LT3868B - Process for the preparation of peptide, use them and process for the preparation of pharmaceutical composition - Google Patents

Abstract

Peptides of the formula I A-B-C-E-F-K-(D)-Phe-G-M-F'-I (1> in which A is hydrogen, alkyl, alkanoyl, alkoxycarbonyl, alkylsulphonyl, cycloalkyl, aryl, aryloyl, arylsulphonyl, heteroaryl or an amino acid, each of which can optionally be substituted, B is a basic amino acid, C is a di- or tripeptide, E is the residue of an aromatic amino acid, F is, independently of one another, an amino acid which is optionally substituted in the side chain or is a direct bond, G is an amino acid, F' is defined as F or can be -NH-(CH2)2-8 or optionally a direct bond, I is -OH, -NH2 or -NHC2H5, and K is a radical -NH-(CH2)1-4-CO- or is a direct bond, act as bradykinin antagonists. Their therapeutic uses comprise all pathological states promoted, induced or assisted by bradykinin and bradykinin-related peptides. The peptides of the formula I are prepared by known methods of peptide synthesis.

Description

The present invention relates to novel peptides having antagonistic effects on bradykinin, and to methods of preparing these peptides.

Antagonistic bradykinin peptides are described in WO 86/07263; for these peptides, the 7-position L-Pro of the bradykinin peptide hormone or other bradykinin analog is replaced by a D-amino acid such as D-Phe, D-Thi, D-Pal, CDF, D-Nal, MDY, D-His, D -Tyr, D-Trp, D-hPhe, D-Val, D-Ala, d-His, D-Ile, D-Leu and DOMT.

The main object of the invention is to find new effective peptides which have antagonistic effect on bradykinin. This problem is solved by the peptide of formula I:

A-B-C-E-F-K- (D) -Phe-G-M-F '- I (I), wherein:

(C.-C _o) alkanoyl,

J. o _η -C _c ) -alkylsulfonyl, o

two or three hydrogen at two or three at one, at such a price, a?

is hydrogen, (C₁-C₆) -alkyl, (C-C _p) -alkoxycarbonyl or (C, if need be, one atom can be replaced by one or different references liekanomi carboxyl, amino / (C i - C₄) - alkyl, (C? -C?) - alkylamino, hydroxy, (C? -C?) - alkoxy, halogen, di (C i -C ₄₎ -alkilaminogrupė, carbamoyl, sulfamoyl, (Ci ^{- C} 4) alkoxycarbonyl, (C ^ Cg) yl ^{_ar as r} ^ ~ ^ 6 12 ^ ^_

-acyl-C 1 -C 4 alkyl;

or wherein, if desired, one hydrogen atom may be replaced by a residue such as:

(C₁-C₈) -cycloalkyl, (C? -C?) - alkylsulfonyl, (C? -C?) - alkylsulfinyl, ^{(α θ -ε ΐ2)} ~ ^{ar (C} l ^-C 4 ^-a lkilsulfonilas (C , -C, -) - aryl- (C 1 -C 4) -alkylsulfinyl, (C 1 -C 4, -J-aryloxy) 12-14 o 12, (C 1 -C 8) -heteroaryl and (C 1 -C 8) - a heteroaryloxy group and one or two hydrogen atoms replaced by one or two identical or different residues, such as:

carboxyl, amino, (C 1 -C 4) -alkylamino, hydroxy, (C 1 -C 4) -alkoxy, halogen, di- (C 1 -C 4) -alkylamino, carbamoyl, sulfamoyl, (C 1 -C 4) ) -alkoxycarbonyl, (C 1 -C 4) -aryl and (C 1 -C 10) -aryl- ₍ C 1 -C 3) -al 12 0 12 1 O;

aryl substituents; (C 1 -C 7) aryl, a,) (C 1 -C 0) -cycloalkyl; carbamoyl, which may be optionally z (C 1 -C 8) -alkyl or (C 1 -C 6) -10 12 (C 1 -C 8) -aryl, (C 8 -C 12) -arylsulfonyl, if necessary at the nitrogen atom (Cg-C _o) -heteroaryl or (C₁-C₆) -heteroariloilas;

in addition, p.ρ. a)) and 32), the residues indicated above, where appropriate, such as aryl, heteroaryl, aroyl, arylsulfonyl and heteroaryl, may be one, two, three or four identical or different substituents such as:

carboxyl, amino, nitro, (C 1 -C 4) -alkylamino, hydroxy, () -alkyl, (C 1 -C 4) -alkoxy, halogen, cyano, di- (C ₁ -C ₄ ) -alkylamino, carbamoyl , sulfamoyl and (C 1 -C 4) alkoxycarbonyl, or a (4)) is a residue of formula (II):

R * - N - CH - C (II), where R · * ^- is as A under a ^) and a ^),

R is hydrogen or methyl,

R ^J is hydrogen or (C 1-4 C 8) alkyl, preferably (C 1-4 C 1-4) alkyl, optionally substituted with one substituent such as amino, substituted amino, hydroxyl, carboxyl, carbamoyl , guanidine group, substituted guanidine group, ureido group, mercapto group, methylmercapto group, phenyl, 4-chlorophenyl, 4-fluorophenyl, 4-nitrophenyl, 4-methoxyphenyl, 4-oxyphenyl, phthalimido group, 4-imidazolyl, 3-indolyl, 2- thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl or cyclonexyl wherein the substituted amino group is -NH-A and the substituted guanidine group is -NH-C (NII) -NH-A, wherein A is as defined a ^) or 3 £) in points;

B is an amino acid of the L- or D-configuration which may be substituted in the side chain;

C are groups of the formulas Illa or Illb:

G ^z - g'- Gly (Illa)

3'-NH - (CH ") - COn '(Illb) wherein G * independently of one another denotes qruoe of formula IV:

- •• N - CH

C

II

(IV), where

R ⁴ and R ⁵ together with the atoms to which they are attached form a heterocyclic mono-, bi- and tricyclic system with 2-15 C atoms and n = 2-8;

E is an aromatic amino acid residue;

F is independently a neutral, acidic or basic, aliphatic or aromatic amino acid residue which may be substituted in the side chain; or is a bond (D) -Phe - is D-phenylalanine which may be optionally substituted on the phenyl moiety;

G is the same as S * having the values given above or is a bond;

F 'is the same as F is a residue of -NH (CH ₂ ) _n ~ where n = = 2-8, or if Θ is not a bond, it may be a bond;

I is -OH, -NH ^ or -HNC ^ H ^;

K is a residue of -NH- (CH 2) -CO- where x = 1-4, or is a bond and

M has the same meanings as F;

and physiologically compatible salts of these compounds.

Unless otherwise noted, truncation of an amino acid without reference to a stereo sign means an L-shaped amino acid residue (see Schrodes, L & bke, The Peptides, Vol. 1, New York, 1965, ΧΧΙΙ-ΧΧΙΙΙ; Huben-v.'eil, Metnoden der Organische Chemie, Bd. XV / I und 2, Studgard, 1974), for example:

Aad, Abu, J'Abu, ABz, 2ABz, £ Aca, Ach, Acp, Adpd, Ahb, Aib, Ala, ^ Ala, oAla, Alg, Ali, Am, Amt, Ape, Apm, Apr, Arg, Asn, Asp,

Asu, Aze, Azi, Bai, Bph, Can, Cit, Cys, Cyta, Daad, Dab, Dada,

Dap, Dapm, Dasu, Djen, Dpa, Dtc, Fel, Gln, Glu, Gly, Guv, hAla,, hArg, hCys, hGln, irGlu, His, hlle, hLeu, hLys, hMet, hPhe, hPro, hSer, hThr , hTrp, hTyr, Hyl, Hyp, 3Hyp, lle, Ise, Iva, Kyn, Lant, Lcn, Leu, Lsg, Lys, j5Lys, ALys, Met, Mim, Min, nArg, Nle, Nva, Oly, Orn, Pan , Pec, Pen, Phe, Phg, Pic, Pro, APro, Pse, Pya, Pyr, Pza, Qin, Ros, Sar, Sec, Ser, Ser, Thi, pThi, Thr, Thy, Thx, Tia, Tie, Tly , Trp, Trta, Tyr, Vai.

As the residue of the heterocyclic system referred to in the description of Formula IV, particular reference is made to the heterocycle residue of the following group:

pyrrolidine-2-carbonic acid, piperidine-2-carbonic acid;

1,2,3,4-Tetrahydroisoquinoline-3-carbonic acid; decahydroisoquinoline-3-carbonic acid; octahydroindole-2-carbonic acid;

decahydroquinoline-2-carbonic acid; octahydrocyclopenta (b) -pyrrole-2-carbonic acid; 2-aza-bicyclo (2.2.2) -octane-3-carbonic acid;

2-Azabicyclo (2.2.1) -heptane-3-carbonic acid;

2-Azabicyclo (3.1.0) -hexane-3-carbonic acid;

2-Azaspiro- (4.4) -nonan-3-cardboard acid;

2-Azaspiro- (4.5) -decane-3-carbonic acid;

spiro // bicyclo (2.2.1) -heptane (-2,3-pyrrolidine-5-carbonic acid);

spiro // bicyclo (2.2.2.) octane (-2,3-pyrrolidine-5-cardboard acid);

2-Azatricyclo (4.3.0.1 '') decane-3-carbonic acid;

decahydrocyclohepta (b) pyrrole-2-carbonic acid; decahydrocycloocta (b) pyrrole-2-carbonic acid;

octahydroisoindole-1-carbonic acid; 2,3,3a, 4,6a-hexahydro-cyclopenta (b) pyrrole-2-carbonic acid;

2,3,3a, 4,5,7a-hexahydroindole-2-carbonic acid; tetrahydrothiazole-4-carbonic acid; isoxazolidine-3-carbonic acid;

pyrazolidine-3-carbonic acid; oxyproline-2-carbonic acid;

which may be substituted if necessary;

Ί

The heterocycles on which the aforementioned residues are based are known, for example, from U.S. Patent No. 4,344,349, U.S. Patent No. 4,374,847, U.S. Patent No. 4,350,704, European Patent No. 4,600,198.

A 50,800, A 31,741, A 51,020, A 49,658, A 49,605, A 29,488,

A 46 553, A 52 870, A 271 865, German Patent No. A 32 26 768,

A 31 .51 690, A 32 10 496, A 32 11 397, A 32 11 676, A 32 27 055,

A 32 42 151, A 32 46 503 and A 32 46 757.

In addition, some of these heterocycles are described in German Patent A 38 18 850.3.

Unless otherwise stated in the particular case, alkyl may be linear or branched. The same is true for alkyl derivative residues such as alkoxy, aralkyl or alkanoyl.

(C 8 -C 12) -aryl preferably represents phenyl, naphthyl or biphenyl. The same applies to residues of its derivatives, such as aryloxy, aralkyl or aroyl.

Halo means fluorine, chlorine, bromine or iodine, preferably chlorine.

Acceptable focal salts include especially alkali or alkaline earth metal salts, physiologically acceptable amine salts, and inorganic or organic acids such as, for example,

HCl, HBr, H2SO4, H ^ PO ^, maleic, fumar, citric, tartaric, acetic acid salts.

Most preferred are peptides of formula I wherein:

B = the residue of Arg, Lys, Orn, 2,4-diaminobutanoyl or L-horaargargine, and in addition, if necessary, the amino or guanidine group in the side chain may be replaced by A as described in a ^) or a ^).

E = a residue of an aromatic amino acid of the L- or D-configuration having 6 to 14 ring C-atoms, such as a phenylalanine which may be optionally substituted in the 2, 3 or 4 position by halogen, O-methyl tyrosine , A residue of 2-thienylalanine, tyrosine, 2-pyridylalanine or naphthylalanine;

F '= the residue of a basic amino acid of the L- or D-configuration, such as Arg or Lys, wherein the guanidine group or amino group of the side chain may be replaced by A as described in a *) and a ^), or -NH- (CH). ₂ ) a residue of _n ~, where n = 2-8;

K = -NH- (CH ₂ ) -CO-, where x = 2-4, a residue or a bond;

(D) -Phe is -D-phenylalanine which may optionally be substituted on the phenyl portion with halogen or (C 1 -C 4) -alkoxyl.

Particularly suitable are peptides of formula I wherein: β = Arg, Orn or Lys, wherein guanidine, or the corresponding amino groups in the side chain, are unsubstituted or may be substituted such as (C 1 -C 8) -alkanoyl, (C 1 -C 8). -C ^ g) -ariloilas (C _.- C) -hetercariloilas (C.-C _o) or jyio alkylsulfonyl (C, .- C) - arylsulfonyl, where the aryl, heteroaryl, aryloyl, aryl sulfonyl 12 and the heteroaryloyl moieties as described in (a) may optionally have 1, 2,3 or 4 substituents, which may be the same or different;

E = phenylalanine, 2-chlorophenylalanine, 3-chlorophenylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, tyrosine, O-methyl tyrosine, or

- (2-thienyl) -aJanine;

K = coupling; fi = a bond and (D) -P'ne is a D-phenylalanine which may optionally be substituted by fluorine, chlorine, bromine or methoxy.

Particularly suitable are peptides of formula I in which:

A = hydrogen, (D) - or (L) -H-Arg, (D) - or (L) -H-Lys or (D) - or (L) -H-Orn;

B = Arg, Orn or Lys, wherein the side chain guanidine or amino group may be substituted such as hydrogen, (C 1 -C 6) alkanoyl, (C 1 -C 4) g -aryl, (C 1 -C 4) -heteroaryl , (C 1 -C 8) -alkylsulfonyl or (C 1 -C 6) -arylsulfonyl whose aryl, heteroaryl, aroyl, arylsulfonyl and heteroarylaryl moieties may optionally have 1, 2, 3 or 4 substituents, such as as methyl, methoxy and halogen;

C = Pro-Pro-Gly, Hyp-Pro-Gly or Pro-Hyp-Gly;

E - Phe or Thia;

F = Ser, Hser, Lys, Leu, Vai, Nle, lle or 'fhr;

K = coupling;

M - connector;

G = a residue of the Heterocyclic System of Formula IV, wherein the preferred moieties are: pyrrolidine-2-carbonic acid; piperidine-2-carbonic acid; 1,2,3,4-Tetrahydroisoquinoline-3-carbonic acid; eis and trans -decahydroisoquinoline-3-carbonic acid; cis-endo-, cis-exo-, trans-octahydroindole-2-carbonic acid; cis-endo-, cis-exo-, trans-octahydrocyclopentane (b) pyrrole-2-carbonic acid or oxyproline-2-carbonic acid;

F '= Arg;

= OH and (D) -Phe = D-phenylalanine.

The invention further relates to a process for the preparation of peptides of formula I, which differs in that

a) The fragment with the free C-terminal carboxyl group or its activated derivative is exposed to the corresponding fragment with the N-terminal amino group '-or

b) the peptide is synthesized step by step, whereupon one or more temporary protecting groups are removed from the compound obtained according to a) or b), if necessary, and the compounds of formula I thus obtained are converted into their physiologically acceptable salts, if necessary.

The peptides of the present invention are prepared by known peptide chemistry techniques (see, e.g., Houben-Weil, Organic Chemistry Methods, 15/2 t), preferably using solid phase synthesis as described, for example, by B. Ilerrfield, J. Amer. Chem. Soc., 85, 2149 (1963), or

B.C. Sheppard, Int. J. Peptide and Protein Res., 21, 118 (1983), or similar methods known in the art. Urethane protecting groups such as, for example, tert-butyloxycarbonyl (Boc) or fluorenylmethyloxycarbonyl (Fmoc) protecting groups are used to protect the o-C-amino group. If side reactions or special peptide synthesis are to be avoided, the side chain functional groups of the amino acids are further blocked using appropriate protecting groups (see, e.g., T. W. Greene, Protsctive Groups in Orcanic Synthesis); most commonly used:

Arg (Tos), Arg (Uts), Arg (Mtr), Arg (Pmc), Asp (OBzl), Asp (OBut),

Cys (4-MeBzl), Cys (Ame), Cys (SBut), Glu (OBzl), Glu (OBut), His (Tos), His (Frnoc), 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 begins with the interaction of the terminal carbon atom of the peptide with an amino acid blocked by the corresponding resin. Starting materials of this type may be obtained by coupling a blocked amino acid with a modified polystyrene or polyacrylamide resin of a modified chloromethyl, oxymethyl, benzhydrylamine (BHA), methylbenzhydrylamine (I-IBHA) to form an ester or amide bond. The resin-bearing resin is sold. BHA and MBHA resins are commonly used when the peptide synthesized must have a free amide group at the C-terminus. If the peptide is to have a secondary amide group at the terminal carbon atom, a chloromethyl or oxymethyl resin is used and the cleavage is carried out with the appropriate amines. If, for example, ethylamide is produced, the resin can be cleaved from the resin with ethylamine; the side-chain protecting groups are resolved with other suitable reagents. If the peptide is to retain the amino-side chain protecting group tert-butyl, then the Fmoc group is used to block the Oc-amino group of the amino acid and the synthesis is carried out according to the procedure described for example in R.C. Sheppard, J. Chem. Soc., Chem. Commun., 1982, 587, and the arginine guanidine group is protected by protonation with pyridine perchlorate; other amino acid side chain functional groups are blocked by the use of benzylic groups which are subsequently cleaved by catalytic trans-hydride (A. Felix et al., J. Org Chem., 13, 4194 (1978) or by use of sodium in liquid ammonia (W. Roberts, J. Arner, Chem. Soc., 7, 6, 6203 (1954).

Once the protecting group which blocks the resin-linked amino acid amino group is removed by a suitable reagent such as trifluoroacetic acid in methylene chloride for the Boc protecting group, or 20% piperidine in dimethylformamide for the Fmoc protecting group, the other blocked amino acids are gradually coupled. The resulting intermediate N-blocked peptide resins are deprotected using the above reagents before being coupled with subsequent amino acid derivatives.

All possible activating reagents for peptide synthesis can be used as the coupling reagent (see, e.g., Houben-Weyl, Organic Chemistry Methods, Vol. 15/2), but carbodiimides such as N, N'-dicyclohexylcarbodiimide are particularly suitable. N, N'-diisopropylcarbodiimide or N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide. Coupling can be accomplished directly by coupling an amino acid derivative with an activating reagent and, if necessary, an additive that inhibits racemization such as, for example, 1-oxybenzotriazole (HOBt) (W. Konig, R. Geiger, Chem. Ber. 103, 708). (1970)) or

3-oxy-4-oxo-3,4-dihydrobenzotriazine (HOObt) (W. Konig, R. Geiger, Chem. Ber., 103, 2054 (1970)), to the resin, or it is possible to separately carry out the initial activation of an amino acid derivative, by converting it into a symmetric anhydride or an HOBt or HOObt ester and passing the solution of the activated derivative in a suitable solvent through a peptidine resin capable of binding to the derivative.

The coupling or activation of the amino acid derivatives with one of the above activating reagents can be carried out in dimethylformamide, N-methylpyrrolidone or methylene chloride, or in a mixture of the above solvents. Generally, an excess of 1.5 to 4 times the activated amino acid derivative is used. In the case of an incomplete coupling, the coupling reaction is repeated without blocking the α-amino group of the peptide resin, which is usually required for the subsequent amino acid attachment.

The success of the coupling reaction can be conveniently controlled by the ninhydrin reaction as described by E. Kaiser et al. al. Anna. Biochem., 34, 595 (1970). Synthesis can be accomplished automated, for example using a peptide synthesizer (Applied Biosystems, model 430;); in this case, either fusion programs provided by the device manufacturer or user-created applications may be used. The latter applications are particularly useful when amino acid derivatives are blocked by Fmoc groups.

After synthesizing the peptides as described above, they can be resolved with resin reagents such as, for example, liquid hydrofluoric acid (preferably for Boc-derived peptides) or trifluoroacetic acid (preferably for Fmoc-derived peptides). These reagents not only cleave the peptide from the resin but also other protecting groups on the side chains of the amino acid derivatives. Thus, using BHA and MBHA resins, a free acid form peptide is obtained. Cleavage with hydrofluoric acid or trifluoromethanesulfonic acid yields, in the case of BHA and MBHA resins, an amide peptide. Other methods for obtaining peptide anhydrides are described in German patent applications P 37 11 866.8 and P 37 43 620.1 Here, peptides are separated from the resin by the action of moderate acids commonly used in peptide synthesis (for example trifluoroacetic acid); cation trapping agents such as phenol, cresol, thiocresol, anisole, thioanisole, ethanethiol, aimethylsulfide, ethylmethylsulfide or the like, e.g. Conventional solid phase synthesis cation traps, whether single or mixed with two or more of these excipients. In addition, trifluoroacetic acid may be used and diluted with other suitable solvents, such as methylene chloride.

If it is necessary to preserve the tertiary-butyl or benzyl protecting groups of the peptide side chains, the separation from the specially modified resin carrier is carried out with 1% trifluoroacetic acid in methylene chloride, for example according to R.C. Sheppard J. Chem. Soc., Chem. Comm., 1982, 587. Where appropriate, a suitable combination of synthesis and resolving techniques may be used to protect the side chains of the tert-butyl or benzyl groups.

A modified resin carrier, as described by Sheppard, is also used for the synthesis of peptides having a C-terminal amide group or an amino group, respectively a co-guanidine alkyl group. The synthesized peptide, which is fully blocked by side chains, is cleaved from the resin and then treated with the corresponding amine,? * - aminoalkylamine or ω-guanidinoalkylamine according to classical synthesis in solutions; in addition, other functional groups may be blocked in known ways, if necessary.

Another method for the preparation of peptides having a > -aminoalkyl group is described in European Patent Λ 264 802.

The peptides of the present invention are preferably synthesized by solid phase synthesis using two universal tactics for protecting groups:

The synthesis is carried out using an automatic peptide synthesizer using Applied Biosystems 430 Model A, a temporary protection of the OC-amino group using Boc- (respectively rmoc-) protecting groups.

Using the Boc-protecting group, the synthesis is performed according to the synthesis cycles programmed by the instrument manufacturer.

Synthesis of peptides with a free carboxyl group at the C-terminus is accomplished using 4- (oxymethyl) -phenylacetamidomethyl-polystyrene resin functionalized with the appropriate Boc-amino acid (R.B. Merrifield,

J. Org. Chem., 43, 2845 (1978), manufactured by Applied Biosystems. MBHA-resin from the same company is used to obtain the peptidamide

The activating reagents used are N, N'-dicyclohexylcarbodiimide or N, N'-diisopropylcarbodiimide. Activation yields symmetric anhydride, HOBt-ester or HOObt-ester in methylene chloride, CF CFC ^-DMF or NMP. 2-4 equivalents of activated amino acid derivative are used for coupling. In cases where the merger is incomplete, the reaction is repeated.

If a Fmoc-protecting group is used to temporarily protect the Oc-amino group, a custom-made program is used to synthesize the Applied Biosystems 430 Model A automated peptide synthesizer. The synthesis is carried out using p-benzyloxybenzyl alcohol type resin (S. Wang, J. Am. Chem. Soc., 1973, 95, 1328), manufactured by Bachem; The resin is esterified with the corresponding amino acid according to known methods (E. Atherton et al., J. Chem. Soc., Chem.Comm. 1981, 336). Activation of the amino acid derivatives to obtain the HOBt- or HOObt-ester is accomplished directly by adding the diisopropylcarbodiimide solution in DMF to a weighed mixture of the amino acid derivative and HOBt or HOObt directly on the device. Alternatively, Fmoc-amino acid-OObt-esters prepared as described in European Patent A 247 573 may also be used as the material. The liberated Fmoc-protecting group is carried out using 20% piperidine in DMF). The excess of the reactive amino acid derivative used is 1.5-2.5 equivalents. If the merge is incomplete, it is repeated as in the Boc method.

The peptides of the invention (alone or in combination) exhibit bradykinin antagonistic activity which can be tested using a variety of models (see Handbook of Exp. Pharmacol., Vol. 25, Springer 1-la, 1970, pp. 53-55), e.g. isolated rat uterus, guinea pig colon, or marine; mumps isolated pulmonary artery.

For testing of the peptides of the invention using isolated Arteria pulmonalis, 400-450 g of guinea-pig (fcDunkin Har tley) (400 g to 450 g) are struck by the occiput, open the thorax and carefully remove Arteria pulmonalis. The surrounding tissue is carefully removed and the pulmonary artery is spirally incised at 45 ° '.

Pieces of blood vessels 2.5 cm long and 3-4 mm wide are fixed in a 10 ml bath for organs filled with Ringer's solution.

Solution composition (mmol / l):

NaCl 154 KCl 5.6

CaCl ₂ 1.9 NaKCO ₃ 2.4 glucose 5.0

The solution is purged with 95% O ₂ and 5% CO ₉ and heated to 37 ° C. pH is 7.4, primary load on vascular strip lg. Isotonic contraction changes are measured with a lever-type attachment and HF-Modem (length gauge) (Hugo Sachs firm) and recorded with a compensation recorder (BEC, Goerz Metrawatt SE 460).

After 1 hour for equilibrium, the test begins. After the vascular strips reach a maximum _7 sensitivity of 2x10 mol / L bradykinin (bradykinin cleaves vascular strips) 5x10 peptides can be added

- At a dose of 1x10 5 mol / l, maintained for 10 minutes and again with bradykinin, a comparable reduction in bradykinin exposure was observed compared to controls.

1x10 ^ -3 is used to determine the partial antagonistic effect

-1x10 mol / L peptide doses.

calculated from the dose-effect curves of the peptides of the invention. The values of θ are given in Table 1.

table

The compound

IC (M)

H- (D) -Arg-Arg-Pro-Hyp-Gly-Phe-Ser- (D) -Phe-Oic-ArgOH 1.4x10

The therapeutic use of the peptides of the invention encompasses all pathological states induced, maintained or assisted by bradykinin and bradykinin related peptides. Such conditions include, but are not limited to, injuries such as wounds, burns, rashes, erythema, edema, angina, arthritis, asthma, allergies, rhinitis, shock, inflammation, low blood pressure, pain, itching and sperm motility.

Therefore, the present invention is also directed to the use of peptides of formula I in medicaments and pharmaceutical preparations containing these compounds.

Pharmaceutical preparations contain an effective amount of a biologically active substance of formula I, either alone or in combination, in combination with an inorganic or organic pharmaceutically acceptable carrier (base).

The compounds can be used for parenteral administration, parenterally, for example, subcutaneously, intramuscularly, intravenously, sublingually, subcutaneously, intranasally, rectally, vaginally or by inhalation. The dose of the biologically active substance depends on the type of warm-blooded animal, body weight and age, and route of administration.

The pharmaceutical preparations of the present invention can be obtained by methods well known in the art, such as dissolving, mixing, granulating, or making a dragee.

ί Ώ J. u »

When formulated for oral administration, or for mucosal application, the active compounds are formulated with suitable excipients such as carriers, stabilizers or inert diluents and conventionally converted into suitable dosage forms such as tablets, dragees, disintegrating capsules; suspensions in water, alcohol, oil or solutions in water, alcohol or oil. Inert carriers which may be used are, for example, gum arabic, magnesium, magnesium carbonate, potassium phosphate, milk sugar, glucose, magnesium stearyl fumarate or starches, in particular maize starch. Preparation can be done by either dry or wet granulation. Oil or carrier oils or vegetable oils, such as sunflower oil and fish oil, for example, are acceptable as oil-type carriers or solvents.

The preparation for topical administration may be a solution in water or oil, lotion, emulsion or gel, ointment or grease, or aerosol, if appropriate, and may be added to the polymer to improve adhesion.

The form for nasal administration may be prepared by admixing with conventional additives for this purpose such as stabilizers or inert diluents and conventionally formulated for use as suspensions in water, alcohol or oil, or solutions in water, alcohol or oil. Aqueous compositions for nasal administration may also contain, as additives, components which form c'nelates, such as ethylenediamine-N, N, N ', N'-tetraacetic acid, citric acid, tartaric acid or their salts. Nasal drip solutions may be pipetted; nasal drops can also be in the form of high viscosity or gel and cream.

Nozzles or compressed gas devices using inert gas carriers may be used for inhalation.

Pharmaceutical formulations for intravenous, subcutaneous, intradermal or topical administration are prepared by dissolving, suspending or emulsifying the active compounds or their physiologically acceptable salts, if necessary in combination with other conventional pharmaceutical auxiliaries, such as, for example, solution isotonic or pH adjusting agents. as well as solvents, emulsifiers or other excipients.

Because of the short half-lives in the body fluids of some of the drugs described, prolonged injection formulations do not make sense. Such dosage forms may be, for example, oil-in-crystalline suspensions, microcapsules, rods or implants; the latter must be fabric-compatible. ^:. polymers, in particular biodegradable polymers such as copolymers of polylactic acid and polyglycolic acid or copolymers of polylactic acid and human albumin.

For topical and inhalation dosage forms, 0.01-5 mg / ml solutions are suitable, for systemic dosage forms 0.01-10 mg / kg.

List of abbreviations:

The amino acid abbreviations used correspond to the three letter code accepted by peptide chemistry as described in Sur. J. Biochem.,

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

ACm - acetamidomethyl? -Ahx -? -Aminohexanoyl

Aoc - cis, endo-2-azabicyclo (3.3.0) -octane-3-S-carbonyl

Boc - tert-butyloxycarbonyl Being - tert.-butyl Bzl - benzyl CDF - chloro- (D) -phenylalanyl Cha - eikiohexylalanil Chg - cyclohexylglycyl Cl-Z - 4-chloro-benzyloxycarbonyl DMF - dimethylformamide DOMT - O-methyl-1- (D) -threonyl Dnp - 2,4-Dinitrophenyl Fmoc - 9-fluorenylmethyloxycarbonyl MDV - M e O-Methyl- (D) -tyrosyl methyl

4-Kebzl -4-methylbenzyl

Mtr - 4-methoxy-2,3,6-trimethylphenylsulfonyl Mts - mesitylene-2-sulfonyl NaS. - naphthalenyl NMP - N-methylpyrrolidine Npg - neopentylglycyl Oic - cis-endo-octahydroindole-2-carbonyl Opr - iscoxazolidin-3-ylcarbonyl Pal - pyridylalanil Pmc - 2,2,5,7,8-pentameth. lchroman-6-su lfonyl Tbg - tert.-beylglycyl TFA - trifluoroacetic acid Tos - 4-methylphenylsulfonyl Thia - 2-way road glass Tie - 1,2,3,4-Tetrahydroisoquinolin-3-ylcarbonyl Trt - trityl

The following examples illustrate the preferred methods for solid phase synthesis of the peptides of the invention; the scope of the invention is not limited thereto.

The following amino acid derivatives are 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-Ssr (tBu) -OObt, Fmoc-(D) -Phe-OH, Fmoc-Gln-OH, Fmoc-Aoc-OH _Z Fmoc-Thia-OH, Fmoc-Opr-OH, Fmoc- (D ) -Asn-OH, Fmoc-B-Ala-OH,

Fmoc-Oic-OH.

example

H- (D) -Arg-Arg-Prp-Hyp-Gly-Phe-Ser- (D) -Phe-Oic-Arg-OH is synthesized using a Fmoc method on a Model 430A peptide synthesizer from Applied Biosystems using the method of Novabiochem. Benzyloxybenzyl alcohol-based resin, esterified with Fmoc-Arg (Mtr) -OH (loading about 0.5 mmol / g resin). 1 g of resin is used and synthesis is carried out according to a modified Fmoc-method synthesis program.

The synthesis cartridge is charged, as appropriate, with 1 mmol of the free carboxylic acid derivative along with 0.95 mmol of HOObt. Pre-activation of these amino acids is done directly in the cartridge by dissolving in 4 mL of DMF and adding 2 mL of 0.55 M diisopropylcarbodiimide solution in DMF.

The HOObt-esters of the other amino acids are dissolved in 6 mL of NMP and then all the pre-activated amino acids in situ are combined with 20% piperidine in DMF-de-blocked resin. Upon completion of the synthesis, the peptide is cleaved using the trifluoroacetic acid, and the thioanisole and the ethanediol as cation traps, together with the removal of the protecting groups from the side chains. After removal of trifluoroacetic acid, the residue is repeatedly triturated with acetic acid at the ester and centrifuged. The remaining residue is chromatographed over (Dsefadeks LH 20, eluting with 10% acetic acid). The fractions containing the pure peptide are mixed and freeze-dried.

MS (FAB): 1292.4 (M + H).

Claims (7)

DEFINITION OF INVENTION 1. A peptide of formula I: A-B-C-E-F-K- (D) -Phe-G - M-F '- I wherein A hydrogen, (C₁-C₆) -alkyl, (C₁-C₆) alkanoyl, (C.-C _o) -alkoxycarbonyl or (C, -C _Q) alkylsulfonyl which se, if necessary, one, two, or three hydrogen atoms may be substituted with one, two or three identical or different residues, such as: carboxyl, amino, (C 1 -C 4) -alkyl, (C 1 -C 1 _A ) -alkylamino, hydroxyl, (C , -C _A ) '1 4 -alkoxy, halogen, di- (C ₁ -C ₄ ) -alkylamino, carbamoyl, sulfamoyl, (C ₁ -C ₄ ) -alkoxycarbonyl, (C ₈ -C ₄ ) -aryl and (-C 1 ') - aryl- ( C 1 -C 1 -) alkyl; or wherein, when required, one hydrogen atom may be replaced by a residue such as: (C 8 -C 8) -cycloalkyl, (C 1 -C 4) -alkylsulfonyl, (C 1 -C 1) -alkylsulfinyl, (C, -C,) - aryl- (C _n -C,) alkylsulfonyl ^(C 6 ^-C i2) (C₁-C₄) - alkylsulfinyl, ^(C 5 ^_c j2 ^{or cks} yl i9 ^{ru Pa} r (Cg-C _o) heteroaryl and (C ~ -C _g) -heteroariloksigrupė, and one or two hydrogen atoms have been replaced with one or two public nodom Arua two different moieties such as carboxyl, amino, (C₁-C₄) -alkilaminogrupė, hydroxy, (C? -C? -alkoxy, halogen, di- (C₁-C₄) - alkylamino, carbamoyl, sulfamoyl, (C, -C.) - alkoxycarbonyl, (C _r C, ") -aryl and 14 6 12 ^(C 6 ^{_c _ari} l i ^(c i ^c ~ 5) alkyl; a _n): (C _O -C _Q) -cycloalkyl; carbamoyl, which may be substituted with (C 1 -C 8) -alkyl or (C ₈ -C ₃₂ ) -aryl at the nitrogen atom; (C, -C ₁₂₎ -aryl, (C ^ -C ^ g) -ariloilas ^{(c _c} i5 ^ j2 ^- -arylsulfonyl, (C 1 -C 8) -heteroaryl or (C 4 -C 4) -heteroaryloyl in addition to the radicals indicated in a ^) and a ₂ ) pp, such as aryl, heteroaryl, aroyl, arylsulfonyl and heteroaryl, optionally be one, two, three or four identical or different substituents such as: carboxyl, amino, nitro, (C 1 -C 4) -alkylamino, hydroxy, (C 1 -C 4) -alkyl, (C 1 -C 4) -alkyl ) alkoxy, halogen, cyano, di- (C 1 -C 4) -alkylamino, carbamoyl, sulfamoyl and (C 1 -C 1) - alkoxycarbonyl, or a (4)): a residue of formula II: R - N - CH - C! I R ² r ³ (II), .1 in which R is as A under (a), (a) and (a) ( ₂ ), R is hydrogen or methyl, R is hydrogen or (C 1 -C 8) -alkyl, preferably C 1 -C 6 -alkyl, which may optionally be substituted with one substituent such as amino, substituted amino, hydroxy, carboxyl, carbamoyl, guanidine , substituted guanidine group, ureido group, mercaptogroup, methylmercaptogroup, phenyl, 4-chlorophenyl, 4-fluorophenyl, 4-nitrophenyl, 4-methoxyphenyl, 4-oxyphenyl, phthalimido group, 4-imidazolyl, 3-indolyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl or cyclohexyl, wherein the substituted amino group is -NH-A and the substituted guanidine group is -NH-C (NH) -NH-A, wherein A is the same as in a ^) and a ₂ ); B is an amino acid of the L- or D-configuration which may be substituted in the side chain; C are groups of formulas IIIa or IIIb: G '- G' - Gly G '- NH - (CH) _n - CO (IIIa) (IIIb) wherein G', independently of one another, represents a group of formula IV: - N - CH - C '4 ¹ 5 »(IV), R R 0 where 4 5. . ... R and R together with the atoms to which they are attached form a heterocyclic mono-, bi- or tricyclic system with 2 to 15 C atoms and n = 2 to 8; E is an aromatic amino acid residue; F is, independently of one another, a neutral, acidic or basic, aliphatic or aromatic amino acid residue which may be substituted in the side chain; or is a bond; (D) -Phe is (D) -phenylalanine which may be optionally substituted on the phenyl moiety; G is a bond, or has the same meanings as G '; F 'is the same as F, is a -NH (CH ₂ ) - moiety where n = = 2-8, or if C is not a bond, it may be a bond; I is -OH, -NH ₂ or -NHC ₂ H ₂ ; K is a residue of -NH- (CH ₂ ) -CO- where x = 1-4, or is a bond and M has the same meanings as F; and a process for preparing the physiologically acceptable salts of these compounds, characterized in that (a) the moiety with the free carboxyl group at the C-terminus or its activated derivative is exposed to the free amino group at the N-terminus, or b) synthesizes with a peptide. gradually separating from the compound obtained according to (a) or (b), optionally, one or more. other functional groups introduced under the protection nrnnl .. and thereby convert the compounds of the formula into their physiologically acceptable salts, if necessary. 2. A process according to claim 1 wherein the peptide of formula I wherein: B = Arg, Lys, Orn, 2,4-diaminobutyryl or 1-homoarginine residue, and additionally, if necessary, the side chain amino or guanidine groups may have a group A substituent as defined in claim 1 of the definition. in a ^ and a ^) parts; E = an aromatic amino acid residue of the L- or D-configuration having an aryl moiety of 6-14 C atoms in the ring as phenylalanine which may be optionally substituted at the 2-, 3- or 4-position; tyrosine, O-methyl tyrosine, 2-thienylalanine, 2-pyridylalanine or naphthalene; F '= a basic amino acid residue of the L- or D-configuration, such as Arg or Lys, wherein the guanidine group or the amino group, respectively, may be substituted by A as described in claim 1. a ^) either in moieties or -NH- (CH ₂ ) - moiety wherein n = 2-8; K = -NH- (CH2) -CO- moiety where x = 2-4, or the bond (D) -Phe is D-phenylalanine, the phenyl moiety of which may optionally be halo or (C1-C4) ) -alkoxy. 3. A process according to claim 1 or 2, wherein the peptide of formula I is obtained in which; B = Arg, Orn or Lys, wherein the guanidine group or the side chain amino group is unsubstituted or may be substituted such as (C 1 -C 8) -alkanoyl, (C 1 -C 8) -arylaryl, (C 8 -C 8) -heteroaryl, (C 8 -C 8) -alkylsulfonyl or (C 1 -C 6) -arylsulfonyl wherein the aryl, heteroaryl, aroyl, arylsulfonyl and heteroaryloyl residues as described in (a) may optionally be one, two, three and four identical. or different substituents; E = phenylalanine, 2-chlorophenylalanine, 3-chlorophenylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, tyrosine, O-methyl tyrosine or β- (2-thienyl) -alanine; K is a bond; M = bond and (D) -Phe = D - phenylalanine which may be optionally substituted, such as chlorine, bromine or methoxy. 4. Gut according to any one of claims 1-3. or a process according to any one of claims 1 to 3, characterized in that it obtains a peptide of formula I in which: C A is hydrogen, (D) - or (L) -H-Arg, (D) - or (L) -H-Lys or (D) - or (L) -H-Orn; . B = Arg, Orn or Lys, wherein the guanidine group, or the side chain amino group, respectively, may have substituents such as hydrogen, (C 1 -C 8) alkanoyl, (C 1 -C 6 g) -aryl, (C ₈ -C ₀₎ ) -heteroariloilas (C.-C _o) alkylsulfonyl or (C, -C. ~) sulfonyl -arilsuli ob 12, wherein the aryl, heteroaryl, aryloyl, arylsulfonyl and heteroariloilo moiety may have one, two, three or four identical or different, substituents such as methyl, methoxy and halogen; C - Pro-Pro-Gly; Hyp-Pro-Gly or Pro-Hyp-Gly; E - Phe or Thia; F - Ser, Hser, Lys, Leu, Vai, Nle, Ile and Thr; K = coupling; M = connector; G = a residue of a heterocyclic system of formula IV, with the following heterocycles being most preferred: pyrrolidine-2-carbonic acid; piperidine-2-carbonic acid; 1,2,3,4-Tetrahydro28 isoquinoline-3-carbonic acid, eis and trans -decahydroisoquinoline-3-carbonic acid; cis-endo-, cis-exo-, trans-octahydrocyclopentane (b) -pyryl-2-carbonic acid or oxyproline-2-carbonic acid; F = A-rg; I = OH and (D) -Phe = D-phenylalanine. A peptide of formula I according to any one or more of claims 1 to 4 for use as a medicament. A peptide of formula I according to any one or more of claims 1 to 4 for use in the treatment of pathological conditions transmitted, maintained or induced by bradykinin and bradykinin-using peptides. 7. A pharmaceutical composition comprising a peptide of the formula I obtained according to claims 1-4 together with a physiologically compatible carrier and, if necessary, other excipients and additives.