| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 2, 2002 |
| PATENT TITLE |
Low molecular weight peptide derivatives as inhibitors of the laminin/nidogen interaction |
| PATENT ABSTRACT |
Low molecular weight peptide derivatives which are able to act as inhibitors of the interaction between laminin and nidogen (laminin/nidogen interaction), a process for their preparation, pharmaceutical compositions prepared therefrom and their use for preparing pharmaceuticals and for identifying inhibitors of the laminin/nidogen interaction. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | February 29, 2000 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Yanaihara et al. Synthetic Study on Human C-Peptide . . . Hoppe-Seyler's Z. Physiol. Chem. vol. 362, pp. 775-797, Jun. 19, 1981.* Zhang, X. et al., "A non-mammalian in vivo model for cellular and molecular analysis of glucose-mediated thickening of basement membranes", Diabetologia, vol. 33, pp. 704-707, (1990). Wilson, R. et al., "2.2 Mb of contiguous nucleotide sequence from Chromosome III of C. elegans", Nature, vol. 368, pp. 32-38, (1994). Veber, D. et al. "The design of metabolically-stable peptide analogs", Trends Neurosci., vol. 8, pp. 392-396, (1985). Stetefeld, J. et al., "Crystal Structure of Three Consecutive Laminin-type Epidermal Growth Factor-like (LE) Modules of Laminin .gamma. 1 Chain Harboring the Nidogen Binding Site", J. Mol. Biol., vol. 257, pp. 644-657. Smola, H. et al., "Dynamics of Basement Membrane Fromation by Keratinocyte-Fibroblast Interactions in Organotypic Skin Culture", Experimental Cell Research, vol. 239, pp. 399-410, (1998). Nicosia, R. et al., "Growth of Microvessels in Serum-Free Matrix Culture of Rat Aorta", Laboratory Investigation, vol. 63, No. 1, pp. 115-122, (1990). Poschl, E. et al., "Site-directed mutagenesis and structural interpretation of the nidogen binding site of the laminin .gamma. 1 chain", The EMBO Journal, vol. 15, No. 19, pp. 5154-5159 (1996). Poschl, E. et al., "Two non-contiguous regions contribute to nidogen binding to a single EGF-like motif of the laminin .gamma. 1 chain", The EMBO Journal, vol. 13, No. 16, pp. 3741-3747, (1994). Pikkarainen, T. et al., "Human Laminim B2 Chain", The Journal of Biological Chemistry, vol. 263, No. 14, pp. 6751-6758 (1988). O'Reilly, M. et al., "Treatment of Murine Hemangioendotheliomas With the Angiogenesis Inhibitor AGM-1470", Journal of Pediatric Surgery, vol. 30, No. 2, pp. 325-330, (1995). Nehls, V. et al., "A Novel, Microcarrier-Based in Vitro Assay for Rapid and Reliable Quantification of Three-Dimensional Cell Migration and Angiogenesis", Microvascular Research, vol. 50, pp. 311-322, (1995). Milner-White, E.J., "Predicting the biologically active conformations of short polypeptides", Trends Pharmacol. Sci., vol. 10, pp. 70-74, (1989). Mayer, U. et al., "A single EGF-like motif of laminin is responsible for high affinity nidogen binding", The EMBO Journal vol. 12, pp. 1879-1885, (1993). Mann, K. et al., "Characterization of proteolytic fragments of the laminin-nidogen complex and their activity in ligand-binding assays", Eur. J. Biochem, vol. 178, pp. 71-80, (1988). Lebl, M. et al., "Screening of Completely Random One-Bead One-Peptide Libraries for Activities in Solution", A Companion to Methods in Enzymology, vol. 6, pp. 381-387, (1994). Lam, K. et al., "A new type of synthetic peptide library for identifying ligand-binding activity", Nature, vol. 354, pp. 82-84, (1991). Krchnak, V. et al., "Noninvasive Continuous Monitoring of Solid-Phase Peptide Synthesis by Acid-Base Indicator", Collection Czechoslovak Chem. Commun., vol. 53, pp. 2542-2548, (1988). Kocis, P. et al., "Symmetrical Structure Allowing the Selective Multiple Release of a Defined Quantity of Peptide from a Single Bead of Polymeric Support", Tetrahedron Letters, vol. 34, No. 45, pp. 7251-7252, (1993). Kadoya, Y. et al., "Importance of nidogen binding to laminin .gamma. 1 for branching epithelial morphogenesis of the submandibular gland", Development 124, pp. 683-691, (1997). Kaiser, E. et al., "Color Test for Detection of Free Terminal Amino Groups in the Solid-Phase Synthesis of Peptides", Anal. Biochem., vol. 34, pp. 595-598 (1969). Jain, R. et al., "Quantitative angiogenesis assays: Progress and problems", Nature Medicine, vol. 3, No. 11, pp. 1203-1208, (1997). Hruby, V.J., "Peptide chemistry: Designing peptides pseudopeptides and peptidomimetics for biological receptors", Petides, Proc. 13.sup.th American Petide Symposium, ESCOM, pp. 3-17, (1994). Grobstein, C., "Trans-Filter Induction of Tubules in Mouse Metanephrogenic Mesenchyme", Experimental Cell Research, vol. 10, pp. 424-440, (1956). Grobstein, C., "Epithelio-Mesenchymal Specificity in the Morphogenesis of Mouse Sub-Mandibular Rudiments in Vitro", J. Exp. Zool, vol. 124, pp. 383-413, (1953). Gerl, M. et al., "Localization of a major nidogen-binding site to domain III of laminin B2 chain", Eur. J. Biochem, vol. 202, pp. 167-174, (1991). Furka, A. et al., "General method for rapid synthesis of multicomponent peptide mixtures", Int. J. Peptide Protein, Res., vol. 37, pp. 487-493, (1991). Engel, J., "EGF-like domains in extracellular matrix proteins: localized signals for growth differentiation?", FEBS Letters, vol. 251, No. 1, 2, pp. 1-7, (1989). Ekblom, P. et al., "Role of mesenchymal nidogen for epithelial morphogenesis in vitro", Development, vol. 120, pp. 2003-2014, (1994). Chi, H. et al., "Primary Structure of the Drosophila Laminin B2 Chain and comparison with Human, Mouse, and Drosophila Laminin B1 and B2 Chains", The Journal of Biological Chemistry, vol. 264, No. 3, pp. 1543-1550, (1989). Burgeson, R. et al., "A New Nomenclature for the Laminins", Matrix Biology, vol. 14, pp. 209-211, (1994). Baumgartner, R. et al., "Structure of the Nidogen Binding LE Module of the Lamin .gamma. 1 Chain in Solution", J. Mol. Biol., vol. 257, pp. 658-668, (1996). Fox, J. et al., "Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV", The EMBO Journal, vol. 10, No. 11, pp. 3137-3146, (1991). Adams, J. et al., "Regulation of development and differentiation by the extracellular matrix", Development 117, pp. 1183-1198, (1993). |
| PATENT CLAIMS |
We claim: 1. A compound of formula I ##STR79## in any stereoisomeric form, or a physiologically tolerable salt thereof, or a mixture thereof, wherein R1 is ##STR80## wherein R4 is --A, --NH.sub.2, --NHR, --NR.sub.2, ##STR81## and R5 is --(CH.sub.2).sub.l COOA, --(CH.sub.2).sub.l CONH.sub.2, --(CH.sub.2).sub.l NH.sub.2, --(CH.sub.2).sub.l --SO.sub.3 H, ##STR82## X is ##STR83## wherein Y is O, S, --N(A)--CO-- or --(CH.sub.2).sub.r --, D is (CH.sub.2).sub.r, O, S, NH, NR, (CH.sub.2).sub.r --O, (CH.sub.2).sub.r --S, (CH.sub.2).sub.r --NH or (CH.sub.2).sub.r NR and R2 is --E--OH, --E--COOH or --E--CONH.sub.2, wherein E is a linear or branched C.sub.1 -C.sub.10 -alkyl chain, which is unsubstituted or substituted by --A, --(CH.sub.2).sub.m --OH, --(CH.sub.2).sub.m --COOH, --(CH.sub.2).sub.m --C(O)NA.sub.2 or by a C.sub.5 -C.sub.10 -cycloalkyl group, or E is C.sub.5 -C.sub.10 -cycloalkyl, which is unsubstituted or substituted by --A, --(CH.sub.2).sub.m --OH, --(CH.sub.2).sub.m --COOH, --(CH.sub.2).sub.m --C(O)NA.sub.2 or by a C.sub.5 -C.sub.10 -cycloalkyl group, R3 is ##STR84## wherein R6 is --H, --COOH, --CONH.sub.2, --CONHR, --CONR.sub.2, --CH.sub.2 OH or ##STR85## and wherein R7 is a linear or branched C.sub.1 -C.sub.10 -alkyl group, which is unsubstituted or substituted by --A, --(CH.sub.2).sub.m --OH, --(CH.sub.2).sub.m --COOH, --(CH.sub.2).sub.m --C(O)NA.sub.2 or by a C.sub.5 -C.sub.10 -cycloalkyl group, or R7 is a C.sub.5 -C.sub.10 -cycloalkyl group, which is unsubstituted or substituted by --A, --(CH.sub.2).sub.m --OH, --(CH.sub.2).sub.m --COOH, --(CH.sub.2).sub.m --C(O)NA.sub.2 or by a C.sub.5 -C.sub.10 -cycloalkyl group, and R is branched or unbranched C.sub.1 -C.sub.6 -alkyl, C.sub.2 -C.sub.6 -alkenyl, C.sub.2 -C.sub.6 -alkynyl, C.sub.5 -C.sub.10 -cycloalkyl, Het or Ar which are unsubstituted or substituted by one or more halogen, C.sub.1 -C.sub.6 -alkyloxy, branched or unbranched C.sub.1 -C.sub.6 -alkyl, C.sub.2 -C.sub.6 -alkenyl, C.sub.2 -C.sub.6 -alkynyl or C.sub.5 -C.sub.10 -cycloalkyl groups or by --C.sub.1 -C.sub.6 -alkyl-Het, --C.sub.1 -C.sub.6 -alkyl-Ar, --O--C.sub.1 -C.sub.6 -alkyl-Het, --O--C.sub.1 -C.sub.6 -alkyl-Ar, Het or by Ar, wherein Het is a monocyclic or bicyclic, 5- to 10-membered aromatic or non-aromatic ring containing 1 or 2 equal or different hetero-atoms as members of said ring, the heteroatoms being selected from the group consisting of nitrogen, oxygen and sulfur, which ring is unsubstituted or substituted by one or more hydroxy or carboxy groups, and wherein Ar is a monocyclic or bicyclic 5- to 10-membered aromatic ring which is unsubstituted or substituted by one or more hydroxy or carboxy groups, Z is (CH.sub.2).sub.m, O, S, NH, NR, N--C(O)--R or NSO.sub.2 R, A is H or C.sub.1 -C.sub.4 -alkyl l, m and r are each independently integers from 0 to 3, n and k are each independently integers from 1 to 2, p is an integer from 0 to 1 and q is an integer from 1 to 3. 2. A compound as claimed in claim 1, wherein n is 1. 3. A compound as claimed in claim 1, wherein R in group X is Het or Ar which are unsubstituted or substituted by --C.sub.1 -C.sub.6 -alkyl-Het, --C.sub.1 -C.sub.6 -alkyl-Ar, --O--C.sub.1 -C.sub.6 -alkyl-Het, --O--C.sub.1 -C.sub.6 -alkyl-Ar, Het or by Ar. 4. A compound as claimed in claim 3, wherein R in group X is Het and Het is ##STR86## 5. A compound as claimed in claim 3, wherein R in group X is Ar and Ar is ##STR87## 6. A compound as claimed in claim 1, wherein X is a group of the formula ##STR88## wherein Y is --(CH.sub.2).sub.r -- and r is 0 or 1. 7. A compound as claimed in claim 1, wherein X is a group of the formula ##STR89## wherein D is --(CH.sub.2).sub.r -- and r is 0 or 1. 8. A compound as claimed in claim 1, wherein R1 is a group of the formula ##STR90## wherein Z is (CH.sub.2).sub.m, m is 0 or 1, R5 is --(CH.sub.2).sub.l --COOH or --(CH.sub.2).sub.l --CONH.sub.2, and R4 is --NH.sub.2 or H, and l is 0. 9. A compound as claimed in claim 1, wherein R1 is a group of the formula ##STR91## wherein Z is --(CH.sub.2).sub.m --, m is 1, R4 is --NH.sub.2, R5 is --(CH.sub.2).sub.l --COOH, and l is 0. 10. A compound as claimed in claim 1, wherein R1 is a group of the formula ##STR92## wherein R5 is --(CH.sub.2).sub.l --COOH and l is 0. 11. A compound as claimed in claim 1, wherein R2 is --CH.sub.2 --COOH, or --CH.sub.2 --OH. 12. A compound as claimed in claim 1, wherein R3 is a group of the formula ##STR93## and k is 2. 13. A compound as claimed in claim 1, wherein R3 is ##STR94## 14. A compound as claimed in claim 1, wherein R3 is a group of the formula ##STR95## wherein R7 is --CH(CH.sub.3).sub.2, --C(CH.sub.3).sub.3, --CH(CH.sub.3)CH.sub.2 --CH.sub.3, or --CH.sub.2 --CH(CH.sub.3).sub.2, and R6 is --H, --COOH, --CONH.sub.2, --CH.sub.2 OH, --CON(CH.sub.3).sub.2, or ##STR96## wherein q is 2. 15. A compound as claimed in claim 1, wherein R3 is a group of the formula ##STR97## wherein R7 is --CH(CH(CH.sub.3).sub.2).sub.2 or --CH.sub.2 C(CH.sub.3).sub.3. 16. A compound as claimed in claim 1, wherein R2 is --CH.sub.2 OH. 17. A compound of formula I ##STR98## in any stereoisomeric form, or a physiologically tolerable salt thereof, or a mixture thereof, wherein R1 is ##STR99## wherein R4 is --A, --NH.sub.2, --NHR, --NR.sub.2, ##STR100## and R5 is --(CH.sub.2).sub.l COOA, --(CH.sub.2).sub.l CONH.sub.2, --(CH.sub.2).sub.l NH.sub.2, --(CH.sub.2).sub.l --SO.sub.3 H, ##STR101## X is ##STR102## wherein Y is O, S, --N(A)--CO-- or --(CH.sub.2).sub.r --, D is (CH.sub.2).sub.r, O, S, NH, NR, (CH.sub.2).sub.r --O, (CH.sub.2).sub.r --S, (CH.sub.2).sub.r --NH or (CH.sub.2).sub.r NR and R2 is --CH.sub.3, R3 is ##STR103## wherein R6 is --H, --COOH, --CONH.sub.2, --CONHR, --CONR.sub.2, --CH.sub.2 OH or ##STR104## and wherein R7 is a linear or branched C.sub.1 -C.sub.10 -alkyl group, which is unsubstituted or substituted by --A, --(CH.sub.2).sub.m --OH, --(CH.sub.2).sub.m --COOH, --(CH.sub.2).sub.m --C(O)NA.sub.2 or by a C.sub.5 -C.sub.10 -cycloalkyl group, or R7 is a C.sub.5 -C.sub.10 -cycloalkyl group, which is unsubstituted or substituted by --A, --(CH.sub.2).sub.m --OH, --(CH.sub.2).sub.m --COOH, --(CH.sub.2).sub.m --C(O)NA.sub.2 or by a C.sub.5 -C.sub.10 -cycloalkyl group, and R is branched or unbranched C.sub.1 -C.sub.6 alkyl, C.sub.2 -C.sub.6 -alkenyl, C.sub.2 -C.sub.6 -alkynyl, C.sub.5 -C.sub.10 -cycloalkyl, Het or Ar which are unsubstituted or substituted by one or more halogen, C.sub.1 -C.sub.6 -alkyloxy, branched or unbranched C.sub.1 -C.sub.6 -alkyl, C.sub.2 -C.sub.6 -alkenyl, C.sub.2 -C.sub.6 -alkynyl or C.sub.5 -C.sub.10 -cycloalkyl groups or by --C.sub.1 -C.sub.6 -alkyl-Het, --C.sub.1 -C.sub.6 -alkyl-Ar, --O--C.sub.1 -C.sub.6 -alkyl-Het, --O--C.sub.1 -C.sub.6 -alkyl-Ar, Het or by Ar, wherein Het is a monocyclic or bicyclic, 5- to 10-membered aromatic or non-aromatic ring containing 1 or 2 equal or different hetero-atoms as members of said ring, the heteroatoms being selected from the group consisting of nitrogen, oxygen and sulfur, which ring is unsubstituted or substituted by one or more hydroxy or carboxy groups, and wherein Ar is a monocyclic or bicyclic 5- to 10-membered aromatic ring which is unsubstituted or substituted by one or more hydroxy or carboxy groups, Z is (CH.sub.2).sub.m, O, S, NH, NR, N--C(O)--R or NSO.sub.2 R, A is H or C.sub.1 -C.sub.4 -alkyl l, m and r are each independently integers from 0 to 3, n and k are each independently integers from 1 to 2, p is an integer from 0 to 1 and q is an integer from 1 to 3. 18. A compound as claimed in claim 1, wherein R1 is ##STR105## and wherein R4 is H; Z is (CH.sub.2).sub.m, where m is 0; and R5 is --(CH.sub.2).sub.l COOA, where l is 0 and A is H. 19. A compound as claimed in claim 1, wherein X is ##STR106## wherein D is (CH.sub.2).sub.r and r is 1; and R is ##STR107## 20. A compound as claimed in claim 1, wherein X is ##STR108## wherein D and Y are both (CH.sub.2).sub.r, r is 0; and R is ##STR109## 21. A compound as claimed in claim 1, wherein R3 is ##STR110## and wherein R6 is ##STR111## where q is 2, and R7 is --(CH)CH.sub.3 CH.sub.3. 22. A compound as claimed in claim 1, wherein R3 is ##STR112## and wherein R6 is --CONH.sub.2 and R7 is --(CH)CH.sub.3 CH.sub.3. 23. A compound as claimed in claim 1, wherein the compound is ##STR113## 24. A compound of the formula ##STR114## 25. A pharmaceutical composition, comprising at least one compound as claimed in claim 1 and at least one pharmaceutically acceptable excipient. 26. A method for the treatment of a disease which is related to an increased or unwanted synthesis of basement membranes, which comprises administering to a host in need of the treatment an effective amount of at least one compound as claimed in claim 1. 27. A method as claimed in claim 26, wherein the disease is a late complication of diabetes mellitus; atherosclerosis; cancer; diabetic retinopathia; fibroplasia retrolentalis; or psoriasis. 28. A method as claimed in claim 26, wherein the disease is a fibrosis accompanied by an increased synthesis of basement membranes or their components. 29. A method as claimed in claim 28, wherein the disease is a fibrosis of the liver. 30. A method as claimed in claim 26, wherein the disease is related to a strong inflammatory component. 31. A method as claimed in claim 30, wherein the disease is rheumatoic arthritis; osteoarthritis; or vasculitis. 32. A method as claimed in claim 26, wherein the disease is related to haemangiomes. 33. A method for identifying a compound that inhibits the interaction of laminin and nidogen, which comprises measuring the inhibition of the compound together with a compound as claimed in claim 1 as a competitive inhibitor. 34. A method as claimed in claim 33, wherein the compound to be identified is formulated in a pharmaceutical acceptable form. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
This application claims the benefit of foreign priority to European Application No. 99103869.6, filed on Mar. 1, 1999. This European priority document is incorporated by reference herein. Objects of the present invention are low molecular weight peptide derivatives which are able to act as inhibitors of the interaction between laminin and nidogen (laminin/nidogen interaction), a process for their preparation, pharmaceutical compositions prepared therefrom and their use for preparing pharmaceuticals and for identifying inhibitors of the laminin/nidogen interaction. The association of laminin (an 800 kDa glycoprotein) and nidogen (a 160 kDa glycoprotein) is regarded as a crucial biomolecular mechanism in the synthesis and stabilization of basement membranes (Mayer, U. and Timpl, R. (1994) in: Extracellular Matrix Assembly and Structure (P. D. Yurchenco, D. Birk and R. P. Mecham, Ed.) S. 389-416, Academic Press, Orlando, Fla.). The ability of nidogen to form ternary complexes with all main constituents of the basement membrane such as, for example, .gamma.1-containing laminin isoforms (for nomenclature see: Burgeson, R. E.; Chiquet, M.; Deutzmann, R.; Ekblom, P.; Engel, J.; Kleinmann, H.; Martin, G. R.; Meneguzzi, G.; Paulsson M.; Sanes, J.; Timpl, R.; Tryggvasson, K.; Yamada, Y.; Yurchenco, P. D. (1994) Matrix Biology 14; 209-211), collagen IV, perlecan and fibulin, and the association structures of each of them, means that it assumes the function of a linker which connects together, spatially organizes and stabilizes the independent macrostructures (Fox, J. W.; Mayer, U.; Nischt, R.; Aumailley, M.; Reinhardt, D.; Wiedemann, H.; Mann, K.; Timpl, R.; Krieg, T., Engel, J.; and Chu, M.-L. (1991) EMBO J. 10, 3137-3146). Basement membranes are highly specialized extracellular structures which are attributed with important functions in the control of cell and tissue functions, tissue architecture, tissue interactions, cell growth, cell transformation, cell migration and in tissue-specific gene expression (Adams, J. C. and Waft, F. M. (1993) Development 117, 1183-1198). Experiments with polyclonal antilaminin antibodies have provided clear evidence of the central function of the laminin/nidogen interaction in the synthesis of a functional basement membrane. The described antibodies were obtained by immunizing rabbits with laminin P1 or with the recombinantly produced nidogen-binding domain of laminin (.gamma.1 III 3-5). The antibodies concentrated by affinity chromatography on laminin P1 or laminin .gamma.1 III 3-5 matrices showed complete inhibition of the laminin/nidogen association in inhibition assays. However, this is based on steric blockade of the access of nidogen to laminin by the antibodies, whose binding regions are located in the vicinity of the nidogen-binding sequences of laminin (Mayer, U.; Nischt, R.; Poschl, E.; Mann, K.; Fukuda, K.; Gerl, M.; Yamada, Y.; Timpl, R. (1993) EMBO J. 12; 1879-1885). In embryonic organ cultures, the described antibodies inhibited both the development of renal tubules, the formation of pulmonary alveoli and the morphogenesis of the embryonic salivary gland. These three models are representative of ontogenesis programs which depend on unimpeded synthesis of new basement membrane (Ekblom, P.; Ekblom, M.; Fecker, L.; Klein, G.; Zhang, H.-Y.; Kadoya, Y.; Chu, M.-L.; Mayer, U.; Timpl, R. (1994) Development 120; 2003-2014). Antibodies directed against the laminin .gamma.1 chain sequence region which is essential for nidogen binding are likewise able to inhibit the laminin/nidogen association. The inhibition is, however, competitive, in contrast to the antilaminin antibodies described above, because they compete directly with the nidogen for the binding site on laminin (WO 98/31709). A monoclonal antibody of the IgM subclass (antilaminin P1 A6/2/4-DSM ACC2327; see WO 98/31709) inhibits the laminin/nidogen interaction in vitro with an IC50 of 30 nM. Like the polyclonal antilaminin antibody preparation described above, it prevents the morphogenesis of the embryonic salivary gland in organ culture. This underlines the specificity of the laminin/nidogen interaction, and the importance of the LE-4 module and of the identified sequence region in the laminin .gamma.1 III 4 domain in this interaction. The nidogen binding domain of laminin has been unambiguously identified and characterized in terms of its location, sequence and its spatial structure (X-ray crystal structure and NMR structure) (Gerl, M.; Mann, K.; Aumailley, M.; Timpl, R. (1991) Eur. J. Biochem. 202; 167-174. Mayer, U.; Nischt, R.; Poschl, E.; Mann, K.; Fukuda, K.; Gerl, M.; Yamada, Y.; Timpl, R. (1993) EMBO J. 12; 1879-1885. Baumgartner, R.; Czisch, M.; Mayer, U.; Poschl, E.; Huber, R.; Timpl, R.; Holak, T. A. (1996) J. Mol. Biol. 257; 658-668. Stetefeld, J.; Mayer, U.; Timpl, R.; Huber, R. (1996) J. Mol. Biol. 257; 644-657). It is located in an "LE module" (laminin type epidermal growth factor-like) of the short arm of the .gamma.1 chain of laminin, in the domain .gamma.1 III 4. "LE modules" are structural motifs of 50-60 amino acids which have a complex folding pattern, analogous to EGF, with 4 disulfide bridges (Bairoch, A.; (1995) Nomenclature of extracellular domains. The SWISS-PROT Protein sequence data bank. release 310. Engel, J. (1989) FEBS Letters 251; 1-7). High-affinity binding of nidogen to the complementary laminin domain has been detected for laminin P1 from the EHS tumor of mice, laminin 2 and laminin 4 from human placenta and laminin from drosophila. The cause of this species-overlapping binding specificity is the extremely large identity of sequences present in the .gamma.1 III 4 domain for the species investigated. It is 97% between human and mouse, 61% between mouse and drosophila and, astonishingly, 51% between mouse and Caenorhabditis elegans when the whole domain is taken into account (Pikkarinen, T.; Kallunki, T.; Tryggvasson, K. (1987) J. Biol. Chem. 263; 6751-6758. Chi, H.-C.; Hui, C.-F. (1989) J. Biol. Chem. 264; 1543-1550. Wilson, R. et al.(1994) Nature 368: 32-38. Poschl, E.; Mayer, U.; Stetefeld, J.; Baumgartner, R.; Holak, T. A.; Huber, R.; Timpl, R. (1996) EMBO J. 15: 5154-5159). Besides the dependency of nidogen binding on an intact three-dimensional structure, unambiguous sequence regions located in the S--S stabilized loops a and c of the domain .gamma.1 III 4 have been identified. Five essential amino acids have been identified, four located inside a section of 7 amino acids in loop a, and a tyrosine side-chain in loop c (Mann, K.; Deutzmann, R.; Timpl, R. (1988) Eur. J. Biochem. 178; 71-80). Synthetic peptides which can be derived from the appropriate regions of the .gamma.1 III 4 domain and are able to inhibit completely the laminin/nidogen binding in specific binding assays have been disclosed by J. W. Fox and R. Timpl (U.S. Pat. No. 5,493,008). The high-affinity binding to the laminin binding site of nidogen is thought to require an interaction with a tyrosine or histidine from a loop (loop c) adjacent to the actual binding sequence. This aromatic interaction was postulated as a precondition for inhibition in the IC50 range <500 nM on the basis of the 3D structure of the laminin .gamma.1 III 3-5 and as a result of the structure/function relations described in the U.S. Pat. No. 5,493,008. The question of whether loop c interacts directly with the nidogen, or whether it makes a contribution to stabilizing the suitable spatial structure of the NIDPNAV (SEQ ID NO:1) sequence region remained unclarified, however (Poschl, E.; Fox, J. W.; Block, D.; Mayer, U.; Timpl, R, (1994) EMBO J. 13; 3741-3747. Baumgartner, R.; Czisch, M.; Mayer, U.; Poschl, E.; Huber, R.; Timpl, R.; Holak, T. A. (1996) J. Mol. Biol. 257; 658-668. Stetefeld, J.; Mayer, U.; Timpl, R.; Huber, R. (1996) J. Mol. Biol. 257; 644-657). The laminin/nidogen interaction is influenced by a strong conformational component (Mayer, U.; Nischt, R.; Poschl, E.; Mann, K.; Fukuda, K.; Gerl, M.; Yamada, Y.; Timpl, R. (1993) EMBO J. 12; 1879-1885. Mann, K.; Deutzmann, R.; Timpl, R. (1988) Eur. J. Biochem. 178; 71-80). The synthetic peptides which can be derived from the nidogen binding site of laminin are not able to form a disulfide linkage pattern as is present in LE modules, but they show an activity in inhibition assays which is about 400-10,000-fold weaker than that of intact laminin P1 or laminin .gamma.1 III 3-5 (Poschl, E.; Fox, J. W.; Block, D., Mayer, U.; Timpl, R, (1994) EMBO J. 13; 3741-3747. J. W. Fox and R. Timpl; U.S. Pat. No. 5,493,008). This decline in activity is not unusual, since it is known that peptides may assume a myriad of different conformations in aqueous solution and that only a certain percentage of peptides is to be found in the biologically active conformation. The most active peptide described to date (IC50 of 22 nM) has a molecular weight of about 2700 Da (.congruent.about 50% of an LE module). It comprises an intact S--S loop which presumably stabilizes the structure of the essential NIDPNAV (SEQ ID NO:1) sequence region (Poschl, E.; Fox, J. W.; Block, D.; Mayer, U.; Timpl, R, (1994) EMBO J. 13; 3741-3747. J. W. Fox and R. Timpl; U.S. Pat. No. 5,493,008). The chemical formula of the sequence NIDPNAV (Asn-lie-Asp-Pro-Asn-Ala-Val) (SEQ ID NO:1) is as follows: ##STR1## Inhibitors of the laminin/nidogen interaction should be suitable for preparing pharmaceuticals for diseases which are related to an increased or unwanted synthesis of basement membranes. Such diseases are e.g. all types of late complications of diabetes which are accompanied by thickening of basement membranes (especially in the kidney, eye, vascular system), hepatic fibrosis, especially alcoholic hepatic fibrosis, characterized by synthesis of a continuous basement membrane in the sinusoids and a capillarization caused thereby, all fibroses (chronic or iatrogenic) in which an increased synthesis of basement membrane or components of the basement membrane can be observed (kidney, lung, skin), atherosclerosis characterized by a limitation of the regulation of lipid metabolism, which may be caused inter alia by impaired filtration of lipoproteins through the partly capillarized liver sinusoids (the pathological changes in the vascular system which can be observed with atherosclerosis may also in part be attributed to modifications of the composition and structure of the basement membranes in the vessels), diseases in which angiogenesis contributes to a deterioration in the clinical picture, for example cancers in which neovascularization is required for tumor growth, diabetic retinopathy, retrolental fibroplasia, disorders with a strong inflammatory component (for example rheumatoid arthritis, osteoarthritis, vasculitis), hemangiomas, psoriasis, and many others. The use of peptides like those described in U.S. Pat. No. 5,493,008 as medicine is however limited to a considerable extent because of their conformational flexibility, their instability to proteases and their poor bioavailability and pharmacodynamics (Milner-White, E. J. (1989) Trends Pharmacol. Sci. 10; 70-74. Verber, D. F.; Freidinger, R. M.; (1985) Trends Neurosci. 8; 392-396. Hruby, V. J. (1994) in: Peptides, Proc. Thirteenth American Peptide Symposium; (Hodges, R. S.; Smith, J. A.; Ed.) S. 3-17; ESCOM: Leiden, Netherlands). An object of this application was thus to find low molecular weight peptide derivatives which are able to interact specifically with the laminin binding site of nidogen and to inhibit competitively the association between laminin and nidogen at low concentration. Therefore, an object of the present invention is a compound of the formula I ##STR2## wherein R1 is a group of one of the following formulae ##STR3## wherein R4 means --A, --NH.sub.2, --NHR, --NR.sub.2, A.sub.2, --NHR1, ##STR4## and R5 means --(CH.sub.2).sub.l COOA, --(CH.sub.2).sub.l CONH.sub.2, --(CH.sub.2).sub.l NH.sub.2 or --(CH.sub.2).sub.l --SO.sub.3 H, ##STR5## and X is a group of one of the following formulae ##STR6## wherein Y means O, S, --N(A)--CO-- or --(CH.sub.2).sub.r --, D means (CH.sub.2).sub.r, O, S, NH, NR, (CH.sub.2).sub.r --O, (CH.sub.2).sub.r --S, (CH.sub.2).sub.r --NH or (CH.sub.2).sub.r NR and R2 means --A, --E--OH, --E--COOH or --E--CONH.sub.2, wherein E means a linear or branched C.sub.1 -C.sub.10 -alkyl chain, which is unsubstituted or substituted by --A, --(CH.sub.2).sub.m --OH, --(CH.sub.2).sub.m --COOH, --(CH.sub.2).sub.m --C(O)NA.sub.2 or by a C.sub.5 -C.sub.10 -cycloalkyl group, or E means C.sub.5 -C.sub.10 -cycloalkyl, which is unsubstituted or substituted by --A, --(CH.sub.2).sub.m --OH, --(CH.sub.2).sub.m --COOH, --(CH.sub.2).sub.m --C(O)NA.sub.2 or by a C.sub.5 -C.sub.10 -cycloalkyl group, and R3 is a group of one of the following formulae ##STR7## wherein R6 means --H, --COOH, --CONH.sub.2, --CONHR, --CONR.sub.2, --CH.sub.2 OH or ##STR8## and wherein R7 means a linear or branched C.sub.1 -C.sub.10 -alkyl group, which is unsubstituted or substituted by --A, --(CH.sub.2).sub.m --OH, --(CH.sub.2).sub.m --COOH, --(CH.sub.2).sub.m --C(O)NA.sub.2 or by a C.sub.5 -C.sub.10 -cycloalkyl group, or R7 means a C.sub.5 -C.sub.10 -cycloalkyl group, which is unsubstituted or substituted by --A, --(CH.sub.2).sub.m --OH, --(CH.sub.2).sub.m --COOH, --(CH.sub.2).sub.m --C(O)NA.sub.2 or by a C.sub.5 -C.sub.10 -cycloalkyl group, and R means branched or unbranched C.sub.1 -C.sub.6 -alkyl, C.sub.2 -C.sub.6 -alkenyl, C.sub.2 -C.sub.6 -alkinyl, C.sub.5 -C.sub.10 -cycloalkyl, Het or Ar which are optionally substituted by one ore more halogen, C.sub.1 -C.sub.6 -alkyloxy, branched or unbranched C.sub.1 -C.sub.6 -alkyl, C.sub.2 -C.sub.6 -alkenyl, C.sub.2 -C.sub.6 -alkinyl or C.sub.5 -C.sub.10 -cycloalkyl groups or by --C.sub.1 -C.sub.6 -alkyl-Het, --C.sub.1 -C.sub.6 -alkyl-Ar, --O--C.sub.1 -C.sub.6 -alkyl-Het, --O--C.sub.1 -C.sub.6 -alkyl-Ar, Het or by Ar, wherein Het means a monocyclic or bicyclic, 5- up to 10-membered aromatic or non-aromatic ring containing 1 or 2 equal or different hetero-atoms as members of said ring, selected from the group consisting of nitrogen, oxygen and sulfur, which is unsubstituted or substituted by one or more hydroxy or carboxy groups, and wherein Ar means a monocyclic or bicyclic 5- up to 10-membered aromatic ring which is unsubstituted or substituted by one or more hydroxy or carboxy groups, and Z means (CH.sub.2).sub.m, O, S, NH, NR, N--C(O)--R or NSO.sub.2 R, A means H or C.sub.1 -C.sub.4 -alkyl and l, m and r are integers from 0 to 3, n and k are integers from 1 to 2, p is an integer from 0 to 1 and q is an integer from 1 to 3, in all its stereoisomeric forms and mixtures thereof in all ratios including all its physiologically tolerable salts. Physiologically tolerable salts are for example salts of inorganic and organic acids, e.g. hydrochloric acid, sulfuric acid, acetic acid, citric acid or p-toluenesulfonic acid, or salts of inorganic and organic bases, such as NH.sub.4 OH, NaOH, KOH, Ca(OH).sub.2, Mg(OH).sub.2, diethanolamine or ethylenediamine, or salts of amino acids, such as arginine, lysine, lysyl-lysine or glutamic acid. One preferred embodiment of the present invention is a compound of formula I wherein n is 1. A further preferred embodiment is a compound of formula I wherein R in group X means Het or Ar which are optionally substituted by --C.sub.1 -C.sub.6 -alkyl-Het, --C.sub.1 -C.sub.6 -alkyl-Ar, --O--C.sub.1 -C.sub.6 -alkyl-Het, --O--C.sub.1 -C.sub.6 -alkyl-Ar, Het or by Ar. More preferably, R in group X means Het. For example Het means ##STR9## A preferred embodiment of the present invention is also compound of formula I wherein R in group X means Ar which is optionally substituted by --C.sub.1 -C.sub.6 -alkyl-Ar, --O--C.sub.1 -C.sub.6 -alkyl-Ar or by Ar. Preferably R in group X means Ar. For example Ar means ##STR10## A preferred embodiment is also a compound of formula I wherein R in group X means ##STR11## In the compound of formula I X is preferably a group of the following formula: ##STR12## Preferably, Y means --(CH.sub.2).sub.r, wherein r is preferably 0 or 1 and k is preferably 1 or 2. A further preferred embodiment of the present invention is a compound of formula I wherein X is a group of the following formula ##STR13## wherein D preferably means --(CH.sub.2).sub.r --, wherein r is 0 or 1. An also preferred embodiment of the present invention compound of formula I wherein R1 is a group of the following formula ##STR14## wherein Z means preferably (CH.sub.2).sub.m and m is 0 or 1. Preferably, R5 means --(CH.sub.2).sub.l --COOA, wherein A means preferably H, or R5 means --(CH.sub.2).sub.l --COONH.sub.2, wherein l is 0. Preferably, R4 means --NH2 or --A, wherein A preferably means H, or preferably, R4 means --NHR1, wherein --NHR1 preferably means ##STR15## and wherein R5 of --NHR1 preferably means ##STR16## and l is preferably 0, or R5 of --NHR1 preferably means ##STR17## and l is preferably 0, or R5 of --NHR1 means preferably (CH.sub.2).sub.l --NH.sub.2 and l is preferably 0. A further preferred embodiment of the present invention is a compound A compound of formula I wherein R1 is a group of the following formula ##STR18## wherein Z means preferably --(CH.sub.2).sub.m -- and m is preferably 1 and wherein R4 preferably means --NH.sub.2, and R5 preferably means --(CH.sub.2).sub.l --COOA, wherein l is preferably 0 and wherein A preferably means H. A further preferred embodiment of the present invention is a compound of formula I wherein R1 is a group of the following formula ##STR19## wherein R5 preferably means --(CH.sub.2).sub.l --COOA, wherein l is preferably 0 and A preferably means H. A further preferred embodiment of the present invention is a compound of formula I wherein R2 means A and A preferably means --CH.sub.3, or wherein R2 means --E--COOH, preferably --CH.sub.2 --COOH, or wherein R2 means --E--OH, preferably --CH.sub.2 --OH. A further preferred embodiment of the present invention is a compound of formula I wherein R3 is a group of the following formula ##STR20## wherein k is preferably 2. A further preferred embodiment of the present invention is a compound of formula I wherein R3 is a group of the following formula ##STR21## A further preferred embodiment of the present invention is a compound of formula I wherein R3 is a group of the following formula ##STR22## wherein R7 is preferably a branched C.sub.1 -C.sub.10 -alkyl group, preferably --CH(CH.sub.3).sub.2, --C(CH.sub.3).sub.3, --CH(CH.sub.3)CH.sub.2 --CH.sub.3 or --CH.sub.2 --CH(CH.sub.3).sub.2, and wherein R6 preferably means --H, --COOH, --CONH.sub.2, --CH.sub.2 OH, --CON(CH.sub.3).sub.2 or, more preferably, wherein R6 means ##STR23## wherein q is preferably 2. A further preferred embodiment of the present invention is a compound of formula I wherein R3 is a group of the following formula ##STR24## wherein R7 preferably means --CH(CH(CH.sub.3).sub.2).sub.2 or --CH.sub.2 C(CH.sub.3).sub.3. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates a differentially cleavable linker. FIG. 2 illustrates the structures of compounds 1 to 4. FIG. 3 illustrates the structures of compounds 5 to 8. FIG. 4 illustrates the structures of compounds 9 to 12. FIG. 5 illustrates the structures of compounds 13 to 16. FIG. 6 illustrates the structures of compounds 17 to 20. FIG. 7 illustrates the structures of compounds 21 to 24. FIG. 8 illustrates the structures of compounds 25 to 28. FIG. 9 illustrates the structures of compounds 29 to 32. FIG. 10 illustrates the structures of compounds 33 to 36. FIG. 11 illustrates the structures of compounds 37 to 40. FIG. 12 illustrates the structures of compounds 41 to 43. The compounds according to the present invention are unnatural (i.e. naturally not occurring), low molecular weight peptide derivatives which are able to inhibit the laminin/nidogen interaction in the nM concentration range. Surprisingly, the low molecular weight structures which have been found are capable of high-affinity binding to the laminin binding site of nidogen without this requiring an interaction with a tyrosine or histidine from a loop (loop c) adjacent to the actual binding sequence. It is all the more surprising that the low molecular weight peptide derivatives, with molecular weights between 550 and 800 Da, described in the present invention show inhibition of the same order of magnitude compared to the most active peptide described to date (IC50 of 22 nM) having a molecular weight of about 2700 Da (.congruent.about 50% of an LE module) and comprising an intact S--S loop which presumably stabilizes the structure of the essential NIDPNAV (SEQ ID NO:1) sequence region (J. W. Fox and R. Timpl; U.S. Pat. No. 5,493,008). The object was achieved by specifically synthesizing, on the basis of structure/function relationships and the published three-dimensional structure of the nidogen binding site, peptide derivatives on resin supports. The building blocks for the peptide syntheses were varied in accordance with suitable criteria to ensure a wide structural diversity and the integration of unnatural building blocks. A suitable, sensitive screening assay was used to test and compare the resulting peptide derivatives for inhibitory activity after they had been cleaved off the support resin. The compounds according to the present invention can be used for preparing a pharmaceutical for the treatment of a disease which is related to an increased or unwanted synthesis of basement membranes. Therefore, possible areas of therapeutic use of the present peptide derivatives and/or the physiologically tolerable salts thereof are: 1. All types of late complications of diabetes which are accompanied by thickening of basement membranes (especially in the kidney, eye, vascular system). 2. Hepatic fibrosis, especially alcoholic hepatic fibrosis, characterized by synthesis of a continuous basement membrane in the sinusoids and a capillarization caused thereby. 3. All fibroses (chronic or iatrogenic) in which an increased synthesis of basement membrane or components of the basement membrane can be observed (kidney, lung, skin). 4. Atherosclerosis characterized by a limitation of the regulation of lipid metabolism, which may be caused inter alia by impaired filtration of lipoproteins through the partly capillarized liver sinusoids. The pathological changes in the vascular system which can be observed with atherosclerosis may also in part be attributed to modifications of the composition and structure of the basement membranes in the vessels. 5. Diseases in which angiogenesis contributes to a deterioration in the clinical picture, for example cancers in which neovascularization is required for tumor growth, diabetic retinopathy, retrolental fibroplasia, disorders with a strong inflammatory component (for example rheumatoid arthritis, osteoarthritis, vasculitis), hemangiomas, psoriasis, and many others. Thus, the compounds according to the present invention and/or their respective physiologically tolerable salts are suitable for use as a pharmaceutical. Therefore, a further object of the present invention is a pharmaceutical composition containing at least one compound according to the present invention and/or its physiologically tolerable salts. The compounds of the formula I and their physiologically tolerable salts and derivatives can be administered according to the invention to animals, preferably to mammals, and in particular to humans, as pharmaceuticals for therapy or prophylaxis. They can be administered per se, in mixtures with one another or in the form of pharmaceutical preparations which permit enteral or parenteral administration and which as active constituent contain an efficacious dose of at least one compound of the formula I and/or its physiologically tolerable salts and derivatives in addition to customary pharmaceutically innocuous excipients and/or additives. The pharmaceuticals can be administered systemically or locally. They can be administered, for example, in the form of pills, tablets, film-coated tablets, sugar-coated tablets, granules, hard and soft gelatin capsules, powders, solutions, syrups, emulsions, suspensions or in other pharmaceutical forms. However, administration can also be carried out vaginally or rectally, for example in the form of suppositories, or parenterally or by implantation, for example in the form of injection solutions or infusion solutions, microcapsules or rods, or topically or percutaneously, for example in the form of ointments, solutions or tinctures, or in another way, for example in the form of nasal sprays or aerosol mixtures or as inhalable dry powder preparations. If solutions are parenterally administered they can be administered, for example, intravenously, intramuscularly, subcutaneously, intraarticularly, intrasynovially or in another manner, e.g. by inhalation of wet aerosols or dry powder preparations. The pharmaceutical preparations according to the invention are prepared in a manner known per se, it being possible to use pharmaceutically inert inorganic and/or organic excipients in addition to the compound(s) of the formula I and/or its/their physiologically tolerable salts and derivatives. For the preparation of pills, tablets, sugar-coated tablets and hard gelatin capsules, it is possible to use, for example, lactose, cornstarch or derivatives thereof, talc, stearic acid or its salts etc. Excipients for soft gelatin capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, polyethylene glycols, natural or hardened oils etc. Suitable excipients for the preparation of solutions, for example injection solutions, or of emulsions or syrups are, for example, water, alcohols, glycerol, diols, polyols, sucrose, invert sugar, glucose, vegetable oils etc. Suitable excipients for microcapsules, implants or rods are, for example, copolymers of glycolic acid and lactic acid. The pharmaceutical preparations normally contain approximately 0.5 to 90% by weight of the compounds of the formula I and/or their physiologically tolerable salts and derivatives. In addition to the active compounds and excipients, the pharmaceutical preparations can additionally contain auxiliaries or additives, such as, for example, fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavorings or aromatizers, thickeners, diluents, buffer substances, solvents or solubilizers, means for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants. They can also contain two or more compounds of the formula I and/or their physiologically tolerable salts and derivatives. Furthermore, they can also contain one or more other therapeutically or prophylactically active substances in addition to at least one compound of the formula I and/or its physiologically tolerable salts and derivatives. The pharmaceutical preparations normally contain 0.2 to 500 mg, preferably 1 to 100 mg, of active compound of the formula I and/or its physiologically tolerable salts and derivatives per dose. If the compounds of the formula I or pharmaceutical preparations containing them are administered as aerosols, for example as nasal aerosols or by wet aerosols or dry powder inhalation, this can be effected, for example, using a spray, an atomizer, a pump atomizer, an inhalation apparatus, a metered inhaler or a dry powder inhaler, respectively. Pharmaceutical forms for administration of the compounds of the formula I as an aerosol can be prepared by the process well known to the person skilled in the art. For their preparation, for example, solutions or dispersions of the compounds of the formula I in water, water-alcohol mixtures or suitable saline solutions using customary additives, for example benzyl alcohol or other suitable preservatives, absorption enhancers for increasing the bioavailability, solubilizers, dispersants and others, and, if appropriate, customary propellants, for example chlorofluorohydrocarbons and/or fluorohydrocarbons are suitable, whereas dry powder preparations of the compounds of the formula I and/or their physiologically tolerable salts may be obtained by freeze drying or preferably spray drying aqueous solutions of the compounds of the formula I and/or their physiologically tolerable salts and of suitable water soluble additives, such as sugars or sugar derivatives and amino acids. The dose when using the compounds of the formula I can vary within wide limits, and as customary it is to be tailored to the individual conditions in each individual case, as is known to the physician. It depends, for example, on the nature and severity of the disease to be treated, on the compound employed or whether an acute or chronic disease state is treated or prophylaxis is conducted or on whether further active compounds are administered in addition to the compounds of the formula I. In general, in the case of oral administration, a daily dose of approximately 0.01 to 100 mg/kg, preferably 0.1 to 10 mg/kg, in particular 0.3 to 2 mg/kg (in each case per kg of body weight) is appropriate in an adult to achieve effective results. In the case of intravenous administration, the daily dose is in general approximately 0.01 to 50 mg/kg, preferably 0.01 to 10 mg/kg of body weight. In particular when relatively large amounts are administered, the daily dose can be divided into a number, for example 2, 3 or 4, of part administrations. If appropriate, depending on individual behavior, it may be necessary to deviate upward or downward from the indicated daily dose. Furthermore, the compounds of the formula I and their salts according to the present invention can be used as intermediates for the preparation of other compounds, in particular of other pharmaceutical active compounds which are obtainable from compounds of the formula I, for example, by modification or introduction of radicals or functional groups, for example by esterification, reduction, oxidation or other conversions of functional groups. The peptide derivatives according to the present invention thus found can on the one hand be used directly as therapeutic agent, but they can also form the basis for related structures, which are also suitable for use as therapeutic agent for treating diseases relating to an increased or unwanted synthesis of basement membranes. A further object of the present invention is a method for identifying a compound that inhibits the interaction of laminin and nidogen wherein the compound according to the present invention is used as a competetive inhibitor. This method may further comprise the formulation of the compound identified in a pharmaceutical acceptable form. It is also an object of the present invention to provide a method for producing a pharmaceutical composition comprising the identification of a compound that inhibits the interaction of laminin and nidogen wherein the compound according to the present invention is used as a competetive inhibitor and furthermore mixing the compound identified and/or its physiologically tolerable salts with a pharmaceutical acceptable carrier. It is also an object of the present invention to provide a method for preparing the compound of the formula I according to the present invention. The compound of formula I ##STR25## according to the present invention is prepared by a fragment condensation of a compound of formula II ##STR26## with a compound of formula III ##STR27## wherein the variables R1, X, n, R2 and R3 have the above-mentioned meanings and whereby the compounds of formulae II and III may be protected at the functional groups defined above by usual protecting groups known in peptide chemistry (see for example Houben-Weyl, Methoden der Organischen Chemie, vol. 15/1 and 15/2, Georg Thieme Verlag, Stuttgart, 1974). Suitable condensation methods are well known in the art (Houben-Weyl, Methoden der Organischen Chemie, vol. 15/1 and 15/2, Georg Thieme Verlag, Stuttgart, 1974). Suitable condensation agents or coupling reagents are for example carbonyl-diimidazoles, carbodiimides, such as di-cyclohexyl-carbodiimide or di-isopropyl-carbodiimide, or O-((cyano(ethoxycarbonyl)methylene)-amino)-N,N,N',N'-tetra-methyl-uronium- tetrafluoro-borate (TOTU) or pyro-phosphoric acid anhydride (PPA). The condensation reactions are carried out under standard conditions. As a rule, it is necessary during peptide condensation to protect amino groups which are not intended to be involved in the coupling reaction by protecting groups which are easily removed under conditions different to the conditions under which coupling occurs. The same applies for the carboxy groups not involved in the coupling reaction, which are preferably protected as C.sub.1 -C.sub.6 -alkyl esters, benzyl esters or tert-butyl esters during the coupling reaction. A protection of the amino groups is not necessary in case the amino groups are still present in the form of amino group precursors, e.g. in form of nitro or cyano groups. The amino groups are then formed by a hydration step subsequent to the condensation reaction. After the condensation step the protecting groups are removed by known suitable methods, e.g. benzyloxy-carbonyl and benzyl groups can be removed by hydration in benzyl esters; protecting groups of the tert-butyl type are in general cleaved under acidic conditions; the 9-fluorenylmethyloxycarbonyl residue is removed by secondary amines. The preparation of the compound of the formula I according to the present invention may also be performed by stepwise addition of the respective components, e.g. natural, unnatural amino acids and their derivatives, on a solid phase, whereby the components may be added in various different sequences. It may also be advantageous in order to produce the compound of formula I not to directly couple the compounds of formulae I and II by a fragment condensation but to couple their respective suitable precursors in order to obtain an intermediate which can be transferred into the compound of the formula I e.g. by derivatization. The above described method for introducing functional groups not directly, but by the way of their respective precursors into the molecule in order to obtain intermediates from which the final product can easily be obtained by transforming the precursor groups into the respective functional groups subsequently to a condensation reaction may also be applied for different parts of the molecule of the compound of formula I, e.g. for the side chain of the compound of formula, I R1- or R1-X-, respectively. EXAMPLES The abbreviations have the following meanings: Agents and solvents: AcOH acetic acid aq aqueous BSA bovine serum albumin DCC N,N'-dicyclohexylcarbodiimide DCM dichloromethane DIPEA N,N-diisopropylethylamine DMAP 4-dimethylaminopyridine DMF N,N-dimethylformamide DMSO Dimethylsulfoxide Et.sub.2 O Diethylether EtOAc Ethylethanoate (acetic acid ethylester) EtOH ethanol Fmoc-OSucc Fmoc-O-succinimide HOBT 1-hydroxybenzotriazole KHMDS potassiumhexamethyldisilazide n-Buli n-butyl-lithium MeOH methanol MTBE methyl tert-butyl ether TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TMEDA tetramethylethylendiamine TMSCI trimethylsilyl chloride TOTU O-((cyano(ethoxycarbonyl)methylene)amino)- N,N,N',N'-tetramethyluronium tetrafluoroborate TrisN.sub.3 trisilyl azide Chemical groups: Me methyl CH.sub.3 -- Et ethyl CH.sub.3 --CH.sub.2 -- nPr n-propyl CH.sub.3 CH.sub.2 CH.sub.2 -- iPr isopropyl (CH.sub.3).sub.2 CH-- nBu n-butyl CH.sub.3 CH.sub.2 CH.sub.2 CH.sub.2 -- iBu isobutyl (CH.sub.3).sub.2 CHCH.sub.2 -- tBu tert-butyl (CH.sub.3).sub.3 C-- Ph phenyl C.sub.6 H.sub.5 -- Fmoc 9-fluorenylmethoxycarbonyl Z benzyloxycarbonyl C.sub.6 H.sub.5 --CH.sub.2 --O--CO-- BOC tert-butyloxycarbonyl (CH.sub.3).sub.3 C--O--CO-- 1. Screening of a library of inhibitors of Laminin/Nidogen interaction The library was designed to find smaller, more potent and more metabolically stable peptides related to the previously known heptapeptide NIDPNAV (SEQ ID NO:1) (Poschl, E.; Fox, J. W.; Block, D.; Mayer, U.; Timpl, R, (1994) EMBO J. 13; 3741-3747. Poschl, E.; Mayer, U.; Stetefeld, J.; Baumgartner, R.; Holak, T. A.; Huber, R.; Timpl, R. (1996) EMBO J. 15: 5154-5159. Baumgartner, R.; Czisch, M.; Mayer, U.; Poschl, E.; Huber, R.; Timpl, R.; Holak, T. A. (1996) J. Mol. Biol. 257; 658-668). The library was synthesized and screened as three sublibraries; pentamer, hexamer and heptamer. Following is a description of the screening strategy for the pentamer sublibrary. The method is representative of the methods employed for the other two sublibraries, except that the hexamers were screened in the first step at about 50 beads per well and the heptamers were screened at about 100 beads per well. 1.1 Screening of the pentamer library. The pentamer library contained 2,160 different compounds. 1) About 8,800 individual beads were suspended in 0.1% HCl and distributed into seven filter bottom 96 well microtiter plates at approximately fourteen beads per well. 2) The beads were washed twice with 200 .mu.l de-ionized water, then 50 .mu.l of 500 mM HEPES, pH 7.0 was added. The linker used in the library releases one aliquot of compound when the pH is increased to 7.0, and this cleavage step was allowed to proceed overnight. 3) The plates were stacked on top of U-bottom filter plates and centrifuged. The mixtures of compounds released from the beads were collected in the bottom plate, while the corresponding beads remain in the original filter plate. 4) 25 .mu.l DMSO per well was added to the beads to wash remaining free compound from the beads, and the plates were centrifuged again to separate the compounds in solution from the beads. The resulting stock was presumably 27 .mu.M per compound in 333 mM HEPES, 33% DMSO. 5) The compound stocks were preincubated with nidogen (10 .mu.l compound stock to 90 .mu.l nidogen solution) and the assay was performed as described in the attached protocol, yielding a final screening concentration of 2.7 .mu.M per compound. 6) In the 25 assay wells where reproducible inhibition of 62% occurred, the corresponding beads from the original filter plates were suspended in 0.05% HCl, 0.1% Tween-20 and pipetted into five new filter plates at 1 bead per well. Two control beads with the parent compound on the same linker were added to each plate as controls. 7) The beads were washed twice with 200 .mu.l de-ionized water, then 25 .mu.l of 50 mM NaOH was added to each well. The linker used in the library releases the second equimolar aliquot of compound when the pH is increased from 7.0 to 10.0 or more. This cleavage step was allowed to proceed for 3 hours. 8) The plates were stacked on top of U-bottom filter plates and centrifuged. The compounds released from the beads were collected in the bottom plate, while the corresponding beads remained in the original filter plate. 9) The beads were washed with 20 .mu.l of 50 mM HEPES (initial pH 7.0) with 50 mM HCl added, and the solution was centrifuged into the lower plate and combined with the first releasate. 10) beads were washed a third time with 25 .mu.l DMSO, which was allowed to equilibrate with the beads for 10 minutes before centrifugation. 11)The resulting releasates were assayed at 1/10th volume, as in Step #5. 12)Solutions which inhibited as well or better than the control beads (about 50% inhibition) were considered hits. 23 hit beads were recovered, with the other two potential hit wells being explainable by additive weak inhibitors in single wells. 13) Hit solutions were subjected to mass spectrometry to determine the molecular weights. 14) The corresponding individual hit beads were subjected to Edman degradation to determine peptide sequences. 15) The combined MS and Edman data was analyzed to identify the hit compound structures. The structures and frequency of their recovery are shown below. G-Hopa=glycine hydroxypropyl amide, the linker remnant. Frequency IC.sub.50, .mu.M 6 D Nal2 N D V G-Hopa (SEQ ID NO: 2) 0.43 4 D Nal2 N A V G-Hopa (SEQ ID NO: 3) 0.37 4 D Nal2 N D I G-Hopa (SEQ ID NO: 4) 0.64 4 D Nal2 N S V G-Hopa (SEQ ID NO: 5) 0.49 3 D Nal2 N S I G-Hopa (SEQ ID NO: 6) 0.81 2 D Nal2 N A I G-Hopa (SEQ ID NO: 7) 0.47 Legende: Nal2 = L-3-(2-naphthyl)-alanyl: ##STR28## G-Hopa = glycine-3-hydroxypropylamide: ##STR29## D = Asp (aspartyl), P = Pro (prolyl), N = Asn (asparaginyl), A = Ala (alanyl), V = Val (valinyl), S = Ser (seryl), I = Ile (isoleucyl). 1.2 Procedures: Preparation of the peptide library Peptide libraries were synthesized by a split/mix synthesis approach (Lam, K. S., Salmon, S. E., Hersh, E. M., Hruby, V. J., Kazmierski, W. M., and Knapp, R. J. (1991) Nature 354, 82; Furka, A., Sebestyen, F., Asgedom, M., and Dibo, G. (1991) lnt. J. Pept. Protein Res. 37, 487) using standard solid-phase peptide Fmoc. chemistry (Stewart, J. M., and Young, J. D. (1984) Solid Phase Peptide Synthesis. Pierce Chemical Co., Rockford, Ill.; Atherton, E., and Sheppard, R. C. (1989) Solid Phase Peptide Synthesis. IRL Press Oxford). Each resin bead was exposed to only a single activated amino acid at each coupling cycle. Therefore, at the completion of the library synthesis, each resin bead expresses only one peptide entity. Since it is not possible to test all compounds separately, we have built the same structure on each resin bead in two copies via differentially cleavable linker, FIG. 1 (Kocis, P., Krchnak, V., and Lebl, M. (1993) Tetr.Lett. 34, 7251; Lebl, M., Krchnak, V., Salmon, S. E., and Lam, K. S. (1994) A Companion to Methods in Enzymolog 6, 381). Release of the peptide from the resin bead can then be carried out in sequential steps using different mechanism of cleavage. Release of the first part of peptide as a hydroxypropylamide is performed in buffer at pH 7-9. The release of the second part of the peptide is achieved by the use of higher pH (Scheme 1). In the peptide libraries, polyethylene glycol-grafted polystyrene beads or TentaGel.RTM.S NH2 were used. In fact, any resin beads that are compatible with peptide synthesis and screening under aqueous conditions are adequate. Penta-, hexa-, and heptamer library were prepared with one fixed position (L-asparagine). Glycine hydroxypropylamide on C-terminus is a part of a linker: H-X4X3-Asn-X2X1-Gly-NH (CH2)3OH (2,160 peptides) H-X5X4X3-Asn-X2X1-Gly-NH (CH2)3OH (25,920 peptides) H-X6X5X4X3-Asn-X2X1-Gly-NH (CH2)3OH (311,040 peptides) X1: N-Fmoc-L-amino acids (9) used in the first randomization: Valine, isoleucine, threonine, phenylalanine, .beta.(2-naphthyl)alanine, 2-azetidinecarboxylic acid, proline, cyclohexylglycine, phenylglycine. X2: N-Fmoc-L-amino acids (4) used in the second randomization: Alanine, glycine, serine, aspartic acid. X3=X5=X6: N-Fmoc-L-amino acids (12) used in the third, fifth and sixth randomization: Pipecolic acid, .beta.(2-naphthyl)alanine, glutamic acid, lysine, 2-azetidinecarboxylic acid, threonine, proline, asparagine, isoleucine, 3,5-diiodotyrosine, citrulline, arginine. X4: N-Fmoc-L-amino acids (5) used in the fourth randomization: Aspartic acid, glutamic acid, 2-aminoadipic acid, O-sulfate tyrosine, .gamma.-carboxyglutamic acid. Resin (PEG-PS.HCl, Millipore.RTM., 20 g, loading 0.58 mmol/g, 220 .mu.m average particle size) was swollen in N,N-dimethylformamide for 2 hours and then neutralized with 10% N,N-diisopropylethylamine in dichloromethane. Resin was washed with dichloromethane and N,N-dimethylformamide. Linker (FIG. 1, 3 eq) was coupled using 1,3-diisopropylcarbodiimide and 1-hydroxybenzotriazole (3 eq each) in N,N-dimethylformamide at room temperature for 12 hours. The reaction was monitored by bromophenol blue method (Krchnak, V., Vagner, J., Safar, P., and Lebl, M. (1988) Collec.Czech.Cem. Commun.53, 2542). Completion of the coupling was then determined by a ninhydrin test (Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1969) Anal. Biochem. 34, 595). After washing with N,N-dimethylformamide, Fmoc protecting group was removed with 50% piperidine in N,N-dimethylformamide for 15 min. Resin was then washed with N,N-dimethylformamide and the amount of released fulvene-piperidine adduct was quantitated by UV spectrometry (302 nm). A stable level of resin loading (mmol/g) determined in this manner throughout the library synthesis served as one of the quality control measures. The resin was divided into 9 equal portions. Nine Fmoc-protected amino acids (X1) were then added separately into each of the resin aliquot and coupled by described procedure for 2 hours. The resin was then pooled in a cylindrical glass vessel fitted with a frit at the bottom. Dry nitrogen was bubbled through for mixing of the resin. Fmoc protecting group was removed as described above. The resin was divided into 4 equal portions. Four Fmoc-protected amino acids (X2) were then added separately into each of the resin aliquot and coupled using the same coupling protocol. Fmoc protecting group was removed and resin loading was determined. In next cycle, L-asparagine was coupled by described-procedure. The resin was then divided into aliquots for another cycle of coupling. After all the randomization steps were completed, the Fmoc group was removed and the side chain protecting groups were cleaved with a mixture of trifluoroacetic acid (82.5%), anisole (5%), water (5%), thioanisole (5%), ethanedithiole (2.5%) during 2,5 hours. The resin was then washed with trifluoroacetic acid, dichloromethane, N,N-dimethylformamide and methanole. The libraries were stored dried at 4.degree. C. To verify the quality of the library, several randomly chosen beads were submitted for sequencing by Edman degradation and mass spectrometric techniques. ##STR30## 1.3 Results (see also FIGS. 2-12) |
| PATENT PHOTOCOPY | Available on request |
|
Want more information ? Interested in the hidden information ? Click here and do your request. |