed in one of claims 1 to as a medicine.

9. The use of a compound as claimed in one of claims 1 to as a diuretic.

10. The use of a compound as claimed in one of claims 1 to as a vasorelaxant agent. I

11. The use of a compound as claimed in one of claims 1 to Sor of a compound of the formula III in which X, C, Z, K, O• L and M are as defined in claim 1, or of their physio- I 4 Logically tolerated salts, as an immunomodulating agent. i

12. A pharmaceutical composition containing a compound as claimed in one of claims 1 to

13. The use of a compound as claimed in one of claims 1 to as an agent for the treatment of glaucoma and/or for re- ducing the intraocular pressure. The use of a compound as claimed in one of claims 1 to for the treatment of renal insufficiency. DATED this 14th day of July 1988. HOECHST AKTIENGESELLSCHAFT EDWD. WATERS SONS PATENT ATTORNEYS QUEEN STREET MELBOURNE. VIC. 3000.