Urokinase inhibitors

Details Urokinase inhibitors Urokinase inhibitors

2002-07-26

2004-01-20

Shah, Mukund J. (Department: 1624)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Having -c-, wherein x is chalcogen, bonded directly to...

C544S388000

Reexamination Certificate

active

06680320

ABSTRACT:

The invention relates to the use of derivatives of 3-amidinophenylalanine as urokinase inhibitors in particular for treating malignant tumors and the formation of metastases or as agents for targeting lymphocytes and for treating disorders of the lymphatic tissue, in particular lymphomas.
The ability of solid tumors to spread and metastasize in surrounding tissue correlates with the degradation or transformation of the extracellular matrix (tumor stroma) in the vicinity of the tumor cell and/or with the ability of said tumors to penetrate the basement membrane. Although the (patho)biochemical connections have not been completely elucidated yet, the plasminogen activator urokinase (uPA) and the urokinase receptor (uPAR) play a central role. uPA mediates the proteolytic cleavage of plasminogen to give plasmin. Plasmin in turn is a protease which has a wide range of actions and is capable of directly breaking down components of the extracellular matrix such as fibrin, fibronectin, laminin and the protein skeleton of proteoglycans. In addition, plasmin can activate “latent” metalloproteases and the inactive proenzyme of uPA, pro-uPA.
Tumor cells and non-malignant cells of the tumor stroma synthesize and secrete the enzymatically inactive proenzyme pro-uPA. Proteases such as, for example, plasmin or the cathepsins B and L cleave pro-uPA by limited proteolysis to give the active serine protease HMW-uPA (HMW=high molecular weight). Pro-uPA and the active protease HMW-uPA bind to the cell surface receptor uPAR (CD87). Plasmin(ogen) likewise binds to specific receptors on the plasma membranes of tumor cells which leads to focused and amplified plasminogen activation in the immediate vicinity of the tumor cells. Invasive cells thus are able to break down the extracellular matrix without finding themselves deprived of the support necessary for directed movement because of proteolysis.
Various cytobiological studies have shown that the cell-associated plasminogen activator system is of particular importance within the cascade-like reaction pathways of tumor-associated proteolytic systems (Wilhelm et al. (1994 The Urokinase/Urokinase receptor system: A new target for cancer therapy? In: Schmitt M., Graeff H., Kindermann G. (eds.): Prospects in Diagnosis and Treatment of Cancer. International Congress Series, Excerpta Medica 1050, Amsterdam, Elsevier 1994, pp 145-156). Cultures of human colon carcinoma cells showed that their ability to migrate through an extracellular matrix depended on the degree of uPA receptor saturation with active uPA. (Hollas et al., Cancer Res. 51 (1991), 3690-3695). The cell culture model likewise showed a reduction in the invasive potential of cells when PAI-1 (Cajot et al., Proc. Natl. Acad. Sci. USA 87 (1990), 6939-6943) or PAI-2 (Baker et al., Cancer Res. 50 (1990), 4676-4684) inhibited the proteolytic activity of uPA. A similar effect was achieved on inhibition of uPA binding to the cell surface by blocking the receptor by means of proteolytically inactive uPA variants (Cohen et al., Blood 78 (1991), 479-487; Kobayashi et al., Br. J. Cancer 67 (1993), 537-544). Transfection of epidermoid carcinoma cells using a plasmid expressing an antisense transcript of a part of uPAR also reduced the invasivity of said cells by suppressing uPAR synthesis (Kook, EMBO J. 13 (1994), 3983-3991). Antibodies directed against uPA and PAI-1 reduced the invasive potential of lung cancer cells in vitro (Liu et al., Int. J. Cancer 60 (1995), 501-506).
Animal models of tumors were also able to show the influence of the plasminogen activator system on the metastasizing process. Thus, addition of anti-uPA antibodies almost completely prevented the formation of lung metastases caused by human carcinoma cells in chicken embryos (Ossowski and Reich, Cell 35 (1983), 611-619). Metastasizing human carcinoma cells were transfected using an expression plasmid which encoded a proteolytically inactive, but uPAR-binding uPA mutant. The mouse model showed that carcinoma cells synthesizing inactive uPA produced a significantly smaller number of metastases after injection than nontransfected cells (Crowley et al., Proc. Natl. Acad. Sci. USA 90 (1993), 5021-5025). Moreover, after administration of uPA antisense oligonucleotides, nude mice showed inhibition of intraperitoneal spreading of human ovarian carcinoma cells (Wilhelm et al., Clin. Exp. Metast. 13 (1995), 296-302).
In recent years, the clinical relevance of factors of the plasminogen activator system (uPA, uPAR, PAI-1 and PA1-2) for the prognosis of patients having solid malignant tumors has been intensively studied. In these studies, the uPA antigen content in various tumors (e.g. breast, ovaries, stomach, lung, kidney) proved to be a strong prognostic factor both for the recurrence-free survival and for the mortality (see for example, Schmitt et al., J. Obstet. Gynaecol. 21 (1995), 151-165; Jaenicke et al., Breast Cancer Res. Treat. 24 (1993), 195-208; Kuhn et al., Gynecol. Oncol. 55 (1994), 401-409; Nekarda et al., Lancet 343 (1994), 117; Pedersen et al., Cancer Res. 54 (1994), 4671-4675). Likewise, increased concentrations of uPAR in lung cancer tissue (Pedersen et al., supra) and breast cancer tissue (Duggan et al., Int. J. Cancer 61 (1995), 597-600; Ronne et al., Breast Cancer Res. Treat. 33 (1995), 199-207) and also in the case of stomach cancer both in the tumor tissue itself (Heiss et, al., J. Clin. Oncol. 13 (1995), 2084-2093) and in tumor cells disseminated into bone marrow (Heiss et al., Nature Medicine 1 (1995), 1035-1039) correlate with a poor prognosis.
The use of synthetic uPA inhibitors makes it possible to suppress invasion and spreading of tumor cells. However, developing specific uPA inhibitors is difficult, since tissue plasminogen activator (tPA) has an identical specificity for cleaving the peptide bond Arg560/Val561 of plasminogen. In most cases therefore, low molecular weight uPA inhibitors also inhibit tPA and thus also tPA-mediated fibrinolysis. In addition, it must be guaranteed that synthetic uPA inhibitors show no strong plasmin inhibition.
Despite these restrictions, some inhibitors are known which have a certain specificity for uPA, but a low inhibition capacity, such as benzamidine derivatives and &bgr;-naphthamidine derivatives, the most effective compound inhibiting uPA with K
i
=2.2 &mgr;mol/l (Stürzebecher and Markwardt, Pharmazie 33 (1978), 599), or amiloride with K
i
=7 &mgr;mol/l (Vassalli and Belin, FEBS. Lett. 214 (1987), 187-191).
DE-A-30 35 086 discloses cyclohexanecarboxylic acid derivatives which have inhibitory effects on proteases such as trypsin, chymotrypsin, thrombin or uPA. However, the compounds studied only show quite weak and, moreover, unspecific uPA inhibition. EP-A-0 183 271 discloses lysine derivatives and the use thereof as protease inhibitors. A benzamidinolysine derivative (compound 108) is also described which inhibits uPA in vitro, but acts comparably on other proteases such as trypsin or plasma kallikrein. WO 95/17885 disloses low molecular weight polypeptides as uPA inhibitors.
Another class of known uPA inhibitors is represented by 4-substituted benzothiophene-2-carboxamidines with K
i
=0.16 mmol/l in the case of benzothiophene 623 (Towle et al., Cancer Res. 53 (1993), 2553-2559). These inhibitors have a significantly higher affinity for uPA than for tPA and plasmin. uPAR-bound uPA, too, is inhibited very effectively. Disadvantageously however, the chemical synthesis of these substances is complicated and few possibilities for structural modifications are present or have been demonstrated until now.
Therefore, the development of further uPA inhibitors is very beneficial for further elucidating the role of uPA and uPAR in various diseases, especially in tumor spreading and metastasizing.
N&agr;-Arylsulfonyl and N&agr;-arylsulfonylaminoacyl derivatives of 3-amidinophenylalanine are known as selective inhibitors of thrombin (Markwardt et al., Thromb. Res. 17 (1980), 425-431) or of coagulation factor Xa (Stürzebecher et al., Thromb. Res. 54 (1989)

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