Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1996-10-30
2002-07-02
Fonda, Kathleen K. (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S008100, C514S009100, C536S001110, C549S200000, C549S356000, C562S459000, C585S275000
Reexamination Certificate
active
06413936
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel mimetics of the tetrasaccharides sialyl-Lewis-X (SLeX) and sialyl-Lewis-A (SLeA) with improved action as inhibitors of cell adhesion, to a process for the preparation of these compounds and to their use as pharmacological active compounds and diagnostic agents.
BACKGROUND OF THE INVENTION
The circulation of blood cells, for example leukocytes, neutrophils, granulocytes and monocytes, is, at the molecular level, a highly complex multistage process of which only individual steps are known (for a review see: Springer,
Cell
76:301-314 (1994)). Recent research has shown that both localization of neutrophils and monocytes at foci of inflammation and lymphocyte recirculation, which is crucial in immune monitoring, respond to very similar molecular mechanisms. Thus, in acute and chronic inflammatory processes leukocytes adhere to endothelial cells and migrate to the focus of inflammation and into the secondary lymphatic organs. This process involves numerous specific signal molecules, for example interleukins, leukotrienes and tumor necrosis factor (TNF), G-protein coupled receptors and, in particular, tissue-specific cell adhesion molecules, which precisely control immune cell and endothelial cell recognition. The most important adhesion molecules involved in this process, designated below as receptors, include the selectins (E-, P- and L-selectins), integrins and the members of the immunoglobulin superfamily.
Adhesion of leukocytes to endothelial cells is mediated by selectin receptors in the initial phase of inflammatory processes, and is a natural and necessary immune response to various inflammatory stimuli and to vascular tissue damage. The course of a variety of acute and chronic disorders, such as rheumatism, reperfusion injuries such as myocardial ischemia/infarct (MI), acute pneumonia following surgery, traumatic shock and stroke, psoriasis, dermatitis, ARDS (adult respiratory distress syndrome) and restenosis following surgical intervention (for example angioplasty and by-pass operations) is, however, adversely affected by excessive leukocyte adhesion and infiltration into affected tissue. Controlling this adhesion process at a very early stage of inflammation is, therefore, a highly attractive and generally applicable concept for the pharmacological control of inflammatory disorders.
The tetrasaccharides sialyl-Lewis-X (SLeX) and sialyl-Lewis-A (SLeA), which occur as substructures of glycosphingolipids and glycoproteins on cell membranes, can function as ligands for all three selectin receptors. A series of glycoproteins, mucins and glycolipids are known to be suitable endogenous ligands for the selecting. These include: Mucosal Vascular Addressin MadCAM-1 (Berg et al.,
Nature
366:695 (1993)) and Sialomucin CD34 (Baumhuter et al.,
Science
262:436 (1993)) for L-selectin: O-linked polylactosamine-sialomucin PSGL-1 on human neutrophils for P-selectin (Moore et al.,
J.Biol.Chem.
269:23318 (1994); and N-linked sialoglycoproteins of the ESL-1 type for E-selectin (Vestweber et al.,
Cell Biol.
121:449 (1993)).
The specificity of these and other potential ligands for selectins in vivo has not yet been elucidated. The tetrasaccharides SLeX and SLeA represent only a substructure of the substantially more complex structures of endogenous selectin ligands and, because of their similar affinity for selecting, cannot alone account for receptor binding specificity. Due to the structural complexity of SLeX and SLeA, the use of simpler, structurally modified mimetics as antagonists for modulating or suppressing excessive leukocyte adhesion is a promising therapeutic starting point for a strategy for alleviating or healing the above-mentioned disorders mentioned.
SLeX has already been used successfully in animal experiments to protect against P-selectin-dependent lung damage (Mulligan et al.,
Nature
364:149 (1993)) and against myocardial reperfusion injuries (Buerke et al.,
J.Clin.Invest.
93:1140 (1994)). In initial clinical trials against acute pneumonia, the compound was employed in a dose of 1-2 grams per day per patient (report by Cytel Corp./La Jolla (Calif.) at the 2nd Glycotechnology Meeting/CHI in La Jolla/USA on May 16-18, 1994).
Some publications and patent applications have also reported efforts to obtain more potent antagonists by structural variation of the ligand. The aim of such work is to provide more effective antagonists that potentially would also be suitable for use in vivo at a relatively low dose. Variation of the fucose and neuraminic acid units regarded as crucial for the structure-activity relationship (Brandley et al.,
Glycobiology
3:633 (1993); Yoshida et al.,
Glycoconjugate J.
10:3 (1993)), did not, however, afford significantly improved inhibition. Only when the glucosamine unit was varied (replacement of GlcNAc by glucose and azido groups and amino groups in position 2 of GlcNAc) was significantly increased affinity for the E-selectin receptor achieved. By contrast, improved binding of the P-selectin receptor was not achieved.
In general, all previous successes have been limited to improving the binding affinity of SLeX and SLeA derivatives for the E-selectin receptor, since at inhibitor concentrations of about 1 mM only weak inhibitory effects with the P-selectin receptor have been found (Nelson et al.,
J.Clin.Invest.
91:1157 (1993)). The binding affinities of modified SLeX/A structures for selecting has been reviewed. See
Pharmacochem
. Libr. 20 (1993))(
Trends in Drug Research
), pp. 33-40.
In addition to their low affinity for selectin binding, these compounds all contain at least one unstable glycosidic linkage, which severely restricts their oral availability as active compounds. This instability also greatly limits the synthesis of various derivatives, since the hydrolytic lability of the glycosidic linkage limits the available reaction conditions. A number of strategies for synthesizing mimetics have been developed to obtain an increase in hydrolytic stability.
For example, stability has been increased by attaching the side chain via a C—C bond to the C-4 carbon of fucose (Floyd et al.,
Tetrahedron Asymmetry
5:2061 (1994).
In this case, however, the linkage to C-4 of fucose caused the orientation of the side chain to differ from that of the natural ligand, and only low affinity for selectin binding was observed.
Use of carbocyclic carbohydrate analogs where the side chain linkage is through a C—C bond to C-1 would give a conformation similar to that of the natural ligand that also would be stable against degradation. Several carbocyclic carbohydrate analogs of monosaccharide units have been prepared. Thus, for example, activated monosaccharides have been reacted with nitromethane (Gross,
Tetrahedron
47:6113 (1991)), allylsilane (Kishi et al.,
J. Am. Chem. Soc
. 104:4976-4978 (1982)), and olefins (Levy et al.,
Tetrahedron Asymmetry
5:2265-2268 (1994)). The functionality introduced into these monosaccharides makes them suitable as a unit for further coupling operations.
Use of a carbocyclic analog as a building block for selectin antagonists has been shown to lead to a mimetic with affinity for selectins. It was possible at the same time, by reacting a fucose unit with allylsilane, to synthesize a specific C-glycosidic unit (1) with an &agr;-orientation at the side chain (WO 95/04751). Selectivities in the allylation are high, with &agr;/&bgr;=14/1, but scale-up of the reaction is difficult due to the conditions employed, particularly the need to use 10 equivalents of volatile and highly corrosive trimethylsilyl triflate. Similarly, chromatographic purification of the product is required, although it is not possible to separate the &agr;/&bgr; mixture.
The terminal acid function of the side chain can be used to synthesize glycopeptide analogs, for example (2). These analogs have IC
50
values of about 1 mM, but are unstable to proteolytic degradation. Consequently, despite stabilization of the sugar unit by the C-glycoside, the oral availability of these compounds c
Klein Robert
Kretzschmar Gerhard
Kunz Horst
Schmidt Wolfgang
Sprengard Ulrich
Foley & Lardner
Fonda Kathleen K.
Glycorex AB
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