EP1315804A2

EP1315804A2 - Novel mmp-2 derivatives for use as inhibitors of integrin alpha vbeta 3

Info

Publication number: EP1315804A2
Application number: EP01980289A
Authority: EP
Inventors: Alfred Jonczyk; Beate Diefenbach; Ulrich Groth; Gunther Zischinsky
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 2000-09-07
Filing date: 2001-08-28
Publication date: 2003-06-04
Also published as: WO2002020566A2; AU2002212172A1; DE10044325A1; JP2004510710A; CA2421283A1; WO2002020566A3

Abstract

The invention relates to novel compounds of the formula (I), which are biologically active as the ligands of integrin αVβ3and 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

New M P-2 derivatives as inhibitors of the integrin α _v ß ₃

The invention relates to novel compounds of the formula I.

X-Y-Z I where

X H, acyl or a peptide fragment with 1 to 20 amino acids selected from the list of naturally occurring ones

Amino acids, Y a peptide fragment selected from the sequence area

466-660 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 list of naturally occurring

Amino acids, A alkyl with 1-10 C atoms,

Acyl alkanoyl with 1-10 C atoms,

mean,

the primary amino groups can also be provided with conventional amino protecting groups, and their physiologically acceptable salts and solvates.

The invention was based on the task of finding new compounds with valuable properties, in particular those which can be used for the production of medicaments.

It has been found that the compounds of the formula I and their salts have very valuable pharmacological properties with good tolerability. Above all, they act as integrin inhibitors, in particular the interactions of the αy-integrin receptors with ligands, e.g.

MMP-2, inhibit. The compounds according to the invention are ligands in particular of

Integrins α ß 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 αvß _ß is known from the literature.

MMP-2 is a matrix-degrading enzyme that facilitates cell migration in invasive processes, e.g. in tumor metastasis and angiogenesis.

There are numerous reviews of matrix metallo-proteinases, e.g. 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 SuppI: S42-51.

MMPs are secreted as inactive proenzymes ("latent enzyme", "zymogen"), so enzymatically inactive Pro-MMP-2 is also known:

Woessner-JF Jr FASEB-J. 1991 May; 5 (8): 2145-54.

Different types of activation from Pro-MM P-2 to MMP-2 are known (autocatalytic, chemical, enzymatic). Activation by tumor cells could be achieved 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 trimer complex are identified

MT1-MMP, TIMP-2 and Pro-MMP-2 also discussed: Teti-A et al. Int J Cancer. 1998 Jul 3; 77 (1): 82-93.

MMPs allow tumor cell invasion and metastasis through at least 3 mechanisms. They remove the physical barrier by breaking down the ECM, they modulate cell adhesion since the cells have to adhere to the ECM but have to break bonds to the ECM again, and they produce other biologically active protein fragments by breaking down proteins.

The inhibition of _ß αvß by the antibody LM609 induced activation of pro-MMP-2 from human Rhabdomyosarcomazellen: 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 the invasive behavior of tumor cells, such as Seftor-RE. et al. in Cancer Res. 1993 Jul 15; 53 (14): 3411-5.

There are various contributions on 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; for 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.

In invasive cells, "invadopodia" and "lamellipodia" on the migration front, which mediate contact with the underlying extracellular matrix (ECM), have different expression and localization of MT1-MMP, MMP-2, and TIMP-2. At the Invadopodia, Pro-MMP-2 and MT1-MMP are responsible for the dismantling of the ECM, since MMP activation takes place here. Although lamellipodia have a similar structure, there is no ECM degradation here, since they are easily accessible to circulating inhibitors such as TIMP-2 on the cellular periphery. It was developed 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 is colocalized on lamellipodia but not on Invadopodia. The authors Nakahara-H; et al. proc

Natl Acad Sci-USA. 1997 Jul 22; 94 (15): 7959-64 reported. MMP-2 occurs in normal and diseased human joints:

Chubinskaya-S et al. Lab Invest. 1999 Dec; 79 (12): 1669-77 and is described in the synovial membrane in rheumatoid arthritis by Konttinen-Y et al.

Ann Rheum Dis. 1999 Nov; 58 (11): 691-7 discussed.

The clinical relevance of MMP-2 as a prognostic marker in cervical

Carcinoma was developed by Davidson-B et al. in Gynecol-Oncol. 1999 Jun; 73 (3):

372-82. 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-

54th

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

The compounds are particularly effective in the case of the integrins' αvß ₃ and vßs- The compounds are particularly effective as adhesion receptor antagonists for the receptor αvß _ß . This effect can be demonstrated, for example, by the method described by B. Diefenbach et al. in Biology of Vitronectins and Their Receptors, Eds. KT Preissner et al., Exerpta Medica, Interntl. Congress Series 1042, Elsevier Sei. Publishers 1993, p. 149-156 is described.

Binding to the vitronectin receptor αvß _ß 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 integrins as adhesion receptors for a wide variety of phenomena and clinical pictures, especially with regard to the receptor αvß ₃ -

The dependence of the development of angiogenesis on the interaction between vascular integrins and extracellular matrix proteins is described by PC Brooks, RA Clark and DA Cheresh in Science 264, 569-71 (1994). The possibility of inhibiting this interaction and thus inducing apoptosis (programmed cell death) of angiogenic vascular cells by a cyclic peptide is from PC Brooks, AM Montgomery, M. Rosenfeld, RA Reisfeld, T.-Hu, G. Klier and DA Cheresh in Cell 79,

1157-64 (1994).

The experimental proof that the compounds according to the invention also prevent the attachment of living cells to the corresponding matrix proteins and accordingly also prevent the attachment of tumor cells to matrix proteins can be provided in a cell adhesion test, which is analogous to the method by F. Mitjans et al. , J. Cell Science 108, 2825-2838 (1995).

PC Brooks et al. describe in J. Clin. Invest. 96, 1815-1822 (1995) αvß ₃ antagonists for combating cancer and for treating 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 diseases, osteoporoses and other osteolytic diseases and for the suppression of angiogenesis.

Compounds of formula I, the interaction of integrin receptors and ligands, such as. B. from fibrinogen to the fibrinogen receptor (glycoprotein llb / llla) prevent GPIIb / llla antagonists from spreading tumor cells through metastasis. This is evidenced by the following observations:

The spread of tumor cells from a local tumor into the vascular system takes place through the formation of micro-aggregates (microthrombi) through the interaction of the tumor cells with platelets. The tumor cells are shielded by the protection in the micro-aggregate and are not recognized by the cells of the immune system. ⁽

The micro-aggregates can attach themselves to the vessel walls, which facilitates further penetration of tumor cells into the tissue. Since the formation of the microthrombi is mediated by fibrinogen binding to the fibrinogen receptors on activated platelets, the GPIIb / llla antagonists are considered to be effective metastasis inhibitors.

Compounds of formula I inhibit the binding of fibrinogen, fibronectin and the Willebrand factor to the fibrinogen receptor

Platelets also bind other adhesive proteins, such as vitonectin, collagen and laminin, to the corresponding receptors on the surface of various cell types. In particular, they prevent the formation of platelet thrombi and can therefore be used to treat thrombosis, apoplexy, heart attack, inflammation and arteriosclerosis.

The properties of the compounds can also be demonstrated by methods which are described in EP-A1-0462 960. The inhibition of fibrinogen binding to the fibrinogen receptor can be demonstrated by the method specified in EP-A1-0 381 033.

The antiplatelet effect can be demonstrated in vitro by the method of Born (Nature 4832, 927-929, 1962). The bone resorption can be inhibited by the compounds according to the invention with the aid of an osteoclast absorption test analogous to WO 95/32710.

Matrix metalloproteinase 2 (MMP-2, also gelatinase A, GelA, 72 kDa type IV collagenase) is a member of the zinc-dependent endoprotease family, which are secreted as pro-enzymes, latent MMPs or zymogens and for the degradation of extracellular matrix proteins and other proteins are responsible to enable cell migration and invasion. The balance between activation from the pro-enzyme and inactivation by the body's own inhibitors, such as TIMP-2, or by extended enzymatic digestion, regulates the activity of MMP-2. The MMPs play a role in physiological as well as pathological situations, in embryonic development, in inflammation, wound healing, tumor cell invasion and metastasis. Inhibitors of the enzymatic

Pocket, expression at the line level or activation from the pro Enzyme can find diagnostic and therapeutic uses in humans and animals.

The enzyme cleaves various types of collagen, elastin, fibronectin and laminin. Binding to collagen takes place by means of 3 repeat units homologous to fibronectin type II modules, which are inserted into the catalytic domain near the active site.

The C-terminus of MMP-2 is known in the literature as "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; von- Schalscha-TL; Friedlander M; Cheresh-DA Cell. 1998 Feb 6; 92 (3): 391-400

Goldberg has described the amino acid sequence of Pro-MMP-2 (72 kDa) in US Pat. No. 4,923,818; the complex with TIMP-2 is documented by Goldberg in EP 404,750.

Soluble Pro-MMP-2 is bound to cell surfaces and is there by the membrane-bound enzyme MT1-MMP by proteolysis of the ProFragmentes in position Asn66-Leu67 in the first step and subsequent MT1-MMP-independent digestion at position Asn109-Tyr110 activated. This activation is not possible if the C-terminus of Pro-MMP-2 is missing. Possible binding partners are MT1-MMP, integrin α _v ß3 and Fetuin.

WO 98/12309, Washington University St. Louis, describes binding sequences from CTD (PEX) to TIMP-2. It is mentioned that the addition of an excess of TIMP-2 (but not TIMP-1) or recombinant CTD (PEX) suppresses the activation of Pro-MMP-2 on the cell surface.

The invention accordingly relates to compounds of the formula I according to claim 1 and / or their physiologically acceptable salts for the preparation of a medicament for use as integrin inhibitors. The invention relates in particular to compounds of the formula I according to Claim 1 and / or their harmless salts for the production of a medicament for combating pathologically angiogenic diseases, tumors, osteoporosis, inflammation and infections.

The compounds of formula I can be used as active pharmaceutical ingredients in human and veterinary medicine, for the prophylaxis and / or therapy of thrombosis, myocardial infarction, arteriosclerosis, inflammation, apoplexy, angina pectoris, tumor diseases, osteolytic diseases such as osteoporosis, hypercalcaemia, pathologically angiogenic diseases such as B. inflammation, ophthalmic diseases, diabetic retinopathy, macular degeneration, myopia, ocular histoplasmosis, rheumatoid arthritis, osteoarthritis, rubeotic glaucoma, ulcerative colitis, Crohn's disease, atherosclerosis, psoriasis, restenosis after angioplasty, infection, with infection, viral infection acute kidney failure and wound healing to support the healing processes.

The compounds of formula I can be used as antimicrobial substances in operations where biomaterials, implants, catheters or pacemakers are used. They have an antiseptic effect. The effectiveness of the antimicrobial activity can be by P.Valentin-Weigund et al., in Infection and Immunity, 2851-2855 (1988).

The invention also relates to the hydrates and solvates, e.g. Alcoholates, these compounds.

The invention furthermore relates to a process for the preparation of compounds of the formula I according to claim 1 and their salts, characterized in that a compound of the formula I is liberated from one of its functional derivatives by treatment with a solvolysing, reducing or hydrogenolysing agent, and / or converts a base or acid of the formula I into one of its salts.

The compounds of the formula I can have one or more chiral centers and can therefore occur in several stereoisomeric forms. All of these forms (e.g. D and L forms) and their mixtures (e.g. the DL forms) are included in Formula I. So-called prodrug derivatives are also included in the compounds according to the invention, i. H. with z. B. alkyl or acyl groups, sugars or oligopeptides modified compounds of formula I, which are quickly cleaved in the organism to the active compounds of the invention.

The abbreviations listed above and below stand for:

Ac Acetyl

BOC tert-butoxycarbonyl

CBZ or Z benzyloxycarbonyl

DCCI dicyclohexylcarbodiimide

DMF dimethylformamide

EDCI N-ethyl-N, N '- (dimethylaminopropyl) carbodiimide

Et ethyl

Fmoc 9-fluorenylmethoxycarbonyl

HOBt 1 -hydroxybenzotriazole Me methyl

Mtr 4-methoxy-2,3,6-trimethylphenyl sulfonyl

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 listed 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

Gin, Q glutamine

Glu, E glutamic acid

Gly. G glycine

His. H histidine, I isoleucine

Leu, L leucine

Lys, K Lysine

Met, M methionine

Phe, F phenylalanine

Pro, P proline

Ser, S Serin

Thr. T threonine

Trp. W tryptophan

Tyr. Y tyrosine Val, V Valin.

The sequence of human Pro-M P-2 (MMP-2) taken from publicly available databases (e.g. available from www.expasy.ch) is as follows:

1 MEALMARGAL TGPLRALCLL GCLLSHAAAA PSPIIKFPGD VAPKTDKELA 51 VQYLNTFYGC PKESCMLFVL KDTLKKMQKF FGLPQTGDLD QNTIETMRKP 101 RCGNPDVANYNFFDPRIETVVDQLF

151 WSDVTPLRFS RIHDGEADIM INFGRWEHGD GYPFDGKDGL LAHAFAPGTG 201 VGGDSHFDDD ELWTLGEGQV VRVKYGNADG EYCKFPFLFN GKEYNSCTDT 251 GRSDGFLWCS TTYNFEKDGK YGFCPHEALF TMGGNAEGQP CKFPFRFQGT 301 SYDSCTTEGR TDGYRWCGTT EDYDRDKKYG FCPETAMSTV GGNSEGAPCV

351 FPFTFLGNKY ESCTSAGRSD GKMWCATTAN YDDDRKWGFC PDQGYSLFLV 401 AAHEFGHAMG LEHSQDPGAL MAPIYTYTKN FRLSQDDIKG lQELYGASPD 451 IDLGTGPTPT LGPVTPEICK QDIVFDGIAQ IRGEIFFFKD RFIWRTVTPR 501 DKPMGPLLVATFWPELPEKI DAVYEAPQEE KAVFFAGNEY WIYSASTLER 551 GYPKPLTSLG LPPDVQRVDA AFNWSKNKKT YIFAGDKFWR YNEVKKKMDP 601 GFPKLIADAW NAIPDNLDAV VDLQGGGHSY FFKGAYYLKL ENQSLKSVKF 651 GSIKSDWLGC

Here, the C-terminal, hemopexin-like region, also known in the literature as PEX, from position 466-660 is marked dark. The integrin α _v β ₃ binding peptide sequences according to the invention are underlined.

It applies to the entire invention that all radicals which occur more than once, such as A, can be the same or different, ie are independent of one another. A is alkyl and has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms and is preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert. Butyl, also for 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 means alkyl substituted by halogen, preferably CF ₃ .

Acyl means alkanoyl with 1-10 C atoms, preferably e.g. Formyl, acetyl, propionyl or butyryl, further e.g. Benzoyl.

Amino protecting group preferably means formyl, acetyl, propionyl, butyryl, phenylacetyl, benzoyl, toluyl, POA, methoxycarbonyl, ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, BOG, 2-iodoethoxycarbonyl, CBZ ("carbobenzoxy"), 4-methoxybenzyloxycarbonyl, FMOC, Mtr or benzyl.

Accordingly, the invention relates in particular to those compounds of the formula I in which at least one of the radicals mentioned has one of the preferred meanings indicated above. Some preferred groups of compounds can be expressed by the following sub-formulas la to le, which correspond to the formula I and in which the radicals not specified have the meaning given for the formula I, but in which

in a) X H, acyl or a peptide fragment with 1 to 20

Amino acids selected from the peptide sequence of human Pro-MMP-2;

in b) X H, acyl or a peptide fragment with 1 to 20

Amino acids selected from the human peptide sequence

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 c) X H, acyl or a peptide fragment with 1 to 20

Amino acids selected from the peptide sequence of human Pro-MMP-2,

Y a peptide fragment selected from the sequence region 480-598 of human Pro-MMP-2,

Z OH, NH _, NHA, NA ₂ or a peptide fragment with 1 to

20 amino acids selected from the human peptide sequence

Pro-MMP-2 mean

in d) X H, 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 human peptide sequence

Pro-MMP-2, mean

in e) X H, acyl or a peptide fragment with 1 to 20

Amino acids selected from the peptide sequence of human Pro-MMP-2, Y a peptide fragment selected from the sequence range 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,

The peptides of the C-terminal sequence of MMP-2 were synthesized as fragments of 8 with 2 amino acid overlaps on paper in an Fmoc strategy.

The quality of the synthesis was assessed using an incident light / fluorescence reader from SLT Tecan and the quantity of the Fmoc signal was evaluated after coupling steps (much fluorescence) and after splitting steps (no fluorescence). These peptide-loaded papers were incubated with fluorescence-labeled integrin α _v β ₃ , washed and evaluated with an incident light fluorescence reader from SLT-Tecan. The peptide sequences of the positive “SPOTs” were prepared conventionally on the resin in separate syntheses and thereby labeled with a C-terminal cysteine. The purified, isolated cysteine peptides were tested in a covalent binding test for binding of biotinylated integrin α _v β ₃ using an anti-biotin / phosphatase Reaction checked, the _v ß ₃ -binding, RGD-independent activity was confirmed.Of the active compounds, 2 peptides are in a consecutive sequence which is derived from the known X-ray structure of the MMP-2 C-terminus the surface of PEX.

The compounds according to the invention prepared by way of example were produced in accordance with the prior art in a Milligen 9050 automatic synthesizer of polystyrene. The process can be understood by a specialist on the basis of textbook knowledge, such as in the appendix to the Novabiochem To find catalog. Chlorine-trityl (o-CI) 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 the Fmoc groups have been cleaved off, the peptide is cleaved from the resin using TFA / water (98: 2), the protective groups of the side chains being cleaved off at the same time.

The compounds of the formula I and also the starting materials for their preparation are otherwise prepared by methods known per se, as described in the literature (for example in the standard works such as Houben-Weyl, Methods of Organic Chemistry, Georg-Thieme-Verlag, Stuttgart; ) are described, namely under reaction conditions which are known and suitable for the reactions mentioned. Use can also be made of variants which are known per se and are not mentioned here in detail.

If desired, the starting materials can also be formed in situ, so that they are not isolated from the reaction mixture, but instead are immediately reacted further to give the compounds of the formula I.

Compounds of formula I can preferably be obtained by liberating compounds of formula I from one of their functional derivatives by treatment with a solvolysing or hydrogenolysing agent.

Preferred starting materials for solvolysis or hydrogenolysis are those which otherwise correspond to the formula I, but instead of one or more free amino and / or hydroxyl groups contain corresponding protected amino and / or hydroxyl groups, preferably those which instead of an H atom, which is connected to an N atom carry an amino protective group, in particular those which carry an R'-N group instead of an HN group, in which R 'represents an amino protective group, and / or those which have one instead of the H atom hydroxy carry a hydroxyl protective group, for example those which correspond to the formula I, but instead of a group -COOH carry a group -COOR "in which R" denotes a hydroxyl protective group.

Several - identical or different - protected amino and / or hydroxy groups can also be present in the molecule of the starting material. If the existing protective groups are different from one another, they can in many cases be split off selectively.

The term "amino protecting group" is generally known and refers to groups which are suitable for protecting (blocking) an amino group from chemical reactions, but which are easily removable after the desired chemical reaction has been carried out at other locations in the molecule. Unsubstituted or substituted acyl, aryl, aralkoxymethyl or aralkyl groups are particularly typical of such groups. Since the amino protective groups are removed after the desired reaction (or reaction sequence), their type and size is otherwise not critical; however, those with 1-20, in particular 1-8, carbon atoms are preferred. The term "acyl group" is to be understood in the broadest sense in connection with the present process. It encompasses acyl groups derived from aliphatic, araliphatic, aromatic or heterocyclic carboxylic acids or sulfonic acids and, 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, BOG, 2-iodoethoxycarbonyl; Aralkyloxycarbonyl such as CBZ ("carbobenzoxy"), 4-methoxybenzyloxycarbonyl, FMOC; Arylsulfonyl such as Mtr. Preferred amino protective groups are BOC and Mtr, furthermore CBZ, Fmoc, benzyl, formyl and acetyl.

The amino protective group is split off, depending on the protective group used, e.g. B. with strong acids, suitably with TFA or perchloric 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-toluenesulfonic acid. The presence of an additional inert solvent is possible, but not always necessary. Suitable inert solvents are preferably organic, for example carboxylic acids such as acetic acid, ethers such as tetrahydrofuran or dioxane, amides such as DMF, halogenated hydrocarbons such as dichloromethane, and also alcohols such as methanol, ethanol or isopropanol, and water. Mixtures of the abovementioned solvents are also suitable. TFA is preferably used in excess without the addition of another solvent, perchloric acid in the form of a mixture of acetic acid and 70% perchloric acid in a ratio of 9: 1. The reaction temperatures for the cleavage are advantageously between about 0 and about 50 °, preferably between 15 and 30 ° (room temperature).

The groups BOC, OBut, Pmc, Pbf and Mtr can e.g. B. preferably with TFA in dichloromethane or with about 3 to 5N HCl in dioxane at 15-30 °, the FMOC group with an about 5 to 50% solution of sec. Amines such as dimethylamine, diethylamine or piperidine in DMF at 15-30 °.

Hydrogenolytically removable protective groups (e.g. CBZ or benzyl) can e.g. B. by treatment with hydrogen in the presence of a catalyst (z. B. a noble metal catalyst such as palladium, advantageously on a support such as coal). Suitable solvents are the above, especially z. B. alcohols such as

Methanol or ethanol or amides such as DMF. The hydrogenolysis is generally carried out at temperatures between about 0 and 100 ° and pressures between about 1 and 200 bar, preferably at 20-30 ° and 1-10 bar. Hydrogenolysis of the CBZ group succeeds e.g. B. good on 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 example, 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 monomethyl 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 such as acetonitrile; Sulfoxides such as dimethyl sulfoxide (DMSO); Carbon disulphide; Carboxylic acids such as formic acid or acetic acid; Nitro compounds such as nitromethane or nitrobenzene; Esters such as ethyl acetate, water or mixtures of the solvents mentioned.

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

It is also possible to replace a conventional amino protecting group with hydrogen by splitting off the protecting group as described above, solvolytically or hydrogenolytically, or by liberating an amino group protected by a conventional protecting group by solvolysis or hydrogenolysis.

A base of the formula I can be converted into the associated acid addition salt using an acid, for example by reacting equivalent amounts of the base and the acid in an inert solvent such as ethanol and subsequent evaporation. In particular, acids that provide physiologically acceptable salts are suitable for this implementation. Thus, inorganic acids can be used, for example sulfuric acid, nitric acid, hydrohalic acids such as hydrochloric acid or hydrobromic acid, phosphoric acids such as orthophosphoric acid, sulfamic acid, and also organic acids, in particular aliphatic, alicyclic, aromatic, heterocyclic, mono- or polycarbonate, sulfone - or sulfuric acids, for example formic acid, acetic acid, propionic acid, pivalic acid, diethyl acetic 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, lauryl sulfuric acid. Salts with physiologically unacceptable acids, for example picrates, can be used for the isolation and / or purification of the compounds of the formula I.

On the other hand, an acid of the formula I can be converted into one of its physiologically acceptable metal or ammonium salts by reaction with a base. Suitable salts here are in particular the sodium, potassium, magnesium, calcium and ammonium salts, and also substituted ammonium salts, e.g. B. the dimethyl, diethyl or diisopropyl ammonium salts, monoethanol, diethanol or diisopropylammonium salts, cyclohexyl, dicyclohexylammonium salts, di- [benzylethylenediammonium salts, z. B. salts with arginine or lysine.

The compounds of the formula I contain one or more chiral centers and can therefore be present in racemic or in optically active form. Racemates obtained can be separated mechanically or chemically into the enantiomers by methods known per se. Diastereomers are preferably formed from the racemic mixture by reaction with an optically active release agent. Suitable release agents are e.g. optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids such as ß-camphorsulfonic acid. Enantiomer separation using a column filled with an optically active separating agent (e.g. dinitrobenzoylphenylglycine) is also advantageous; a suitable solvent is e.g. a mixture of hexane / isopropanol / acetonitrile, e.g. in the volume ratio 82: 15: 3.

Of course, it is also possible to obtain optically active compounds of the formula I by the methods described above by using starting materials which are already optically active. The invention includes not only the compounds mentioned, but also mixtures and preparations which, in addition to these compounds according to the invention, also include other pharmacological active ingredients or

Contain adjuvants which can influence the primary pharmacological action of the compounds according to the invention in a desired manner. These can be used as therapeutic agents, diagnostic agents or as reagents

Find use. They can be given to humans or animals locally or systemically, orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, transdermally, nasally, buccally, or iontophoretically, which includes formulations in suspensions, emulsions or solutions, liposomes, ointments, pastes, biodegradable polymers or as nanoparticles, tablets, capsules or pills, granules or powder, as an aerosol for inhalation, as intranasal drops or sprays. It is also possible to combine the new products with other technologies such as surgery, radiation, diagnosis, radiotherapy, photodynamic therapy and gene therapy, as well as with other medications.

Such drugs can e.g. from the areas of cardiovascular,

Central nervous system or oncology. It can

Tumor agents, such as angiogenesis inhibitors or cytostatics,

Chemotherapeutics of the groups alkylating agents, antibiotics,

Antimetabolites, biologicals and immunomodulators, hormones and their antagonists, mustard gas derivatives, alkaloids and others, these

Substances can be low molecular weight and high molecular weight. It can

Lipids, carbohydrates or proteins. This includes cytokines,

Toxins, fusion proteins, monoclonal antibodies and vaccines.

The invention accordingly relates to compounds of the formulas defined above and below and in the claims, including their physiologically acceptable salts, as medicaments, diagnostic agents or reagents. In particular, these are drugs for combating diseases based on an interaction of ligands, for example of MMP-2 with the _v ß ₃

Integrin receptor based.

The drugs are suitable for fighting thrombosis,

Heart attack, coronary heart disease, arteriosclerosis, tumors,

Osteoporosis, fibrosis, inflammation, infections, psoriasis and for

Influencing wound healing processes.

Corresponding pharmaceutical preparations which contain at least one medicament of the formula I and, if appropriate, carriers and / or auxiliaries are also an object. The invention furthermore relates to the use of the compounds according to the invention and / or their physiologically harmless

Salts and / or solvates for the manufacture of a medicament for inhibiting the integrin receptor α _v ß3-

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 manufacture of a medicament for combating diseases which are based on an interaction of a ligand, for example the matrix metalloproteinase (MMP-2) with the _v ß ₃ integrin receptor based. - '

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 pathological processes which are maintained or propagated by angiogenesis.

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 manufacture of a medicament for combating thromboses, heart attacks, coronary heart diseases, arteriosclerosis, tumors, osteoporosis, fibrosis, inflammation, Infections, psoriasis and to influence wound healing processes.

The medicaments according to the invention) or pharmaceutical preparations containing them can be used in human or veterinary medicine. Suitable carriers are organic or inorganic substances which are suitable for enteral (e.g. oral), parenteral, topical application or for application in the form of a

10 inhalation sprays are suitable and do not react with the new compounds, for example water, vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols, glycerol triacetate, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc, petroleum jelly. For oral

^ _C application are used in particular tablets, pills, dragees, capsules, powders, granules, syrups, juices or drops, for rectal application positorien Sup-, for parenteral application of solutions, preferably oily or aqueous solutions, furthermore suspensions, emulsions or implants, and for topical application ointments, creams or powder. The new

20 compounds can also be lyophilized and the resulting lyophilisates e.g. can be used for the production of injectables. The specified preparations can be sterilized and / or auxiliaries such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers.

25 gators, salts to influence the osmotic pressure, buffer substances, coloring, flavoring and / or contain several other active ingredients, for. B. one or more vitamins. Sprays can be used for the application as an inhalation spray,

OQ which contain the active substance either dissolved or suspended in a propellant gas or propellant gas mixture (e.g. C0 ₂ or chlorofluorocarbons). The active ingredient is expediently used in micronized form, with one or more additional physiologically tolerable ones

Solvents may be present, e.g. B. ethanol. inhalation solutions

35 can be administered using standard inhalers. The substances according to the invention can generally be administered in analogy to other known, commercially available preparations (for example described in US Pat. No. 4,472,305), preferably in doses between about 0.05 and 500 mg, in particular between 0.5 and 100 mg per dosage unit. The daily dosage is preferably between about 0.01 and 20 mg / kg body weight. However, the specific dose for each patient depends on a variety of factors, for example on the effectiveness of the particular compound used, on the age, body weight, general health, sex, on the diet, on the time and route of administration, on the rate of elimination, combination of drugs and severity the respective disease to which the therapy applies. Parenteral administration is preferred.

All temperatures above and below are given in ° C.

The HPLC analyzes (retention time Rt) were carried out in the following system:

Column 250x4 mm Lichrospher 100RP 18 (Merck KGaA) with a 50 minute gradient from 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): El (electron impact ionization) M ⁺

FAB (Fast Atom Bombardment) (M + H) ⁺

Examples

Production and purification of peptides according to the invention: In principle, production and purification is carried out by means of the Fmoc strategy while protecting acid-labile side chains on acid-labile resins using a commercially available "continuous flow" peptide synthesizer in accordance with the information provided by Haubner et al. (J. Am. Chem. Soc. 118, 1996, 17703).

The following compounds were obtained

The following designations are synonymous

KDRFIWRTVC MMP-2 (489-497) -Cys

AAFNWSKNKKTYIFAGC MMP-2 (570-585) -Cys

FWRYNEVKKKC MMP-2 (588-597) -Cys

The following compounds were also obtained

Single letter code synonymous

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 (478483) -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 ß ₃ can

Placenta can be cleaned as described by Smith et al. J. Biol. Chem. 263, 18726-

18731 (1988) with the modifications according to F.C. Mitjans et al. J. Cell Sei.

108, 2825-2838 (1995). Soluble α _v ß ₃ is like in RJ

Mehta et al. Biochem. 330, 861-869 (1998), there is also labeling with biotin, carrying out a ligand

'Binding test and competition assays performed, another quote for that

Binding test is S. Kraft et al. J. Biol. Chem. 274, 1979-1985 (1999).

1. The receptor binding test on covalently bound peptides 5 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.

In the receptor binding test, fluorescein-labeled integrin αvß ₃ 0 was used, which interacts directly with the cellulose-bound peptide. The evaluation was carried out by measuring the fluorescence. In another variant, BSA coated in microtiter plates was activated with sulfo-SMPB and the thiopeptides were covalently bound to it. After 5 incubations with biotinylated αvß ₃ , bound receptor with alkaline-phosphatase-coupled goat anti-biotin antibody and coloring by a phosphatase substrate was detected.

0

Peptide spot sequence humMMP-2 EC ₅₀ [μM]

No. sren: 7 cyclo- (RGDfK (mercapto- - 0.2! Propionyl))

1 53-57 AAFNWSKNKKTYIFAGC 570-585 8

2 62-63 FWRYNEVKKKC 588-597 5 5 3 12-13 KDRFIWRTVC 489-497 10 The pharmacological data demonstrate the activity of the compounds according to the invention as ligands for the ctvß ₃ receptor.

2. The binding of the PEX peptides to αvß ₃ is an RGD-independent binding. This was verified 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 αvß _{3 was} inhibited by cyclo- (Arg-Gly-Asp-D-Phe-N-Me-Val), the binding was of the PEX peptide AAFNWSKNKKTYIFAGC regardless of this.

The following examples relate to pharmaceutical preparations:

Example A: Injection glasses

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

Example B: Suppositories

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

Example C: solution

A solution is prepared from 1 g of Lys-Asp-Arg-Phe-Ile-Trp-Arg-Thr-Val-Cys, 9.38 g of NaH ₂ P0 ₄ ^■ 2 H ₂ 0, 28.48 g Na ₂ HP0 ₄ • 12 H ₂ 0 and 0.1 g

Benzalkonium chloride in 940 ml of double distilled water. It is adjusted to pH 6.8, made up to 1 I and sterilized 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 with 99.5 g of petroleum jelly 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 in the usual way pressed into tablets such that each tablet contains 10 mg of active ingredient. Example F: coated tablets

Analogously to Example E, tablets are pressed, which are then coated in a conventional manner with a coating of sucrose, potato starch, talc, tragacanth and colorant.

Example G: Cape

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

Example H: ampoules

A solution of 1 kg of Lys-Asp-Arg-Phe-Ile-Trp-Arg-Thr-Val-Cys in 60 l of double-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 I of isotonic NaCl solution and the solution is filled into commercially available spray vessels with a pump mechanism. The solution can be sprayed into the mouth or nose. One spray (approximately 0.1 ml) corresponds to a dose of approximately 0.14 mg.

Claims

claims

1. Compounds of formula I I

X-Y-Z I

wherein

Amino acids, Y a peptide fragment selected from the sequence area

Amino acids selected from the list of naturally occurring

Amino acids, A alkyl with 1 -10 C atoms, acyl alkanoyl with 1-10 C atoms,

mean

2. Compounds according to claim 1,

wherein

X H, acyl or a peptide fragment with 1 to 20 amino acids selected from the peptide sequence of human Pro-MM P-2

means the primary amino groups can also be provided with conventional amino protective groups, and their physiologically acceptable salts and solvates.

3. Compounds according to claim 1, ^~

wherein

X H, acyl or a peptide fragment with 1 to 20 amino acids selected from the peptide sequence 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,

the primary amino groups can also be provided with conventional amino protective groups, and their physiologically acceptable salts and solvates.

4. Compounds according to claim 1,

wherein

X H, 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 area

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,

where the primary amino groups can also be provided with conventional amino protecting groups, and their physiologically acceptable salts and solvates.

5. Compounds according to claim 1,

wherein

Y is a peptide fragment selected from the sequence area

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,

6. Compounds according to claim 1, woπn,

Y is a peptide fragment selected from the sequence area

489-497 and / or 570-585 and / or 588-597 of human Pro-MMP-2,

Z OH, NH ₂ , NHA, NA ₂ or Cys

mean,

0 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 5 and their physiologically acceptable salts and solvates.

8. A process for the preparation of compounds of formula I according to _Q claim 1 and their salts, characterized in that

releases a compound of formula I from one of its functional derivatives by treatment with a solvolysing, reducing or hydrogenolysing agent, 5 and / or converts a base or acid of the formula I into one of its salts.

9. Compounds of formula I according to claims 1-6 and the compounds according to claim 7 and their physiologically acceptable salts and solvates as medicaments.

10. Medicament according to claim 9 as an inhibitor of the integrin receptor α _v ß3.

11. Medicament according to claim 10 for combating diseases which are based on an interaction of ligands with the α _v ß3 integrin receptor.

12. Medicament according to claim 11 for combating diseases which are based on an interaction of the matrix metalloproteinase (MMP-2) with the α _v ß ₃ integrin receptor.

13. Medicament according to claim 12 for combating thrombosis, heart attack, coronary heart disease, arteriosclerosis, tumors, osteoporosis, fibrosis, inflammation, infection, psoriasis and for influencing wound healing processes.

14. Pharmaceutical preparation containing at least one medicament according to one of claims 10 to 13 and optionally carriers and / or auxiliaries and optionally other active substances.

15. Use of compounds according to claims 1 to 7 and / or their physiologically acceptable salts for the manufacture 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 preparation a drug for combating diseases based on an interaction of ligands with the α _v ß ₃ integrin receptor.

17. Use according to claim 16 for the manufacture of a medicament for combating diseases which are 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 manufacture of a medicament for combating pathological processes which are maintained or propagated by angiogenesis.

19. Use according to claim 18 for the manufacture of a medicament for combating thromboses, myocardial infarction, coronary heart diseases, arteriosclerosis, tumors, osteoporosis, fibrosis, inflammation, infections, psoriasis and for influencing wound healing processes.