DE10044325A1 - New MMP-2 derivatives as inhibitors of the integrin avbeta3 - Google Patents

Abstract

The invention relates to novel compounds of the formula (I), which are biologically active as the ligands of integrin alpha V beta 3 and wherein X represents H, an acyl or a peptide fragment of 1 to 20 amino acids, selected from the list of the naturally occurring amino acids, Y represents a peptide fragment selected from the sequence range 466-660 of human Pro-MMP-2, Z represents OH, NH2, NHA, NA2 or a peptide fragment of 1 to 20 amino acids, selected from the list of the naturally occurring amino acids, A represents an alkyl having 1-10 C atoms, and acyl represents an alkanoyl having 1-10 C atoms, wherein the primary amino groups can be also provided with conventional amine protecting groups. The invention further relates to the physiologically acceptable salts and solvates of these novel compounds.

Description

The invention relates to novel compounds of the formula I.

XYZ I

wherein
XH, acyl or a peptide fragment of 1 to 20 amino acids selected from the list of naturally occurring amino acids,
Y is a peptide fragment selected from the sequence region 466-660 of human pro-MMP-2,
Z is OH, NH ₂ , NHA, NA ₂ or a peptide fragment of 1 to 20 amino acids selected from the list of naturally occurring amino acids,
A is alkyl having 1-10 C atoms,
Acyl alkanoyl with 1-10 C atoms,
mean,
wherein the primary amino groups may also be provided with conventional amino protective groups,
and their physiologically acceptable salts and solvates.

The invention was based on the object, new compounds with wertvol len properties, especially those for the production of medicines can be used.

It has been found that the compounds of formula I and their salts with good compatibility zen very valuable pharmacological properties besit. Above all, they act as integrin inhibitors, in particular the interactions of the α _v integrin receptors with ligands, eg. As MMP-2 inhibit.

The compounds according to the invention are ligands, in particular of the integrin α _v β ₃ , which do not contain the sequence RGD and were derived from the C-terminal fragment PEX of the matrix metalloproteinase 2 (MMP-2).

The binding of PEX to the integrin α _v β ₃ is known from the literature.

MMP-2 is a matrix-degrading enzyme that inhibits cell migration facilitates invasive processes, such as B. in tumor metastasis and Angiogenesis.

There are numerous reviews of matrix metalloproteinases such. B. Birkedahl-Hansen H. Crit. Rev. Oral Biol. Med. 4, 197-250 (1993), Borkakoti N. Curr. Opin. Drug Descovery & Development 2, 449-462 (1999), Whittaker M. et al. Chem. Rev. 99, 2735-2776 (1999), Stetler-Stevenson W.G.J. Clin. Invest. 103, 1237-1241 (1999), Siletti S. & Cheresh D.A. Fibrinolysis & Proteolysis 13, 226-238 (1999), Kleiner-DE & Stetler Stevenson WG in Cancer-Chemother-Pharmacol. 1999; 43 Suppl: pp. 42-51.

MMPs are called inactive proenzymes ("latent enzyme", "zymogen") secreted, enzymatically inactive pro-MMP-2 is also known: Woessner-JF Jr FASEB-J. 1991 May; 5 (8): 2145-54.

Various modes of activation of pro-MMP-2 to MMP-2 are known (autocatalytic, chemical, enzymatic). The activation by tumor cells could, with the help of osteopontin, bone sialoprotein or RGD peptides. Here, the role of the C Terminus of MMP-2 and the formation of the trimeric complex MT1-MMP, TIMP-2 and Pro-MMP-2 discussed: Teti-A et al. Int J Cancer. 1998 Jul 3; 77 (1): 82-93.

MMPs facilitate tumor cell invasion and metastasis through at least 3 mechanisms. They eliminate the physical barrier  By degrading the ECM, they modulate cell adhesion, as the cells attach to ECM but have to break ties to the ECM again, and they produce other biologically active protein fragments by degradation of proteins.

The inhibition of α _v β ₃ by the antibody LM609 induced the activation of pro-MMP-2 of human rhabdomyosarcoma cells: Kubota-S. et al. Int J Cancer. 1997 Jan 6; 70 (1): 106-11.

The expression of integrins modulates the expression and secretion of MMP-2 and invasive behavior of tumor cells, such as seftor RE. et al. in Cancer Res. 1993 Jul 15; 53 (14): 3411-5.

There are different contributions to the relevance of MMPs. A selection shows the importance in embryonic development, Kinoh-H. et al., J-Cell Sci. 1996 May; 109 (Pt 5): 953-9; in teeth: Heikinheimo-K; Salo-T J- Dent Res. 1995 May; 74 (5): 1226-34; and in the development of the kidney: Kanwar YS; et al. Am J Physiol. 1999 Dec; 277 (6 Pt 2): F934-47.

Invasive cells have "invadopodia" and "lamellipodia" at the Migration front, which makes contact with the underlying extracellular Matrix (ECM) mediate different expression and localization of MT1-MMP, MMP-2, and TIMP-2. At the invadopodia are pro-MMP-2 and MT1-MMP responsible for dismantling the ECM, as MMP Activation takes place. Although Lamellipodien are similarly constructed, takes place There is no ECM leakage here, as they circulate on the cellular periphery Inhibitors such as TIMP-2 are readily available. It was designed by Chen-WT et al. in Ann-N-Y-Acad-Sci. 1999 Jun 30; 878: 361-71 showed that TIMP-2 with MMP-2 and MT1-MMP on lamellipodia but not on invadopodia colocalized. The same is said by the authors Nakahara-H; et al. proc Natl Acad-Sci-U-S-A. 1997 Jul 22; 94 (15): 7959-64.  

MMP-2 occurs in normal and diseased human joints: Chubinskaya-S et al. Lab Invest. 1999 Dec; 79 (12): 1669-77 and will be in synovial membrane in rheumatoid arthritis of Konttinen-Y et al. Ann Rheum Dis. 1999 Nov; 58 (11): 691-7.

The clinical relevance of MMP-2 as a prognostic marker in cervix Carcinoma was reported by Davidson-B et al. in Gynecol-Oncol. 1999 Jun; 73 (3): 372-82 shown.

MMP-2 and its activation play a role in neointima formation in restenosis: Wang-H; Keizer-JA J-Vasc-Res. 1998 Jan-Feb; 35 (1): 45-54.

MMP-2 modulates glial porcelain adhesion and migration: Deryugina-El; et al. J- Cell-Sci. 1997 Oct; 110 (Pt 19): 2473-82.

The compounds show particular effectiveness in the case of integrins α _v β ₃ and α _v β ₅ . The compounds are particularly effective as adhesion receptor antagonists for the receptor α _v β ₃ .

This effect can z. B. can be detected by the method as they by B. Diefenbach et al. in Biology of Vitronectins and Their Receptors, Eds. K. Preissner et al., Exerpta Medica, lnterntl. Congress Series 1042, Elsevier Sci. Publishers 1993, p. 149-156 is described.

The binding to the vitronectin receptor α _v β ₃ has been experimentally proven for some peptides according to the invention.

B. Felding-Habermann and DA Cheresh describe in Curr. Opin. Cell. Biol. 5, 864 (1993) the meanings of the integrins as Adhäsionsrezep factors for a variety of phenomena and clinical pictures, especially with respect to the receptor α _v β. ₃

The dependence of the origin of angiogenesis on the change effect between vascular integrins and extracellular matrix Proteins is from P.C. Brooks, R.A. Clark and D.A. Cheresh in Science 264, 569-71 (1994).  

The possibility of inhibiting this interaction and thus the Initiation of apoptosis (programmed cell death) angiogenic vascular Cells by a cyclic peptide is of 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 experimental proof that the inventive compounds The attachment of living cells to the corresponding Matrix proteins prevent and, accordingly, the attachment of Preventing tumor cells from matrix proteins may result in cell adhesion Mitsubishi et al., J. Cell Science 108, 2825-2838 (1995).

PC Brooks et al. describe in J. Clin. Invest. 96, 1815-1822 (1995) α _v β ₃ antagonists for the fight against cancer and for the treatment of tumor-induced angiogenic diseases.

The compounds of the formula I according to the invention can therefore be used as Active pharmaceutical ingredients, in particular for the treatment of tumor cancer diseases, osteoporosis and other osteolytic diseases as well used to suppress angiogenesis.

Compounds of formula I, the interaction of integrinreceptors and ligands such. B. fibrinogen to the fibrinogen receptor (glycoprotein IIb / IIIa) block prevent as GPIIb / IIIa antagonists the spread of tumor cells by metastasis. This is evidenced by the following observations:
The spread of tumor cells from a local tumor into the vascular system occurs through the formation of microaggregates (micro thrombi) through interaction of tumor cells with platelets. The tumor cells are shielded by protection in the microaggregate and are not recognized by the cells of the immune system.

The microaggregates can attach to vessel walls, causing further penetration of tumor cells into the tissue is facilitated. Since the formation of microthrombi by fibrinogen binding to the fibrino The gene receptors on activated platelets can be mediated  GPIIb / IIIa antagonists are considered as effective metastasis inhibitors become.

Compounds of the formula I inhibit addition to the binding of fibrinogen, Fibronectin and von Willebrand factor to the fibrinogen receptor of Platelets include the binding of other adhesive proteins, such as Vitro nectin, collagen and laminin, to the corresponding receptors on the Surface of different cell types. In particular, they prevent the Formation of platelet thrombi and therefore can be used for treatment thrombosis, apoplexy, heart attack, inflammation and arterio sclerosis are used.

The properties of the compounds can also be determined by methods be detected, which are described in EP-A1-0 462 960. The Inhibition of fibrinogen binding to the fibrinogen receptor may be after be detected by the method described in EP-A1-0 381 033 is specified.

The platelet aggregation-inhibiting effect can be detected in vitro of the Born method (Nature 4832, 927-929, 1962). The Inhibition of bone resorption by the compounds of the invention can by means of an osteoclastic resorption test analogous to WO 95/32710 respectively.

Matrix metalloproteinase 2 (MMP-2, also gelatinase A, GelA, 72 kDa type IV Collagenase) is a member of the zinc-dependent endoproteases family secreted as pro-enzymes, latent MMPs or zymogens and for the degradation of extracellular matrix proteins and others Proteins are responsible for cell migration and invasion. The balance between activation from the pro-enzyme and the inak activation by endogenous inhibitors, such as TIMP-2, or by extension enzymatic digestion, regulates the activity of MMP-2. The MMPs play a role in physiological as well as pathological situation in embryonic development, inflammation, wound healing, Tumor cell invasion and metastasis. Inhibitors of enzymatic Pocket, expression at the cellular level or activation from the pro  Enzyme may be diagnostic and therapeutic use in humans and find animal.

The enzyme cleaves different types of collagen, elastin, fibronectin and laminin. Binding to collagen occurs by means of 3 to fibronectin type II modules homologous repeating units in the catalytic Domain are inserted near the active site.

The C-terminus of MMP-2 is referred to in the literature as a "C-terminal domain" CTD (Goldberg et al., Overall et al.) Or PEX (Cheresh et al.).

literature

Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3.
Brooks PC; Stromblad-S; Sanders-LC; of-Schalscha-TL; Aimes RT; Stetler-Stevenson WG; Quigley JP; Cheresh DA
Cell. 1996 May 31; 85 (5): 683-93.
Disruption of angiogenesis by PEX, a noncatalytic metalloproteinase fragment with integrin binding activity. Brooks PC; Silletti-S. from Schalscha TL; Friedlander M; Cheresh-da Cell. 1998 Feb 6; 92 (3): 391-400.
In US 4,923,818 Goldberg has described the amino acid sequence of Pro-MMP-2 (72 kDa), the complex with TIMP-2 is documented by Goldberg in EP 404,750.
Soluble pro-MMP-2 is bound to cell surfaces and is activated there by the membrane-bound enzyme MT1-MMP by proteolysis of the pro-fragment in position Asn66-Leu67 in the first step and subsequent MT1-MMP-independent self-digestion at position Asn109-Tyr110. This activation is not possible if the C-terminus of Pro-MMP-2 is missing. Suitable binding partners are MT1-MMP, integrin α _v

β ₃

and fetuin.
In WO 98/12309, Washington University St. Louis, binding sequences of CTD (PEX) to TIMP-2 are described. It is noted that the addition of an excess of TIMP-2 (but not TIMP-1) or recombinant CTD (PEX) suppresses activation of pro-MMP-2 at the cell surface.

The invention accordingly compounds of formula I. according to claim 1 and / or its physiologically acceptable salts for Preparation of a medicament for use as an integrin inhibitor. The invention relates in particular to compounds of the formula I. according to claim 1 and / or its harmless salts for the preparation of a drug for controlling pathologically angiogenic Diseases, tumors, osteoporosis, inflammation and infections.

The compounds of the formula I can be used as active pharmaceutical ingredients in the Human and veterinary medicine are used for prophylaxis and / or Therapy of thrombosis, myocardial infarction, arteriosclerosis, inflammation apoplexy, angina, tumors, osteolytic Diseases such as osteoporosis, hypercalcaemia, pathologically angiogenic Diseases such. As inflammation, ophthalmological diseases, diabetic retinopathy, macular degeneration, myopia, ocular Histoplasmosis, rheumatoid 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 in wound healing Support of the healing processes.

The compounds of the formula I can be used as antimicrobial sub punching operations are used where biomaterials, implant tate, catheter or pacemaker. They work antiseptic. The effectiveness of the antimicrobial activity can be improved by the  by Valentin P. Weigund et al., in Infection and Immunity, 2851-2855 (1988).

The invention also relates to the hydrates and solvates, z. B. Alcoholates, these compounds.

The invention further provides a process for the preparation of compounds of the formula I according to claim 1 and their salts, characterized in that
releasing a compound of formula I from one of its functional derivatives by treatment with a solvolyzing, reducing or hydrogenolyzing agent,
and / or converting a base or acid of the formula I into one of its salts.

The compounds of formula I may be one chiral or several chiral Centers can and therefore possess several stereoisomeric forms occur. All these forms (eg D- and L-forms) and their mixtures (eg, the DL forms) are included in Formula I.

In the compounds of the invention are also called prodrug Derivatives included, d. H. with z. As alkyl or acyl groups, sugars or oligopeptides modified compounds of the formula I, which in Organism rapidly to the effective compounds of the invention be split.

The abbreviations above and below stand for:
Ac acetyl
BOC tert-butoxycarbonyl
CBZ or Z benzyloxycarbonyl
DCCl dicyclohexylcarbodiimide
DMF dimethylformamide
EDCl N-ethyl-N, N '- (dimethylaminopropyl) carbodiimide
Et ethyl
Fmoc 9-fluorenylmethoxycarbonyl
HOBt 1-hydroxybenzotriazole
Me methyl
Mtr 4-methoxy-2,3,6-trimethylphenylsulfonyl
HONSu N-hydroxysuccinimide
OBut tert-butyl ester
Oct octanoyl
OMe methyl ester
OEt ethyl ester
Pbf 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl
Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl
POA phenoxyacetyl
TFA trifluoroacetic acid
Trt trityl (triphenylmethyl).

The abbreviations of amino acid residues given above and below stand for the residues of the following naturally occurring amino acids:
Ala, A Alanine
Asn, N asparagine
Asp, D aspartic acid
Arg, R arginine
Cys, C cysteine
Gln, Q Glutamine
Glu, E glutamic acid
Gly, G glycine
His, H histidine
Ile, I isoleucine
Leu, L Leucin
Lys, K Lysine
Met, M methionine
Phe, F phenylalanine
Pro, P Proline
Ser, S serine
Thr, T threonine
Trp, W tryptophan
Tyr, Y tyrosine
Val, V Valin.

The sequence of human pro-MMP-2 (MMP-2) taken from publicly available databases (eg available at www.expasy.ch) is as follows:

Here, the C-terminal, hemopexin-like, also known in the literature as PEX range of position 466-660 is dark labeled. The inventive integrin α _v β ₃ binding peptide sequences are underlined.

For the entire invention, it is true that all the radicals which are multiply occur, such. B. A, may be the same or different, d. H. are independent of each other.  

A is alkyl and has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 C atoms and is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert. Butyl, furthermore also pentyl, 1-, 2- or 3-methylbutyl, 1,1-, 1,2- or 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-, 2-, 3- or 4 Methylpentyl, 1,1-, 1,2-, 1,3-, 2,2-, 2,3- or 3,3-dimethylbutyl, 1- or 2-ethylbutyl, 1-ethyl-1-methylpropyl, 1 Ethyl 2-methylpropyl, 1,1,2-, 1,2,2-trimethylpropyl, heptyl, octyl, nonyl or decyl, undecyl. A also denotes halogen-substituted alkyl, preferably CF ₃ .

Acyl is alkanoyl having 1-10 C atoms, preferably z. Formyl, Acetyl, propionyl or butyryl, also z. B. benzoyl.

Amino protecting group is preferably formyl, acetyl, propionyl, Butyryl, phenylacetyl, benzoyl, toluyl, POA, methoxycarbonyl, Ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, BOC, 2-iodoethoxycarbonyl, CBZ ("carbobenzoxy"), 4-methoxybenzyloxycarbonyl, FMOC, Mtr or Benzyl.

Accordingly, the invention relates in particular diejeni gene compounds of formula I, in which at least one of said radicals has one of the preferred meanings given above. Some preferred groups of compounds can be expressed by the following partial formulas Ia to Ie, which correspond to the formula I and in which the unspecified radicals have the meaning given in the formula I, but in which
in a) XH, acyl or a peptide fragment with 1 to 20 amino acids,
selected from the peptide sequence of human pro-MMP-2
means;
in b) XH, acyl or a peptide fragment with 1 to 20 amino acids,
selected from the peptide sequence of human pro-MMP-2,
Z is OH, NH ₂ , NHA, NA ₂ or a peptide fragment with 1 to 20 amino acids,
selected from the peptide sequence of human pro-MMP-2,
mean;
in c) XH, acyl or a peptide fragment with 1 to 20 amino acids,
selected from the peptide sequence of human pro-MMP-2,
Y is a peptide fragment selected from the sequence region 480-598 of human pro-MMP-2,
Z OH, NH ₂ NHA, NA ₂ or a peptide fragment with 1 to 20 amino acids,
selected from the peptide sequence of human pro-MMP-2
mean,
in d) XH, acyl or a peptide fragment with 1 to 20 amino acids,
selected from the peptide sequence of human pro-MMP-2,
Y is a peptide fragment selected from the sequence region 489-497 and / or 570-585 and / or 588-597 of human pro-MMP-2,
Z OH, NH ₂ NHA, NA ₂ or a peptide fragment with 1 to 20 amino acids,
selected from the peptide sequence of human pro-MMP-2,
mean,
in e) XH, acyl or a peptide fragment with 1 to 20 amino acids,
selected from the peptide sequence of human pro-MMP-2,
Y is a peptide fragment selected from the sequence region 489-497 and / or 570-585 and / or 588-597 of human pro-MMP-2,
Z is OH, NH ₂ , NHA, NA ₂ or Cys
mean,
wherein the primary amino groups may also be provided with conventional amino protective groups,
and their physiologically acceptable salts and solvates.

The peptides of the C-terminal sequence of MMP-2 are as Ber fragments each with 2 amino acids overlap on paper in a Fmoc strategy been synthesized.

The quality of the synthesis was assessed by means of a reflected light / fluorescence reading device of SLT-Tecan and so the quantity of the Fmoc signal after coupling steps (much fluorescence) and after cleavage steps (no fluorescence) evaluated. These peptide-loaded papers were incubated with fluorescence-labeled integrin α _v β ₃ , washed and evaluated with a SLT-Tecan incident light / fluorescence reader. The peptide sequences of the positive "SPOTs" were conventionally synthesized on the resin in separate syntheses and labeled with a C-terminal cysteine. The purified, isolated cysteine peptides were tested in a covalent binding assay for binding of biotinylated integrin α _v β ₃ by means of an anti-biotin / phosphatase reaction, thereby confirming the α _v β ₃ -binding, RGD-independent activity. Of the active compounds, there are two peptides in a consecutive sequence located on the surface of PEX, derived from the known X-ray structure of the MMP-2 C-terminus.

The compounds of the invention prepared by way of example were according to the prior art in a synthesizer Milligen 9050 Polystyrene produced. The method is for a person skilled in the art from Textbook knowledge comprehensible, such. In the Annex to the Novabiochem  To find a catalog. Chloro-trityl (o-Cl) anchors were used. Fmoc- protected amino acids with acid labile side protection were coupled. The side-protected derivatives Fmoc-Ser (But), Thr (But), Tyr (But), His (Trt), Asn (Trt), Gln (Trt), Arg (Pmc) or Arg (Pbf), Lys (Boc), Asp (OBut), Glu (OBut), Cys (Trt) are used. After splitting off the Fmoc groups, the peptide is split off from the resin with TFA / water (98: 2), wherein at the same time the protective groups of the side chains with cleaved become.

The compounds of the formula I and also the starting materials for their Her Otherwise, they are prepared by methods known per se, as in the literature (eg in the standard works such as Houben-Weyl, Methods of Organic Chemistry, Georg-Thieme-Verlag, Stuttgart;) are described, under reaction conditions, the ge known implementations are known and suitable. You can also do that of known per se, not mentioned here variants use do.

The starting materials can, if desired, also be formed in situ, so they are not isolated from the reaction mixture, but immediately further converts to the compounds of formula I.

Compounds of the formula I can preferably be obtained by compounds of the formula I from one of their functional derivatives by treating with a solvolysing or hydrogenolyzing Sets funds in freedom.

Preferred starting materials for the solvolysis or hydrogenolysis are those which otherwise correspond to the formula I, but instead of one or correspondingly more free amino and / or hydroxy groups contain protected amino and / or hydroxy groups, preferably those which, instead of an H atom linked to an N atom, bear an amino protecting group, especially those which replace one HN group carry an R'-N group, wherein R 'is an amino protecting group means, and / or those which instead of the H atom of a hydroxy group  carry a hydroxy protecting group, e.g. For example, those of the formula I. but instead of a group -COOH, a group -COOR " wherein R "represents a hydroxy protecting group.

It is also possible to use several identical or different protected amino acids. and / or hydroxy groups present in the molecule of the starting material his. If the existing protective groups are different from each other, they can be selectively split off in many cases.

The term "amino protecting group" is well known and relates to groups that are capable of an amino group before chemical Um protection (blocking), but which are easily removable, after the desired chemical reaction at other points of the Molecule has been performed. Typical of such groups are in particular unsubstituted or substituted acyl, aryl, aralkoxymethyl or aralkyl groups. Since the amino protecting groups are according to the desired Reaction (or reaction sequence) is their type and size in the other not critical; however, preferred are those with 1-20, esp in particular 1-8 C atoms. The term "acyl group" is related to be understood in the broadest sense with the present process. He around excludes aliphatic, araliphatic, aromatic or hetero cyclic carboxylic acids or sulfonic acids derived acyl groups as well as in particular alkoxycarbonyl, aryloxycarbonyl and especially Aralkoxycarbonyl groups. Examples of such acyl groups are formyl or alkanoyl such as acetyl, propionyl, butyryl; Aralkanoyl such as phenylacetyl; Aroyl such as benzoyl or toluyl; Aryloxyalkanoyl such as POA; alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, BOC, 2-iodoethoxycarbonyl; Aralkyloxycarbonyl such as CBZ ("carbobenzoxy"), 4- Methoxybenzyloxycarbonyl, FMOC; Arylsulfonyl such as Mtr. Preferred Amino protecting groups are BOC and Mtr, furthermore CBZ, Fmoc, benzyl, Formyl and acetyl.

The cleavage of the amino protecting group succeeds - depending on the used Protective group - z. B. with strong acids, useful with TFA or Per chloric acid, but also with other strong inorganic acids such as Hydrochloric acid or sulfuric acid, strong organic carboxylic acids such as  Trichloroacetic acid or sulfonic acids such as benzene or p-toluenesulfone acid. The presence of an additional inert solvent is possible, but not always required. Suitable as inert solvents preferably organic, for example carboxylic acids such as vinegar acid, ethers such as tetrahydrofuran or dioxane, amides such as DMF, haloge hydrocarbons such as dichloromethane, and also alcohols such as Methanol, ethanol or isopropanol, as well as water. Further come Mixtures of the aforementioned solvents in question. TFA is preferred Use in excess without the addition of another solvent Det, 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 Spal tion are suitably between about 0 and about 50 °, preferably you work between 15 and 30 ° (room temperature).

The groups BOC, OBut, Pmc, Pbf and Mtr can, for. B. preferably with TFA in dichloromethane or with about 3 to 5n HCl in dioxane at 15-30 ° be split off, the FMOC group with about 5 to 50% Solution of sec. Amines, such as dimethylamine, diethylamine or piperidine in DMF at 15-30 °.

Hydrogenolytically removable protecting groups (eg CBZ or benzyl) can z. By treating with hydrogen in the presence of a kata lysators (eg of a noble metal catalyst such as palladium, expedient a carrier such as coal) are split off. Suitable solvents while the above, in particular z. For example, alcohols such as Methanol or ethanol or amides such as DMF. The hydrogenolysis is in usually at temperatures between about 0 and 100 ° and printing between about 1 and 200 bar, preferably at 20-30 ° and 1-10 bar by guided. Hydrogenolysis of the CBZ group succeeds z. B. good at 5 to 10% Pd / C in methanol or with ammonium formate (instead of Hydrogen) on Pd / C in methanol / DMF at 20-30 °.

Suitable inert solvents are, for. As hydrocarbons such as hexane, Petroleum ether, benzene, toluene or xylene; chlorinated hydrocarbons such as Trichlorethylene, 1,2-dichloroethane, carbon tetrachloride, chloroform or dichloromethane; Alcohols such as methanol, ethanol, isopropanol, n-propanol,  n-butanol or tert-butanol; Ethers, such as diethyl ether, diisopropyl ether, Tetrahydrofuran (THF) or dioxane; Glycol ethers, such as ethylene glycol mono methyl or monoethyl ether (methyl glycol or ethyl glycol), ethylene glycol dimethyl ether (diglyme); Ketones such as acetone or butanone; amides such as acetamide, dimethylacetamide or dimethylformamide (DMF); Nitriles like acetonitrile; Sulfoxides such as dimethylsulfoxide (DMSO); Carbon disulphide; Carboxylic acids such as formic acid or acetic acid; Nitro compounds like Nitromethane or nitrobenzene; Esters such as ethyl acetate, water or Mixtures of the solvents mentioned.

Furthermore, it is possible to saponify an ester of the formula I. This is conveniently done by solvolysis or hydrogenolysis, as above indicated, z. B. with LioH in methanol, NaOH or KOH in dioxane-water at temperatures between 0 and 60 ° C, preferably between 10 and 40 ° C.

Further, it is possible to use a conventional amino protecting group Replacing hydrogen by the protecting group as described above, split off solvolytically or hydrogenolytically or that one by a conventional protecting group protected amino group Solvolysis or hydrogenolysis sets free.

A base of the formula I can react with an acid in the associated acid addition salt can be converted, for example by reacting equi valent amounts of the base and the acid in an inert solvent like ethanol and then evaporation. For this implementation In particular acids come into question, the physiologically harmless Deliver salts. Thus, inorganic acids can be used, for. B. Sulfuric acid, nitric acid, hydrohalic acids such as chlorine hydrofluoric or hydrobromic acid, phosphoric acids such as ortho phosphoric acid, sulfamic acid, furthermore organic acids, in particular aliphatic, alicyclic, araliphatic, aromatic or heterocyclic mono- or polybasic carboxylic, sulfonic or sulfuric acids, eg. B. 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 ethanesulfone acid, ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfone acid, p-toluenesulfonic acid, naphthalene mono- and disulfonic acids, lauryl sulfuric acid. Salts with physiologically unacceptable acids, eg. B. Picrates, can be used to isolate and / or purify the compounds of formula I can be used.

On the other hand, an acid of the formula I by reaction with a Base in one of its physiologically harmless metal or ammonium be converted salts. Salts are in particular the Sodium, potassium, magnesium, calcium and ammonium salts in Consider further substituted ammonium salts, e.g. As the dimethyl, diethyl or diisopropylammonium salts, monoethanol, diethanol or diiso propylammonium salts, cyclohexyl, dicyclohexylammonium salts, di benzylethylenediammonium salts, further z. As salts with arginine or Lysine.

The compounds of formula I contain one or more chiral centers and therefore may be in racemic or optically active form. Obtained racemates mecha according to methods known per se nisch or chemically separated into the enantiomers. Preferably be from the racemic mixture by reaction with a optically active release agent formed diastereomers. Suitable as a release agent z. As optically active acids, such as the D and L forms of tartaric acid, Diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, milk acid or the various optically active camphorsulfonic acids such as β- Camphor sulfonic acid. A separation of enantiomers is also advantageous Help one with an optically active release agent (eg Dinitrobenzoyl phenylglycine) filled column; as the eluent z. B. a mixture Hexane / isopropanol / acetonitrile, e.g. B. in the volume ratio 82: 15: 3.

Of course, it is also possible to optically active compounds of the formula I. according to the methods described above, by selecting Off used, which are already optically active.  

The invention includes not only the compounds mentioned, but also mixtures and preparations, which in addition to these invention According to compounds other pharmacological agents or Containing adjuvants that have the primary pharmacological activity of Influence compounds of the invention in the desired manner can. These can be used as therapeutics, diagnostics or as reagents Find use. They can be local or human or animal systemic, oral, intravenous, intraperitoneal, intramuscular, subcutaneous, transdermal, nasal, buccal, or iontophoretic, that includes formulations in suspensions, emulsions or solutions, Liposomes, ointments, pastes, biodegradable polymers or as Nanoparticles, tablets, capsules or pills, granules or powders, as Aerosol for inhalation, as intranasal drops or sprays. Also a combination of new products with other techniques, like Surgery, radiation, diagnosis, radiotherapy, photodynamic Therapy and gene therapy, as well as other medications is possible. Such drugs can be used for. B. from the areas of cardiovascular, Central nervous system or oncology come. It can Tumor agents, such as angiogenesis inhibitors or cytostatics, Chemotherapeutic agents of groups alkylating agents, antibiotics, Antimetabolites, biologics and immunomodulators, hormones and their Antagonists, mustard gas derivatives, alkaloids and others, these being Substances can be low molecular weight and high molecular weight. It can Be lipids, carbohydrates or proteins. These include cytokines, Toxins, fusion proteins, monoclonal antibodies and vaccines.

The invention accordingly compounds of the above and Formulas defined below as well as in the claims including theirs physiologically acceptable salts as medicaments, diagnostics or Reagents.  

In particular, these are drugs for combating diseases that are based on an interaction of ligands, eg. B. of MMP-2 with the α _v β ₃ integrin receptor based.

The medicaments are suitable for controlling thromboses, Heart attack, coronary heart disease, arteriosclerosis, tumors, Osteoporosis, fibrosis, inflammation, infections, psoriasis and Influence of wound healing processes.

The subject matter also relates to corresponding pharmaceutical preparations, which at least one drug of formula I and optionally Contain carriers and / or excipients.

The invention further relates to the use of the compounds according to the invention and / or their physiologically acceptable salts and / or solvates for the preparation of a medicament for inhibiting the integrin receptor α _v β ₃ .

The invention furthermore relates to the use of the compounds according to the invention and / or their physiologically acceptable salts and / or solvates for the production of a medicament for combating diseases which are based on an interaction of a ligand, eg. As the matrix metalloproteinase (MMP-2) with the α _v β ₃ integrin receptor based.

The invention further relates to the use of the invention according to the compounds and / or their physiologically harmless Salts and / or solvates for the manufacture of a medicament for Combating pathological processes caused by angiogenesis be entertained or propagated.

The invention further relates to the use of the invention according to the compounds and / or their physiologically harmless Salts and / or solvates for the manufacture of a medicament for Fight thrombosis, myocardial infarction, coronary heart disease, Atherosclerosis, tumors, osteoporosis, fibrosis, inflammation,  Infections, psoriasis and to influence wound healing processes.

The medicaments according to the invention or pharma containing them Ceutical preparations may be used in human or veterinary medicine be used. Suitable carriers are organic or inorganic ganic substances which are suitable for enteral (eg oral), paren terale, topical application or for application in the form of a Inhalation sprays are suitable and do not react with the new compounds ren, for example, water, vegetable oils, benzyl alcohols, alkylene kole, polyethylene glycols, glycerol triacetate, gelatin, carbohydrates such as Lactose or starch, magnesium stearate, talc, petrolatum. For oral In particular tablets, pills, dragees, capsules, Pul serve ver, granules, syrups, juices or drops, for rectal use Sup positorien, for parenteral use solutions, preferably oily or aqueous solutions, furthermore suspensions, emulsions or implant tate, topical ointments, creams or powders. The new Compounds may also be lyophilized and the resulting lyophilisates z. B. be used for the preparation of injection preparations. The Preparations may be sterilized and / or auxiliaries such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers gators, salts for influencing the osmotic pressure, Buffersub punching, color, flavor and / or several other active ingredients ent hold, z. B. one or more vitamins.

For application as an inhalation spray, sprays may be used which contain the active ingredient either dissolved or suspended in a propellant gas or propellant gas mixture (eg CO ₂ or chlorofluorocarbons). It is expedient to use the active ingredient in micronized form, one or more additional ones being used physiologically acceptable solvents may be present, for. For example, ethanol. Inhalation solutions can be administered using standard inhalers.

The substances according to the invention can, as a rule, be analogous to other known commercially available preparations (eg described in US-A-4,472,305), preferably in dosages between about 0.05 and 500 mg, especially between 0.5 and 100 mg administered per dosage unit. The daily dosage is preferred between about 0.01 and 20 mg / kg of body weight. The special one However, dose for each patient depends on a variety of factors from, for example, the effectiveness of the particular used Connection, age, body weight, general health, Sex, diet, time and route of administration, of the diet Excretion rate, drug combination and severity of respective disease to which the therapy applies. The parenteral Application is preferred.

Above and below, all temperatures are given in ° C. The HPLC analyzes (retention time Rt) were carried out in the following system:
Column 250 x 4 mm Lichrospher 100RP 18 (Merck KGaA) with a 50 minute gradient of 0-80% 2-propanol in water (with 0.3% trifluoroacetic acid) at a flow of 1 ml / min and detection at 215 nm.
Mass spectrometry (MS): EI (electron impact ionization) M ⁺
FAB (Fast Atom Bombardment) (M + H) ⁺

Examples Preparation and purification of peptides according to the invention

In principle, the preparation and purification is carried out by Fmoc strategy under protection of acid-labile side chains on acid-labile resins Use of a commercially available "continuous flow" peptide synthesizers as described by Haubner et al. (J. Am. Chem. Soc. 118, 1996, 17703).

The following compounds were obtained

Synonymous with these are the following terms

KDRFIWRTVC MMP-2 (489-497) -Cys AAFNWSKNKKTYIFAGC MMP-2 (570-585) -Cys FWRYNEVKKKC MMP-2 (588-597) -Cys

Further, the following compounds were obtained

letter code synonym VKKKMDPGC MMP-2 (594-603) -Cys, M 1005.23 LERGYPKPLTSLGLC MMP-2 (548-561) -Cys, M 1646.94 KPMGPLLVC MMP-2 (502-509) -Cys, M 957.24 IAQIRGC MMP-2 (478-483) -Cys, M 759.94 LKSVKFGSIC MMP-2 (645-653) -Cys, M 1081.34 AGDKFWRYNC MMP-2 (585-593) -Cys, M 1259.42 RGYPKPLTSLC MMP-2 (550-559) -Cys, M 1234.5 PGFPKLIAC MMP-2 (600-607) -Cys, M 945.19 WRTVTPRDC MMP-2 (494-501) -Cys, M 1133.31 AYYLKLENC MMP-2 (635-642) -Cys, M 1116.31

Examples of materials and methods

Recombinant MMP-2 is available from Chemicon. Integrin α _v β ₃ can be purified from placenta as described by Smith et al. J. Biol. Chem. 263, 18726-18731 (1988) with the modifications according to FC Mitjans et al. J. Cell Sci. 108, 2825-2838 (1995). Soluble α _v β ₃ is as described in RJ Mehta et al. Biochem. J. 330, 861-869 (1998), there also labeling with biotin, performing a ligand binding assay and competition assay is carried out, another citation for the binding assays is S. Kraft et al. J. Biol. Chem. 274, 1979-1985 (1999).

• 1. The receptor binding test on covalently bonded peptides can be carried out analogously to: B. Diefenbach et al. in Biology of Vitronectins and Their Receptors; eds. KT Preissner et al. from Exerpta Medica International Congress Series 1042, Elsevier Science Publishers Amsterdam 1993, p. 149-156.
  The receptor binding assay used fluorescein-labeled integrin α _v β ₃ , which interacts directly with the cellulose-bound peptide. The evaluation was carried out by measuring the fluorescence.
  In another variant, BSA coated with microtiter plates was activated with sulfo-SMPB and the thiopeptides covalently attached thereto. After incubation with biotinylated α _v β ₃ , bound receptor was detected with alkaline phosphatase-coupled goat anti-biotin antibody and stained with a phosphatase substrate.
  The pharmacological data prove the activity of the compounds according to the invention as ligands for the receptor α _v β ₃ .
• 2. The binding of PEX peptides to α _v β ₃ is an RGD-independent binding. This was demonstrated in a further test, which was carried out analogously to 1..
  The receptor was incubated with and without the soluble peptide cyclo- (Arg-Gly-Asp-D-Phe-N-Me-Val). While the binding of the cyclo-RGD peptide "cyclo- (RGDfK (mercapto-propionyl))" to α _v β _{3 was} inhibited by cyclo- (Arg-Gly-Asp-D-Phe-N-Me-Val) the binding of the PEX peptide AAFNWSKNKKTYIFAGC independent thereof.

The following examples relate to pharmaceutical preparations:

Example A injection Vials

A solution of 100 g Lys-Asp-Arg-Phe-Ile-Trp-Arg-Thr-Val-Cys and 5 g Disodium hydrogen phosphate is in 3 liters of double distilled water with 2 N hydrochloric acid adjusted to pH 6.5, sterile filtered, abge in injection glasses abge filled, lyophilized under sterile conditions and sealed sterile. each Injection jar contains 5 mg active substance.

Example B suppositories

A mixture of 20 g of Lys-Asp-Arg-Phe-Ile-Trp-Arg-Thr-Val is melted. Cys with 100 g soy lecithin and 1400 g cocoa butter, pouring into molds and lets cool. Each suppository contains 20 mg of active ingredient.

Example C solution

A solution of 1 g of Lys-Asp-Arg-Phe-Ile-Trp-Arg-Thr-Val-Cys, 9.38 g of NaH ₂ PO ₄ .2H ₂ O, 28.48 g of Na ₂ HPO _{4 is} prepared. 12 H ₂ O and 0.1 g of benzalkonium chloride in 940 ml of bidistilled water. Adjust to pH 6.8, make up to 1 liter and sterilize by irradiation. This solution can be used in the form of eye drops.

Example D ointment

500 mg of Lys-Asp-Arg-Phe-Ile-Trp-Arg-Thr-Val-Cys are mixed at 99.5 g Vaseline under aseptic conditions.

Example E tablets

A mixture of 1 kg Lys-Asp-Arg-Phe-Ile-Trp-Arg-Thr-Val-Cys, 4 kg Lactose, 1.2 kg potato starch, 0.2 kg talc and 0.1 kg magnesium stearate is compressed in a conventional manner into tablets, such that each tablet Contains 10 mg of active ingredient.  

Example F dragees

As in Example E tablets are pressed, which are then in the usual With a coating of sucrose, potato starch, talc, tragacanth and dye are coated.

Example G capsules

2 kg Lys-Asp-Arg-Phe-Ile-Trp-Arg-Thr-Val-Cys are in the usual way in Filled hard gelatin capsules, so that each capsule 20 mg of the active ingredient contains.

Example H ampoules

A solution of 1 kg Lys-Asp-Arg-Phe-Ile-Trp-Arg-Thr-Val-Cys in 60 l twice distilled water is sterile filtered, filled into ampoules, lyophilized under sterile conditions and sealed sterile. each Ampoule contains 10 mg of active ingredient.

Example I Inhalation Spray

14 g of Lys-Asp-Arg-Phe-Ile-Trp-Arg-Thr-Val-Cys are dissolved in 10 l of isotonic NaCl solution and filled with the solution in commercial spray vessels A pump mechanism. The solution can be sprayed in the mouth or nose become. One spray (about 0.1 ml) corresponds to a dose of about 0.14 mg.

Claims (19)

1. Compounds of the formula I
XYZ I
wherein
XH, acyl or a peptide fragment of 1 to 20 amino acids selected from the list of naturally occurring amino acids,
Y is a peptide fragment selected from the sequence region 466-660 of human pro-MMP-2,
Z is OH, NH ₂ , NHA, NA ₂ or a peptide fragment of 1 to 20 amino acids selected from the list of naturally occurring amino acids,
A is alkyl having 1-10 C atoms,
Acyl alkanoyl with 1-10 C atoms,
mean
wherein the primary amino groups may also be provided with conventional amino protecting groups, and their physiologically acceptable salts and solvates. 2. Compounds according to claim 1,
wherein
XH, acyl or a peptide fragment of 1 to 20 amino acids selected from the peptide sequence of human pro-MMP-2
means
wherein the primary amino groups may also be provided with conventional amino protective groups,
and their physiologically acceptable salts and solvates. 3. Compounds according to claim 1,
wherein
XH, acyl or a peptide fragment of 1 to 20 amino acids selected from the peptide sequence of human pro-MMP-2,
Z is OH, NH ₂ , NHA, NA ₂ or a peptide fragment of 1 to 20 amino acids selected from the peptide sequence of human pro-MMP-2
mean,
wherein the primary amino groups may also be provided with conventional amino protective groups,
and their physiologically acceptable salts and solvates. 4. Compounds according to claim 1,
wherein
XH, acyl or a peptide fragment of 1 to 20 amino acids selected from the peptide sequence of human pro-MMP-2,
Y is a peptide fragment selected from the sequence region 480-598 of human pro-MMP-2,
Z is OH, NH ₂ , NHA, NA ₂ or a peptide fragment of 1 to 20 amino acids selected from the peptide sequence of human pro-MMP-2,
mean,
wherein the primary amino groups may also be provided with conventional amino protective groups,
and their physiologically acceptable salts and solvates. 5. Compounds according to claim 1,
wherein
XH, acyl or a peptide fragment of 1 to 20 amino acids selected from the peptide sequence of human pro-MMP-2,
Y is a peptide fragment selected from the sequence region 489-497 and / or 570-585 and / or 588-597 of human pro-MMP-2,
Z is OH, NH ₂ , NHA, NA ₂ or a peptide fragment of 1 to 20 amino acids selected from the peptide sequence of human pro-MMP-2,
mean,
wherein the primary amino groups may also be provided with conventional amino protective groups,
and their physiologically acceptable salts and solvates. 6. Compounds according to claim 1,
wherein
XH, acyl or a peptide fragment of 1 to 20 amino acids selected from the peptide sequence of human pro-MMP-2,
Y is a peptide fragment selected from the sequence region 489-497 and / or 570-585 and / or 588-597 of human pro-MMP-2,
Z is OH, NH ₂ , NHA, NA ₂ or Cys,
wherein the primary amino groups may also be provided with conventional amino protective groups,
and their physiologically acceptable salts and solvates. 7. Compounds according to claim 1

• a) Lys-Asp-Arg-Phe-Ile-Trp-Arg-Thr-Val-Cys,
• b) Ala-Ala-Phe-Asn-Trp-Ser-Lys-Asn-Lys-Lys-Thr-Tyr-Ile-Phe-Ala-Gly-Cys,
• c) Phe-Trp-Arg-Tyr-Asn-Glu-Val-Lys-Lys-Lys-Cys

and their physiologically acceptable salts and solvates. 8. A process for the preparation of compounds of the formula I according to claim 1 and their salts, characterized in that
releasing a compound of formula I from one of its functional derivatives by treatment with a solvolyzing, reducing or hydrogenolyzing agent,
and / or converting a base or acid of the formula I into one of its salts. 9. Compounds of the formula I according to claim 1-6 and the Compounds according to claim 7 and their physiological harmless salts and solvates as pharmaceuticals. 10. Medicament according to claim 9 as an inhibitor of the integrin receptor α _v β ₃ . 11. A pharmaceutical composition according to claim 10 for combating diseases based on an interaction of ligands with the α _v β ₃ integrin receptor. 12. Medicament according to claim 11 for combating diseases based on an interaction of the matrix metalloproteinase (MMP-2) with the α _v β ₃ integrin receptor. 13. Medicament according to claim 12 for controlling thromboses, Heart attack, coronary heart disease, arteriosclerosis, tumors, Osteoporosis, fibrosis, inflammation, infections, psoriasis as well for influencing wound healing processes. 14. A pharmaceutical preparation containing at least one drug Agent according to one of claims 10 to 13 and optionally Carriers and / or adjuvants and optionally other active ingredients. 15. Use of compounds according to claims 1 to 7 and / or their physiologically acceptable salts for the preparation of a medicament for inhibiting the integrin receptor α _v β ₃ . 16. Use of compounds according to claims 1 to 7 and / or their physiologically acceptable salts for the production  a medicine to combat diseases that affect one Interaction of ligands with the V integrin receptor based. 17. Use according to claim 16 for the preparation of a medicament for combating diseases based on an interaction of the matrix metalloproteinase (MMP-2) with the α _v β ₃ integrin receptor. 18. Use according to claim 16 or 17 for the preparation of a Medicament for combating pathological processes caused by Angiogenesis be entertained or propagated. 19. Use according to claim 18 for the preparation of a medicament to combat thrombosis, myocardial infarction, coronary heart diseases, arteriosclerosis, tumors, osteoporosis, fibrosis, Inflammation, infections, psoriasis and to influence Wound healing processes.