Pentasaccharides processes for their preparation and...

Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai

Reexamination Certificate

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C514S025000, C536S123100, C536S122000

Reexamination Certificate

active

06670338

ABSTRACT:

The present invention relates to penta-saccharides, to processes for their preparation and to pharmaceutical compositions containing them.
Heparin is a polysaccharide of the glycosamino-glycan family which is known for its anticoagulant properties. It is known (I. Björk and U. Lindahl, Molecular and Cell Biochemistry, 1982, Dr. W. Junk Publishers-Netherlands) that blood clotting is a complex physiological phenomenon. Certain stimuli, such as contact activation and tissue factor, trigger the subsequent activation of a series of clotting factors present in blood plasma. Irrespective of the nature of the stimulus, the final steps are identical, the activated factor X (Xa) activates factor II (also known as prothrombin), which, in its activated form (factor IIa, also known as thrombin), gives rise to partial proteolysis of soluble fibrinogen with release of insoluble fibrin, which is one of the main constituents of a blood clot.
Under normal physiological conditions, the activity of the clotting factors is regulated by proteins such as antithrombin III (ATIII) and heparin cofactor II (HC II), which are also present in plasma. AT III exerts inhibitory activity on a certain number of clotting factors, and in particular on factors Xa and IIa.
Inhibition of factor Xa or of factor IIa is thus the preferred means for obtaining anticoagulant and antithrombotic activity, since these two factors are involved in the final two steps of clotting, which are independent of the triggering stimulus.
A pentasaccharide such as the one described by P. Sinay et al., Carbohydrate Research 1984, 132 C5 represents the minimum heparin sequence required for binding to AT III. This compound was obtained about fifteen years ago by total chemical synthesis.
Since then, a certain number of synthetic oligosaccharides, obtained by total chemical synthesis and having antithrombotic anticoagulant activity, have been described in the literature.
Patent EP 0,084,999 describes derivatives consisting of uronic acid (glucuronic or iduronic acid) monosaccharide units and glucosamine which have advantageous antithrombotic properties. Besides substituents consisting of hydroxyl groups, these compounds contain N-sulphate groups, N-acetyl groups and, in certain cases, the anomeric hydroxyl groups are replaced with methoxy groups.
Application EP 0,165,134 also describes synthetic oligosaccharides with antithrombotic activity. These compounds, consisting of uronic acid monosaccharide units and glucosamine and containing an O-sulphate group in position 3 of the glucosamine unit, are also described in application EP 0,301,618. These compounds have powerful antithrombotic and anti-coagulant properties. Patent EP 0,454,220 describes uronic acid derivatives and glucose derivatives which contain O-alkyl or O-sulphate groups as substituents. These latter compounds also have antithrombotic and anticoagulant properties.
Sulphated glycosaminoglycanoid derivatives in which the N-sulphate, N-acetate or hydroxyl functional groups have been replaced with alkoxy, aryloxy, aralkyloxy or O-sulphate groups are also described in patent EP 0,529,175. These compounds have advantageous antithrombotic properties. The latter compounds are also inhibitors of smooth muscle cell proliferation.
Oligosaccharides, and in particular penta-saccharides, which are analogous to the minimum heparin sequence required for binding to AT-III are described in Angew. Chem. Int. Ed. Engl. 1993, 32, 3, 434-436. These compounds contain glucuronic acid or glucose units whose hydroxyl functions have been replaced with O-sulphate or O-methyl groups.
Many studies have since been carried out on pentasaccharides, and it has been indicated in the literature that the conformation of the L-iduronic acid unit G plays an important role in the activity of the products. Several conformational states for the unit G have been described (
4
C
1
,
1
C
4
,
2
S
0
) and it has been suggested that this conformational flexibility is essential for the biological activity of products containing L-iduronic acid (B. Casu, M. Petitou, A. Provasoli and P. Sinay, Conformational flexibility: a new concept for explaining binding and biological properties of iduronic acid-containing glycosamino-glycans. Trends Biochem. Sci. 1988, 13, 221-225).
It has now been found, surprisingly, that by replacing one of the O-alkyl groups with an alkylene bridge, and thus by locking the conformation of the L-iduronic acid, oligosaccharides are obtained which have advantageous biological properties although lacking in conformational flexibility. The reason for this is that the compounds of the present invention differ from the other synthetic heparinoids described in the literature, by virtue of their novel structures and their powerful and unexpected biological properties. The compounds of the invention are pentasaccharides in which the L-iduronic unit G is in the so-called “locked”
2
S
0
conformation and in which the D-glucuronic acid unit E optionally has an ethyl group in position 5. These compounds have very great anti-factor Xa activity and great affinity for AT III.
The subject of the present invention is, more particularly, a pentasaccharide in acidic, form and its pharmaceutically acceptable salts, with one or more pharmaceutically acceptable cations, the anionic form of which has the formula (I):
in which:
R represents hydrogen or an —SO
3

, (C
1
-C
3
)alkyl or (C
2
-C
3
)acyl group;
T represents hydrogen or an ethyl group; and
n represents 1 or 2.
The invention encompasses pentasaccharides in acidic form, or in the form of a pharmaceutically acceptable salt. In the acidic form, the —COO and —SO
3
functions are in the form —COOH and —SO
3
H, respectively.
The expression “pharmaceutically acceptable salt of the pentasaccharides of the invention” is intended to refer to pentasaccharides in which one or more of the —COO and/or —SO
3
functions are ionically bonded to a pharmaceutically acceptable metal cation.
The preferred salts according to the invention are those in which the cation is chosen from the cations of alkali metals and, even more preferably, those in which the cation is Na
+
or K
+
.
The subject of the present invention is also a process for preparing the pentasaccharides of the invention, characterized in that a precursor of the unit G is prepared, which is coupled with a precursor of the unit H to give a precursor of GH, the pentasaccharide finally being obtained: either by coupling a precursor of GH with a precursor of DEF, or by coupling a precursor of GH with a precursor of EF, followed by addition of D.
Any precusor of G, of H, of EF or of DEF can be used. This means that it is possible, according to s these processes, to prepare an entire family of pentasaccharides having the unit G of locked configuration in common.
The process described above is the preferred process of the invention. However, the pentasaccharides of the invention can be prepared by other known methods of sugar chemistry, and in particular by reacting a monosaccharide containing protective groups such as described by T. W. Green, in Protective Groups in Organic Synthesis (Wiley, N.Y. 1981), on the hydroxyl radicals and optionally on the carboxyl radicals, if present, with another protected monosaccharide, to form a disaccharide which is then reacted with another protected monosaccharide to form a protected trisaccharide, from which a protected tetrasaccharide and then a protected pentasaccharide can be obtained.
The protected pentasaccharides are then deprotected and optionally sulphated, or partially deprotected, then sulphated and then deprotected, in order to obtain the compounds of the invention.
Such processes are known in carbohydrate chemistry, and are described in particular by G. Jaurand et al. in Bioorganic and Medicinal Chemistry Letters 1992, 2, 9, 897-900, by J. Basten et al., in Bioorganic and Medicinal Chemistry Letters 1992, 2, 9, 905-910 and by M. Petitou and C. A. A. van Boeckel in “Chemical synthesis of heparin fragment and analogues” 203-210—Process in the chemistry of organic

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