WO2001000660A1

WO2001000660A1 - INHIBITORS OF THE INTEGRIN αvβ¿6?

Info

Publication number: WO2001000660A1
Application number: PCT/EP2000/005404
Authority: WO
Inventors: Alfred Jonczyk; Beate Diefenbach; Ulrich Groth; Gunther Zischinsky
Original assignee: Merck Patent Gmbh
Priority date: 1999-06-26
Filing date: 2000-06-13
Publication date: 2001-01-04
Also published as: CN1358195A; PL352374A1; SK18722001A3; AU6263000A; EP1189930A1; DE19929410A1; HUP0201729A2; NO20016341L; KR20020015704A; CZ20014484A3; MXPA01013247A; HUP0201729A3; NO20016341D0; BR0011954A; ZA200200673B; AU771099B2; AR024472A1; CA2377224A1; JP2003503422A

Abstract

The invention relates to novel peptides of formula (I) Ac-Arg-X?1-Asp-X2-X3-X4-X5-X6-NH¿2 which are biologically active as ligands of the integrin αvβ6, wherein: Ac represents Acetyl, X1 represents Ser, Gly, Thr, Asp, Arg, Val, Tyr, His or Ala; X2 represents Leu, Ile, Nle, Val or Phe; X3 represents Asp, Glu, Lys, Phe, Aib, Nal, Gly, Ala, Bgl or Phg; X4 represents Gly, Ala, Ser, β-Ala or φ-Abu; X5 represents Leu, Ile, Nle, Val or Phe, and; X6 represents Arg, Har, Lys, Leu, Orn, Phe, Ala, Tyr, Gly, Ser or Asp, whereby the aforementioned amino acids can be derivatized. The amino acid radicals are linked to one another in a peptide-like manner via the α-amino groups and the α-carboxy groups. The dextrorotatory forms and the levorotatory forms of the optically active amino acid radicals are contained as well as the salts of said peptides. Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH¿2? is excluded. The inventive peptides can also be used as effective inhibitors of the αvβ6 integrin receptor and thus for treating different diseases and pathological findings.

Description

Inhibitors of the integrin α _v ßβ

The invention relates to novel peptides of the formula I which are biologically active as ligands of the integrin α _v β _δ ,

Ac-Arg-X ¹ -Asp-X ² -X ³ -X ⁴ -X ⁵ -X ⁶ -NH ₂ I

wherein

Ac Acetyl,

X ¹ Ser, Gly, Thr, Asp, Arg, Val, Tyr, His or Ala,

X ² Leu, Ile, Nie, Val or Phe,

X ³ Asp, Glu, Lys, Phe, Aib, Nal, Gly, Ala, Bgl or Phg,

X ⁴ Gly, Ala, Ser, ß-Ala or ω-Abu,

X ⁵ Leu, Ile, Nie, Val or Phe,

X ⁶ denotes Arg, Har, Lys, Leu, Orn, Phe, Ala, Tyr, Gly, Ser or Asp,

wherein the amino acids mentioned can also be derivatized, the amino acid residues are peptide-like linked via the α-amino and α-carboxy groups, the D- and the L-forms of the optically active amino acid residues are included, as well as their physiologically acceptable salts, and wherein Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH _{2 is} excluded.

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 according to the invention and their salts have very valuable pharmacological properties with good tolerability.

The peptides according to the invention can be used as effective inhibitors of the α _v ß integrin receptor and thus for the treatment of various diseases and pathological findings. Other inhibitors of the integrin α _v ß ₆ are in DE 19858857 and by S.Kraft et al. in J. Biol. Chem. 274, 1979-85 (1999). The compounds according to the invention are to be regarded as selection inventions in relation to the application mentioned.

Integrins belong to the family of heterodimeric class I transmembrane receptors, which play an important role in numerous cell matrix or cell-cell adhesion processes (Tuckwell et al., 1996, Symp. Soc. Exp. Biol. 47) , They can be roughly divided into three classes: the β-integrins, which are receptors for the extracellular matrix, the β ₂ integrins, which can be activated on leukocytes and are "triggered" during inflammatory processes, and the α _v integrins which influence the cell response in wound healing and other pathological processes (Marshall and Hart, 1996, Semin. Cancer Biol. 7, 191).

The integrins αsß-i, αu ß3, aßi, α _v ßι, α _v ß3, α _v ßs, α _v ßs and α _v ß6 all bind to the Arg-Gly-Asp (RGD) peptide sequence in natural ligands such as fibronectin or vitronectin. Soluble peptides containing RGD are capable of the interaction of each of these integrins with the corresponding natural one

Inhibit ligands. α _v ßβ is a relatively rare integrin (Busk et al., 1992 J. Biol. Chem. 267 (9), 5790), which is increasingly formed in epithelial tissue during repair processes and preferentially binds the natural matrix molecules fibronectin and tenascin (Wang et al ., 1996, Am. J. Respir. Cell Mol. Biol. 15 (5), 664). The physiological and pathological functions of α _v ß _δ are not yet exactly known, but it is believed that this integrin is important in physiological processes and diseases (e.g. inflammation, wound healing, tumors) in which epithelial cells are involved Role play. Thus, α _v ß _{δ is expressed} on keratinocytes in wounds (Haapasalmi et al., 1996, J. Invest.

Dermatol. 106 (1), 42), from which it can be assumed that, in addition to wound healing processes and inflammation, other pathological events of the skin, such as, for example, B. psoriasis, can be influenced by agonists or antagonists of said integrin. Furthermore, α _v ßβ plays a role in the respiratory epithelium (Weinacker et al., 1995, Am. J. Respir. Cell Mol. Biol. 12 (5), 547), so that corresponding agonists / antagonists of this integrin in respiratory pathological diseases such as bronchitis, asthma, pulmonary fibrosis and respiratory tumors could be used successfully. Ultimately, it is known that α _v β6 also plays a role in the intestinal epithelium, so that corresponding integrin agonists / antagonists could be used in the treatment of inflammation, tumors and wounds of the gastrointestinal tract.

The dependence of the development of angiogenesis on the interaction between vascular integrins and extracellular matrix proteins has been reported by P.C. Brooks, R.A. Clark and D.A. Cheresh in Science 264, 569-71 (1994).

It was therefore the task, in addition to the previously known natural high molecular weight ligands and antibodies, which are difficult to handle therapeutically and diagnostically, to find potent, specific or selective low molecular weight ligands for α _v β, preferably peptides, but for the therapeutic areas mentioned can also be used as a diagnostic or reagent.

It has been found that the peptide compounds according to the invention and their salts act as soluble molecules on cells which carry the receptor mentioned or, if they are bound to surfaces, are artificial ligands for α _v β ₆ -mediated cell attachment. Above all, they act as α _v ß ₆ integrin inhibitors, in particular inhibiting the interactions of the receptor with other ligands, such as. B. the binding of fibronectin. This effect can be demonstrated, for example, by the method described by JW Smith et al. in J. Biol. Chem. 265, 12267-12271 (1990).

It was also found that the new substances have very good pharmacological properties and are well tolerated and can be used as medicaments. This is described in more detail below. The peptide compounds according to the invention can also be used as diagnostics for the detection and localization of pathological conditions in the epithelial system in vivo if they are equipped with appropriate markers (for example the biotinyl residue) according to the prior art.

The invention also includes combinations with at least one other active ingredient and / or conjugates with other active ingredients, such as cytotoxic active ingredients and conjugates with radio markers for X-ray therapy or PET diagnosis, but also fusion proteins with marker proteins such as GFP or antibodies, or therapeutic proteins such as IL-2.

Some preferred groups of compounds can be expressed by the following sub-formulas Ia to If, 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 ^{1 is} Ser, Gly or Thr;

in b) X ¹ Ser, Gly or Thr,

X ^{2 means} Leu;

in c) X ¹ Ser, Gly or Thr,

X ² Leu,

X ^{3 represents} Asp or D-Asp;

in d) X ¹ Ser, Gly or Thr,

X ² Leu,

X ³ Asp or D-Asp,

X ^{4 represents} Gly, Ala or Ser; in e) X ¹ Ser, Gly or Thr,

X ² Leu,

X ³ Asp or D-Asp,

X ⁴ Gly, Ala or Ser,

X ^{5 represents} Leu;

in f) X ¹ Ser, Gly or Thr,

X ² Leu,

X ³ Asp or D-Asp,

X ⁴ Gly, Ala or Ser,

X ⁵ Leu,

X ^{6 represents} Arg; as well as their salts.

The invention relates in particular to peptide compounds selected from the group

Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Gly-Asp-Leu-Asp-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Ser-Asp-Leu-Asp-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Asp-Asp-Leu-Asp-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Ala-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-D-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Arg-NH ₂ ,

Ac-D-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-D-Ala-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Aib-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-D-Nal-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Gly-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Ala-Ser-Leu-Arg-NH ₂ , Ac-Arg-Thr-Asp-Nle-D-Asp-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Ile-D-Asp-Ser-Leu-Arg-NH ₂ , Ac-Arg-Thr-Asp-Leu-Asp-D-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Asp-Ala-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Asp-Gly-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Har-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Lys-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-D-Asp-D-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Ala-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-D-Asp-Gly-Leu-Arg-NH ₂ , and their physiologically acceptable salts.

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

Abu 4-aminobutyric acid

Aha 6-aminohexanoic acid, 6-aminocaproic acid

Aib α-amino isobutyric acid

Ala alanine

Asn asparagine

Asp aspartic acid

Arg arginine

Bgl C-alpha-tert-butylglycine

Cys cysteine

Dab 2,4-diaminobutyric acid

Dap 2,3-diaminopropionic acid

Gin glutamine

Glp pyroglutamic acid

Glu glutamic acid

Gly Gly a

Har homoarginine

His histidine homo-Phe homo-phenylalanine

Ile isoleucine

Leu leucine

Lys lysine

With methionine Nal Naphth-2-yl alanine

Never norleucine

Orn 0 therefore

Phe phenylalanine

Phg phenylglycine

4-Hal-Phe 4-halophenylalanine

Pro Proliπ

Ser Serin

Thr threonine

Trp tryptophan

Tyr tyrosine

Val valine.

Furthermore, below mean:

Ac Acetyl

BOC tert-butoxycarbonyl

BSA Bovine Serum Albumin

CBZ or Z benzyloxycarbonyl

DCCI dicyclohexylcarbodiimide

DMF dimethylformamide

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

Et ethyl

FCA fluorescein carboxylic acid

FITC fluorescein isothiocyanate

Fmoc 9-fluorenylmethoxycarbonyl

FTH fluoresceinthiourea

HOBt 1-hydroxybenzotriazole

Me methyl

MBHA 4-methyl-benzhydrylamine

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

Sal salicyloyl

TBS ++ Tris buffered saline with divalent cations

TBSA TBS + BSA

TBTU 2- (1 H-benzotriazol-1-yl) -1, 1, 3-tetramethyluronium tetrafiuoroborate

TFA trifluoroacetic acid

Trt trityl (T phenylmethyl).

If the above-mentioned amino acids can occur in several enantiomeric forms, all of these forms and also their mixtures (for example the DL forms) are included above and below. Furthermore, the amino acids can be used with corresponding known per se

Protecting groups.

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 that are quickly cleaved in the organism to the active compounds of the invention. This also includes biodegradable polymer derivatives of the compounds according to the invention, as described, for. B. in Int. J. Pharm. 115, 61-67 (1995).

The amino acids and amino acid residues mentioned, such as, for example, the NH functions or the C-terminal amide function, can also be derivatized, the N-methyl, N-ethyl, N-propyl, N-benzyl or C _α - Methyl derivatives are preferred. Further preferred are derivatives of Asp and Glu, in particular the methyl, ethyl, propyl, butyl, tert-butyl,

Neopentyl or benzyl ester of the side chain carboxy groups, also derivatives of Arg, which may be substituted on the -NH-C (= NH) -NH ₂ group with an acetyl, benzoyl, methoxycarbonyl or ethoxycarbonyl radical. In addition to the compounds of the formula I which carry an acetyl group at the N-terminal, the compounds according to the invention also include compounds in which Ac is replaced by another acyl function, such as propionyl, butyryl or also benzoyl.

Furthermore, the compounds according to the invention also include derivatives which consist of the actual peptides according to the invention and known marker compounds which make it possible to easily detect the peptides. Examples of such derivatives are radio-labeled, biotinylated or fluorescence-labeled peptides.

Fluorescent dye residue preferably means 7-acetoxycoumarin-3-yl, fluorescein-5- (and / or 6-) yl, 2 ', 7'-dichlorofluorescein-5- (and 6-) yl, dihydrotetramethylrosamin-4-yl, tetramethylrhodamine 5- (and / or 6-) yl, 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3-ethyl or 4,4-difluoro-5,7 diphenyl-4-bora-3a, 4a-diaza-s-indacene-3-ethyl.

Suitable functionalized fluorescent dye residues that can serve as reagents for the preparation of the compounds of formula I according to the invention are, for. B. described in "Handbook of Fluorescent Probes and Research Chemicals, 5th Edition, 1992-1994, by R.P. Haughland, Molecular Probes, Inc.".

In general, the peptides according to the invention are linear, but they can also be cyclized. The invention includes not only the peptides mentioned but also mixtures and preparations which, in addition to these compounds according to the invention, also contain other pharmacological active ingredients or adjuvants which can influence the primary pharmacological action of the peptides according to the invention in a desired manner.

The compounds of the invention and also the starting materials for their preparation are otherwise known and common methods used, as they are in the literature (e.g. in the

Standard works such as Houben-Weyl, methods of organic chemistry,

Georg-Thieme-Verlag, Stuttgart) are described under

Reaction conditions which are known and suitable for the reactions mentioned. You can also use known variants

Make use.

The peptides according to the invention can preferably be prepared by means of solid phase synthesis and subsequent cleavage and purification, as is the case for by Jonczyk and Meienhofer (Peptides, Proc. 8th

At the. Pept. Symp., Eds. V. Hruby and D.H. Rieh, Pierce Comp. III, p. 73-77,

1983, or Angew. Chem. 104, 1992, 375) or according to Merrifield (J. Am.

Chem. Soc. 94, 1972, 3102).

The peptides according to the invention can be prepared on a solid phase (manually or in an automatic synthesizer) in an Fmoc strategy with acid-labile side-protecting groups and purified by RP-HPLC. Peak uniformity can be measured by RP-HPLC and substance identity using FAB-MS.

In addition, the peptides can be prepared by conventional methods of amino acid and peptide synthesis, such as. B. from Novabiochem - 1999 Catalog & Peptide Synthesis Handbook from Calbiochem-Novabio-chem GmbH, D-65796 Bad Soden, is known from numerous standard works and published patent applications.

Gradual couplings and fragment condensation can be used. Different N-terminal, C-terminal and side protection groups can be used, which are preferably selected to be orthogonally cleavable. Coupling steps can be carried out with different condensation reagents such as carbodiimides, carbodiimidazole, those of the uronium type such as TBTU, mixed anhydride methods, and acid halide or active ester methods. activated Esters are conveniently formed in situ, e.g. B. by adding HOBt or N-hydroxysuccinimide.

A cyclization of a linear precursor molecule with side protecting groups can also be carried out with such condensation reactions, such as. B. in DE 43 10 643 or in Houben-Weyl, I.e., Volume 15/11, pages 1 to 806 (1974).

Different resins and anchor functions can be used in solid phase peptide synthesis. Resins can e.g. on polystyrene or

Polyacrylamide based, anchor functions such as Wang, o-chlorotrityl are for the production of peptide acids, aminoxanthenoxy anchors e.g. usable for the production of paptidamides.

Biotinylated or fluorescence-labeled peptides / proteins can also be produced using standard methods (e.g. EA Bayer and M. Wilchek in Methods of Biochemical Analysis Vol 26 The Use of the Avidin-Biotin Complex as a Tool in Molecular Biology; and Handbook of Fluorescent Probes and Research Chemicals , 6 ^th Edition, 1996, by Haugland RP, Molecular Probes, Inc .; or WO 97/14716).

Of course, the peptides according to the invention can also be released by solvolysis, in particular hydrolysis, or by hydrogenolysis of their functional derivatives. Preferred starting materials for solvolysis or hydrogenolysis are those which contain corresponding protected amino and / or hydroxyl groups instead of one or more free amino and / or hydroxyl groups, preferably those which instead of an H atom which is connected to an N atom is an amino protective group or which carry a hydroxy protective group instead of the H atom of a hydroxy group. The same applies to carboxylic acids which can be protected by substitution of their -CO-OH hydroxy function by means of a protective group, for example as an ester. 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 at other locations on the

Molecule has been carried out. The term "hydroxyl protecting group" is also generally known and refers to groups which are suitable for protecting a hydroxyl group against chemical reactions, but which are easily removable after the desired chemical reaction has been carried out elsewhere in the molecule. The in freedom

Depending on the protective group used, setting the compounds from their functional derivatives succeeds, for. 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. 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).

Typical ^• protecting groups for N-termini and for pendant amino groups are Z, BOC, Fmoc, those for C-termini or the Asp or Glu side chains are O-prim.-alkyl (eg OMe or OEt), O-tert .-Alkyl (e.g. OBut) or OBenzyl. Z, BOC, N0 ₂ , Mtr, Pmc or Pbf are suitable for the guanidino function of Arg. Alcoholic functions can be protected by tert-alkyl radicals or trityl groups.

The groups BOC, OBut 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 dimethylamine, diethylamine or piperidine in DMF at 15 -30 °. The trityl group is used, for example, to protect the amino acids histidine, asparagine, glutamine and cysteine. Depending on the desired end product, the cleavage is carried out with TFA / 10% thiophenol, the trityl group being cleaved from all the amino acids mentioned; when using TFA / anisole or TFA / thioanisole, only the trityl group of His, Asn and Gin is cleaved, whereas they are remains on the Cys side chain.

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 °.

As already mentioned, the peptides according to the invention comprise their physiologically acceptable salts, which can also be prepared by standard methods. For example, a base of a compound according to the invention can be converted with an acid into the associated acid addition salt, 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. For example, 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, araliphatic, aromatic or heterocyclic mono- or poly-based carbon atoms. , Sulfonic or sulfuric acids, e.g. formic acid, acetic Acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, lactic acid, tartaric acid, malic acid, citric acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methane or ethanesulfonic acid, 2-dodesulfonic acid, ethanesulfonic acid, ethanesulfonic acid, ethanesulfonic acid, 2-sulfonic acid, 2-diacid, , p-toluenesulfonic acid, naphthalene mono- and disulfonic acids, laurylsulfuric acid. Salts with physiologically unacceptable acids, such as picrates, can be used

Isolation and / or purification of the compounds according to the invention can be used. On the other hand, an acid of the compounds according to the invention can be converted into one of their 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 diisopropylammonium salts, monoethanol, diethanol or diisopropylammonium salts, cyclohexyl, dicyclohexylammonium salts, dibenzylethylenediammonium salts, z. B. salts with arginine or lysine.

As already mentioned, the peptide compounds according to the invention can be used as active pharmaceutical ingredients in human and veterinary medicine, in particular for the prophylaxis and / or therapy of diseases in which epithelial cells are involved. Of particular note here are diseases or inflammations or wound healing processes in the skin, respiratory organs and stomach and intestinal area, such as apoplexy, angina pectoris, tumor diseases, osteolytic diseases such as osteoporosis, pathologically angiogenic diseases such as B. Inflammation, pulmonary fibrosis, ophthalmic diseases, diabetic retinopathy, macular degeneration, myopia, ocular histoplasmosis, rheumatoid arthritis, osteoarthritis, rubeotic glaucoma, ulcerative colitis, Crohn's disease, atherosclerotic disease, psoriasis angiosis, psoriasis disease kidney failure,

Inflammation of the kidneys, microbial infections and multiple sclerosis. The invention accordingly relates to peptide compounds of the formulas defined above and below and in the claims, including their physiologically acceptable salts, as medicaments, diagnostics or reagents.

The invention relates in particular to corresponding medicaments as inhibitors for combating diseases which are based directly or indirectly on expression of the α _v β ₆ integrin receptor, in particular in the case of pathologically angiogenic diseases, thromboses, heart attacks, coronary heart diseases, arteriosclerosis, tumors, osteoporosis. Inflammation, infections and to influence wound healing processes.

The invention also relates to corresponding pharmaceutical preparations which contain at least one medicament of the formula I and, if appropriate, carriers and / or auxiliaries. The invention furthermore relates to the use of the peptide compounds and / or their physiologically acceptable salts according to the claims and the description for the manufacture of a medicament for combating diseases which are based, directly or indirectly, on expression of the α _v β ₆ integrin receptor, in particular thus in the case of pathologically angiogenic diseases, thromboses, heart attacks, coronary heart diseases, arteriosclerosis, tumors, osteoporosis, inflammation, infections and for influencing wound healing processes. The pharmaceuticals according to the invention or pharmaceutical preparations containing them can be used in human or veterinary medicine. Suitable carrier substances are organic or inorganic substances which are suitable for enteral (for example oral), parenteral, topical application or for application in the form of an inhalation spray and which do not react with the new compounds. ren, for example water, vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols, glycene triacetate, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc, petroleum jelly. Tablets, pills, dragees, capsules, powders, granules, syrups, juices or drops are used in particular for oral use, supositoses for rectal use, solutions, preferably oily or aqueous solutions, and also suspensions, emulsions or implants for parenteral use , for topical application of ointments, creams or powder. The new compounds can also be lyophilized and the lyophilizates obtained used, for example, for the production of injectables. The specified preparations can be sterilized and / or auxiliaries such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffer substances, coloring, flavoring and / or one or more further active substances included, e.g. B. one or more vitamins. For the application as an inhalation spray, sprays can be used which contain the active ingredient 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, it being possible for one or more additional physiologically acceptable solvents to be present, for. B. ethanol. Inhalation solutions can be administered using standard inhalers.

The substances according to the invention can generally be administered analogously to other known, commercially available peptides (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, gender, on the diet, on the time and route of administration, on which Elimination rate, drug combination and severity of the disease to which the therapy applies. Parenteral administration is preferred.

Furthermore, the new compounds of formula I in the analytical

Biology and molecular biology are used. The new compounds of formula I, wherein X is a -CONH-, -COO-, -NH-C (= S) -NH-, -NH-C (= 0) -NH-, -S0 ₂ NH- or -NHCO- linked fluorescent dye residue means can be used as diagnostic markers in FACS (Fluorescence Activated Cell cultivar technique and fluorescence microscopy.

The use of labeled compounds in fluorescence microscopy is e.g. B. described by Y.-L. Wang and D. L. Taylor in "Fluorescence Microscopy of Living Cells in Culture, Part A + B, Academic Press, Inc.

1989 ".

The new compounds according to the invention can also be used as integrin ligands for the preparation of columns for affinity chromatography for the purification of integrins. The complex of an avidin-derivatized carrier material, e.g. Sepharose and the new compounds are formed by methods known per se (e.g. E.A. Bayer and M. Wilchek in Methods of Biochemical Analysis Vol 26 The Use of the Avidin-Biotin Complex as a Tool in Molecular Biology). Suitable polymeric carrier materials are the polymeric solid phases known per se in peptide chemistry with preferably hydrophilic properties, for example cross-linked poly sugars such as cellulose,

Sepharose or Sephadex ^R , acrylamides, polymer based on polyethylene glycol or tentacle polymers ^R.

Finally, the invention also comprises recombinant DNA sequences which contain sections which code for peptide regions which have the peptide structural motifs according to the invention. Such DNA can be transferred to cells by particles, as described in Ch. Andree et al. Proc. 91, 12188-12192 (1994), or the transfer to cells can be increased by other means such as liposomes (Al Aronsohn and JA Hughes J. Drug Targeting, 5, 163-169 (1997)).

The transfer of such DNA could thus be used in yeast, by means of bacculoviruses or in mammalian cells for the production of the peptide substances of this invention.

If an animal or human organism is infected with such a recombinant DNA, the peptides according to the invention ultimately formed by the infected cells themselves can bind directly to the α _v ß ₆ integrin receptor, for example of tumor cells, and block it.

Corresponding recombinant DNA, which can be provided by known and customary techniques, can also be present, for example, in the form of virus DNA which contains sections which code for the virus coat protein. By infection of a host organism with such recombinant, preferably non-pathogenic viruses, host cells which express the integrin α _v ß ₆ can be attacked (targeting).

Suitable viruses are, for example, adenovirus types which have been used several times as vectors for foreign genes in mammalian cells. A number of traits make them good candidates for gene therapy, such as S.J. Watkins et al. Gene Therapy 4, 1004-1012 (1997) can be found (see also J. Engelhardt et al. Hum. Gene Ther. 4, 759-769 (1993)).

As in A. Fasbender et al. J. Clin. 102, 184-193 (1998) is a common problem in viral and non-viral gene therapy Vectors the limited efficiency of gene transfer. With the additional ligand sequence for α _v β ₆ integrin described above in the coat protein of the adenoviruses, an improvement in the transfer, for example of cystic, can be achieved

Fibrosis transmembrane conductance regulator (CFTR) cDNA can be achieved.

Similar to the work of T. Tanaka et al. Cancer Research 58, 3362-3369 (1998), instead of the DNA for angiostatin, the DNA for the sequences of this invention can also be used for cell transfections by means of retroviral or adenoviral vectors.

The peptides according to the invention can also be used within a liposome complex made of lipid / peptide / DNA for a transfection of cell cultures together with a liposome complex consisting of lipid / DNA (without peptide) for use in gene therapy in humans. The production of a liposome complex from lipid / DNA peptide is described, for example, by Hart SL, et al 1998: Lipid-Mediated Enhancement of Transfection by a Non-Viral Integrin-Targeting Vector. Human Gene Therapy 9, 575-585. A liposome complex of lipid / pepid / DNA can be prepared, for example, from the following stock solutions: 1 μg / μl lipofectin (equimolar mixture of DOTMA (= N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethyl- ammonium chloride) and DOPE (dioleyl phosphatidylethanolamine)), 10 μg / ml plasmid DNA and 100 μg / ml peptide. For this purpose, both DNA and peptide are dissolved in cell culture medium. The liposome complex is produced by mixing the three components in a specific weight ratio (lipid: DNA: peptide, for example 0.75: 1: 4). Liposome DNA complexes for gene therapy in humans have already been described (Caplen NJ, et al 1995: Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis Nature Medicine 1, 39-46). The invention thus also relates to the use of appropriately modified recombinant DNA from gene-releasing systems, in particular virus DNA, for combating diseases which are based directly or indirectly on expression of α _v β ₆ integrin receptors, in particular in the case of pathologically angiogenic diseases,

Thrombosis, heart attack, coronary heart disease, arteriosclerosis,

Tumors, osteoporosis, inflammation, infections and to influence wound healing processes.

All temperatures above and below are given in ° C.

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

systems:

Column 5 μm LichroSpher 60 RP-Select B (250-4), with a 50 minute

Gradients from 0 to 80% 2-propanol in water / 0.3% trifluoroacetic acid, at 1 ml / min flow and detection at 215 nm.

Mass spectrometry (MS): El (electron impact ionization) M ⁺

FAB (Fast Atom Bombardment) (M + H) ⁺

example 1

Production and purification of peptides according to the invention:

In principle, the production and purification was 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 from Haubner et al. (J. Am. Chem. Soc. 118, 1996, 17703).

Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NH ₂

2 g of amino-xanthenyloxy-polystyrene resin (Novabiochem 0.37 mmol / g) were successively 2 x with 0.50 g each in a double coupling technique TBTU, 0.53 ml of ethyldiisopropylamine and Fmoc-amino acid in DMF in a commercial synthesizer and a typical procedure (device and manual Milligen 9050 PepSynthesizer ™, 1987), each for 30 minutes, subjected to a coupling step. Washing steps were carried out in DMF for 10 minutes, splitting steps in piperidine / DMF (1: 4 vol) for 5

Minutes, N-terminal acetylations (capping) were carried out with acetic anhydride / pyridine / DMF (2: 3: 15 vol) for 15 minutes. There came the amino acids Fmoc-Arg (Pmc), then Fmoc-Leu, then Fmoc-Ser (But), then Fmoc-D-Asp (OBut), then Fmoc-Leu, then Fmoc-Asp (OBut), then Fmoc- Gly and finally Fmoc-Arg (Pmc) to

Commitment. After the Fmoc protective group had been removed from the Fmoc-Arg (Pmc) -Gly-Asp (OBut) -Leu-D-Asp (OBut) -Ser (But) -Leu-Arg (Pmc) - aminoxanthenyloxy polystyrene resin, acetylation was carried out again. After washing with DMF and isopropanol and subsequent drying in vacuo at room temperature, 2.8 g of Ac-Arg (Pmc) -Gly-Asp (OBut) -Leu-D-

Asp (OBut) -Ser (But) -Leu-Arg (Pmc) aminoxanthenyloxy polystyrene resin obtained.

Treatment of this peptidyl resin with 20 ml of trifluoroacetic acid / water / TIS (94: 3: 3 vol) for 2 hours at room temperature, filtration, concentration in vacuo and trituration with diethyl ether gave 0.47 g

Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NH ₂ (code EMD 272974).

The product was purified by RP-HPLC on Lichrosorb RP 18 (250 - 25, 7 μm, Merck KGaA) in 0.3% TFA with a gradient from 4% to 24% 2-propanol in one hour at 10 ml / min and assessment of the eluate using a UV flow photometer at 215 and 254 nm. 168 mg of product were obtained; Rt 15.5 min; FAB 973. The following products were manufactured analogously:

Nomenclature for amino acids according to Eur.J.Biochem. 138, 9-37 (1984) small letter = D-amino acid

Example 2: c _v esse / fibronectin receptor binding test:

The peptides according to the invention which were produced were bound to the immobilized α _v β ₆ receptor in solution together with fibronectin having a competitive effect and the Q value was determined as a measure of the selectivity of the binding of the peptide to be tested to α _v β β. The Q value is calculated from the quotient of the IC ₅ o values of test peptide and a standard. The linear Ac-RTDLDSLR-NH ₂ (code EMD 271293) was used as the standard (lit./patent, see Pytela et al. Science 231, 1559, (1986)). The binding test was carried out in detail as follows: The immobilization of soluble α _v β ₆ receptor on microtiter plates was carried out by diluting the protein solution in TBS ++ and then incubating overnight at 4 ° C. (100 μl / well). Unspecific binding sites were blocked by incubation (2 h, 37 ° C) with 3% (w / v) BSA in TBS ++ (200 μl / well). Excess BSA was removed by washing three times with TBSA ++. Peptides were serially (1:10) diluted in TBSA ++ and incubated together with biotinylated fibronectin (2 μg / ml) with the immobilized integrin (50 μl peptide + 50 μl ligand per well; 2 h; 37 ° C). Unbound fibronectin and peptides were removed by washing three times with TBSA ++. The bound fibronectin was detected by incubation (1 h; 37 ° C) with an alkaline phosphatase-coupled anti-biotin antibody (Biorad) (1: 20000 in TBSA ++; 100 ul / well). After washing three times with TBSA ++, the colohmetric detection was carried out by incubation (10-15 min; 25 ° C, in the dark) with substrate solution (5 mg nitrophenyl phosphate, 1 ml ethanolamine, 4 ml H ₂ 0; 100 μl / well). The enzyme reaction was stopped by adding 0.4 M NaOH (100 ul / well). The color intensity was determined at 405 nm in the ELISA measuring device and compared to the zero value. Wells that were not coated with receptor served as zero value. Ac-RTDLDSLR-NH _{2 was} used as the standard. The IC ₅ o values for the peptides tested were read off from a graph and determines together with the _IC50 of the standard peptide, the Q value of the peptide according to the invention.

Q value = IC ₅₀ test peptide / IC ₅₀ standard

Q values from repeated tests were averaged.

The results of the test described are summarized in Table 1 below:

Table 1

Results of the α ßε / fibronectin receptor binding test

The following examples relate to pharmaceutical preparations:

Example A: Injection glasses

A solution of 100 g of Ac-RGDLdSLR-NH ₂ and 5 g of disodium hydrogenphosphate is adjusted to pH 6.5 in 3 l of double-distilled water with 2N hydrochloric acid, sterile filtered, filled into injection glasses, lyophilized and sterile sealed under sterile conditions. Each injection jar contains 5 mg of active ingredient.

Example B: Suppositories A mixture of 20 g Ac-RGDLdSLR-NH _{2 is} melted with 100 g soy lecithin and 1400 g 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 Ac-RGDLdSLR-NH ₂ , 9.38 g 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 double distilled water. It is adjusted to pH 6.8, made up to 1 I and sterilized by irradiation. This solution can take the form of

Eye drops can be used.

Example D: ointment

500 mg of Ac-RGDLdSLR-NH _{2 are} mixed with 99.5 g of petroleum jelly under aseptic conditions.

Example E: tablets

A mixture of 1 kg of Ac-RGDLdSLR-NH ₂ , 4 kg of lactose, 1, 2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearate is compressed into tablets in a conventional manner 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: capsules

2 kg of Ac-RGDLdSLR-NH ₂ are filled into hard gelatin capsules in a conventional manner, so that each capsule contains 20 mg of the active ingredient.

Example H: ampoules A solution of 1 kg of Ac-RGDLdSLR-NH ₂ 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 Ac-RGDLdSLR-NH _{2 are dissolved} in 10 I of isotonic NaCI 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. A spray

(about 0.1 ml) corresponds to a dose of about 0.14 mg.

Claims

claims

1. Peptide compounds of the formula I.

Ac-Arg-X -Asp-X ² -X ³ -X ⁴ -X ⁵ -X ⁶ -NH ₂ I

wherein

Ac Acetyl,

X ¹ Ser, Gly, Thr, Asp, Arg, Val, Tyr, His or Ala, X ² Leu, Ile, Nie, Val or Phe,

X ³ Asp, Glu, Lys, Phe, Aib, Nal, Gly, Ala, Bgl or Phg,

X ⁴ Gly, Ala, Ser, ß-Ala or ω-Abu,

X ⁵ Leu, Ile, Nie, Val or Phe,

X ⁶ Arg, Har, Lys, Leu, Orn, Phe, Ala, Tyr, Gly, Ser or Asp

mean,

where the amino acids mentioned can also be derivatized, the amino acid residues are linked in peptide fashion via the α-amino and α-carboxy groups, the D- and the L-forms of the optically active amino acid residues are included, as well as their salts, and where Ac- Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH _{2 is} excluded.

Peptide compounds according to claim 1 selected from the group

Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Gly-Asp-Leu-Asp-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Ser-Asp-Leu-Asp-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Asp-Asp-Leu-Asp-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Ala-NH _2l Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-D-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Arg-NH ₂ ,

Ac-D-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-D-Ala-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Aib-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-D-Nal-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Gly-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Ile-D-Asp-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Asp-D-Ser-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Asp-Ala-Leu-Arg-NH ₂ , Ac-Arg-Thr-Asp-Leu-Asp-Gly-Leu-Arg-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Har-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Lys-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-D-Asp-D-Ser-Leu-Arg-NH _2l

Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Ala-NH ₂ ,

Ac-Arg-Thr-Asp-Leu-D-Asp-Gly-Leu-Arg-NH ₂ ,

and their physiologically acceptable salts.

5 3. Peptide compounds of formula I according to claim 1 and the

Compounds according to claim 2 and their physiologically acceptable salts as medicaments.

_Q 4. Medicament according to claim 3 as an inhibitor for combating

Diseases based on expression and pathological function of α _v β β integrin receptors.

5. Medicament according to claim 4 for combating thrombosis, 5 heart attack, coronary heart disease, arteriosclerosis, tumors, Osteoporosis, fibrosis, inflammation, infections, psoriasis and for influencing wound healing processes.

6. Pharmaceutical preparation containing at least one medicament according to one of claims 4 and 5 and optionally

Carriers and / or auxiliary substances and optionally other active substances.

7. Use of peptide compounds according to claims 1 and 2 and / or their physiologically acceptable salts for

Production of a medicament for combating diseases which are based on the expression and pathological function of α _v β _δ integrin receptors.

Use according to claim 7 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.