SK115696A3 - Biotinylated peptide derivative, preparation method and use thereof and pharmaceutical composition containing them - Google Patents

Abstract

Peptide derivatives from the group comprising Bit-Gly-Gly-Gly-Arg-Gly-Asp-Ser-Pro-Lys-OH, Bit-Gly-Gly-Gly-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-OH, cyclo-(Arg-Gly-Asp-D-Phe-Lys(N'epsilon'-Bit)), cyclo-(Arg-Gly-Asp-D-Phe-Lys(N'epsilon'-Aha)), cyclo-(Arg-Gly-Asp-D-Phe-Lys(N'epsilon'-Bit-Aha)-Gly), cyclo-(Arg-Gly-Asp-D-Phe-Val-Lys(N'epsilon'-Bit-Aha)), cyclo-(Arg-Gly-Asp-D-Phe-N-Me-Lys(N'epsilon'-Bit-Aha)), cyclo-(Arg-Gly-Asp-D-Phe-Lys(N'epsilon'-Bit-Aha)), cyclo-(Arg-Gly-Asp-D-Phe-Lys(BOC-Aha)) and pharmaceutically acceptable salts thereof, which are as integrin inhibitors usable for production of pharmaceutical compositions and for other purposes in biology and molecular biology.

Description

A biotinylated peptide derivative, a process for its preparation and use, and a pharmaceutical composition containing the same

Technical field

The invention relates to a biotinylated peptide derivative that is well tolerated and has valuable pharmacological properties since it is an integrin inhibitor and is thus suitable for the manufacture of pharmaceutical compositions. The invention also relates to a process for its preparation and to a pharmaceutical composition containing it.

BACKGROUND OF THE INVENTION

Similar biotinylated peptide derivatives are described, for example, in WO 9415956 (biotinylated endothelin receptor antagonists), in WO 9413313 (biotinylated LHRH-antagonists) and in WO 9418325 (biotinylated necrosis factors).

Biotinylation of peptides during solid phase synthesis. on a resin to improve purification, described by Lobl et al., Anal. Biochem., 170, p. 502, 1988].

Similar cyclic and linear peptide compounds are known from DE 43 10643, DE 43 36 75θ, EP 0 406 428 and WO 89/05150.

SUMMARY OF THE INVENTION It is an object of the invention to provide novel compounds having valuable properties, in particular compounds and their physiologically acceptable salts, which can be used for the manufacture of medicaments.

SUMMARY OF THE INVENTION

The present invention provides a novel peptide of the general formula of a derivative of a biotinyl I derivative

where

Q is absent or is -NH- (CH 2) _n -CO- or -NH- (CH ₂ ) _n -NH-.

about

R represents the group X-Arg-Gly-Asp-Y, A-cys- (R9-BU or cyclo- (Arg-Gly-Asp-Z), wherein Z in the side chain is bound to Qja to biotin if Q ' a mistake,

X and Y are each independently an amino acid residue or a di-, tri-, tetra- or pentapeptide residue, wherein the amino acids are independently selected from the group consisting of Ala, Asn, Asp, Arg, Cys, Gln, Glu, Gly, 4Hal -Phe, His, homo-Phe, Ile, Leu, Lys, Met, No, Phe, Phg, Pro, Ser, Thr, Trp ,. Tyr and Val, these acids being optionally derivatized,

A is absent or is an Asp or peptide fragment selected from the group consisting of

Lys-Thr-Ala-Asp, Lys-Thr-Ala-Asp, Lys-Thr-Ala-Asp, Lys-Thr-Ala-Asp, Lys-Thr-Ala-Asp, Arg-Thr-Ala-Asp, Ser-Ala-Asp,

Gl-Ser-Ala-Asp, Gl-Ser-Ala-Asp, Gl-Lys-Thr-Ala-Asp, Gl-Lys-Thr-Ala-Asp, Ile-Ser-Ala-Gly, Arg-Ser-Ala-Gly, Cys-Asn-Gly-Lys-Thr-Ala-Asp, Tyr-Cys-Asn-Gly-Lys-Thr-Ala-Asp,

Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Ala-Asp, Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Ala-Asp, Gly-Lys-Thr-Cys (Trt) -Asp Met-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Ala-Asp, Asp-Met-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Ala-Asp;

B is absent or is OH, Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Orn, Phe, 4-Hal-Phe, Pro, Ser, Thr, Trp, Tyr and Val, or a nitrogen-methylated derivative of these amino acid residues, or is a fragment of a peptide selected from the group consisting of Pro-Arg, Pro-Arg-Asn,

Pro-Arg-Asn-Pro, Pro-Arg-Asn-Pro-His, Pro-Arg-Asn-Pro-His-Lys, Pro-Arg-Asn-Pro-His-Lys-i

Pro-Arg-Asn-Pro-His-Lys-Gly-Pro, Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala, Pro-Arg-Asn-Pro-Hi-Lys-G 1y -Pro-A1a-Thr, 19 'ar wherein when R is A-Cys (R JBU), only one of the groups A or B may be absent, and R ^ is hydrogen, C 1 -C 6 alkyl alkyl,

Trt, Dpm, or Bzl,

U OH, OR ⁹ , NH 2, NHR ⁹ or N (R ⁹ J 2,

Z is each independently an amino acid residue or a di-, tri- or tetrapeptide residue, wherein the amino acids are independently selected from the group consisting of Ala, Asn, Asp, Arg, Cys, Gln, Glu, Gly, His, Ile, Leu , Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val or M, wherein these amino acids are optionally derivatized, and the amino acid residues are peptide-linked to one another via

- 4 d-amino and t-carboxyl groups and M is always present,

M is NH (R ⁸⁾ -CH (R ³⁾ -COOH,

R ³ -R ⁵ -R ⁴ , R ⁶ -R ⁴ , -R ⁷ -R ⁴ ,

R ⁴ OH, NH _2, SH or COOH,

K /

R (C 1 -C 6) alkylene, R (C 7 -C 14) alkylenephenyl,

R 'is an alkylenephenylalkylene group having 8 to 15 carbon atoms,

R ⁸ is hydrogen, C 1 -C 6 alkyl.

or C 7 -C 12 alkylenephenyl, C 1 -C 6 alkyl, C 1 -C 6 alkyl, Hal, fluorine, chlorine, bromine or iodine; and n, 1, 2, 3, 4, 6, 7, 8, 9, or 10 and when the derivative comprises optically active amino acid residues and optically active amino acid derivatives, the D and L forms of the derivative and its pharmaceutically acceptable salts.

Indeed, it has been found that the novel compounds of the formula I and their physiologically acceptable salts possess valuable pharmacological properties with good tolerability. They act especially as integrin inhibitors, which inhibit in particular the interactions of _the d ~, too "or N -integríno'vých receptors with ligands, such as the binding of fibrinogen to the receptor N -integrínový. In particular, the compounds of the formula I have a particularly high activity in the case of integrins, <χ _ν β _τ oC _at ^ ₆ ^ q q.

This action can be demonstrated by the method of J. W. Smith et al. / J. Biol. Chem. 265, p. 12267-12271, 1990].

The dependence of angiogenesis on the interaction between vascular integrins and extracellular matrix proteins has been described by P. C. Brooks, R. A.. Clark and D. A. Cheresh / Science 264, p. 569 to 571].

The possibility of inhibiting the interaction and thereby inducing apoptosis / programmed cell death / angiogenic vascular cells by cyclic peptide has been described by P.C. Brooks, A.M. Montgomery, M.R. Rosenfeld, R.A. Reisfeld, T.-Hu, G. Klier, and D.A. Cheresh / Cell 79, p. 1157-1164, 1994].

Compounds of formula I that block the interaction of integrin receptors and ligands such as fibrinogen at the fibrinogen receptor / glycoprotein IIb / IIIa7 prevent, as GPIIb / IIIa-antagonists, the spread of tumor cells via matestases. This observation is supported by the following observations:

The dissemination of tumor cells from one local tumor to the vascular system is effected by the binding of microaggregates / micro-thrombi / interaction of tumor cells and platelets. Tumor cells are shielded. protection in microaggregates and not recognized by immune cells.

The microaggregates can be firmly attached to the vessel walls, thereby facilitating further penetration of tumor cells into the tissue. Since the binding of microthrombi by fibrinogen binding to fibrinogen receptors is mediated on activated platelets, GPIIa / IIIb-antagonists can be considered to be an effective brake on metastasis.

The compounds of the formula I according to the invention can be used

- 6 to be used as active substances in human and veterinary medicine, in particular in the prophylaxis and / or treatment of thromboses, myocardial infarction, arteriosclerosis, inflammation, apoplexy, angina pectoris, cancer, osteolytic diseases such as osteoporosis, pathological angiogenic diseases such as angiogenic diseases for example inflammation, ophthalmological diseases, diabetic retinopathy, muscle degeneration, myopia, ocular histoplasmosis, rheumatoid arthritis, osteoarthritis, rubeotic glaucoma, tumor colitis, Crohn's disease, atherosclerosis, psoriasis, restenosis after angioplasty, bacterial infection, viral infection, viral infection, viral infection, viral infection renal failure and wound healing to support the healing process.

The compounds of formula (I) may be used as antimicrobial active agents in operations using biological materials, implants, catheters or heart stimulators. They act antispetically. The efficacy of antimicrobial activity can be demonstrated as described by P. Valentin Weigund et al. Infection and Immunity, p. 2851-2855, 1988].

Since the compounds of formula I are inhibitors of fibrinogen binding and thus fibrinogen receptor ligands on platelets, they can be used as diagnostics for the detection and localization of thrombi in the vesicular system in vivo, since the biotinyl residue is a residue detectable by ultraviolet rays.

The compounds of formula (I) may also be used as active adjuvants in the study of platelet metabolism at various activation stages or in the study of intracellular signaling mechanisms as fibrinogen binding inhibitors:. fibrinogen receptor antagonists. The detectable biotin label unit, after binding to the receptor, allows the mechanisms to be investigated.

The amino acid abbreviations used have the following meanings: Abu 4-Aminobutyric acid I see 6-amino-hexanoic acid, 6-aminocaproic acid, Ala alanine same time asparagine Asp Arg Cys Dab arginine, aspartic acid, cysteine ^and 2,4 -diaminomaslová Dap gin 2, 3-diaminopropionic acid glutamine Glp Glu Gly His glutamic acid glycine histidine

homo-Phe homo-phenylalanine

Ile isoleucine Leu leucine Lys Met lysine methionine Not norleucine Orn ornithine Phe phenylalanine PHG phenylglycine

4-Hal-Phe 4-halo-phenylalanine

, Pro proline Ser Thr serine threonine Trp Tyr wall tryptophan tyrosine valine ^H H tn bit f H ^H 0

It also means

BOC t, -butoxykarbony1 CBZ benzyloxycarbonyl DCC1 dicyclohexylcarbodiimide DMF et dimethylformamide ethyl Fmoc 9-fluorenylmethoxycarbonyl HOBt M e 1-hydroxybenzotriazole methyl MBHA 4-methylbenzhydrylamine Mtr 4-methoxy-2,3,6-trimethylphenylsulfonyl OBu tert. -buty1ester OMe OEt methyl ester ethyl ester POA TFA fenoxyacetvl trifluoroacetic acid Trt trityl (three phenylmethyl)

If the aforementioned acids can exist in several enantiomeric forms, the invention always encompasses all these forms and mixtures thereof (e.g. DL-forms), for example as moieties of a compound of formula I. In addition, they may have amino acids, for example as moieties of compounds of formula I , appropriate protecting groups known per se.

The invention also relates to prodrug derivatives, i.e. compounds of formula I which are altered by alkyl or acyl groups, carbohydrates or oligopeptides, which are rapidly cleaved in the body to release the active compound of formula I according to the invention.

The process for the preparation of the compounds of the formula I and their salts consists in reacting and / or reacting a compound of the formula II

HQR ¹

II wherein Q and R are as defined above, by acylation reaction with a compound of formula III

where it means

L is a chlorine, bromine, iodine atom or a free or reactive functionally modified hydroxyl group, or b) reacting a compound of formula IV

HR ¹

wherein R ¹ is as defined above, by acylation reaction with a compound of formula V ^H H T 0 / x> ° (v) or • · 5 A H ^{H H} 0

where Q is as defined above and where is

L is a hydrogen, chlorine, bromine, iodine or free or reactive functionally modified hydroxyl group, or c) the compound of formula I is released from its functional derivative by treatment with a solvolyzing or hydrogenolyzing agent, and / or a basic or acidic compound of formula I by treatment with acid or base into its salt.

The groups Q, R ¹ and L in the formulas I, II and III are as defined above unless otherwise stated.

In formula I, the alkyl group is preferably methyl, ethyl, isoprolyl and tert-butyl.

The alkylene is preferably methylene, ethylene, propylene, butylene, pentylene or hexylene.

Alkylenephenyl. is preferably benzyl or phenethyl.

The alkylene-phenylalkylene is preferably 4-methylene-benzyl or 4-ethylene-benzyl.

-R 1 -R 1 is preferably 2-, 3- or 4-hydroxybenzyl, 2-, 3- or 4-aminobenzyl, 2-, 3- or 4-mercaptobenzyl, 2-, 3- or 4-carboxybenzyl, further preferably 2-,. 3- or 4-hydroxyphenethyl, 2-, 3- or 4-aminophenethyl, 2-, 3- or 4-mercaptophenethyl, 2-, 3- or 4-carboxyphenethyl.

The symbol Q is preferably a residue of 6-aminohexanoic acid (6-aminocaproic acid) or is absent.

Preferably M is Dap, Ser, Cys, Asp, D-Asp, Dab > homoserine, homocysteine, Glu, D-Glu, Thr, Orn, Lys, D-Lys, 4 aminomethyl-Phe or 4-aminomethyl-D-Phe .

X is preferably Ala, Asn, Asp, Arg,

Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,

Thr, Trp, Tyr or Val and further preferably Lys-Gly, Lys-Ala, Lys-g-Ala, Tyr-Gly, Tyr-Ala, Tyr-p-Ala, Phe-Gly, Phe-Ala, Phe-p -Ala, Tyr-Gly-Gly, Phe-Gly-Gly, Lys-Gly-Gly, Tyr-Gly-Ala, Phe-Gly-Ala, Lys-Gly-Ala, Arg-Gly-Asp, Lys-Gly-Gly -Gly, Tyr-Gly-Gly-Gly, Phe-Gly-Gly-Gly, Lys-Gly-Gly-Ala, Tyr-Gly-Gly-Ala, Phe-Gly-Gly-Ala, Lys-Gly-Gly-p -Ala, Tyr-Gly-Gly-p-Ala, Phe-Gly-Gly-p-Ala, and "Lys-Gly-Gly-Gly-Gly, Tyr-Gly-Gly-Gly-Gly, Phe-Gly" -Gly-Gly-Gly, Lys-Gly-Gly-Al-Gly, Tyr-Gly-Gly-A (a-Gly, Phe-Gly-Gly-Ala-Gly, Lys-Gly-Gly-p-Ala -Gly, Tyr-Gly-Gly-3-Al and -Gly or Phe-Gly-Gly-p-Ala-Gly.

Y is preferably Ala, Asn, Asp, Arg,

Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,

Thr, Trp, Tyr or Val and further preferably Tyr-Ala, Tyr-Asn, Tyr-Asp, Tyr-Arg, Tyr-Cys, Tyr-Gln, Tyr-Glu, Tyr-Gly, Tyr-His, Tyr-lle, Tyr-Leu, Tyr-Lys, Tyr-Met, Tyr-Ph, Tyr-Pro, Tyr-Ser, Tyr-Thr, Tyr-Trp, Tyr-Tyr.Tyr-Val, Phe-Ala, Phe-Asn, Phe- Phe-Arg, Phe-Arg, Phe-Cys, Phe-GIn, Phe-Glu, Phe-Gly, Phe-His, Phe-Ile, Phe-Leu, Phe-Lys, Phe-Met, Phe-Phe, Phe-Pro, Phe-Ser, Phe-Thr, Phe-Trp

Php-Tyr, Php-Val, Trp-Ala, Trp-Asn, Trp-Asp, Trp-Arg, Trp-Cys, Trp-Gln, Trp-Glu, Trp-Gly, Trp-His, Trp-lle, Trp Trp-Lys, Trp-Met, Trp-Phe, Trp-Pro, Trp-Ser, Trp-Thr, Trp-Trp, Trp-Tyr, Trp-Val, Asp-Ala, Asp-Asn, Asp-Asp Asp-Arg, Asp-Cys, Asp-Gln, Asp-Glu, Asp-Gly, Asp-His, Asp-Ile, Asp-Leu, Asp-Lys, Asp-Met, Asp-Phe, Asp-Pro, Asp-Asp -Ser, Asp-Thr, Asp-Trp, Asp-Tyr, Asp-Val, Ser-Pro-Lys, Tyr-Pro-Lys, Phe-Pro-Lys, Trp-Pro-Lys, Asp-Pro-Lys, Ser-Gly-Lys, Phr-Gly-Lys, Php-Gly-Lys, Php-Gly-Lys, Asp-Gly-Lys, Phr-Ala-Lys, Php-Ala-Lys, Trp- Ala-Lys, Asp-Ala-Lys, Ser-Pro-Ala, Ser-Leu-Lys, Tyr-Leu-Lys, Phe-Leu-Lys, Trp-Leu-Lys, Asp-Leu-Lys, Ser-lle- Lys, Tyr-lle-Lys, Php-lle-Lys, Trp-lle-Lys, Asp-He-Lys, Ser-Pro-Ala-Ser, Tyr-Pro-Ala-Ser, Phe-Pro-Ala-Ser, Ser-Gly-Ala-Ser, Ser-Gly-Ala-Ser, Ser-Gly-Ala-Ser, Ser-Gly-Ala-Ser, Ser-Gly-Ala-Ser, Asp- Gly-Ala-Ser, Sec-Ala-Ala-Ser, Phe-Ala-Ala-Ser, Trp-Ala-Ala-Ser,

Cys,

Ser.

Ser-Val-Ala-Ser, Ser-Val-Ala-Ser, Asp-Val-Ala-Ser, Asp-Val-Ala-Ser, Asp-Val-Ala-Ser, Ser -Leu-Ala-Ser, Phe-Leu-Ala-Ser, Asp-Leu-Ala-Ser, Ser-lle-Ala-Ser, Tyr-lle -Ala-Ser, Phe-lle-Ala-Ser, Trp-lle-Ala-Ser, Asp-lle-Ala-Ser, also Ser-Pro-Ala-Ser-Ser.Tyr-Pro-Ala-Ser Ser, Phe-Pro-Ala-Ser-Ser, Trp-Pro-Ala-Ser-Ser, Asp-Pro-Ala-Ser-Ser. Ser-Gly-Ala-Ser-Ser, Phr-Gly-Ala-Ser-Ser, Asp-Gly-Ala-Ser-Ser, Asp-Gly-Ala-Ser-Ser, Ser-Ala-Al-Ser-Ser, Phr-Ala-Ala-Ser-Ser, Asp-Ala-Ala-Ser-Ser, Asp-Ala-Ala-Ser-Ser, Ser-Val-Ala-Ser-Ser, Phr-Val-Ala-Ser-Ser, Asp-Val-Ala-Ser-Ser, Asp-Val-Ala-Ser-Ser, Ser-Leu-Ala-Ser-Ser, Phr-Leu-Ala-Ser-Ser, Asp-Leu-Ala-Ser-Ser, Asp-Leu-Ala-Ser-Ser, Ser-lle-Ala-Ser-Ser, Tyr-lle-Ala-Ser-Ser, Phe-lle-Ala-Ser-Ser, Trp-lle-Ala-Ser-Ser or Asp-lle-Al-Ser- Ser.

The amino acids and amino acid residues indicated by X, Y and Z may be in the form of derivatives, with N-methyl, N-ethyl, N-propyl, N-benzyl or Coc-methyl derivatives being preferred. Further preferred derivatives are Asp and Glu, in particular methyl, ethyl, propyl, butyl, tert-butyl, neopentyl or N-benzyl ester derivatives of the side chain carboxy groups, furthermore an Arg derivative substituted on -NH-C (= NH). The -NH ₂ group is an acetyl, benzoyl, methoxycarbonyl or ethoxycarbonyl radical.

In addition, the amino acids and their residues, referred to as X and Y, may have appropriate protecting groups per se.

The symbol Z is preferably M, further preferably D-Phe-M, D-Trp-M, D-Tyr-M, D-Phe-Lys, D-Phe-D-Lys, D-Trp-Lys, D. -Trp-D-Lys, D-Tyr-Lys, D-Tyr-D-Lys, D-Phe-Orn, D-Phe-Dab, Q-Phe-Dap, D-Phe-D-Orn, D-Phe -D-Dab, D-Phe-D-Dap, D-Phe-4-aminomethyl-Phe, D-Phe-4-aminomethyl-D-Phe, D-Trp-4-aminomethyl-Phe, D-Trp-4- aminomethyl D-Phe, D-Tyr-4-aminomethyl-Phe, D-Tyr-4-aminomethyl-D-Phe, D-Phe-Asp, D-Phe-D-Asp, D-Trp-Asp, D-Trp-D- Asp-D-Tyr-Asp, D-Tyr-D-Asp, D-Phe-Cys, D-Phe-D-Cys, D-Trp-Cys, D-Trp-D-Cys, D-Tyr-Cys, D-Tyr-D-Cys, D-Lys, D-Lys, D-Lys, D-Lys, D-Lys, D-Lys, D-Lys, D-Lys, D-Lys, D-Lys, D-Lys, Tyr-Om, Tyr-Dab, Tyr-Dap, Phe-4-aminomethyl-D-Phe, Trp-4-aminomethyl-D-Phe, Tyr-4-aminomethyl-D-Phe, Phe-D-Asp, Trp-D-Asp, Tyr-D-Asp, Ph-D-Cys, D-Phe-Lys-Gly, D-Php-Lys-Gly, D-Trp-Lys-Gly, D-Trp- M-Gly, D-Tyr-Lys-Gly, D-Tyr-M-Gly, D-Phe-Val-Lys, D-Phe-Gly-Lys, D-Phe-Ala-Lys, D-Phe-lle- Lys, D-Phe-Leu-Lys, D-Trp-Val-Lys, D-Trp-Gly-Lys, D-Trp-Ala-Lys, D-Trp-Lle-Lys, D-Trp-Leu -Lys, D-Tyr-Val-Lys, D-Tyr-Gly-Lys, D-Tyr-Ala-Lys, D-Tyr-lle-Lys, D-Tyr-Leu-Lys, and also M-Pro-Ala- Ser-Ser.

The compounds of formula (I) may have one or more chiral centers and therefore may be in different stereoisomeric forms, all of which are included in formula (I).

In particular, the invention relates to compounds of the formula I in which at least one of the abovementioned groups has at least one of the aforementioned preferred meanings. Some preferred groups of compounds may be represented by the following formulas (Ia) to (Ia), which belong to formula (I), wherein the unspecified groups are as defined in formula (I) but are absent from formula (Ia) and (Q).

R ¹ X-Arg-Gly-Asp-Y,

Ib Q -NH- (CH ₂ ) ₅ -CO- a

R ¹ X-Arg-Gly-Asp-Y.

j _c q -NH- (CH 2) s "CO 4" ^and r ¹ cyclo- (Arg-Gly-Asp-Z)

Id Q -NH- (CH 2) s -CO · ^a .

r ¹ Cyclo- (Arg-Gly-Asp-M) le Q -NH- (CH ₂ ) ₅ -CO- ar ¹ A-Cys (R ² ) -B 1 Q -NH- (CH 2) n "CO" »r ¹ X-Arg-Gly-Asp-Y n 1,2,3,4,5 or 6th

The compounds of formula (I) and the starting materials for their preparation are prepared by methods known per se as described in the literature (for example, in standard publications such as Houben-Weyl, Methoden der organischen Chemie, Georg-Thieme Verlag, Stuttgart). reaction conditions that are known and suitable for the above reactions. It is also possible to use variants known per se, not described here in greater detail.

Alternatively, the starting materials can also be formed in situ by not isolating from the reaction mixture but reacting readily to the compounds of formula (I).

The compounds of formula (I) are preferably prepared by reacting compounds of formula (II) with compounds of formula (III), the symbols being as defined above.

Compounds of formula (II) and (III) are generally known. If not known, they can be prepared by methods known per se.

In the compounds of formula III, the -CO-L moiety is a moiety of a pre-activated carboxylic acid, preferably a carboxylic acid halide, symmetrical or unsymmetrical anhydride or active ester. Such residues for the activation of the carboxyl group in typical acylation reactions are described in the literature (for example, in standard publications such as Houben-Weyl, Methoden der organischen Chemie, Georg-Thieme ^; Verlag, Stuttgart /. The activated esters are conveniently formed in situ, for example by the addition of HOBt or N-hydroxysuccinimide. L is preferably hydrogen, chlorine, bromine, iodine or -ON-succinimide.

The reaction is generally carried out in an inert organic solvent in the presence of an acid binding agent, preferably in the presence of an organic base such as triethylamine, dimethylaniline, pyridine or quinoline, or with an excess of the carboxyl component of formula III. The addition of an alkali metal or alkaline earth metal hydroxide, carbonate or bicarbonate or other weak alkali metal or alkaline earth metal salt, preferably potassium, sodium, calcium or cesium, may be favorable. The reaction time is, depending on the reaction conditions, a few minutes to 14 days, the reaction temperature being about -30 to 140 ° C, generally -10 to 90 ° C, and especially about 0 to about 70 ° C.

Suitable inert solvents are, for example, hydrocarbons, such as hexane, petroleum ether, benzene, toluene or xylene; chlorinated hydrocarbons such as trichlorethylene, 1,2-dichloroethane or carbon tetrachloride, chloroform or dichloromethane; alcohols such as methanol, ethanol, isopropyl alcohol, n-propanol, n-butanol or tert-butanol; ethers such as diethyl ether, dipropropyl ether, tetrahydrofuran (THF) or dioxane; glycol ethers such as ethylene glycol monomethyl ether or ethylene glycol monoethyl ether (methyl glycol or ethyl glycol), ethylene glycol dimethyl ether (diglyme); ketones such as acetone or butanone; amides such as acetamide, dimethylacetamide, dimethylformamide (DMF); nitriles such as acetonitrile; sulfoxides such as dimethyl sulfoxide (LMSO); hydrogen sulfide; organic carboxylic acids such as formic or acetic acid; nitro compounds such as nitromethane or nitrobenzene; esters such as ethyl acetate. Mixtures of these solvents are also suitable.

Compounds of formula (I) may be obtained by reacting compounds of formula (IV) with compounds of formula (V). The starting compounds of formula (IV) and formula (V) are generally known. If they are not known, they can be prepared by methods known per se.

In the compounds of formula V, the -CO-L moiety is a moiety of a pre-activated carboxylic acid, preferably a carboxylic acid halide or a mixed anhydride or an active ester. Such radicals for the activation of the carboxyl group ⁱⁿ typical acylation reactions are described, for / example, in the standard works, such as Houben-Weyl, Methods of Organic Chemistry, Georg _R Thieme Verlag, Stuttgart /. L is preferably chlorine, bromine or -ON-succinimide.

The reaction of a compound of formula IV with a compound of formula V is carried out under the same conditions with respect to reaction time, temperature and solvents as the reaction of a compound of formula II with a compound of formula III.

Further, the compounds of formula I with a straight open chain in which R is X-Arg-Gly-Asp-Y or A-Cys- (R) -BU can be obtained by reacting biotin in the final step of the solid phase synthesis. in the same cycle, it couples as a normal amino acid with a protected N-terminus as the last component and the biotin-peptide is normally cleaved from the resin.

Solid phase synthesis, cleavage and purification are carried out as described by A. Jonczyk and J. Meienhofer / Peptides, Proc. 8th Am. Peptidase. Symp. Ed. V. Hruby and DH Rich, Pierce Comp. III, p. 73-77 (1983), or similar methods as described in Angew. Chem. 104, p. 375-391 (1992).

The compounds of formulas II and IV with a straight open chain can be prepared by methods known per se for the synthesis of amino acids and peptides as described, for example, in the aforementioned standard publications and patent literature, for example the solid phase synthesis method described by Merrifield / B. F. Gysin and R. B. Merrifield, J. Am. Chem. Soc. 94, pp. 3102 et al., 1972].

Cyclic compounds of formulas II and IV wherein r is cyclo- (Arg-Gly-Asp-Z), can be prepared by cyclization of linear compounds such as described in DE 43 10 643 and in HouberrWeyl, Methoden der organischen Chemie, I.c., Vol. 15/11, p. 1 to 806 (1974) ·

The compounds of the formula I can also be obtained by liberating by solvolysis, in particular by hydrolysis or hydroganolysis, from their functional derivatives.

Preferred compounds for solvolysis or hydro-hydrogenolysis are those which otherwise correspond to the general formula I, but have, instead of one or more free amino and / or hydroxyl groups, the corresponding protected amino and / or hydroxyl groups, preferably those which substitute for an atom. hydrogen, which is bonded to a nitrogen atom, have an amino-protecting group, for example compounds of formula I having an NHR 'group instead of the N'-group (where R * is an amino-protecting group, such as BOC or CBZ).

Further preferred compounds are those having a hydroxyl-protected group instead of a hydroxyl group, for example compounds of the formula I but having an R''θ-phenyl group instead of a hydroxyl group (where R '' stands for a hydroxyl protecting group) .

There may also be several of the same or different protecting groups in the molecule of the starting material. If such groups are different from each other, they can in many cases be selectively cleaved.

The term amino protecting group is well known and refers to groups which are suitable for protecting (blocking) the amino group from chemical reactions but which are readily removable if the desired reaction is carried out elsewhere in the molecule. Typically, such groups are unsubstituted or substituted acyl, aryl, aralkoxymethyl or aralkyl groups. Since the amino-protecting groups are removed after the desired reaction (or reaction sequence), their type and size are not critical. However, groups having 1 to 20, and especially 1 to 8, carbon atoms are preferred. As used herein, the term acyl is always meant in its broadest sense. It includes acyl groups derived from aliphatic, araliphatic, aromatic or heterocyclic carboxylic or sulfonic acids, such as, in particular, alkoxycarbonyl, aryloxycarbonyl and, in particular, aralkoxycarbonyl groups. Examples of such acyl groups include alkanoyl groups such as acetyl, propionyl, butyryl; aralkanoyl such as phenylacetyl; aroyl, such as benzoyl or toluyl; aryloxyalkanoyl such as phenoxyacetyl (POA); alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl (BOC), 2-iodoethoxycarbonyl; aralkoxycarbonyl, such as CBZ / CBZ / / carbobenzoxy "/, 4-methoxybenzyloxycarbonyl and ^9-d, luórenylmetoxykarbonylová / Fmoc / group; arylsulfonyl such as the 4-methyl-2,3,6-trimethylphenylsulfonyl group (Mtr./). Preferred amino protecting groups are BOC and Mtr, further CBZ, FMOC, benzyl and acetyl.

The term "hydroxy protecting group" is also generally known and refers to groups which are suitable for protecting (blocking) the hydroxy group from chemical reactions but which are readily removable when the desired reaction is carried out elsewhere in the molecule. Typical of such groups are the above-mentioned unsubstituted or substituted aryl, aralkyl or acyl groups, furthermore alkyl groups.

Since the hydroxyl protecting groups are removed after the desired reaction (or reaction sequence), their species and size are not critical. Preferred are, however, groups having 1 to 20 and in particular 1 to 10 carbon atoms. Examples of hydroxyl protecting groups include benzyl, p-nitrobenzoyl, p-toluenesulfonyl, tert-butyl and acetyl, with benzyl and tert-butyl being particularly preferred. The COOH groups in aspartic acid and glutamic acid are preferably protected in the form of a tert-butyl ester (e.g. Asp (OBut)).

Depending on the protecting group used, the liberation of the compounds of the formula I from their functional derivatives is successful, for example with strong acids, preferably trifluoroacetic acid or perchloric acid, but also other strong inorganic acids such as hydrochloric or sulfuric acids with strong organic carboxylic acids such as trichloroic acid. or with sulfonic acids such as benzenesulfonic acid or toluenesulfonic acid. The presence of an additional inert solvent is possible but not necessary. Suitable inert solvents are organic, for example, carboxylic acids such as acetic acid, ethers such as tetrahydrofuran or dioxane, amides such as dimethylformamide (DMF), halogenated hydrocarbons such as dichloromethane, and also alcohols such as methariol, ethanol or the like. isopropyl alcohol as well as water. Mixtures of these solvents may also be contemplated. Trifluoroacetic acid is preferably used in excess without addition of other solvents, perchloric acid in the form of a 9: 1 mixture of acetic acid and 70% perchloric acid. The reaction temperature for the cleavage is expediently about 0 to about 50 ° C, preferably 15 ° C. to 30 ° C (room temperature).

BOC groups. For example, Obut and Mtr may be conveniently cleaved with trifluoroacetic acid in dichloromethane or with approximately 3N to 5N hydrochloric acid in dioxane at 15 to 30 ° C, FMOC with a 5 to 50% solution of dimethylamine, diethylamine or piperidine in dimethylformamide at 15 to 30 ° C. C.

The trityl group is used to protect amino acids: histidine, arginine, glutamine and cysteine. Cleavage protection

The 21-group is carried out according to the desired end product with trifluoroacetic acid / 10% thiophenol, cleavage. a trityl group of all said amino acids. Using the trifluoroacetic acid / anisole or trifluoroacetic acid / thioanisole system, the trityl group is only cleaved from histidine, asparagine and glutamine, while the protecting group on the side chain of the cysteine remains intact.

Hydrogenolytically removable protecting groups (e.g., CBZ or benzyl) may be cleaved, for example, by treatment with hydrogen in the presence of a catalyst (e.g., a noble metal catalyst such as palladium, suitably supported on a carrier such as coal). Suitable solvents include the abovementioned solvents, in particular, for example, alcohols, such as methanol or ethanol, or amides, such as dimethylformamide. As a rule, the hydrogenolysis is carried out at a temperature of about 0 to 100 ° C and at a pressure of about 0.1 to 20 MPa, preferably at a temperature of 20 to 30 ° C, at a pressure of about 0.1 to 1 MPa. Hydrogenolysis of the CBZ group thrives, for example, well on 5-10% palladium on carbon in methanol or ammonium formate (instead of hydrogen) in the presence of palladium on carbon in a methanol / dimethylformamide system at 20-30 ° C.

The base of formula (I) may be acidic for. to an appropriate acid addition salt, for example by reacting an equivalent amount of base and acid in an inert solvent such as ethanol, followed by evaporation of the solvent. Suitable acids for this reaction are, in particular, acids. which provide physiologically acceptable salts. Inorganic acids such as sulfuric, nitric, hydrohalic acids such as hydrochloric or hydrobromic acids, phosphoric acids such as trihydrogenphosphoric acid, sulfamic acid and organic acids, especially aliphatic, alicyclic, araliphatic, aromatic or heterocyclic acids can be used. . monohydric or polybasic carboxylic, sulfonic or sulfuric acids such as formic, acetic, propionic, pivalic, diethylacetic, malonic, succinic, pumelic, fumaric, maleic, lactic, tartaric, malic, citric, gluconic, ascorbic, nicotonic, nicotonic, , ethanesulfonic, ethanedisulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, p-toluenesulfonic, naphthalene monosulfonic and naphthalenedisulfonic and laurylsulfonic acids. Salts with physiologically unacceptable acids can be used for the isolation and / or purification of the compounds of formula I.

On the other hand, the compounds of formula I can be converted into physiologically acceptable metal or ammonium salts by reaction with a base. Suitable salts are, in particular, sodium, potassium, magnesium, calcium and ammonium salts, and also substituted ammonium salts, for example dimethylammonium, diethylammonium, diisopropylammonium, monoethanolammonium, diethanolammonium or diisopropylammonium, cyclohexylammonium, cyclohexylammonium, cyclohexylammonium, cyclohexylammonium, cyclohexylammonium, cyclohexylammonium; with lysine.

The compounds of the formula I and their physiologically acceptable salts can be used for the production of pharmaceutical compositions, in particular by a non-chemical route. For this purpose, they can be converted into a suitable dosage form with at least one solid or liquid and / or semi-solid carrier or excipient and optionally in admixture with one or more other active substances.

The invention therefore also relates to compositions, in particular pharmaceutical compositions comprising at least one compound of the formula I and / or a physiologically acceptable salt thereof.

The compositions of the invention may be used as medicaments in human and veterinary medicine. Suitable excipients are organic or inorganic substances, ⁱⁿ Torah are suitable for enteral / oral e.g. / or pare.nterálne or topical administration or for administration as an inhalation spray and do not react with the novel compounds, for example water, vegetable oils , benzyl alcohols, alkylene glycols, polyethylene glycols, glycerol triacetate, gelatin, carbohydrates such as lactose or starches, magnesium stearate, talc and petrolatum. Especially suitable for oral use are tablets, pills, dragees, capsules, powders, granules, syrups, juices or drops. rectal use suppositories, for parenteral use solutions, in particular oily or aqueous solutions, other suspensions, emulsions or implants, for topical use ointments, creams or powders. The compounds of the invention can also be lyophilized and the lyophilizates obtained used, for example, for the preparation of injectables. The compositions may be sterilized and / or contain adjuvants such as glidants, preservatives, stabilizers, or wetting agents, emulsifiers, salts for affecting the osmotic pressure, buffers, coloring agents, flavoring agents and / or flavoring agents. Alternatively, they may contain one or more other active ingredients, such as vitamins. For administration in the form of inhalation sprays, the active ingredient is dissolved or suspended in a propellant or propellant mixture (such as, for example, carbon dioxide or fluorocarbon). In this case, the active ingredient is used in micronized form, and at least one physiologically compatible solvent, for example ethanol, can be added. Inhalable solutions may be administered using devices known in the art for this purpose.

The compounds of formula I and their physiologically acceptable salts can be administered as integrin inhibitors in the fight against diseases such as, in particular, pathological angiogenic diseases, thromboses, heart attacks, coronary heart diseases, arteriosclerosis, tumors, osteoporosis, inflammations and infections.

The compounds of the formula I according to the invention are generally used in doses similar to commercially known peptides, in particular analogously to the compounds of U.S. Pat. No. 4,472,305, preferably at a dose of about 0.05 to 500 mg, in particular 0.5 to 100 mg per dosing. unit. The daily dose is preferably about 0.01 to 2 mg / kg body weight. The dose for each individual depends on a variety of factors, such as the efficacy of the particular compound used, age, body weight, general health, sex, diet, time and route of administration, elimination rate, drug combination, and severity of the disease. . Parenteral administration is preferred.

The compounds of formula I may be used. in the analytical laboratory and in molecular biology. Their ability to form complexes between the biotinyl residue and the avidin glycoprotein is utilized. The use of the biotin-avidin complex [E.A. Bayer and M. Wilchek, Methods of Biochemical Analysis 25, p. 1 to 45, 1980 [lit. 1J / ·

The novel compounds of formula I can be used as integrin ligands for affinity chromatography columns for pure preparation of integrins. A complex of avidin-derivatized carrier material, for example Sepharose, and the novel compounds of formula I is formed in a manner known per se, as described (for example in Lit. 1 /. For this reason, this method is not described in detail here and reference is therefore made to the relevant literature, for example lit. 1 ,.

Suitable polymeric carrier materials are polymeric solid phases known per se from peptide chemistry with preferred hydrophilic properties, for example cross-linked polysaccharides such as cellulose, Sepharose or SephaP dex ¹ , acrylamides, polymers based on polyethylene glycol or p

Tentacelpolymers.

The novel compounds of formula I can be used as diagnostic markers for anti-biotin-antibody responses in ELISA and in FACS (fluorescence activated cell sorter analysis).

The use of antibiotic antibodies to detect biotin / M is known. Berger, Biochemistry 14, p. 2338-2342, 1975].

Use of an IgG derivatized; biotin in the enzyme-linked ELISA assay (described by U. Holmkov-Nielsen et al.) / Journal of Chromatography 297, p. 225-233, 1984].

J. Gao and S. J. Shattil / J. Immunol. Methods 181, p. 55-64 (1995) have described an ELISA assay demonstrating substances that inhibit integrin activation. In this case, biotinylated fibrinogen is used as evidence.

The use of flow cytometry in the clinical diagnosis of cells has been described by G. Schmitz and G. Rothe / DG Klinische Chemie

Mitteilungen 24, workbook 1, p. 1-14, 1993].

In addition, the compounds of formula I may be used in atomic force microscopy AFM to measure the strength of ligand-receptor interaction. By ligand is meant in particular a complex of avidin and a novel compound of formula I. The receptor is preferably an integrin receptor. E. -L. Florin et al. Science 264, p. 415-417 (1994) describe the measurement of adhesion forces between avidin-functionalized microscope and biotinylated agarose.

DETAILED DESCRIPTION OF THE INVENTION

The invention is illustrated, but not limited, by the following examples. Temperatures are always given in degrees Celsius. In the following examples, the expression conventional processing means:

Optionally, water is added, the pH of the final product is optionally adjusted to 2-10, the reaction mixture is extracted with ethyl acetate or dichloromethane, separated, dried over the organic phase with sodium sulfate, evaporated and purified by silica gel chromatography and / or crystallization. Rf values are on silica gel, eluent is ethyl acetate-methanol 9: 1, RZ is retention time (in minutes) for HPLC in the following systems:

. [A] column:

eluent A: eluent B: flow:

gradient:

Nucleosil 7C18 250 x 4 mm 0.1% TFA in water

0.1% TFA in acetonitrile ml / min to 50% B / 30 min min [B]

50-minute gradient of 0 to 80% 2-propanol in water with 0.3% TFA at 1 mL / min on a Lichrosorb ^R RP Select B column (7 µπι)

250 x 4mm.

27 [C]

column: Lichrospher 5 pm 100RP8 125 x 4 mm eluent A: 0.01 M Na-phosphate pH 7.0

eluent B: 0.005 M Na-phosphate pH 7.0 / v / v 60% 2

flow: gradient: propanol 0.7 ml / min 1-99% B / 50 min

Mass spectrometry (MS): EI / electron impact ionization (M)

FAB (fast atom bombardment) / (M + H) ⁺

The term DMPP resin refers to 4- (2 ', 4'-dimethoxyphenylhydroxymethyl) phenoxy resin, which allows the synthesis of side chain protected peptides.

Example 1

0.6 g of Fmoc-Lys (BOCl-OH in 100 ml of dichloromethane) was dissolved, treated with 1.2 equivalents of DMPP resin, 1.4 equivalents of phobt · DCCL and stirred for 12 hours at room temperature. After removal of the solvent, Fmoc-Lys (BOC) -DMPP-resin was obtained.

In the peptide synthesizer, Fmoc-Pro-OH is condensed with H-Lys (BOC) -DMPP-resin [released from Fmoc-Lys (BOC) -DMPP-resin with piperidine-dimethylformamide (20%) J system using a triple excess of protected proline . Coupling with the DCCl / HOBt system is performed at room temperature. The Fmoc-Pro-Lys (BOC) -DMPP-resin was obtained. Similarly, it is obtained by subsequent cleavage of the Fmoc protecting group and subsequent coupling with Fmoc-Ser (.But) -OH, Fmoc-Gly-OH, Fmoc-Arg (Mtr) -OH, FmocGly-OH, Fmoc-Gly-OH, Fmoc- Gly-OH and Bit-OH under repeated reaction conditions at each coupling

- release of the α-amino group by the piperidine system (IMF / 20%)

- washing with dimethylacetamide

- reaction with Fmoc-amino acid, optionally with Bit-OH Bit-Gly-Gly-Gly-Arg (Mtr) -Gly-Asp (OBut) -Ser (But) -Pro-Lys (BOC) MDPP-resin.

The resin was washed with CF 3 SO 4 -H ₂ / CH ₂ Cl ₂ / H ₂ O to give Bit-Gly-Gly-Gly-Arg (IR) -Gly-Asp (OBut) -Ser (But) -Pro-Lys (BOC) ) OH. After cleavage of the protecting groups with 2N hydrochloric acid in dioxane, removal of the solvent, removal of the residue into the trifluoroacetic acid / dichloromethane system and precipitation with diethyl ether, the product is purified by RP-HPLC. Bit-Gly-Gly-Gly-Arg-Gly-Asp-Ser-Pro-Lys-OH x 2 TFA was obtained; RZ [B] = = 12.14-; FAB 1056.

Example 2

Similar to Example 1, DMPP resin is obtained by subsequent coupling of Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH , Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH and Bit-OH Bit-Gly-Gly-Lys (BOC) -Ala Asp (OtBu) -Cys (Trt) -ProDMPP resin.

Cleavage from the resin, removal of the protecting groups and purification gave Bit-Gly-Gly-Gly-Lys-Thr-Ala-Asp-Cys (Trt) -Pio-OH x 2 TFA; RZ [B] = 27.6; FAB 1273.

Similarly, it is obtained by condensation of DMPP-resin.

with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH, Fmoc-Lys (BOC) -OH, and Bit OH; Bit-Lys-Thr-Ala-Asp-Cys (Trt) -Pro-OH;

with. Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Lys (BOC) -OH, and Bit-OH: bit-Lys-Ala-Ala-Asp-Cys (Trt) -Pro-OH;

Fmoc-Pro-OH; Fmoc-Cys (Trt) -OH; Fmoc-Asp (OBut) -OH; Fmoc-Ala-OH; Fmoc-Thr (But) -OH; Fmoc-Arg (Mtr) -OH. Bit-OH: Bit-Arg-Thr-Al and Asp-Cys (Trt) -Pro-OH;

with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Ser (But) -OH and. Bit-OH: Bit-Ser-Ala-Asp-Cys (Trt) -Pro-OH;

with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Ser (But) -OH, Fmoc-Gln (Trt) -OH and Bit -OH: Bit-Gln-Ser-Ala-Asp-Cys (Trt) -Pro-OH;

with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Ser (But) -OH, Fmoc-Glp-OH and Bit-OH: bit-Glp-Ser-Ala-Asp-Cys (Trt) -Pro-OH;

- 30 with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH,

Fmoc-Ala-OH, Fmoc-Ser (But) -OH, Fmoc-lle-OH and ‘Bit-OH:

Bit-Ile-Ser-Ala-Gly-Cys (Trt) -Pro-OH;

with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH,

Fmoc-Ala-OH, Fmoc-Ser (But) -OH, Fmoc-Arg (Mtr) -OH, and Bit-OH:

Bit-Arg-Ser-Ala-Gly-Cys (Trt) -Pro-OH;

with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, and Bit-OH:

Bit-Lys-Gly-Gly-Asp-Cys (Trt) -Pro-OH;

with Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, ...

Fmoc-Thr (But) -OH; Fmoc-Lys (BOC) -OH; Bit-OH:

Bi-Lys-Thr-Ala-Asp-Cys (Trt) -O H;

with Fmoc-Thr (But) - OH, Fmoc - Ala - OH, Fmoc - Pro - OH, Fmoc - Gly - OH,

Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro-OH,

Fmoc-Asn (Trt) - OH, Fmoc - Arg (Mtr) - OH, Fmoc - Pro - OH, Fmoc - Cys (Trt) - OH, Fmoc - Asp (OBut) - OH, Fmoc - Ala - OH · and Bit OH:

(a) for cleavage of the protecting group by TFA and 10% thiophenol:

Bit-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-OH;

b) Cleavage of the protecting group with TFA and 10% thioanisole.

Bit-Ala-Asp-Cys (Trt) -Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-OH;

with Fmoc-Thr (But) -OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro- OH, Fmoc-Asn (Trt), OH, Fmoc-Arg (Mtr) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Cys (Trt) - OH, Fmoc - Thr (But) - OH, Fmoc - Lys (BOC) - OH, Fmoc - Gly - OH and Bit - OH:

(a) when cleavage of the protecting group with TFA and • 10% thiophenol:

Bit-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala

(b) in the case of cleavage of the protecting group with TFA and% thioanisole:

Bit-Gly-Lys-Thr-Cys (Trt) -Asp-Cys (Trt) -Pro-Arg-Asn-Pro-His-LysGly-Pro-Ala-Thr-OH;

with Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH - Pro - OH, Fmoc - Cys (Trt) - OH, Fmoc - Asp (OBut) - OH, Fmoc - Ala - OH, Fmoc - Thr (But) - OH, Fmoc - Lys (BOC) - OH. bit-OH:

(e) when cleaving the protecting group with. TFA and% thiophenol:

Bit-Lys-Thr-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-OH;

(b) cleavage of the protecting group with TFA; and

1-0% thioanisole:

Bit-Lys-Thr-Ala-Asp-Cys (Trt) -Pro-Arg-Asn-Pro-His-Lys-Gly-OH;

with Fmoc-Thr (But) -OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro- OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH; Fmoc-Lys (BOC) -OH; Bit-OH:

(a) in the case of cleavage of the protecting group by TFA and 0% thiophenol:

Bit-Lys-Thr-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-ThrOH;

(b) · when cleavage of the protecting group by TFA and .10 thioanisole:

Bit-Lys-Thr-Ala-Asp-Cys (Trt) -Pro-Arg-Asn-Pro-His-Lys-Gly-ProAla-Thr-OH;

32 with Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Mtr) - OH, Fmoc-Pro-OH, Fmoc-Cys (trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH, Fmoc-Lys (BOC) -OH, Fmoc-Gly-OH. and Bit-OH:

(a) for cleavage of the protecting group with TFA and% thiophenol:

Bit-Gly-Lys-Thr-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-OH;

(b) when cleavage of the protecting group by TFA and 10% thioanisole:

Bit-Gly-Lys-Thr-Ala-Asp-Cys (Trt) -Pro-Arg-Asn-Pro-His-Lys-Gly-OH;

with Fmoc-Thr (But) -OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro- OH

Fmoc-Asn (Trt) -OH; Fmoc-Arg (Mtr) -OH; Fmoc-Pro-OH; Fmoc-Gys (Trt) -OH; Fmoc-Asp (OBut) -OH; Fmoc-Ala-OH; Thr (But) -OH a. Bit-OH:

Bit-Thr-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-OH;

^p . Fmoc-Thr (But) -OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro-OH, Fmoc-Pro-OH .

Fmoc-Asn (Trt) -OH; Fmoc-Arg (Mtr) -OH; Fmoc-Pro-OH; Fmoc-Cys (Trt) -OH; Fmoc-Asp (OBut) -OH; Bit-OH:

Bit-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-OH;

Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro-OH,

Fmoc-Asn (Trt) -OH; Fmoc-Arg (Mtr) -OH; Fmoc-Pro-OH; Fmoc-Cys (Trt) -OH; Fmoc-Asp (OBut) -OH; Fmoc-Ala-OH; Bit-OH:

Bit-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-OH;

with. Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OH) -OH, Fmoc- Ala-OH, Fmoc-Thr (But) -OH, Fmoc-Lys (BOC) -OH, and Bit-OH:

Bit-Lys-Ala-Tbr-Asp-Cys (Trt) -Pro-Arg-Asn-Pro-OH;

Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Mtr) -OH, Fmoc-Pro- OH, Fmoc-Cys (Trt) -OH, Fmpc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH, Fmoc-Lys (BOC) -OH, and Bit-OH:

Bit-Lys-Thr-Ala-Asp-CysCTrt) -Pro-Arg-Asn-Pro-His-Lys-OH;

s ^; . Fmoc-His (Trt) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc- Asp (OBut) OH, Fmoc - Ala - OH, Fmoc - Thr (But) - OH, Fmoc - Lys (BOC) - OH ⁸ Bit - OH:

Bit-Lys-Thr-Ala-Asp-Cys (Trt) -Pro-Arg-Asn-Pro-His-OH;

with Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH, Fmoc -Lys (BOC) -OH- and Bit-OH: Bit-Lys-Thr-Ala-Asp-Cys (Trt) -Pro-Arg-OH;

with . Fmoc-Lys (BOC) - OH, Fmoc - His (Trt) - OH, Fmoc - Pro - OH, Fmoc - Asn (Trt) - OH, Fmoc - Arg (Mtr) - OH, Fmoc - Pro - OH, Fmoc - Cys (Trt) -OH, Fmoc-Asp (OBut) -OH ⁸ 'Bit-OH: Bit-Asp-Cys (Trt) -Pro-Arg-Asn-Pro-His-Lys-OH;

_with Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH and. Bit-OH: Bit-Ala-Asp-Cys (Trt) -Pro-Arg-OH;

_p . Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, FmocAsp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH, and Bit-OH: bit-Thr-Ala-Asp-Cys (Trt) -Pro-Arg-OH;

with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-AlaOH, Fmoc-Thr (But) -OH, Fmoc-Lys (BOC) -OH, and. Bit-OH: Bit-Lys-Thr-Ala-Asp-Cys (Trt) -Pro-OH;

with Fmoc-NMeAla-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH, Fmoc-Lys (BOC) -OH, and Bit -OH: Bit-Lys-Thr-Ala-Asp-Cys (Trt) -N-MeAla-OH;

- 34 Example 3

One solution of 3.05 g of cyclo-CArg-Gly-Asp-D-Phe-Lys) [Obtainable by cyclization of H-Arg (Mtr) -Gly-Asp (OBut) -D-Phe-Lys (BOC) -OH to cyclo- (Arg (Mtr) -Gly-Asp (OBut) -D-Phe-Lys- (BOC)) followed by cleavage of the protecting groups in 100 ml of dichloromethane is added 1.7 g of the cheaply obtainable (+) - biotinyl-N-succinimidyl ester and 0.5 g triethylamine. Stirring is carried out for 5 hours at room temperature and conventional work-up gives cyclo- (Arg-Gly-Asp-D-Phe-Lys- (N-Bit)) x TPA; RZ [B] = 11.32; FAB 830.

Example 4

Analogously to Example 3, 3.5 g of cyclo- (ArgGly-Asp-D-Phe-Lys) and 2.3 g of the cheaply obtainable N-succinimidyl ester of (+) - biotinyl-6-aminocaproic acid (A) are obtained. e 0.5 g of triethylamine cyclo- (Arg-Gly-Asp-D-Phe-Lys (N-Bit-Aha)) x TFA; R 2 [C] = 23.67; FAB 943.

Similarly, it is obtained from ”A” and the following cyclic compounds

Cyclo- (Arg-Gly-Asp-D-Trp-Lys) Cyclo- (Arg-Gly-Asp-D-Tyr-Lys) Cyclo- (Arg-Gly-Asp-D'Phe-D-Lys) Arg-Gly-Asp-D-Phe-Cys) Cyclo- (Arg-Gly-Asp-D-Phe-Dab) Cyclo- (Arg-Gly-Asp-D-Trp-D-Cys) -Gly-Asp-D-Tyr-D-Cys) Cyclo- (Arg-Gly-Asp-Phe-D-Lys) CyAlo- (Arg-Gly-Asp-Trp-D-Lys) Cyloo (Arg-Gly- Asp-Tyr-D-Lys) Cyllo- (Arg-Gly-Asp-Phe-D-Cys) CyMo- (Arg-Gly-Asp-Phe-Dab) Cylo- (Arg-Gly-Asp-Trp-D-Cys) ) CyMo- (Arg-Gly-Asp-Ty-D-Cys) Cytlo- (Arg-Gly-Asp-D-Trp-Orn)

- 35 Cy4lo- (Arg-Gly-Asp-D-Tyr-Orn)

Cyclo- (Arg-Gly-Asp-D-Phe-Orn) Cyclo- (Arg-Gly-Asp-D-Trp-D-Orn) - (Arg-Gly-Asp-D-Phe-D-Orn)

Cyclo- (Arg-Gly-Asp-D-Trp-Dab) Cyclo- (Arg-Gly-Asp-D-Trp-Dap) Cyclo- (Arg- Gly-Asp-D-Trp-Dab) Gly-Asp-D-Tyr-Dap) Cyclo- (Arg-Gly-Asp-D-Phe-Dap) Cyclo- (Arg-Gly-Asp-D-Trp-D-Dap) Cyclo- (Arg-Gly-Asp) -D-Tyr-D-Dap) Cyclo- (Arg-Gly-Asp-D-Phe-D-Dap) the following compounds

Cyclo- (Arg-Gly-Asp-D-Trp-Lys (N ^e -Bit-Aha)) cyclo- (Arg-G (y-Asp-D-Tyr-Lys (N ^c -Bit-Aha)) cyclo- (Arg-Gly-Asp-D-Phe-D-Lys (N ^c -Bit-Aha)) Cyldo- (Arg-Gly-Asp-D-Phe-Cys (S-bit-Aha)) cyclo- (Arg Gly-Asp-D-Phe-Dab (N ^T -Bit-Aha)) cyclohexyl o- (Arg-Gly-Asp-D-Trp-D-Cys (S-Aha-bit) j cyclo- (Arg-Gly Asp-D-Tyr-D-Cys (S-bit-Aha)) Cylío- (Arg-Gly-Asp-Phe-D-Lys (N ^e -Bit-Aha)) CyJílo-cargo-Gly-Asp-Trp -D-Lysine-N-Bit-Aha) Cyclo- (Arg-Gly-Asp-Tyr-D-Lys (N'-Bit-Aha)) Cyclo- (Arg-Gly-Asp-Phe-D-Cys (S) -Bit-Aha)) cyclohexyl | o- (Arg-Gly-Asp-Phe-Dab (N ^T -Bit-Aha)) cyclo- (Arg-Gly-Asp-Trp-D-Cys (S-Aha-bit) ) Cyclo- (Arg-Gly-Asp-Tyr-D-Cys (S-Bit-Aha)) Cylio- (Arg-Gly-Asp-D-Trp-Orn (N ⁵ -Bit-Aha)) Cyclo- (Arg Gly-Asp-D-Tyr-Orn (N ⁵ -Bit-Aha)) cyclo- (Arg-Gly-Asp-D-Phe-Orn (N ⁸ -Bit-Aha)) cyclo- (Arg-Gly-Asp -D-Trp-D-Orn (N ⁵ -Bit-Aha)

- 36 Cyclo- (Arg-Gly-Asp-D-Tyr-D-Orn (N ⁵ -Bit-Aha))

Cy1 {lo- (Arg-Gly-Asp-D-Phe-D-Orn (N ⁵ -Bit-Aha))

Cyclo- (Arg-Gly-Asp-D-Trp-Dab (N ^T -Bit-Aha)) Cytlo- (Arg-Gly-Asp-D-Tyr-Dab (N ^T -Bit-Aha)) CyHo- (Arg -Gly-Asp-D-Trp-Dap (N ^p -Bit-Aha)) cyclo- (Arg-Gly-Asp-D-Tyr-Dap (N ^p -Bit-Aha)) cyclo- (Arg-Gly-Asp -D-Phe-Dap (N ^p -Bit-Aha)) cyclo- (Arg-Gly-Asp-D-Trp-D-Dap (N ^p -Bit-Aha)) Cyldo- (Arg-Gly-Asp D-Tyr-D-Dap (N ^p -Bit-Aha)) cyclo- (Arg-Gly-Asp-D-Phe-D-Dap (N ^p -bit-Aha))

Example 5

To a solution of 3.05 cyclo- (Arg-Gly-Asp-D-Phe-Lys) in 40 mL

6% Boc-Aha-N-succinimidyl ester was added to a 5% aqueous solution of sodium bicarbonate and 40 ml of tetrahydrofuran. Stir 4 hours, work-up in the usual way to give cyclo- (Arg-Gly-Asp-D-Phe-Lys (BOC-Aha)); RZ [c] = 27.7; FAB 817.

After removal of the BOC protecting group in the hydrochloric acid / dioxane system, the cyclo (Arg-Gly-Asp-D-Phe-Lys (N-Aha)) x 2 TFA is obtained after conventional work-up; Rf [c] = 14.76; FAB 717.

Similar to Example 1, cyclo- (Arg-Gly-Asp-D-PheLys (1H-Bit-Aha)) x TFA was obtained by subsequent reaction with (+) - Biotinyl-N-succinimidyl ester; LC LC] = 23.67; FAB 943.

Example 6

Similarly, and ⁱⁿ Example 4, it is obtained from cyclo- (Arg-Gly-Asp-D-Phe-Lys-Gly) obtainable by cyclization of H-Arg (Mtr) -Gly-Asp (OBut) -D-Phe-Lys (BOC) -Gly -H on the cyclo- (Arg (Mtr) -Gly-Asp (OBut) -D-Phe-Lys- (BOC) -Gly) followed by cleavage of the protecting groups) and from the N-succinimidyl acid ester. (+) - biotinyl-6-aminoVapron cyclo- (Arg-Gly-Asp-D-Phe-Lys (N, Bi-Aha) -Gly) x TFA; Rf [Aj = 10.97; FAB 1000.

Similarly, it is obtained from cyclo- (Arg-Gly-Asp-D-Phe-Val-Lys) [obtainable by cyclization of H-Arg (Mtr) -Gly-Asp (OBut) -D-Phe-Val-Lys (BOC)]. -OH on cyclo- (Arg (Mtr) -Gly-Asp (OBut) -D-Phe-Val-Lys- (BOC)) followed by cleavage of the (+) - biotinyl-6-amino-acid protecting group from the N-succinimidyl ester. apronovej cyclo- (Arg-Gly-Asp-D-Phe-Val-Lys ^(Ν ε -Bit-Aha)) x TFA; RZ [A] = 16.11; FAB 1042.

Similarly, (+) - biotinyl-6-aminoVapronic acid N-succinimidyl ester (Arg-Gly-Asp-D-Phe-N-) is obtained from cyclo-Arg-Gly-Asp-D-Phe-M-Me-Lys). Me-Lys (N ^f -Bit-Aha)).

Example 7

Similar to Example 1, it is obtained by subsequent couplings on MBHA resin and with the addition of 1.4 equivalents of HOBt and 1.4 equivalents of DCCl with Fmoc-Pro-OH, Fmoc-CysITT) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Lys (BOC) -OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Bit-OH, Bit-Gly-Gly- Gly Lys (BOC) -Thr (But) -Ala-Asp (OBut) -Cys (Trt} -Pro-MBHA-Resin) Cleavage of the resin with TFA and cleavage groups protecting the piperidine / dimethylformamide system yields

Bit-Gly-Gly-Gly-Lys-Thr-Ala-Asp-Cys (Trt) -Pro-NH _second

Similarly obtained by condensing MBHA resin with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH, Fmoc-Lys (BOC) -OH and Bit-OH:

Bit-Lys-Thr-Ala-Asp-Cys (Trt) _-Pro-NH2;

with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Lys (BOC) -OH, and Bit-OH:

Bit-Lys-Ala-Ala-Asp-Cys (Trt) _-Pro-NH2;

- 38 with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, and Fmoc-Asp (OBut) -OH,

Fmoc-Ala-OH, Fmoc-Thr (But) -OH, Fmoc-Arg (Mtr) -OH and Bit-OH:

Bit-Arg-Thr-Ala-Asp-Cys (Trt) _-Pro-NH2;

with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Ser (But) -OH, and Bit-OH: Bit-Ser-Ala- Asp-Cys (Trt) _-Pro-NH2;

with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Ser (But) -OH, Fmoc-Gln (Trt) -OH and Bit -OH: Bit-Gln-Ser-Ala-Asp-Cys (Trt) -Pro-NH ₂ ;

with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Ser (But) -OH, Fmoc-Glp-OH and Bit-OH: Bit-Gl-Ser-Ala-Asp-Cys (Trt) -Pro-NH ₂ ;

Fmoc-Pro-OH; Fmoc-Cys (Trt) -OH; Fmoc-Gly-OH; Fmoc-Ala-OH; Fmoc-Ser (But) -OH; OH-bit: Bit-Ile-Ser-Ala-Gly-Cys (Trt) _-Pro-NH2;

with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ser (But) -OH, Fmoc-Arg (Mtr) -OH and Bit-OH: bit-Arg-Ser-Ala-Gly-Cys (Trt) _-Pro-NH2;

with Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, and Bit-OH: bit-Lys-Gly-Gly-Asp-Cys (Trt) _-Pro-NH2;

- 39 with Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH,

Fmoc-Thr (But) -OH, Fmoc-Lys (BOC) -OH, and Bit-OH:

Bit-Lys-Thr-Al-αsp-Cys (T rt) -NH ₂ ;

Fmoc-Thr (But) -OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH,

Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Ág (Mtr) -OH, Fmoc-Pro-OH, Fmoc- Cys (Trt) - OH, Fmoc - Asp (OBut) - OH, Fmoc - lala - OH. Bit-OH:

a). for cleavage of the protecting group with TFA and% thiophenol:

_{Bit-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-NH2;}

b), at cleavage, a protecting group with TFA and% thioanisole;

Bit-Ala-Asp-Cys (Trt) _{-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-NH2;}

with Fmoc-Thr (But) -OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro- OH

Fmoc-Asn (Trt) -OH, Fmoc-Arg (Mtr) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Cys (Trt) -OH Fmoc-Thr (But) -OH, Fmoc-Lys (BOC) -OH, Fmoc-Gly-OH, and Bit-OH:

^(a ) for cleavage of the protecting group with TFA and 10% thiophenol:

_{Bit-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-AlaThr-NH2;}

(b) For cleavage of the protecting group of TFA and% thioanisole:

Bit-Gly-Lys-Thr-Cys (Trt) -Asp-Cys (Trt) _{-Pro-Arg-Asn-Pro-His-LysGly-Pro-Ala-Thr-NH2;}

with . Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Mtr) -OH, Fmoc- Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH,

- 40 Fmoc-Lys (BOC) -OH and Bit-OH:

a), at the cleavage of the protecting group by TFA-a • 10% of thiophenol:

_{Bit-Lys-Thr-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-NH2;}

(b) at 0‘ cleavage, protecting groups by TFA and 10% thioanisole:

Bit-Lys-Thr-Al-Asp-Cys (Trt) -Pfo-Arg-Asn-Pro-His-Lys-Gfy-NH ₂ ;

with. Fmoc-Thr (But) -OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro-OH, Fmoc-Pro-OH Fmoc-Asn (Trt) -OH, Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc -Thr (But) -OH, Fmoc-Lys (BOC) -OH, and Bit-OH:

(a) for cleavage of the protecting group with TFA and 10% thiophenol:

Bit-Lys-Thr-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-AlaNH ₂ ;

(b) for cleavage of the protecting group by TFA and 10% thioanisole:

Bit-Lys-Thr-Ala-Asp-Cys (Trt) _{-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-NH2;}

^of _Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-HisCTrt) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Mtr) -OH, Fmoc- Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH,

Fmoc-Lys (BOC) -OH, Fmoc-Gly-OH, and Bit-OH:

^(a ) for cleavage of the protecting group ... with TFA and 10% thiophenol:

_{Bit-Gly-Lys-Thr-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-NH2;}

(b) ',. for cleavage of the protecting group with TFA and% thioanisole:

Bit-Gly-Lys-Thr-Ala-Asp-Cys (Trt) _{-Pro-Arg-Asn-Pro-His-Lys-Gly-NH2;}

with Fmoc-Thr (But) -OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro- OH

Fmoc-Asn (Trt) -OH; Fmoc-Arg (Mtr) -OH; Fmoc-Pro-OH; Fmoc-Cys (Trt) -OH; Fmoc-Asp (OBut) -OH; Fmoc-Ala-OH; Thr (But) -OH and Bit-OH: Bit-Thr-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-NH ₂ ;

with Fmoc-Thr (But) -OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro- OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH and Bit-OH: Bit-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-NHa:

Fmoc-Gly-OH, Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH; Fmoc-Cys (Trt) -OH; Fmoc-Asp (OBut) -OH; Fmoc-Ala-OH; Bit-OH:

_{Bit-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-NH2;}

With Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc -Ala-OH, Fmoc-Thr (But) -OH, Fmoc-Lys (BOC) -OH, and Bit-OH:

Bit-Lys-Thr-Ala-Asp-Cys (Trt) _{-Pro-Arg-Asn-Pro-NH2;}

with Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH, Fmoc -Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH, Fmoc-Lys (BOC) -OH, and Bit-OH:

Bit-Lys-Ala-TŤir-Asp-Cys (Trt) -Pro-Arg-Asn-Pro-His-Lys-NH 2;

with Fmoc-His (Trt) -OH; Fmoc-Pro-OH; Fmoc-Asn (Trt) -OH; Fmoc-Arg (Mtr) -OH; Fmoc-Pro-OH; Fmoc-Cys (Trt) -OH; Fmoc -Asp (OBut) OH, Fmoc-Ala-OH, Fmoc-Tlir (But) -OH, Fmoc-Lys (BOC) -OH, and Bit-OH: Bit-Lys-Thr-Ala-Asp-Cys (Trt) -Pro-Arg-Asn-Pro-His-NH _2;

with Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH, Fmoc -Lys (BOC) -OH and Bit-OH:

Bit-Lys-Thr-Ala-Asp-Cys (Trt) _{-Pro-Arg-NH2;}

with Fmoc-Lys (BOC) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro-OH,

Fmoc-Asn (Trt) -OH, Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, and Bit-OH:

Bit-Asp-Cys (Trt) _{-Pro-Arg-Asn-Pro-His-Lys-NH2;}

with Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH: and Bit-OH:

Bit-Ala-Asp-Cys (Trt) _{-Pro-Arg-NH2;}

with Fmoc-Arg (Mtr) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, FmocAsp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH, and Bit-OH :

Bit-Thr-Ala-Asp-Cys (Trt) _{-Pro-Arg-NH2;}

with. Fmoc-Pro-OH, Fmoc-CysCrrt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Alar, Fmoc-Thr (But) -OH, Fmoc-Lys (BOC) -OH, and Bit-OH:

Bit-Lys-Thr-Ala-Asp-Cys (Trt) _-Pro-NH2;

. with Fmoc-NMeAla-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OBut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH, Fmoc-Lys (BOC) -OH, and Bit OH:

Bit-Lys-Thr-Ala-Asp-Cys (Trt) _-NMeAla-NH2;

Example 8

A process for preparing a suitable affinity chromatography material for purifying integrins

Sepharose is activated by the method of lit. 1, p. 14. Then, to 10 kg of activated Sepharose, 20 mg of avidin in 20 ml of a 0.1 M sodium bicarbonate solution is added. The suspension was stirred at 4 ° C for 12 hours and then washed. The product is suspended in water with several sodium azide crystals. An avidin complex with biotinylated compounds of formula I, for example with cyclo- (Arg-Gly-Asp-D-Phe-Lys (N-Bit)) x TFA, is formed by dissolving 1.1 equivalents of the peptide in

43 sodium acetate buffer, the solution is added to the avidin-Sepharose suspension and stirred for 4 hours. Excess peptide is removed by washing.

The following examples illustrate pharmaceutical compositions:

Example A. Injection ampoules

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

Example B. Suppositories

A mixture of 20 g of an active compound of the formula I is melted with 100 g of soya 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 of 1 g of an active compound of the formula I is prepared, 9.38 g of sodium dihydrogenphosphoricane dihydrate.

28.48 g of disodium hydrogen phosphate with 12 molecules of water and 0.1 g of benzalkonium chloride in 940 ml of double-distilled water. The pH of the solution was adjusted to 6.8, made up to 1 liter and sterilized by irradiation. This solution can be used as eye drops.

Example D. Ointment

500 mg of an active compound of the formula I are mixed with

99.5 g of petroleum jelly under aseptic conditions.

Example E. Tablets

From a mixture of 1 kg of active compound of the formula I, 4 kg of lactose, 1.2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearate, tablets are compressed in a conventional manner such that each tablet contains 10 mg of the active ingredient.

Example F. Dragees

Analogously to Example E, tablets are compressed and then coated in a customary manner with a coating of sucrose, potato starch, talc, tragacanth and dye.

Example G. Capsules

In a manner known per se, 2 kg of the active ingredient of the formula I are filled into hard gelatine capsules such that each capsule contains 20 mg of the active ingredient.

Example H. Ampoules

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

Example I. Inhalation Spray

14 g of an active compound of the formula I are dissolved in

1 isotonic sodium chloride solution and filled into conventional commercial spray canisters with a pump mechanism. The solution may be sprayed into the mouth or nose. Each spray (approximately 0.1 ml) corresponds to a dose of approximately 0.14 mg.

Industrial usability

The biotinylated peptide derivative and pharmaceutically acceptable salts thereof are useful as integrin inhibitors for the manufacture of pharmaceutical compositions and for various purposes in biology.

Claims (10)

PATENT CLAIMS 7V A biotinylated peptide derivative of the formula I X R ¹ (I) wherein Q is absent or is -NH- (CH ₂ ) _n -CO- or -NH- (CH ₂ ) _n -NH- _; 1 2 R is X-Arg-Gly-Asp-Y, A-cys- (R JBU or cyclo- (Arg-Gly-Asp-Z), wherein Z in the side chain is linked to Qa to biotin as Q is absent . X and Y are each independently an amino acid residue or a di-, tri-, tetra- or pentapeptide residue, wherein the amino acids are independently selected from the group consisting of Ala, Asn, Asp, Arg, Cys, Gln, Glu, Gly, 4Hal -Phe, His, homo-Phe, Ile, Leu, Lys, Met, Nle, Phe, Phg, Pro, Ser, Thr, Trp, Tyr and Val, these acids being optionally derivatized, A is absent or is an Asp or peptide fragment selected from the group consisting of Lys-Thr-Ala-Asp, Lys-Thr-Ala-Asp, Lys-Thr-Ala-Asp, Lys-Thr-Ala-Asp, Lys-Thr-Ala-Asp, Arg-Thr-Ala-Asp, Ser-Ala-Asp, Gln-Ser-Ala-Asp, Glp-Ser-Ala-Asp, Gly-Lys-Thr-Al-Asp, Asn-Gly-Lys-Thr-Ala-Asp Arg-Ser-Ala-Gly-Cys-Asn-Gly-Lys-Thr-Ala-Asp Tyr-Cys-Asn-Gly-Lys-Thr-Ala-Asp, Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Ala-Asp, Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Ala-Asp, Gly-Lys-Thr-Cy (Trt) -Asp, Met-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Al-Asp, Asp-Met-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Ala-Asp, B is absent or is OH, Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Orn, Phe, 4-Hal-Phe, Pro, Ser, Thr, Trp , Tyr and Val, or a methylated derivative of these amino acid residues _t on a nitrogen atom, or is a fragment of a peptide selected from the group consisting of Pro-Arg, Pro-Arg-Asn, Pro-Arg-Asn-Pro, Pro-Arg-Asn-Pro-His, Pro-Arg-Asn-Pro-His-Lys, Pro-Arg-Asn-Pro-His-Lys-Gly, Pro-Arg-Asn- Pro-His-Lys-Gly-Pro, Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala, Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr, Wherein when B is A-Cys (R 1) -B-U, only one of A or B may be absent, and is further hydrogen, C 1 -C 6 alkyl, Trt, Dpm, or Bzl, U OH, OR ⁹ , NH 2, NHR ⁹ or n (R ⁹ J 2, Z is each independently an amino acid residue or a di-, tri- or tetrapeptide residue, wherein the amino acids are independently selected from the group consisting of Ala, Asn, Asp, Arg, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys , Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val or M, wherein these amino acids are optionally derivatized, and the amino acid residues are peptide-linked to one another via - 48 .alpha.-amino and o (-carboxyl groups and M are always present, M is NH (R ⁸⁾ -CH (R ³⁾ -COOH, R ³ -R ⁵ -R ⁴ , R ⁶ -R ⁴ , -R ⁷ -R ⁴ , R ⁴ OH, NH _2, SH or COOH, 5 is a C 1 -C 6 alkylene group, R ⁸ alkylenephenyl group having 7 carbon atoms až.14, R ⁷ alkylénfenylalkylénovú having 8 to 15 carbon atoms, R ⁸ is hydrogen, C 1 -C 6 alkyl, or C 7 -C 12 alkylenephenyl. Q, R is C 1 -C 6 alkyl, Hal is a fluorine, chlorine, bromine or iodine atom and 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and if the derivative contains both optically active amino acid residues and optically active amino acid derivatives, D and L forms of the derivative; pharmaceutically acceptable salts thereof. A biotinylated peptide derivative according to claim 1 of formula (I) a) Q is missing and where is I R ¹ cyclo- (Arg-Gly-Asp-D-Phe-Lys); b) Q is missing and where is R ¹ Gly-Gly-Gly-Arg-Gly-Asp-Ser-Pro-Lys-OH, c) Q is missing and where is R ¹ Gly-Gly-Gly-Lys-Thr-Ala-Asp-Cys (Trt) -Pro-OH, d) Q is -NH- (CH 2) 5 -CO a R ¹ cyclo- (Arg-Gly-Asp-D-Phe-Lys); e) Q is -NH- (CH 2) 5 -CO a R ¹ cyclo- (Arg-Gly-Asp-D-Phe-Lys-Gly) f) Q is -NH- (CH 2) 5 -CO a R ¹ cyclo- (Arg-Gly-Asp-D-Phe-Val-Lys), g) Q is -NH- (CH 2) 5 -CO a R ¹ cyclo- (Arg-Gly-Asp-D-Trp-Lys) h) Q is -NH- (CH 2) s -CO a R ¹ cyclo- (Arg-Gly-Asp-D-Tyr-Lys), and pharmaceutically acceptable salts thereof. A process for the preparation of a biotinylated peptide derivative according to claim 1, wherein the individual symbols are as defined in claim 1, and salts thereof, characterized in that a compound of formula II is reacted and / or reacted. HQR ^{1 (} CII), wherein Q and R ^ are as defined in nation 1, by acylation reaction with a compound of formula III wherein L is a chlorine, bromine, iodine or free or reactive functionally modified hydroxyl group, or b) reacting a compound of formula IV HR ¹ (IV) wherein R ¹ is as defined in claim 1, by acylation reaction with a compound wherein Q has. in claim 1, said meaning and who is L is a hydrogen, chlorine, bromine, iodine or free or reactive functionally modified hydroxyl group; or c) the compound of formula I is released from its functional derivative by treatment with a solvolyzing or hydrogenogenizing agent, and / or a basic or acidic compound of formula It is also converted by acid or base treatment into its salt. ; 4. A process for the manufacture of a pharmaceutical composition, wherein the biotinylated peptide derivative according to claim 1, wherein the individual symbols are as defined in Nation 1, or a physiologically acceptable salt thereof, is treated together with at least one solid. a semi-solid or liquid carrier or excipient to a suitable dosage form. Pharmaceutical composition, characterized in that it contains as active ingredient at least one biotinylated peptide derivative according to claim 1 of the general formula (I), wherein the individual symbols are as defined in claim 1, or a physiologically acceptable salt thereof. • f ty The biotinylated peptide derivative according to claim 1, wherein the individual symbols are as defined in claim 1, or a physiologically acceptable salt thereof as integrin inhibitors for the control of pathological angiogenic diseases, thromboses, heart attack, coronary heart disease, arteriosclerosis, tumors, osteoporosis, inflammation and infections. Use of a biotinylated peptide derivative according to claim 1, wherein the individual symbols are as defined in claim 1 1 or a physiologically acceptable salt thereof for the manufacture of a medicament. Use of a biotinylated peptide derivative according to claim 1, wherein the individual symbols are as defined in claim 1 1, for the purification of integrins by affinity chromatography. Grafine. Use of a biotinylated peptide derivative according to claim 9 1, wherein the individual symbols are as defined in claim 1 1, as diagnostic markers for anti-biotin antibody responses in ELISA and FACS analysis. Use of a biotinylated peptide derivative according to claim 1, wherein the individual symbols are as defined in claim 1 1, meaning for force field microscopy to measure the strength of ligand-receptor interaction.