CN1034416Y - Preparation method of peptide with bradykinin antagonism - Google Patents

Abstract

Peptides of formula I with bradykinin antagonism are disclosed: A-B-C-E-F-K-(D)-Tic-G-M-F'-I(I) wherein the definitions of the symbols are detailed in the specification. Their therapeutic applications include all pathological conditions mediated, induced or contributed by bradykinin or bradykinin-related peptides. The peptides of formula I can be prepared by known methods of peptide synthesis.

Description

Preparation method of peptide with bradykinin antagonism

The present invention relates to a peptide with bradykinin antagonism and a preparation method thereof.

International patent No. WO86/07263 describes the bradykinin antagonistic peptide, characterized in that L-Pro in position 7 of the peptide hormone bradykinin or other bradykinin analogs is replaced by D-amino acids such as D-Phe, D-Thia, D-PalCDF, N-Nal, MDY, D-Phg, D-His, D-Trp, D-Tyr, D-hPhe, D-Val, D-Ala, D-His, D-Ile, D-Leu and DOMT replaced.

[003] The object of the present invention is to find a new active peptide that has the effect of antagonizing bradykinin.

This object is achieved by making the peptides of the following formula I and their physiologically tolerable salts:

ABCEFK-(D)-Tic-GMF'-I (I) wherein A is al) hydrogen, (C1-C8)-alkyl, (C1-C8)-alkanoyl, (C1-C8)- Alkoxycarbonyl or (C1-C8)-alkylsulfonyl, wherein 1, 2 or 3 hydrogen atoms in each group may be replaced by 1, 2 or 3 of the same or different residues listed below; Carboxyl, Amino, (C1-C4)-Alkyl, (C1-C4)-Alkylamino, Hydroxy, (C1-C4)-Alkoxy, Halogen, Di-(C1-C4)-Alkylamino, Amino formyl, sulfamoyl, (C1-C4)-alkoxycarbonyl, (C6-C12)-aryl and (C6-C12)-aryl-(C1-C5)-alkyl; or each of these One hydrogen atom of the group may be replaced by a residue selected from the group consisting of:

(C3-C8)-cycloalkyl, (C1-C4)-alkylsulfonyl,

(C1-C4)-alkylsulfinyl, (C6-C12)-aryl-

(C1-C4)-alkylsulfonyl, (C6-C12)-aryl-

(C1-C4)-alkylsulfinyl, (C6-C12)-aryloxy

base, (C3-C9)-heteroaryl and (C3-C9)-heteroaryloxy

base, and 1 or 2 carbon atoms are replaced by 1 or 2 identical or different residues selected from the group consisting of: carboxyl, amino, (C1-C4)-alkylamino, hydroxyl, (C1-C4 )-alkoxy, halogen, di-(C1-C4)-alkylamino, carbamoyl, sulfamoyl, (C1-C4)-alkoxycarbonyl, (C6-C12)-aryl and (C6 -C12)-Aryl-(C1-C5)-Alkyl, a2)(C3-C8)-Cycloalkyl, may be replaced by (C1-C6)-alkyl or (C6-C12)-aryl on the nitrogen atom substituted carbamoyl, (C6-C12)-aryl, (C7-C13)-aroyl, (C6-C12)-arylsulfonyl, (C3-C9)-heteroaryl or (C3-C9 )-heteroaroyl, wherein each of the residues defined under a1) and a2), aryl, heteroaryl, aroyl, alkylsulfonyl and heteroaroyl can be selected from the following 1, 2, 3 or 4 identical or different residues: carboxyl, amino, nitro, (C1-C4)-alkylamino, hydroxyl, (C1-C4)-alkyl, (C1-C4)-alkoxy, Halogen, cyano, di-(C1-C4)-alkylamino, carbamoyl, sulfamoyl and (C1-C4)-alkoxycarbonyl substituted, or a3) residues having the following formula II

Wherein R' is defined as A under a1) or a2), R2 is hydrogen or methyl, R3 is hydrogen or can be amino, substituted amino, hydroxyl, carboxyl, carbamoyl, guanidino, substituted guanidine group, urea group, mercapto group, methylthio group, phenyl group, 4-chlorophenyl group, 4-fluorophenyl group, 4-nitrophenyl group, 4-methoxyphenyl group, 4-hydroxyphenyl group, phthaloyl group imino, 4-imidazolyl, 3-indolyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl or cyclohexyl monosubstituted (C1-C6)-alkyl, and most Preferably (C1-C4)-alkyl, wherein the substituted amino is -NH-A and the substituted guanidino is -NH-C(NH)-NH-A;

A in the formula is the same as the A defined under a) and a), B is a basic amino acid in L or D configuration and can be substituted in the side chain; C is the connecting arm G' of formula IIIa or IIIb -G'-Gly G'-NH-((CH2)n-CO (IIIa) (IIIb) where G' is independently a residue of formula N, respectively

wherein R4 and R5 together with the atoms carrying them form a heterocyclic mono-, bi- or tricyclic ring system of 2-5 carbon atoms, and n is 2 to 8; E is a neutral, acidic or basic aliphatic or aliphatic Cyclo-aliphatic amino acid residues; F are each a residue of a neutral, acidic or basic aliphatic or aromatic amino acid that may be substituted in the side chain, or a direct bond; (D)-Tic is the following formula V The residues of:

G is as defined above as G', or is a direct bond; F' is as defined as F and is a residue -NH-(CH2)n- where n is 2 to 8

Or when G is not a direct bond it can be a direct bond and I is -OH, -NH2 or -NHC2H5, K is a residue -NH-(CH2)X-CO- wherein X=1 to 4 , or a direct bond, and M is defined as F, unless otherwise specified, the abbreviations of amino acid residues without stereoscopic description symbols represent L-shaped residues (see Schröder, Lübke, The Peptides, Volume I, New York1965, pages XXII-XXIII; Houben-Weyl, Methoden der Organischen Chemie (Methods of Organic Chemistry), Volume XV/1 and 2, Stuttgart 1974), such as: Aad, Abu, γAbu, ABz, 2ABz, εAca, Ach, Acp , Adpd, Ahb, Aib, βAib, Ala, BAla, ΔAla, Alg, All, Ama, Amt, Ape, Apm, Apr, Arg, Ash, Asp, Asu, Aze, Azi, Bai, Bph, Can, Cit, Cys , Cyta, Daad, Dab, Dadd, Dap, Dapm, Dasu, Djen, DpaDtc, Fel, Gln, Glu, Gly, Guv, hAla, hArg, hCys, hGln, hGlu, His, hIle, hLeu, hLys, hMet, hPhe , hPro, hSer, hThr, hTrp, hTyr, Hyl, Hyp, 3Hyp, Tle, Ise, Iva, Kyn, Lant, Lcn, Leu, Lsg, Lys, βLys, ΔLys, Met, Mim, Min, nArg, Nle, Nva , Oly, Orn, Pan, Pec, Pen, Phe, Phg, Pic, Pro, APro, Pse, Pya, Pyr, Pza, Qin, Ros, Sar, Sec, Sem, Ser, Thi, βThi, Thr, Thy, Thx , Tia, Tle, Tly, Trp, Trta, Tyr, Val.

Residues suitable as heterocyclic systems of formula IV, in particular heterocyclic residues selected from the following compounds: pyrrolidine-2-carboxylic acid; piperidine-2-carboxylic acid; 1,2,3 , 4-Tetrahydroisoquinoline-3-carboxylic acid; Decahydroisoquinoline-3-carboxylic acid; Octahydroindole-2-carboxylic acid; Decahydroquinoline-2-carboxylic acid; Octahydrocyclopenta[ b) pyrrole-2-carboxylic acid; 2-azabicyclo[2,2,2]octane-3-carboxylic acid; 2-azabicyclo(2,2,1]heptane-3-carboxylic acid 2- Azabicyclo[3,1,0]hexane-3-carboxylic acid 2-azaspiro[4,4]nonane-3-carboxylic acid 2-azaspiro[4,5]decane-3-carboxylate acid; spiro[(bicyclo[2,2,1]heptane)-2,3-pyrrolidine-5-carboxylic acid]; spiro[(bicyclo[2,2,2]octane)-2,3-pyrrole alkane-5-carboxylic acid]; 2-azacyclo[4,3,0,16.9]decane-3-carboxylic acid; decahydroarhepto[b]pyrrole-2-carboxylic acid; [b]pyrrole-2-carboxylic acid; octahydroarhepto[c]pyrrole-2-carboxylic acid; octahydroisoindole-1-carboxylic acid [b]pyrrole-2-carboxylic acid; 2,3,3a,4,5,7a-hexahydroindole-2-carboxylic acid; tetrahydrothiazole-3-carboxylic acid; pyrazolidine-3-carboxylic acid ; Hydroxyprohydrogen-2-carboxylic acid; they can be substituted;

The heterocycles forming the above-mentioned residues have been disclosed in the literature, such as US-A 4,344,949, US-A 4,374,847, US-A 4,350,704, EP-A 29 488, Ep-A 31 741, Ep-A 46 953, EP-A 49 605, EP-A 49 658, EP-A 50 800, EP-A 51 020, EP-A 52 870, EP-A 79 022, EP-A 84 164, EP-A 89 637, EP-A A 90 341, Ep-A 90 362, EP-A 105 102, EP-A 109 020, EP-A 111 873, EP-A 271 865 and EP-A 344 682.

[016] Except where indicated in the specific examples, alkyl groups can be straight chain or branched. The same applies to residues derived therefrom, such as alkoxy, aralkyl or alkanoyl.

[017] (C6-C12)-aryl is preferably phenyl, naphthyl or biphenyl. Residues derived therefrom, such as aryloxy, aralkyl or aroyl, can be defined accordingly.

Halogen is fluorine, chlorine, bromine or iodine, and preferably chlorine.

Particularly suitable salts are alkali metal or alkaline earth metal salts, salts with physiologically tolerable amines and with inorganic or organic acids such as HCl, BBr, H2SO4, H3PO4, maleic acid, fumaric acid, A salt of citric acid, tartaric acid and acetic acid.

Preferred peptides of formula I are wherein

B is Arq, Lys, Orn, 2,4-diaminobutyryl or L-homo

Arginine residues, wherein the amino or guanidino group of the side chain of each residue can be

replaced by A as defined under a1) or a2);

E is an aliphatic or

Alicyclic-aliphatic amino acid residues; such as alanine, serine, threonine, O-

(C1-C6)-alkyl- or O-(C6-C10)-aryl is protected

Serine or threonine, valine, norvaline, leucine, isoleucine

acid, norleucine, neopentylglycine, tert-butylglycine or (C3-

C7)-cycloalkyl(C1-C3)-alkylglycine;

F' is a basic amino acid residue in L or D configuration, such as Arq or Lys,

wherein the guanidino or amino group of the side chain can be represented by A as described under a1) or a2).

Substitution, or a residue where n=2 to 8

-NH-(CH2)n-;

K is a residue -NH-(CH2)x-CO- with X=2 to 4 or is

[035] Peptides with Direct Bonds.

More preferred peptides of formula I are wherein

B is the guanidino or amino group of its side chain can be by (C1-C8)-alkanoyl, (C3-C7)-aroyl, (C3-C9)-heteroaroyl, (C1-C8 )-alkylsulfonyl or (C6-C12)-arylsulfonyl unsubstituted or substituted Arq, Orn or Lys, wherein these aryl, heteroaryl, aroyl, arylsulfonyl and heteroaroyl residues Where appropriate, it may be substituted by 1, 2, 3 or 4 of the same or different residues described under a2); E is leucine, isoleucine, norleucine, tert-butylglycine, Serine, threonine or cyclohexylalanine; K is a direct bond and M is a direct bond peptide. Particularly preferred peptides of formula I are those wherein A is hydrogen, (D)- or (L)-H-Arg, (D)- or (L)-H-Lys or (D) or (L)-Orn and B is The guanidino or amino group in its side chain can be replaced by (C1-C8)-alkanoyl, (C7-C13)-aroyl, (C3-C9)-heteroaroyl, (C1-C8)-alkyl Sulfonyl or (C6-C12)-arylsulfonyl substituted Arg, Orn or Lys, wherein the aryl, heteroaryl, aroyl, arylsulfonyl and heteroaroyl residues may in turn be selected from methyl, 1, 2, 3 or 4 identical or different residues of methoxy and halogen are substituted; C is Pro-Pro-Gly, Hyp-Pro-Gly or Pro-Hpy-Gly, E is Leu, Ile, Tbg or Cha, F is Ser, Hser, Lys, Leu, Val, Nle, Ile or Thr, K is a direct bond, M is a direct bond, G is the residue of a heterocyclic system of formula IV, of which heterocyclic compounds are preferred Pyrrolidine-2-carboxylic acid; pyridine-2-carboxylic acid; tetrahydroisoquinoline-3-carboxylic acid, cis- and trans-decahydroisoquinoline-3-carboxylic acid; cis-endo-, cis- Exo-, trans-octahydroindole-2-carboxylic acid; cis-endo-, cis-exo-, trans-octahydroarhepta[b]pyrrole-2-carboxylic acid or hydroxyproline-2-carboxylate Acid residues, peptides where F' is Arg and I is OH. An example of a particularly preferred peptide of formula I is: H-(D)-Arg-Arg-Pro-Hyp-Gly-Leu-Ser-(D)Tic-Oic-Arg-OHH-(D)-Arg-Arg-Pro -Hyp-Gly-Cha-Ser-(D)-Tic-Oic-Arg-OHH-(D)-Arg-Arg-Pro-Hyp-Gly-Tbg-Ser-(D)-Tic-Oic-Arg-OH

The present invention relates to a method for preparing a peptide of formula I, comprising

a) reacting a fragment having a C-terminal free carboxyl group or an activated derivative thereof with a corresponding fragment having an N-terminal free amino group, or

b) stepwise synthesis of the peptide,

Remove - one or more protective groups temporarily introduced in the compound prepared as described in (a) or (b) to protect other appropriate functional groups, and the prepared Compounds of formula I are converted to their appropriate physiologically tolerable salts.

The peptides of the invention can be prepared according to commonly known methods of peptide chemistry, e.g. see Houben-Weyl, Methodend der Organischen Chemie, Vil. 15/2, but preferably using e.g. B. Merrifield (J.Am.ChemSoc.85 , 2149 (1963)) or RC Sheppard (Int. J. Peptide Rrotein Res. 21118 (1983)) described in solid-phase synthesis or a known equivalent method. Useful as a-amino protecting groups are urethane protecting groups such as tert-butoxycarbonyl (DOC) or fluorenylmethoxycarbonyl (Fmoc) protecting groups. In order to prevent side chain reactions or to synthesize specific peptides, appropriate protecting groups can be used to additionally protect functional groups in amino acid side chains if necessary (see, for example, TW Greene, "Protective Groups in Organic Synthesis"), protecting groups mainly used for different amino acids The groups are Arg(Tos), Arg(Mts), Arg(Mtr), Arg(PMC), Asp(OBzl), Asp(OBut), Cys(4-MeBzl), Cys(Acm), Cys(SBut), Glu (OBzl), Glu(OBut), His(Tos), His(Fmoc), His(Dnp), His(Trt), Lys(Cl-z), Lys(Boc), Met(O), Ser(Bzl) , Ser(But), Thr(Bzl), Thr(But), Trp(Mts), Trp(CHO), Tyr(Br-Z), Tyr(Bzl) or Tyr(But).

Solid phase synthesis was initiated from the C-terminus of the peptide by coupling a protected amino acid to an appropriate resin. The starting materials for this synthesis can be linked via ester or amide linkages to protected amino acids with chloromethyl, hydroxymethyl, benzhydrylamino (BHA) or methylbenzylamino (MBHA) Group-modified polystyrene or polyacrylamide resin. Resins used as carrier materials are commercially available. BHA- and MBHA-resins are commonly used when the peptide being synthesized must contain a free carbamoyl group at the C-terminus. If the peptide contains a second carbamoyl group at the C-terminus, a chloromethyl- or hydroxymethyl-resin can be used and cleaved with the appropriate amine. If it is desired to contain acetamide, the peptide can be cleaved from the resin with ethylamine, in which case the side chain protecting group can then be removed with an appropriate reagent. If the tert-butyl protecting group in the amino acid side chain is to be retained on the peptide, the synthesis can be accomplished with the Fmoc protecting group, using methods already described (see, e.g., RC Sheppard, J. Chem. Soc., Chem. Comm. 1982, 587) The α-amino group of the amino acid is temporarily blocked, in this case the guanidine group of arginine can be protected by protonation with pyridinium perchlorate, and the guanidine group of arginine can be protected with the aid of catalytic transfer hydrogenation (A. Felix et al., J. Org. Chem. 13, 4194 (1978)) or sodium in ammonia (W. Roberts, J. Am. Chem. Soc. 76, 6203 (!(%$)) removal of benzyl groups Protecting groups to protect other amino acids functionalized in the side chain.

The amino protecting group of the amino acid coupled on the resin can be removed using a suitable reagent, such as trifluoroacetic acid dissolved in dichloromethane for the Boc protecting group and difluoroacetic acid for the Fmoc protecting group. A 20% strength solution of piperidine in methylformamide followed by sequential coupling of the following protected amino acids according to the desired sequence. The N-terminal protected peptide-resin (as an intermediate) was deblocked with the above reagents for religation of subsequent amino acid derivatives.

All possible activators for peptide synthesis can be used as coupling reagents, e.g. see Houben-Weyl, Methoden der Organischem Chemie, Vol. 15/2, but preferably carbodiimides such as N, N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide or N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. In addition, amino acid derivatives can be added directly to the resin with the aid of an activator, with the addition of an additional meso-inhibiting activator, such as 1-hydroxybenzotriazole (HOBt) (W. Konig, R. . Geiger, Chem. Ber. 103, 708 (1979)) or 3-hydroxy-4-oxo-3,4-dihydrobenzotriazole (HOBt) (W. Konig, R. Geiger, Cehm. Ber. 103, 2054 (1979)) for coupling, or the amino acid can also be preactivated as a symmetrical anhydride or HOBt or HOObt ester, respectively, and a solution of the activated amino acid derivative in a suitable solvent can be added to the compound ready for coupling. in the peptide-resin.

Coupling or activation of amino acid derivatives can be carried out with one of the above-mentioned reactive reagents in dimethylformamide, N-methylpyrrolidone or dichloromethane, or in a mixture of said solvents. Typically, activated amino acid derivatives are used in 1.5 to 4-fold excess. In the case of incomplete coupling, the coupling reaction can be repeated without prior deblocking of the alpha-amino group on the peptide-resin, which is necessary to couple the next amino acid in the sequence.

[047] The success of the coupling reaction can be checked using the ninhydrin reaction already described (eg see E. Kaiser et al., Anal. Biochem. 34, 595 (1979)). Automated synthesis is also possible, eg, using a peptide synthesizer, Model 430A, produced by Applied Biosystems, in which case a synthesis program provided by the instrument manufacturer or a synthesis program developed by me can be used. The latter is used in particular when using amino acid derivatives protected by the Fmoc group.

After the peptide has been synthesized as described above, it can be cleaved from the resin using reagents such as liquid hydrogen chloride (particularly suitable for peptides prepared by the Boc method) or trifluoroacetic acid (particularly suitable for peptides synthesized by the Fmoc method). These reagents not only cleave peptides from resins, but also other side chain protecting groups of amino acid derivatives. The peptides obtained in this way are in the free acid form, except for those using BHA- and MBHA-resins. In the case of BHA- and MBHA-resins, cleavage with hydrogen chloride or trifluoromethane-sulfonic acid yields the amide-shaped peptide. Further processing methods for the preparation of peptide amides are described in EP-A 287,882 and EP-A 322,348. In this case, moderate-strength acids commonly used in peptide synthesis (eg, trifluoroacetic acid) can be used, plus phenol, methoxycresol, methoxythiophenol, anisole, thioanisole, ethanedithiol, ethyl Cation capture species such as methyl sulfide or similar cation capture species commonly used in solid phase synthesis (these coagents may be used alone or in combination of two or more) are treated to cleave the peptide amide from the resin. In this case, trifluoroacetic acid can also be used after dilution with a suitable solvent such as dichloromethane.

If the tert-butyl or benzyl side chain protecting group on the peptide is retained, it can be used according to the method described (for example, referring to RC Sheppard, J.Chem.Soc., Chem.Comm., 1982, 587). 1% trifluoroacetic acid in dichloromethane cleaved peptides that had been synthesized on specially modified support resins. If the individual tert-butyl or benzyl side chain protecting groups are retained, the appropriate combination of synthetic and cleavage methods is used.

The modified carrier resins introduced by Sheppard were also used to synthesize peptides with a C-terminal carbamoyl group and a W-amino or -guanidinoalkyl group. After synthesis, peptides with fully protected side chains are cleaved off the resin and then combined in classical synthesis solution with the appropriate amine or W-aminoalkylamine or W-guanidinylamine, in which case it is possible to press Other functional groups present are temporarily protected in known manner.

Another method for preparing peptides with W-aminoalkyl groups is described in EP-A264802.

The peptides of the present invention are preferably synthesized using solid-phase techniques with two general protecting groups:

Synthesis was carried out using an automatic peptide synthesizer model 430A produced by Applied Biosystems, wherein the α-amino group was temporarily blocked with Boc and Fmoc protecting groups.

When using a Boc protecting group, it can be synthesized using synthetic cycles preprogrammed by the instrument manufacturer.

4-(hydroxymethyl) phenylacetamidomethyl polystyrene resin (RB Merrifield, J. Org. Chem., 43, 2845) functionalized with appropriate Boc-amino acids, produced by Applied Biosystems (1978)) to synthesize peptides with a free carboxyl group at the C-terminus. Peptide amides were prepared using MBHA-resin from the same company. The activator used was N,N'-dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide. Symmetrical anhydride, HOBt or HOObt ester activation in CH2Cl, CH2Cl/DMF mixtures or NMP. Coupling was performed using 2-4 equivalents of the activated amino acid derivative. In the case of incomplete coupling, the reaction must be repeated.

When the α-amino gene was temporarily protected with the Fmoc protecting group, our own synthesis procedure was introduced for synthesis with the aid of an Applied Biosystems Model 430A automated peptide synthesizer. Parabenzyloxybenzyl alcohol-resin (S. Wang, J. Am) of Bachem which has been esterified with the appropriate amino acid according to known methods (E. Atherton et al., JCS Chem. Comm. 1981, 336) Chem. Soc. 95, 1328 (19873)). In the amino acid cartridge provided by the instrument manufacturer, a solution of diisopropylcarbodiimide in DMF was added to the mixture of amino acid derivative and HOBt or HOObt that had been weighed in as the HOBt or HOObt ester And direct activation of amino acid derivatives. It is also possible to use Fmoc-amino acid OObt esters for batch production as described in EP-A247573. The Fmoc protecting group was removed using a strong 20% solution of piperidine in DMF in the reaction vessel. The excess reactive amino acid derivative used is 1.5 to 2.5 equivalents. If the coupling is not complete, the coupling reaction can be repeated according to the Boc method.

The peptides of the present invention all show bradykinin antagonism when used alone or in combination, and this effect can be tested by various experimental models (see Handbook of Exp.Pharmacol., Vol.25, SpringerVerlag, 1970, pp.53- 55), such as using isolated rat uterus, guinea pig ileum or isolated guinea pig pulmonary artery.

[058] To test the peptides of the invention with isolated pulmonary arteries, guinea pigs (Dunkin Hartley) weighing 400-450 g were sacrificed by inflating the back of the neck. Open the chest cavity and carefully dissect the pulmonary artery. The surrounding tissue was removed and the arteries were dissected into a spiral at 45°C.

[059] Vessel strips 2.5 cm long and 3-4 mm wide were fixed in a 10 ml organ bath filled with Ringer's solution.

Wherein the composition (mmol/l) of solution is:

NaCl 154

KCl 5.6

CaCl 1.9

NaHCO 2.4

Glucose 5.0 The solution was heated to 37°C and 95% O and 5% CO were blown through the solution. The pH was 7.4, and the load of the vessel strip was 1.0 g.

Changes in isometric contractions were detected with a lever attachment and an HF demodulator (stroke measuring device) (HugoSachs) and recorded on a potentiometric recorder (BEC, Goerz Metrawatt SE460).

[067] The experiment was started after equilibration for 1 hour. Once the strips have reached their maximum sensitivity to 2 x 10-7 mol/1 bradykinin - bradykinin can cause vasoconstriction - doses of 5 x 10-8 to 1 x 10-5 mol/l of the peptide are administered Acted for 10 minutes, and after replacement of bradykinin, the increase in bradykinin effect was compared with the control group.

[068] In order to test the partial antagonistic effect, the peptide dose used was 1×10-5-1×10-3 mol/1.

The IC50 values of the peptides of the invention calculated from dose-effect plots are listed in Table 1.

[070] Table 1: Compound IC50 [M]H-(D)-Arg-Arg-Pro-Hyp-Gly-Leu-Ser-(D)-Tic-Oic-Arg-OH5.9×10-9H-( D)-Arg-Arg-Pro-Hyp-Gly-Cha-Ser-(D)-Tic-Oic-Arg-OH3.7×10-8H-(D)-Arg-Arg-Pro-Hyp-Gly-Tbg -Ser-(D)-Tic-Oic-Arg-OH6.0×10-6

Therapeutic applications of the peptides of the invention include all pathological conditions mediated, induced or contributed to by bradykinin and bradykinin-related peptides. These pathological conditions include trauma such as trauma, burns, rash, rash, edema, tonsillitis, arthritis, asthma, allergy, rhinitis, shock, inflammation, hypotension, pain, pruritus and altered sperm motility.

The present invention therefore also relates to the use of the peptides of formula I as medicaments, and medicinal products containing these compounds.

[073] The medicinal product may contain only an effective amount of the active compound of formula I, or may also incorporate pharmaceutically available inorganic or organic excipients.

Administration can be enteral or parenteral, such as subcutaneous, intramuscular or intravenous injection, or sublingual, epidermal, nasal, rectal, intrauterine, oral or inhalation. The dose of active compound depends on the warm-blooded species, body weight, age and mode of administration.

[075] The pharmaceutical products of the present invention can be prepared by known processes such as dissolving, mixing, granulating or coating.

For dosage forms for oral administration or transmucosal administration, the active compound can be mixed with auxiliary additives commonly used for this purpose, such as excipients, stabilizers or inert diluents, and converted into suitable Dosage forms such as tablets, coated tablets, hard gelatine capsules, aqueous, alcoholic or oily suspensions or solutions. Inert carriers that may be used include acacia, magnesium oxide, magnesium carbonate, potassium phosphate, lactose, dextrose, magnesium stearoyl fumarate or starch, especially corn starch. The formulations may be in the form of dry or wet granules. Suitable solvents in the form of oily vehicles may be vegetable or animal oils, such as sunflower oil and cod liver oil.

For topical administration products, can be aqueous, oily solutions, lotions, emulsions or jellies, in the form of soft or ointments, if necessary in the form of a spray, and possibly by adding a polymer substances to improve drug adhesion.

For dosage forms for intranasal administration, the active compound can be admixed with conventional additives such as stabilizers or inert diluents and converted by conventional methods into suitable dosage forms such as aqueous, alcoholic or oily suspensions or solutions. It is possible to add chelating agents such as ethylenediamine-N,N,N',N'-tetraacetic acid, citric acid, tartaric acid or salts thereof to aqueous intranasal formulations. Nasal solutions can be administered in a graduated nebulizer, or as nasal drops, nasal gels, or nasal creams with viscosity-increasing contents.

[079] Administration by inhalation can be carried out using a nebulizer or a compressed gas pack utilizing an inert carrier gas.

For the preparation of intravenous, subcutaneous, epidermal or intradermal administration, active compound or its pharmaceutically tolerable salt can be added to maintain isotonicity or adjust pH according to the need. into a solution, suspension or emulsion.

[081] The short half-life in body fluids of some of the mentioned pharmaceutical substances means that injectable depot formulations are necessary. Pharmaceutical dosage forms that can be used for this are oily crystalline suspensions, microcapsules, rods or implants, the latter of which may contain histocompatible polymers, especially biodegradable polymers, such as those based on polyacetic/polyglycolic acid Copolymers or biodegradable polymers of human albumin.

Appropriate dosage ranges for topical or inhalation dosage forms are solutions containing 0.01-5 mg/ml of the active compound, and 0.01-10 mg/kg for systemic administration.

The amino acid abbreviation symbols used correspond to the three-letter codes customary in peptide chemistry as described in Europ. J. Biochem. 138, 9 (1984). Other abbreviations are listed below: Acm acetamidomethyl ε-Ahx ε-aminocaproyl Aoc cis, endo-2-azabicyclo[3,3,0]octane-

3-S-carbonyl Bcc tert-butoxycarbonyl But tert-butyl Bzl benzyl CDF chloro-(D)-phenylalanyl Cha cyclohexyl alanyl Chg cyclohexyl glycyl cl-z 4-Chlorobenzyloxycarbonyl DMF Methylformamide DOMF O-methyl-(D)-threonyl Dnp 2,4-dinitrophenyl Fmoc 9-Fluorenylmethoxycarbonyl MDY O-methyl Alkyl-(D)-acylaminoacyl Me Methyl 4-Mebzl 4-methylbenzyl Mtr 4-methoxy-2,3,6-trimethylsulfonyl Mts 1,3,5-trimethylbenzene- 2-sulfonyl Nal 1- or 2-naphthylalanyl NMP N-methylpyrrolidine Npg neopentylglycyl Oic cis-endo-octahydroindole-2-carbonyl Opr isoxazolidine-3 -ylcarbonyl Pal 2- or 3-pyridyl alanyl Pmc 2,2,5,7,8-pentamethylchroman-

6-sulfonyl Tbg tert-butylglycyl TFA trifluoroacetic acid Thia 2-thienyl alanyl Tcs 4-toluenesulfonyl Tic 1,2,3,4-tetrahydroisoquinoline-3- Carbonyl Trt Trityl

The following examples are intended to further illustrate the preferred method of solid phase synthesis of peptides according to the invention without constituting a limitation of the invention.

The following amino acid derivatives were used: Fmoc-Arg(Mtr)-OH, Boc-(D)-Arg-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Hyp-OH, Fmoc-Pro-OObt, Fmoc-Gly-OObt, Fmoc-Phe-OObt, Fmoc-Ser(tBu)-OObt, Fmoc-(D)-Tic-OH, Fmoc-Gln-OH, Fmoc-Aoc-OH, Fmoc-Thia-OH, Fmoc -Opr-OH, Fmoc-(D)-Asn-OH, Fmoc-β-Ala-OH, Fmoc-Oic-OH.

Embodiment 1

Peptide Synthesizer Model 430A from Applied Biosystems was used and the Fmoc method was used on parabenzyloxybenzyl alcohol-resin esterified with Fmoc-Arg(Mtr)-OH (Novabiochem) (added per gram resin). The sample amount is about 0.5 mmol) to synthesize H-(D)-Arg-Arg-Hyp-Pro-Gly-Leu-Ser-(D)-Tic-Oic-Arg-OH step by step. One gram of resin was used in the synthesis and a synthetic procedure adapted for the Fmoc method was used.

In each case, 1 mmol of amino acid derivative with free carboxyl group and 0.95 mmol of HOObt were added exactly to the synthesizer cartridge. These amino acids were dissolved in 4 ml DMF before use and preactivated directly by adding 2 ml of a 0.55 mol/l solution of diisopropylcarbodiimide in DMF. HOObt esters of other amino acids were dissolved in 6 ml of NMP and the preactivated amino acids were coupled in situ to a resin previously deblocked with 20% piperidine in DMF. After the synthesis was completed, the peptide was cleaved from the resin, while the side chain protecting groups were removed with trifluoroacetic acid using thioanisole and ethanedithiol as cation capture species. After removal of trifluoroacetic acid, the resulting residue was digested several times with ethyl acetate and centrifuged. The remaining residue was chromatographed on a Sephadex LH20 column and eluted with 10% acetic acid. Fractions containing pure peptides were pooled and lyophilized. MS (FAB): 1264 (M+H) The following peptides were prepared in a similar manner to Example 1. Example 2H-(D)-Arg-Arg-Hyp-Pro-Gly-Cha-Ser-(D)-Tic-Oic-Arg-OHMS(FAB): 1304 (M+H) Example 3H-(D) -Arg-Arg-Hyp-Pro-Gly-Tbg-Ser-(D)-Tic-Oic-Arg-OHMS(FAB): 1264(M+H)

Claims (1)

1. A method for preparing the following peptides of formula I and pharmaceutically tolerable salts thereof, A-B-C-E-F-K-(D)-Tic-G-M-F′-I (I) where A is H-(D)-Arg, B is Arg, C is Pro-Hyp-Gly, E is Leu, Tbg or Cha, F is Scr, K and M are direct keys, G is Oic, and F' is Arg, and I is OH; (D)-Tic is a residue of the following formula V: The method includes: a) reacting a fragment having a C-terminal free carboxyl group or an activated derivative thereof with a corresponding fragment having an N-terminal free amino group, or b) stepwise synthesis of the peptide, removing one or more protecting groups temporarily introduced into the compound prepared as described in (a) or (b) to protect other appropriate functional groups, and will have prepared in this way The compounds of formula I are converted into their appropriate physiologically tolerable salts.