Dextran derivatives, preparation and medicinal applications

Details Dextran derivatives, preparation and medicinal applications Dextran derivatives, preparation and medicinal applications

2000-09-26

2003-11-11

Wilson, James O. (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C514S012200, C530S399000

Reexamination Certificate

active

06646120

ABSTRACT:

This application claims priority from PCT/FR98/02699 filed Dec. 11, 1998, and from French patent application 97/15702 filed Dec. 11, 1997.
The present invention relates to dextran derivatives, and to their applications as medicines with specific biological action, such as a cicatrizing action, an anti-complement action (substitute for plasma), a proliferation modulating action or an anticoagulant action, and more specifically an anti-thrombotic action, as well as to a process for their preparation.
Various dextrans substituted with side chains bearing carboxylate and sulfonate groups have been described. In particular, French patent 2,461,724 and French patent 2,555,589 describe dextrans substituted with said groups, which respectively display anticoagulant properties and anticoagulant and anti-inflammatory properties; European patent 0,402,194 describes the cell and tissue regeneration properties of such substituted dextrans.
It has also been shown that such substituted dextrans can have other biological activities, depending on the degree of substitution with said groups; in particular, European patent 0,514,449 describes dextrans (D) substituted with carboxymethyl (CM) and carboxymethylbenzylamide sulfonate (BS) groups of general formula D
X
CM
Y
BS
Z
, in which X, which represents the average number of unsubstituted saccharide units per 100 saccharide units, is less than or equal to 50, Y, which represents the average number of carboxymethyl groups per 100 saccharide units, is between 10 and 90, and Z, which represents the average number of carboxymethylbenzylamide sulfonate groups per 100 saccharide units, is between 15 and 35; to give an agent for inhibiting the growth of tumor cells.
These various derivatives, the structure of which is summarized in
FIG. 1
, are generally obtained by random substitution of dextran with three different groups: carboxymethyl (CM), carboxymethylbenzylamide (B) and carboxymethylbenzylamide sulfonate (S) (sulfonation on the aromatic ring with chlorosulfonic acid).
More specifically:
a) the carboxymethylation of dextran (production of CMD) is carried out in basic aqueous medium, by the action of monochloroacetic acid. Three successive carboxymethylation reactions are required to obtain a degree of substitution (ds) of the dextran, expressed relative to the number of free hydroxyl functions in a glucoside unit of the dextran, of between 0.7 and 1.1;
b) the coupling of benzylamine to the carboxymethyl groups (production of CMDB) is based on the ability of the carboxylate function to form an unstable mixed anhydride capable of reacting with a reagent bearing a primary amine function (R—NH
2
). Two different processes or activation reactions were used to achieve the formation of a mixed anhydride:
action of isobutyl chloroformate (IBC) or
action of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ).
In both cases, the reaction is carried out in a heterogeneous medium: water/dimethylformamide or water/ethanol, respectively.
These two coupling processes give similar degrees of substitution (ds), of about 0.08 to 0.12, in a single step. In the case of coupling with EEDQ, the ds can reach 0.25 to 0.30, in a single step, when the intermediate product is activated at a temperature of 30 to 40° C.
As with the carboxymethylation, to achieve high ds values for benzylamine, it is not possible to increase the concentrations of the various reagents. It is thus necessary to perform successive couplings in order to improve the reaction yield. The CMDB precipitated, washed and dried after the first coupling undergoes a second and/or a third coupling under exactly the same conditions as the first, without consideration of the substitutions due to the first coupling.
c) the sulfonation or sulfation takes place by the action of monochlorosulfonic acid on the CMDB, in anhydrous organic medium (for example dichloromethane) and in heterogeneous phase, the CMDB not being soluble in dichloromethane. In such a heterogeneous medium, the distribution of the sulfates in the saccharide units occurs unequally (Ricketts, C. R.,
J. Chem. Soc
., 1956, 3752-3756). It is necessary to perform the reaction in excess monochlorosulfonic acid, while at the same time avoiding acid hydrolysis of the polysaccharide chain.
The [HSO
3
Cl]/[bound B units] ratios for the CMDBs and [HSO
3
Cl]/[free OH] ratios for the CMD range between 0.8 and 3.
In order essentially to obtain a sulfonation of the aromatic rings (B units), the [HSO
3
Cl]/[bound B units] molar ratio should be equal to 3, whereas, in order to obtain a sulfation of the hydroxyl functions borne by the glucoside units (production of sulfate functions), the [HSO
3
Cl]/[free OH] molar ratio is about 1. In any case, the concentration of chlorosulfonic acid in the reaction medium should not exceed 0.15 M. Under these conditions, the percentage of units bearing a sulfonate function depends on the percentage of units substituted with benzylamide groups. A recent study (Maiga-Revel O. et al.,
Carbohydrates Polymers
, 1997, 32, 89-93) has shown that the anticoagulant activity of the CMDSu (=carboxymethyldextran sulfate) and CMDBS (=carboxymethyldextran benzylamide sulfonate) dextran derivatives of the prior art depends on their sulfur content; however, CMDBSs have better anticoagulant activity than that obtained with CMDSu's, for an identical sulfur content.
The dextran derivatives obtained under the conditions defined above have the drawback of having irregular distribution of the chemical groups and of the sizes of the polysaccharide chains, leading to a heterogeneous final product whose properties are difficult to control.
The inventors have developed a novel process for preparing dextran derivatives, which allows better control of the production of specifically defined products (controllable degree of substitution, homogeneity of the distribution of the charged or uncharged chemical groups and size homogeneity of the polysaccharide chains in the final product, selection and better reproducibility of the desired activity).
A subject of the present invention is dextran derivatives of general formula DMC
a
B
b
Su
c
S
d
, in which:
D represents a polysaccharide chain, preferably consisting of concatenations of glucoside units,
MC represents methylcarboxylate groups,
B represents carboxymethylbenzylamide groups,
Su represents sulfate groups (sulfation of the-free hydroxyl functions borne by the glucoside units),
S represents sulfonate groups (sulfonation of the aromatic rings),
a, b, c and d represent the degree of substitution (ds), expressed relative to the number of free hydroxyl functions in a dextran glucoside unit, with groups MC, B, Su and S, respectively; a being equal to 0 or ≧0.3, b being equal to 0 or ≧0.1, c being equal to 0 or ≧0.1 and d being equal to 0 or ≧0.15, with the proviso that when d=0, a and/or b are ≠0,
which products display:
homogeneity of the size distribution of the chains, illustrated by an elution profile of symmetrical Gaussian type in high performance steric exclusion chromatography, and
homogeneity of the distribution of charged chemical groups, illustrated by an elution profile as a single symmetrical peak in low-pressure ion exchange chromatography.
These products are considered as being copolymers consisting of fictive subunits R—OH and R—OX, it being possible for X to be a methylcarboxylate (MC) benzylamide (B), sulfate (Su) or sulfonate (S) group, the polysaccharide chain of the unsubstituted dextran being considered as consisting of 300 fictive R—OH subunits, instead of 100 glucoside units, with regard to the fact that an unsubstituted glucoside unit comprises three free hydroxyl groups. Thus, a dextran methylcarboxylate (DMC) with a degree of substitution (ds) with methylcarboxylate groups of 1.2 contains 1.20 substituted groups (R—MC) and 1.80 free hydroxyl groups (R—OH), per glucoside unit.
This thus gives, in contrast with the heterogeneous products of the prior art, homog

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