Cyclopeptide, the way they are received, containing the pharmaceutical composition, its preparation


(57) Abstract:

The invention relates to new cyclopeptides formula cyclo-(pad-nGly-nAsp-nD-nE), where n, D, and E have the meanings indicated in the claims, and to pharmaceutical compositions based on them having inhibitory activity againstv3and/orv5-integrins and to a method for producing a pharmaceutical composition having inhibitory activity. Cyclopeptide act as inhibitors of integrins and can be used for the treatment and prevention of tumors and/or metastasis. 4 C. and 6 C.p. f-crystals.

The invention relates to new cyclopeptides formula I

Cyclo-(nArq-nGly-nAsp-nD-nE) (I),

where D and E are each independently of one another denotes Gly, Ala, -Ala, Asn, Asp, Asp(OR), Arg, Cha, Cys, Gln, Glu, His, Ile, Leu, Lys, Lys(Ac), Lys(AcNH2), Lys(AcSH), Met, Nal, Nle, Orn, Phe, 4-Hal-Phe, Homo-Phe, Phg, Pro, Pya, Ser, Thr, Tia, Tic, Trp, Tyr or Val

moreover, these amino acid residues can be derivatization,

R is alkyl with 1-18 C atoms, Hal, F, Cl, Br, I,

Ac alkanoyl with 1-10 atoms, aroyl with 7-11 C atoms or arkanoid with 8 to 12 C-atoms,

n does not denote any Deputy or denotes the remainder R is alkyl, benzyl or Uralkaliy residue with 7 to 18 C-atoms in the alpha-AMINOPHENYL corresponding tel n, and since we are talking about the balance of optically active amino acids and derivatives of amino acids, are covered as D-and L-forms, as well as their physiologically acceptable salts.

Such compounds, without, however, N-alkyl substituents, are known for example from EP 0406428 and FEBS Lett. 291, 50-54 (1991).

The basis of the invention lies task is to find new compounds with valuable properties, in particular those that can be applied to obtain drugs.

It has been unexpectedly found that the compounds of formula I and their salts have very valuable properties. First of all, they act as inhibitors of integrin, and they specifically inhibit the interaction of integrin receptors3or5with ligands. Particularly active compounds exhibit in the case of integrinsv3,v5or113but also in relation to the receptorsv3v6andv8. These actions can be proven, for example, by the method described by J. W. Smith et al in J. Biol. Chem. 265, 12267-12271 (1990). Additionally manifest anti-inflammatory effects.

The dependence of the occurrence of angiogenesis from the interaction between vascular integrins and Valet is for interaction and thereby start apotosis (programmed cell death) of angiogenic vascular cells cyclic peptide described P. C. Brooks, A. M. Montgomery, M. Rosenfeld, R. A. Reisfeld, T.-Hu, G. Klier and D. A. Cherish in the Cell. 79, 1157-64 (1994).

The compounds of formula I, blocking the interaction of integrin receptors with ligands, such as fibrinogen at the fibrinogen receptor (cicloprofen IIb/IIIa), prevent as GPIIb/IIIa-antagonists spread of tumor cells metastasis. This is confirmed by the following observations:

Compounds can inhibit the adherence of metalloproteinases to integrins and inhibit so that the cells can use the enzymatic activity of proteases. An example of this can be seen in zatrudnyaet joining MMP-2 (matrixmetalloproteinase-2) to vitronectin-receptorv3cyclo-RGD-peptide, as described by P. C. Brooks et al. in Cell 85, 683-693 (1996).

The spread of tumor cells from the local tumor in the vascular system occurs through the formation of microaggregates (microthrombi) due to the interaction of tumor cells with blood platelet. Tumor cells escaped from the protection microaggregate and are not recognized by cells of the immune system.

Microaggregate can be fastened in the walls of blood vessels, making easier the further penetration of tumor cells in tigerbunny blood plates, the GPIIa/IIIb antagonists can be considered as effective inhibitors of metastasis.

The compounds of formula I can further be used as antimicrobial substances in operations that are applied biomaterials, implants, catheters or cardiac stimulants. They act bacteriostatically. Antimicrobial effectiveness can be proven by the method described P. Valentin-Weigund et al. in Infection and Immunity, 2851-2855 (1988).

Since the compounds of formula I are inhibitors of the binding of fibrinogen and thereby ligand-receptor fibrinogen to blood platelets, they can be used as diagnostic tools for the detection and localization of thrombi in the vascular system in vivo, since they will be replaced by the residue labeled with an isotope or detected using UV rays. The visualization method allows to detect and localize with their help, even tumors (visualization of tumors; PET).

The compounds of formula I can be used as inhibitors of the binding of fibrinogen as an effective auxiliary substances for metabolic studies of blood platelets in various stages of activation or intracellular signaling mechanisms of receptor for fibrinogen. OBN is to sledovat these mechanisms.

The compounds have the property to inhibit the binding of natural or synthetic ligands to integrins, especially integrinsv3,v5,11b3but alsov1,v6andv8.

In comparison with the level of technology they have in addition the advantage that due to the N-alkylation of one or more peptide linkages is achieved metabolic stabilization and increased solubility in fats. Due to the reduction potential of hydrogen bridges as N-alkyl cannot be H-donor for C=O, improving the ability of penetration through the membrane, so that it may be received high oral absorbability, it can also increase the formation of plasma protein.

N-Alkylation of the peptide bond raises the inhibitory potency of compounds and increases the selectivity of inhibition against certain integrins. In particular, the selectivity can be influenced by the choice of the position and the number of N-alkyl groups.

The compounds can be applied as the actual beginning of medicines in medicine and veterinary medicine, in particular for the prevention and treatment of diseases of the circulation, thrombosis, heart attack the problems in particular osteoporosis, angiogenesis and is caused by angiogenesis diseases, such as diabetic retinopathy eye, macular degeneration, myopia, ocular histoplasmosis, rheumatoid arthritis, osteoarthritis, robotically glaucoma, but also colitis, Crohn's disease (Morbus Crohn), multiple sclerosis, psoriasis, and restenosis after angioplasty. Further, the compounds can be used to improve and support the healing process during microbial infections and acute renal failure.

These compounds can be detected, for example, using known literature methods described, for example, P. C. Brooks et al. in the Cell, 79, 1157-1164 (1994) or Science 264, 569-571 (1994).

Above - and below abbreviations amino acid residues indicate residues of the following amino acids.

Abu 4-Aminobutyric acid

Aha 6-Aminohexanoic acid

Ala Alanine

Asn Asparagine

Asp Aspartic acid

Asp (OR), Aspartic acid ( -ester)

Arg Arginine

Cha 3-Cyclohexylamin

Cit Citroen

Cys Cysteine

Dab 2,4-Diaminopentane acid

Dap 2,3-Diaminopropionic acid

Gln Glutamine

Glu Glutamic acid

Gly Glycine

His Giganology

Lys(AcSH) N-Mercaptoquinoline

Met Methionine

Nal 3-(2-Naphthyl)-alanine

Nle Norleucine

Orn Ornithine

Phe Phenylalanine

4-Hal-Phe, 4-Halogen-phenylalanine

The Phg Phenylglycine

Pro Proline

Pya 3-(2-Pyridyl)-alanine

Sar Sarcosine (N-methylglycine)

Ser serine

Tia 3-(2-Thienyl)-alanine

Tic of Tetrahydroisoquinoline-3-carboxylic acid

Thr Threonine

Trp Tryptophan

Tyr Tyrosine

Val Valine.

Next, indicate below:

Boc tert.-Butoxycarbonyl

Brl Benzyl

DCCl Dicyclohexylcarbodiimide

DMF Dimethylformamide

EDCI N-Ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride

Et Ethyl

Fmoc 9-Fluorenylmethoxycarbonyl

HoBt 1-Hydroxybenzotriazole

Me Methyl

Mtr 4-Methoxy-2,3,6-trimetilfenil-sulfonyl

NMe N-methylated-amino group

OBut Complex tert.-butyl ether

OMe methyl ester

OEt Complex ethyl ester

POA Phenoxyacetyl

i-Pr is Isopropyl

n-Pr n-Propyl

TBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-tetrafluoroborate

TFA Triperoxonane acid

Since the above-mentioned amino acids occur in several enantiomeric forms, above and below all these forms and also their mixtures (for example as an integral part of the compounds of formula I, can be supplied with corresponding known in themselves protective groups.

Further, the invention covers also such peptides, amino acid residues which are partially or completely derivatization. Under "derivatization" it should be understood that also includes so-called prodrugs, such as N-guanidino-ACI-derivatives of Arg, beta-esters Asp, N-alkanoyl, aminoalkanoic, mercaptoethanol-lysine derivative, (here are just a few of them). In addition, amino acid residues can be partially alpha-alkylated or, for example, for diagnostic purposes, the state of isotopes. Further included are such compounds of formula I, in which side chains of links D and E additionally derivatization amino-, carboxy - or mercaptopropyl as such derivatives are valuable starting compounds for more macromolecular conjugates, for example for the purpose of immunization and generate antibodies. Further, it is possible to apply the functional group in the side chain of certain amino acid residues or derivatizing amino acid residues for immobilization of peptides on polymer materials with the aim of obtaining offenderinfoservices columns sludge is then: fluorescent substituents.

A further object of the invention is a method of obtaining the compounds of formula I on p. 1 or one of its salts, characterized in that it is isolated in free form from one of its functional derivatives by treatment solvolysis or hydrogenolysis tool or the fact that the peptide of formula II

H-Z-OH (II),

where Z denotes-nArg-nGly-nAsp-nD-nE-nGly-nAsp-nD-nE-nArg-nAsp-nD-nE-nArg-nGly-nD-nE-nArg-nGly-nAsp - or-nE-nArg-nGly-nAsp-nD-

or a reactive derivative of such a peptide is treated collisium means,

or that cyclopeptide, itself corresponding to the formula I, but which have one or more free amino groups, acid groups and/or activated C-atoms, derivateservlet alkylation, acylation or etherification

and/or basic or acidic compound of formula I by treatment with an acid or base is translated into one of its salts.

Above and below the residues D, E and n have the above in formulas I and II values, not otherwise noted. Used letters for the corresponding residues are not in any connection with a single-letter code for amino acids.

In the above formulas, the alkyl refers mainly methyl, ethyl, isopropyl, n-butyl, sec.-butyl or tert.-booth is also Trp, Tyr, Homo-Ph, Nal or Phg, and equally preferred and also the D-form.

E is predominantly hydrophobic amino acid residue, in particular Gly, Ala, Val, Leu, Nle or Ile. The variable n denotes mainly N-methyl, N-ethyl, N-propyl-, N-benzyl - or N-isopropyl-substituted-amino group of the peptide, and several amino acid residues may be N-substituted by identical or different alkyl residues.

In accordance with this are the subject of the invention, in particular, those compounds of formula I in which at least one of these residues has one of the abovementioned preferred meanings.

A preferred group of compounds may be expressed by private formula Ia corresponding to the formula I, in which, however, mean:

D D-Phe, Phe, D-Trp, Trp, D-Tyr, Tyr, D-Phe, Phe, D-Nal, Nal, D-Phg-or 4-HAl-Phe (D - or L-form) and

E Val, Gly, Ala, Leu, Ile or Nle.

Another preferred group of compounds may be expressed by private formula Ib corresponding to the formula I, in which, however,

D represents D-Phe, and

E denotes Gly, Ala, Val, Leu, Ile or Nle and one of the amino acid residues Arq, Gly, or Asp at the amino group has an alkyl substituent.

Another site is, is the formula I, in which, however, one of the amino acid residues D, or E is alkylated in the amino group.

Further especially preferred all physiologically tolerated salts of the compounds falling under private formulas Ia, Ib and Ic.

The compounds of formula I and also the source materials for their production however get by known methods described in the literature (for example in standard works such as Houben-Weyl, Methods der organischen Chemie, Georg-Thieme-Verlag, Stuttgart), namely under the reaction conditions which are known and suitable for the mentioned transformations. Thus it is possible to use known in detail here, don't fix options.

Source materials can, if desired, be formed in situ so that they are not isolated from the reaction mixture, and immediately subjected to further interaction with obtaining the compounds of formula I.

The compounds of formula I can be obtained by selecting them in the free form of their functional derivatives by solvolysis, in particular hydrolysis, or by hydrogenolysis.

The preferred initial agents for the solvolysis or hydrogenolysis are those which contain instead of one or more free amino and/or Gintama, connected to the N-atom, are one aminosidine group, for example those which correspond to the formula I, but instead of NH-groups contain other'group (where R' denotes aminosidine group, for example BOC or CBZ).

Further preferred initial matter, bearing instead of the H atom of the hydroxyl group hydroxyamino group, for example those which correspond to the formula I, but instead hydroxyproline groups contain R"O-phenyl group (where R' denotes hydroxyamino group).

In the molecule of the original substance may be several identical or different protected amino and/or hydroxyl groups. If the existing protective groups differ from one another, they can in many cases be selectively split.

The expression "Aminosidine group" generally known and relates to groups which are suitable for protecting (blocking) an amino group from chemical reactions but which can easily be removed after the desired chemical reaction carried out elsewhere in the molecule. Typical of such groups - in particular unsubstituted or substituted acyl, aryl, Alcoceber or Uralkaliy. As aminosidine group after completion of the desired reaction (or series d is a group containing 1-20, in particular 1-8 C-atoms. The expression "Acyl group" should be understood in connection with the present method in its broadest sense. It includes acyl groups derived from aliphatic, alifaticheskih, aromatic or heterocyclic carboxylic acids or sulfonic acids, and, in particular, alkoxycarbonyl, aryloxyalkyl and primarily alcoxycarbenium group. Examples of such acyl groups are alkanoyl, such as acetyl, propionyl, butyryl; arkanoid, as phenylacetyl; aroyl, such as benzoyl or toluoyl; aryloxyalkanoic, such as POA; alkoxycarbonyl, as methoxycarbonyl, etoxycarbonyl, 2,2,2-trichlorocyanuric, BOC, 2-iodoxybenzoic; arelaxation, such as CBZ ("carbobenzoxy"), 4-methoxybenzeneboronic, Fmoc; arylsulfonyl, such as Mtr. Preferred aminosidine groups are BOC and Mtr, then CBZ, Fmoc, benzyl and acetyl.

The expression "Hidroxizina group" is also generally known and relates to groups which are suitable for the protection of the hydroxy-group from chemical reactions but which can easily be removed after the desired chemical reaction carried out elsewhere in the molecule. Typical of such groups are the above-mentioned resumes the protective groups are not critical, since after completing the desired chemical reaction or series of reactions they again removed; preferred group with 1-20, especially with 1-10 C-atoms. Examples hydroxyamine groups are, inter alia, benzyl, p-nitrobenzoyl, p-toluensulfonyl, tert.-butyl and acetyl and benzyl and tert.-butyl is particularly preferred. COOH-group in aspartic and glutamic acids are preferably protected in the form of their tert.-mutilation (for example, Asp(OBut)).

Functional derivatives of compounds of formula I used as starting substances can be obtained by conventional methods of amino acid and peptide synthesis, as described, for example, called the standard works and patent applications, as well as, for example, solid-phase method and Merrifield (B. F, Gysin and R. B. Merriffield, J. Am. Chem. Soc. 94, 3102 ff. (1972)).

The selection in the form of free compounds of the formula I from their functional derivatives is possible - depending on the applicable protective groups for example, with a strong acid, preferably TFA or perchloric acid, but also with other strong inorganic acids, such as hydrochloric or sulfuric acid, strong organic carboxylic acids, such as trichloroacetic acid, or sulf the indicator may but not always required. As inert solvents are suitable, usually organic, for example carboxylic acids, such as acetic acid, ethers, such as tetrahydrofuran or dioxane, amides, such as dimethylformamide, halogenated hydrocarbons such as dichloromethane, then also alcohols, such as methanol, ethanol or isopropanol, and also water. Next you can apply a mixture of the above solvents. TFA is used, typically, in excess without the addition of another solvent, perchloric acid in the form of a mixture of acetic acid and 70% perchloric acid in the ratio 9:1. The reaction temperature for the removal of suitable selectable between 0 and approx. 50oC, typically operate at temperatures from 15 to 30oC (room temperature).

Group BOC, OBut and Mtr can preferably be chipped off using TFA in dichloromethane or approx. 3 - 5 N. HCl in dioxane at 15 - 30oC, the Fmoc group using approx. 5 - 50%-aqueous solution of dimethylamine, diethylamine or piperidine in dimethylformamide at 15 - 30oC.

Hydrogenations removable protective group (for example, CBZ or benzyl) can be derived, for example, by treatment with hydrogen in the presence of a catalyst (for example, a noble metal is quite suitable to the above, in particular, for example, alcohols, such as methanol or ethanol, or amides, such as dimethylformamide. The hydrogenolysis is carried out, generally at temperatures from 0 to 100oC and pressures from 1 to 200 bar, preferably at 20 to 30oand 1-10 bar. Hydrogenolysis of CBZ groups can, for example, on 5-10% Pd-C in methanol or with ammonium formate (instead of H2) on Pd-C in methanol/dimethylformamide at 20 - 30oC.

The compounds of formula I can be obtained by cyclization of compounds of formula II under conditions of peptide synthesis. While working appropriate according to conventional methods of peptide synthesis as described, for example, in Houben-Weyl, 1.c. Band 15/11, Seiten 1 bis 806 (1974).

Carrying out the reaction, usually in the presence of digitalisierung means, such as a carbodiimide, such as: DCCl or EDCl, then anhydride papapostolou acid (cp. Angew. Chem. 92, 129 (1980)), diphenylphosphinite or 2-ethoxy-N-etoxycarbonyl-1,2-dihydroquinoline, in an inert solvent such as halogenated hydrocarbon, such as dichloromethane, ether, such as tetrahydrofuran or dioxane, amide, such as dimethylformamide or dimethylacetamide, a nitrile such as acetonitrile, or in mixtures of these solvents, at temperatures between approx. -10 and 40oC, Priazovye peptide, it is advisable to work in diluted solutions (principle of dilution).

Instead of the compounds of formula II can be used in the reaction are also suitable reactive derivatives of these substances, for example those in which reactive groups are blocked intermediate protective groups. Amino acid derivatives of compounds of formula II can be, for example, applied in the form of their activated esters, expediently produced in situ, for example by addition of HOBt or N-hydroxysuccinimide.

Educt of the formula II, as a rule, new. They can be obtained by known methods, for example the above methods peptide synthesis and cleavage of the protective groups.

As a rule, first synthesize protected pentapeptidnogo esters of the formula R'-Z-OR", for example, BOC-Z-OMe or BOC-Z-OEt, which then amyraut in acid of the formula R'-Z-OH, for example BOC-Z-OH; last otscheplaut protective group R', receiving the free peptides of the formula H-Z-OH (II).

The derivatization of cyclopeptide, which corresponds to the compound of formula I, are also known methods, for example, by a method of alkylation of amines, the esterification of carboxylic acids or the nucleophilic semadeni the N. Y. (1985).

The basis of the formula I can be converted by acid into the corresponding salt accession acid. For this reaction the exchange of decomposition are particularly suitable acids which give physiologically not doubtful salt. So, can be used inorganic acids, for example sulfuric acid, nitric acid, halogen acids such as florodora acid or bromatologia acid, phosphoric acids such as orthophosphoric acid, sulfamic acid, then the organic acids, in particular aliphatic, alicyclic, analiticheskie, aromatic or heterocyclic one - or polybasic carboxylic, sulfonic or sulfuric acids, e.g. formic acid, acetic acid, procinema acid, pavlikova acid, diethyloxalate acid, malonic acid, succinic acid, Emelyanova acid, fumaric acid, maleic acid, lactic acid, tartaric acid, malic acid, benzoic acid, salicylic acid, 2 - or 3-phenylpropionate acid, citric acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinamide acid, methane - or econsultancy acid, ethicality acid, 2-hydrocone acid, lauriola acid. Salts with physiologically faulty acids, such as the picrate, can be used for isolation and/or purification of the compounds of formula I.

On the other hand, the acid of formula I by the interaction with the substrate can be transferred in one of its physiologically portable metal or ammonium salts. As the salts thus, in particular, discusses the salts of sodium, potassium, magnesium, calcium and ammonium, next, substituted ammonium salts, e.g. salts of dimethyl-, diethyl-, or Diisopropylamine, salt monoethanol-, diethanol - or triethanolamine, salt cyclohexyl-, dicyclohexylamine, salts of dibenzylethylenediamine, further, for example, salts with N-methyl-D-glucamine or with arginine or lysine.

The new compounds of formula I and their physiologically not objectively salt can be used to obtain pharmaceutical preparations by transferring them together with at least one carrier or auxiliary substance and, if necessary, together with one or more other active substances in a suitable dosage form. Thus obtained combination can be used as drugs in medicine or veterinary medicine. Substances vehicles is high), parenteral (e.g. intravenous injection) or local (e.g., local, dermal, ophthalmic or nasal) applications or for use in the form of inhalation aerosols and do not react with the new compounds, for example water or an aqueous isotonic solution of sodium chloride, lower alcohols, vegetable oils, benzyl alcohols, polyethylene glycols, glyceryltrinitrate and other fatty acid glycerides, gelatin, soya lecithin, carbohydrates such as lactose or starch, magnesium stearate, talc, cellulose, vaseline. For oral administration are, in particular, tablets, coated tablets, capsules, syrups, juices or drops; interest special lacquer tablets and capsules with a coating or, respectively, with shells that are resistant to gastric juice. For rektalnogo of use suppositories, for parenteral application solutions, mainly oily or aqueous solutions, then suspensions, emulsions or implants. For local use are suitable, for example, solutions that can be applied in the form of eye drops, further, for example, suspensions, emulsions, creams, ointments or comprimate. For use as inhalation of aerosols may apply sprayable rest gases (for example, CO2or substance-substitutes perchloroethane) state. It is advisable to use this active principle in micronized form, and can be one or more physiologically tolerated solvents such as ethanol. Inhalation solutions can be administered to a patient using a conventional inhalers. The new compounds can also be lyophilized and the resulting lyophilizate, for example, be used to obtain solutions for injection. Injections may be made in the form of injections or as a continuous infusion (e.g., intravenously, intramuscularly, subcutaneously or podvoloshino). These compositions can be sterilized and/or contain auxiliary substances, such as preservatives, stabilizers and/or wetting agents, emulsifiers, salts for regulating the osmotic pressure, buffer substances, dyes and/or fragrances. They can, if desired, also contain one or more other active substances, for example one or more vitamins.

The substances according to the invention can be administered to a patient, as a rule, similar to other known, commercially available peptides, in particular likewise opisannymi unit. The daily dosage is preferably between approx. 0.01 and 2 mg per kg of body weight. Special dose for each particular patient depends, however, on various factors, such as effectiveness of the specific compound, the age, body weight, General health, sex, diet, time and route of administration of the medicinal product, on the rate of excretion, combination of drugs and the severity of the corresponding disease. Preferred parenteral form of administration of medication.

One connection of the present invention, in which Arg or DArg replaced by Orn or DOrn, can be used as a precursor for the synthesis of peptides that constitute the subject of the present invention, as Orn can be transformed into an Arg by guanidine. This method is particularly suitable for the production of Arg-containing peptides labeled with isotopes11C or14C.

Furthermore, the new compounds of formula I can be used as integrilligent to get columns for affinity chromatography with the aim of obtaining integrins in its purest form.

The ligand, i.e. the derivative of the peptide of formula I, with covalently attached to the polymer carrier via anchor the property hydrophilic properties, for example, cross-linked, polisher, such as cellulose, sepharose or Sephadexacrylamide, polymers on polietilenglikoli basis or Tentakelpolymere.

As the anchor of the functions associated with polymeric media, mainly suitable linear alkylene chain with 2-12 C atoms, attached at one end directly to the polymer and at the other end having a functional group, such as hydroxy, amino, mercapto, maleinimide or COOH group, and will be able to connect with C - or N-terminal section of the corresponding peptide.

It is possible that the peptide was directly or via a second anchor function is connected to the anchor of the polymer. Then it is possible that peptides containing amino acid residues with functionalized side chains were connected through them with the anchoring function of the polymer.

In addition, certain amino acid residues included in the peptides of formula I, can be modified in their side chains that they can connect through groups of SH-, OH-, NH2- or COOH with an anchor polymer.

Possible with this unusual amino acids, such as, for example, derivatives of phenylalanine, are what is at the end of the chain.

Examples of amino acid residues, the side chain which can directly serve as the anchor is, for example, Lys, Orn, Arg, Dab, Dap, Asp, Asn, Glu, Gln, Ser, Thr, Cys, Cit or Tyr.

Examples of N-terminal anchors are remnants, such as: -CO-CnH2-NH2, -CO-CnH2n-OH, -CO-CnH2n-SH or-CO-CnH2n-COOH with n = 2-12, and the length of alkalinous circuit is not critical and it can optionally be substituted, for example, the appropriate aryl or alcylaryl remains.

C-terminal anchors can be for example-O-CnH2n-SH-, -O-CnH2n-OH, -O-CnH2n-NH2, -O-CnH2n-COOH, -NH-CnH2n-SH, -NH-CnH2n-OH, -NH-CnH2n-NH2or-NH-CnH2n-COO, and n, as well as for alkalinous chain rightly been said in the previous paragraph.

N - and C-terminal anchors can also serve as an anchor link for the already functionalized side chain amino acid residue. Particularly suitable, for example, amino acid residues, such as Lys(CO-C5H10-NH2), Asp(NH-C3H6-COOH) or Cys(C3H6-NH2), and the anchor always connect the harsh conditions of integrins is carried out under conditions normal for condensation of amino acids and are known in themselves and are already discussed in the section about how to obtain the compounds of formula I.

Along with the use of cyclopeptides for immobilization on polymeric materials for affinity chromatography columns it is possible to use compounds with their functionalized side chains for further derivatization of the diagnostic auxiliary reagents, such as fluorescent substituents.

Then perhaps the additional introduction of side chains of residues D and E of functional groups such as amino-, mercapto - or carboxypropyl, through which can then be obtained conjugates with proteins or other macromolecular substances, such as, for example, for the purposes of immunization and/or production of antibodies.

All temperatures are listed inoC. In the following examples, "Conventional treatment" means add, if necessary, water, neutralized, extracted with ether or dichloromethane, separated, the organic phase is dried over sodium sulfate, filtered, evaporated and purified via chromatography on kieselgel and/or crystallization. RZ = retention time (minutes). An analytical study was carried out port A: 0,3% TFA in water; eluent B: 0,3% TFA in 2-propanol/water (8:2) the gradient of 1-99% B in 50 min at a flow of 1 ml/min and determining at 215 nm. M+= molecular peak in the mass spectrum, obtained by the method of fast atom bombardment (FAB).

Example 1

A solution of 0.6 g of H-NMe-Arg(Mtr)-Gly-Asp(OBut)-D-Phe-Val-ONa /for example, derived from Fmoc-NMe-Arg(Mtr)-Gly-Asp(OBut)-D-Phe-O-Wang, and o-Wang means used in the modified methods of Merrifield balance 4-oxymethyl-phenoxymethylpenicillin resin, removal of Fmoc group with piperidine/DMF (dimethylformamide) and the removal of the resin using TFA/CH2Cl2(1:1) in 15 ml of dimethylformamide diluted with 85 ml of dichloromethane and mixed with 50 mg NaHCO2. After cooling in a mixture of dry ice with acetone to add 40 ál diphenylphosphinite. After 16 hours of standing at room temperature the solution is concentrated. The concentrate is filtered (column Sephadex G-10 in isopropanol/water 8:2) and then purified by liquid chromatography high pressure. Get after processing through TFA/H2O (98:2) cyclo-(NMe-Arg-Gly-Asp-D-Phe-Val); RZ = 18,1; FAB-MS (M + H): 589.

Similarly obtained by cyclization of the corresponding linear peptides and cleavage of the protective groups. (See at the end of the description.

Example 2

RAS is 0.9 ml thiophenol leave to stand for 4 hours at room temperature, then concentrate and thinned with water, dried by freezing. Using gelfiltration on Sephadex G 10 (acetic acid/water 1:1) and subsequent purification by preparative liquid chromatography high pressure under specified conditions get cyclo-(Arg-Gly-Asp-NMePhe-to dval); FAB-MS (M + H): 589.

Similarly, get:

from cyclo-(Arg(Mtr)-Gly-NMeAsp-DPhe-LEU), cyclo-(Arg-Gly-NMeAsp-DPhe-LEU); FAB-MS (M + H): 603;

from cyclo-(D-Arg(Mtr)-NMeGly-Asp(OBut)-DPhe-Nle): cyclo-(D-Arg-NMeGly-Asp-DPhe-Nle);

from cyclo-(NMeArg(Mtr)-Gly-D-Asp(OEt)-DPhe-LEU), cyclo (NMeArq-Gly-D-Asp-DPhe-LEU);

from cyclo-(NMeArg(Mtr)-Gly-Asp-Phe-DIle): cyclo-(NMeArg-Gly-Asp-Phe-DIle);

from cyclo-(Arg(Mtr)-Gly-NMeAsp-Phe-DLeu): cyclo-(Arg-Gly-NMeAsp-Phe-DLeu);

from cyclo-(Arg(Mtr)-NMeGly-Asp-Phe-DSer): cyclo-(Arg-NMeGly-Asp-Phe-DSer);

from cyclo-(Arg(Mtr)-NMeGly-Asp-DNal-Leu), cyclo-(Arg-NMeGly-Asp-DNal-Leu);

from cyclo-(NMeArg(Mtr)-Gly-Asp-Hal-DIle): cyclo-(NMeArg-Gly-Asp-Nal-DIle);

from cyclo-(Arg(Mtr)-Gly-Asp-NMePhg-to dval): cyclo-(Arg-Gly-Asp-NMePhg-to dval);

from cyclo-(Arg(Mtr)-Gly-NMeAsp-Trp-to dval): cyclo-(Arg-Gly-NMeAsp-Trp-to dval).

Example 3

80 mg of cyclo-(Arg-Gly-Asp-DPhe-NMeVal) five or six times dissolved in 0.01 M HCl and after each dissolution dried by freezing. Subsequent purification using liquid chromatography high pressure allows to obtain cyclo-(Arg-Gly-Asp-DPhe-NMeVal)HCl; FAB-MS (M + H): 589.

Similarly, get:

from cyclo-(NMeArg-Gly-Asp-DPhe-Valsp-DPhe-Val): cyclo-(Arg-Gly-NMeAsp-DPhe-Val)HCl;

from cyclo-(Arg-Gly-Asp-NMeDPhe-Val): cyclo-(Arg-Gly-Asp-NMeDPhe-Val)HCl;

from cyclo-(Arg-Gly-Asp-Phe-DNMe-Val): cyclo-(Arg-Gly-Asp-Phe-DNMe-Val)HCl; RZ = 18,2; FAB-MS (M + H): 589.

Similarly receive treatment with acetic acid (AcOH): cyclo-(Arg-Gly-NMeAsp-DPhe-Val): cyclo-(Arg-Gly-NMeAsp-DPhe-Val)AcOH; RZ = 15,4; FAB-MS (M + H): 589.

Similarly, the receive processing methanesulfonic acid (MeSO3H):

from cyclo-(Arg-Gly-Asp-DPhe-NMeVal): cyclo-(Arg-Gly-Asp-DPhe-NMeVal)MeSO3H; RZ = 17,8; FAB-MS (M + H): 589.

Example 4

To obtain phase affinity suspended 0.9 g N-maleimido-(CH2)5-CO-NH-(CH2)3-polymer (obtained by condensation of N-maleimido-(CH2)5-COOH H2N-(CH2)3-polymer / 10 ml 0.1 M nutrifaster buffer at pH 7 and add in 4oC one equivalent of cyclo-(Arg-Gly-Asp-DPhe-NMeLys(CO(CH2)2SH). Stirred for 4 hours while heating the reaction mixture to room temperature, the solid residue is filtered off and washed twice each time with 10 ml of buffer solution (pH 7) and then three times with water to 10 ml each time. Get cyclo-(Arg-Gly-Asp-DPhe - NMeLys(CO(CH2)2S-3-(N-maleimido-(CH2)5- CONH-(CH2)3-polymer)).

Example 5

Analogously to example 4 is obtained by condensation polymer-O-(CH2)2)4- CO-NH-(CH2)3-O-polymer).

Similarly obtained by condensation of cyclo-(NMe-Arg-Gly-Asp-DPhe-Lys-(CO-(CH2)5-NH2)) HOOC-CH2-O-polymer: cyclo-(NMe-Arg-Gly-Asp-DPhe-Lys-(CO-(CH2)5-NH-CO-CH2-O-polymer)).

The following examples relate to pharmaceutical compositions.

Example a: Injection flask

A solution of 100 g of cyclopeptide formula I and 5 g of secondary sodium phosphate in 3 l of double-distilled water was adjusted to 2 N. hydrochloric acid to a pH of 6.5, sterile filtered, dispensed into injection vials, lyophilizer in sterile sterile conditions and closed. Each injection vial contains 5 mg of active substance.

Example B: Suppositories

Melt a mixture of 20 g of the active substance of the formula I with 100 g of soya lecithin and 1400 g of cocoa butter, poured into moulds and allow to cool. Each suppository contains 20 mg of active substance.

Example C: a Solution of

Prepare a solution of 1 g of the active substance of the formula I, 9,38 g NaH2PO42H2O, 28,48 g Na2HPO412H2O and 0.1 g of benzylaniline in 940 ml of double-distilled water. Bring pH to 6.8, made up to 1 l and sterilized by irradiation. This solution can be applied in the form and aseptic conditions.

Example E: Tablets

A mixture of 100 g of cyclopeptide formula I, 1 kg of lactose, 600 g of microcrystalline cellulose, 600 g of corn starch, 100 g of polyvinylpyrrolidone, 80 g of talc and 10 g of magnesium stearate pressed in the usual way in the tablets, so that each tablet contains 10 mg of active substance.

Example F: Bean

Tablets are pressed as described in example E, and then cover in the usual way by a coating of sucrose, corn starch, talc, tragant and dye.

Example G: Capsules

As usual capsules of hard gelatin fill the active substance of the formula I, so that each capsule contains 5 mg of active substance.

Example H: Inhalation aerosol

Dissolve 14 g of the active substance of the formula I in 10 l of isotonic NaCl solution and the solution is poured through a pumping mechanism in the usual trade aerosol vessels. The solution can be sprayed into the mouth or nose. Each portion of the spray solution (approx. 0.1 ml) corresponds to a dose of approx. 0,14 mg

1. Cyclopeptide formula I

cyclo-(nArg - nGly - nAsp - nD - nE),

where D represents D-Phe, Phe, D-Trp, Trp, D-Tyr, Tyr, D-Phe, Phe, D-Nal, Nal, D-Phg-or 4-Hal-Phe (D - or L-form);

E denotes Val, Gly, Ala, Leu, Ile or Nle;

skinny residue with 7 - 18 C-atoms in alphalinolenic the corresponding amino acid residue, with the proviso that at least one amino acid residue has one Deputy n, as well as their optically active D - and L-forms,

and physiologically tolerated salts.

2. Enantiomer or diastereoisomer of cyclopeptide formula I under item 1.

3. Cyclopeptide formula I on p. 1, representing: a) cyclo-(NMeArg-Gly-Asp-DPhe-Val), b) cyclo-(Arg-Gly-Asp-DPhe-NMeVal), in a) cyclo-(Arg-NMeGly-Asp-DPhe-Val), cyclo-(Arg-Gly-NMeAsp-DPhe-Val), cyclo-(Arg-Gly-Asp-NMeDPhe-Val), (e) cyclo-(Arg-Gly-Asp-DPhe-NMeGly),

and their physiologically tolerated salts.

4. Cyclopeptide formula I under item 1 or their physiologically acceptable salts for obtaining medicines.

5. Cyclopeptide formula I under item 1 and their physiologically acceptable salts for the treatment and prevention of tumors and/or metastasis.

6. Cyclopeptide formula I under item 1 and their physiologically acceptable salts for obtaining immobilized ligands for affinity column chromatography.

7. Cyclopeptide formula I under item 1 and their physiologically acceptable salts for purification of integrins by affinity chromatography.

8. The method of producing cyclopeptide formula I under item 1 or one of it with the-nAgr-nGly-, -nD-nE-nArg-nGly-nAsp- -nE-nArg-nGly-nAsp-nD,

or its reactive derivative, process collisuem means and, if necessary, cyclopeptide formula I by treatment with an acid or base is translated into one of its salts.

9. A method of obtaining a pharmaceutical composition having inhibitory activity againstv3and/orv5- integrins, characterized in that an effective amount of the compounds of formula I according to p. 1 and/or one of its pharmaceutically portable salts together with at least one solid, liquid or semi-liquid carrier or auxiliary substance is transferred into a suitable dosage form.

10. Pharmaceutical composition having inhibitory activity againstv3and/orv5- integrins, characterized in that the active substance it contains at least one compound of formula I under item 1 and/or one of its physiologically-tolerated salt in an effective amount.


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