NO314694B1 - Biotin derivatives, pharmaceutical preparations containing them, and uses of the compounds - Google Patents

Abstract

Biotin compounds of formula I. wherein Q lacks or is -NH- (CH) „- CO- or -NH- (CH)„ - NH-, and Rer X-Arg-Gly-Asp-Y ,. A-Cys (R) -BU- or cyclo- (Arg-Gly-Asp-Z), wherein Z in the side chain is bound to Q or, if Q is deficient in biotin, and A, B, U, X, Y, Z and n have they in requirements in specified meanings, as well as their salts, can be used as integrin inhibitors, especially for prophylaxis and treatment of diseases of the circulation, in thrombosis, myocardial infarction, coronary heart disease, arteriosclerosis, angiogenic diseases and in tumor therapy.

Description

The invention relates to the following biotin compounds:

and the salts thereof.

Similar compounds of biotinylated peptides are e.g. described in WO 9415956 (biotinylated endothelin receptor antagonists), in WO 9413313 (biotinylated LHRH antagonists) or in WO 9418325 (biotinylated necrosis factor). The biotinylation of peptides during the solid-phase synthesis on resin, for the sake of the improved purification possibility, is described by TJ. Lobl et al. in Anal. Biochem., 770, 502 (1988). Similar compounds of cyclic and linear peptides are known from DE 43 10 643, DE 43 36 758, EP 0 406 428 and WO 89/05150.

The invention was based on the task of finding new compounds with valuable properties, especially those that can be used for the production of pharmaceuticals.

It was found that the above-mentioned compounds and their salts have very valuable pharmacological properties with good compatibility. Above all, they act as integrin inhibitors, as they particularly inhibit the interaction between o^-, ft- or /?5-integrin receptors and ligands, such as e.g. the binding of fibrinogen to the β-integrin receptor. The compounds show particular activity in the case of the integrins Oyft, 0^85, Gfobft as well as o^, o^ fa and o^Ss. This effect can e.g. is detected according to the method described by J.W. Smith et al. in J. Biol. Chem., 265, 12267-12271 (1990). The dependence of angiogenesis on the interaction between vascular integrins and extracellular matrix proteins has been described by P.C. Brooks, R.A. Clark and D.A. Cheresh in Science, 264, 569-71 (1994).

The possibility of the inhibition of this interaction and thus of the initiation of apoptosis (programmed cell death) in angiogenic vascular cells by means of a cyclic peptide is described by P.C. Brooks, A.M. Montgomery, M. Rosenfeld, R.A. Reisfeld, T.-Hu, G. Klier and D.A. Cheresh in Cell, 79, 1157-64 (1994).

The compounds according to the invention which block the interaction between integrin receptors and ligands, such as e.g. of fibrinogen on the fibrinogen receptor (glycoprotein Ilb/HIa), as GPIIb/IIIa antagonists prevent the spread of tumor cells through metastasis. This is substantiated by the following observations: The spread of tumor cells from a local tumor in the vascular system occurs through the formation of microaggregates (microthrombi) through interaction between tumor cells and platelets. The tumor cells are shielded by the protection in the microaggregate and are not recognized by the cells of the immune system.

The microaggregates can attach to vessel walls, thereby facilitating a further penetration of tumor cells into the tissue. As the formation of microthrombi is mediated through fibrinogen binding to the fibrinogen receptors on activated platelets, the GPIIa/IIIb antagonists can be considered active metastasis inhibitors.

The compounds according to the invention can be used as active medicinal compounds in human and veterinary medicine, in particular for the prophylaxis and/or treatment of thrombosis, myocardial infarction, arteriosclerosis, inflammation, stroke, angina pectoris, tumor diseases, osteolytic diseases such as osteoporosis, pathological angiogenic diseases such as e.g. inflammation, ophthalmological diseases, diabetic retinopathy, macular degeneration, myopia, ocular histoplasmosis, rheumatic arthritis, osteoarthritis, rubeotic glaucoma, ulcerative colitis, Crohn's disease, atherosclerosis, psoriasis, restenosis after angioplasty, viral infection, bacterial infection, fungal infection, in acute renal failure and wound healing to support the healing process.

The compounds according to the invention can be used as antimicrobially active substances in operations where biological materials, implants, catheters or pacemakers are used. The effect of the antimicrobial activity can be demonstrated through the method described by Valentin-Weigund et al., Infection and Immunity, 2851-2855 (1988).

As the compounds according to the invention are inhibitors of fibrinogen binding and thus ligands for fibrinogen receptors on platelets, they can be used as diagnostics for the detection and localization of thrombi in the vascular system in vivo, since the biotinyl residue is a UV detectable residue.

The compounds according to the invention can be used as inhibitors of fibrinogen binding and also as active aids for the study of the metabolism of blood platelets in different stages of activation, or of intracellular signaling mechanisms at the fibrinogen receptor. The detectable entity in "biotin markers" allows, after binding to the receptor, investigation of the aforementioned mechanisms.

The above and below abbreviations for amino acid residues stand for the rest of the following amino acids:

If the above-mentioned amino acids can appear in several enantiomeric forms, then above and below, e.g. as a constituent of the compounds according to the invention, all these forms and also their mixtures (e.g. the DL forms) are included. Furthermore, the amino acids, e.g. as a component in compounds according to the invention be provided with corresponding, per se known protective groups.

The compounds according to the invention and also the starting compounds for their preparation are prepared according to methods known per se, as they have been described in the literature (e.g. in standard works such as Houben-Weyl, Methoden der organischen Chemie, Georg-Thieme-Verlag, Stuttgart ), and then under reaction conditions that are known and suitable for the aforementioned conversions. In this way, you can also make use of variants that are known in and of themselves, and are not explained in more detail here.

If desired, the starting compounds can also be formed in situ, so that they are not isolated from the reaction mixture, but are immediately converted further into the compounds according to the invention.

The compounds according to the invention can also be obtained by freeing them from their functional derivatives by solvolysis, particularly hydrolysis, or by hydrogenolysis.

Preferred starting compounds for the solvolysis or hydrogenolysis are those which instead of one or more free amino and/or hydroxy groups contain correspondingly protected amino and/or hydroxy groups, preferably those which instead of an H atom is bound to an N atom , carries an amino protecting group, e.g. such as correspond to the compounds according to the invention, but which instead of an NH2 group contain an NHR group (where R' means an amino protecting group, e.g. BOC or CBZ).

Furthermore, starting compounds are preferred which instead of the H atom in a hydroxy group carry a hydroxy protecting group, e.g. such as correspond to the compounds according to the invention, but which instead of a hydroxyphenyl group contain an R"O-phenyl group (where R" means a hydroxy protecting group).

Several, identical or different, protected amino and/or hydroxy groups may also be present in the molecule of the starting compound. If the protective groups present are different from each other, they can in many cases be selectively cleaved off.

The term "amino protecting group" is generally known and refers to groups which are suitable to protect (to block) an amino group against chemical reactions, but which are however easily removable after the desired chemical reaction has been carried out elsewhere in the molecule. Typical of such groups are particularly unsubstituted or substituted acyl, aryl, aralkyl or aralkyl groups. As the amino protecting groups are removed after the desired reaction (or series of reactions), their type and size are otherwise not of decisive importance; however, those with 1-20, especially 1-8, C atoms are preferred. The term "acyl group" is to be understood in its broadest sense in the context of the present method. It includes acyl groups which are derived from aliphatic, araliphatic, aromatic or heterocyclic carboxylic acids or sulphonic acids, as well as in particular alkoxycarbonyl, aryloxycarbonyl and, above all, aralkylcarbonyl groups. Examples of such acyl groups are alkanoyl such as acetyl, propionyl, butyryl; aralkanoyl such as phenylacetyl; aroyl such as benzoyl or toluoyl; aryloxyalkanoyl as POA; alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, BOC, 2-iodoethoxycarbonyl; aralkyloxycarbonyl such as CBZ ("carbobenzoxy"), 4-methoxybenzyloxycarbonyl, FMOC; arylsulfonyl as Mtr. Preferred amino protecting groups are BOC and Mtr, further CBZ, Fmoc, benzyl and acetyl.

The term "hydroxy protecting group" is also generally known and refers to groups which are suitable to protect a hydroxy group against chemical reactions, but which are however easily removable after the desired chemical reaction has been carried out elsewhere in the molecule. Typical of such groups are the above-mentioned, unsubstituted or substituted aryl, aralkyl or acyl groups, further also alkyl groups. The type and size of the hydroxy protecting groups are not of critical importance, as they are again removed after the desired chemical reaction or series of reactions; however, groups with 1-20, especially 1-10, C atoms are preferred. Examples of hydroxy protecting groups include benzyl, p-nitro-benzoyl, p-toluenesulfonyl, tert-butyl and acetyl, benzyl and tert-butyl being particularly preferred. The COOH groups in aspartic acid and glutamic acid are preferably protected in the form of their tert-butyl esters (e.g. Asp (OBut)).

The release of the compounds according to the invention from their functional derivatives is successful, depending on the protective group used, e.g. with strong acids, suitably with 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 sulphonic acids such as benzene or p-toluenesulphonic acid. The presence of an additional inert solvent is possible, but not necessary. As an inert solvent, organic ones are preferably suitable, e.g. carboxylic acids such as acetic acid, ethers such as tetrahydrofuran or dioxane, amides such as DMF, halogenated hydrocarbons such as dichloromethane, also alcohols such as methanol, ethanol or iso-propanol, as well as water. Furthermore, there are mixtures of the previously mentioned solvents. TFA is preferably used in excess without the addition of a further solvent, perchloric acid in the form of a mixture of acetic acid and 70% perchloric acid in the ratio 9:1. The reaction temperatures for the cleavage are suitably between approx. 0 and approx. 50 °C, preferably working between 15 and

30 °C (room temperature).

The groups BOC, OBut and Mtr can e.g. preferably cleaved with TFA

in dichloromethane or with approx.' 3-5 N HCl in dioxane at 15-30 °C, the Fmoc group with an approx. 5-50% solution of dimethylamine, diethylamine or piperidine in DMF at 15-30 °C.

The trityl group is used to protect the amino acids histidine, asparagine, glutamine and cysteine. The cleavage takes place, depending on the desired end product, with TFA/10% thiophenol, the trityl group being cleaved from all the mentioned amino acids; when using TFA/anisole or TFA/thioanisole, only the trityl group is cleaved from His, Asn and Gin, while it remains on the Cys side chain.

Hydrogenolytically removable protecting groups (e.g. CBZ or benzyl) can be cleaved off e.g. by treatment with hydrogen in the presence of a catalyst (eg a noble metal catalyst such as palladium, conveniently on a support such as carbon). As a solvent, those indicated above are suitable, especially e.g. alcohols such as methanol or ethanol, or amides such as DMF. Hydrogenolysis is usually carried out at temperatures between approx. 0 and 100 °C and pressure between approx. 1 and 200 bar, preferably at 20-30 °C and 1-10 bar. A hydrogenolysis of the CBZ group takes place e.g. well on 5-10% Pd-C in methanol or with ammonium formate (instead of hydrogen) on Pd-C in methanol/DMF at 20-30 °C.

A base according to the invention can be transferred with an acid to the associated acid addition salt, e.g. by reaction of equivalent amounts of the base and the acid in an inert solvent such as ethanol, and by subsequent evaporation. For this turnover, acids that give the physiologically acceptable salts come into question in particular. Thus, inorganic acids can be used, e.g. sulfuric acid, nitric acid, hydrohalic acids such as hydrochloric acid or hydrobromic acid, phosphoric acids such as ortho-phosphoric acid, sulfamic acid, further organic acids, especially aliphatic, alicyclic, araliphatic, aromatic or heterocyclic mono- or polybasic carboxylic, sulphonic or sulfuric acids, e.g. formic acid, acetic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, lactic acid, tartaric acid, malic acid, citric acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methane- or ethanesulfonic acid, ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid , p-toluenesulfonic acid, naphthalene mono- and -disulfonic acids, and laurylsulphuric acid. Salts with physiologically unacceptable acids, e.g. picrates, can be used for isolation and/or purification of the compounds according to the invention.

On the other hand, an acid according to the invention can be converted by reaction with a base into one of its physiologically acceptable metal or ammonium salts. As salts, sodium, potassium, magnesium, calcium and ammonium salts, further substituted ammonium salts, e.g. the dimethyl, diethyl or diisopropylammonium salts, the monoethanol, diethanol or diisopropylammonium salts, the cyclohexyl or dicyclohexylammonium salts, the dibenzylethylenediammonium salt, also e.g. salts with arginine or lysine.

The object of the invention is further the use of the compounds according to the invention and/or their physiologically acceptable salts for the production of pharmaceutical preparations, particularly in a non-chemical way. Hereby, they can be brought together with at least one solid, liquid and/or semi-liquid carrier or auxiliary substance, and possibly in combination with one or more additional active compounds, in a suitable dosage form.

Subject matter of the invention are further pharmaceutical preparations containing at least one compound according to the invention and/or one of its physiologically acceptable salts.

These preparations can be used as pharmaceuticals in human or veterinary medicine. Carriers include organic or inorganic substances which are suitable for enteral (e.g. oral), parenteral, topical application or for an application in the form of an inhalation spray, and which do not react with the new compounds, e.g. . water, vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols, glycerol thyroacetate, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc or petrolatum. For oral use tablets, pills, dragees, capsules, powders, granules, syrups, juices or drops are particularly useful, for rectal use suppositories, for parenteral use solutions, preferably oil or water solutions, further suspensions, emulsions or implants, for topical use ointments , creams or powders. The new compounds can also be lyophilized and the lyophilisates obtained are used, e.g. for the production of injection preparations. The indicated preparations may be sterilized and/or contain auxiliary substances such as lubricants, preservatives, stabilizers and/or wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffer substances, colouring, flavoring and/or several additional active substances , e.g. one or more vitamins. For application as an inhalation spray, sprays containing the active compound either dissolved or suspended in a propellant gas or propellant gas mixture (e.g. CO2 or chlorofluorohydrocarbons) can be used. Appropriately, the active compound is then used in micronized form, as one or more additional physiologically compatible solvents may be added, e.g. ethanol. Inhalation solutions can be administered using regular inhalers.

The compounds according to the invention and their physiologically acceptable salts can be used as integrin inhibitors in the fight against diseases, especially pathological angiogenic diseases, thrombosis, heart attacks, coronary heart diseases, arteriosclerosis, tumors, osteoporosis, inflammations and infections.

The compounds according to the invention can then, as a rule, be administered analogously to other known peptides that are available in the trade, particularly, however, analogously to the compounds described in US-A-4,472,305, preferably at dosages between approx. 0.05 and 500 mg, especially between 0.5 and 100 mg per dosage unit. The daily dosage is preferably between approx. 0.01 and 2 mg/kg body weight. However, the particular dose for each patient depends on a wide variety of factors, e.g. of the activity of the particular compound used, of age, body weight, general state of health, heredity, of diet, of the time and route of administration, of the excretion rate, the drug combination and the severity of the individual disease for which the treatment applies. The parenteral application is preferred.

Furthermore, the new compounds according to the invention can be used in analytical biology and molecular biology. This utilizes the ability to form a complex between the biotinyl residue and the glycoprotein avidin. Use of biotin-avidin complexes is known from E.A. Bayer and W. Wilchek in Methods of Biochemical Analysis, 26,1-45(1980) (lit. 1).

The new compounds according to the invention are used as integrin ligands for the preparation of columns for affinity chromatography for the pure preparation of integrins. The complex of an avidin derivatized carrier material, e.g. Sepharose, and the new compounds according to the invention are formed according to methods known per se, such as are described, e.g. in literature 1. For this reason, this procedure is not discussed in more detail here, and reference is made to the relevant literature, e.g. literature 1.

Polymeric solid phases with preferably hydrophilic properties known in and of themselves within peptide chemistry are suitable as polymeric carrier materials, e.g. cross-linked polysaccharides such as cellulose, Sepharose or Sephadex, acrylamides, polyethylene glycol-based polymer or tentacle polymers.

The new compounds according to the invention can also be used as a diagnostic marker for antibiotin-antibody reactions in ELISA-type analyses, and by FACS (fluorescence-activated cell sorter) analysis. The use of antibiotin antibody for the detection of biotin is known from M. Berger, Biochemistry, 14, 2338-2342 (1975). The use of immunoglobulin IgG, which is derivatized with biotin, in an enzyme immunoassay (ELISA) is described by U. Holmkov-Nielsen et al. in Journal of Chromatography, 297, 225-233 (1984).

IJ. Immunol. Methods, 181, 55-64 (1995), it is by J. Gao and S.J. Shattil described an ELISA test that detects substances that inhibit the integrin-Gfobftn activation. For the detection, biotinylated fibrinogen is used here.

The use of flow cytometry in clinical cell diagnostics is described by G. Schmitz and G. Rothe in DG Klinische Chemie Mitteilungen 24 (1993), volume 1, pp. 1-14.

Furthermore, the compounds according to the invention can be used for measuring

the strength of ligand-receptor interactions in the force field microscopy (atomic force microscopy, AFM). Ligand preferably means a complex of avidin and the new compounds according to the invention. Receptor preferably means an integrin receptor. Measurements of adhesion forces between an avidin-functionalized force field microscope and biotinylated agarose are described by E.-L. Florin et al. in Science, 264, 415-417 (1994).

Pharmacological report

Receptor inhibin<g>sanalvse

Purified human integrins GPIIbllla (identical to cillbfi3) from platelets and avP3 from term placenta were absorbed into microtiter wells and challenged with biotinylated complementary ligands - vitronectin (VN) for avP3, and figrinogen (FGN) for ctllbp3 in the presence of increasing amounts of test compounds.

Method: 1 µg/ml biotin ligand was incubated with 1 µg/ml coated receptor in the presence of serially diluted peptides. After 3 hours at 30°C, bound ligand was measured by antibiotin-alkaline phosphatase detection.

References: Charo, I.F., Nannizzi, L., Smith, J.W. and Cheresh, D.A., J.Cell. Biol. 111, 2795-2800(1990).

The compounds tested exhibited higher inhibitory activity on OvpVvitronectin interaction and on cdIbp3-fibrinogen interaction compared to the comparison compounds in both assay systems.

Above and below, all temperatures are given in °C. In the following examples, "usual work-up" means: If necessary, water is added, if necessary, the pH value is adjusted between 2 and 10, depending on the constitution of the final product, it is extracted with ethyl acetate or dichloromethane, separated, the organic phase is dried over sodium sulfate, evaporated and purified by chromatography on silica gel, and/or by crystallization. Rf value on silica gel. Eluent: ethyl acetate:methanol = 9:1. RZ = retention time (minutes) by HPLC in the following systems:

[A]

Column: Nucleosil 7C18, 250 x 4 mm

Eluent A: 0.1% TFA in water

Eluent B: 0.1% TFA in acetonitrile

Flow rate: 1 ml/minute

Gradient: 20-50% B/30 minutes.

[B]

50 minute gradient of 0-80% 2-propanol in water with 0.3% TFA at 1 ml/minute on a column, Lichrosorb RP Select B (7 fim), 250 x 4 mm.

[C]

Column: Lichrosorb (5 µm) 100RP8.125 x 4 mm Eluent A: 0.01 M Na-phosphate, pH 7.0

Eluent B: 0.005 M Na phosphate, pH 7.0/60 vol% 2-propanol Flow rate: 0.7 ml/minute

Gradient: 1-99% B/50 minutes.

Mass spectrometry

(MS): EI (electron deionization) M<+>

FAB (fast atom bombardment) (M+H)<+ >DMPP resin stands for 4-(2',4'-dimethoxyphenylhyo^oxymethyl)phenoxy resin5 a super acid labile resin that allows the synthesis of side chain protected peptides.

Example 1

0.6 g of Fmoc-Lys(Boc)-OH is dissolved in 100 ml of dichloromethane, 1.2 equivalents of DMPP resin, 1.4 equivalents of HOBt and 1.4 equivalents of DCC1 are added, and the mixture is stirred for 12 hours at room temperature. After removal of the solvent, Fmoc-Lys(Boc)-DMPP resin is obtained. In the peptide synthesis apparatus, Fmoc-Pro-OH is condensed with H-Lys(Boc)-DMPP resin [release from Fmoc-Lys(BOC)-DMPP resin with piperidine/DMF (20%)], using a three times excess of protected proline. The coupling is carried out with DCCl/HOBt at room temperature. Fmoc-Pro-Lys(BOC)-DMPP resin is obtained. Analogously, subsequent removal of the Fmoc protecting group and subsequent couplings with Fmoc-Ser(But)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Gly-OH, Fmoc-Arg(Mtr)-OH, Fmoc -Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH and Bit-OH under reaction conditions that are repeated at each coupling

- release of the amino group with piperidine/DMF (20%)

washing with dimethylacetamide

-reaction with the Fmoc amino acid respectively Bit-OH Bit-Gly-Gly-Gly-Arg(Mtr)-Gly-Asp(OBut)-Ser(But)-Pro-Lys(BOC)-DMPP resin.

The resin is washed with CF3SO3H/CH2Cl2/H2O, and Bit-Gly-Gly-Gly-Arg(Mtr)-Gly-Asp(OBut)-Ser(But)-Pro-Lys(BOC)-OH is obtained. After removal of the protecting groups with 2 N HCl in dioxane, removal of the solvent, absorption of the residue in TFA/CH 2 Cl 2 and precipitation with Et 2 O, it is purified over RP-HPLC. Bit-Gly-Gly-Gly-Arg-Gly-Asp-Ser-Pro-Lys-OH x 2 TFA is obtained; RZ [B] =12.14; FAB 1056.

Example 2

Analogous to example 1, one obtains by consecutive connections of DMPP resin with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr( But)-OH, Fmoc-Lys(BOC)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH and Bit-OH: Bit-Gly-Gly-Gly-Lys(BOC)-Thr (But)-Ala-Asp(OBut)-Cys(Trt)-Pro-DMPP resin.

Through cleavage of the resin, cleavage of the protective groups and purification, Bit-Gly-Gly-Gly-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-OH x 2 TFA is obtained; RZ [B] 27.6; FAB 1273.

Example 3

To a solution of 3.05 g of cyclo-(Arg-Gly-Asp-D-Phe-Lys) [which can be obtained by cyclization of H-Arg(Mtr)-Gly-Asp(OBut)-D-Phe-Lys (BOC)-OH to cyclo-(Arg(Mtr)-Gly-Asp(OBut)-D-Phe-Lys(BOC)) and subsequent removal of the protecting groups] in 100 ml of dichloromethane is added 1.7 g of cheaply obtainable (+) -biotinyl-N-succinimidyl ester and 0.5 g of triethylamine. It is stirred for 5 hours at room temperature and is obtained after usual work-up cyclo-(Arg-Gly-Asp-D-Phe-Lys(N<£->Bit)) x TFA; RZ [B] 11.32; FAB 830.

Example 4

Analogously to example 3, 3.05 g of cyclo-(Arg-Gly-Asp-D-Phe-Lys) and 2.3 g of cheap (+)-biotinyl-6-aminocaproic acid-N-succinimidyl ester ("A") are obtained and 0.5 g triethylamine cyclo-(Arg-Gly-Asp-D-Phe-Lys(N<É->Bit-Aha)) x TFA; RZ [C] 23.67; FAB 943.

Example 5

6 g of Boc-Aha-N-succinimidyl ester are added to a solution of 3.05 g of cyclo-(Arg-Gly-Asp-D-Phe-Lys) in 40 ml of 5% aqueous NaHCO 3 and 40 ml of THF. It is stirred for 4 hours, worked up in the usual way and cyclo-(Arg-Gly-Asp-D-Phe-Lys(BOC-Aha)) is obtained; RZ [C] 27.7; FAB 817. After cleavage of the BOC group in HCl/dioxane, cyclo-(Arg-Gly-Asp-D-Phe-Lys(N<e->Aha)) x 2 TFA is obtained after normal work-up; RZ [C] 14.76; FAB 717.

Analogous to example 1, subsequent reaction with (+)-biotinyl-N-succinimidyl ester gives cyclo-CArg-Gly-Asp-D-Phe-LysQ^-Bit-Aha)) x TFA; RZ [C] 23.67; FAB 943.

Example 6

Analogous to example 4, one obtains from cyclo-(Arg-Gly-Asp-D-Phe-Lys-Gly) [which can be obtained by cyclization of H-Arg(Mtr)-Gly-Asp(OBut)-D-Phe-Lys (BOC)-Gly-OH to cyclo-(Arg(Mtr)-Gly-Asp(OBut)-D-Phe-Lys(BOC)-Gly) and subsequent removal of the protecting groups] and (+)-biotinyl-6-aminocaproic acid -N-succinimidyl ester cyclo-(Arg-Gly-Asp-D-Phe-Lys(N<e->Bit-Aha)-Gly) x TFA; RZ [A] 10.97; FAB 1000.

Analogously, cyclo-(Arg-Gly-Asp-D-Phe-Val-Gly) [which can be obtained by cyclization of H-Arg(Mtr)-Gly-Asp(OBut)-D-Phe-Val-Lys( BOC)-OH to cyclo-(Arg(Mtr)-Gly-Asp(OBut)-D-Phe-Val-Lys(BOC)) and subsequent removal of the protecting groups] and (+)-biotinyl-6-aminocapixic acid-N- succinidyl ester cyclo-(Arg-Gly-Asp-D-Phe-Val-Lys(N<e->Bit-Aha)) x TFA; RZ [A] 16.11; FAB 1042.

Analogously, cyclo-(Arg-Gly-Asp-D-Phe-N-Me-Lys) and (+)-biotinyl-6-arninocaporic acid-N-succinimidyl ester give cyclo-(Arg-Gly-Asp-D-Phe- N-Me-Light(N<*->Bit-Aha)).

Example 7

Preparation<g> of a material suitable for integrin purification for affinity chromatography

The activation of Sepharose takes place as described in literature 1, p. 14. Then 20 mg of avidin in 20 ml of 0.1 M sodium bicarbonate solution is added to 10 g of activated Sepharose. The suspension is stirred for 12 hours at 4 °C and then washed. The material is then suspended in water with a few crystals of sodium azide. The formation of the avidin complex with the biotinylated compounds according to the invention, e.g. cyclo-(Arg-Gly-Asp-D-Phe-Lys(N<É->Bit)) x TFA, is done by dissolving 1.1 equivalent in sodium acetate buffer, adding the solution to the avidin-Sepharose suspension and stirring for 10 hours at 4 °C. Excess peptide is removed by washing.

The following examples relate to pharmaceutical preparations.

Example A

Injection glass

A solution of 100 g of an active compound of formula I and 5 g of disodium hydrogen phosphate in 3 l of double-distilled water is adjusted with 2 N hydrochloric acid to pH 6.5, sterile filtered, filled into injection vials, lyophilized under sterile conditions and closed sterile. Each vial contains 5 mg of active compound.

Example B

Suppositories

A mixture of 20 g of an active compound of formula I with 100 g of soy lecithin and 1400 g of cocoa butter is melted, poured into molds and allowed to cool. Each suppository contains 20 mg of active compound.

Example C

Resolution

A solution of 1 g of an active compound with the formula 1.9.38 g NaH2PO4-2 H20, 28.48 g Na2HP04-12 H20 and 0.1 g benzalkonium chloride in 940 ml double-distilled water is made. It is adjusted to pH 6.8, filled up to 11 and sterilized by irradiation. This solution can be used in the form of eye drops.

Example D

Ointment

500 mg of an active compound of formula I is mixed with 99.5 g of vaseline under aseptic conditions.

Example E

Pills

A mixture of 1 kg of active compound with the formula 1.4 kg of lactose, 1.2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearate is pressed in the usual way to

tablets, so that each tablet contains 10 mg of active compound.

Example F

Dragees

Analogously to example E, tablets are pressed which are then coated in the usual way with a coating of sucrose, potato

starch, talc, tragacanth and colouring.

Example G

Capsules

2 kg of active compound of formula I is filled in the usual way into hard gelatin capsules, so that each capsule contains 20 mg of the active compound.

Example H

Ampoules

A solution of 1 kg of active compound of formula I in 60 1 of double-distilled water is sterile filtered, filled into ampoules, lyophilized under sterile conditions and closed sterilely. Each ampoule contains 10 mg of active compound.

Example I

Inhalation spray

14 g of active compound of formula I are dissolved in 101 isotonic NaCl solution, and the solution is filled into commercially available syringe containers with a pump mechanism. The solution can be sprayed into the mouth or nose. One syringe stroke (approx. 0.1 ml) corresponds to a dose of approx. 0.14 mg.

Claims (7)

1. Connections, characterized by the fact that they are selected from the group consisting of: and the salts thereof. 2. Pharmaceutical preparation, characterized by a content of at least one compound according to claim 1, and/or one of its physiologically acceptable salts. 3. Compounds according to claim 1 and their physiologically acceptable salts, as integrin inhibitors for combating pathological angiogenic diseases, thrombosis, heart attacks, coronary heart diseases, arteriosclerosis, tumors, osteoporosis, inflammation and infections. 4. Use of compounds according to claim 1 and/or their physiologically acceptable salts for the preparation of a medicinal product. 5. Use of compounds according to claim 1 for purification of integrins by affinity chromatography. 6. Use of compounds according to claim 1 as a diagnostic marker for antibiotin-antibody reactions in ELISA-type analyses, and in the FACS analysis. 7. Use of compounds according to claim 1 within force field microscopy for measuring the strength of ligand-receptor interactions.