UA79070C2 - Novel lhrh-antagonists, production and use thereof as medicament - Google Patents

Abstract

The invention relates to peptides, comprising an N-methylated amino acid component and improved water solubility. According to the invention, medicaments containing the said peptides can be used for treatment of hormone-dependant tumors and hormone-influenced non-malignant disease states.

Description

Description of the invention

The invention relates to NSCLC antagonists with improved solubility properties, the method of obtaining these 2 compounds, medicinal products containing these compounds, as well as the use of medicinal products for the treatment of hormone-dependent tumors and the use of hormonal drugs in non-malignant diseases, such as benign prostatic hyperplasia (BPH ) and endometriosis.

The nomenclature used to identify the peptides is consistent with the nomenclature being interpreted

Commission of ISRAS-IOV on biochemical nomenclature (Eogoreap 9. Viospet. 1984, 138, 9-37), according to which, according to 70 traditional ideas, the amino groups at the M-end are located from right to left, and the carboxyl group at the C-end - from left to right . And H-KN antagonists, such as peptides according to the invention, include natural and synthetic amino acids, and the former include AiYa, MaiYi, Hec, Pe, Zeg, TAg, Huz, Ago, Avr, Avp, Sim, Sp, Suv,

Mei, Re, Tug, Rgo, Tgtr and Niz. Abbreviations for individual amino acid residues are based on the traditional names of amino acids: AIa-alanine, Ago-arginine, SiIu-glycine, IeI-leucine, Iuvz-lysine, RaI(3)-3-(3-pyridyl)alanine, 12 Ma/( 2)-3-(2-naphthyl)alanine, Rpe-phenylalanine, Cpa-4-chlorophenylalanine, P-proline, Zep-serine, TPg-threonine,

Tgr-tryptophan, Tug-tyrosine and Zag-sarcosine. All amino acids described by the authors originate from the I-series, unless otherwise indicated. For example, O-Ma|(2) is short for 3-(2-naphthyl)-O-alanine, and Zeg is short for

II-serine. Substitution in the ε-amino group in the side chain of lysine is represented by the I bond in parentheses, not necessarily in the form of a contraction.

Other abbreviations:

Ac Acetyl in 4-(4-amidino-phenyl)-amino-1,4-dioxo-butyl

Vos Tert-butyloxycarbonyl p

Vor Benzotriazole-1-oxy-tris-(dimethylamino)-phosphonium-hexafluorophosphate Ge)

ОСС Dicyclohexylcarbodiimide

OSM Dichloromethane ra; Dimethoxyphenyl-dimethylmethyleneoxy-carbonyl (Dimethoxy-dimethyl-7)

BIS Diisopropylcarbodiimide o

PIREA M,M-diisopropylethylamine u

OME Dimethylformamide

Ethos Fluorenylmethyloxycarbonyl o

NO Hydrofluoric acid Ge)

NEW 1 tidroxybenzotriazole them

NRIS Liquid chromatography of high pressure

Me Methyl

TEA Trifluoroacetic acid 7 Benzyloxycarbonyl « 70 Peptides according to the invention are analogues of the luteinizing hormone-releasing hormone (LH-KN), which has the following structure: -Rho-C1u-MH", (CH-KH, gonadorelins. :z" For more than 20 years, researchers have been looking for selectively active antagonists of I H-KH-decapeptides | M. Kagep pa

U.E. Kimieg, Epdosgipe Kemiemuz 7, 44-66 (1986)).

Great interest in such antagonists is explained by their usefulness in the fields of endocrinology, gynecology, - 15 pregnancy prevention and oncology. A large number of such compounds were obtained as potential

And nH-KN-antagonists. The most interesting compounds that have been discovered so far are compounds whose structure is (Ce) a modification of the I H-KN structure. o The first series of effective antagonists was obtained through the introduction of aromatic amino acid residues in positions 1, 2, З and 6 or 2, З and 6. The usual writing of compounds looks like this: first, the amino acids are indicated, which -and 20 in the peptide chain of И Н-КН occupy the place amino acids that were originally there, and the positions in which the exchange took place are indicated by superscript numbers. In addition, due to the notation "/H-KN" it was demonstrated that we are talking about analogues of I H-KN, on which the exchange takes place.

Known antagonists:

IAs-O-Sra!?, O-Ttr3b) IN-VN (ON.Sou ey a). ip: Ogoz5, E. apa Meieppoieg, .. (Eavs) Reriidev;

Rgoseeedipodz oii (pe boi Ategisap Reriide Zutrovisht, 5.775-779, Riegse Spet.So., Koskmime PI. (1979)|;

F) IAs-Rho", O-Sra?, O-Mai(23391 IN-VEN (US Patent Mo4.419.347| and ka IU.. Ripeaa, ei a!., 9. Siip. Epadoshipoi. Meiab. 56, 420, 1983).

To improve the action of antagonists, basic amino acids, such as O-Aga, would be introduced into the position later. for example (As-O-Sra" 2, O-Tgtr3, O-Agoye, O-AiIa"9| GN-VN. (020-30276) (ON. Sou, ei ai., Epaosgipoiyudu 100, 1445, 19821; and (As-O-Ma(2)1, O-Rne(4-)?, O-Tgtr3, O-Ag9dbI IN-EN (OBE 18260) Yu.E. Vimieg ei ai., ip: Miskegu V .N.

Meziog, 9U.3., Naie, E.5.E (Edav). ИНКН апа її Apaiodv, 5.11-22 MTR Rgezzv, I apsavieg, OK1984).

Other effective I H-KN antagonists are described in MO 92/19651, UMO 94/19370, MO 92/17025, MO 94/14841, UMO 94/13313, 05-A 5,300,492, O5-A 5,140,009, ER 0413209 A1 and OE JSC 19544212). 65 The latter describes compounds with a modified ornithine or lysine link in position b, which correspond to the following formula: As-O-Ma|(2)-O-Sra?-p-RaI(3)3-Zeg-Tugo-O-Xxxb -I ey "-AgudZ-Rgo?-O-AIa"9-MN»o, where -D-

Or-Xxx is an amino acid group of the general formula (MI) pon -0 ram chn sva-n

Other known I H-KN antagonists are Apiageii, Sapigeii, and Seigogeii. Apiageiich E (IMM: Temegeiich):

As-0-MaI(2)1-0-Sra?-O-RaI(3)3-beg'-Tugo-O-Nsib-I ei 7-I uv(iRg)8-Rgo9-O-AIa9-MN » Sapigeiih: 70 As-0-MaI(2)1-O-Sra?-O-RaI(3)3-beg"-Tug?-O-pAg(Ebd»b-I ey "-PAga(EV»Z -Rgo9-O-Aia"9-MN"

Segogeyih:

As-O-Map(2)-O-Sra?-O-RaI(3)2-Zeg"-Tug?-O-Si-I ey"-Agde-Rho?-O-AIa"9-MNo

The purpose of the invention is to provide new IN-KN-antagonists, which have increased enzyme stability and significantly improved solubility in water.

This problem is solved thanks to the compounds of the general formula (I)

А-Ххх 1-Ххх2-Ххх З-Ххх 4-Ххх 5-Ххх 6-Ххх 7-Ххх 8-Ххх 9-Ххх 19-МНо (И) in which

A means acetyl group,

Xxx" 0-Ma((2),

Xxx2O-Sra,

ХххЗ р-Ра/(3),

Xxx" Veg cm'

Hhhh? M-Me-Tug, i)

Ххх5 p-sii, p-Hsi or O-(6-M'-4-(4-amidinophenyl)-amino-1,4-dioxo-butylI-I bond (abbreviated: O-I uv(B)),

XXX" Gets or Mieye,

XxxZ Agu or I uv(iRg), Fr

Ho Ro, and them-

Ххх "o r-Aia or Zag, under the condition, o what in the case when Ххх? means О-И ув(В), then Ххх" is Mie, (o) from when Ххх? means O-Sii, then Xxx" is Mie and Xxx "9 is O-Aa, or M when Xxx? means O-Hsi, then Xxx" is Ie and Xxx"9 is O-Aia, and their salts with pharmaceutically acceptable acids, in particular acetates, embonates and trifluoroacetates.

According to another aspect of the invention, particular preference is given to the following compounds, as well as their salts with pharmaceutically acceptable acids:

As-0-MaI(2)1-0-Sra?-O-RaI(3)3-Beg'-M-Me-Tugo-O-I uv(V) 9-Me "-AgodZ-Rgo?-Zago -MNo - with As-0-MaI(2)1-O-Sra?-O-RaI(3)3-Beg'-M-Me-Tugo-O-I uv(V) 9-Mie "-AgodZ- Rgo?-0-AIa"9-MN" "with As-0-MaI(2)1-O-Sra?-O-RaI(3)3-Beg'-M-Me-Tugo-O-I uv( B) 9-Mie I uv(iRg)8-Rgo?-Zag'9-MN" " As-0-MaI(2)1-0-Rpe(4-SI)2-O-Rai(3)3- Zeg'-M-Me-tug?-O-I uv(B) 9-Mie -I uv(iIR r) 8-Rgo9-O-AIa"9-MN"

As-0-Ma(2)1-O-Sra?-O-RaI(3)3-Zeg"-M-Me-Tug-O-sSib-Me"-AgaZ-Rho?-O-AIa"9- MN? - And what As-O-Ma(2)-O-Sra?-O-RaI(3)3-Zeg'-M-Me-Tug?-O-Nsib-I ey "-Agdb-Rgo?- 0-AIIIa 79-MN»

As-0-MaI(2)1-0-Sra?-O-RaI(3)3-Beg'-M-Me-Tug-O-Sib-Me"-I uv(iRg)8-Rgo92-O- AIa"9-MN" o As-0-MaI(2)1-O-Sra?-O-RaI(3)3-Beg'-M-Me-Tugo-O-Neig-I ei7-I uv(iRg )8-Pho?-O-AIa"9-MH" o According to another aspect of the invention, the compounds according to the invention exist in the form of acetate, trifluoroacetate 5p or embonate salts. According to another aspect of the invention, the compounds according to the invention are used as medicinal means or pharmaceutical compositions.

According to another aspect of the invention, pharmaceutical compositions are obtained which include at least one of the compounds according to the invention and traditional carriers and excipients.

According to another aspect of the invention, a method of obtaining compounds according to the invention of the general formula I is proposed, in which fragments of units of XXX" with appropriate protective groups, where t means an integer from 1 to 10, and XXX" is acetylated, are built in a traditional way on a solid phase or in solution, immediately after that the fragments are connected to the solid phase by means of a segmental connection and after the connection of the compound of the general formula I is separated from the solid phase by the traditional method with amidation on the XXX 9 chain. According to another aspect of the invention, it is proposed to use the compounds according to the invention for the preparation of drugs for the treatment of hormone-dependent tumors, in particular, carcinoma of the prostate gland or breast cancer, as well as non-malignant indications, the treatment of which requires inhibition of the I H-KN hormone.

According to another aspect of the invention, a method of obtaining pharmaceutical compositions is proposed, in which at least one compound according to one of paragraphs from 1 to 10 are mixed with traditional carriers and excipients and packaged as a medicinal product.

According to another aspect of the invention, a method of treating hormone-dependent tumors, in particular carcinomas of the prostate gland, breast cancer or uterine fibroids, as well as non-malignant indications, the treatment of which requires inhibition of the I H-KN hormone, such as endometriosis, benign prostatic hyperplasia (BPH ), as well as the treatment of female or male fertility disorders in mammals, in particular humans, through the administration of an active dose of at least one compound according to the invention.

Compounds according to the invention can be used for the treatment of hormone-dependent tumors, in particular carcinoma of the prostate gland, breast cancer or uterine myoma, as well as non-malignant indications, the treatment of which requires inhibition of the I H-KN hormone, such as endometriosis or benign prostatic hyperplasia (BPH). . In addition, they can be used for the treatment of fertility disorders in women and 7/0 men, for example, for controlled ovarian superstimulation within artificial insemination (insemination ip miigo). For this purpose, they are usually mixed with traditional carriers and excipients in ways known to specialists and packaged as medicinal products.

The synthesis of compounds according to the formula (I) is carried out both through the sequential construction using in the side chain already acetylated with a carboxylic acid of the general formula К"-СООН O-lysine by classical 75 fragment condensation or through the solid-phase synthesis according to Merrifield, and through the transformation of the decapeptide link with by corresponding carboxylic acids through an amide bond in the side chain

O-lysine? Thus, the introduction of the K7-CO group can occur at three different moments of the method: before the condensation of individual links to the peptide, after the insertion of lysine or ornithine into the peptide chain, but before the condensation of the next link, or after the condensation of all links.

Compounds of the formula (I) can be synthesized by known methods, for example, purely by a solid-phase method, partially by a solid-phase method (so-called fragment condensation) or through the classic combination of solutions | see M. Vodapzak, "Rhypsiriyez oi Reriide Zupipeviv", Zrhipdeg Megiad 1984).

For example, the methods of solid-phase synthesis are described in the works of |epgrisp "Zoiid Rnase Reriide Zupipneziv",

U.M. eemzhshai apa 9. O. Moipo, Riegse Spet. Sotrapu, Koskooga, Ii, 1984, and S. Vagapu apa K. V. Meigtiyei "Te em

Reriidev", Sp. 1, 5.1-285, 1979, Asadetis Rgezz Ips.). Classical methods of synthesis in solutions are described in detail in the work ("Meiyodep deg Ogdapizspep Spetie (Noirep-UUeui!), Zupipeze mop Reriidev" E. Umiipzsp ( Negaizdebeg) 1974,

Seogud Tpiete Megiad, ZishNdat, VKO.

Step-by-step construction is carried out, for example, as follows: first, the carboxy-terminal amino acid, the o-constant amino group of which is protected, is covalently bound to the insoluble carrier usually used for this purpose, | "c) which cleaves the o-amino-protecting group of this amino acid, the next protected amino acid binds to the free amino group formed in this way through its carboxy group, and in this way the remaining amino acids of the peptide to be synthesized are gradually connected in the proper sequence, and after the connection of all (α) amino acids, side functional protective groups are ready. Step-by-step condensation takes place in the traditional way through synthesis with the corresponding amino acids protected in the usual way.

C5 The connection of individual amino acids to each other usually occurs by the following methods, in particular: - - the method of symmetrical anhydride in the presence of dicyclohexylcarbodiimide or diisopropylcarbodiimide (cc, IC) - the carbodiimide method in general " - the carbodiimide-hydroxybenzotriazole method | see Te Reriides, Moiishte 2, Ed. E. Sgov5 apa 3.

Meijeprogeg). t c When connecting fragments, in the optimal version, an azide connection is used, which is carried out without racemization, or ros-1-hydroxybenzotriazole or ros-3-lhydroxy-4-oxo-3,4-dihydro-1,2, 3-benzotriazine method. Activated esters of fragments can also be used.

Activated esters of M-protected amino acids, such as, for example, M-hydroxysuccinimide ester or 2,4,5-trichlorophenyl ester, are particularly suitable for stepwise condensation of amino acids. Aminolysis is well catalyzed by M-hydroxy compounds that have the acidity of acetic acid, for example, 1-hydroxybenzotriazole. o As intermediate amino protecting groups, dehydrating groups are suitable, such as, for example, - 20 benzyloxycarbonyl residue (-7-residue) or weak acid leaving groups. As protective groups for

A-permanent amino groups are suitable, for example: m) tertiary butyloxycarbonyl groups, fluorenylmethyloxycarbonyl groups, carbobenzoxy groups or carbobenzthio groups (optionally with a corresponding p-bromo- or p-nitro-benzyl residue), trifluoroacetyl group, phthalic residue, o-nitrophenoxyacetyl group , trityl group, p-toluenesulfonyl group, 22 benzyl group, benzyl residues (p-bromo- or p-nitro-benzyl residue) o and a-phenylethyl residue substituted in the benzene skeleton. They are also indicated in the works (ezze R. Sgeepvivip ipa Miyop Umipi:, Spetivigu oi Atipo Asidz, Mem ZhMogk 1961", doip UmPeu apa 5opv, Ips., Moishte 2, for example, p.883 ff., "Rgipsiriev yu oi Reriide Zupipevziv", Zrgipdeg Megpad 1984, "Bod Rnaze Reriide Zupipeviv" 9.M.5iezhshay apa 4.0.Moypa,

Riegse Spet. Sotrapu, Koskioga, I, 1984, b. Vagapu apa k.V. Megitiyei "Te Reriidev", Sp. 1, 5.1-285, 1979, 60 Asadetis Rgezz Ips. and also Te Reriides, Moishte 2, Ed. E. Sgovzv apa u. Maieppoieg, Asadetis Rgezz, Mem

MogKk)|. In principle, these protective groups are also suitable for protecting other functional side groups (OH-groups, MNO-groups) of the corresponding amino acids.

The available hydroxy groups (serine, threonine) are optimally protected by benzyl and similar groups. Other non-o-constant amino groups (for example, amino groups in the o-position, guanidino group of arginine) are orthogonally protected in the optimal version.

Individual amino acid links, with the exception of lysine modified with a 1-CO group, are produced serially.

A possible order of implementation of the method of obtaining lysine modified by the B -CO group is as follows: 1. the o-carboxylic acid group is appropriately protected, for example, by esterification. 2. the ε-amino group is protected, for example, by a 2-group, 3. the o-amino group (for example, the Bos group) is protected in such a way that selectivity is ensured with respect to the subsequent cleavage of amino-protecting groups. 4. The 7-group on the ε-amino group is removed. 5. The required K7-CO group is added to the β-amino group. 6. The protective group on the o-amino group is removed. 7. the o-amino group is not necessarily subjected to reversible derivatization, for example, with a 2-group.

For the introduction of the B "-CO group through the transformation of the amino group of lysine with a suitable carboxylic acid or carboxylic acid derivative, the same method as described above for the connection of 75 amino acids is suitable in principle. However, condensation using a carbodiimide, for example 1-ethyl -3-(3-dimethylaminopropyl)-carbodiimide, and 1-hydroxybenzotriazole.

The reaction for the connection of amino acids takes place in a solvent or suspending agent usually used for this purpose (for example, dichloromethane), and dimethylformamide is optionally added to improve solubility.

As a synthetic base, insoluble polymers are suitable, for example, polystyrene resin in the form of granules, which swells in an organic solvent (for example, a copolymer of polystyrene and 195 divinylbenzene).

Construction of a protected decapeptidamide on a methyl-benzhydrylamine resin (MVNA-resin, i.e., a polystyrene resin having methyl-benzhydrylamino groups), which provides the desired C-terminal amide function of the peptide after

Non-E-splitting of the carrier is carried out according to the following scheme of the sequence of operations: p

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Mo-Boc-protected amino acids are usually combined in a three-fold molar excess in the presence of diisopropylcarbodiimide (IS) and 1-hydroxybenzotriazole (NOVO in SNSO/OME for 9 minutes, and the Boc-protecting group -I is cleaved by half-hour action of 5095 trifluoroacetic acid (TRA) in SNiC.” The chloranil test according to Christensen and the ninhydrin test according to Is) Kaiser can be used to control the complete transformation. The remaining free amino functions are blocked through acetylation in a five-fold excess of acetylimidazole in СНоСі. The sequence of reaction stages for the construction of peptides on resin is presented in the table. To cleave the resin-bound peptides, the corresponding end product of solid-phase synthesis is dried in a vacuum over RoCOv and treated in a 500-fold excess on HE/anisole 10:1/vol:vol at 020. o After distillation of HE and of anisole in a vacuum through extraction with anhydrous ethyl ether in the form of a white solid, peptides are obtained, the separation of the polymer carrier obtained in parallel is carried out by washing with 5095 aqueous solution of acetic acid. By careful narrowing of solutions in acetic acid in a vacuum, the corresponding peptides are obtained in the form of highly viscous oils, which are transformed after the addition of abs. of ether in the refrigerator to a white solid. (F. Further purification is carried out by standard methods of preparative high-pressure liquid chromatography (HPLC).

Conversion of peptides into their acid addition salts is carried out using acids known to specialists in a way. | in contrast, free peptides are obtained by converting their acid addition salts with bases.

Peptidoembonates can be obtained through the conversion of trifluoroacetic acid salts (TEA salts) of the peptide with free embonic acid (pamamic acid) or the corresponding disodium salt of embonic acid. To do this, the peptide-TEA salt in an aqueous solution is mixed with a solution of disodium embonate in a polar aprotic medium, ideally - dimethylacetamide, and the light yellow precipitate formed in 5 is separated.

The following examples illustrate the invention without limiting its scope.

Example 1 (0-68968):

As-0-MaI(2)1-0-Sra?-O-RaI(3)3-Beg'-M-Me-Tugo-O-I uv(V) 9-Me "-AgodZ-Rgo?-Zago - MNO

The synthesis of the decapeptide takes place on a polymer carrier with a loading density of 0.55 mmol/g (aminomethyl-substituted resin, Etos-protection, type O-1675, Ra. Vaspet). Lysine was connected as Ethos-O-I uv(Vos)-OH,

Ethos-defense groups were exposed to 2095 piperidine in OME. After simultaneous cleavage of all protective groups of the side chains and separation of the polymer carrier, the selected crude peptide was purified by preparative NRI C. After freeze-drying, 98.595 decapeptide was obtained.

Substitution on the β-nitrogen of ХО-lysine with 4-(4-aminophenyl)-amino-1,4-dioxobutyric acid was carried out using RuVor in OME with the addition of SIREA. Purification of the isolated unprocessed peptide was carried out by preparative NRI C. Immediately after this, freeze-drying provided approximately the 9995th product (trifluoroacetate) of the total formula SvoNiovSIM19O45 with the correct RGAV-M5 1633 (MAN) (calc. 1631.78096)

Example 2 (0-68969):

As-0-MaI(2)1-O-Sra?-O-RaI(3)3-Beg'-M-Me-Tugo-O-I uv(V) 9-Mie "-AgodZ-Rgo?-0 - AIa "9-MN"

The synthesis of the decapeptide was carried out on a polymer carrier with a loading density of 0.55 mmol/g (aminomethyl-substituted resin, Etos-protection, type ХО-1675, Ra. Vaspegp). Lysine was connected as Ethos-O-I uv(Vos)-OH,

Ethos-defense groups were exposed to 2095 piperidine in OME. After simultaneous cleavage of all protective groups of the side chains and separation of the polymer carrier, the isolated crude peptide with a content of approximately 7190 (NRI C) was further converted without purification.

Substitution in the side chain of SO-lysine 4-(4-aminophenyl)-amino-1,4-dioxo-butyric acid was carried out using RuVor in OME with the addition of SIREA. The isolated crude peptide was purified by preparative NRI C. After immediate freeze-drying, a 98.8905-th product (trifluoroacetate) of the general formula SvONiobSIM9O35 with the correct RBAV-M5 1633 (MAN) was obtained (calc. Se 1631.78096) (5)

Example C (0-68971):

As-O-Ma(2)-O-Sra?-O-RaI(3)3-Zeg'-M-Me-Tug?-O-I uv(B)9-Mie "-I uv(iRg)8 -Rho?-Zapo-MNo

The synthesis of the decapeptide was carried out on a polymer carrier with a loading density of 0.55 mmol/g (aminomethyl-substituted resin, Etos-protection, type ХО-1675, Ra. Vaspegp). Lysine was connected as Etos-O-I uv(Vos)-OH, o

Ethos protection groups split 2095 piperidine in OME. After simultaneous cleavage of all the protective groups of the rib chains and separation of the polymer carrier, the isolated crude peptide (Sepaii sa. 5995, NRI C) was purified by preparative NRI C. After freeze-drying, the 9590th decapeptide was obtained. at

Substitution in the side chain of O-lysine with 4-(4-aminophenyl)-amino-1,4-dioxo-butyric acid was carried out using RuVor in OME with the addition of SIREA. The isolated crude peptide was purified by

From the preparative NRI of S. After immediate freeze-drying, 96.6905th was obtained - a product (trifluoroacetate) of the total formula Sv5H.442SIM-7O35 with the correct RGAV-M5 1648 (MAN) (calc. 1645.8218)

Example 4 (0-68987) «

As-O-Mai(2)1-0-Rpe(4-SI)2-O-RaI(3)3-Veg"-M-Me-Tug?-O-I uv(B) 9-Mie 7- I uv(iR r) 8-Rgo?-O-AIa"0-MN"

The synthesis of 0-I uz-6-unsubstituted decapeptide was carried out on 9.09 g of a polymer carrier with a loading density of 0.55 mmol/g, lysine? connected as Etos-O-I uv(Vos)-OH. After the separation of the resin, 8.15 g of the unprocessed peptide was isolated. Purification of the unprocessed peptide was carried out by preparative NRI C.

Substitution in the side chain of O-lysine with 4-(4-aminophenyl)-amino-1,4-dioxo-butyric acid was carried out 79 using RuVor in OME with the addition of SIREA. The isolated crude peptide was purified by preparative NRI C. After immediate freeze-drying, a 94.6905-th (Ce) product (trifluoroacetate) of the general formula Sv5H.412SIM47O45 with the corresponding BAV-M5 was obtained: 1646.8 (MAN; cal.: about Macau report 5: -about 70 As-0-MaI(2)1-O-Sra?-O-RaI(3)3-Beg'-M-Me-Tug-O-Sib-Me"-AgudZ-Rgo ?-O-AIa"9-MN" o Example 6:

As-0-MaI(2)1-O-Sra?-O-RaI(3)3-Beg'-M-Me-Tugo-Or-Nsi? | ей"-Agdv-Rgo?-O-AIa 79-МНо

Example 7:

As-O-Ma(2)1-O-Sra?-O-RaI(3)3-Zeg'-M-Me-Tug?-O-Si-Mie 7-I uv(iRg2)-Rgo?-O -AIa"9-MNo

Example 8:

IF) As-0-MaI(2)1-0-Sra?-O-RaI(3)3-Beg'-M-Me-Tugo-O-Nsi?- I ey 7-I uv(iRgU)-Rgo9 -O-AIa"9-MH" ko General technological instructions for obtaining peptides according to Examples 5 to 8:

Decapeptides can be obtained both by Merrifield solid-phase synthesis (ZRRB) and by 60 classic fragment condensation in solution. The construction of a peptide sequence on a polymer carrier is preferred for economic reasons, and it is carried out according to the choice of methods (1) Vos or (2) Ethos; accordingly, use either methyl-benzhydrylamine resin (for 1), or

Ethos-2,4-dimethoxy-4-(carboxymethyloxy)-benzhydrylamine resin (for 2) for the C-terminal addition of p-alanine. 65 Solid phase synthesis. Merrifield's method:

Decapeptides are synthesized under standardized reaction conditions (scheme of the sequence of operations, Table 1) for solid-phase synthesis according to the Ethos methodology using 5 grams of a polymer carrier

Ethos-2,4-dimethoxy-4-(carboxymethyloxy)-benzhydrylamine resin, Ra. Vaspegp 01675, loading density approximately O0.55mmol/g, particle size 200-400mesh.

Step-by-step construction of the sequence on the resin is carried out with the help of Mo-Ethos-protected amino acids according to the following scheme of the sequence of operations: o iv in prin 0 (volronyu 111111111102e|; ho in zedinya 000 Voleon new antimony zbe) lh in Promyanya 000 ME 00010000 15. control test |purging with sarenium chloride| | sch y Nilsnnnnvvnnanayashny o

D see T. SNgiviepzep, Ada Spet. Zsapa In 33, 763-766, 1979)

Typical for the method (repetitive) parameters of the reaction of the solid-phase synthesis of decapeptides according to the above scheme: o z o - cleavage of the Ethos-protecting group 20 95 with piperidine in OME, 2 x 5 min at room temperature (Step 2). - the connection in threefold molar excess amounts of Ethos-amino acids with diisopropylcarbodiimide - (GIS) in the presence of hydroxybenzotriazole (NEW) (Step 8). o - C-terminal cleavage of the polymer carrier, including removal of protective groups of amino acid side chains with trifluoroacetic acid (TEA). Me

After cleavage of the polymer carrier, when using 5 grams of resin, approximately 5-6 grams of unprocessed peptide mixture with a content of approximately 70-80,905 of the required components are obtained; they are obtained by means of preparatory NRI S carried out immediately afterwards.

Purification of decapeptides by preparative NRI C: chromatography conditions: 19 Prep. NRI S, Ra. 5 pitaighs, YuOupatakh column ER18.12μm, ZOOA, I -250mm, 10-41 4mm «

Gradient system with software control, 4090 V--»9090 o V, 5 Ohv shsh s Solvent A: 970ml NoOZOml SNUSMnTml SEZSOON and Solvent B: ZOobml NgoZh-700ml SNZSMTmMl SEZSOON i"" UV detection, 5-22Ohm, flow rate bOml/min

The obtained fractions are narrowed in a vacuum and lyophilized. Decapeptides are formed as a light, colorless material. Conversion to the form of acetate salt -I required for further pharmacological processing is carried out immediately after this by means of chromatographic ion exchange.

Study of biological action: i-o Compounds according to the invention according to the formula | investigate their receptor binding. The method is based on (as described in the work "VeskKegz" ей а!., Eig. 9). Viospet. 231,535-543 (1995)) methods. Obtained by the above-described -1 50 synthesis of Seigogei iodide (| 22) (Ategzpat; specific activity 80.5Bq/fmol) using the reagent

IodosSep (Riegse). The reaction mixture is purified by reversed-phase high-performance liquid chromatography 62, and mono-iodinated Seigogei without unlabeled peptide is obtained. Suitable for specific receptor binding are approximately 8095 I"22I|-Seigogei and unlabeled compounds according to the invention.

Compounds according to the invention are tested for their activity by the following methods and 2, and 22 binding affinity is determined in a binding analysis with | 1251|Y-Seigogeikh (Method 1) and functional

F! activity with Thyriogeip as an agonistic stimulus (Method 2).

Method 1 (determination of Ko using the example of Seigogei): o Analysis of receptor binding according to (Veskege, T., Magpeiipeke, K., Keyapaeg, N., Niidaga R. (1995) "Beiesiop apa spagasieegilaiop oi tattayap sei! pev om /y viabie omegehrgezviop oi Ppitap rishyagu 60 teseriogv og dopadoiyregip (SPEN)" Eng. 9. Viospet. 231, 535-543.

For the study of receptor binding of Seygogei using the reagent IosdosSep (Riegse) iodized

Г25II (Atezhpat; specific activity 80.5 Bq/fmol). The reaction mixture is purified by high-performance liquid chromatography with phase reversal, and mono-iodinated Seygogeichi without an unlabeled peptide is obtained. Approximately 8095 I"22I| Secgogei were suitable for specific receptor binding. 65 Receptor binding assays are performed with intact cells under physiological conditions as described

IVeskKege ei aYi. 1995). Subconfluent cultures of stably transfected I TC cells expressing

Human ICHN receptor, isolated by incubation in Mass/P; (137mM Mass, 2.7mM KCl, 8.1mM Ma»HPO,, 11.47mM KnoRODimMM EOTA and collected by centrifugation. The cell pellet is resuspended in binding buffer (OMEM without HCO), with 4.5 g/l glucose, 10 mM Nerev, pH 7.5, 0.595 (mass/volume) VZA, Tg/l bacitracin,

O, g/l 5VTI, 0.196 (mass/volume) Mother"). For displacement assays, 0.25 x 10 cells/1000 μl are incubated with approximately 225 pM |25I|Y-SeigogeiH (specific activity 5-10x10"art/pmol) and various concentrations of an unlabeled compound according to the invention as a competitive compound. A layer of cell suspension in 10 Оmcl binding medium is applied in 400 μl tubes to 200 μl 84 90 (by volume) silicone oil (MegskK Tour 550)/1695 (by volume) paraffin oil 70. After incubation for 3 hours at 37 °C with slow continuous shaking of the cells by centrifugation for 2 min at 9000 rpm (Rotor type НТА13.8; Negayiz Zeraiyes, Oviegode/Segtapu) is separated from the incubation medium. The ends of the tubes containing the cell pellet are cut off. The cell pellet and supernatant are immediately analyzed by y-radiation counting The amount of non-specifically bound material is determined by the inclusion of unlabeled Seigogei with a final concentration of 1 µM, and it is, as a rule, 1095 of the total amount of binding. their binding is carried out using the EVOAL idapa analytical program (Viozoy M3.0).

Seygogeich shows a Cro value of 170 picomoles per liter (pM) (number of independently performed tests: 21).

Method 2 (Functional analysis to determine the antagonistic efficiency (IC values)):

The analysis is carried out with the following modification, as described in the work (VesKegv5, T., Keiyapaeg, N.,

Niidaga, R. (1997) "Snagasiegigayop oi dopadoigorip-geieeavipd pogtope apayudvz Brazei op a zeppoyme seyishag

Isitegase heropeg depe azzau", Apaiui Viospet. 251, 17-23 (VesKege ei a). 1997) І. 10,000 cells per well, which express the human NKN receptor and the luciferase reporter gene, are cultured for 24 hours in microtitre plates with with OMEM supplemented with 195 (vol:vol) EC5. Cells were immediately stimulated for 2 h using 1 nM (O-TgrU| and NEN. Before stimulation, antagonistic c 29 compounds according to the invention were added, and the cells were finally lysed for quantification of cellular I is activity. Ge)

Calculation of ISvo values on the basis of dose-effectiveness curves is carried out through nonlinear regression analysis using the Nii-model (Rgodgatt EX 2.0 from S OgipmzhaId, AggpeitShShemetk Ogevzdep). Quantification

His activities are carried out essentially as described by |IRgoted Thesppis! VyPeipe 2Zh101/161) using the appropriate luciferase analytical system (Rgoteda E4030) in duplicate. Due to the addition of coenzyme A (CoA), oxidation of luciferyl-CoA occurs with the required kinetics. After removing the culture medium from the microtiter plate, the cells are lysed by adding 100 μl of lysis buffer (25 mM Tgiv phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM o 1,2-diaminocyclohexane-M,M,M',M'-tetraacetic acid (SOTA), 1095 (volume:volume) glycerin, 1956 (volume) du

Tiiyop X-100). After 15 min of incubation at room temperature, 1 μm of cell lysate is transferred to a suitable 39 for luminometric detection of a white microtiter tablet (Oupaisn). in

Enzymatic reaction is caused by adding 5 μl of analytical buffer (20 mM tricine, pH 7.8, 1.07 mM (MoCO3)Mo(OH)2), 2.67 mM Ma9zoO, 0.1 mM ethylenediamine-tetraacetic acid (EOTA), 33.9 mM dithiothreitol , 27 µM coenzyme A, 470 µM firefly luciferin (Rpoiipiz rugaiiv), 530 µM gATRMa"). After one minute "for a total time of one second, the luminescence is determined with a half-time of the signal of five minutes with 50 application of ES Vegiioia Misgoi itas I V 96 R. s Table 2 collects the physico-chemical and ip migo data of the compounds according to the invention . IT refers to the functional

From" activity, and pM means the number of picomoles per liter. Solubility in water was determined by the method described in note 2):

IN.

F o z o ov

F)

GI Determination of solubility by the Atizbiai method in Ringer's solution:

To determine the solubility by the Atizriai method, the tested substance is mixed in excess with an inert carrier, such as sand, and placed in a glass column (volume approximately 10 ml). At the bottom of the column, filters made of cotton wool and fiberglass were installed in advance. The mixture of the substance and sand is soaked for 1 hour in 1.0 ml of solvent, in which its solubility must be determined. Immediately after this, 1 Oml of the solvent is poured into a glass column. The solution is pumped cyclically using a hose pump. Determination is carried out at room temperature (approximately 202C). Solubility is determined when the mass concentration of successively taken fractions is 65 unchanged. Determination of mass concentration is carried out using the NRI C-method described below.

NRI C-Method:

Equipment

NRI sS-system: Nemlek RasKaga 1100; Oeseiesiop Nemieyi Raskaga SA Zegievz 1100

Column:

Column material: Mysieovikyu 120-3 St

Particle size: µm

Column size: 125 mm

Manufacturer: MasNegeu 8. Made!

Equipment parameters: 70 Injection volume: 15 μl

Flow rate: 1.Oml/min

Furnace temperature: 4596

Wavelength: 22 nm

Stopping time: 15 min. 5576 mobile phase A: mix 97 ml of Mg-O-H2O, 3 ml of acetonitrile and 1 ml of trifluoroacetic acid.

The final pH level is approximately 1.9. 45956 of mobile phase B: mix 300 ml of Mg-2-H20, 700 ml of acetonitrile and 1 ml of trifluoroacetic acid.

The final pH level is approximately 1.8.

The introduction of compounds according to the invention can take place in various forms acceptable for peptide active substances. Suitable methods of administration are well known to those skilled in the art. Administration can take place, for example, by injection. Administration can take place, for example, parenterally. Preference is given to subcutaneous (5.c), intramuscular (i.t.), intravenous (i.m.-), buccal (for example, sublingual) or rectal administration. Particular preference is given to i.5. and i.p. introduction

The compounds according to the invention are suitable for obtaining various forms of application, for example, for Ha lyophilizates, solutions or suspensions. Suitable forms of application and their preparation are known to specialists.

Suitable excipients and fillers are, for example, hexites such as mannitol, in particular O-mannitol, o

Y-mannite or O, -mannite, sorbitol such as Y-sorbitol, Y- or I-altrite, ydite, glucite and dulcite. The preparation is carried out by methods known to specialists, for example, by stirring, suspending or lyophilization. at

Example A: Lyophilisate for 1 mg of the compound according to Example 1 to obtain a subcutaneous injection solution (corresponding to 0.26-0.27 mg of acetate salt); 0-16.9 mass parts of SO-mannitol, in the optimal version 0.1-7 - mass parts, all - in relation to Example 1, and water for injection purposes (for obtaining an injection solution from a lyophilizate).

Preparation: 1.62 g of the composition according to Example 1 is dissolved in 30965 acetic acid (approximately 1.5 liters of water for injection purposes and 91.17 g of acetic acid). The solution is diluted with water in the amount of 1.5 liters. Add 82.2 g of chmannite, filter through a sterile filter, pack into 2 ml sterile injection ampoules under aseptic conditions and freeze-dry. 1 mg of lyophilisate of the compound according to Example 1 is obtained.

The compounds according to the invention are suitable, for example, for the treatment of malignant or non-malignant hormone-dependent diseases, for example, for the treatment of breast carcinoma, prostate carcinoma, endometriosis, uterine fibroids, benign prostatic hyperplasia (BPH), as well as for the treatment of female or male fertility disorders, for example, for the prevention of premature ovulation in patients subjected to controlled ovarian stimulation followed by egg retrieval and artificial insemination. The above examples of treatment can be applied to mammals, in particular, humans. 1. Compounds of the general formula 1 - 20 А-Ххх -Ххх?-ХххЗ-Ххх"-Ххх?-Хххб-Ххх /-ХххЗ-Ххх?-Ххх 9-МН" (1), where c A represents an acetyl group,

Xxx" represents O-Ma|(2),

Hhhh? represents O-Sra, 52 XxxZ represents O-Ra/(3),

F! Xxx" represents 5eg, with Xxx? represents M-Me-Tug,

Hhhh? represents O-Si or O-(E-M'-4-(4-amidinophenyl)-amino-1,4-dioxo-butyl-I uv (abbreviated: О-I uv(B)), vo Xxx" represents myself Mie,

Hhhh? is Ag or I uv(IRg),

Hhhh? is Ro, and

Xxx "o represents O-Alya or Zag, with the exception of the compound in which Xxx? represents O-Csi and Xxx "o represents Zag, as well as their salts with pharmaceutically acceptable acids.

Claims (1)

2. The compound according to claim 1, which differs in that it has the formula: As-0-MaI(2)1-0-Sra?-O-RaI(3)3-Beg'-M-Me-Tugo-O-I uv(B) 9-Me "-AgodZ-Rho?-Zagpo-MH", or its salt with pharmaceutically acceptable acids. Z. The compound according to claim 1, which differs in that it has the formula: As-0-Ma(2)1-O-Sra?-O-RaI(3)3-Zeg"-M-Me-Tug?-O- And uv(B) 9-Mie "-Agob-Rho?-O-AIa"0-MH", or its salt with pharmaceutically acceptable acids. 4. The compound according to claim 1, which differs in that it has the formula: As-0-MaI(2)1-O-Sra?-O-RaI(3)3-Beg'-M-Me-Tugo-O-I uv(B) 9-Mie and uv(iRg)8-Rgo?-Zag'9-MH", or its salt with pharmaceutically acceptable acids. 5. The compound according to claim 1, which differs in that it has the formula: As-O-Mai(2)1-0-Pne(4-СИ)2-O-Ra(3)3-Zeg"-M-Me- Tug?-O-I uv(B)9-Mie 7-I uv(iP)Z-Rgo?-O-AiIa"9-MH", or its salt with pharmaceutically acceptable acids. 6. The compound according to claim 1, which differs in that it has the formula: 19 As-0-MaI(2)1-O-Sra?-O-RaI(3)3-Beg'-M-Me-Tug-O- Sib-Me"-AgudZ-Rgo?-O-Alya"9-MN", or its salt with pharmaceutically acceptable acids. 7. The compound according to claim 1, which differs in that it has the formula: As-0-MaI(2)1-O-Sra?-O-RaI(3)3-Beg'-M-Me-Tug-O-Sib -Me"-I uv(iRg)8-Rgo?-O-AIa"9-MH", or its salt with pharmaceutically acceptable acids. 8. The compound according to any one of claims 1 - 7, which differs in that the salt is acetate, trifluoroacetate or embonate. 9. The compound according to any of claims 1-8, which is characterized by the fact that it is used as a medicine. 10. Pharmaceutical composition, which includes at least one compound according to any of claims 1 - 8, as well as traditional carriers and excipients. ch » 11. The method of obtaining compounds of the general formula 1 according to any of claims 1 - 7, in which the fragment from the units of Xxx "(o) with appropriate protective groups, where t corresponds to a whole number from 1 to 10, and Xxx is acetylated , are synthesized by generally accepted methods on a solid support or in solution, then the fragments are condensed on a solid support by means of segmental coupling, and after completion of the coupling of compounds of the general formula 1, they are cleaved from the solid phase by the generally accepted method of amidation along the Xxx 9 chain, 12. Use of compounds according to any of claims 1-34 for the preparation of medicinal products for the treatment of - hormone-dependent tumors, in particular prostate carcinoma, breast cancer or uterine myomas, as well as non-malignant indications, the treatment of which requires inhibition of IH-KN- hormone, such as endometriosis, benign prostatic hyperplasia (BPH) or for the treatment of female or male fertility disorders in mammals, in particular humans. uh- 13. The method of obtaining a pharmaceutical composition according to claim 10, which is characterized by the fact that at least one compound according to any of claims 1-8 is mixed with traditional carriers and excipients and formulated as a medicine. 14. The method of treatment of hormone-dependent tumors, in particular carcinoma of the prostate gland, cancer of the breast or uterine fibroids, as well as non-malignant diseases, the treatment of which requires suppression of the INH-KN hormone, such as endometriosis, benign prostatic hyperplasia (BPH), and the treatment of female or male fertility disorders in mammals, in particular humans, which differs in that it is administered;" an effective dose of at least one compound according to any one of claims 1-8. Official bulletin "Industrial Property". Book 1 "Inventions, useful models, topographies of integrated -I microcircuits", 2007, M 7, 25.05.2007. State Department of Intellectual Property of the Ministry of Education and Science of Ukraine. se) («c) - 50 (42) F) ime) 60 b5