Antagonists releasing factor, luteinizing hormone (lh - rf) (options), the retrieval method (variants), pharmaceutical composition and method of preparation of medicines

 

(57) Abstract:

The invention relates to novel antagonists of LH-RF, and above all, peptidomimetics and peptide with a modified side chain, of the formula V, where D-Xxx, R1- R8described in the description, and their salts with pharmaceutically acceptable acids, as well as it describes their therapeutic use as analogue releasing factor, luteinizing hormone (LH-RF) with high antagonistic activity and not detecting unwanted side effects, especially not promote swelling. Also described is a method of obtaining the compounds of formula V by solid-phase method or partially solid-phase method and therapeutic composition with high affinity to the receptor releasing factor, luteinizing hormone, comprising as active substance connections PP. 1 to 4. It also describes the method of preparation of pharmaceutical preparations containing the compounds according to paragraphs.1 to 9, characterized by the fact that these compounds are mixed with auxiliary substances. 6 C. and 11 C.p. f-crystals, 7 tab., 6 Il.

Ac-D-Nal(2)1-D-(pCl)Phe2-D-Phe(3)3-Ser4-Tyr5-D-Xxx6-heu7-Arg
p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, [LH-RF, gonadorelin].

Research potential selective antagonists Decapeptide LH-RF conducted for more than 20 years [M. Karten und J. E. Rivier, Endocrine Reviews 7, 44-66 (1986)]. The increased interest in such antagonists is associated with the need for these drugs in some areas of medicine: endocrinology, gynecology, contraception and Oncology. Was synthesized by a large number of compounds representing potential antagonists of LH-RF. The biggest interest among synthesized to date, compounds cause connection with the modified structure of LH-RF.

The first series of potential antagonists was obtained by the introduction of esters of aromatic amino acids in positions 1, 2, 3, and 6 or 2, 3, and 6. A common way to describe such modified analogues is the following: first of all indicate amino acid residue, which replace the existing balance of LH-RF, and numerals in Superscript index indicates the position number Eoisode replacement you will use the abbreviation "LH-RH" (LH-RF).

Known antagonists are:

[Ac-D-Phe(4-Cl)1,2, D-Trp3,6] LH-RH (D. H. Coy et al., In: Gross, E. and Meienhofer, J. (Eds) Peptides: Proceedings of the 6th American Peptid Symposium, S. 775-779, Pierce Chem.Co., Rockville III. (1979);

[Ac-Pro1, D-Phe(4-Cl)2, D-Nal(2)3,6] LH-RH (US-Patent Nr.4.419.347) and [Ac-Pro1, D-Phe(4-Cl)2, D-Trp3,6] LH-RH (J. L. Pineda, et al., J. Clin. Endocrinol.Metab. 56, 420, 1983).

Later, with the aim of increasing the solubility of the antagonists in water was carried out introduction residues of basic amino acids such as D-Arg in position 6. For example, [Ac-D-Phe(4-Cl)1,2, D-Trp3D-Arg6, D-Ala10] LH-RH (ORG-30276) (D. H. Coy, et al., Endocrinology 100, 1445, 1982) and [Ac-D-Nal(2)1, D-Phe(4-F)2, D-Trp3D-Arg6] LH-RH (ORF 18260) (J. E. Rivier et al., in: Vickery, B. H. Nestor, Jr.J.J., Hafez, E. S. E (Eds). LHRH and its Analogs, S. 11-22 MTP Press, Lancaster, UK 1984).

Such analogs are characterized not only expect high solubility in water, but high antagonistic activity. However, these highly hydrophilic analogues containing D-Arg, and other basic side chain at position 6, cause temporary swelling of the face and extremities after subcutaneous administration to rats at a dose of 1.25 or 1.5 mg/kg (F. Schmidt, et al., Contraception 29, 283, 1984: J. E. Morgan, et al., Int.Archs.Allergy Appl. lmmun. 80, 70 (1986). Other potentially is">

Due to stimulation of swelling, found in the test some of these antagonists in rats have doubts about the safety of use of these compounds in relation to man and therefore delayed their introduction into clinical practice in the quality of medicines.Thus there is a need for antagonistic peptides, which have no side effects.

According to the invention this problem is solved with the use of compounds of General formula I

< / BR>
where n = 4, R1- alkyloxy, aryl group, kalkilya group, heteroalkyl group, aralkylated group or heteroaromatic group, in each case unsubstituted or substituted, R2and R3- independently of each other depending on the necessity mean a hydrogen atom, alkyl group, aracelio group or heteroalkyl group, depending on the need unsubstituted or substituted, with the substitution, in turn, may include aryl group or heteroaryl group, or-NR2R3represents an amino acid residue, and R4group of the formula (II)

< / BR>
where p is an integer from 1 to 4, R5- water is the Deputy in turn, may include aryl group or heteroaryl group, or R4is a ring of General formula (III):

< / BR>
in which q is a number 1 or 2, R7and R8are hydrogen atoms, and X represents a sulfur atom, and an aromatic or heteroaromatic residues can exist in a completely gidrirovannoe form, and chiral carbon atoms are of L-configuration; and salts of these compounds with pharmaceutically acceptable acids.

Preferred combinations of residues R1to R4are the following:

a) R1- benzyloxy, R2is hydrogen and R3is hydrogen,

b) R1- benzyloxy, R2is hydrogen and R4group of the formula II in which p is 2 or 3, R5is hydrogen and R6- 4-amidinophenoxy group, and

in R2is hydrogen, R3is hydrogen and R4group of the formula II in which p is 2, R5is hydrogen and R6- 4-amidinophenoxy group.

Alkyl groups are preferably methyl-, ethyl-, n-propyl-, isopropyl-, n-butyl-, isobutyl-, tert-butyl, 2-ethylhexyl, dodecyl-and hexadecyl group.

Aryl groups are preferably phenyl-, naphthyl-, Henan 4 pirimidil-, imidazolyl, imidazopyridine-, 5 - and 6-indolyl-, 5 - and 6-indazole-, triazolyl-, tetrazolyl-, benzimidazole-, chinolin-, 2,6 - dichloropurine-3-yl - and furyl group.

Gidrirovanie heteroaryl groups are preferably piperidine, piperazinil, morpholino and pyrrolidinyl group.

Uralkalij groups and heteroalkyl groups are groups that are connected in a suitable position through alkylenes group, preferably methylene, ethylene, n-propylene or n-butylene group.

Preferred substituents along with the aforementioned aryl - and heteroaryl groups are halogen atoms such as fluorine, chlorine, bromine and iodine, and methyl-, ethyl-, isopropyl-, tert-butyl, cyano-, nitro-, residues of carboxylic acids, carboxylic acid amide, methyl ester carboxylic acid, ethyl ester of carboxylic acid, ethyl ester of crotonic acid, trifluoromethyl-, benzoyl-, methoxy-, benzyloxy-, pyridyloxy-, amino-, dimethylamino-, isopropylamino-, amidino and chinaillon group.

Further, according to the invention, use of the compounds of General formula (V)

[Ac-D-Nal(2)1-D(pCl)Phe2-D-Pal(3)3-Ser4- Tyr5-D-Xxx6-Leu7-Arg8-ProSUP>4
, R5, R6, R7, R8and X is as defined above, and their salts with pharmaceutically acceptable acids with the above properties.

Claimed according to the invention the compounds are characterized by high antagonistic activity and do not have undesirable side effects, first of all do not cause swelling. If they are not in the form of salts with membrane not easily soluble in water, pharmaceutically acceptable acids, these compounds also have a high solubility in water. These compounds are characterized by high affinity to the receptor LH-RF, that is, are highly active in inhibiting the release of gonadotropins from the pituitary gland of mammals, including humans: when tested on rats reveal prolonged inhibition of release of testosterone and cause minimal release of histamine in vitro.

Compounds of General formula (1) preferably are: - N-Z-[-N-4-(4-amidinophenoxy)-amino-1,4-DIOXOLANYL]-osiname, - N-Z-[N'-(imidazolidin-2-he-4-yl)-formyl]- osiname.

Peptides of the formula V are preferably such compounds, in which Xxx - [- N-4-(4-amidinophenoxy)-amino-1,4 - DIOXOLANYL]-lysyl-group or [-poorly soluble in water. Particularly preferred salts of 4,4'-methylene-bis(3-hydroxy-2-Neftyanoi acid), also known as albanova or amoeba acid.

The nomenclature used to refer to peptide corresponds to the rules of biochemical nomenclature, published by the Administrative Council IUPAC(IUPAC-IUB) (European J. Biochem., 1984, 138, 9-37), according to which the standard method of presenting peptide is N - terminal amino acid residue on the left and the C-terminal on the right. Claimed according to the invention, the peptides and peptide mimetics, which are antagonists of LH-RF, contain natural amino acids and synthetic amino acids, and the former include Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met, Phe, Tyr, Pro, Trp and His. Abbreviation of single amino acid residues is based on the trivial names of the amino acids: Ala, alanine, Arg is arginine, Gly is glycine, Leu is leucine, Lys is lysine, Pal(3) - 3-(3-pyridyl)alanine, Nal(2) - 3-(2-naphthyl)alanine, Phe is phenylalanine, (pCl)Phe - 4-chlorophenylalanine, Pro is Proline, Ser is serine, Thr is threonine, Trp - tryptophan and Tyr - tyrosine. All the above amino acids are the L-series, if not specified otherwise. For example, the abbreviation D-Nal(2) is by reduction from 3-(2-naphthyl)-D-alanine and Ser - L-serine. Below other is hexaphosphate; DCC - dicyclohexylcarbodiimide; DCM is dichloromethane; Ddz - acid-dimethylethylenediamine (dimethoxymethyl-Z), DIC diisopropylcarbodiimide; DIPEA IS N,N-diisopropylethylamine, DMF is dimethylformamide; Fmoc - fluorenylmethoxycarbonyl; HF - liquid anhydrous hydrofluoric acid; HOBt is 1-hydroxybenzotriazole; HPLC - liquid chromatography high pressure (ghvd); Rover - benzotriazol-1-yl-oxy-Tris-pyrrolidinone hexaphosphate; TFA - triperoxonane acid; Z - benzyloxycarbonyl.

According to the invention compounds of General formula (I) are obtained as follows: first of all protect two of the three functional groups (amino, - -amino group and carboxyl group), and free unprotected group involved in the condensation reaction. Under certain conditions, if this is possible and leads to better results in the first stage, introducing an intermediate protective groups are removed after the second stage to obtain the desired functional group. Suitable protective groups and methods for their introduction are known in the literature. For example, protective groups described with the following "Principles of Peptide Synthesis", Springer Verlag 1984), Lehrbuch "Solid Phase Peptide Synthesis" J. M. Steward and J. D. Young, Pierce Chem.Company, R is resolved or the classic method of condensation of fragments, either by using the solid phase method of Merrifield, with a gradual build-up of the peptide chain and used as a starting compound D-lysine acylated on the side of the group of carboxylic acid of General formula VII, as well as by providing reaction Decapeptide unit with the corresponding carboxylic acids with formation of the amide bond in the side chain of D-lysine6. In this regard, according to the invention, there are two alternative ways to obtain the compounds of General formula (V).

The first alternative method for obtaining compounds of General formula (V) includes the following stages:

a) binding of D-alanine containing blocked amino group, with a suitable substrate for solid-phase synthesis,

b) removing the protective group from the amino group of alanine,

C) securing the condensation reaction of alanine associated with the substrate, with the Proline containing a protected nitrogen atom,

g) removing the protective group from the nitrogen atom of Proline,

d) repeating stages C) and d) using the amino acids from the first to the eighth according to the General formula (V) in order from the eighth to the first,

(e) removal of the compound obtained in stage d), the substrate,

g) providing the reaction of conden the C) optionally, providing the condensation reaction with a pharmaceutically acceptable acid, preferably monowai acid, with formation of salts.

The second alternative method of synthesis of compounds of General formula (V) includes the following stages:

a) binding of D-alanine containing blocked amino group, with a suitable substrate for solid-phase synthesis,

b) removing the protective group from the amino group of alanine,

C) securing the condensation reaction of alanine associated with the substrate, with the Proline containing a protected nitrogen atom,

g) removing the protective group from the nitrogen atom of Proline,

d) repeating the stages b) and d) using the amino acids from the sixth to the eighth according to the General formula (V) in order from eighth to sixth,

e) removing the protective group of the amino group of D-lysine or D-ornithine in position 6 and the provision of condensation with a carboxylic acid of formula (VII)4-COOH, where R4matter, as stated above,

g) removing the protective group of the amino group of D-lysine or D-ornithine,

C) repeating the stages b) and d) using the amino acids from the first to the fifth according to the General formula (IV) in order from fifth to first,

I) removal of the compound obtained in stage C), with the substrate and cleaning (for example, ghvd),

K) optionally, providing the reaction is Oli.

The third alternative method of synthesis of compounds of General formula (V) includes the following stages:

a) binding of D-alanine containing blocked amino group, with a suitable substrate for solid-phase synthesis,

b) removing the protective group from the amino group of alanine,

C) securing the condensation reaction of alanine associated with the substrate, with the Proline containing a protected nitrogen atom,

g) removing the protective group from the nitrogen atom of Proline,

d) repeating the stages b) and d) using the amino acids from the sixth to the eighth according to the General formula (V) in order from eighth to sixth,

e) removing the protective group of the amino group of D-lysine or D-ornithine in position 6 and the provision of condensation with a carboxylic acid of formula (Vll)4-COOR, where R4matter, as stated above,

g) removing the protective group of the amino group of D-lysine or D-ornithine,

C) repeating the stages b) and d) using the amino acids from the first to the fifth according to the General formula (IV) in order from fifth to first,

I) removal of the compound obtained in stage C), with the substrate and cleaning (for example, ghvd),

K) optionally, providing the condensation reaction with a pharmaceutically acceptable acid, preferred is (VII) are: imidazolidin-2-he-4-carboxylic acid and N-(4-amidinophenoxy)-amino-4-oxomalonate acid.

The compounds of formula (V) are synthesized in accordance with known techniques, for example, when using a solid-phase method, a partially solid-phase method or the classical synthesis in solution (see M. Bodanszky, "Principles of Peptide Synthesis" Springer Verlag 1984). For example, solid-phase synthesis methods described in the manuals Lehrbuch "Solid Phase Peptide Synthesis" J. M. Steward and J. D. Young, Pierce Chem.Company, Rockford, III, 1984, and G. Barany and R. B. Merrifild "Peptides", Ch.1, S. 1-285, 1979, Academic Press Inc. The methods of classical synthesis in solution is described in detail in the manual "Methods der Organischen Chemie (Houben-Weyl), Synthese von Peptiden" E. Wunsch (Herausgeber) 1974, Georg Thieme Verlag, Stuttgart, BRD.

Stepwise synthesis is carried out, for example, as follows: first C-terminal amino acid with a protected amino group covalently attached to a well-known insoluble substrate, removing the protective group from the amino group, this amino acid and thus obtained the free amino group attached the following amino acid with blocked amino group, then in the same way, stage by stage, attach the remaining amino acids in the correct order, and after joining all the amino acids of the finished peptide is removed from the substrate. Stepwise condensation is carried out according to standard methods with the use of the use of automatic peptide synthesizer, for example, type Labortec SP 650 (Bachem, Switzerland) using commercial preparations of protected amino acids.

The binding of amino acids to each other is carried out in accordance with conventional methods, in particular can be used the following methods:

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

- General carbodiimide method;

- carbodiimide-hydroxybenzotriazole method (see The Peptides, Volume 2, Ed. E. Gross and J. Meienhofer). To attach arginine preferably using carbodiimide method. For other amino acids is mainly used methods symmetrical or mixed anhydrides.

To connect fragments preferably used azide method, not involving racemizations, or DCC-1-hydroxybenzotriazole, or DCC-3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine methods. To connect fragments can also be used and the method of activated esters.

At step condensation is particularly preferable to apply the activated esters of N-protected amino acid, for example, N-hydroxysuccinimidyl ether or 2,4,5 - trichlorophenoxy ether. For the catalysis of aminolysis effective, for example, 1-hydroxybenzotriazole.

As an interim protective groups use group removed by hydrogenolysis, for example, benzyloxycarbonyl group (Z radical) or group otsepleniya in a slightly acidic environment. As the protective groups of the amino groups are used, for example, tert - butyloxycarbonyl group, carbobenzoxy group or carbobenzoxy group (optionally, in each case containing n-bromo - or p-nitrobenzyl-group), TRIFLUOROACETYL-, phthalyl-about-nitrophenoxyacetic, trityl-, p-toluensulfonyl-, benzyl group, substituted in besolova the nucleus of the benzyl groups (p-bromo or p-nitrobenzyl), - phenylethyl groups (see guidance Jesse P. Greenstein and Milton Winitz, Chemistry of Amino Acids, New York, 1961, John Wiley and Sons, Inc., Volume 2, p. 883, and following, The Peptides, Volume 2, Ed. E. Gross and J. Meienhofer, Academic Press, New York). These protecting groups used to block functional groups of the side chains (OH-, NH2groups) corresponding to amino acids.

To protect the hydroxy groups of amino acids (serine, threonine) preferably use benzyl or similar group. To protect other side groups (e.g. amino group, a guanidinium moiety of arginine) preferably using the orthogonal method.

the AI (for example, in dichloromethane), and under certain conditions to increase the solubility can be added dimethylformamide.

For the introduction of R4-CO-group by condensation of the amino group of lysine with a carboxylic acid of General formula (VII) can be used the above methods of condensation of amino acids. However, it is particularly preferable to carry out the condensation in the presence of a carbodiimide, such as 1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide and 1-hydroxybenzotriazole.

As synthetic substrates using insoluble polymers, for example, swelling in organic solvent of polystyrene resin in the form of granules (e.g., a copolymer of polystyrene and divinylbenzene (1%)). Synthesis of protected amide of Decapeptide spend on methylbenzhydrylamine resin (MVNA-resin, i.e. polystyrene, containing methylbenzhydrylamine group), and after removal from the substrate in the presence of HF receive a peptide with the desired C-terminal amide group, in accordance with A scheme (see below).

For the synthesis of peptide amino acids with N--Boc-protective groups add in a threefold molar excess in the presence of diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in CH22Cl2within 30 minutes To monitor the completeness of the conversion use chloranilic test Christensen and ninhydrin test Kaiser. The remnants of free amino groups blocked by acetylation using acetylimidazole (a five-fold excess) in CH2Cl2.

The diagram shows the sequence of stages of peptide synthesis. At the stage of cleavage of the peptide from the resin final product solid-phase synthesis is dried in vacuum over P2O5and process it after drying 500-fold excess of a mixture of HF/anisole at a ratio of 10:1/(./about.) for 60 min at 0oC.

After removal of HF and anisole in vacuum and processing of the remainder of anhydrous ethyl ether amide peptide is precipitated in the form of a white solid product, the separation of the peptide from the fall together with the polymer carrier is conducted by washing the precipitate with 50% solution of acetic acid in water. With careful evaporation of acetic acid solutions in vacuum it is possible to obtain the corresponding peptide in the form of a highly viscous oil which after adding thereto absolute ether in the cold turns into a white solid product.

Further purification is carried out using preparative ghvd.

Peluchetti can be obtained by processing their acid salts with bases. Embonate peptides can be obtained by treatment of the salts triperoxonane acid (TFA-salt) free monowai (Paveway) acid or the corresponding disodium salt monowai acid. For this purpose an aqueous solution of TFA-salt of the peptide is mixed with a solution of the disodium salt monowai acid in a dipolar aprotic solvent, preferably dimethylacetamide, and allocate the resulting light yellow precipitate.

For a better understanding of the invention, the following examples do not limit the scope of the invention.

Example 1

Obtaining Ac-D-Nal(2)-D(pCl)Phe-D-Pal(3)-Ser-Taut-D - [N'- (imidazolidin-2-he-4-yl)formyl]-Lys-Leu-Arg-Pro-D-Ala-NH2< / BR>
The synthesis is carried out in accordance with the above scheme using 5 g of mBHA resin (loading polymer growing peptide chain is 1.08 mmole of peptide per 1 g of polymer). Lysine is added to the polymer in the form of Fmoc-D-Lys(Boc)-OH, which after removal of the Boc-protective group in the side chain of lysine acelerou threefold excess imidazolidin-2-he-4-carboxylic acid. Then remove the Fmoc-protective group in the presence of 20% piperidine/DMF and increasing peptide chain towards the N end of the scheme. Synthesized peptide otscheplaut from polymer and receive 5,2 is eptide, which according to jhud is a homogenous product of General formula C74H97N18O15Cl molecular weight, adjusted according to the FAB-mass spectroscopy - 1514 (M+H+) (calculated - 1512,7) and the associated1H-NMR spectrum.

1H-NMR (500 MHz, DMSO - d6in ppm): 8,56, m, arene.H; 8,08 m, 1H, arene. H; 7,81, m, 1H, arene.H: 7,73 m, 2H, arene.H; 7,66, m, 1H, arene.H; 7,60, s, 1H, arene.H; 7,44, m, 2H, arene.H; 7,30, d, 1H, arene.H; 7,25, 7,18 and, 2 d, 2 x 2H, arene.H p-Cl-Phe; 6,97 and 6,60, 2 d, 2 x 2H, arene.H Tyr; and 9.2 to 6.3 some signals, amide-NH; 4,8-4,0, numerous m, C - H and aliphatic.H; 2,1-1,1, numerous m, the residual aliphatic.H; 1,70, s, 3H, acetyl: 1,22, d, 3H, Ala H: 0,85, dd, C - H Leu.

Example 2

Obtaining Ac-D-Nal(2)-D(pCl) Phe-D-Pal(3)-Ser-Tyr-D-[ -N-4-(4 - amidinophenoxy)-amino-1,4-DIOXOLANYL]-Lys-Leu-Arg-Pro-D-Al-N2< / BR>
0.7 mmole (1,03 g) Decapeptide Ac-D-Nal-D(pCl)Phe-D-Pal-Ser-Tyr-D-Lys-Leu-Arg-Pro-D-Ala-NH2condense with 0.1 mmole (0.27 g) (4-amidinophenoxy)amino - 4-oxomalonate acid in the presence of 1.0 mmole (0.16 g) and 1-ethyl-3- (3-dimethylaminopropyl)-carbodiimide and 1.0 mmole (0.16 g) of 1 - hydroxybenzotriazole in fresh DMF. After incubation of the reaction mixture for 24 h the solvent is removed in vacuo, the obtained residue is dissolved in water and freeze-dried. The resulting crude product (fleet a homogenous product total formula C81H104N19O15Cl molecular weight, adjusted according to the FAB-mass spectroscopy - 1618,7 (M+H+) (calculated - 1617,7) and the associated1H-NMR spectrum.

1H-NMR (500 MHz, DMCO-d6in ppm):

10,4, s, 1H. 9,15, s, 2H, u 8,8, s, 1H, NH group 4-amidinophenoxy; 8,60 m, 2H, arene. H; 8,20 m, 1H, arene. H; 7,80 m, 1H, arene.H; 7,73, m, arene. H; to 7.61, s, 1H, arene.H; 7,44, m, 2H, arene.H; 7,30, d, 1H, arene.H; 7,25 u 7,20, 2d, 4H, arene. H (pCl)Phe; 7,0 u 6,6, 2d,4H, arene.H Tyr: 8,3-7,2, some signals, amide-NH; 4,73-4,2, some multiplets, C-H; 4,13, m, 1H, C-H; Ala; 3,78-2,4, some multiplets, C-H and aliphatic.N; 1,72, s, 3H, acetyl; 1,22, d, 3H, C Ala: 0,85, dd, 6N, Leu C.

Example 3

To 0.5 g (0.3 mmole) of peptide antagonists of LH-RF, obtained according to example 1, dissolved in 50 ml of water, add a solution 0,130 g (0.3 mmole) disodium salt monowai acid in 2 ml of water, while having embonate peptide, which quickly falls out of solution in the form of a yellow precipitate. Get 0,281 g fine crystalline powder yellow - green color. Content monowai acid is 33%.

Example 4

To 0.3 g (0,17 mmole) peptide antagonists of LH-RF, obtained according to example 2, dissolved in 5 ml of dimethylacetamide, add a solution of € 0.195 g (0.45 mmole) disodium salt monopole add 50 ml of water. Get 0,330 g fine crystalline powder yellow. Content monowai acid is 20%.

Compounds of General formula I can be obtained according to the following schemes 1, 3, 4 and 5, with three functional groups R1, R3and R4systematically vary. Scheme 1 shows the synthesis of compounds in accordance with example 1.

General principles of synthesis of compounds of General formula I according to scheme 1.

Carboxylic acid, R4-COOH, containing the substituted residue R4and underlying formulas I and scheme of the synthesis of 1, and in which case the primary residue R4there may also be in the form of a salt, for example, in the form of hydrochloride, hydrosulfate or acetate, is dissolved or suspended in an anhydrous medium with stirring in a non-polar or dipolar aprotic organic solvent, for example tetrahydrofuran, dioxane, methyl tert-butyl ether, toluene, dimethylformamide, dimethylacetamide, N-organic, dimethyl sulfoxide or methylene chloride and combined under stirring with the base, which is used to remove the acid, for example, Diisopropylamine, triethylamine, N-methylmorpholine, dimethylaminopyridine or pyridine. Zavoral, used for dissolving the carboxylic acid R4-COOH containing substituent R4. Then the pH of the reaction mixture is supported by the base, which adds to bind the acid, in the range of 6.5 to 9.0, preferably 7.0-8.5 in, especially 7,0-7,5. Finally, to the reaction mixture with stirring, a solution of a condensing agent, for example, benzotriazol-1-yl-oxy-Tris(dimethylamino)- phosphodiesterase (THIEF) or benzotriazol-1-yl-oxy-Tris - pyrrolidinedithiocarbamate (Rumor) or DICYCLOHEXYL carbodiimide (DCC) and after a short period of time again bring the pH of the reaction mixture in the above range. The suspension is stirred, for example, for 1-15 h at 0-80oC, preferably at 10-50oC, especially at 20-30oC, then filtered, the solid product is washed and the filtrate dried in vacuum. The resulting residue is crystallized by trituration in an organic solvent, for example toluene, tetrahydrofuran, methyl ethyl ketone or isopropyl alcohol, or purify by recrystallization, distillation or column or flash chromatography on silica gel or aluminium oxide. As the mobile phase used, for example, the mixture containing the chloride is (25%) in the ratio 80:25:5 (b/b).

Synthesis of triptoreline

Connection, purified as described above, is dissolved in a proton or an aprotic solvent, for example alcohols, such as methanol, ethanol, isopropanol, or cyclic ethers, for example tetrahydrofuran or dioxane, and the pH of the medium was adjusted to 10-11 with a solution of 2 N. sodium hydroxide. Precipitated solid product is filtered off, washed, dried in vacuum and solution in ethanol is mixed with a stoichiometric quantity or 2-4-fold excess triperoxonane acid at a temperature of from 10 to 80oC, preferably from 20 to 40oC. After 24 h incubation the reaction mixture at 0 to 4oC triptorelin precipitates, which is filtered off and dried in vacuum.

Compounds in table 1 are synthesized as described in example 5 according to the above General method, which is the basis of the synthesis of 1.

Example 5

Receipt - N-[benzyloxycarbonyl]- N-[5-[(4-amidinophenoxy)amino]-5-oxopentanoic]-L-linename triptorelin

5 g (of 17.5 mmole) of the hydrochloride of 5-[[4-(aminoiminomethyl)phenyl]-amino]- 5 - oxopentanoic acid are suspended with stirring in 200 ml of anhydrous dimethylformamide and type of 3.85 ml (35,0 mmole) N - methylmorpholine, and C is adjusted to 7.0-7.5 to N-methylmorpholine (NMM). At the final stage add a solution 9,73 g (of 21.9 mmole) benzotriazol-1-yl - oxy-Tris(dimethylamino)phosphonium-hexaphosphate (THIEF) and after incubation for 10-15 minutes again establish the pH of the medium to 7.0 and 7.5. The yellow suspension is stirred while controlling the pH value, which should be between 7.0 and 7.5, for 3-4 h at room temperature, filtered colorless precipitate. The precipitate is washed twice with dimethylformamide and the yellow filtrate evaporated to dryness. The oily residue is treated with ethyl ketone (5 x 40 ml) and after each of the five treatments of the solvent phase of methyl ethyl ketone was separated and discarded. Released in crystalline form crude product is washed with 30 ml of methyl ethyl ketone and dried in vacuum at room temperature.

The obtained solid product is dissolved in approximately 50 ml of ethanol and the pH of the solution was adjusted to 10-11 2 N. a solution of sodium hydroxide. The precipitated base is filtered off, washed with water and ethanol, dried in vacuum at 35oC.

Yield: 5.5 g (62% of theoretical)

Triptorelin: a Suspension of 5.5 g of the base in ethanol is mixed with a fivefold molar excess triperoxonane acid at 60oC. the Solution was stored over night at 4< from theory)

Melting point: 185oC

Elemental analysis data:

Calculated: C 53,84 H 5,65 N 13,45

Found: C 54,11 H 5,74 N 13,33

The structure of the compounds with the General formula (I) synthesized by the above method are shown in table 1, and for all examples, n is equal to 4.

< / BR>
The melting point of the compounds obtained according to the above examples, are shown in table 2.

Precursors for the synthesis of compounds of General formula I obtained according to the synthesis scheme 1, the structure of which is shown in table 1.

As a starting substance for the synthesis of compounds in accordance with examples 5-34 use a commercial preparation of Z-(L)-liinamaa. Substituted derivatives of "aryl" or "heteroelement-oxo-limit acids obtained according to scheme 1 and used for the further synthesis as an intermediate derivatives can be synthesized similarly to scheme 2 by known literature method (P. R. Bovy, J. Organ.Chem. 58, 7948 (1993)).

As an aromatic or heteroaromatic amines (A-NH2) starting compounds for the synthesis according to scheme 2, using commercial preparations. Aminoimidazo[1,2 a] pyridine, which is the starting compound for the synth is

"Aryl" or "heteroelement-oxo-limiting acid used as starting compounds can also be synthesized through reaction of aminolysis nanometrology ether limiting dicarboxylic acid, for example, nanometrology ether subernova acid and nanometrology ester of azelaic acid with aromatic or heteroaromatic amine in boiling ethanol or butanol, or by well-known methods in an aromatic solvent, e.g. toluene or xylene at the boiling temperature of the solvent or in an autoclave at the boiling temperature of the solvent under a pressure of 50 bar. Then the reaction mixture was concentrated in vacuo and the residue crystallized from methanol or ethanol or purified by the method of column chromatography. As the mobile phase used, for example, a mixture comprising methylene chloride, methanol, ammonia (25%), in the ratio of 85:15:1 (b/b) or methylene chloride, methanol, ammonia (25%) in the ratio 80: 25:5 (b/b).

An alternative method of obtaining compounds of General formula I, where R1- benzyloxycarbonyl group, a R2and R3the hydrogen atom is the following:

1. Lidiruyut - carboxyl group.

2groups.

4. Remove the Z-group with the amino group.

5. In the amino group enter the necessary R4-CO-Deputy.

6. S - amino remove the Boc-protective group.

7. In the amino group introduced the Z - protective group.

Other compounds of General formula I get according to scheme 3, which presents the synthesis of the compounds described in example 35.

General principles of synthesis of compounds of General formula I according to scheme 3.

stage 1

As a dipolar aprotic or nonpolar organic solvent used, for example, tetrahydrofuran, dimethylsulfoxide, dimethylformamide, acetonitrile, ethyl ester acetic acid, dimethylacetamide, N - organic, dioxane, toluene, simple ether, methylene chloride or chloroform at temperatures between -30oC to 30oC, preferably from -20oC to 20oC, especially from -15oC to 5oC in the case of Z-Lys(BOC)-OH, and a base, such as triethylamine, Diisopropylamine, N-methylmorpholine, N-ethylpiperidine and aliphatic or aromatic chloranhydride carboxylic acid, for example, acetylchloride, isobutyrophenone, isovalerianic, pivaloyloxy, benzoyl chloride, or 4-IU the 0oC the solution or suspension of an amine in a dipolar aprotic solvent or non-polar organic solvent, such as tetrahydrofuran, dimethylsulfoxide, dimethylformamide, acetonitrile, ethyl ether, acetic acid, dimethylacetamide, N-organic, dioxane, toluene, simple ether, methylene chloride or chloroform with vigorous stirring. The suspension is stirred at a temperature of from -30oC to 30oC, preferably from -20oC to 20oC, in particular from -15oC to 5oC during the period of time from 1 h to 2 h After completion of the reaction the base in the form of the acid chloride is filtered off under vacuum and the solvent is distilled off. The oily residue is treated aprotic or nonpolar organic solvent, for example ether, diisopropyl ether, methyl tert-butyl ether, petroleum ether, toluene, xylene, pentane, hexane. The solution is stirred, for example, in the period from 30 minutes to 3 hours to fall a white powdery precipitate. The precipitate is filtered off and dried.

stage 2

Z-Lys(BOC)-amide, obtained in the 1st stage, as described above, dissolved in triperoxonane acid at a temperature of from -20oC to 30oC, 1 h prepost Excessive triperoxonane acid is distilled off and the oily residue is mixed with dipolar aprotic or nonpolar organic solvent, such as dimethylformamide, methylene chloride, tetrahydrofuran, acetonitrile, N-methylpyrrolidinone, ethyl ester acetic acid. Then add the desired acid, base, for example, diisopropylethylamine, N-methylmorpholine and suitable condensing agent, for example, a THIEF, PyBOP, DCC in dipolar aprotic or nonpolar organic solvent, for example dimethylformamide, methylene chloride, tetrahydrofuran, acetonitrile, N-methylpyrrolidone, ethyl ester acetic acid. The reaction is carried out at a temperature of from -10oC to 100oC, preferably from 0oWith up to 80oWith, in particular in the range between 10oC and 35oC. After incubation the reaction mixture at the specified temperature conditions during the period of time from 1 to 5 h and at room temperature for 24 h the solvent is distilled off. The residue is treated with water or an organic solvent, such as isopropanol, methylene chloride, or a simple ester. The crude product is subjected to chromatographic purification on a column of silica gel.

Sogla, in table 3, and for all connections, without exception, n = 4.

< / BR>
Example 35:

Obtain N-[N-[benzyloxycarbonyl] -- N-[4-[(4-amidinophenoxy)-amino] -4-oxo - butanoyl]]-L-lysine-N-(3-pyridylmethyl))-amide

stage 1

Obtain N - [N-[benzyloxycarbonyl]-- N-[tert-butyloxycarbonyl]]- L-lysine-N-(3-pyridylmethyl))-amide

To 60 ml of tetrahydrofuran added at -15oC 4 g (10 mmol) of Z-Lys(BOC)-OH (commercial preparation), 1 g (10 mmol) of triethylamine and 1.26 g (10 mmol) of pivaloate. After 30 min, to this mixture with vigorous stirring pre-cooled to -10oC solution of 1.08 g (10 mmol) 3-(aminomethyl) of pyridine in 20 ml of tetrahydrofuran. The suspension is stirred at -15oC for 1 to 2 hours, the triethylamine Hydrochloride is filtered at a low temperature and the tetrahydrofuran is distilled off. The oily residue is mixed with 100 ml of diethyl ether. The solution is stirred until precipitation of a white powdery precipitate, the precipitate is filtered off and dried. Yield: 4 g (85% of theory).

stage 2

Obtain N - [N-[benzyloxycarbonyl] --N-[4-[(4-amidinophenoxy)-amino] -4-oxo - butanoyl]]-L-lysine-N-(3-pyridylmethyl))-amide

2 g (4.25 mmole) of N - [N-benzyloxycarbonyl] -- N-[tert - butyloxycarbonyl is for 20 minutes The excess TFA is evaporated and the oily residue is treated with 10 ml DMF. After that add 4.6 ml (42.5 mmole) N-methylmorpholine and 1.15 g (4.25 mmole) 4-[[4- (aminoiminomethyl)phenyl] amino] -4-oxomalonate acid hydrochloride, 2.35 g (5.3 mmole) is a THIEF and 20 ml of DMF. The resulting mixture was stirred at room temperature for 24 hours of DMF is distilled off, the residue is treated twice with 40 ml of water, filtered and dried. The crude product is subjected to chromatographic purification on a column of silica gel using the eluent 89b (70% HCCl3, 40% MeOH, 10% CH3COO-Na+in a solution of NH4OH (1 Mol/l, 25%). Yield: 340 mg (14% of theoretical).

The synthesis of compounds according to examples 36-55 was performed by analogy with example 35.

The results are given in table. 4.

Compounds of General formula I get the following schemes 4 and 5.

The acylation of carboxylic acids or esters of anhydrides of formic acid according to schemes 4 and 5:

H-Lys(Boc)-NH2condensed with a carboxylic acid in a dipolar aprotic solvent (DMF, DMSO) in the presence of a base (DIPEA, N) and a condensing agent (DCC, DIC, EDCI) and get amide derivative. After removal of solvent the residue is mixed with water, is canola or isopropanol) or an ether (e.g. ethyl ether, acetic acid) or a ketone (e.g. methyl ethyl ketone).

The interaction of H-Lys(Boc)-NH2c carboxylic acid anhydrides is carried out in an aqueous-alkaline solution (conditions Schotten's-Bauman), the yield of the target products is 90-95%. The crude product is filtered off and purified by recrystallization from alcohol (methanol/ethanol/isopropanol) or ethyl acetate or methyl ethyl ketone.

2. Removal of the BOC-protective group using TFA.

Quantitative removal of the BOC-protective group is carried out at room temperature in a mixture of dichloromethane : FA (2: 1) for 60 min Obtained R1-Lys-NH2used immediately without further purification.

3. The acylation of the hydrochloride of 4-((4-aminoiminomethyl)phenyl)amino)- 4-oxomalonate acid.

The reaction with the corresponding carboxylic acid (R4) is carried out in a dipolar aprotic solvent (DMF, DMSO) at room temperature in the presence of a base (NMM, DIPEA) and a condensing agent such as EDCI, Thief or Rumor). After removal of solvent the residue is treated with water to give the crude product as a precipitate. The resulting product overview the ode, acetonitrile and TFA. The final product is a TFA-salt.

According to the General principles of synthesis presented in schemes 4 and 5, receive connections, the synthesis of which is described in example 56, and the structure in table 5.

Example 56

To 120 ml of dry degassed N,N-dimethylformamide (DMF) is added at room temperature, 32 mmol hydrochloride of Z-liinamaa and 32 mmol hydrochloride 4-((4-(aminoiminomethyl)phenyl)-amino)-4-oxo - butyric acid. Added reagents are quickly dissolved under stirring; after adding 104 mmol of diisopropylethylamine and 40 mmol THIEF, the reaction mixture was stirred at room temperature for 16 hours the Solvent and excess DIPEA evaporated on a rotary evaporator at a residual pressure of about 10 mbar and heated in a water bath at 50-55oC. the Oily residue is treated with 250 ml of water, homogenized in an ultrasonic bath and cooled. The resulting crude product is filtered on a suction filter and washed with water.

After drying under vacuum over calcium chloride receive approximately 16 g of beige powder product, which is a hydrochloride, the purity of which sostavljaet ml of water and the suspension added 32 mmol (2,45 ml) TFA (99%). Excess acid is removed on a rotary evaporator in a short period of time, then aqueous suspension lyophilizer.

After recrystallization of the crude product from ethanol (ethanol/methanol) obtained product can again be liofilizirovanny to increase solubility.

Output: 5,26,

So pl.: 210-213oC.

Data1H-NMR (500 MHz, DMSO-d6in ppm):

10,47, C. , 6, 1H, NH anilide, 9,14 and 8,82 S., NH amidon, of 7.82, m, 1H, Lys--NH and 7,79 and 7,46, 2C., aromatic. H, 7.27 and 6,93 2C., 2H, CONH2, 7,20, D., 1H, urethane. NH, 5,0, s, 2H, benzyl., H, 3,89, m, 1H, C - H, 3.0, and 2,58 and 2.40 a, 3 meters, all 6H, alipac.H 1,60 - 1,20, 4 m, all 6H, residual aliphatic.H.

< / BR>
Salts of compounds of General formula I

Obtained according to the invention the compounds may exist as salts of various acids, for example, in the form of salts of mineral acids, such as, for example, hydrochloric, sulfuric, phosphoric acids, salts of organic acids, such as, for example, acetic, triperoxonane, lactic, malonic, maleic, fumaric, gluconic, glucuronic, lemon, monowai, methansulfonate, hydroxyethanesulfonic, pyruvic and succinic. Compounds of General formula I, as well as their salts, exhibit biological activity. Connections about the. is soedineniya can be administered orally, parenteral, intravenous, subcutaneous or inhalation route.

The invention also relates to a method for preparation of pharmaceutical preparations containing antagonists of LH-RF, corresponding to the invention. For preparing pharmaceutical preparations of the compounds according to the invention is mixed with auxiliary substances. As auxiliary substances can be used conventional fillers and other optional components, traditionally used in the prior art.

The invention also relates to pharmaceutical preparations containing at least one compound of General formula I or its salt formed with physiologically acceptable inorganic or organic acid or its mixture with a pharmaceutically acceptable carrier and/or diluent/auxiliary material.

Example 83.

The affinity of cetrorelix, compounds obtained according to examples 1, 2, 5, 35 and 56, to the receptor LH-RF on human cells (Cetrorelix: Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Cit-Leu-Arg - Pro-D-Ala-NH2).

The method for determining the affinity for the receptor (dissociation constants Kd): Determination of the affinity of the peptides to the receptor wire the use of [125J] cetrorelix (specific activity of 5-10 x 105dpm/mcmol; the solution contains 20% (b/b) acetonitrile, and 0.2% (V/o) albumin, 0.1 percent (V/o) TFA, 80% (b/W) water)). The ability to bind to the receptor at yugirovannykh peptides is 60 and 85%. As unlabeled test compounds used cetrorelix (compounds obtained according to examples 1, 2 and 56). Compounds are used in the concentration range of 0.01 nm to 1000 nm (cetrorelix, the compounds obtained according to examples 1 and 2) and 0.01 μm - 10 μm (compounds obtained according to examples 5, 35, 56).

In the experiment using human cells (clone 3.5 L/78), sverkhekspressiya receptor LH-RF. Cells were washed off under density monolayer cell layer buffer PBS/EDTA (PBS without Ca2+/Mg2+/1mM EDTA), determine the number of cells and resuspended in the appropriate volume of incubation medium (medium Needle in the modification Dulbecco: 4.5 g/l glucose, 10 mm HEPES, pH 7.5, 0.5% of (EC/b) BSA, 1 g/l bacitracin, 0.1 g/l SBTI, 0,1% (EU/b) NaN3). In a special reaction tube 400 ál (Fa.Rerner Tour Beckman) placed 200 μl silicone/paraffin mixture (84/16% (by volume)) and using the pipette, add 50 ál of cell suspension (2.5 x 105cells). Then to the cell suspension in silicone-partienlarly. The mixture is incubated at slow rotation in thermostat at 37oC for 60 minutes and Then centrifuged (Heraeus Biofuge 15R, rotor NTA 13,8) for 2 min at 9000 rpm at room temperature. Cells pass through the silicone/paraffin layer and thus are separated from the solution with ligands. After centrifugation of the reaction tube quickly frozen with liquid nitrogen and the lower part of the tube with sediment cut nippers. The lower part of the tube with sediment (linked ligand [125J] -cetrorelix) and top layer (unbound ligand [125J]-cetrorelix) is placed in the cuvette for the pulse counter. When determining the maximum binding (Bo) competitive compounds do not add. To determine nonspecific binding add 1 μm of unlabeled cetrorelix as competing connections. The nonspecific binding is not more than 10% of the total binding (In). Counting pulses carried out using a gamma counter, and the results processed in accordance with the program EBDA/Ligand V3.0 (McPherson, J. Pharmacol. Methods 14, 213-228, 1985). The chart dose/effect estimate IC50(the concentration that causes 50% inhibition of the receptor binding). On the basis of these data with Ponoko binding (see Fig. 1) it is obvious that all tested compounds compete with radioactively labeled ligand, [125J] cetrorelix for binding to the receptor LH-RF.

Also shown are values of binding (% of full-linking In), depending on the concentration of competing substances. For connections, shown in Fig. 1, the calculated dissociation constants Kd [nm], reflecting the degree of affinity for the receptor: cetrorelix (SB-75): 0,214 nm, the compound obtained according to example 1: 0,305 nm, the compound obtained according to example 2: in 0.104 nm, the compound obtained according to example 5: 108 nm, the compound obtained according to example 35: 300 nm and the compound obtained according to example 56: 1082 nm. The average values of the affinity of the receptor are shown in table 7.

Example 84

Functional testing of the antagonistic action of the compounds obtained according to examples 2 and 56 in relation to the receptor LH-RF human cells.

The way to determine IP3(D-monoset-1,3,5-triphosphate): Under the condition of submonolayer culture of human cells (clone 3.5 L/78) expressing the receptor for LH-RF, washed once by PBS, the cells suspended in PBS/EDTA and collect the cell suspension. The cells are then suspended in sooty) BSA, 5 mm LiCl, 1 g/l bacitracin, 0.1 g/l SBTI), aliquot taken part in a test tube with a volume of 1.5 ml and incubated at 37oC for 30 minutes In each dimension using 4 x 106cells in 500 ál. After pre-incubation in cell suspension make of LH-RF (0.5 mm initial solution in buffer: 10 mm Tris, pH 7.5, 1 mm dithiothreitol, 0,1% (EU/W) BSA/Bachem Art # H4005) to a final concentration of 10 nm. Simultaneously to the cell suspension, add the antagonists in the appropriate concentration (the compound obtained according to example 2: 0.0316; 0.1; 0,316 and so on up to 100 nm). As a control using cell suspension, incubated in the absence of LH-RF. After incubation of the samples for 15 min at 37oC generated IP3allocate extraction trichloroacetic acid (TFA). To the cell suspension is added 500 μl) cooled to 0oC 15% (EU/W) THU. The precipitate was separated by centrifugation (Biofuge 15R Heraeus) at 2000 g and 4oC for 15 minutes the Supernatant volume of 950 μl three times extracted with 10 volumes of chilled water diethyl ether in a test tube with a volume of 15 ml After the final extraction, the pH of the solution was adjusted to 7.5 with dilute NaHCO3.

The concentration of IP3performed using Chur>H] -IP3and unlabeled IP3. When measured using the test set TRK 1000 production Amersham. Use a standard methodology that came with the set. After all necessary operations are add 2 ml of scintillator for water samples (Rotiszint Ecoplus), thoroughly mixed with resuspendable sediment containing bound [3H]-IP3and carry out measurement using a beta scintillation counter. The number of intracellular IP3calculated by the calibration curve and build a graph of dose-response relationships. IC50calculate end point on the graph of dose-response relationships.

Results: In Fig. 1 shows the corresponding plots of the dose dependence for peptide antagonists mentioned in example 2 (Fig.2), as well as peptidomimetics, mentioned in example 56 (Fig.3). Defined stimulatory effect of 10 nm LH-RF and inhibitory effect on the formation of IP3depending on the concentration of the substance. The compounds mentioned in examples 2 and 56, do not show properties of agonists, i.e., the action of these substances does not stimulate synthesis IP3. Not shown here control experiments it was shown that LH-RF does not stimulate the formation of IP3netresult with values corresponding to those for unstimulated cells. Thus in examples 2 and 56 we are talking about functional antagonists of LH-RF. In addition, the test substance differ in activity. In the experiment IC50for the compounds obtained according to example 2, is approximately 0.4 nm; compounds obtained according to example 56, IC50approximately 4 μm. This value activity correlates well with data affinity receptor (Kd=0,109 nm for compound obtained according to example 2, and Kd = 1,08 μm for compound obtained according to example 56), established in experiments in vitro competitive binding in the presence of [125J]-cetrorelix.

Example 85

Hormone-suppressor effect of the compounds obtained according to examples 1, 2 and 56 defined in the experiments in healthy male rats.

To determine the suppression of testosterone in the blood of healthy male rats test compound is injected subcutaneously into the right side. The dose in the case of compounds obtained according to examples 1 and 2, 1.5 mg/kg, and in the case of compound obtained according to example 56, 10 mg/kg To determine the concentration of testosterone in experimental animals take samples of blood from the sublingual vein in a volume of 300 µl after 0, 2, 4, 8 (only to connect the Ni testosterone (not more than 1 ng/ml) after administration of the compound, obtained according to example 1, stored up to 264 hours (one animal), up to 336 hours (2 animals), up to 384 hours (one animal) (Fig. 4). To compound obtained according to example 2, the level of testosterone decreases in the period of time up to 408 hours (two animals), up to 648 hours (4 animals) (Fig.5). To compound obtained according to example 56 (administration of 10 mg/kg subcutaneously), the level of testosterone decreases in all five animals after two hours, and this effect lasts for 8 hours. In the following time point (24 h) level testoterone again increases (Fig.6).

Table 7 Biological data

The affinity to the receptor LH-RF human cells (expressed through dissociation constants Kd [nm] ; data analysis calculated by the program EBDA/Ligand. The average values obtained in different experiments (the number of experiments indicated in parentheses).

In table. 7 shows the results of in vivo studies on the suppression of testosterone, the determination of histamine release in vitro and the water solubility of various compounds in comparison with cetrorelix SB-75.

1. The compound of General formula V

Ac-D-Nal(2)1-D-(pCl)Phe2-D-Pal(3)3-Ser4-Tyr5-D-Xxx6-Leu7-Arg8-Pro9-D-Ala
R4group of the formula II

< / BR>
where p = 1 - 4, integer, R5is a hydrogen atom or an alkyl group, R6is unsubstituted or substituted aryl or heteroaryl group, or R4- ring structure of General formula III

< / BR>
where q = 1 or 2, R7and R8is a hydrogen atom, X is a sulfur atom,

and their salts with pharmaceutically acceptable acids.

2. Connection on p. 1, characterized in that the Xxx - [-N-4(4-amidinophenoxy)-amino-1,4-DIOXOLANYL]-lysyl-group.

3. Connection on p. 1, characterized in that the Xxx - [N-(imidazolidin-2-he-4-yl)-formyl]-lysyl-group.

4. Connection PP.1 to 3, characterized in that it is a salt monowai acid.

5. The compound of General formula I

< / BR>
where n = 4;

R1- aryl-, alkyloxy, aralkyl, heteroalkyl, aralkylated or heteroarylboronic, unsubstituted or substituted;

and R2and R3independently from each other can be represented by a hydrogen atom, alkyl group, aranceles group or heteroalkyl group, unsubstituted or substituted, and the Deputy may be represented by aryl or heteroaryl group, or-NR2R3is the amino acid g is a hydrogen atom or an alkyl group;

R6is unsubstituted or substituted aryl or heteroaryl group, and the substituents can be represented aryl or heteroaryl group;

or R4- ring structure of General formula III

< / BR>
where q = 1, 2;

R7and R8is a hydrogen atom;

X is a sulfur atom, and aromatic residues can be completely gidrirovanny and can be represented chiral carbon atoms of configuration L,

as well as their salts with pharmaceutically acceptable acids.

6. Connection on p. 5, characterized in that a represents-N-[benzyloxycarbonyl] --N-[5-[(4-amidinophenoxy)amino]-5-oxo-pentanoyl]-L-osiname-triptorelin.

7. Connection on p. 5, characterized in that a represents-N-[benzyloxycarbonyl] --N-[5-[(4-amidinophenoxy)amino] -4-oxo-butanoyl]-L-osiname-triptorelin.

8. Connection on p. 5, characterized in that a represents-N-[benzyloxycarbonyl] --N-[4-[(4-amidinophenoxy)amino] -4-oxo-butanoyl]-L-lysine-N-(3-pyridylmethyl)amide-triptorelin.

9. Connection PP.5 to 8, characterized in that it is a salt monowai acid.

10. Connection PP.1 to 9, characterized in that it is obtained for the breast, as well as non-malignant tumors, the treatment of which requires suppression of LH-RF.

11. Pharmaceutical composition having high affinity to the receptor releasing factor, luteinizing hormone, containing as active substance connections PP.1 to 4.

12. A method of obtaining a connection on p. 1, formula V, comprising the following stages: (a) the binding of D-alanine containing blocked amino acid, with a suitable substrate for solid-phase synthesis, (b) removing the protective group from the amino group of alanine, (C) ensuring the condensation reaction associated with the substrate alanine with Proline containing a protective group on the nitrogen atom, (d) removing the protective group from the nitrogen atom of Proline, (l) repeating steps (C) and (d) amino acids 1 to 8 according to the formula (V) in order from eighth to first, (e) the removal of the compound obtained in stage (d), the substrate (W) ensuring the condensation reaction with a carboxylic acid of General formula VII

R4-COOH, (VII)

where R4matter under item 1,

(C) when required to ensure the condensation reaction with a pharmaceutically acceptable acid, preferably monowai acid, with formation of salts.

13. The method of obtaining connection right substrate for solid-phase synthesis, (b) removing the protective group from the amino group of alanine, (C) ensuring the condensation reaction associated with the substrate alanine with Proline containing a protective group on the nitrogen atom, (d) removing the protective group from the nitrogen atom of Proline, (l) repeating steps (C) and (d) with amino acids 6 to 8 according to the General formula (V) in order from eighth to sixth, (e) the removal of the protective group of the amino group of D-lysine or D-ornithine in position 6 and the provision of condensation with a carboxylic acid of General formula VII

R4-COOH, (VII)

where R4matter in accordance with p. 1,

(g) removing the protective group of the amino group of D-lysine and D-ornithine, (C) repeating steps (C) and (d) amino acids 1 to 5 according to the General formula (IV) in order from fifth to first, (and) the removal of the compound obtained in stage (C), with the substrate and cleaning, in particular by the method ghvd, (K) if necessary, ensure the condensation reaction with a pharmaceutically acceptable acid, preferably monowai acid, with formation of salts.

14. The method according to PP.12 and 13, characterized in that carboxylic acids of General formula VII is used N-(4-amidino-phenyl)-amino-4-oxo-butyric acid.

15. The method according to PP.12 and 13, characterized in that the qualities of the b-PP.12 - 15, characterized in that as the pharmaceutically acceptable acid is used albanova acid.

17. The method of preparation of pharmaceutical preparations containing the compounds according to paragraphs.1 to 9, characterized by the fact that these compounds are mixed with auxiliary substances.

 

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