ZA200200673B - Inhibitors of the integrin avbeta6. - Google Patents

Description

- EY

Ce .2002/0673

WO 01/00660 PCT/EP00/05404 - 1 =

Inhibitors of the integrin o.Pe

The invention relates to novel peptides of the formula

I which, as ligands of the integrin of, are biologically active

Ac-Arg-X'-Asp-X?-x3-X*-X°-X°-NH, I in which

Ac is acetyl, x! is Ser, Gly, Thr, Asp, Arg, Val, Tyr, His or Ala, x? is Leu, Ile, Nle, val or Phe, x3 is Asp, Glu, Lys, Phe, Aib, Nal, Gly, Ala, Bgl or

Phg, x! is Gly, Ala, Ser, P-Ala or mw-2bu, x° is Leu, Ile, Nle, Val or Phe, x® is Arg, Har, Lys, Leu, Orn, Phe, Ala, Tyr, Gly,

Ser or Asp, where the amino acids mentioned can also be derivatized, the amino acid residues are linked to one another in peptide fashion via the oO-amino and o-carboxyl groups, the D and the L forms of the optically active amino acid residues are included, and their physiologically acceptable salts, and where Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH; is excluded.

The invention is based on the object of finding novel compounds having valuable properties, in particular those which can be used for the production of medicaments.

It has been found that the compounds according to the invention and their salts have valuable pharmacological properties together with good tolerability.

® ,

The peptides according to the invention can be employed as efficacious inhibitors of the o,f¢ integrin receptor and thus for the treatment of various diseases and pathological findings.

Other inhibitors of the integrin o.fs are described in

DE 19858857 and by S. Kraft et al. in J. Biol. Chem. 274, 1979-85 (1999). The compounds according to the invention are to be considered as a selection invention with respect to the application mentioned.

Integrins belong to the family of heterodimers of Class

I - transmembrane receptors which play an important role in numerous cell-matrix or cell-cell adhesion processes (Tuckwell et al., 1996, Symp. Soc. Exp. Biol. 47). They can be roughly divided into three classes: the B1 integrins, which are receptors for the extracellular matrix, the f; integrins, which are activatable on leucocytes and are “triggered” during inflammatory processes, and the oo, integrins, which influence the cell response during wound-healing and other pathological processes (Marshall and Hart, 1996,

Semin. Cancer Biol. 7, 191).

The integrins osfi, Omfs, 0sfi, Pi, Ps, ofs, oP and o.fs all bind to the Arg-Gly-Asp (RGD) peptide sequence in natural ligands, such as, for example, fibronectin or vitronectin. Soluble RGD-containing peptides are able to inhibit the interaction of each of these integrins with the corresponding natural ligands. ops is a relatively rare integrin (Busk et al., 1992 J.

Biol. Chem. 267(9), 5790), which is formed to an increased extent in repalr processes in epithelial tissue and preferably binds the natural matrix molecules fibronectin and tenascin (Want et al., 1996,

Am. J. Respir. Cell Mol. Biol. 15(5), 664). The physiological and pathological functions of offs are still not precisely known; it is suspected, however, that this integrin plays an important role in ’

- @ a physiological processes and disorders (e.g. inflammation, wound healing, tumours) in which epithelial cells are involved. Thus of is expressed on keratinocytes in wounds (Haapasalmi et al., 1996, J.

Invest. Dermatol. 106(1l), 42), from which it is to be assumed that in addition to wound-healing processes and inflammation other pathological skin events, such as, for example, psoriasis, can also be influenced by agonists or antagonists of the said integrin. ofBs furthermore plays a role in the respiratory tract epithelium (Weinacker et al., 1995, Am. J. Respir. Cell

Mol. Biol. 12 (5), 547), so that appropriate agonists/antagonists of this integrin could be successfully employed in respiratory tract disorders, such as bronchitis, asthma, pulmonary fibrosis and respiratory tract tumours. Finally, it is known that oes also plays a role in the intestinal epithelium, so that appropriate integrin agonists/antagonists could be used in the treatment of inflammation, tumours and wounds of the stomach/intestinal tract.

The dependence of the formation of angiogenesis on the interaction between vascular integrins and extra- cellular matrix proteins is described by P.C. Brooks,

R.A. Clark and D.A. Cheresh in Science 264, 569-71 (1994) .

The object was therefore, in addition to the previously known natural high molecular weight ligands and antibodies, which are therapeutically and diagnos- tically difficult to handle, to find potent, specific and selective low molecular weight ligands for of, preferably peptides, which can be used for the therapeutic areas mentioned, but also as diagnostic or reagent.

It has been found that the peptide compounds according to the invention and their salts, as soluble molecules, exert an effect on cells which carry the said receptor,

® or if they are bound to surfaces, are artificial ligands for oyPs-mediated cell adhesion. Above all, they act as O.fs integrin inhibitors, where they particularly inhibit the interactions of the receptor with other ligands, such as, for example, the binding of fibro- nectin. This action can be detected, for example, by the method which is described by J.W. Smith et al. in

J. Biol. Chem. 265, 12267-12271 (1990).

It has furthermore been found that the novel substances have very valuable pharmacological properties together with good tolerability and can be employed as medicaments. This is described more precisely further below.

The peptide compounds according to the invention can furthermore be used in vivo as diagnostics for the detection and localization of pathological conditions in the epithelial system, if they are equipped with appropriate markers (e.g. the biotinyl radical) according to the prior art.

The invention also comprises combinations with at least one other active compound and/or conjugates with other active compounds, such as cytotoxic active compounds and conjugates with radiolabels for X-ray therapy or

PET diagnosis, but also fusion proteins with marker proteins such as GFP or antibodies, or therapeutic proteins such as IL-2.

Some preferred groups of compounds can be expressed by the following subformulae Ia to If, which correspond to the formula I and in which the radicals not designated in greater detail have the meaning indicated in the formula I, but in which in a) xt is Ser, Gly or Thr; in b) x! is Ser, Gly or Thr, x? is Leu;

: C Ce in ¢) x is Ser, Gly or Thr, x? is Leu, x3 is Asp or D-Asp; in d) xt is Ser, Gly or Thr, x? is Leu, x3 is Asp or D-Asp, x? is Gly, Ala or Ser; in e) xt is Ser, Gly or Thr, x? is Leu, x3 is Asp or D-Asp, x4 is Gly, Ala or Ser, x> is Leu; in f) xt is Ser, Gly or Thr, x* is Leu, x3 is Asp or D-Asp, x* is Gly, Ala or Ser, x> is Leu, x8 is Arg; and their salts.

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

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

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

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

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

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

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

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

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

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

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

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

Ac-Arg-Thr-Asp-Leu-Gly-Ser-Leu-Arg-NH;y,

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

® - 6 -

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

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

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

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

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

Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Hax-NH;,

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

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

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

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

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

Abu 4-aminobutyric acid

Aha 6-aminohexanoic acid, 6-aminocaproic acid

Aib o-aminoisobutyric acid

Ala alanine

Asn asparagine

Asp aspartic acid

Arg arginine

Bgl C-alpha-tert-butylglycine

Cys cysteine

Dab 2,4-diaminobutyric acid

Dap 2,3-diaminopropionic acid

Gln glutamine

Glp pyroglutamic acid

Glu glutamic acid

Gly glycine

Har homoarginine

His histidine homo-Phe homo-phenylalanine

Ile isoleucine

Leu leucine

Lys lysine

Met methionine

Nal naphth-2-ylalanine i C

Nle norleucine

Orn ornithine

Phe phenylalanine

Phg phenylglycine 4-Hal-Phe 4-halophenylalanine

Pro proline

Ser serine

Thr threonine

Trp tryptophan

Tyr tyrosine val valine.

In addition, the following have the meanings below:

Ac acetyl

BOC tert-butoxycarbonyl

BSA bovine serum albumin

CBZ or Z benzyloxycarbonyl

DCC1 dicyclohexylcarbodiimide

DMF dimethylformamide

EDC1 N-ethyl-N,N’- (dimethylaminopropyl) - carbodiimide

Et ethyl

Fca fluoresceincarboxylic acid

FITC fluorescein isothiocyanate

Fmoc 9-fluorenylmethoxycarbonyl

FTH fluoresceinthiourea

HOBt 1-hydroxybenzotriazole

Me methyl

MBHA 4-methylbenzhydrylamine

Mtr 4-methoxy-2,3,6-trimethylphenylsulfonyl

HONSu N-hydroxysuccinimide

OBut tert-butyl ester

Oct octanoyl

OMe methyl ester

OEt ethyl ester

Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5- sulfonyl

Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl

} C Ca

POA phenoxyacetyl

Sal salicyloyl

TBS++ tris buffered saline with divalent cations

TBSA TBS + BSA

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

TFA trifluoroacetic acid

Trt trityl (triphenylmethyl).

If the abovementioned amino acids can occur in two or more enantiomeric forms, all these forms and their mixtures (for example the DL forms) are included above and below. In addition, the amino acids can be provided with appropriate protective groups which are known per se.

So-called prodrug derivatives are included in the compounds according to the invention, that is compounds modified with, for example, alkyl or acyl groups, sugars or oligopeptides, which are cleaved rapidly in the body to give the active compounds according to the invention. These also include biodegradable polymer derivatives of the compounds according to the invention, as is described, for example, in Int. J.

Pharm. 115, 61-67 (1995).

The amino acids and amino acid residues mentioned, such as, for example, the NH functions or alternatively the

C-terminal amide function, can also be derivatized, the

N-methyl, N-ethyl, N-propyl, N-benzyl or Cyg-methyl derivatives being preferred. Derivatives which are additionally preferred are those of Asp and Glu, in particular the methyl, ethyl, propyl, butyl, tert- butyl, neopentyl or benzyl esters of the side chain carboxyl groups, and in addition also derivatives of

Arg, which can be substituted on the -NH-C(=NH)-NH; group by an acetyl, benzoyl, methoxycarbonyl or ethoxycarbonyl radical.

Claims (16)

Patent Claims

1. Peptide compounds of the formula I Ac-Arg-X'-Asp-X?-x3-x%-X°-X°-NH, I in which Ac is acetyl, x! is Ser, Gly, Thr, Asp, Arg, Val, Tyr, His or Ala, X* is Leu, Ile, Nle, Val or Phe, x® is Asp, Glu, Lys, Phe, Aib, Nal, Gly, Ala, Bgl or Phg, x! is Gly, Ala, Ser, B-Ala or w-Abu, X° is Leu, Ile, Nle, Val or Phe, x® is Arg, Har, Lys, Leu, Orn, Phe, Ala, Tyr, Gly, Ser or Asp, where the amino acids mentioned can also be derivatized, the amino acid residues are linked to one another in peptide fashion via the o-amino and o-carboxyl groups, the D and the L forms of the optically active amino acid residues are included, and their salts, and where Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH, is excluded.

2. Peptide compounds according to Claim 1 selected from the group consisting of Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NH;, Ac-Arg-Gly-Asp-Leu-Asp-Ser-Leu-Arg-NH;, Ac-Arg-Ser-Asp-Leu-Asp-Ser-Leu-Arg-NH;, Ac-Arg-Asp-Asp-Leu-Asp-Ser-Leu-Arg-NH;, Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Ala-NH;,

@ Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-D-Arg-NHz, Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Arg-NH, . Ac-D-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH;, Ac-Arg-Thr-Asp-Leu-D-Ala-Ser-Leu-Arg-NHz, Ac-Arg-Thr-Asp-Leu-Aib-Ser-Leu-Arg-NHy, Ac-Arg-Thr-Asp-Leu-D-Nal-Ser-Leu-Arg-NHz, Ac-Arg-Thr-Asp-Leu-Gly-Ser-Leu-Arg-NH;, Ac-Arg-Thr-Asp-Leu-Ala-Ser-Leu-Arg-NHz, Ac-Arg-Thr-Asp-Nle-D-Asp-Ser-Leu-Arg-NH;, Ac-Arg-Thr-Asp-Ile-D-Asp-Ser-Leu-Arg-NHz, Ac-Arg-Thr-Asp-Leu-Asp-D-Ser-Leu-Arg-NH;, Ac-Arg-Thr-Asp-Leu-Asp-Ala-Leu-Arg-NH;, Ac-Arg-Thr-Asp-Leu-Asp-Gly-Leu-Arg-NH;, Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Har-NH;, Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Lys-NHz, Ac-Arg-Thr-Asp-Leu-D-Asp-D-Ser-Leu-Arg-NHy, Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Ala-NHy, Ac-Arg-Thr-Asp-Leu-D-Asp-Gly-Leu-Arg-NH;, and their physiologically acceptable salts.

3. Peptide compounds of the formula I according to Claim 1 and the compounds according to Claim 2, and their physiologically acceptable salts as medicaments.

4. Medicament according to Claim 3 as an inhibitor for the control of disorders which are based on an expression and pathological function of Ps integrin receptors.

5. Medicament according to Claim 4 for the control of thromboses, cardiac infarct, coronary heart disorders, arteriosclerosis, tumours, osteo- porosis, fibroses, inflammation, infections, psoriasis and for influencing wound-healing processes.

EE -32- PCT/EP00/05404

6. Pharmaceutical preparation, comprising at least one medicament according to one of Claims 4 and 5 and, if appropriate, vehicles and/or excipients and, if appropriate, other active compounds.

7. Use of peptide compounds according to Claims 1 and 2 and/or their physiologically acceptable salts for producing a medicament for the control of disorders which are based on expression and pathological function of «8s integrin receptors.

8. Use according to Claim 7 for producing a medicament for the control of thromboses, cardiac infarct, coronary heart disorders, arteriosclerosis, tumours, osteoporosis, fibroses, inflammation, infections, psoriasis and for influencing wound-healing processes.

9. A substance or composition for use in a method for the control of disorders which are based on expression and pathological function of o,f integrin receptors, said substance or composition comprising peptide compounds according to Claims 1 and 2 and/or their physiologically acceptable salts, and said method comprising administering said substance or composition.

10. A substance or composition for use in a method of treatment according to claim 9 for the control of thromboses, cardiac infarct, coronary heart disorders, arteriosclerosis, tumours, osteoporosis, : fibroses, inflammation, infections, psoriasis and for influencing wound-healing processes. AMENDED SHEET :

-33- PCT/EP00/05404

11. A compound as claimed in claim 1, substantially as herein described and illustrated.

12. A medicament as claimed in claim 4, substantially as herein described and illustrated.

13. A preparation as claimed in claim 6, substantially as herein described and illustrated.

14. Use as claimed in claim 7, substantially as herein described and illustrated.

15. A substance or composition for use in a method of treatment as claimed in claim 9, substantially as herein described and illustrated.

16. A new compound, a new medicament, a new preparation, a new use of a peptide compound as claimed in claim 1 or claim 2 or a physiologically acceptable salt thereof, or a substance or composition for a new use in a method of treatment, substantially as herein described. AMENDED SHEET ’