BG64386B1 - New lh-rh antagonists with improved effectiveness - Google Patents

Abstract

The invention relates to a compound with a general formula Ac-D-Nal(2)1-D(pCl)Phe2-D-Pal(3)3-Ser4-Tyr5-D-Xxx6-Leu7-Arg8-Pro9-D-Ala10-NH2 wherein the meanings of the substituents are disclosed in the description, to a method for their preparation and usage for the preparation of medicamentous forms for the treatment of hormone-independent tumours, especially carcinoma of the prostate gland or cancer of the breast, as well as non-malignant indications, the treatment of which demands LH-RH hormone suppression.

Description

Technical field

The invention relates to novel LHRH antagonists, in particular peptidomimetics and side chain modified peptides, to their salts with pharmaceutically acceptable acids, and to a method for preparing LH-RH antagonists and their salts. The peptides of the invention are analogues of the luteinizing hormone releasing hormone (LH-RH-), with the following structure p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-ProGly-NH ₂ , [LH-RH, Gonadorelin ]

BACKGROUND OF THE INVENTION

For more than 20 years, researchers have been searching for selective potent antagonists of LH-RH-decapeptides [M. Karten, J.E. Rivier, Endocrine Rewiews 7, 44-66 (1986)]. The great interest in such antagonists is based on their utility in the field of endocrinology, gynecology, pregnancy prevention and cancer. A number of compounds have been obtained as potential LH-RH antagonists. The most interesting compounds that have been discovered so far are those whose structure is a modification of the LHRH structure.

The first series of potent antagonists was obtained by introducing aromatic amino acid radicals at positions 1, 2, 3 and 6 or 2, 3 and 6. The usual way of describing the compounds is as follows: first, the amino acids that are listed in the peptide chain of the LH-RH are inserted at the site of the amino acids originally present, where the substitution sites are indicated by numbers affixed above. Further, by the designation “LH-RH” after them, it is stated that this is an LHRH analogue to which a replacement has been made.

Known antagonists are:

[Ac-D-Phe (4-Cl) ¹ · ² , D-Trp ³ · ⁶ ] LH-RH (DH Coy et al., In: Gross, E. and Meienhofer, J. (Eds) Peptides; Proceedings of the 6 ^{th American Peptid Symposium, p.775-779),} Pierce Chem.Co., Rockville III ( 1979);

[Ac-Pro ', D-Phe (4-Cl) ² , D-Nal (2) ³ · ⁶ ] LHRH (US 4,419,347) and [Ac-Pro', D-Phe (4Cl) ² , D-Trp ³ · ⁶ ] LH-RH (JL Pineda et al., J. Clin. Endocrinol. Metab, 56, 420, 1983).

To increase the water solubility of the antagonists, basic amino acids, for example D-Arg, were introduced at position 6. For example, [Ac-D-Phe (4-Cl) '· ² , D-Trp ³ , DArg ⁶ , D-Ala ¹⁰ ] LH-RH (ORG-30276) (DH Coy et al., Endocrinology 100, 1445,1982); and [Ac-D-Nal (2) ', D-Phe (4-F) ² D-Trp ³ , DArg ⁶ ] LH-RH (ORF 18260) (JE Rivier et al., in: Vickery V. H. Nestor, Jr. JJ, Hafez, ESE (Eds. LH-RH and its Analogs, pp. 11-22 MTP Press, Lancaster, UK 1984).

Such analogs not only show the expected improved solubility in water, but also show improved antagonistic activity. These externally potent hydrophilic analogues with D-Arg ⁶ and other basic side chains at the 6th site cause transient facial and limb edema when administered to rats at doses of 1.25 or 1.5 mg / kg subcutaneously. (F. Schmidt et al., Contraception 29, 283, 1984; JEMorgan et al., Archs. Allergy Appl. Immun.80, 70 (1986). Other potent LH-RH antagonists are described in WO 1992/019651, WO 1994 / 019370, WO 1992/017025, WO 1994/014841, WO 1994/13313, US-A 5,300,492, USA 5,140,009 and EP O 413 209 Al.

The occurrence of edematogenic effects after administration of some of these antagonists to rats raises doubts about their safety in human administration and thus delays the administration of drugs to clinical use. Therefore, there is a great need for peptide antagonists that have no side effects.

SUMMARY OF THE INVENTION

The problem is solved according to the invention with compounds of general formula (V):

Ac-D-Nal / 2 / '- D / pCl / Phe ² -D-Pal / C / ³ Ser ⁴ - Tyr ³ -D-Xxx ⁶ - Leu ⁷ -Arg * -Pro' -D-Ala '° NH ₂ / V / in which D-Xxx means an amino acid group of the general formula / VI /

NH-CH-COI / CH2 Cl

And NH

I CO - R * / VI / in which n denotes an integer from 1 to 4, R ⁴ denotes a group of general formula II

O II / CH ^ --N-R *

IR * (II) where p is an integer from 1 to 4, R ⁵ is a hydrogen atom or a lower alkali group and R ⁶ is an unsubstituted or substituted phenyl group, or R ⁴ is a ring of general formula III

R ⁷ /

wherein q is an integer of 1 to 2, R ⁷ represents a hydrogen atom or a lower alkyl group, R ⁸ represents a hydrogen atom or a lower alkyl group and X represents an oxygen atom or a sulfur atom, and their pharmaceutically acceptable salts. Preferred alkyl groups are methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl. The compounds according to the invention exhibit high antagonistic activity and are devoid of undesirable side effects, in particular having no edematogenic properties. If they are in the form of salts with poorly soluble pharmaceutically acceptable acids in water, they also show improved water solubility. In addition, the compounds have high affinity for the human LH-RH receptor, i.e. with high suppression of gonadotrophin release from the pituitary gland in mammals, including humans, exhibit long-term suppression of testosterone in rats and cause minimal histamine release in vitro. Preferred peptides of formula (ν) xxx [ε-Ν'-4- (4-amidino-phenyl) -amino-1,4-dioxo-butyl] -lysyl group or [ε-Ν ^1- yl) -formyl] -Lysyl group. Pharmaceutically acceptable acid salts are preferably difficult to dissolve in water. Salts of 4,4'-methylene-bis (3-hydroxy-2-naphthoic acid), also of emboic acid or pamoic acid, are particularly preferred.

The nomenclature attached to the definition of the peptides is in accordance with that clarified by the IUPAC-IUB Committee on the Biochemical Nomenclature / Europen J. Biochem. 1984,138,9-37 /, where, in accordance with the usual representation, the amino groups at the N-terminus appear to the left and the carboxyl groups at the extremity appear to the right. LH-RH antagonists such as peptides of the invention and peptidomimetics comprise naturally occurring synthetic amino acids, the former including Ala, Vai, Leu, lie, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gin, Cys, Met, Phe, Tyr, Pro, Trp and His. The abbreviations for the individual amino acid residues are based on the trivial names of the amino acids and are A1a = alanine, Arg = arginine, Gly = glycine, Eee = leucine, Lys = nH3HH, Pa1 / 3 / = 3- (3pyridyl / 1) alanine, respectively. 2) = 3- (2-naphthyl) alanine, Phe = phenylalanine, (pCl) Phe = 4-chlorophenylalanine, Pr = proline, Ser = serine, Thr = threonine, Trp = tryptophan, and Th = tyrosine. All amino acids described herein are derived from the L series unless otherwise stated. For example, D-Nal (2) is the abbreviation for 3- (2-naphthyl) - D-alanine and Ser is the abbreviation for Lserine. Other abbreviations used are:

Boc = tert-butyloxycarbonyl

Thor = benzotriazol-1-yl-oxy-tris- (dimethylamino) phosphonium hexafluorophosphate

DCC = dicyclohexylcarbodiimide DCM = dichloromethane

Ddz = dimethoxyphenyl-dimethylmethylenoxy-carbonyl (dimethoxydimethyl-Z)

DIC = diisopropylcarbodiimide DIPEA = Ν, Ν-diisopropylethylamine DMF = dimethylformamide

Fmoc = fluorenylmethyloxycarbonyl HF = liquid, anhydrous hydrofluoric acid

HPLC = high performance liquid chromatography

PyBop = benzotriazol-1-yl-oxy-trispyrrolidino-phosphonium hexafluorophosphate

TFA = trifluoroacetic acid

Z = benzyloxycarbonyl.

According to the invention, compounds of general formula V are prepared such that the first two of the three functions (α-amino group, ε-amino group and α-carboxyl group) are protected by protecting groups and then the free third function is suitably transformed. In one case, intermediate protecting groups may also be introduced in the first step, which in turn leads to better results, which are then replaced by the desired functions in the second stage. Suitable protecting groups and methods for introducing them are known in the art. Examples of protecting groups are described in "Principles of Peptide Synthesis" (Springer Verlag, 1984), in the textbook "Solid Phase Peptide Synthesis" by J.M. Steward and J.D.Young, Pierce Vhem. Company, Rockford, III, 1984, and in G. Barany and R.B. Merrifield “The Peptides,” Ch.l, p. 1-285, 1979, Academic Press Ins. In the process of preparing a compound of the general formula (V) according to the invention, three alternatives can be used. The first option covers the stages of:

(a) protecting the A-amino group and the carboxyl group of D-lysine or D-ornithine with suitable protecting groups / b) reacting the protected with the protecting groups Dlysine or D-ornithine with a carboxylic acid of general formula (VII):

R ⁴ is COOH (VII) where R ⁴ has the above meanings; (c) cleavage of the protecting group at the acarboxylic group of the compound obtained in step (b) for incorporation into position 6 at step (h), / d (coupling the D-alanine protected by the amino group protecting group to a solid carrier in the form of a resin / Merrifield synthesis), / e) cleaving the protecting group at the alanine amino group, (f) reacting the alanine-bound with the solid carrier proline which is protected by a protecting group at the nitrogen atom, (g) cleavage of the protecting group at the nitrogen atom at prolys of, (h) repeating steps f) and g) with the amino acids 1 to 8 of the general formula (V) in sequence from 8 to 1 using the modified D-lysine or D-ornithine described in step (c) position 6, (i) cleavage of the compound obtained in step (h) from the carrier and optionally purification (e.g. HPLC), (i) optionally reacting with a pharmaceutically acceptable acid, preferably emboic acid.

According to the second alternative, the method of preparing a compound of general formula (V) comprises the steps of (a) coupling a protected group at the amino group D-alanine with a suitable solid-phase carrier, (b) cleaving the protecting group at the amino group alanine, (c) interaction of the resin-bound alanine with proline protected by a protecting group at the nitrogen atom, (d) cleavage of the protecting group at the proline nitrogen atom, (e) repeating steps c) and d) with amino acids 1 to 8 of general formula (V), in sequence from 8 to 1, (f) (g) reacting with a carboxylic acid of formula (VII)

R ⁴ is COOH (VII) in which P ⁴ is defined above, (h) optionally reacting with a pharmaceutically acceptable acid, preferably emboic acid.

The third embodiment of the process for preparing a compound of general formula (V) comprises the steps of (a) coupling an amino-protected group with a D-alanine protecting group to a carrier suitable for solid phase synthesis, (b) cleaving the protecting group at the amino group of alanine, (c) interaction of the resin-bound alanine with proline having a protecting group at the nitrogen atom, (d) cleavage of the protecting group at the nitrogen of the proline atom, (e) repeating steps c) and d) with the amino acids 6 to 8 of general formula (V), in sequence from 8 to 6, (f) cleavage of the ε-amino Protecting group of D-lysine or D ornithine in position 6 and reacting with a carboxylic acid of formula (VII)

R ⁴ is COOH (VII) in which R ⁴ is defined above, (g) cleavage of the protecting group at the α-amino group of D-lysine or D-ornithine, (h) repeating steps c) and d) with the amino acids 1 to 5 of general formula (IV), in sequence from 5 to 1, (i) cleavage of the compound obtained in step (h) from the resin and purification (eg by HPLC), (i) optionally reacting with a pharmaceutical acceptable acid, preferably emboic acid.

Preferred carboxylic acids of formula (VII) are imidazolidin-2-one-4-carboxylic acid and N- (4-amidinophenyl) amino4-oxobutyric acid.

The compounds of formula (V) are synthesized by known methods, such as by pure solid-phase technique, partially solid-phase technique or by classical binding in solutions (see M. Bodanszky, “Principles of Peptide Synthesis”, Springer Verlag, 1984). The methods for solid phase synthesis are described, for example, in the textbook Solid Phase Peptide Synthesis, J. M. Steward and J. M. Steward and J. D. Young, Pierce Chem. Company, Rockford, III, 1984 and in G. Barany and R. B. Merrifield ”The Peptides” Ch.l, p. 1-285, 1979, Academic Press Inc. The classical syntheses in solutions are described in detail in the tract "Methoden der Organischen Chemie (Houben-Weyl), Synthese von Peptiden" E.Wunsch (Herausgeber) 1974, Georg Thieme Verlag, Stuttgart, BRD.

The gradual construction is carried out, for example, by first linking the amino acid covalently at the carboxy terminus, whose disposed amino group is protected, with a customary insoluble carrier, and the amino-protecting group of this acid cleaves, to the resulting free amino acid group by means of its carboxyl group, and thus step by step linking together the remaining amino acids of the peptide to be synthesized. After binding of all amino acids, the finished peptide is cleaved from the carrier and optionally cleaved by the protecting groups of others on personal side functional groups. The gradual condensation is accomplished by the synthesis of the corresponding normally protected amino acids in a conventional manner. It is also possible to use an automatic peptide synthesizer, such as the Labortec SP 650 type from Bachem, Switzerland, when applying commercially available protected amino acids.

The binding of the individual amino acids to one another is carried out according to conventional methods, in particular taking into account:

- symmetric anhydride methods in the presence of dicyclohexylcarbodiimide or diisopropylcarbodiimide (DCC, DIC), - carbodiimide methods in general,

- carbodiimide-hydroxybenzotriazole methods (see The Peptids, volume 2, Ed. E. Gross and J. Meienhofer). A predominantly carbodiimide method is used to bind arginine. For the remaining amino acids, methods with symmetric or mixed anhydrides are generally used.

Preferably, the non-racemic azide coupling or DCC-1-hydroxybenzotriazole flowing, respectively, DCC-3-hydroxy4-oxo-3,4-dihydro-1,2,3-benzotriazine method is used in the fragment binding. Activated fragment esters may also be used.

Well-activated esters of N-protected amino acids, such as N-hydroxy-succinimidoester or 2,4,5-trichlorophenyl ester, are particularly suitable for the gradual condensation of amino acids. Aminolysis is very well catalyzed by N-hydroxyl compounds, which to some extent have the acidity of acetic acid, such as 1-hydroxybenzotriazole.

Dehydrogenating groups such as benzyloxycarbonyl moiety (= Z-ocTarbk) or leaving in slightly acidic groups are used as intermediate amino protecting groups. Protective groups for the disposed amino groups include: tert-butoxycarbonyl groups, carbobenzoxy groups, respectively carbobenzthio groups (optionally also with p-bromo or p-nitrobenzyl moiety), trifluoroacetyl group, phthalyl moiety, o-nitrophenyl group, toluenesulfonyl group, benzyl group, benzoyl residues substituted in the benzene nucleus (p-bromo or p-nitrobenzyl residue) and α-phenylethyl residue. The book of Jesse P. Greenstein and Milton Winitz, Chemistry of Amino Acids, New York 1961, John Wiley and Sons, Ins, is also recommended for this purpose. Volume 2, for example, page 883 et seq., And The Peptides, Volume 2, Ed. E.Gross and J. Meienhofer, Academic Press, New York. These protecting groups can in principle also be considered for protecting other functional side groups (OH groups, NH1 groups) of the corresponding amino acids.

Available hydroxy groups (serine, threonine) are protected mainly by benzyl and similar groups.

Available hydroxy groups (serine, threonine) are protected mainly by benzyl groups and the like. Other non-α-positioned amino groups (e.g. amino groups at the ω position, guanidine group of arginine) are protected primarily orthogonally.

The amino acid coupling reaction is carried out in a known indifferent solvent or suspending agent (eg dichloromethane), wherein dimethylformamide is added optionally to improve solubility.

For the introduction of R ⁴ -CO-pyrene by the interaction of the carboxylic acid lysine amino group of the general formula (VII), the same methods as those described above for amino acid binding are generally considered. Particularly preferred, however, is condensation using carbodiimide, for example 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 1-hydroxybenzotriazole.

As a synthetic carrier, insoluble polymers, for example beads swellable in organic solvents in the form of pearls (for example copolymerized from polystyrene and 1% divinylbenzene), are considered. The construction of a protective decapeptidamide on methylbenzhydrylamide 20 resin (MBHA-resin, i.e. provided with methylbenzhydrylamide groups of polystyrene resin), on which the desired C-terminal amide function of the peptide is obtained after HF-cleavage from the carrier, but can be carried out25 the following diagram:

Flowchart

Leptide Synthesis Protocol

Stage Action Solvent / Reagent (v / v) Time 1 Washing Methanol 2x2 min 2 Washing □ SEE 3x3 min 3 Cleavage DCM / TFA (1: 1) 1 x 30 min 4 Washed Ijaopropanol 2x2 min 5 Washing Methanol 2x2 min 6 Washing DCM 2x3 min 7 Neutralization DCM / DIPEA (8 1) 3x5 min IN Washing Methanol S; x 2 min 9 Washing DCM 3x3 min 10 STOP Add Boc-Ab in DCM + D1C + HOB1 11 Connectivity - ca.90 min 12 Washing Methanol 3x2 min 13 Washing DCM 2x3 min

The β-α-Boc-protected amino acids were combined in triple molar excess in the presence of diisopropyl-carbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in CH ₂ C1 ₂ / DMF for 90 min and the Boc-protecting group was cleaved by half-hour exposure to 50% trifluoroacetic acid (TFA) in CH ₂ Cl ₂ Chloraniline test may be used to control complete conversion

Christensen and the Kaiser ninhydrin test. Free amino acid residues are blocked by acetylation in five times the excess of acetylimidazole in CH ₂ Cl ₂ .

The sequence of the steps of the reaction of peptide formation on the resin can be seen in the process diagram. For cleavage of the resin-bound peptide, the final product of the solid phase synthesis was dried in vacuo over P ₂ O ₅ and treated in a 500 fold excess of HF / anisole 10: 1 / v: v, 60 min at 0 C.

After distillation of HF and anisole in vacuo, the peptidamides precipitate under stirring with anhydrous ethyl ether as a white solid, the removal of the precipitated polymeric carrier is also carried out by washing with 50% aqueous acetic acid. Concentration under mild conditions of the acetic acid solution in vacuo afforded the desired peptides as a high-viscosity oil which was converted to a white solid by addition of absolute ether in the cold.

Further purification is carried out by routine preparative high performance liquid chromatography (HPLC) methods.

The conversion of the peptide to its acid addition salts can be accomplished by reacting it with acids in a known manner. In contrast, free peptides can be prepared by reacting their acid addition salt with a base. Peptidembonates can be prepared by reacting salts of the peptide with trifluoroacetic acid (TFA salts) with free emboic acid (pamoic acid) or from the corresponding disodium salt of emboic acid. To this end, the peptide TFA salt in aqueous solution is reacted with a solution of disodium embonate in a dipolar aperture medium, preferably dimethylacetamide, and the resulting light yellow precipitate is isolated.

Examples of carrying out the invention

The following examples illustrate the invention without limiting it.

Example 1.

Ac-D-Nal (2) -D- (pCl) Phe-D-Pal (3) -SerTug-D- [-Ν'-ε (imidazolidin-2-one-4-yl) -full] -Lys- Leu-Arg-Pro-D-Ala-NH ₂ The synthesis was carried out according to the process diagram on 5 g mBHA-resin (loading density 1.08 mmol / g). Lysine binds as Fmoc-D-Lys (Boc) -OH and after cleavage of the Vosgroup in the side chain is acetylated with imidazoline-2-one-4-carboxylic acid in 3x excess. After cleavage of the Fmoc protecting group with 20% piperidine / DMF, it is extended to the N-terminus according to the process diagram. After cleavage of the polymer carrier, 5.2 g of polypeptide were purified, which was purified by standard preparative HPLC method. Subsequent lyophilisation yielded a single HPLC product of the total formula C ₇₄ H ₉₇ N ₁ O ₁₀ Cl with correct FAB-M5 1514 (M + H ⁺ ) (ref 1512.7) and its corresponding NMR spectrum. Ή NMR (500 MHz, DMSO-d6, δ in ppm): 8.56 m, 2H, arom. Η; 8.08 m, 1H, arom. H; 7.81 m, 1H, arom. H .; 7.73 m, 2H, arom. H; 7.66 m, 1H, arom. H; 7.60 d, 1H, arom. H; 7.44 m, 2H, arom. H; 7.30 d, 1H, arom. H; 7.25 and 7.18 2d, 2x2H, arom. H p-ClPhe; 6.97 and 6.60 2d, 2x 2H, arom. H Tyr; 9,26,3, multiple signals, amide-NH; 4.8-4.0 several m, Ca-H and aliph. H; 2,1-1,1 several tons, Ost. alif. H; 1.70, s, 3H, acetyl; 1.22, d, 3H, CP-H Ala; 0.85, dd, 6H, Sat-H Leu.

Example 2

Ac-D-Nal (2) -D- (pCl) Phe-D-Pal (3) -SerTyr-D- [ε-Ν '-4- (4-amidinophenyl) amino-1,4-dioxo-butyl] -Lys-Leu-Arg-Pro-D-Ala-NH ₂

0.7mmol (1.03 g) decapeptide Ac-D-NalD- (pCl) Phe-D-Pal-Ser-Tyr-D-Lys-Leu-Arg-Pro-DAla-NH ₂ react with 1.0 mmol ( 0.27 g) (4-amidinophenyl) amino-4-oxobutyric acid in the presence of 1.0 mmol (0.16 g) 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide and 1.0 mmol (0.16 g) ) 1-hydroxybenzotriazole in freshly distilled DMF. After 24 h, the solvent was removed in vacuo, the residue dissolved in water and lyophilized. The crude reaction product obtained (1.63 g) was purified by preparative reverse phase HPLC; a total of 0.61 g of HPLC-single product of the total formula C _gl H ₁₀₄ N ₁₉ O ₁₅ Cl with correct FAB-MS is obtained: 1618.7 (M + H ⁺ ) (ref. 1617.7) and the corresponding NMR- spectrum.

Ή NMR (500 MHz, DMSO-d6, δ ppm): 10.4, s, 1H and 9.15, s, 2H, and 8.8, s, 1H, NHs of 4-amidinoaniline; 8.60, m, 2H, arom.H; 8.20, m, 1H, arom.H; 7.80, m, 1H, arom.H; 7.73, m, arom.H; 7.61, s, 1H, arom.H; 7.44, m, 2H, arom.H; 7.30, d, 1H, arom.H; 7.25 and 7.20, 2d, 4H, arom. H (pCl) Phe; 7.0 and 6.6, 2d, 4H, arom.H Tyr; 8.3-7.2 several signals, amide NH; 4,734.2 several multiplets, Ca-H; 4.13, m, 1H, Ca-H; Ala; 3.78-2.4, several multiplets, CPH and aliph. H; 1.72, s, 3H, acetyl; 1.22, d, 3H, CP Ala; 0.85, 66, 6H, Sat Leu.

Example 3.

0.5 g (0.3 mmol) of the peptide LH-RH antagonist according to Example 1 dissolved in 50 ml of H ₂ O was converted by reaction with 0.130 g (0.3 mmol) of the disodium salt of the emboic acid in 2 ml of aqueous solution in peptide-embonate, which is rapidly removed from the solution as a yellow precipitate. 0.281 g of a fine crystalline yellow-green powder are obtained, with an emboic acid content of 33%.

Example 4.

0.3 /0.17 mmol / peptide LH-RH - ANC Scheme 1 sample according to Example 2 dissolved in 5 ml of dimethylacetamide is converted by reaction with 0.195 g / 0.45 mmol / disodium salt of emboic acid in 2 ml of aqueous solution. in 5 peptide-embonate which, after the addition of 50 ml of water, precipitates as a yellow precipitate. 0.330g of a fine crystalline yellow product are obtained with an emboic acid content of -20%.

The following diagram illustrates the construction of the compound according to Example 1:

HQQC

V Bvizotriasl-1-yloxy-tris * (diethylamio) phosphonem x xc Flurosphosphate (BQP)

2) N-methylmorphol

3} 3nNaOH

4) CFjCOOH

The compounds of the invention may also exist in the form of acid addition salts, for example salts with mineral acids, such as hydrochloric, sulfuric, phosphoric, and salts with organic acids, for example acetic, trifluoroacetic lactic, malonic, maleic, fumaric, gluconic, glucuronic, citric, embon, methanesulfonic, hydroxyethanesulfonic, pyrogene days and succinic.

The compounds of the invention, as well as their salts, are biologically active. The compounds of the invention may be administered in the free form or in the form of salts with physiologically acceptable acid. Administration is orally, parenterally, intravenously, transdermally or by inhalation.

The invention also relates to pharmaceutical preparations containing at least one compound of the general formula (V), or a salt thereof, with physiologically acceptable inorganic or organic acids and optionally pharmaceutically acceptable carriers and / or diluents, respectively auxiliaries.

The binding affinity of Cetrorelix, a compound obtained in Example 1 and Example 2, respectively, with the human LH-RH receptor. / Cetrorelix: Ac-D - Nal / 2 / - D -r - Cl- Phe - D- Pal / 3 / - Ser - Tyr - D - Cit -Leu - Arg - Pro - D Ala - NH ₂ /.

The method for determining binding affinity (dissociation constant Kd): is performed by competitive displacement binding experiment; Beckers et al., Eur. J. Biochem., 231,535-43, 1995 /. [ ¹²⁵ J] Cetrorelix / specific activity 5-10 x 10 ⁵ dpm / pmol was used as the radiolabeled ligand; dissolved in 20% v / v acetonitrile, 0.2% w / v albumin, 0.1% w / v TFA about 80% v / v water /. The binding capacity of the iodized peptide ranges from 60% to 85%. Cetrorelix, the compound of Example 1 and the compound of Example 2, in solution form, were used as labeled test compounds. The substances were used at concentrations of 0.01 nM-1000 nM (Cetrorelix, compound of Example 1 and Example 2).

The cells used for the binding test of human LH-RH receptor overexpressing clones of single cells L 3.5 / 78 were carefully separated with PBS / EDTA / PBS without Ca ²⁺ / Mg ²⁺ / 1 mM EDTA / from chalets with non-confluent cell growth. culture, determine cell number and resuspend with appropriate cell density in incubation medium / Dulbecco's modified Eagle Medium 10 mM Hepes, pH 7.5, 0.5% w / v BSA, 1g / 1 bacitracin, 0.1 g / 1 SBTI, 0.1% w / v NaN ₃ . In special 400 μΐ reaction vessels (Fa Renner type Beckman), 200 μΐ of silicone / paraffin oil mixture (84/16 vol%) was placed and then pipetted with 50 μΐ of the cell suspension (2.5 x 10 ⁵ cells). To the cell suspension on a layer of silicone / paraffin oil was rapidly added 50 μΐ medium for binding to [ ^l25 J] Cetrorelix and the compound to be tested at the appropriate concentrations. It was then incubated by rotation in an oven at 37 ° C for 60 min. After this step, it was centrifuged on Heraeus Biofuge 15 with a rotor HTA 13.8 for 2 min at 9000 upm at room temperature. The cells are then pelleted through a layer of silicone / paraffin oil and thus separated from the binding medium. After centrifugation, the reaction vessels were frozen in shock in liquid N ₂ and the tips of the reaction vessels (cell pellets) were cut off with tweezers and tips with cell pellets (bound ligand [ ¹²⁵ J] Cetrorelix) as well as standing above cell pellets (bound ligand [ ¹²⁵ J] as well as the rest (unbound, free ligand [ ¹²⁵ J] Cetrorelix) are transferred to enumeration tubes No competitor is added to determine maximum binding (B0) To determine nonspecific binding, 1 μΜ unlabeled Cetrorelix is added to competition physical binding is <10% lower than total binding (B0). Quantification is performed on a γ counter, evaluation is performed with the EBDA / Ligand V3.0 program (McPherson, J. Pharmacol. Methods, 14,213-228, 1985) Dosage plotting makes it possible to estimate IC 50 (Concentration that causes a 50% inhibition of the reaction), the EBDA / Ligand program calculates from it the dissociation constants Kd [nM]. Results: From the competition curves (see FIG. 1) it can be seen that all test compounds compete with the radiolabelled ligand [ ¹²⁵ J] Cetrrelix) for binding to the human LH-RH receptor. The binding (in% of maximum binding) was applied to the concentration of the competitor. For those shown in FIG. 1 compounds can be calculated the following binding affinities calculated as the dissociation constant Kd [nM]: Cetrorelix / SB -75 /: 0.214 nM, compound of example 1: 0.305 nM, compound of example 2: 0.104 nM. The binding affinities as averages of the various compounds are shown in Table 1. The antagonistic action of the compound of Example 2 in a human LH-RH receptor functional test. Methods for Determining IP3 / D-Myo-1,3,5 - Triphosphate: Subconfluent Culture of Human LH-RH Receptor Over-Expression Cell Clones / L3.5 / 78 / Cultured Once in PBS, Cells Is PBS / EDTA and the cell suspension was pelleted. cells were resuspended in incubation medium / Dulbecco's modified eagle medium with 4.5 g / l glucose, 10 mM Hepes, pH 7.5, 0.5% w / v. BSA, 5 mM LiCL; - 1 g (1 bacitracin, 0.1 SBT1), aliquoted into 1.5 ml reaction vessels and incubated for 30 min. at 37 C. 4 x 10 ⁶ cells in 500 μΐ volume are required for each measurement point. After pre-incubation, LH-RH (stock solution 0.5 mM in 10 mM Tris pH 7.5, 1 mM dithiothreitol, 0.1% w / v BSA / Bachem Art # H4005) was added to the cell suspension with a final concentration of 10 nM. The action of an antagonist is tested by the simultaneous addition of the appropriate concentration (e.g. 0.0316, 0.1, 0.316, etc. up to 100 nM for example 2). Cells were incubated as a negative control without the addition of LH-RH. After 15 min incubation at 37 C, the formed 1P3 was isolated from the cells by trichloroacetic acid (TCA) extraction. Then, 500 μΐ ice-cold 15% (w / v) solution of TCA was added to the cell suspension. The resulting precipitate was pelleted by centrifugation at 4 C at 2000 xg for 15 min in Haereus Biofuge 15R. The 950 μΐ supernatant was extracted 3 x with 10 volumes of cold, saturated diethyl ether in a 15 ml vessel placed over ice. After the last extraction, the solution was adjusted to a pH of 7.5 with a 0.5 M solution of NaHCO ₃ .

Determination of 1P3 concentration in cell extracts was performed by a sensitive competitive binding assay with 1P _3- binding protein, labeled [Ή] -1P3 and unlabeled 1P ₃ . An Amersham instrument (TRK 1000) is used for this purpose; the implementation is carried out as prescribed in the test protocol. After carrying out the various steps, 2 ml of Rotiszint Ecoplus for each aqueous sample was finally added, the resuspended pellet with the bound [Ή] 1P3 mixed carefully with it and measured in a β-scintillator. The amount of cellular 1P3 was calculated using a standard curve and a dose-response curve was constructed. IC ₅₀ can be determined from the turning point of this curve.

Results: In FIG. 1 shows the corresponding dose-response curve for the peptide antagonist of Example 2 / FIG. 2 /. It is stimulated with 10 nM LH-RH and determines the inhibition of 1P ₃ formation depending on the concentration of the substance. For example 2, the antagonistic activity cannot be determined, i. the substance alone does not lead to stimulation of the 1P _3- synthesis. In control experiments not presented here, it was shown that non-transfecting cells could not be stimulated to IL- ₃ synthesis by LH-RH. The measured at the highest concentrations still measurable 1P ₃ concentrations correspond to those of unstimulated cells. Therefore, the compound of Example 2 is a functional LH-RH antagonist. Substances, however, differ in their strength. Under selected test conditions, the IC ₅₀ of the compound of Example 2 is about 0.4 nM. These activities correlate very well with those determined in the in vitro competitive binding test with [ ¹²³ J] - Cetrorlix binding affinities = 0.109 nM for the compound of Example 2. The hormonal suppressive effect of the compound of Example 1 and that of Example 2 in healthy male rats . To determine the inhibition of testosterone in the blood in healthy male rats, the substance is injected subcutaneously into the animal in the right flank. The dosage ranges from the compound of Example 1 and that of Example 2

1.5 mg / kg. To control testosterone levels, approximately 300 μ 300 sublingual blood vein is collected from animals at intervals of 0,

2, 4, 24, 48, 72, 96 hours and then every three days until the end of suppression. Suppression continued at 1 ng / Ι testosterone after administration of the compound of Example 1 in one animal up to 164 h, in two animals up to 336 h, and in one animal up to 384 h / FIG. 3 /. After administration of the compound of Example 2, testosterone in the blood was suppressed in one animal up to 408 h, in four animals up to 648 h / FIG. 4 /.

TABLE 7: Biological data

Human LH-RH receptor binding affinities (expressed as dissociation constants Kd [ηΜ]; Determination by EBDA / Ligand assay program. Mean values from different experiments are given (number of experiments in brackets), as well as suppression of testosterone in vivo, histamine release in vitro, and water solubility compared to SB-75.

Substance Human LH-RHreceptor affinity [nmol / L] (1.5 mfl / ko, once) Testosterone suppression in rats IhJ (ic ₅₀ ) I'm not histamine (i9 / t1) n _d oraztvori bridge [mg / ml] Cetrorelix SB-75 0.202 (10) 144 9J 9 Example 1 0.306 (2) 336 31.9 27 Example 2 0.109 (2) 648 17,1 23 Example 3 0.170 (2) 864 *) *) Example 4 0.206 (2) 696 *) ·)

*) the difficulty of dissolution cannot be determined.

Claims (13)

Claims 1. Compound of General Formula / V / Ac-D-Nal / 2 / '- D / pCl / Phe ² -D-Pal / ^3/3 Ser ⁴ - Tyr ³ -D-Xxx ⁶ - Leu ⁷ -Arg ⁸ -Pro' -D-Ala ¹⁰ NH ₂ (V) in which D-Xxx denotes an amino acid group of the general formula (VI) -NH-CHCOI / CHA I NH I CO - R * W in which n denotes an integer from 1 to 4, R ⁴ means a group of general formula II O U / CH ^ - N-R * I R * / II / 25 where p is an integer from 1 to 4, R ⁵ represents a hydrogen atom or a lower alkyl group and R ⁶ represents an unsubstituted or substituted phenyl group, or R ⁴ represents a ring of general formula 30 or III R ⁷ / 40 W in which q is an integer from 1 to 2, R ⁷ is a hydrogen atom or a lower alkyl group, R ⁸ is a hydrogen atom or a lower alkyl group and X is an oxygen 45 atom or a sulfur atom, and their pharmaceutically acceptable salts. A compound according to claim 1, wherein Xxx represents a [εΝ-4- (4-amidinophenyl) amino-1,4-dioxobutyl] lysyl group. 50 A compound according to claim 1, wherein Xxx represents a [is N - [- imidazolidin2-one-4-yl] formyl] lysyl group. A compound according to claims 1 to 3, the salt of which is an embonate. A pharmaceutical agent comprising, as an active ingredient, a compound according to claim 1 to 4. A process for the preparation of a compound according to claim 1, characterized in that it comprises the following steps: (a) protecting the α-amino group and the carboxyl group of D-lysine or D-ornithine with suitable protecting groups, (b) reacting the protected with D-lysine or D-ornithine with a carboxylic acid of general formula / VII / : R ₄ is COOH where R ⁴ has the above meanings (c) cleavage of the protecting group at the α-carboxyl group of the compound obtained in step (b) for incorporation into position 6 in step (b), (d) binding of the D-alanine protected by the protecting group at the amino group to a solid carrier in the form of a resin (Merrifield synthesis), / is the cleavage of the protecting group at the amino group of the alanine, (f) interaction of the solid-bound alanine with proline which is protected by a protecting group at the nitrogen atom, (g) cleaving the protecting group at the nitrogen atom of proline , (h) repeating steps f) and g) with the amino acids 1 to 8 of the general formula (V) in sequence from 8 to 1 using the modified D-lysine or D-ornithine for position 6 described in step (c) , (i) cleavage of the compound obtained in step (h) from the carrier and optionally purification (e.g. HPLC), (j) optionally reacting with a pharmaceutically acceptable acid, preferably emboic acid. A process for the preparation of a compound according to claim 1, characterized in that it comprises the following steps: (a) coupling of a protected group at the amino group D-alanine with a suitable solid phase carrier synthesis, (b) cleavage of the protecting group at the alanine amino group, (c) interaction of the resin bound alanine with a proline which is protected with a protecting group at the nitrogen atom, (d) cleaving the protecting group at the nitrogen atom of proline, (e) repeating steps c) and d) with amino acids 1 to 8 of general formula (V), in a sequence of 8 to 1, (f) cleavage of the compound obtained in step e) a compound from the carrier, (g) reaction with carboxylic acid with ether of the formula (VII) R ⁴ is COOH (VII) in which R ⁴ is defined above, (h) optionally reacting with a pharmaceutically acceptable acid, preferably emboic acid. A process for the preparation of a compound according to claim 4, characterized in that it comprises the following steps: (a) linking the amino group protected with the protecting group D-alanine to a carrier suitable for solid phase synthesis, (b) cleaving the protecting group at the alanine amino group, (c) reacting the resin bound alanine with a proline having a nitrogen atom protecting group, (d) cleavage of the proline nitrogen atom protecting group, (e) repeating steps c) and d) with amino acids 6 to 8 of general formula (V), in a sequence of 8 to 6, ( f) cleavage of the ε-amino protecting group of D-lysine or Dornitine at position 6, and reaction with carboxylic acids and of formula (VII) R ⁴ is COOH (VII) in which R ⁴ is defined above, / g / cleavage of the protecting group at the α-amino group of D-lysine or D-ornithine, / b / repeat steps / c / and / d (with amino acids 1 to 5 of general formula (IV), in sequence from 5 to 1) (i) cleavage of the compound obtained in step (h) from the resin and purification by HPLC, (j) optionally reacting with a pharmaceutically acceptable acid, preferably emboic acid. The process according to claims 6 to 8, characterized in that b1- (4-amidinophenyl) amino-4-butyric acid is used as the carboxylic acid of the general formula (VII). The process according to claims 6 to 8, characterized in that imidazolidin-2-one-4-carboxylic acid is used as the carboxyl-5 acid of the general formula (VII). A method according to claims 6 to 10, characterized in that emboic acid is used as a pharmaceutically acceptable acid. Use of the compounds according to claims 1 to 4 for the preparation of medicaments for the treatment of hormone-dependent tumors, in particular prostate cancer or breast cancer, and for non-malignant indications whose treatment requires LHRH hormone suppression. Method for the preparation of medicaments containing compounds according to claims 1 to 4, characterized in that the compound according to claims 1 to 4 is mixed with the usual carriers and excipients and is prepared in the form of a galenical form.