SK62998A3 - New lh-rh antagonists with improved effectiveness - Google Patents

Abstract

New LH-RH antagonists of formula (I) are disclosed, in particular peptidomimetics and peptides modified in a side chain, their salts with pharmaceutically acceptable acids and a process for preparing these LH-RH antagonists and their salts. The disclosed peptides represent analogues of the luteinising hormone releasing hormone (LH-RH). The disclosed compounds have a high antagonistic power and are free of undesirable side effects, in particular edematogenic effects.

Description

Novel LH-RH antagonists with improved activity

Technical field

The invention relates to novel LH-RH antagonists, in particular peptidomimetics and peptides modified in their side chain, their salts with pharmacologically acceptable acids, and a process for the preparation of LH-RH antagonists and their salts. The peptides of the invention are analogues of a luteinizing hormone releasing hormone (LH-RH) having the following structure: p-Glu-HisTrp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ , [LH-RH, gonadorelin] .

BACKGROUND OF THE INVENTION

For over 20 years, researchers have been looking for selectively acting LH-RH-decapeptide antagonists [M. Karten and J.E. Riviera, Endocrine Reviews 7, p. 44-66 (1986)]. The reason for their great interest in such antagonists is their usefulness in the fields of endocrinology, gynecology, protection against conception and cancer. A large number of compounds were prepared as potential LH-LR antagonists. Interesting compounds that have been discovered so far are those whose structure represents a modification of the structure of LH-RH.

The first series of potent antagonists was obtained by introducing aromatic amino acid residues at positions 1, 2, 3 and 6 or 2, 3 and 6. The usual notation of compounds is as follows: the amino acids that have entered the LH-RH peptide chain at the site of the previously existing amino acids , the positions where the exchange occurred are indicated by the numbers placed above. In addition, the following designation LH-RH indicates that these are LH-RH analogues that have been exchanged.

Known antagonists are:

[Ac-D-Phe (4-Cl) ^1/2 , D-Trp ³ ' ⁶ ] LH-RH (DH Coy et al., Gross, E. and Meienhofer J. (eds.) Peptides; Proceedings of the 6th American Peptide Symposium, pp. 775-779, Pierce Chem. Co., Rockville III (1979):

[Ac-Pro ¹ , D-Phe 4-Cl) ² , D-Nal (2) ³ ' ⁶ ] LH-RH (U.S. Pat. No. 4,419,347) and [Ac-Pro ¹ , D-Phe (4- Cl) ^2, D-Trp ^{3 '6]} LH-RH (JLPineda, et al., J. Clin. Endocrinol. Metab. 56, p. 420, 1983).

In order to increase the solubility of the antagonists in water, basic amino acids such as D-Arg at position 6 were introduced later. For example:

[Ac-D-Phe (4-Cl) ¹ ' ² , D-Trp ³ , D-Arg ⁶ , D-Ala ¹⁰ ] LH-RH (ORG-30276) (DH Coy et al., Endocrinology 100, p. 1445, 1982) and [Ac-D-Nal (2) ¹ , D-Phe (4-F) ² , D-Trp ³ , D-Arg ⁶ ] LH-RH (ORF 18260) (Rivier et al., Vickery BH Nestor, Jr., JJHafez,

R.S.E (Publishers). LHRH and their analogs, p.11-22 MTP Press. Lancaster, UK 1984).

Such analogs not only exhibited the expected improved water solubility but also exhibited improved antagonist activity. However, these highly potent, hydrophilic analogs with D-Arg ⁶ and other basic side chains in position 6 cause transient edema in the kidney and limbs when administered subcutaneously to rats at doses of 1.25 or 1.5 mg / kg (F. Schmidt et al Contraception 29, 283 (1984): JEMorgan et al Int Archs Allergy Appl.Immun 80: 70 (1986) Other useful LH-RH antagonists are described in WO 92/19651, WO 94/19370, WO 92/17025, WO 94/14841, WO 94/13313, US-A 5 300 492, US-A 5 140 009, and EP 0413209 A1.

The occurrence of endematogenic effects following administration of some of these antagonists in rats has raised doubts about the safety of their use in humans, thus delaying the introduction of these drugs into clinical use. Therefore, there is a great demand for antagonist peptides without side effects.

SUMMARY OF THE INVENTION

The present invention provides a compound of formula I

R ¹ - CO - NH - CH - CO

N - R ³

NH

CO-R ⁴ where n is 3 or 4, R ^{1 is} optionally substituted alkyl, alkyloxy, aryl, heteroaryl, aralkyl, heteroaralkyl, aralkyloxy or an aralkyloxy heteron, R and R independently of each other hydrogen, optionally substituted alkyl, aralkyl or heteroaralkyl, wherein aryl or heteroaryl are suitable as substituents, or -NR ² R ³ is an amino acid group, and R ⁴ is a group of formula II

II

WHAT

(II)

where p is an integer from 1 to 4, R ⁵ is a hydrogen atom or an alkyl group and R ^{6 is an} optionally substituted aryl or heteroaryl group, wherein aryl or heteroaryl are as substituents again or R ⁴ represents a ring of formula III µ '

(III) * 7 wherein q is 1 or 2, R is hydrogen or alkyl, R ⁸ is hydrogen or alkyl and X is oxygen or sulfur, wherein the aromatic or heteroaromatic radicals may be partially or fully hydrogenated and the chiral carbon atoms may be in the D or L configuration and salts thereof with pharmaceutically acceptable acids.

«I

Preferred combinations of the groups R ¹ to R ⁴ are:

a) R ¹ is benzyloxy, R ² is H, and R ³ is H,

b) R ^x is benzyloxy, R ² is H, and R ⁴ group of formula II, wherein p is 2 or 3, R ⁵ is H and R ⁶ 4-amidinophenyl group, and

c) R ² represents a hydrogen atom, R ³ a hydrogen atom and R ^{4 a} group of the general formula II wherein p is 2, R 6 is a hydrogen atom and R ⁶ a 4-amidinophenyl group.

Preferred alkyl groups are: methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, 2-ethylhexyl, dodecyl and hexadecyl.

Preferred aryl groups are: phenyl, naphthyl, phenanthrenyl and fluorenyl.

Preferred heteroaryl groups are 2-, 3-a

4-pyridyl, 2- and 4-pyrimidyl, imidazolyl, midzopyridyl, 5- and 6-indolyl, 5- and 6-imidazolyl, triazolyl, tetrazolyl, benzimidazolyl, quinolyl, 2,6-dichloropyrid-3-yl and furyl.

Preferred hydrogenated heteroaryl groups are piperidine, piperazinyl, morpholine and pyrrolidinyl groups.

Aralkyl and heteroaralkyl groups are groups which are bonded to the respective binding site by an alkylene group, preferably a methylene, ethylene, n-propylene or n-butylene group.

In addition to the above-mentioned aryl and heteroaryl groups, preferred substituents for halogen atoms such as fluorine, chlorine, bromine and iodine are also methyl, ethyl, i-propyl, tert-butyl, cyano and nitro, carboxylic acid, amide, methyl ester, ethyl ester, ethyl ester, ethyl ester, ethyl ester, ethyl ester, of ethyl crotonic acid and trifluoromethyl, benzoyl, methoxy, benzyloxy, pyridyloxy, amino, dimethylamino, isopropylamino, amidino and quinolylmethoxy.

The invention also relates to compounds of the general formula V

Ac-D-Nal (2) - (DCl) Phe ² -D-Pal (3) ³ -Ser ⁴ -Tyr ⁵ -D-Xxx ⁶ -Leu ⁷ -Arg ⁸ Pro ⁹ -D-Ala ¹⁰ -NH ₂ (V) wherein D-Xxx is an amino acid group of formula VI

- HN-CH-CO-0 *

I

J ( ^CH ₂ ) n

I (VI)

NH

I CO - R ⁴ in which n, p, q, R ^4, R ^5, R ^6, R ^7, R ⁸ and X are as defined above, and their salts with pharmaceutically acceptable acids.

The compounds of the invention exhibit a high antagonistic potency and are free of unwanted side effects, in particular they are. without edematogenic effects. If they are not in the form of salts of sparingly water-soluble pharmaceutically acceptable acids, they also exhibit improved water solubility. Furthermore, the compounds have a high affinity for the human LH-RH receptor, thus they are highly effective in inhibiting the release of gonadotropins of the pendant gland in mammals, including humans, exhibit long-acting suppression of testosterone in rats and cause minimal histamine release in vitro.

and

Suitable compounds of formula (I) are: alpha-NZ [epsilon-N '-4- (4-amidinophenyl) amino-1,4-dioxo-butyl] lysamide, alpha-NZ- [epsilon-N ^1- (imidazolidine- 2-on-4-yl) formyl] lysinamide. Preferred peptides of formula (V) are peptides wherein Xxx is [epsilon-N'-4- (4-amidinophenyl) amino-1,4-dioxobutyl] -lysyl or [epsilon-N '- (imidazolidin-2-one) 4-yl) formyl] lysyl. Salts with pharmaceutically acceptable acids are preferably sparingly water soluble. Especially preferred are the salts of 4,4'-methylene-bis (3-hydroxy-2-naphthoic acid, also known as embonic or pamoic acid).

The nomenclature used to define peptides agrees with the nomenclature explained by the IUPAC-IUB-Commission on Biochemical Nomenclature (European J. Biochem. 138, pp. 9-37, 1984), with the amino groups at the N-terminus on the left and carboxyl groups consistent with the present illustration. at the C-terminus to the right. LH-RH antagonists such as peptides and peptidomimetics of the invention include naturally occurring and synthetically produced amino acids, naturally including Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met , Phe, Tyr, Pro, Trp, and His. The abbreviations of the individual amino acids are based on the trivial amino acid names and mean: Ala alanine, Arg arginine, Gly glycine, Leu leucine, Lys lysine, Pal (3) -3- (3-pyridyl) alanine, Nal (2) 3- (2- naphthyl) alanine, Phe phenylalanine, (pCl) Phe 4-chlorophenylalanine, Pro proline, Ser serine, Thr threonine, Trp tryptophan and Tyr tyrosine. Unless otherwise stated, amino acids of the series are described

L. For example, D-Nal (2) is an abbreviation for 3- (2-naphthyl) -D-alanine and Ser is an abbreviation for L-serine. Other abbreviations used have the following meanings:

Boe tert-butyloxycarbonyl

Bop benzotriazol-1-yloxy-tris- (dimethylamino) DCC

DCM

ddz

DIC

DIPEA

DMF phosphonium hexafluorophosphate, dicyclohexylcarbodiimide dichloromethane dimethoxyphenyldimethylmethylenoxycarbonyl (dimethoxydimethyl-Z) diisopropylcarbodiimide

N, N-diisopropylethylamine dimethylformamide

Fmoc

HF

HOBt

HPLC

PyBop

TFA Fluorenylmethyloxycarbones1 Liquid anhydrous hydrofluoric acid 1-Hydroxybenzotriazole High pressure liquid chromatography Chromatography Benzotriazol-1-yloxy-tris-pyrrolidinophosphoniumhexafluorophosphate Trifluoroacetic acid Benzyloxycarbonyl

The process for the preparation of the compounds of the formula I according to the invention consists in that the functional groups (alphaamino, epsilon-amino and alpha-carboxylic acid) are first protected with a protective group and then the free third functional group is reacted in a suitable manner. Alternatively, in the case of better results, it is also possible to introduce transient protecting groups in the first operation, which are exchanged for the desired functional group after the second operation. Suitable protecting groups and methods for introducing them are well known in the art. Examples of suitable protecting groups are described in Principles of Peptide Synthesis by Spinger Verlag 1984, in Solid Phase Peptide Synthesis J.M. Stewart and J.D.Young, Pierce Chem. Company, Rpckford, III, 1984; and G. Barany and R.B. Merrifield The Peptides, Vol. 1, p. 1 to 285 (1979), Academic Press Inc.

The preparation of compounds of the formula IV can be carried out either by the classical condensation of fragments or by solid phase synthesis according to Merrifield by successive adjustments using D-lysine acidified with a carboxylic acid of the formula VII in the side chain as well as reaction of the decapeptide D-lysine ⁶ .

In this process, compounds of formula (I) are prepared for the preparation

Three alternatives are available according to the invention.

According to a first possibility for the preparation of a compound of formula V (a), suitable protecting groups are introduced into the alpha-amino group and the carboxylic acid group of D-lysine or D-ornithine, (b) reacting the protected groups with D-lysine or D-ornithine. a carboxylic acid of formula VII

R ⁴ - COOH (VII) wherein R ⁴ is as defined above, (c) the alpha-carboxylic acid protecting groups of the compound obtained in step b) are cleaved off for inclusion in the 6-position by operation (h), (d) coupling D amino-protected alanine to a solid support in the form of a resin (Merrifield synthesis), (e) deprotecting the amino groups of the alanine, (f) reacting the alanine bound on the solid support with proline, protected nitrogen, (g) cleaving (h) the operation f) and g) are repeated with amino acids 1 to 8 of the general formula V in the order of 8 to 1 using the modified D-lysine or D-ornithine described in operation (c) for position 6, (i) cleaving the compound obtained in operation (h) from the carrier and optionally purifying it, in particular by HPLC chromatography, (j) optionally reacting the compound with a pharmaceutically acceptable acid, preferably embonic acid.

According to a second possibility of preparing a compound of formula (V) (a), the amino-protected D-alanine is coupled to a carrier suitable for solid phase synthesis, (b) the amino-protecting groups of the alanine are cleaved, (c) (d) the protecting group is removed from the proline nitrogen atom, (e) operation (c) and (d) are repeated with amino acids 1 to 8 of the formula V in the order of 8 to 1, (f) cleaved off in step (e) reacting the obtained compound with a carrier; (g) reacting it with a carboxylic acid of the formula VII

R ⁴ - COOH (VII) wherein R ⁴ is as defined above, (h) optionally, the compound is reacted with a pharmaceutically acceptable acid, preferably embonic acid.

According to a third possibility of preparing a compound of formula (V) (a), the amino-protected D-alanine is coupled to a carrier suitable for solid phase synthesis, (b) the amino-protecting group of the alanine is cleaved, (c) (d) the protecting group is removed from the proline nitrogen atom, (e) the operation (c) and (d) are repeated with amino acids 6 to 8 of the formula V in the order of 8 to 6, (f) the protecting group is split off epsilon-amino group of Dlysine or D-ornithine at the 6-position and reacted with a carboxylic acid of formula (VII)

R ⁴ - COOH (VII) wherein R ⁴ is as defined above, (g) the protecting group is removed from the alpha-amino group of Dlysine or D-ornithine, (h) the operation (c) and (d) are repeated with amino acids 1 to 5 of formula IV in the order of 5 to 1, (i) cleaving the compound obtained in operation (h) from the resin and purifying in particular by HPLC chromatography, (j) optionally reacting the compound with a pharmaceutically acceptable acid, preferably embonic acid.

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

Compounds of formula (V) are prepared by known methods, for example, solid-phase technique, semi-solid phase technique, or classical solution coupling (see

M. Bodanszky Principles of Peptide Synthesis Springer Verlag 1984). Solid phase synthesis methods are described in the Solid Phase Peptide Synthesis J.M. Stewart and J.D.Young, Pierce Chem. Company, Rockford, III, 1984 and in G. Barany and R.B. Merrifield The Peptides, Vol. 1, p. 1 to 285 (1979), Academic Press Inc. Classical solution syntheses are described in detail in Methoden der Organischen Chemie (Houben-Weyl),

Synthese von Peptiden Publisher E. Wunsch 1974, Georg Thieine Verlag, Stuttgart, BRD.

For example, the sequential construction is by covalently bonding to a conventional insoluble carrier a carboxyl terminal amino acid whose amino group at the alpha position is protected, the alpha-amino protecting group of that amino acid is cleaved and the free amino group thus formed binds the nearest protected amino acid via its carboxyl group; thus, step by step, the other amino acids of the synthesized peptide are coupled in the correct order and, after coupling of all the amino acids, the finished peptide is cleaved from the carrier and, optionally, further existing protecting groups of the side groups are cleaved. Gradual condensation occurs by synthesis from the corresponding conventional protected amino acids in a known manner. It is also possible to use automatic peptide synthesizers, for example of the Labortec SP 650 type from the company. Bachem, Switzerland using commercially available protected amino acids.

The coupling of the individual amino acids to each other is carried out by conventional methods, and is particularly suitable

- method of symmetrical anhydrides in the presence of dicyclohexylcarbodiimide or diisopropylcarbodiimide (DCC, DIC)

- carbodiimide method in general

- carbodiimide hydroxybenzotriazole method (The Peptides, vol. 2, edited by R. Gross and J. Meienhofer). The carbodiimide method is preferably used for coupling arginine. For other amino acids, the method of symmetrical or mixed anhydrides is generally used.

In the coupling of the fragments, azide coupling without racemization or DCC-1-hydroxybenzotriazole or DCC-3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine is preferably used. Activated fragment esters may also be used.

Activated esters of N-protected amino acids such as the N-hydroxysuccinimide ester or 2,4,5-trichlorophenyl ester are particularly well suited for the progressive condensation of amino acids. Aminolysis can be well catalyzed by N-hydroxy compounds having approximately acetic acid, such as 1-hydroxybenzotriazole.

Suitable transient amino protecting groups are hydrogen-removable groups such as benzyloxycarbonyl (Z = residue) or weak-acid-cleavable groups. Suitable protecting groups for amino groups in the alpha position include, for example: tertiary butyloxycarbonyl, carbobenzoxy, optionally carbobenzothio (optionally with bromine or nitrobenzyl in the p-position), trifluoroacetyl, phthalyl, o-nitrophenoxyacetal, -toluolsulfonyl, benzyl, substituted benzyl groups in the benzol nucleus (with a bromine atom or nitrobenzyl group in the p-position) and alpha-phenylethyl (Jesse P. Greenstein and Milton Winitz, Chemistry of Amino Acids, New York 1961, John Wiley and Sons , Inc. vol. 2, or p. 883, and also The Peptides, vol. 2, edited by E. Gross and J. Meienhofer, Academic Press, New York). In principle, these protecting groups are also suitable for protecting other functional side groups (hydroxyl groups, amino groups) of the respective amino acids.

The contained hydroxyl groups (serine, threonine) are preferably protected with benzyl or similar groups. Other non-alpha amino groups (e.g., omega amino groups, arginine guanidine group) are preferably orthogonally protected.

The reaction to join the amino acids occurs in an inert solution or suspension medium customary for such reactions (e.g., dichloromethane), and dimethylformamide may be added to improve solubility.

To incorporate the R ⁴ -CO group by reaction of the amino group of the lysine with a carboxylic acid, essentially the same procedures as described above for amino acid coupling are possible. However, condensation using carbodiimide, for example 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 1-hydroxybenzotriazole, is particularly preferred.

Possible insoluble carrier materials are insoluble polymers, for example pearl-shaped polystyrene resin, swellable in organic solvents (for example, a copolymer of polystyrene and 1% divinylbenzol). The formation of the protected decapeptidamide on the methylbenzhydrylamide resin (MBHA resin, i.e. polystyrene resin provided with methylbenzhydryl groups), which gives the desired C-terminal amide group of the peptide after HF-cleavage from the support can be carried out according to the following flow chart:

Flowchart

Peptide synthesis protocol

degree feature Solvent / reagent v / v Time 1 wash methanol 2x2 min 2 wash DCM 3x3 min 3 secession DCM / TFA (1: 1) 1x30 min 4 wash isopropanol 2x2 min

5 wash methanol 2x2 min 6 wash DCM 2X3 min 7 neutralization DCM / DIPEA 9: 1 3X5 min 8 wash methanol 2X2 min 9 wash DCM 3X3 min 10 STOP Boc-As v DCM + DIC + HOBt 11 coupling - pribi.90 min 12 wash methanol 3X2 min 13 wash DCM 3x2 1 min

The N-alpha-Boc protected amino acids are coupled in a triple molar excess in the presence of diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in dichloromethane / dimethylformamide over 90 minutes and the Boe protecting group is cleaved by half-hour in trifluoroacetic acid. Christensen's chloroanil test and Keiser ninhydrin test can be used to check for complete reaction. The free amino residues are blocked by acetylation in a five-fold excess of acetylimidazole in dichloromethane.

The sequence of the resin-forming reaction reactions is based on a flow chart. In order to cleave the resin-bound peptides, the final solid synthesis product is always dried in phosphorus pentoxide under vacuum and treated in a 500-fold excess of 10: 1 hydrogen fluoride / anisole system (v / v) for 60 minutes at 0 ° C.

After distillation of the hydrogen fluoride and the anisole, the peptide amides are precipitated by mixing with anhydrous ethyl ether as white solids, separation from the precipitated polymeric carrier by washing with 50% aqueous acetic acid. By carefully concentrating the solutions acidified with acetic acid in vacuo, the corresponding peptides can be obtained in the form of highly viscous oils, which in the cold are converted to white solids by the addition of absolute ether.

Further purification is by routine preparative high pressure liquid chromatography (HPLC) methods.

Conversion of peptides to their acid addition salts can be accomplished by their reaction with acids in a known manner. Conversely, it is possible to obtain free peptides by reacting their salts with base addition acids. Peptidembonates can be obtained by reacting salts of trifluoroacetic acid (TFA salts) of peptides with free embonic acid. For this purpose, the TFA peptide salt in aqueous solution is reacted with a solution of disodium disodium in dipolar aprotic medium, preferably in dimethylacetamide, and the light yellow precipitate formed is isolated.

The invention is illustrated, but not limited, by the following examples. Percentages and parts are by weight unless otherwise indicated. The invention is illustrated by the accompanying drawings.

List of pictures

In FIG. 1 is the binding of test compounds to the human receptor relative to the concentration of the competitor, on the x-axis the concentration of the competitor, on the y-axis the percentage of specific binding, the curve with wheels applies to the compound of example 56, with squares for cetrorelix the compound of Example 1 and with diamonds for the compound of Example.

In FIG. 2

In FIG. 3 is the inhibition of formation of IP ₃ depending on the concentration of the test substance according to Example 2.

is the inhibition of IP ₃ formation depending on the concentration of the test substance according to Example 56.

In FIG. 4 is the testosterone suppression in ng / ml test compound of Example 1 at 1.5 mg / kg over time in hours, on the x-axis is time in hours, on the y-axis testosterone in ng / ml, the empty-wheel curve is for animal 1, with empty squares for animal 2, with commas for animal 3, with full castors for animal 4 and with full squares for animal 5.

In FIG. 5 is the testosterone suppression in ng / ml test compound of Example 2 at 1.5 mg / kg over time in hours, on the x-axis is time in hours, on the y-axis testosterone in ng / ml, the empty-wheel curve is for animal 1, with empty squares for animal 2, with commas for animal 3, with full castors for animal 4 and with full squares for animal 5.

In FIG. 6 is the testosterone suppression in ng / ml test compound of Example 56 at 10 mg / kg on time in hours, on the x-axis is time in hours, on the y-axis testosterone in ng / ml, the empty-wheel curve is for animal 1 , with empty squares for animal 2, with commas for animal 3, with full castors for animal 4 and with full squares for animal 5.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Ac-D-Nal (2) -D (pCl) Phe-D-Pal (3) -Ser-Tyr-D- [epsilon-N '(imidazolidin-2-on-4-yl) formyl] -Lys- Leu-Arg-Pro-D-Ala-NH ₂

Synthesis proceeds according to the flow chart on 5 g mBHA resin (density 1.08 mmol / g). Lysine is coupled as Fmoc-D-Lys (Boe) -OH and, after cleavage of the Boc group, acylated in the side chain with imidazolidin-2-one-4-carboxylic acid in a 3-fold excess. After cleavage of the Fmoc protecting group with 20% piperidine / DMF, the common flow chart is extended to the N-terminus. After cleavage of the polymeric carrier, 5.2 g of crude peptide precipitated, which was purified by standard preparative HPLC. After subsequent freeze-drying, it is obtained

2.1 g of HPLC-unitary product of formula C ₇₄ H _{g 7} N ₁₈ O ₁₅ Cl with correct FAB-MS 1514 (M + H ⁺ ) (calcd 1512.7) and the corresponding spectrum 1 H-NMR.

¹ H-NMR (500 MHz, DMSO-d 6, δ ppm):

8.56 m, 2H, arom; 8.08, m, 1H, arom; 7,81 m 1H, arom; 7,73 m 2H, arom; 7,66 m 1H, arom; .7,60, s, 1H, arom; 7.44 m 2H, arom; 7,30 d, 1H, arom; 7,25 and 7 18. 2d, 2 x 2H, aroma .H p- Cl-Phe; 6.97 and 6 60. 2d, 2 x 2H, aroma. H Tyr 9,2-

6.3 numerous signals, amide-NH; 4.8-4.0 numerous m, C-alpha-H and alifat.H; 2.1-1.1 Numerous m, remaining aliphatic.H; 1.70, s, 2H, acetyl; 1.22, d, 3H, Cbeta H Ala; 0.85; d; 6H; Cdelta-H Leu.

Example 2

Ac-D-Nal (2) -D (pCl) Phe-D-Pal (3) -Ser-Tyr-D- [epsilon-N-4- (4-amidinophenyl) amino-1,4-dioxo-butyl] - Lys - Leu - Arg - Pro - D - Ala - NH ₂

0.7 mmol (1.03 g) of the decappetide Ac-D-NalD- (pCl) Phe-D-Pal-Ser-Tyr-D-Lys-Leu-Arg-Pro-D-Ala-NH _{2 was} reacted with 1.0 mmol (0.27 g) of (4-amidinophenyl) amino-4-oxo-butyric acid in the presence of 1.0 mmol (0.16 g) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 1, 0 mmol (0.16 g) of 1-hydroxybenzotriazole in freshly distilled DMF. After 24 hours the solvent was removed in vacuo, the precipitated residue was dissolved in water and freeze-dried. The precipitated crude product (1.63 g) was purified by preparative reverse phase HPLC. Total precipitation was 0.61 g of uniform product purified by HPLC empirical formula C ₈₁ H ₁₀₄ N ₁₅ O C _ig with correct FAB MS: 1618.7 (M + H ⁺⁾ (calculated 1617.7) and the corresponding spectra ^X H-NMR ¹ H-NMR (500 MHz, DMSO-d 6 in ppm):

10.4, s, 1H and 9.15 s, 2H and 8.8, s, 1H, NH-4-amidinoaniline; 8.60, m, 2H, aromatic H; 8.20, m, 1H, aromatic H; 7.80, m, 1H, aromatic H; 7.73, m, aromatic H; 7.61, s, 1H, aromatic H; 7.44, m, 2H, aromatic H; 7.30, d, 1H, aromatic H; 7.25 and 7.20, 2d, 4H, aromatic H (pCl) -Phe; 7.00 and 6.60, 2d, 4H, aromatic H Tyr; 8.3-7.2 numerous signals, amide-NH; 4.73-4.2 multiple multiplets, C-alpha-H, Ala; 3,78-

2.4 numerous multiplets, C-beta H and alifat.H; 1.72, 3, 3H, acetyl; 1.22; d; 3H, C-beta Ala; 0.85, dd, 6H, C-delta Leu.

Príklad'3

0.5 g (0.3 mmol) of the peptide LH-RH antagonist of Example 1, dissolved in 50 mL of water, is reacted with 0.130 g (0.3 mmol) of embryonic acid disodium salt in 2 mL of aqueous solution to give the peptide embonate which rapidly precipitates from the solution as a yellow precipitate. 0.281 g of finely crystalline yellow-green powder with a 33% embonic acid content is obtained.

Example 4

0.3 g (0.17 mmol) of the peptide LH-RH antagonist of Example 2, dissolved in 5 ml of dimethylacetamide, is reacted with 0.195 g (0.45 mmol) of embonic acid disodium salt in 2 ml of an aqueous solution to obtain the peptide embonate. which precipitates after the addition of 50 ml of water as a yellow precipitate. 0.330 g of a finely crystalline yellow product with a 20% embonic acid content is obtained.

The compounds of the formula I can be obtained according to the following schemes 1, 3, 4 and 5, the meanings of the three symbols R ¹ , R ³ and R ⁴ being systematically changed. Scheme 1 shows the construction of the compound of Example 1:

Scheme 1

O nh-ch-co-nh ₂ x HCl

HOOC

NH

NH

HCl

NH ₂

1) Benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP)

2) N-methylmorpholine

3) 2n NaOH

4) CF ₃ COOH

General formula for the preparation of compounds of formula I according to Scheme 1

The carboxylic acid of formula R ⁴ -COOH substituted with an R ⁴ group which forms the basis of the compound of formula I and the process of Scheme 1, which may exist as a salt, for example hydrochloride, hydrosulphate, in the case of a basic radical R ⁴ , is dissolved or suspended or acetate, excluding moisture with stirring in a non-polar or dipolar aprotic organic solvent such as tetrahydrofuran, dioxane, methyl tert-butyl ether, toluene, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide or methylene chloride and allowed to react with stirring with an acid-binding base such as diisopropylamine, triethylamine, N-methylmorpholine, dimethylaminopyridine or pyridine. A mixture of Z- (L) -lysinamide hydrochloride in a diluent is added, and diluents which may be used are the solvents used to dissolve the carboxylic acid of the formula R @ ⁴ -COOH substituted by R @ ⁴ . The pH of the reaction mixture is adjusted with an acid-binding base, for example 6.5 to 9.0, preferably 7.0 to 8.5, especially 7.0 to 7.5. Finally, a solution of a coupling reagent such as benzotriazol-1-yl-oxytris- (dimethylamino) phosphonium hexafluorophosphate (BOP), or benzotriazol-1-yloxy-trispyrrolidinophosphonium hexafluorophosphate (PyBOP) or dicyclohexyl dicyclohexyl (dBi) is added to the stirred reaction mixture. the pH of the solution sets to the above range. The suspension is stirred, for example, for 1 to 15 hours at 0 to 80 ° C, preferably at 10 to 50 ° C, especially 20 to 30 ° C, suctioned off, the solid is washed and the filtrate is concentrated to dryness in vacuo. The residue is crystallized by rubbing with an organic solvent such as toluene, tetrahydrofuran, acetone, methyl ethyl ketone, optionally with isopropanol, or purified by recrystallization, distillation or column or flash chromatography on silica gel or alumina. The eluent used is, for example, methylene chloride, methanol, ammonia (25%) (85: 15: 1 by volume) or methylene chloride, methanol, ammonia (25%), 80: 25: 5 by volume.

Synthesis of trifluoroacetate

The purified compound obtained above is dissolved in a protic or aprotic solvent, for example in an alcohol such as methanol, ethanol, isopropanol or in cyclic ethers such as tetrahydrofuran or dioxane, and the pH is adjusted to 10-11 with 2N sodium hydroxide solution. The precipitated solid is filtered off with suction, washed, dried under vacuum and in ethanol solution at a temperature of 10 to 80 ° C, preferably 20 to 40 ° C, and reacted with a molar equivalent or a 2- to 4-fold excess of trifluoroacetic acid. After standing for 24 hours at 0-4 ° C, the desired trifluoroacetate crystallizes, which is suctioned off and dried in vacuo.

Following this general formula, which is the basis for Scheme 1, the compounds illustrated in Example 5 and the following Table I are synthesized.

Example 5

Alpha-N- [benzyloxycarbonyl] -epsilone-N- [5 - [(4-amidinophenyl) amino] -5-oxo-pentanoyl] -L-lysine amide trifluoroacetate

While stirring and excluding moisture, 5 g (17.5 mmol) of 5 - [[4- (aminoiminomethyl) phenyl] amino] -5-oxopentanoic acid hydrochloride is suspended in 200 ml of dimethylformamide and treated with 3.85 ml (35, 0 mmol) of N-methylmorpholine. A mixture of 5.53 g (17.5 mmol) of Z- (L) -lysinamide hydrochloride in 100 ml of dimethylformamide is added and the pH is adjusted to 7.0-7.5 with N-methylmorpholine. Finally, a solution of 9.73 g (21.9 mmol) of benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP) is added and the pH is again adjusted to 7.0 to 7.5 after 10 to 15 minutes. The yellow-colored suspension is stirred for 3 to 4 hours while monitoring the pH to 7.0 to 7.5 at room temperature, the colorless precipitate is filtered off with suction, washed twice with dimethylformamide and the yellow-colored filtrate is evaporated to dryness. The oily residue was digested with a total of 5x40 ml of methylene ketone by decanting and discarding the methyl ketone phase after each of the five solvent treatments. The remaining crude product in the crystalline state is suctioned off, washed with 30 ml of methyl ketone and dried under vacuum at room temperature.

The solid is then dissolved in approximately 50 ml of ethanol and the pH is adjusted to 10-11 with sodium hydroxide solution. The precipitated base is filtered off with suction, washed with water and ethanol and dried under vacuum at 35 ° C. Yield: 5.5 g (62% of theory).

trifluoroacetate:

5.5 g of base in ethanol suspension are reacted with a 5-fold amount of trifluoroacetic acid at 60 ° C. The solution is kept at 4 ° C overnight, the trifluoroacetate obtained is filtered off with suction and dried under vacuum at 35 ° C. Yield: 5.9 g (87.7% of theory). Mp 185 ° C.

analysis:

calculated C 53.84 H 5.65 found C 54.11 H 5.74

N, 13.45

N 13.33 described above

In the above process, the other compounds of Table I are prepared in which n is 4.

Table I

Alpha, epsilon-N-substituted L-lysinamide derivatives according to Scheme 1 and Formula I (n = 4 in all examples)

The melting points of the compounds of the example are given in Table II

Table II

Melting points of the compounds of Examples 5 to 34

Pr. t.top. [° C] Pr. t.top. [° C] Pr. t.top. [° C] 5 185 15 225 25 syrup, the rest 6 185 16 211-214 26,205 - 210 7 216-220 17 183-186 27 172-177 8 225 18 (Oil) 28 227-230 9 217-220 19 syrup, rest 29 225-229 10 218-222 20 (Oil) 30 233-235 11 208-212 21 (Oil) 31 215-218 12 (Oil) 22 (Oil) 32 155 13 232-236 23 syrup, the rest 33 (Oil) 14 194-198 24 (Oil) 34 (oil

The starting steps of the compounds of the formula I prepared according to Scheme 1 resulting from Table I.

The lysamide Z- (L) used as the starting material for the final preparation steps of Examples 5-34 is commercially available. The substituted aryl- or heteroarylamino-oxoalkanoic acids used as starting materials according to Scheme 1 can be prepared analogously to Scheme 2 by methods known in the literature (P.R. Boy, J. Organ. Chem. 58, 7948, 1993).

ABOUT

Scheme 2

*

A = Aryl, Heteroaryl p = 2-5

The aromatic or heteroaromatic amines A-NH ₂ suitable for the process of Scheme 2 are commercially available; the aminoimidazole [1,2-a] pyridine, which is the basis for the compound of Example 28, can be prepared by methods known in the literature (R. Westwood, J. Med. Chen. 31, p. 1098 (1988)).

Said aryl- or heteroarylamino-oxoalkanoic acids as starting materials may further be prepared by reacting an alkanonomethyl dicarboxylic acid ester, for example a suberic acid monomethyl ester and azelaic acid monomethyl ester as the starting material with an aromatic or heteroaromatic amine in an aminolysis reaction, e.g. boiling ethanol or butanol, or optionally in an aromatic solvent such as toluene or xylene at the boiling point or in an autoclave at the boiling point of the solvent at a pressure of 5 MPa, the reaction solution is concentrated in vacuo and the residue purified by crystallization from methanol or ethanol; or by column chromatography. The eluent is, for example, a mixture of methylene chloride, methanol, ammonia (25%) in a ratio of 85: 15: 1, or a mixture of methylene chloride, methanol, ammonia (25%) in a ratio of 80: 25: 5.

An alternative embodiment of a process for preparing compounds of formula (I) wherein R ^{1 is} benzyloxycarbonyl and R ² and R ³ is hydrogen is as follows:

1. an alpha-carboxylic acid group is amidated,

2. the epsilon-amino group is protected with a Z-group,

3. The alpha-amino group is protected with a Boc-group, thus producing selectivity to the later cleavage of the amino-protecting groups.

4. the Z-group of the epsilon-amino group is cleaved,

5. the desired R ⁴ -CO group is introduced on the epsilon-amino group;

6. cleavage of the Boc group from the alpha-amino group,

7. the Z-group is introduced into the alpha-amino group.

Further compounds of formula I can be obtained according to the following Scheme 3, illustrated by the method for preparing the compound of Example 35:

Scheme 3

Stage 1

THF / EtgN

Stage 2

i DMF INMM

HO

NH

NH,

HCl nmm / bop

DMF

NH,

Y

General process for the preparation of compounds of formula I according to Scheme 3.

Stage 1

To an aprotic or non-polar organic solvent such as tetrahydrofuran, dimethylsulfoxide, dimethylformamide, acetonitrile, ethyl acetate, dimethylacetamide, N-methylpyrrolidone, dioxane, toluene, ether, methylene chloride or chloroform is added at -30 to 30 ° C, preferably -20 ° C. up to 20 ° C and in particular -15 to 5 ° C Z-Lys (BOC) -OH and a base such as triethylamine, diisopropylamine, N-methylmorpholine, N-ethylpiperidine and an aliphatic or aromatic carboxylic acid chloride such as acetyl chloride, isobutyroyl chloride, isovaleroyl chloride, pivaloyl chloride, benzoyl chloride or 4-methoxybenzoyl chloride. After a period of time, for example 30 minutes to 3 hours, a solution or suspension of the amine cooled to -10 ° C in a dipolar aprotic or nonpolar organic solvent such as tetrahydrofuran, dimethylsulfoxide, dimethylformamide, acetonitrile, ethyl acetate, dimethylacetamide, N-methylpyrrolidone, dioxane, toluene, ether, methylene chloride or chloroform. The suspension is stirred for 1 to 2 hours at -30 to 30 ° C, preferably -20 to 20 ° C, especially -15 to 5 ° C. After completion of the reaction, the hydrochloride base is filtered off with suction and the solvent is concentrated. The oily residue is mixed with an aprotic or non-polar organic solvent such as ether, diisopropyl ether, methyl tert-butyl ether, petroleum ether, toluene, xylene, pentane, hexane. The solution is stirred for a period of time, for example 30 minutes to 3 hours, until a white powder precipitates. The precipitate is filtered off with suction and dried.

Stage 2

The Z-Lys (BOC) -amide obtained in step 1 is dissolved in trifluoroacetic acid at -20 to 30 ° C, preferably -20 to 20 ° C, and in particular -15 to 5 ° C, and stirred for 15 minutes up to 1 hour. Excess trifluoroacetic acid is evaporated and the oily residue is mixed with a dipolar aprotic or nonpolar organic solvent such as dimethylformamide, methylene chloride, tetrahydrofuran, acetonitrile, N-methylpyrrolidone and ethyl acetate. The desired acid, base such as diisopropylamine, N-methylmorpholine and a suitable coupling agent such as BOP, PyBOP, DCC in a dipolar aprotic or nonpolar organic solvent such as dimethylformamide, methylene chloride, tetrahydrofuran, acetonitrile, N-methylpyrrolidone and N-methylpyrrolidone are then added. acetate. The reaction is carried out at a temperature of -10 to 100 ° C, preferably 0 to 80 ° C, and in particular 10 to 35 ° C. After a reaction time of 1 to 5 hours and standing at room temperature for 24 hours, the reaction mixture is concentrated. The residue is precipitated with an organic solvent such as isopropanol, methylene chloride or ether. The crude product was purified by silica gel chromatography.

In a general manner according to Steps 1 and 2, the compounds illustrated in Table III below, where n is 4, are synthesized according to the method of Scheme 3.

Table III

Alpha, epsilon N-substituted L-lysinamide derivatives according to Scheme 3 and Formula I (n is 4 each)

Example 35

N- [alpha-N- [benzyloxycarbonyl] -1-epsilone-N- [4 - [(4-amidinophenyl) amino] -4-oxobutanoyl] -L-lysine-N- (3-pyridylmethyl)] amide

Stage 1

N- [alpha] - N- [benzyloxycarbonyl] -epsilone-N- [tert. -butyloxycarbonyl] -L-lysine-N- (3-pyridylmethyl)] -amide

4 g (10 mmol) of commercially available Z-Lys (Boc) -OH, 1 g (10 mmol) of triethylamine and 1.26 g (10 mmol) of pivaloyl chloride are added to 60 ml of tetrahydrofuran at -15 ° C. After 30 minutes, a pre-cooled solution of 1.08 g (10 mmol) of 3- (aminomethyl) pyridine in 20 ml of tetrahydrofuran was added at -10 ° C with vigorous stirring. The suspension is then stirred for 1 to 2 hours at -10 ° C. The triethylamine hydrochloride is suctioned off at low temperature and then the tetrahydrofuran is evaporated. The oily residue is mixed with 100 ml of diethyl ether. The solution was further stirred until a white powder precipitated. The precipitate is filtered off with suction and dried. Yield: 4 g (85% of theory).

Stage 2

N- [alpha-N- [benzyloxycarbonyl] -epsilone-N- [4 - [(4-amidinophenyl) amino] -4-oxobutanoyl] -L-lysine-N- (3-pyridylmethyl)] amide

2 g (4.25 mmol) of N- [alpha-N- [benzyloxycarbonyl] -epsilone N- [tert-butyloxycarbonyl] -L-lysine-N- (0 [deg.] C.) are dissolved in 20 ml of trifluoroacetic acid (TFA). 3-pyridylmethyl) amide and stirred for 20 minutes. The excess trifluoroacetic acid is evaporated and the oily residue is mixed with 10 ml of dimethylformamide. Add 4.6 ml (42.5 mmol) of N-methylmorpholine, 1.15 g (4.25 mmol) of 4 - [[4-aminoiminomethyl) phenyl] amino] -4-oxomasic acid hydrochloride, 2.35 g ( 5.3 mmol) of BOP and 20 ml of dimethylformamide. The reaction mixture was stirred at room temperature for 24 hours. The dimethylformamide is evaporated, the residue is digested twice with 40 ml of water and then filtered off with suction and dried. The crude product was purified by silica gel chromatography eluting with 89b (70% HClCl 2, 40% MeOH, 10% CH ₃ CO ₃ Na ⁺ in 1 mole per liter NH 4 OH 25%). Yield: 340 mg (14% of theory).

Analogously to Example 35, the compounds of Examples 36-55 were obtained.

Table IV

Melting points of the compounds of Examples 35-55

Pr. t.top. [° C] Pr. t.top. [ ⁰ C] Pr. t.top. [° C] 35 190-198 42 190 49 189 36 218-220 43 198 50 197 37 209 44 213 51 38 195 45 52 39 189-191 46 175 53 40 215-220 47 196 54 194 41 183 48 217 55 12 (Oil) 22 (Oil) 32 155 13 232-236 23 syrup, the rest 33 (Oil) 14 194-198 24 (Oil) 34 (oil

Further compounds of formula I are prepared according to Schemes 4 and 5 below

Scheme 4: Reaction with carboxylic acids

Diisopropylcarbodiimide ------------------------> N-Methylaorfoline

+ TFA

ABOUT

- Boe

Scheme 5: Reaction with chloroformic acid esters

ci

Schotten-Baumanrv

---------------- ► Conditions

+ TFA

- Boe

1. Acylation with carboxylic acids or chloroformic acid esters according to Schemes 4 and 5

The resulting amide is treated with H-Lys (Boc) -NH ₂ at room temperature in a dipolar solvent (dimethylformamide, dimethylsulfoxide) in the presence of a base (DIPEA, NMM) and a coupling reagent (DCC, DIC, EDCI) with a carboxylic acid. After removal of the solvent, the residue is mixed with water and the poorly soluble crude product is filtered off with suction. The product can be purified by crystallization from an alcohol (e.g. methanol, ethanol or isopropanol) or an ester (e.g. ethyl acetate) or a ketone (e.g. methyl ethyl ketone).

Reaction of H-Lys (Boe) -NH ₂ with carboxylic acid chlorides leads in 90% to 95% yield in aqueous alkaline solution (Schotten-Baumann conditions) to the desired derivatives. The crude product is isolated by suction and purified by recrystallization from alcohol (methanol / ethanol) or ethyl acetate or methyl ethyl ketone.

2. Cleavage of the TFA Boc protecting group

The cleavage of the Boc protecting group at room temperature in dichloromethane / trifluoroacetic acid (2: 1) is quantitative after about 60 minutes. The isolated crude product R ¹ -Lys-NH ₂ reacted rapidly without further purification.

3. Acylation of 4 - ((4- (aminoiminomethyl) phenyl) amino) 4-oxobutyric acid hydrochloride

The reaction with another carboxylic acid (R ⁴ ) is carried out in dipolar aprotic solvents (dimethylformamide, dimethylsulfoxide) in the presence of a base (NMM, DIPEA) with coupling reagents such as EDCI, Bop or PyBop. After removal of the solvent, the product precipitated with the addition of water. Purification was by preparative HPLC on a pure RP ₁₈ "column using a mixture of water, acetonitrile and trifluoroacetic acid as eluent. The product precipitated as a TFA salt.

According to this general formula, based on Schemes 4 and 5, the compounds illustrated in Example 56 and the following Table V are synthesized.

Example 56

To 120 ml of dried, degassed N, N-dimethylformamide (DMF) was added at room temperature 32 mmol of Z-lysine amide hydrochloride and 32 mmol of 4 - ((4- (aminomethyl) phenyl) amino-4-oxobutyric acid hydrochloride). After the addition of 104 mmol of diisopropylethylamide and 40 mmol of BOP, the mixture was stirred at room temperature for 16 hours.

The solvent and excess DIPEA are drawn off on a rotary evaporator at a bath temperature of 50-55 ° C and at a pressure of 10 bar. The oily residue is mixed with 250 ml of water, homogenized in an ultrasonic bath and cooled. The precipitated end product is filtered off with suction and washed with water in a suction cup.

After drying in vacuo of calcium chloride, about 16 g of a beige powder with a purity of about 90% (HPLC) are obtained as the hydrochloride salt.

To prepare the corresponding trifluoroacetate, the product is suspended in 100 mL of water and mixed with 32 mmol (2.45 mL) of trifluoroacetic acid (99%). To remove excess acid, the mixture is evacuated on a rotary evaporator and then the aqueous suspension is lyophilized. After recrystallization from alcohol (EtOH / MeOH), the product obtained can be lyophilized again for better solubility. Yield 5.26 g, mp 210-213 ° C.

¹ H-NMR (500 MHz, DMSO-d 6, δ in ppm):

10.47, s, 1H anilide, 9.14 and 8.82 s, NH amidine, 7.82 m, 1H, Lys-epsilon-NH, 7.79 and 7.46, 2s, aromatic H, 7, 27 and 6.93 2s, 2H, _CONH2, 7.20 d, 1 H, urethane NH, 5.0, s, 2H, benzyl.H, 3.89, m, 1H, calf-H, 3.0 and 2.58 and 2.40, 3m, total 6H, aliphatic.H, 1.60-1.20, 4m, total 6H, aliphatic residue. H

Tabuika 5

Alpha, epsilon-N-substituted L-lysinamide derivatives according to Schemes 4 and 5 and Formula I (n = 4 each)

Table 6

Melting points of the compounds of Examples 56-82

Pr. t.top. [° C] Pr. t.top. [° C] Pr. t.top. [’C] 56 210-213 65 218-222 74 191-193 57 220-223 66 216-219 75 186-188 58 213-215 67 235-238 76 220-222 59 223-226 68 to 218 77 210-215 60 to 233 69 205-208 78 to 223 61 to 237 70 168-170 79 to 226 62 to 221 71 197-202 80 194-197 63 to 220 72 221-226 81 215-222 .64 230-236 73 225-228 82 219-222

Note: The data up to indicates that the substances, after freeze-drying, form an amorphous foam with corresponding physical properties. The melting point does not strictly take place, rather slow sintering until liquefaction.

Salts of compounds of formula I

The compounds of the invention may be in the form of an acid addition salt, for example, as a mineral acid salt such as hydrochloric, sulfuric, phosphoric, organic acid salts such as acetic, trifluoroacetic, lactic, malonic, maleic, fumaric, gluconic, glucuronic, citric, embonic, methanesulfonic, hydroxyethanesulfonic, pyruvic and amber.

Both the compounds of formula I and their salts are biologically active. The compounds of formula I may be administered in free form or as salts with physiologically acceptable acids. Administration can be oral, parenteral, intravenous, transdermal or inhalation.

The invention further relates to pharmaceutical compositions comprising at least one compound of the formula I or a salt thereof with physiologically acceptable inorganic or organic acids and, optionally, pharmaceutically acceptable carriers and / or diluents, optionally with auxiliaries.

Example 83

Cetrorelix Binding Affinities, Example 1, Example 2, Example 5, Example 35 and Example 56 at the Human LH-RH Receptor (Cetrorelix: Ac-D-Na (2) -Dp-Cl-Phe-D-Pal (3) -Ser _{Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2)}

Method of determining binding affinity (dissociation constant Kd):

Binding affinity is determined by a competition binding assay (displacement binding experiment, Becker et al. Eur. J. Biochem. 231: 535-543, 1995). [ ¹²⁵ J] Cetrorelix (specific activity 5-10x10 ⁵ dpm / pmol; dissolved in 20% v / v acetonitrile, 0.2% w / v albumin, 0.1% w / v TFA) is used as the radiolabeled ligand. (about 80% water by volume). The binding ability of the iodinated peptide is 60 to 85%. As unlabeled test compounds, the compound of Example 1, Example 2 and Example 56 in solution were used. The substances are used at concentrations of 0.01 nM to 1000 nM (Cetrorelix, Example 1, Example 2) or 0.01 µΜ to 10 µΜ (Example 5, Example 35, Example 56).

The cells of the single cell L3.5 / 78 clone displacing the human LH-RH receptor used for the binding assay were separated from PBS with the aid of PBS / EDTA (PBS without Ca ²⁺ / Mg ²⁺ / 1mM EDTA) and counted. cells at the corresponding cell density, are resuspended in incubation medium (modified Eagle Medium Dulbecco with 4.5 g / l glucose, 10 mM Hepes, pH 7.5, 0.5% w / v BSA, 1 g / l Bacitracin, 0). , 1 g / 1 SBTI, 0.1% w / v NaNg) 200 μΐ of silicone / paraffin oil (84/16 v / v) are added to special 400 μΐ reaction vessels (Renner type Beckman) and then Pipette 50 μΐ of cell suspension (2.5x10 ⁵ cells). To the cell suspension on the silicone / paraffin oil layer is then immediately added 50 μΐ of [ ¹²⁵ J] Cetrorelix binding medium and test compounds at the appropriate concentration. It is now incubated for 60 minutes in a heated cabinet at 35 ° C while rotating. This operation is followed by centrifugation in Heraeus Biofuge 15 in an HTA rotor 13.8 2 minutes at 9000 rpm / room temperature). Due to the silicone / paraffin oil layer, the cells pellet and are thus separated from the binding medium. After centrifugation, the reaction vessels are snap frozen in liquid nitrogen and the tip of the reaction vessel (cell pellet) is cut off with ticks and the cell pellet tip (bound [ ¹²⁵ J] Cetrorelix ligand) as well as excess cell pellet (bound [J] Cetrorelix ligand) and excess The cell pellet (unbound, free ligand of [? ² ? J] Cetrorelix) is transferred to a counting tube. A competitor is not added to determine non-specific binding (Bo). To determine non-specific binding, 1 μ 1 of unlabelled Cetrorelix is added to the competition. The non-specific binding is equal to or less than 10% of the total Bo formed and is thus low. The gamma counter, quantified with EBDA / Ligand V3.0 (McPherson, J. Pharmacol. Methods 14, 213-228, 1985), serves to quantify. Plotting the dose effect allows the IC ₅₀ concentration (the concentration that causes 50% inhibition of the receptor response) to be assessed, and the EBDA / Ligand program calculates a dissociation constant K d [nM] therefrom.

Result: It is evident from the competition curves (see Figure 1) that all test compounds with the radiolabeled [ ¹²⁵ J] ligand (Cetrorelix) competed for binding to the human LH-RH receptor. The binding (in% of total Bo binding) to the concentration of the competitor is always plotted. For the compounds shown in FIG. 1, the following binding affinities calculated as the dissociation constant Kd [nM:] could be calculated: Cetrorelix (SB-75): 0.214 nM, Example 1: 0.305 nM, Example 2: 0.104 nM, Example 5: 108 nM, Example 35: 300 nM and Example 56: 1082 nM. The binding affinities as the mean of the various assays are shown in Table VII.

Example 84

Antagonist activity of the compound of Example 2 and Example 56 in the human LH-RH receptor functional assay

IP ₃ (D-myo-1,3,5-triphosphate) assay method: Subconfluent culture of the human LH-RH receptor expressed cell clone (L 3.5 / 78) is washed once with PBS, cells are released with PBS / EDTA and cellular the suspension is pelleted. Cells are resuspended in incubation medium (modified ulle medium Dulbecco s

4.5 g / l glucose, 10 mM Hepes, pH 7.5, 0.5% w / v. BSA, 5 mM LiCl, 1 g / L bacitracin, 0.1 g / L SBTI), aliquoted in 1.5 mL reaction flasks and preincubated at 37 ° C for 30 min. 4x10 ⁶ cells in a volume of 500 μΐ per measurement point are needed. The preincubation operation is followed by the addition of LH-RH (0.5 mM stock solution in 10 nM Tris, pH 7.5, 1 mM dithiothreitol, 0.1 w / v BSA / Bachem Art # H4005) to the cell suspension at the final concentration 10 nM. The antagonist effect is tested by simultaneous addition of the appropriate concentration (e.g. 0.0316, 0.1, 0.316 and the like to 100 nM for Example 2). As a negative control, cells without added LH-RH are incubated. After incubation for 15 minutes at 37 ° C, the generated IP _{3 is} isolated from the cells by extraction with trifluoroacetic acid (TCA). Add 500 μΐ of ice-cold 15% (w / v) TCA solution to the cell suspension. The resulting precipitate is pelleted by centrifugation at 4 ° C for 15 minutes at 2000 xg in a Heraeus 15R bio-centrifuge. Excess 950 μΐ is extracted three times with 10 volumes of cold water saturated diethyl ether in a 15 ml flask by standing on ice. After the last extraction operation, the pH of the solution was adjusted to 7.5 with 0.5M sodium bicarbonate solution.

The determination of the IP ₃ concentration in the cell extracts is performed using a sensitive competitive binding assay with the IP ₃ binding protein, labeled with [ ³ H] -IP3 and unlabeled IP3. For this purpose, an Amersham test kit (TRK 1000) is used, according to the test protocol described. After performing the appropriate operations, 2 ml of water scintillator (Rotiszint Ecoplus) is finally added, the suspended pellet bound [ ³ H] -IP3 is mixed gently and measured by a beta-scintillation counter. The amount of cellular IP ₃ is calculated using a standard curve and a dose efficiency curve is constructed. IP ₃ can be estimated from the inflection point of the curve.

Result: In FIG. 1, the corresponding dose-effectiveness curves for both the peptide antagonist of Example 2 (FIG. 2) and the peptidomimetic of Example 56 (FIG. 3) are shown. 10nM LHRH is simulated and the inhibition of IP ₃ formation is determined in dependence on substance concentration. No agonist activity could be determined for Example 2 and Example 56, i.e. the substances themselves do not lead to a simulation of synthesis ΙΡ _3. In control experiments not reported here, it has been shown that untransfected cells cannot be simulated with LH-RH for IP ₃ synthesis. Even measurable peak concentrations of IP ₃ correspond to those of unstimulated cells. Thus, Example 2 and Example 56 are functional LH-RH antagonists. However, the substances differ in potency. The selected test conditions, the _IC50 of Example 2 of approximately 0.4 nM, the _IC50 of Example 56, however, is about 4μπι. These activities are very well correlated with Kd = 0.109 nM for Example 2 and Kd = 1.08 µ l for Example 56 as determined by the Cetrorelix [ ¹²⁵ J] competitive binding assay.

Example 85

Hormones suppressing the effect of the compounds of Example 1, Example 2 and Example 56 in healthy male rats.

To determine the suppression of testosterone in the blood of healthy male rats, the substance is injected subcutaneously into the right valve. The dosage is 1.5 mg / kg for Example 1 and 2, and 10 mg / kg for Example 56. To control testosterone values, 300 μΐ of blood is taken from the vein of the sublingualis vein at 0, 2, 4, 8 (only for Example 56), 24, 48, 72, and 96 hours, and then each

Day 3 until the end of suppression. Suppression below 1 ng / ml of testosterone lasted up to 264 hours in a single animal for up to 264 hours, up to 336 hours in two animals and 384 hours in one animal (Fig. 4). After dosing of the compound of Example 2, the testosterone level was suppressed in one animal for 408 hours, in 4 animals up to 648 hours (Fig. 5). Example 56 (10 mg / kg s.c.) suppressed testosterone levels in all five animals as early as 2 hours and this effect was maintained for up to 8 hours. At the next test time (24 hours), testosterone levels increased again (Fig. 6).

Table 7

Biological values

Human LH-RH receptor binding affinity (expressed as dissociation constant Kd [nM]; evaluation by EBDA / ligand analysis program. Mean values from various experiments (number of trials in parentheses) as well as testosterone suppression in vivo, histamine release in vivo and water solubility compared to SB-75:

affinity (1.5 mg / kg ^h 2 ° human one dose) release solubility LH-RH-recipe. suppression histamine [Mg (ml] [Nmol / 1] testosterone in rats | ^ G / ml] [H]

cetrorelix SB-75 0.202 (10) 144 9.7 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 N.B. * N.B. Example 4 0,206 (2) 696 N.B. N.B. Example 5 108 (2) - - - Example 35 300 (2) - - - Example 56 1082 (2) -  -

* not determined for solubility

Industrial usability

A compound for the manufacture of pharmaceutical compositions for the treatment of hormone-dependent tumors, especially prostate or breast cancer, and for the treatment of benign indications whose treatment requires the inhibition of LH-RH.

Claims (18)

PATENT CLAIMS A compound of formula I R ¹ - CO - NH - CH - CO - N - R ³ I (CH ₂ ) _n (I) I NH CO-R ⁴ where n is 3 or 4, R ^{1 is} optionally substituted alkyl, alkyloxy, aryl, heteroaryl, aralkyl, heteroaralkyl, aralkyloxy or heteroaralkyloxy, R ² and R ³ are independently hydrogen) optionally substituted alkyl , aralkyl or heteroaralkyl, with aryl or heteroaryl being suitable as substituents, or -NR ² R ³ is an amino acid group, and R ⁴ is a group of formula II (CH ₂ ) _p -CO (II) wherein p is an integer of 1 to 4, R ⁵ is a hydrogen atom or an alkyl group and R 0 is an optionally substituted aryl or heteroaryl group, whereby the aryl or heteroaryl group is, in turn, or R ⁴ represents a ring of the formula III _χ * 7 where q is q 1 or 2, R is H or alkyl, R ⁸ is H or alkyl and X is O or p hole, wherein the aromatic or heteroaromatic radicals can be partially or completely hydrogenated and chiral carbon atoms may be in the D or L configuration, and their salts with pharmaceutically acceptable acids. Compound according to claim 1, which is alpha-N- [benzyloxycarbonyl] -epsilone-N- [5 - [(4-amidinophenyl) amino] -5-oxo-pentanoyl] -L-lysinamide trifluoroacetate. A compound according to claim 1, which is alpha-N- [benzyloxycarbonyl] -epsilone-N- [5 - [(4-amidinophenyl) amino] -4-oxo-butanoyl] -L-lysine amide trifluoroacetate. 4. A compound according to claim 1, which is alpha-N- [benzyloxycarbonyl] -epsilone-N- [5 - [(4-amidinophenyl) amino] -4-oxobutanoyl] -L-lysine-N- ( 3-pyridylmethyl) amide trifluoroacetate. A compound according to any one of claims 1 to 4 in the form of an embonate salt. A compound according to claim 1 of formula V Ac-D-Nal (2) ¹ -D (pCl) Phe ² -D-Pal (3) ³ -Ser ⁴ -Tyr ⁵ -D-Xxx ⁶ -Leu ⁷ -Arg ⁸ Pro ⁹ -D-Ala ¹⁰ -NH ₂ (V) wherein D-Xxx is an amino acid group of Formula VI - HN - CH - CO - 0 I ( ^CH 2> n I (VI) NH I CO - R ⁴ where n is 3 or 4, R ^{4 is a} group of formula II < ^CH 2> p CO (II) wherein p represents an integer from 1 to 4, R ⁵ represents a hydrogen atom or an alkyl group and R ^{6 represents an} optionally substituted aryl or heteroaryl group, or R ⁴ represents a ring of formula III (III) wherein q represents 1 or 2, R R @ ⁷ is hydrogen or alkyl, and R @ ⁸ is hydrogen or alkyl, and X is oxygen or sulfur, and salts thereof with pharmaceutically acceptable acids. A compound according to claim 6, wherein Xxxx is [epsilon-N-4- (4-amidinophenyl) amino-1,4-dioxobutyl] lysyl. 8. A compound according to claim 6, wherein Xxxx is [epsilon-N- (imidazolidin-2-on-4-yl) formyl] lysyl. Compound according to claims 6 to 8 of the general formula V in the form of an embonate salt. A process for the preparation of a compound according to claim 6, wherein the individual symbols are as defined in claim 6, characterized in that (a) they are introduced into the alpha-amino group and into the carboxylic acid group of D-lysine or D-ornithine (b) reacting D-lysine or D-ornithine with protected carboxylic acid groups of formula VII R ⁴ - COOH (VII) wherein R ⁴ is as defined above,> (c) cleaving the alpha-carboxylic acid protecting groups of the compound obtained in step b) for incorporation at the 6-position by operation (h), (d) binding the amino-protected D-alanine to a solid resin carrier; (e) the amino groups of the alanine are cleaved (f) reacting the alanine bound on the solid support with a proline, protected nitrogen atom, (g) deprotecting the proline nitrogen atom, (h) repeating operation f) and g) with amino acids 1-8 of formula V in the order of 8 to 1 using the modified D-lysine or D-ornithine described in operation (c) for position 6, (i) cleaving the compound obtained in operation (h) from the carrier and optionally (j) optionally reacting the compound with a pharmaceutically acceptable acid, preferably embonic acid. A process for the preparation of a compound according to claim 6, wherein the individual symbols are as defined in claim 6, characterized in that (a) the amino-protected D-alanine is coupled to a carrier suitable for solid phase synthesis, (b) (a) reacting the alanine bound to the proline with a protected nitrogen atom; (d) removing the protecting group from the proline nitrogen atom; (e) repeating operation (c) and (d) with amino acids (F) cleaving off the compound obtained in step (e) from the carrier; (g) reacting with a carboxylic acid of formula (VII) R ⁴ - COOH (VII) wherein R ⁴ is as defined above, (h) optionally, the compound is reacted with a pharmaceutically acceptable acid, preferably embonic acid. A process for the preparation of a compound according to claim 6, wherein each symbol is as defined in claim 6, characterized in that (a) the amino-protected D-alanine is coupled to a carrier suitable for solid phase synthesis, (b) the amino-protecting group of alanine is removed, (c) the alanine bound to the proline-protected proline resin is reacted, (d) the proline nitrogen protecting group is removed, (e) operation c) and d) are repeated with amino acids 6 (f) cleaving the epsilon-amino protecting group of Dlysine or D-ornithine at the 6-position, and reacting with the carboxylic acid of formula (VII) R ⁴ - COOH (VII) wherein R ⁴ is as defined above, (g) the alpha-amino protecting group of D-lysine or D-ornithine is cleaved, (h) operation (c) and (d) are repeated with amino acids 1 to 5 of general (i) cleaving the compound obtained in operation (h) from the resin and purifying, in particular by HPLC chromatography, (j) optionally reacting the compound with a pharmaceutically acceptable acid, preferably embonic acid. 13. The process according to claim 10, wherein N- (4-amidinophenyl) amino-4-oxobutyric acid is used as the carboxylic acid of formula VII. 14. The process according to claim 10, wherein imidazolidin-2-one-4-carboxylic acid is used as the carboxylic acid of formula (VII). A process according to claims 10 to 14, characterized in that embonic acid is used for the preparation of a pharmaceutically acceptable salt. 16. A pharmaceutical composition comprising, as an active ingredient, a compound according to claims 1-9. Use of a compound according to claims 1 to 9 for the manufacture of pharmaceutical compositions for the treatment of hormone-dependent tumors, in particular prostate or breast cancer, and for the treatment of benign indications whose treatment requires LH-RH suppression. A process for the manufacture of pharmaceutical compositions comprising as active ingredient a compound according to claims 1 to 9, characterized in that the compound according to claims 1 to 9 is mixed with conventional carriers and auxiliary agents and the mixture is formulated as a pharmaceutical composition.