DK174348B1 - Pentasaccharides, tetrasaccharides and intermediates in the form of pentasaccharides, tetrasaccharides and disaccharides for the preparation of pentasaccharides, and the process for the preparation of pentasaccharides - Google Patents

Description

DK 174348 B1

The present invention relates to novel tetra and pentasaccharides having therapeutic activity and to novel intermediates thereof. The invention further relates to a process for the preparation of pentasaccharides. The end products in question have particular biological properties which make them particularly interesting as drugs and / or useful e.g. as laboratory reagents. The end products in question are particularly applicable in the biological and biochemical fields.

The term "mucopolysaccharide acids" is understood to mean compounds which may also be referred to as glycosaminoglycuron glycans. These are oligosaccharides and polysaccharides which are particularly encountered in the chains of biologically active derivatives, such as hepatine-type derivatives and of the heparan sulfate type.

In natural products, mucopolysaccharides are composed mainly of alternating components, namely amino sugar-uronic acid or vice versa. The amino sugar species, hereinafter referred to as A, exhibits in particular such components (repeat groups) a D-glucosamine structure. The uronic acid, hereinafter referred to as U, in particular exhibits a D-glucuronic acid structure or L-iduronic acid structure.

The basic structure of A corresponds to the following formula a, and the basic structure of U corresponds to the following formulas b and c:

25 6r0H

(A) Drglucosamine

SO COOH

Y — o A_0 \

/ \ / COOH V ^ OH

i ^ / 0 "" r /

In OH

Oil (b) D-glucuronic acid (c) L-iduronic acid 2 DK 174348 B1

The various components in the natural products concerned are interconnected in a stereospecific manner, which is generally done by the bonds of 1- * 4 (a) and 1-4> β (β).

5

Thus, for example, heparin bonds of type 1-> 4 (a) (between groups c and a, a and b and a and c) and of type 1-> 4 (β) (between groups b and a) are found. 10 It is further noted, in the case of natural products, that the above-mentioned components contain specific substitutions, that is, certain substitutions in certain positions. Thus, for example, the chains of natural products contain components that are -O-substituted, such as 2-O-sulfate-L-iduronic acid, 3-O-sulfate-D-glucosamine, 3,6-di-O-sulfate-D-glucosamine , 6-O-sulfate-D-glucosamine, as well as non-O-substituted groups such as, for example, D-glucuronic acid groups, L-iduronic acid groups and D-glucosamine groups. In addition, the groups denoted by a are N-substituted at the 2-position with -N-acetyl groups and / or -N-sulfate groups.

The utility of the above-mentioned mucopolysaccharide acids in therapy is well known, in particular for the prevention and treatment of coagulation and vascular problems, especially thrombosis, as well as atherosclerosis and arterial sclerosis.

25 Furthermore, numerous works by the applicant are known to obtain high affinity fragments against AT III and with biological activity from heparin chains. The basic inventions of these works are disclosed in various patent applications, including European Patent Application No. 80 40 1425.6, cf. EP 270089 A1 and French Patent Application No. 81 30 08604, publication number FR 2504928 B1.

3 DK 174348 B1 In the European patent application mentioned, in particular, an octasaccharide, referred to as ABCDEFGH, which possesses great anti-thrombosis properties having the structural formula, is mentioned:

'c0 ° p OR p OR ^ 0 ° r- OR rOR

5 Lo Lo.-O Lo. J-oA / Lo / -o / o,

OSO "KHSO" OH NHAc OH NHSO "OSO ~ NHSO ~ 10A B CDE FG H

In this formula, R represents an S (V group or a hydrogen atom).

French patent application mentioned above discloses a homogeneous hexasaccharide compound having the structure C'DEFGH, which also possesses potent anti-thrombosis properties. The structure of this compound is similar to the formula:

r- OR cno ”j- OR r OR

20 II OH NHAc Oli NHSO "0S ° 3 IJHSO ~

C D E F G H

wherein R represents a SO 3 group or a hydrogen atom.

25

The methods described so far for obtaining products of this type employ extraction techniques from heparin or products obtained during the preparation of heparin, or also depolymerization technique applied to heparin chains under the action of a chemical or enzymatic agent followed by specific fractionation. especially through affinity chromatography.

4 DK 174348 B1

Further research in this area has focused on new approaches to obtaining this product type, in particular to explore the possibilities of achieving such by the synthetic route.

5 Mention should be made here of the large number of problems encountered in such a synthesis.

Such products, on the one hand, contain in their chains several components of type A and U. The bonds between these components, on the other hand, correspond to a defined stereochemistry, since these are 1.4-type bonds which, as is well known, are difficult to produce. In addition, each component carries one or more specific substitutions depending on the product type in question. The glucosamine components of the natural products also contain two mutually different nitrogenous groups, an N-acetyl group and an N-sulfate group.

15 It follows that such syntheses have so far practically never been discussed in scientific literature, especially as regards L-iduronic acid.

All of these details establish limited requirements, which makes it easy to understand the difficulties encountered in the preparation of a general process and a synthetic process.

In the exploration of the interlacing synthesis conditions suitable for the preparation of such type of compounds, a strategy has now been developed through the selection of certain particular types of protection for the starting materials used.

The studies carried out show that with such starting materials protected in such a way it is possible to carry out a stereospecific chain formation and then, if desired, introduce substitutions into predetermined positions on the sequences thus formed.

The process of the invention exhibits a very high degree of flexibility, which is a feature of great importance. Thus, with certain advantages, particularly in terms of specificity and purity, it is possible to obtain numerous oligosaccharide derivatives containing specific substitutions found in natural products, or various substitutions and / or components of analog structure, but with different configurations.

5

Pentasaccharides having particularly valuable medical properties, especially with high anti-thrombosis activity, have been obtained using the method of the invention. In the process according to the invention, a large number of particularly valuable pentasaccharides can also be obtained, which can be used especially as biological reagents and / or as a reference compound for structural studies.

Accordingly, the present invention relates to novel therapeutically active tetra- and pentasaccharides and intermediates in the form of tetra- or penta- or disaccharides, and to a process for the preparation of some of the therapeutically active pentasaccharides.

In the process of the invention, certain functional groups are introduced into the components of the glycoside chain, especially specific substituents, such as meet them with the chains of biologically active molecules, especially compounds of the type heparin and heparan sulfate. The process is used to prepare pentasaccharides having the structure of the natural products mentioned above.

In a first aspect of the present invention, the pentasaccharides disclosed have the formula: - υΟΟϊ'ΟΟϊ I-O-jp i-Oi> p

Ni 0i> N-L ° * ρ N3 6 wherein M is hydrogen or alkyl, e.g. methyl;

Sp and P are identical or different and are selected from acetyl, benzoyl, aryl, benzyl, hydrogen and sulfate, with Sp and P not being benzoyl simultaneously, Sp and P being preferably acetyl, hydrogen or sulfate and most preferably P being H The drug or benzyl and Sp acetyl, hydrogen or sulfate;

Ni, N2, and N3 are independently selected from NH2, -NH-COO-benzyl, -NH-COO-acetyl, -NH-SO3 "and -NH-acetyl, preferably -NH-COO-benzyl, -NH-SO3 ' and -NH-acetyl and most preferably NH 2 and -NH-COO-benzyl; T is hydrogen, benzyl, acetyl, monochloroacetyl or trichloroacetyl, or p-methoxybenzoyl, preferably hydrogen, acetyl, monochloroacetyl or trichloroacetyl, p-methoxybenzoyl, and most preferably hydrogen or benzyl; and R is selected from acetyl, alkyl, for example methyl, and benzyl, preferably benzyl; or -OR is halogen, for example Br, or imidoyl; and their salts where M is a alkali metal.

In another aspect of the present invention, the penta-saccharides of the present invention have the formula: o - - co ° 'roso' 'rOSOi' f HH50, ° f N, ISUi '· OB0' NHSO> 25 where P is selected from acetyl, benzoyl , aryl, benzyl, hydrogen and sulfate, preferably hydrogen, -NHSO 3 'can be replaced by -NH-acetyl, and -OH can be replaced by alkoxy, e.g. methoxy.

In a third aspect of the present invention, the present tetra-saccharides have the formula: ('T | 4vwf ^ O' ° s 'm'

Op W 0 05 p where M is hydrogen or alkyl, e.g. methyl, preferably alkyl, Sp and P are identical or different and are selected from acetyl, benzoyl, aryl, benzyl, hydrogen and sulfate, with Sp and P not being benzoyl simultaneously, preferably Sp being acetyl, hydrogen or sulfate and P benzyl or hydrogen,

Ni and N2 independently selected from -NH-COO-benzyl, -NH-COO-acetyl, NH2 or -NH-SO3, preferably -NH-COO-benzyl, -NH-COO-acetyl or NH2, T is hydrogen, benzyl , acetyl, monochloroacetyl or trichloroacetyl, or p-methoxybenzoyl, preferably hydrogen, monochloroacetyl or benzyl and R is selected from acetyl, alkyl, e.g. methyl, and benzyl; or OR is halogen, e.g. Br, or imidoyl, and their salts, wherein M is an alkali metal.

A fourth aspect of the present invention comprises intermediates in the form of pentasaccharides of formula XXIV or in the form of tetrasaccharides of formula XXIII, wherein the substituents are as defined above for the corresponding final compounds of the same formula and wherein at least one of the groups Ni, N2 and / or N3 are azido.

A fifth aspect of the present invention relates to intermediates in the form of disaccharides of the formula: 30 8 DK 174348 B1

C O OM

i r ° Rl jcVvØ- “

5 where uRj W

M is hydrogen or alkyl, e.g. methyl, N is selected from -NH-COO-benzyl, azido, -NH-COO-acetyl, -NH-SO3 'and -NH-acetyl, T is hydrogen, benzyl, acetyl, monochloroacetyl or trichloroacetyl, or p-methoxybenzoyl ; and R is selected from acetyl, alkyl, e.g. methyl, and benzyl, or OR is halogen, e.g. Br, or imidoyl,

R 1 is acetyl, benzyl, hydrogen or sulfate, or R and R 1 together form the divalent radical -CH 2 -O-, and their salts where M is an alkali metal.

In a preferred embodiment, these disaccharide intermediates of the present invention are of formula I wherein M is Na + or alkyl, T is monochloroacetyl, hydrogen, benzyl or acetyl, R is alkyl, or -OR is bromine,

R 1 is acetyl, benzyl, hydrogen or sulfate, or R and R 1 together form the divalent radical -CH 2 -O-, N is azido or acetylamino.

25

A sixth aspect of the present invention relates to intermediates in the form of disaccharides of the formula: p-o fc 1 t-o -o 30 / '(CcrOM f \ OR. Ύ ^ (VIII)

OA, N

Wherein T, R1f M, and R are defined as for Formula I and wherein M is preferably alkyl or hydrogen, 5N is preferably -NH-COO-benzyl or azido, T is preferably hydrogen, monochloroacetyl or benzyl,

R 1 is preferably benzyl or acetyl, R 1 is preferably benzyl, or

Preferably, R 1 and R together form the divalent radical -CH 2 -O-.

10

A final aspect of the invention relates to a process for preparing a pentasaccharide of formula XXXII of the present invention wherein P is hydrogen which comprises a compound of formula XXIV of the present invention wherein Sp is acetyl, P is benzyl. Nt and N2 are azido, N3 is -NH-COO-benzyl, M is alkyl, and T is benzyl is subjected to the following reactions: - removal of the O-acyl blocking groups, - sulfation of the liberated OH groups. using a trimethylamine / SO3 complex as a sulfating agent, - releasing the OH groups protected by benzyl groups and converting the nitrogen-containing groups into functional amino groups by catalytic hydrogenation, and - sulfating free OH groups and of functional amino groups using a trimethylamine / SO3 complex as a sulfating agent.

In one embodiment of the process of the present invention, the sulfation may be carried out in a solvent, e.g. dimethylformamide, at a temperature higher than room temperature and close to 50 ° C.

In another embodiment of the process of the present invention, the catalytic hydrogenation can be carried out under hydrogen pressure in the presence of a Pd / D type catalyst in an organic solvent.

5

The process of the present invention may further comprise that the compound of formula XXIV used is prepared by reacting a compound of formula XXIII of the invention wherein Sp is acetyl, P is benzyl, T is Η, M is alkyl, Ni is azido and N 2 is -10 NHCOO-benzyl having a monosaccharide of the formula: (44) In a preferred embodiment of this process, it further comprises compound of formula XXIII is prepared by a condensation reaction between the following disaccharides: -OAc pOAc -_α

OBn Ντ I

J OAc nh (20) (40 t 30 where MCA represents monochloroacetyl and subsequently -O-dechloroacetylation, or alternatively, the compound of formula XXIII used is prepared by a condensation reaction between the following disaccharides: 11 DK 174348 B1

.-OAC

iOOM '—0AC I

5 +

I I OA c NH

OBn Mj t = 0 (97) (41) (> Bn 10

End products and intermediates of the invention are prepared by establishing covalent bonds between components of structure A and U according to the stereochemistry present in this type of chain formation within the desired biologically active molecules.

15

The desired chain formation is carried out in a given order and / or with a given stereospecific ratio.

Thus, in particular, a type 1 -> 4 (a) bond between a D-20 glucosamine component and either D-glucuronic acid or L-iduronic acid, a "1-tA (β) type bond between a D-glucuronic acid component and a D-glucosamine component as well as a type 1-4 (a) bond between an L-iduronic acid component and a D-glucosamine component.

The mono- or oligo-saccharides acting as intermediates in this synthesis are semi-open or open compounds. The term half-open to the right refers to a compound which is activated or which can be activated by its anomeric carbon atom, whereby it can be transferred to the non-reducing end of a monosaccharide or an oligosaccharide. The term "semi-open left compound" refers to a monosaccharide or an oligosaccharide having a single OH group which is free or potentially free, allowing specific glycoside formation. By way of illustration, the following Formula 1 shows an example of a compound that is half-open to the left, and Formula 2 shows an example of a compound that is half-open to the right: rrO! \ C cool ^ F \ o6> \,

N

10 1 2

As a result, as open connections, a connection is referred to as being simultaneously half-open to the left and right as defined above, such connections allowing for a chain extension in both directions. For example, a compound of such type may correspond to Formula 3: thu * · / 1 20 (MtAO y --- j \

rV

In the case of closed compounds, these are compounds whose groups are unable to lead to a chain extension due to the nature of the incoming substituents,

Components A or U of the resulting sequence should, for the addition of additional components to the sequence AU or UA obtained in the preceding processing step, contain temporary protecting groups, that is, groups capable of selectively blocking a position in the component. A or U, which is intended to form part of an additional glycoside formation reaction. These groups can be removed in the presence of other groups present in the starting components during recovery of an alcohol, which, by repeating the preceding glycoside formation step, allows extension of the glycoside backbone.

5

Thus, the method provides access to the synthesis of oligosaccharides with varying chain configurations, whether in stereospecificity α or β and / or the order of chain formation between components a and / or b and c and / or d, this extension being carried out at will. .

In another embodiment, the glycoside chain thus prepared is subjected to one or more chemical reactions for introducing a given type of functional group or successively several types of groups upon which derivatives of these functional groups can be formed, if desired.

This treatment step for introducing functional groups can be accomplished by removing only some of the protecting groups and / or some of the precursor groups for the amino group-containing derivatives, or also the total amount of protecting groups and / or precursor groups, and instead of these, a given type of substituent or successively introduces various substituents, upon which, if desired, part or all of the permanently blocked OH groups are released.

It is to be understood that the different groups present in the chain repeat groups are compatible with the substituents introduced at each step.

The chemical reaction (s) used during the various processing steps to introduce functional groups is carried out in such a way that they do not alter the structure of the chain, or the groups which it is desired to maintain and / or such, which has already been introduced.

14 DK 174348 B1

In a preferred method for obtaining pentasaccharides with specific substituents, as defined above, advantageously, starting components containing multiple types of protecting groups, i.e. (1) one or more semipermanent groups and (2) one or more permanent 5 groups, are used. . By the term "semipermanent groups" is meant groups which can be eliminated in the first step after the glycoside formation reaction when the glycoside backbone contains the desired number of components, without excluding or altering the other groups present, thereby enabling the introduction of the desired functional groups in the positions that they occupy.

"Permanent groups" are those which are capable of maintaining the protection of the OH groups during the introduction of the functional groups instead of the semi-permanent groups.

15

These groups are selected from those compatible with the functional groups introduced after the elimination of the semi-permanent groups.

In addition, these are groups which are inert to the reactions carried out to bring these functional groups into place and which can be removed without changing the functional groups mentioned.

Advantageously, these systems can produce a glycoside chain wherein components A and U are selectively substituted.

In particular, for the preparation of oligosaccharides containing repeating groups A and / or U of the above-mentioned biologically active molecules, protecting groups such as acyl groups, optionally substituted alkyl groups or aryl groups may be used.

The type A components put into use contain in the 2-position a nitrogen-containing group which allows the presence of a nitrogen-containing functional group during the treatments carried out by the process. The nitrogen-containing group preferably consists of such groups as -N3 or -NHCOO-CH2-C6H5, or any other group which forms a precursor of the amino group or an amino derivative, especially -NHSO3 · or -NH-acyl , in particular -NH-COCH3.

As for the carboxyl groups of components U, they are blocked by groups which are inert to the reactions used to replace the protecting groups and which can be removed at the end of the synthesis to release these carboxyl groups, possibly for the purpose of on the salt formation. These protecting groups for the carboxyl group are advantageously selected from alkyl groups or aryl groups.

The structure of the starting materials used in the glycosylation reaction is selected depending on the desired components of the glycoside backbone as well as the desired substituents.

15

For example, if one wishes to form a -U-A- type disaccharide, two compounds having uronic acid structure and amino sugar structure, respectively, are used, which otherwise correspond to the above definitions.

Moreover, these compounds, as used to prepare the disaccharide in question, contain, for the purpose of extending the chain, a temporary moiety in the position intended to be included in the subsequent glycoside formation reaction. In order to extend the disaccharide U-A to the left, the temporary group in question is present on the repeat group U, while for a right extension it is on the repeat group A.

In this way it is possible to obtain in particular chain configurations Uw Αχ UY Az, in which the sum of the specified index values is from 2 to 12, 30, since w and y cannot simultaneously have the value zero. Regular chain configurations are of the type U (AU) n (AU) nA, (UA) n or (AU) n, with 1 <n <6.

You can modify the change between the type AU or UA encountered in the structures of the natural products, using one or the other of the repeat groups A or U, a sugar which constitutes a structural analogue for the repeat groups A or U , such as a neutral sugar or a deoxy sugar type, or other uronic acid or amino sugar type components U or A of various configurations.

5

For example, one transfers. the alcohol described above with a reactive derivative such as a halide, an imidate or an orthoester. Such condensations are carried out under anhydrous conditions.

The condensation reaction between the halide and the alcohol is most advantageously the so-called Koenigs-Knorr type. The halide is preferably a bromide or a chloride due to their easy availability.

Work is carried out in a solvent medium, especially in an organic solvent medium, particularly of the chloromethane or dichloroethane type.

A catalyst, generally a silver salt or a mercury salt, is advantageously used, for example silver trifluoromethanesulfonate, which is frequently referred to as silver triflate, silver carbonate, silver oxide, mercuribromide or mercuricyanide. A proton acceptor such as symcollidin is also used as well as an acceptor for the water present and / or for the hydrogen halide acid formed, e.g. and 4 Å molecular sieves

Examination of the reaction conditions has shown that it is suitable to operate at ambient temperature or at a lower temperature, however, at least at 0 ° C, and under atmospheric pressure of an inert gas such as nitrogen or argon.

Under these conditions, it is possible, after the desired stereochemical configuration, to condense components of structure a and b or c (or vice versa). It is also possible to establish covalent bonds with neutral sugars or with deoxy sugars.

A variant involving the use as a catalyst of mercurial derivatives, especially mercuricyanide and / or mercuribromide, has been found suitable for establishing covalent bonds between alcohols of varying structure and starting compounds for an L-idose precursor for a component of structure c (L-iduronic acid). This variant also uses a 4 Å molecular-5 screen sieve. The organic solvent is selected depending on the reactivity of the alcohol. Thus, a solvent of the nitrobenzene type is advantageously used when the condensation requires a temperature higher than 100 ° C. Solvents such as benzene or dichloromethane are used at lower temperatures.

10

The mixture of solvents is also suitable for carrying out the condensation reaction.

It is advantageous to use a so-called orthoester for components of the type U, especially of the type designated as c. Preferably, for this reaction, a temperature above 100 ° C is used.

The solvent medium is of the chlorobenzene type or any other solvent, the boiling point of which exceeds 100 ° C and which is advantageously between 100 and 150 ° C. To activate the reaction, a catalyst such as 2,6-dimethylpyridine perchlorate is used.

This embodiment of the condensation step has been found to be of great interest in providing an interglycoside bond between a component of structure c (L-iduronic acid) and a component of structure a (D-glucosamine).

By the use of an orthoester group, a further advantage is obtained.

30

Thereby one obtains, on the one hand, the anomeric carbon atom in c the reactivity necessary for the glycoside formation reaction. The opening of this group, on the other hand, ensures the position in the 2-position of c of a protecting group which can be selectively removed and replaced by a specific substituent group.

Thus, by reacting a 1,2-O-methoxyethylidene group in a component c with the OH group of a component a at once 5, it is possible to create an interglycoside bond between the two starting products used and in the 2-moiety of c to have an O-Ac group available (Ac denotes an acetyl group) which may be selectively removed upon completion of the introduction of a given functional group, e.g. - $ 03 '. This precaution also gives complete freedom in the treatment of 4 position in component 10.

By these particularly advantageous precautions, a 2-O-iduron sulfate repeat group can be produced, such as, for example, in the heparin chains.

15

It has been found appropriate when employing an imidoyl group as a reactive group, to operate at low temperatures, especially at a temperature lower than or equal to approx. OX, in solvent environment, such as in dichloromethane, in the presence of a 4 Å molecular sieve and of a catalyst such as boron trifluoride etherate.

In the starting alcohol, the free OH group repeats the position one wishes to use in the glycoside bond.

Thus, by selecting an appropriate alcohol, it is possible to establish bonds of the types 1-2, 1-3, 1-4 or 1-6.

From the sequence formed at the end of the condensation reaction, a chain containing the desired number of repeating groups is repeated, repeating the glucoside formation step.

The alcohol group in one of repeat groups A or U, which is implicated in the already prepared glycoside sequence, is then released from its temporary protecting group. The choice of such a group is easy to determine for one skilled in the art, depending on the nature of the other groups present in the glycoside chain.

Among the various groups which can be used are the allyl group which, for example, is treated first with an isomerizing agent such as derivatives of Pd, Rh and Ir, especially rhodium tris-triphenylphosphine (I) or potassium tertiobutoxide, and subsequent treatment , especially under acidic conditions, with a mixture of mercuric oxide and mercuric chloride, easily leads to the recovery of an alcohol in the position it occupies.

10

It is also possible to obtain an OH group by saponifying an O-acyl group, in particular an O-acetyl group or O-chloroacetyl group.

These groups can then, for example, be eliminated to release an OH-15 group, e.g. by means of thiourea in solvent environment, especially at a temperature above 80 ° C, preferably in the order of 100 ° C.

The arrangements described above permit the obtaining of a glycosidic chain with alternating repeat groups A-U or U-A.

20

This regular alternation can be modified by the use of appropriate starting materials in the glycoside formation reaction. Thus, it is possible to produce an irregular structure incorporating components different from U or A, especially neutral sugars or deoxy sugars. Another type of irregular structure can be obtained by adding tens of consecutive components A or U between two components of structure A-U or U-A.

It is to be understood that the various arrangements of the invention described for components A and U can also be applied to other components which may be contained in the glycoside chain, such as neutral sugars or deoxy sugars.

As previously stated, the various groups present on components A and U are selected in such a way that they provide these latter with a sufficient reactivity to establish the glycoside bond in question.

Generally, the protecting groups for the OH groups, other than the temporary protecting groups already mentioned, are selected within the group comprising acyl groups, especially acetyl, alkyl and substituted alkyl such as benzyl, and for two neighboring positions within the group consisting of acetals or ketals, e.g. eg benzylidene. Another form of protection consists in blocking two OH groups in the form of an epoxide or of a 1,6-anhydro bridge.

The starting products used in the glycoside formation reaction advantageously contain several types of protecting groups, whereby during the treatment step for introducing functional groups one or more functional groups can be successively introduced and release one or more OH groups if desired.

In general terms, the protecting groups may already occupy predetermined positions in the starting products used in the glycoside formation reaction.

They can also be introduced from other groups once the glycoside backbone has been created. In this variant, for example, for the glycoside moiety, a component A may be used in which the OH groups in the 2 and 3 positions as well as in the 1- and 6 positions are blocked in anhydride form, respectively as 2,3-epoxide and as , 6-anhydro. Thanks to this blockage, an element has been made available during the preparation of the glycoside backbone, which potentially constitutes a repeat group A; but which does not interfere with the reactions used during the synthesis. This arrangement has the advantage of having a high degree of freedom to carry out the desired reactions with the groups of the other components.

21 DK 174348 B1

Moreover, it should be noted in the present case that the opening of the epoxide group by means of sodium azide permits introduction into the 2-position of an N3 group, thus constituting a precursor for the amino group.

Preferably, in order to be able to dispose of a glycoside chain which allows the selective introduction of one or more types of substituents during treatment to introduce functional groups, especially the specific substitutions mentioned above, starting products containing various types of protecting groups , namely both semi-permanent and permanent groups, as defined above.

The substituents in the natural products concerned, with the exception of the substituents in the 2-positions of repeat groups A, as already indicated, consist mainly of sulphate groups.

15

Studies carried out to determine the appropriate sulfation conditions have shown that it is possible and even advantageous to carry out a sulfation reaction in the presence of benzyl groups. The removal of permanent benzyl groups in the presence of O-sulfate groups can be carried out contrary to what has been hitherto thought.

Preferably, the OH groups in the starting material which are intended to be sulfated are protected by acyl groups, especially acetyl groups, while the OH groups which are intended to be released at the end of the synthesis are protected by a permanent group such as a benzyl group.

It is possible, thanks to the great flexibility associated with the process, to subject the entire glycoside chain thus produced to a given chemical reaction for introducing a particular type of substituent.

This treatment may, for example, consist of an esteritization, especially a sulfation, by an appropriate agent, this treatment being carried out under conditions which do not alter the interlaced structure. This sulphation can be carried out specifically or unspecifically and if desired on the total glycoside liberated for protecting groups.

The treatment step for introducing functional groups may e.g. is carried out selectively in such a way that successively introduces several types of substituents and then releases certain OH groups.

Under particularly advantageous conditions which allow the introduction of sulphate groups at certain positions in the components, the release of OH groups in 10 other positions, that in the 2-position of A-components an amino derivative is formed and in the 6-position of U-components form acid derivatives, components which are in accordance with the following characteristics are used:

The semi-permanent groups in these components occupy positions which are intended to be sulfated and consist of -O-acetyl groups,

As for the positions corresponding to an OH group which is intended to be released, they are occupied by semi-permanent groups consisting of benzyl groups, the 2-positions of the A components being substituted by groups such as or -NH-COO-CH2-C6-H5, and the 6-positions of the U components are occupied by carboxylic acid groups protected by an alkyl group, especially methyl.

These conditions allow the completion of the treatment step to introduce functional groups, for example, as follows: First, the sulfate groups are selectively introduced after removing the -OH-acetyl groups used for blocking. This reaction is carried out so as not to affect the benzyl groups, nitrogen-containing groups or carboxyl groups present.

To this end, a saponification reaction is advantageously carried out by a strong base such as sodium hydroxide.

This reaction is preferably carried out at a temperature lower than ambient temperature, especially in the vicinity of O'C.

5

The reaction product thus prepared can be subjected to hydrolysis by the action of an alkylating agent to introduce onto the carboxyl group those alkyl protecting groups which will be removed during the hydrolysis.

In this way, by reaction with a sulfating agent, sulfate groups are introduced into the positions liberated by hydrolysis and which remain free after the interaction with the alkylating agent.

In order to obtain satisfactory reaction conditions for carrying out the sulfation, a sulfating agent such as a trimethylamine / SO3 complex is used. This reaction is advantageously carried out in solvent medium, especially in a solvent such as dimethylformamide. It is preferred to operate at a temperature higher than the ambient temperature, generally at approx. 50 ° C, which corresponds to a reaction time of approx. 12 hours.

20

Following the introduction of the sulfate groups on the alcohol groups, the release of the OH groups blocked by benzyl groups is continued.

Advantageously, removal of the benzyl groups by catalyzed hydrogenation is carried out under reaction conditions which are compatible with the maintenance of the sulfate groups and with the conversion of the nitrogen-containing groups into functional amino groups.

Preferably, hydrogen is operated in the presence of a Pd / C type catalyst.

This reaction is advantageously carried out in an organic solvent medium. especially alcoholic to which water is added.

24 DK 174348 B1

The reaction is advantageously carried out to obtain the hydrogenation of the nitrogenous precursor groups and the removal of the groups protecting the OH groups with a reaction time of approx. 3 - 4 days, 5 As already indicated, the functional amino groups are present in the biologically active molecules concerned in the form of N-acetyl or N-sulfate derivatives.

For the formation of N-acetyl groups, the reaction product obtained by the hydrogenation reaction is subjected to the action of an acetylation agent. Acetic anhydride is a particularly suitable agent for this purpose.

In order to carry out this selective acetylation reaction without affecting the other substituents present on the components, it is especially convenient to operate at basic pH, especially near the value of 8 and in aqueous medium.

It is also desirable to form N-sulfate groups, which can be carried out by means of a sulfating agent as indicated above. In this sulphation, pH values greater than 9 are used, especially on the order of 9- 20 10.

Addition of a strong base after the sulfation reaction allows the release of the carboxyl groups.

The reaction products thus formed are readily converted to salts by suitable cation exchange resins. This cation consists in the natural products, especially sodium. Therefore, ion exchange resins containing sodium ions are advantageously used.

You can also form salts with potassium, lithium, magnesium and calcium.

Here, a proton-ion exchange resin is then utilized to neutralize the acid thus formed with the base corresponding to the cation.

The present invention also relates to compounds which are synthetic intermediates at the various steps of the synthetic process described above.

Intermediates in the form of penta, tetra, and disaccharides according to the invention 5 contain e.g. at least one fully protected binary repeat group AU and UA having either a reactive group on the anomeric carbon of the component at the reducing end, or a single free OH group on the component at the non-reducing end, said OH group occupies the position 3, 4 or 6 in the case of a component of type A, or the position 2, 3 or 4 in the case of a component of the type U.

Intermediates may also consist of fully protected repeat groups as obtained at the end of the glycoside formation step, or of products in which one or more OH groups are released.

These various oligosaccharides contain a chain based on binary repeat groups of structure (A-U) n or (U-A) n wherein n is a number from 1 to 6.

20

These oligosaccharides correspond to a chain formation of type a-b or a-c.

The glycoside chain may also consist of a single type of these binary repeat groups.

25 In other cases, several of these types are present.

Corresponding oligosaccharides in the chains contain repeating groups a-b and a-c.

30

It will be appreciated that the order of chain formation described above within one or more of the binary repeat groups may be exchanged within the scope of the invention.

26 DK 174348 B1

According to a variant, intermediates according to the invention contain one or more consecutive components a or b or c.

According to another variant in the structure, the intermediates contain one or more components consisting of neutral sugars and / or several deoxy sugars. The various protecting groups for these sugars correspond to the definitions given above for components A and U.

The components included in these compounds are interconnected with 10 bonds of the types 1-2, 1-3, 1-4 or 1-6 depending on the nature of the alcohol used in the glycoside formation step.

The compounds having the structure as heparm fragments or heparan sulfate fragments contain bonds d (a) 1-4a, a (a) 1-4b, a (a) 1-4c and 15b (p) 1 -4a.

Preferred compounds of this group contain in the 2-position of these components a -NHSCV group. Other compounds contain an -NH-acyl group, especially -NFI-acetyl 20

The esters hereinafter preferably are in the form of salts with an inorganic or organic cation, especially a metal cation, especially an alkali metal cation, or a cation derived from a nitrogenous organic base, for example the triethylammonium ion.

25

The cations used are preferably sodium. Other cations are also suitable, such as potassium, magnesium or calcium.

Compounds of the invention containing carboxyl groups in components b or c may be free, or are preferably in the form of salts with an organic or inorganic cation as defined above. They may also be protected as mentioned above.

The compounds of the invention can be used as biological reagents, which can be used in the laboratory, especially as comparative compounds in the study of other compounds whose anticoagulation activity is desired to be tested, especially in relation to inhibition of factor Xa and the determination of antithrombin. III (AT III).

5

An oligosaccharide for anticoagulation activity (anti-Xa activity) can be tested by using factor Xa, as marketed by the company SIGMA, in a solution containing 8 U / ml in physiological saline solution, the concentration of the substrate being 1.33 mM.

10

The procedure for this test is as follows:

Mix 10 µl of the solution to be measured and 300 μΙ of human plasma diluted with a buffer (Tris-maleate 0.02 M, pH 5).

15

Allow to incubate for 1 minute. 37 ° C.

Then 100 μΙ of the above factor Xa (8 U / ml) is added, and one minute later the solution thus obtained is injected into the substrate.

20

Certain compounds have the advantage that they do not have an effect on activating platelet aggregation and that they do not cause thrombocytopenia. They also have the advantage of being virtually devoid of any effect on bleeding time, eliminating the risk of bleeding. These two properties are extremely important in the medical application.

In addition, an extended pharmacokinetics have been observed at subcutaneous administration, which also gives these compounds great importance.

In addition, such compounds show no toxicity.

Therefore, these compounds are particularly valuable in the manufacture of drugs which are particularly useful in the prevention and treatment of thrombo- bosses.

Such compounds can also be used in the preparation of pharmaceutical formulations containing compounds with high anti-Xa activity, especially the pentasaccharides described above.

5

They are particularly suitable for the preparation of pharmaceutical formulations which do not contain pyrogenic ingredients and which contain an effective amount of the active ingredients together with conventional pharmaceutical excipients.

They also find use in formulations in which the pharmaceutical carrier is suitable for oral administration. Such formulations suitable for oral administration may be gastroresistant gelatin capsules, tablets or pills, or they may be present as liposomes.

Still other pharmaceutical formulations containing these compounds together with appropriate excipients may be intended for rectal administration.

The corresponding forms may consist of suppositories.

Other forms of administration for the compounds in question consist of aerosols or 20 pomades

The compounds in question may also be available as injectable formulations which are sterile or sterilizable and which are suitable for intravenous as well as intramuscular or subcutaneous administration.

Such solutions advantageously contain 1000 to 100,000 units (Yin-Wessler) / ml of oligosaccharides, preferably 5,000 to 50,000, e.g. 25,000 U / ml when these solutions are intended for subcutaneous injection. For example, they may contain 500 - 10,000, especially 5,000 u / ml oligosaccharides, when they are intended for intravenous injection or perfusion.

Such pharmaceutical formulations may advantageously be available as disposable syringes ready for use.

29 DK 174348 B1

The compounds of the present invention may also be included in pharmaceutical formulations containing such compounds together with another active ingredient which are particularly useful for the prophylaxis and treatment of thromboses such as a venetonic agent such as dihydroergotamine, a nicotinic acid salt or a thrombolytic agent. agent such as urokinase.

The pharmaceutical formulations in question are particularly suitable for the regulation (preventive or curative) of certain steps of blood coagulation in humans or in animals, especially in such cases where the patient is at risk of hypercoagulant ability arising in particular from surgical operations. atheromatous processes, the development of tumors as well as disease states with regard to coagulation due to bacterial or enzymatic activators etc.

15 In order to illustrate the use of such compounds, the following is an example of posology as it is applicable to humans: Such a posology includes, for example, administration to the patient of 1,000 to 25,000 units (Yin-Wessler) in a subcutaneous manner to three times a day, depending on the degree of risk of hypercoagulability or the patient's thrombotic state, 1,000 - 25,000 units / 24 hours, respectively, by intravenous administration at regular intervals or continuously through perfusion or also 1,000 - 25,000 units (three times per day). week) by intramuscular or subcutaneous route (all of these units being expressed as Yin-Wessler units). Of course, such doses can be regulated for each individual patient, depending on the results obtained and the prior blood tests performed, depending on the nature of the patient's condition and the general condition of his or her health.

In addition to containing oligosaccharides, the pharmaceutical formulations in question may also contain at least one compound, as defined above, linked by covalent bonding to a soluble or insoluble carrier, advantageously by a sugar of the terminally reducing group.

30 DK 174348 B1

Preferred conjugates of the soluble carrier compounds are compounds bound to AT III.

Other preferred conjugates with soluble carriers are compounds attached to a carrier such as a protein, especially polylysine or bovine serum albumin.

These products are useful as immunogenic substances which in themselves are source of antibody circulation products in vivo or monoclonal antibodies which are cloned in vitro by appropriate techniques.

The present compounds are bound to insoluble carriers by other preferred conjugates. Here you will advantageously use the classic carriers. These conjugates can be used as immunoabsorbents, e.g. by a highly specific purification of AT III, and by the determination thereof, or in the preparation of novel blood-compatible athrombotic polymers, by fixing the compounds on biologically compatible polymers.

The present oligasaccharides also find use in nuclear medicine as radiopharmaceutical products. In that case, these products are marked by a tracer, selected from those commonly used in this field, especially by technetium 99 m.

For this purpose, technetium 99 m obtained from commercially available generators in the form of non-reactive sodium per-technetate with valence 7 is converted into valence to technetium which is reduced in valence to 4, which is the most responsive form of technetium. . This conversion is carried out with a reducing system formed from tin salts (stannous chloride), iron salts (ferrous sulfate), titanium salts (titanium trichloride) or other salts.

In the vast majority of cases, this simple reduction of technetium is sufficient to allow fixation of technetium on the molecule in question under predetermined pH conditions.

31 DK 174348 B1

The compounds of this invention, which in some way constitute a carrier, can be used for measurements in the order of 100 - 200 units Yin-Wessler.

In further development of these radiopharmaceutical reaction components, one can proceed according to the method set forth by P.V.

KULKARNI et al. in The Journal of Nuclear Medicine 21, No. 2, pages 117-121.

Advantageously, the thus-labeled products are used in in vivo tests to detect and diagnose the extent of thrombosis and 10 of thrombotic conditions.

The compounds of the invention can also be used for the determination of the specificity of numerous enzyme systems involved in the metabolism of glucosaminoglucuric glycans.

15 In the years 2001 and 2002, a large number of articles have been published, all documenting the therapeutic and other beneficial effects of the following compound, also referred to as Fondaparinux sodium (a specific embodiment of formula XXIV) and sold under the name "ARIXTRA" : 20 ° S °, - coo- C! O °! '

Lh 25 O "" "JHS0 OH NHSOj- OSO," NHSO, ·

Fondaparinux sodium specifically inhibits factor Xa. This is well documented in, inter alia: Petitou et al. Semin. Thromb. Hemost. (2002) the synthetic pentasaccharide Fondaparinux: First in the class of antithrombotic agents that selectively inhibit coagulation facor Xa 28 (4): 393-402; Bauer et al. PMID (2002)

Factor Xa inhibition in the prevention of venous thromboembolism and treatment of venous thromboembolism 8 (5): 398-404; Bauer et al.

Cardiovasc. Drug Rev. (2002) Fondaparinux, a synthetic pentasaccharide: the first in a new class of antithrombotic agents - the selective factor Xa inhibitors 20 (1): 37-52. Review, 5

In addition, a number of articles have been published that document that Fondaparinux can be used successfully for orthopedic surgery to prevent thrombosis, for example: Bauer et al. N. Eng. J. Med. Fondaparinux compared with enoxapahn for the prevention of venous throm-10 embolism after elective major knee surgery 18: 1305-10; Eriksson et al. N.

Engl. J. Med. (2001) Fondaparinux compared with enoxapahn for the prevention of venous thromboembolism after hip fracture surgery 18: 1298-304.

The following examples illustrate the invention. Figures 1-32 illustrate some of the products used in the syntheses described.

In these figures, the numbering of formulas is also used in the examples to describe uniform products.

20 In the formulas in question, the following abbreviations are used: A: allyl

Ac: acetyl

Me: methyl Bn: benzyl

Bz: benzoyl MCAO. monochloroacetyl

Tr: trityl P; propenyl but but: butyl and S represent: enSO3 group 33 DK 174348 B1 EXAMPLE 1 (Starting material 5 Synthesis of derivative 13: methyl- (prop-1 '-enyl) -2,3-di-Q-benzyl-gD- glucopyranoside uronate of the formula: COOMe H ON-βΡ OBn 15 This synthesis is carried out from glucose using the following steps (a) to (m): (a) preparation of the allyl derivative; (b) blocking positions 4 and 6 in (c) introducing benzyl groups at positions 2 and 3; (d) unblocking positions 4 and 6 upon removal of the benzylidene group; (e) introducing a trityl group at position 6 followed by an acetyl. (f) removal of the trityl group from the 6 position; (g) oxidation of the primary alcohol group at the 6 position; (h) methylation of the carboxyl group at the 6 position; ( i) introducing the propenyl group at the 1 position; 5 (j) removing the acetyl group at the 4 position.

These steps are carried out as shown below (see Figures 1 and 2): (a) Preparation of allyl-α-D-glycopyranoside (Compound 1)

A solution of gaseous hydrogen chloride (18 g) in allyl alcohol (600 ml) is heated at 70 ° C. Anhydrous glucose (300 g) is then added and the temperature is maintained for 3 hours. The reaction is followed by thin layer chromatography (TLC) in the solvent mixture methanol / chloroform (1/4, v / v). The brown solution obtained after 3 hours is evaporated to dryness under vacuum, neutralized with a solution of concentrated ammonia water (50 ml) and then evaporated to dryness again. To the evaporation residue thus obtained is added the acetone (500 ml) which is heated to boiling and the boiling is maintained until complete dissolution. The liquid is decanted off after cooling. Evaporation residue is again subjected to the same treatment until TLC analysis of the extract shows a depletion of the compound 1 in the evaporation residue, or excessive contamination of the extract with impurities, respectively.

25

Part of the first extracted fraction (12 g) is chromatographed over silica. Compound 1 is obtained, which can be recrystallized in a mixture of acetone / ether (6.5 g; mp 95 - 99 ° C). The rest of the manufactured product can be purified by the same procedure.

(B) Blocking of the 4- and S-stillinos in the allyl derivative leading to allyl-4,6-O-benzylidene-α-D-glucopyranoside (compound 2)

Compound 1 (37 g) is dissolved in dimethylformamide (200 ml). Then, dimethoxytoluene (41 g) is added followed by ptoluene sulfonic acid with crystal water (130 mg). After 2 hours of heating (water bath) under vacuum and reflux, the reaction is complete (TLC; methanol / chloroform, 2/25, v / v). The solvent is evaporated. The syrupy liquid is dissolved in methanol (at least 5 possible amounts) and this solution is poured drop by drop into an aqueous solution of sodium bicarbonate (6.3 g in 320 ml of water). The precipitate thus obtained is crystallized from ethanol (21 g; mp 120 -121 ° C). The mother liquor leads to additional amounts of the reaction product 2.

Total yield (37 g; 71.4%).

(C) Introduction of the benzyl group leading to allyl-2,3-di-0-benzyl-4,6-o-benzylidene-α-D-glucopyranazide (Compound 31

The compound 2 (45 g) is dissolved in anhydrous DMF (500 ml). Sodium hydride (28 g 50% dispersion in oil) is added.

After 30 minutes, the mixture is cooled to 0 ° C, and drop by drop of benzyl bromide (52 ml) is added dropwise. The reaction is followed by TLC (ether / hexane, 1/1, v / v). Then methanol (150 ml) is slowly added which is evaporated to dryness and redissolved in chloroform. The chloroform phase is washed with water, dried over sodium sulfate. After evaporation of the solvent, the residue is recrystallized from an ether / hexane mixture (36.5 g: mp 83-84 ° C). This reaction product is more readily contaminated with an impurity that moves faster by TLC (ether / hexane; 1/1; v / v).

(D) Removal of the benzylidene group of allyl-2,3-di-O-benzyl-g-D-glucopyranoside (Compound 4)

To a solution of the compound 3 (56 g) in methanol (1 liter) water 30 (450 ml) and then p-toluenesulfonic acid with crystal water (17 g) is added.

After 2 hours at 80 ° C, the mixture is cooled, the solvent is evaporated and the residue is redissolved in chloroform (1 liter). The chloroform solution is washed with water to neutral pH and dried over sodium sulfate.

36 DK 174348 B1 In this way a light yellow syrup (48 g) is obtained which is used in subsequent steps (synthesis of compound 5).

(θ ') Introduction of a trityl group at the 6-position followed by an acetylation-5 reaction at the 4-position leading successively to allyl-2,3-di-0-benzyl-6-O-trityl-α-D-gluco-pyranoside ( compound 5) and its 4-Q-acetyl analogous compound (compound 6a) (R = acetyl in Figure 1-2)

The above-obtained derivative 4 (48 g) is dissolved in pyridine (250 ml) and trityl chloride (28.5 g) is added. After 1 hour at 100 ° C, the reaction is complete (TLC, ether / hexane, 1/1, v / v). Ti) the above solution is added acetic anhydride (200 ml). After one night, the reaction is complete (TLC, ether / hexane, 1/2, v / v). It is evaporated to dryness, the residue is redissolved in chloroform (500 ml), the chloroform phase is washed with a 10% potassium hydrogen sulfate solution, with water and dried over sodium sulfate.

The chloroform is evaporated. In this way, the compound 6a used as it is obtained in the reaction to produce the compound 7a is obtained.

(F) Removal of the trityl group leading to allyl-4-O-acetyl-2,3-di-Q-benzyl-α-D-glucopyranoside (Compound 7a)

The thus obtained derivative 6a is dissolved in chloroform (500 ml). 25 drops drop by drop is added to this solution, which is cooled to 0 ° C, a solution of boron trifluoride in methanol (12%, 120 ml). The reaction is followed by TLC (toluene / acetone, 10/2, v / v).

The reaction mixture is transferred to a separatory funnel. The chloroform phase is washed with water (2 x 100 ml) with a saturated sodium bicarbonate solution and then with water to neutral pH. The evaporation residue obtained after drying and evaporation is transferred to a silica column (500 g) which is equilibrated with toluene. After eluting the first portion of the impurities by pure toluene, the reaction product is eluted with a toluene / acetone mixture (10/2, v / v). In this way, 48 g of compound 7a is obtained which is used directly in the synthesis of compound 8a. Part of the compound 7a was prepared in the pure state: (a] 20o = + 11 * (chloroform). The structure was confirmed by IR and NMR spectra as well as by elemental analysis.

(G) Oxidation of the primary alcohol syrup in 6-stillina leading to 4-allyl-Q-acetyl-2,3-di-O-benzyl-α-D-alucopyranoside uronic acid (compound 8a)

A solution of the compound 7a (48 g) in acetone (800 ml) is cooled to -5 ° C. Then, drop by drop, a solution of chromium trioxide (30 g) in sulfuric acid (3.5 M; 125 ml) is added drop by drop. The mixture is allowed to reach ambient temperature. The reaction is monitored with TLC (methanol / chloroform, (1/10, v / v). After completion of the reaction, the reaction mixture is poured into water (500 ml). The reaction product is extracted with chloroform (3 x 250 ml)) and the chloroform phase is washed with water until neutral pH, dried over sodium sulfate and evaporated to dryness.

The syrup thus obtained (83 g) is used, as it is, in the preparation of compound 9a.

(H) Methylation of the carboxyl group at the 6-position leading to methyl (4-allyl-0-acetyl-2,3-di-O-benzyl-g-D-glucopyranoside uronate compound 9a)

The syrup prepared in the step of preparing compound 8a is dissolved in 25 ether (300 ml). Then a solution of diazomethane in ether is added until compound 8a disappears (TLC, ether / hexane, 1/1, v / v). The solvents are removed after being acidified with acetic acid.

The obtained evaporation residue (53 g) is dissolved in ethanol in the heat. The compound 9a crystallizes on cooling. After recrystallization, this compound 9a is obtained in a pure state (18.4 g, mp 85-86 ° C, [α] 20 ° = + 12 ° (1.2 chloroform).

This product is characterized by IR and NMR spectra as well as by elemental analysis.

An additional 7.6 g of compound 9a is obtained from the filtrate of crystallization.

The total yield of 9a prepared from compound 2 is 38%.

(i) Introduction of the propylene group at the 1-position leading to the methyl (prop-1'-envyl-4-O-acetyl-2,3-di-O-benzyl-qD-glucopyranoside) uronate (compound Oa) The derivative 9a (4 g) is dissolved in a mixture of ethanol (119 ml) L and benzene (51 ml) and water (17 ml). Then add diazabicyclooctane (170 mg and reflux. To the boiling solution is added tris- (tri-phenylphosphine) -rhodium (I) (550 mg) The boiling is maintained for 4 hours (TLC, ether / hexane, 1/1, v / v).

15

At the end of the reaction, the solution is filtered and the solvents removed. The residue is chromatographed over silica gel (150 g) with a mixture of ethyl acetate / chloroform (1/50, v / v). The compound 10a (3.25 g; 81%) is obtained, which recrystallizes from ethanol. [α] 20ο = + 12 ° (1, chloroform). Melting point 90 ° C. The structure of the compound is confirmed by elemental analysis as well as by IR and NMR spectra, (i) Removal of the acetyl group from the 4-position leading to methyl-fprop-1 '-enyl-2,3-di-Q-benzvi-aD-qluco-pyranoside ) -uronate (Compound 13) 25

The compound 10a (350 mg) is dissolved in methanol (5 ml). Sodium methanolate (0.2 ml, 2 M) is added. After 1 hour at ambient temperature, the reaction is stopped by the addition of the ion exchange resin "Dowex 50-Η +". After filtration, the reaction product 13 is obtained, which is contaminated with some of the reaction product originating from the α-β elimination.

In a variant of the step (s), instead of carrying out an acetylation reaction, a benzylation reaction can be carried out which leads to 4-allyl-O - benzoyl-2,3-di-0-benzyl-4 -O-benzoyl-6-O-trityl-α-D-glucopyranoside (compound 6b) (R6 = benzyl in Figure 1-2), from which the trityl group is then removed, allowing 4-allyl-0 to be obtained. -benzoyl-2,3-di-O-benzyl-α-D-glucopyranoside (compound 7b).

These reactions are carried out as follows:

Preparation of Compounds 6b and 7b

To the solution in pyridine of compound 5 is added benzoyl chloride (1.5 equivalents) and the reaction is followed by TLC (ethyl acetate / benzene, 1/20, v / v). The excess benzoyl chloride is destroyed by the addition of an excess of methanol. After evaporation to dryness, the residue, which is redissolved in chloroform, is washed with a 10% solution of KHSO 4 with water, dried and evaporated to dryness. The syrup thus obtained is used, as it stands, in the synthesis of compound 7b. This syrup (105 g, obtained from 30 g of compound 3) is dissolved in chloroform (300 ml). P ~ toluenesulfonic acid (76 g of the monohydrate in 100 ml of methanol is added. After a night, the reaction is complete (TLC, ethyl acetate / chloroform, 1/20, v / v). The chloroform phase is washed with water until neutral pH is dried) The concentrate thus obtained is chromatographed over a column of silica gel (1.2 kg), eluted with chloroform (0.6 liter) and then with an ethyl acetate / chloroform mixture (1/20, v / v). In this way, the derivative 7b is obtained in the pure state (30 g), which is used, as it is, in the step of preparing the compound 8b.

25

It is possible from the compound 7b to oxidize the -CH 2 OH group at the 6-position and then introduce a methyl group on the carboxyl group thus formed, successively forming 4-allyl-O-benzoyl-2,3-di-O-benzyl -αD-glyco-pyranoside uronic acid (compound 8b) and the corresponding methyl ester (compound 9b). These derivatives are prepared by proceeding as follows:

Preparation of compound 8b and of ester 9b

Compound 7b (27 g) is treated analogously to that described for 7a in the preparation of 8a. The syrup obtained by the treatment contains the compound 8b, which is methylated with diazonethane, as described for the compound 8a.

The evaporation residue obtained at the end of the methylation is purified over silica gel (200 g; ether 1 / hexane I). In this way the compound 9b (21 g; 77.5%) is obtained. The structure is confirmed by IR and NMR spectra.

You make out. from compound 9b, the corresponding propenyl derivative 10b, 10 proceeding analogously to 10a. Then, the derivative 13 is obtained from 10b by the same reaction given for 10a.

According to another variant, allyl-2,3-di-O-benzyl-α-D-glucopyranoside uronic acid and methyl (allyl-2,3-di-O-benzyl-α-D-15-glucopyranoside uronate (compounds 11 and 12) are prepared) ) by dissolving compound 8b (1.9 g) in methanol (40 ml), then adding 5N sodium hydroxide in sufficient quantity to give a 1M concentration with respect to sodium hydroxide. The reaction is monitored by TLC (methanol / chloroform, 1/4, v / v) When completed, water (100 ml) is added, washed with 20 ether, acidified, and the reaction product extracted with ether. The acidic ether phase is washed with water to neutral pH. by an ether solution of diazomethane which thus leads to compound 12 (900 mg, 56%) which is then purified over a column of silica (ether / hexane, 1/1, v / v) [o] 20d = + 35.2 ° (1.3, chloroform) The structure is confirmed by elemental analysis as well as by IR and NMR spectra. the derivatives 11 and 12 thereon from 9a or 9b.

The compound 13 can be obtained from the compound 12, proceeding as follows: 30

Compound 12 is treated with the rhodium complex as described in connection with 9a. Compound 13 is obtained with a yield of 90%. It is characterized by its IR and NMR spectra. In addition, by treatment with acetic anhydride (1 ml to 180 mg 9a) for compound 10a.

41 DK 174348 B1

The derivative 13 can be obtained from yet another variant from 10a or 10b, proceeding as described in the preparation of 17 from 9a or 9b.

EXAMPLE 2 (Intermediate of Formula I)

Synthesis of the disaccaride 20 or 1-bromo-3,6-di-O-acetyl-2-azido-4-O [methyl-2,3-di-O-benzyl-4-O-chloroacetyl-8-D-glucopyranosyl-uronate-1 D-Glucopyranose of the formula: COOMe i-Q Ac M3Bn / \ / oAc)

15 MCA0 \ _Y

OBn N3 (20)

This synthesis consists of the following steps (see Figures 3 and 4): 20 (A) The preparation from derivative 13 of Example 1 of the monosaccharide 16 or methyl- (1-bromo-2,3-di-0-benzyl-4-O) (B) Condensation of the compound 16 with the monosaccharide 17 leading to the di-saccharide 18; (C) Acetolysis of the compound 18 leading to the disaccharide 19; and (D) Bromination leading to the disaccharide 20.

Preparation of the monosaccharide 17

This synthesis is carried out from the monosaccharide 13 or methyl (prop-1'-enyl-2,3-di-O-benzyl-α-D-glycopyranoside uronate according to the following three steps: 1: Chloroacetylation of compound 13; 5 2: unblocking of the anomeric carbon atom; 3: bromination of the anomeric carbon atom.

1: Chloroacetylation of compound 13 leading to compound 14, i.

10-methylprop-1-enyl-2,3-di-0-benzyl-4-O-chloroacetyl-α-D-glucopyranoside uronate

2.8 g of compound 13 is dissolved in 30 ml of pyridine (6.56 mmol). After cooling to 0 ° C, drop by drop of 10 ml of a solution of 2 ml of chloroacetyl chloride in 20 ml of dichloromethane is added dropwise. After 30 minutes, evaporate to dryness, dissolve the residue in 200 ml of chloroform, wash with a 10% solution of KHSO 4, then with water and dry and evaporate. The obtained syrup is chromatographed over silica gel (200 g; eluant AcOEt / hexane; 1/3, v / v). In this way, 2.7 g of compound 14 is obtained in pure 20 form in the form of a syrup (yield: 80%). [α] 20 O = + 2 '(c 1.5, chloroform). The expected structure is confirmed by elemental analysis and by the NMR spectrum.

2: Deblocking of the anomeric carbon atom leading to compound 15 or methyl (2,3-di-O-benzyl-4-Q-chloroacetyl-D-qyucopyranoside) uronate 25

2.7 g (5.3 mmol) of the derivative 14 is dissolved in 80 ml of a mixture of acetone / water (5/1; v / v). Then mercury dioxide (3.1 g) is added followed by a solution of mercuric chloride (3.9 g) in acetone (27 ml). After 5 minutes, the salts are removed by filtration. After concentration to dryness, the evaporation residue is redissolved in chloroform. The chloroform phase is washed with a 10% KI solution and then with water. The reaction product is recrystallized after evaporation from a mixture of ethyl acetate / hexane. 2 g of a solid are obtained, m.p. 105 - 077; [α] 20 D = -4.7 * ((eq, 1, chloroform). Elemental analysis and NMR study confirm the structure (yield 80%).

43 GB 174348 B1 3: Bromination of the anomeric carbon atom leading to compound 16 or methyl (1-bromo-2,3-di-O-benzyl-4-Q-chloroacetyl-o-D-quiucopyranoside) uronate 5

2 g (4.30 mol) of compound 15 are dissolved in 50 ml of dichloromethane. Then add 4.8 ml (34.4 mmol) of symcollidine at 0 ° C, followed by bromomethylene-dimethylammonium bromide (17 mmol) prepared after HEPBURN D.R. and HUDSON H.R., J. Chem. Soc. Perkin I (1976) 754-757.

10

The mixture is diluted after 4 hours with 100 ml of dichloromethane and poured into ice water. After washing with ice water, the solvent is evaporated. After chromatography on silica gel (20 g, eluent hexane / ethyl acetate, 2/1, v / v), 2.06 g of compound 16 is obtained in the form of a syrup (yield: 90%), [α] 20 D = + 82.5 ° (c = 1.5; chloroform). The structure is confirmed by elemental analysis and a study of NMR.

(B) Preparation of the disaccharide 18 or 3-O-acetyl-1,6-anhydro-2-azido-4-O-methyl-2,3-di-O-benzyl-4-O-chloroacetyl-gD-glucopyranosyl uronate BD-20 qluco-pyranose

This synthesis is based on the condensation of monosaccharides 16 and 17 with 78 mg (3.8 mmol).

To a solution of 870 mg (3.8 mmol) of compound 17 in dichloromethane is added 1 g of drierite, 0.5 g of molecular sieve 4 Å in powder form and 0.525 g of freshly prepared silver carbonate. After 2 hours of stirring, drop by drop at 0 ° C is added 670 mg (1.3 mmol) of compound 16. After 6 hours, the solids are removed by filtration. The syrup thus obtained is chromatographed after evaporation over silica gel (50 g; eluent: chloroform / ethyl acetate, 4/1, v / v). The disaccharide 18 is obtained in the form of a foam (421 mg; 50%). [α] 20 D = -17 ° (c = 1, chloroform). Elemental analysis confirms the structure. Investigation by NMR confirms the configuration of the interglycosidic bond.

44 DK 174348 B1 (C) Preparation of disaccharides having the structure 19 by acetolysis of the disaccharide 18 The disacharides 19 are prepared, subjecting the disaccharide 18 to an acetolysis reaction as shown below.

300 mg of compound 19 is dissolved in a mixture of 4 ml of acetic anhydride and 0.5 ml of freshly distilled trifluoroacetic acid. The reaction mixture is stirred for 0 hours at 18 ° C, then evaporated to dryness and re-evaporated together with toluene. The residue is chromatographed over a silica gel column (15 g). Elution is obtained by eluting with a mixture of dichloromethane and ethyl acetate (19/1, v / v) 282 mg of a mixture of the anomeric acetates of structure 19 in the form of a colorless syrup (yield: 86%). The ratio of α-forms to β-forms determined by NMR analysis is 4/1.

The NMR spectrum confirms the expected structure.

(D) Preparation of the disaccharide 20 by brominating the disaccharides 19 20

The mixture of the acetates of structure 19 is subjected to the action of TiBr4: A solution of 140 mg of the acetate mixture 19 in a mixture of 3 ml of dichloromethane and 0.3 ml of ethyl acetate at 17-18 ° C under a dry argon atmosphere is stirred under stirring. of 140 mg TiBr (~ 2 equivalents) for 2 hours. After cooling to 0 ° C and diluting with 30 ml of dichloromethane, the mixture is washed with ice water and then with an aqueous 5% potassium bromide solution and finally with water and dried over sodium sulfate, filtered and evaporated. The residue is chromatographed over a silica gel column (10 g). By elution with a mixture of dichloromethane and ethyl acetate 30 (19/1, v / v) the following is eluted in the elution order: • the bromide 20 (74 mg; yield 50%) in the form of a colorless syrup which is unstable (and which is used immediately) in subsequent reaction); NMR spectrum confirms the expected structure.

A fraction (28 mg; yield 20%) corresponding to unreacted starting compound; A slower-moving fraction corresponding to partial O-debenzylation reaction products.

EXAMPLE 3 (Starting Material)

Synthesis of the monosaccharide 22 or benzyl-6-Q-acetyl-3-Q-benzyl-2-benzyloxocarbonylamino-2-desoxy-α-D-glucopyranoside of the formula: 15 µg / g

hoNlYW

2q WHCoo Βγ »

This derivative is prepared from benzyl-3-O-benzyl-2-benzyloxy-carbonylamino-2-deoxy-α-D-glucopyranoside (derivative 21) to proceed as follows (see Figure 5):

Reflux of the compound 21 (987 mg, 2 mM, stirred for 30 hours, this compound was prepared in accordance with PC WYSS and J. KISS, Helv. Chim. Acta, 58 (1975) 1833-1847) in anhydrous 1 2-Dichloroethane (15 ml) in the presence of N-acetyl-imidazole (2.5 mM, freshly prepared). After cooling and diluting with chloroform (50 ml), the organic phase is washed with an ice-cooled solution of 1 M hydrogen chloride, with water, with a saturated solution of sodium hydrogen carbonate, with water which is dried (sodium sulfate), filtered and evaporated. The residue is chromatographed over a silica column (50 g). Elution with a mixture of dichloromethane (15/1, v / v) leads to derivative 22 in the form of a syrup which crystallizes from a mixture of ethyl acetate and hexane (759 mg, 71%). mp: 114-115 ° C; [α] D = + 88 ° (c = 1, chloroform).

EXAMPLE 4 (Starting Material)

Synthesis of the monosaccharide 33 of formula: 10 /; ° \ / COOMe

Aob "/ 15 ~ l

OH

This synthesis is carried out from Compound 23 using the following steps (see Figure 6): (1) introducing a benzoyl group at the 5-position (2) methylene of the carboxyl group at the 6-position (3) isomerizing the OH group to 5 position (4) formation of the pyran ring.

(1) The benzoylation reaction

63 g of 3-Obenzyl-1,2-o-isopropylidene-α-D-glucufuranoside (compound 23) is dissolved in 500 ml of anhydrous pyridine. Then 85 g of trityl chloride are added and 30 heated to 80 ° C for one hour. In this way the compound 24 is obtained.

Optical rotation: (α] 20ο = ~ 34.7 \ chloroform.

The structure of this compound has been confirmed by IR and NMR spectra, and elemental analysis has proved correct.

The mixture is then cooled to 0 ° C and 45 ml of benzoyl chloride is added. After one night, excess quantities of reaction components are destroyed by the addition of 300 ml of methanol. The thus obtained mixture, which is evaporated to dryness, is redissolved in chloroform. The chloroform phase is washed with water, dried over sodium sulfate and evaporated. In this way the compound 25 is obtained.

The syrup thus obtained is dissolved in 400 ml of chloroform. After adding 100 ml of a solution of p-toluene-sulfonic acid 5 M in methanol, the solution is allowed to stand at 4 ° C overnight. After washing with water of the organic phase, 215 g of a mixture is obtained. The compound is obtained by chromatography of this mixture over silica gel with a solvent ether-hexane 2/1 (v / v). In this way, 36 g of compound 26. is obtained. Optical rotation: [α] 20 D = 65.3 °, chloroform. The structure of compound 26 is confirmed by IR and NMR spectra.

(21 Methylation of the carboxyl group at 6-position 20

The compound 26 (1.88 g) is dissolved in acetone (20 ml). Then, drop by drop at -50 ° C, 3.5 ml of a solution of CrO 3 (13 g) in 3.5 M H2 SO4 (29 ml) is added. The temperature is allowed to rise and left for 1 hour under these conditions. The reaction mixture is then poured over ice and the product is extracted with chloroform. After washing with water and drying, evaporate to dryness.

The compound 27 is obtained.

The compound thus obtained is dissolved in methanol (20 ml), then 10 m 1 of 1 N sodium hydroxide is added and allowed to stand overnight at ambient temperature. The reaction mixture then passes through a column (25 ml) of the "Dowex 50" resin of the f-Γ form, which has previously been rinsed with methanol. · The reaction product is obtained by concentrating the eluate. In this way the compound 28 is obtained.

48 DK 174348 B1

This compound is dissolved in ether and methylated in a classical manner by diazomethane. The compound 29 is obtained after evaporation (1.08 g; 70.4%).

Optical rotation: [α] 20 ° = -27 ', chloroform.

The elemental analysis found for compound 29 is correct. The structure thereof is further confirmed by IR and NMR spectra.

(3) Isomerization of the QH group at the 5-position

Ten! a solution of trifluoroacetic anhydride (0.8 ml) in dichloromethane (16 ml) cooled to -20 ° C is added drop by drop a solution of pyridine (0.8 ml) in dichloromethane (8 ml). Then, at -10 ° C, drop by drop of 15 800 mg of compound 29 dissolved in dichloromethane (8 ml) is added. After one hour at -50 ° C, the reaction mixture is poured into a mixture of water and ice (8 ml) containing 160 mg of sodium bicarbonate. Stir until the separation of the two phases, the organic and the aqueous. The organic phase is washed with 3% HCl, with H 2 O, with saturated NaCl, dried and evaporated.

20 In this way, compound 30 is obtained.

The syrup is redissolved in DMF (10 ml). Sodium trifluoroacetate (1.6 g) is added and heated to 80 ° C for 3 hours. In this way, the compound is obtained 31. After evaporation, redissolving in dichloromethane, washing with water and drying, the evaporation residue is redissolved in methanol and the solvent is evaporated after one hour. After chromatography over a column of the solvent, etherhexane 2/1 compound 32 (450 mg; 56.2%) is obtained.

Optical rotation: [α] 20 D = -33 °, chloroform.

30

The structure of compound 32 is confirmed by IR and NMR spectra. The eementary analysis found is correct.

(4) The formation of the pyran ring 49 DK 174348 B1

This synthesis is carried out from Compound 32. Compound 32 (200 mg) is dissolved in a 9: 1 trifluoroacetic acid / water mixture.

After 15 minutes, the solvents are evaporated. Evaporation residue 5 is recrystallized from ethyl acetate / hexane. In this way, 110 mg of compound 33 is obtained.

The characteristics of this compound are as follows: 10 IR spectrum: in CHCl, γ in cm * 1: 3450 (OH), 3080. 3060, 3030 (CH2: benzyl) and 1740 (COOCH3).

NMR spectrum: δ in ppm relative to TMS: 3.75 (s, 3H +, COOMe) 4.98 (1H *) 7.30 (s, 5H +, C6H5)

Optical rotation: [a] 20o = + 13 *. methanol.

20

Elemental Analysis for C14H1807:

Calculated: C 56.37 - H 6.08 Found: C 56.17 - H 5.85.

Melting point: 125-126 ° C.

EXAMPLE 5 (Starting material) 30

Synthesis of the derivative 38 or 3-O-benzyl-4-O-chloroacetyl-1,2-O-tert-butoxylidene-α-methyl-idopyranuronate of the formula: 50 DK 174348 B1 / ÆoMe ° \ \ 0Βη Λ MCAOV - / x0 Uj · —But but CH3

This synthesis (see Fig. 7) is carried out from the derivative 33 with the iduronic acid structure, by (a) subjecting the derivative 33 to an acetylation reaction, 10 (β) subjecting the mixture of the anomeric acetates 34 thus obtained and the effect of a brominating agent to introduce of a bromine atom on the anomeric carbon atom, 15 (y) forms an orthoester at positions 1,2 and (δ) performs a monochloroacetylation at the 4-position of the orthoester (a) acetylation reaction leading to 1,2,4-tri-O-acetyl -3-O-benzyl-α, β-1-20 methyl-idopyranuronates (derivatives 34 and 35).

A solution of the compound 33 (3 g) in a mixture of anhydrous pyridine (20 ml) and acetic anhydride (10 ml) is stirred at 0 ° C without the presence of humidity for 5 hours. The reaction mixture is evaporated to dryness. dryness, md-25 is evaporated together with toluene (4 x 20 ml) and dried under vacuum. The evaporation residue is chromatographed over a silica gel column (150 g).

Elution with a mixture of toluene / ethyl acetate (4/1, v / v) leads in the elution order: 30 · a pre-fraction consisting of furan derivatives, • compound 34 (anomer a), syrup (170 mg, 4%), [ a] D -43 '; (c: a, chloroform), NMR (CDCl 3) δ: 6.23 (s, 1H, H-1), compound 35 (anomer β) crystallizing from a mixture of ether and hexane (2,688 g, 63%), m.p .: 112 - 113 ° C, [α] D = + 9 ° (c: 1, chloroform), NMR (CDCl 3) δ: 6.08 (d, 1 H. H-1, J .2: 1.5 Hz).

5 The anomeric compounds α and β 34 and 35, respectively, are not separated when proceeding with the described sequence of syntheses. The mixture thereof is used directly in the form of a syrup for the subsequent reactions, (3) Bromination reaction leading to compound 36 or 2,4-di-Q-acetyl-3-Q-10 benzyl-g-L-methyl-idopyranuronyl bromide

A mixture of the acetates 34 and 35 (212 mg; 0.5 mM) is dissolved in anhydrous dichloromethane (5 ml) and in anhydrous ethyl acetate (0.5 ml). Titanium tetrabromide (250 mg, 0.7 mM) is added at once, and the reaction mixture is stirred for 24 hours at ambient temperature without the presence of moisture. After cooling to 0 ° C and diluting with dichloromethane, the organic phase is washed with ice water (three times), dried (sodium sulfate), filtered and evaporated to give the derivative 36 as a slightly colored syrup (217 mg, 96%). NMR (CDCl 3) δ: 6.41 (s, 1H, H-1). This highly unstable compound is used immediately in the subsequent reaction.

(y) Preparation of the orthoester or 4-O-acetyl-3-O-benzyl-1. 2-O-tert-butoxyethylidene-BL-methyl-ido-pyranuronate 25 A solution of the bromide 36 [freshly prepared from 2.122 g, 5 mM, of the mixture of acetates 34 and 35 in anhydrous dichloromethane (20 ml)] is stirred. at ambient temperature under an atmosphere of dry argon. 7-collidine (2.65 ml, 20 mM) and anhydrous tart are added successively. butanol (3 mL; 30 mM) and the reaction mixture is stirred for 15 hours under these conditions.

After dilution with dichloromethane (50 ml), the organic phase is washed with a saturated aqueous sodium hydrogen carbonate solution, with water dried over sodium sulfate, filtered and evaporated. The residue is chromatographed over a silica gel column (120g). Elution with a mixture of hexane / ethyl acetate (2/1, v / v containing 0.5% triethylamine) gives compound 37 in the form of a pure syrup (1.542 g, 70% from 34 and 35). [α] = -23 ° (c: 1, chloroform), NMR (CDCl 3) δ: 5.48 (d, 1H, H-1, J, i2: 2.5 Hz).

(5) Monochloroacetylation of the orthoester 37 5

A solution of the ortho ester 37 (220 mg, 0.5 mM) in anhydrous methanol (10 ml) is cooled to -20 ° C with stirring and a dry argon atmosphere. Anhydrous potassium carbonate (40 mg) is added and the reaction mixture is stirred for 5 hours under these conditions. The solids are extracted, the filtrate is evaporated, and the residue is redissolved in chloroform (50 ml). The organic phase is washed rapidly with ice-water (3 times), dried (sodium sulfate), filtered and evaporated. The residue is immediately dissolved in anhydrous pyridine (4 ml) and anhydrous dichloromethane (2 ml). After cooling to -20 ° C under a dry argon atmosphere, drop by drop is added a solution of chloroacetyl chloride (0.1 ml, 1.24 mM, freshly distilled) in anhydrous dichloromethane (1 ml). The reaction mixture is stirred under these conditions for 30 minutes, then poured into a mixture of ice and water (100 ml). After stirring for 15 minutes, the mixture is extracted in chloroform (3 x 20 ml). The organic phases are washed with ice water, with an aqueous 2% sodium bicarbonate solution, with water, dried (sodium sulfate), filtered and evaporated. The residue is rapidly chromatographed over a silica gel column (12 g). Elution with a mixture of hexane / ethyl acetate (5/2, v / v, containing 0.2% triethylamine) results in the elution order: 25 · an unsaturated compound 39 (15 mg, 8%), the orthoester 38 syrup (145 mg, 61% from 12), [α] D = + 19 ° (c: 1, chloroform), NMR (CDCl 3) δ: 5.45 (d, 1 Η, H-1, J 2): 2.5 Hz), 5.24 (d of d, 1 Η, H-4, J3. <: 2.5 Hz, J4.5: 1.5 Hz), 4.00 (s, 2H; CI-CH -COO-).

EXAMPLE 6 (Intermediate of Formula VIII) Synthesis of the disaccharide 41 or benzyl-6-Q-acetyl-3-O-benzyl-2-benzyloxycarbonylamino-2-deoxy-4-Q- (2-o-acetyl) -3-0-Benzyl-αL-methyl-idopyranuronol) -αD-glucopyranoside I-OAc OAc NHCOOBn 15 step β, leading to the desired disaccharide 41 (see Fig. 8): Step a: Preparation of the disaccharide 40 or benzyl-6-O-acetyl-3-O-benzyl-2-benzyloxycarbonylamino-2-desoxy-4- 0- (2-0-acetyl-3-0-benzyl-4-0-chloro-acetyl-.alpha.-methyl-idopvranuronvP-.alpha.-D-glucopyranoside

A solution of the orthoester 38 (284 mg, 0.6 mM) and the alcohol 22 (214 mg, 0.4 mM) in anhydrous chlorobenzene (12 ml) is heated to 140 ° C with stirring and a slight stream of dry argon. After slow distillation of 10 ml of the solvent, drop by drop, a solution of 2,6-dimethyl-pyridinium perchlorate (0.006 mM, freshly prepared) in chlorobenzene (4 ml) is added over 30 minutes under simultaneous distillation of the solvent (4 ml). The reaction mixture is stirred for 1 hour with the addition of fresh solvent (10 ml) and simultaneous distillation, so that the reaction volume remains constant and equals approx. 4 ml. After cooling and dilution with chloroform, the organic phase is washed with a saturated sodium hydrogen carbonate solution, with water dried over sodium sulfate, filtered and evaporated. The evaporation residue is chromatographed over a silica gel column (40 g). Elution with a mixture of hexane and ethyl acetate (4/3, v / v) in the elution order results in: • the product 22 (120 mg, 56%), 5 • the disaccharide 40 which is crystallized from a mixture of ether and hexane (112 mg , 30%, m.p .: 144-145 ° C, [α] 20 D = +35 '(c: 1, chloroform), NMR (CDCl 3) consistent with the expected structure.

10

Step β: Removal of the monochloroacetyl group

A mixture of the disaccharide 40 (56 mg, 0.06 mM) and thiourea (7 mg, 0.1 mM) in pyridine (2.5 ml) and absolute ethanol (0.5) was stirred for 30 minutes at 100 ° C. mL). After cooling and evaporation to dryness, the residue is redissolved in a mixture of water and chloroform (1/1, v / v, 40 ml). The organic phase is washed with water, dried (sodium sulfate), filtered and evaporated. The residue is chromatographed over a silica gel column (2 g).

Elution with a mixture of ethyl acetate and hexane (2/1, v / v) leads to the disaccharide 41, which is recrystallized from ether (46 mg, 30%), mp: 146 -147 ° C, [α] D = 44 1 (c: 1, chloroform), NMR (CDCl 3): consistent with expected structure.

EXAMPLE 7 (Intermediate of Formula XXIII)

Synthesis of the disaccharide 43 of the formula: 30 Q1c OAc

OBn N OAc, NH

3 c = o 5 The tetrasaccharide 43 is prepared by performing: in step (a) the condensation of the disaccharides 20 and 41, the synthesis of which is described in Examples 2 and 6, respectively, and subjecting to step (b) the resulting tetrasaccharide 42 a selective -0 demonochloroacetylation at the 4-position (see Fig. 9, cf. also claim 21): 1a) The condensation reaction

Stir for half an hour at ambient temperature under a dry-atmosphere atmosphere a mixture of freshly prepared bromide 20 mg (80 μΜ), 51 mg (60 μΜ) of compound 141 and 80 ml of 4 Å powdered molecular sieve. in 1.5 ml of anhydrous dichloroethane, then cool to -20 ° C. Then 20 ml (150 μΜ) of symcollidine and 31 mg (120 μΜ) of silver triflate are added successively. The reaction mixture is kept under stirring at -20 ° C for 1 hour, then the temperature is allowed to rise to ambient temperature over 15 minutes.

After dilution with 50 ml of dichloromethane, the solids are suctioned off and the filtrate is washed with a 1 M hydrogen chloride solution in ice water, then with water (2 times). It is then dried over sodium sulfate, filtered and evaporated.

The residue is chromatographed over a silica gel column (8 g, gel 230-400 mesh). Elution with a mixture of hexane and ethyl acetate (4/3, v / v) results in the recovery of 37 mg of the tetrasaccharide 42 (yield 39% in the form of a colorless glass. [A] 2 + = + 56 ° (c = 0 , 6; CHCl 3); NMR spectrum confirms the expected structure.

30

Eluting the column with the ethyl acetate-hexane mixture (2/1, v / v) gives 23 mg of the starting product 41 (yield 44%).

fb) The O-Dichloroacetylation reaction 56 B1

A solution of 36 mg (23 μΜ) of tetrasaccharide is heated in 42 ml of 1.25 ml of a mixture of pyridine and 0.25 ml of absolute ethanol to 100 ° C in the presence of 7 g (100 μΜ) of thiourea for 20 minutes. After cooling and evaporating to dryness, the solid evaporation residue is redissolved in 20 ml of water and extracted with chloroform (5x5 ml). The organic phases are washed with a 10% aqueous sodium hydrogen sulfate solution, with water, dried over sodium sulfate, filtered and evaporated. . The residue is chromatographed over a silica gel column (3 g). Elution with a mixture of ethyl acetate and hexane (3/2; 10 v / v) gives 27 g of the derivative 43 (yield 80%) as a colorless glass.

[α] 20ο = -6Γ (c = 0.8; chloroform); The NMR spectrum confirms the expected structure (see Fig. 31).

EXAMPLE 8 (Intermediate of Formula XXIV)

Synthesis of the pentasaccharide 45 of the formula.

20 rOSO; «, + COOMi.-OSO '/ Wi' * · pOSo / M, - * -

A A / »A A„ A

A? 1 * '/ Λ? 8 "/ Α ° Ά ° \? Βη / Atln A

'------- {I ..... I I · "I

25 n3 oli "N} oso; w, * KHCOe6

A condensation reaction is carried out between the tetrasaccharide 43 and the monosaccharide 44 leading to the pentasaccharide 45.

30

Stir for half an hour at ambient temperature and under a dry argon atmosphere a mixture of 27 mg (54 µΜ) of the bromide 48 prepared according to B. PAULSEN and W. STENZEL, Chem. Calcd. 111 (1978) 2334-2347, 26 mg (18 μΜ) of the tetrasaccharide 43 and 50 mg molecular sieve 4 Å in 57 ml of 17748 B1 powder form in 0.8 ml of dichloroethane, then cool to -20 ° C. 16 ml (120 μΜ) sym-collidin and 26 mg (100 μΜ) silver triflate are added successively and the reaction mixture is stirred for 18 hours, slowly allowing the temperature to rise to ambient temperature.

5

After dilution with 50 ml of dichloromethane, the solids are suctioned off and the filtrate is washed with a 1 M hydrogen chloride solution in ice water and then with water (2 times). Then, dry over sodium sulfate, which is filtered and evaporated.

The residue is chromatographed over a silica gel column (5 g, gel 230-400 mesh). Elution is obtained with a mixture of hexane and ethyl acetate (4/3, v / v) 30 g of pentasaccharide 45 in the form of a colorless glass (90% yield). [α] 20 ° = + 67 ° (c: 1, chloroform). The NMR spectrum confirms the expected structure. In particular, for the anomeric protons in the glucosamine-15 units, shifts (δ, TMS) of 5.36 and 5.52 ppm for the protons of H, F and D are found, respectively.

EXAMPLE 9 (End product of formula XXIV)

Preparation of pentasaccharide 50 (see Figs. 10 and 11)

The following steps are used here: (a) Removal of acetyl groups - (pentasaccharide 46) (b) sulfation of the OH groups thus released (pentasaccharide 47) 30 (c) Hydrogenation to release OH groups protected by benzyl groups and for conversion of the N3 group to the NH2 group (pentasaccharide 48) (d) Sulfation of the NH2 groups (pentasaccharide 49), subsequent saponification of the -COOMe groups at the 6-position (pentasaccharide 50).

These steps are carried out as follows: (a) Removal of the acetyl groups from the derivative 45

With stirring to 0 ° C, a solution of 28 mg of pentasaccharide 45 is cooled in a mixture of 2.5 ml of 1,2-dimethoxyethane and 0.8 ml of methanol. Then drop by drop over 10 minutes add 1 ml of a 1 M solution of 10 sodium hydroxide. The reaction mixture is stirred for 1 hour at 0 ° C and then for 12 hours at ambient temperature. After cooling to 0 ° C, 3 ml of 1 M hydrochloric acid solution is added and the milky mixture is immediately extracted with chloroform (5x5 ml). The organic phases are washed with water, dried over sodium sulfate, filtered and evaporated, The residue is redissolved in 2 ml of methanol and treated with a solution of diazomethane in ether (in excess until pale yellow staining) for half an hour.

After evaporation to dryness, the residue is chromatographed over a 20 silica gel column (2 g, gel 230-400 mesh). Elution is obtained with a mixture of dichloromethane and methanol (15/1, v / v) 18 mg of pentasaccharide 46 (yield: 72%) as a colorless glass. [α] 20 D + 57 ° (c = 1; chloroform).

The expected structure is confirmed by the NMR spectrum.

(B) Sulfation of the OH groups

To a solution of compound 46 (22 mg) in dimethylformamide (0.5 mf) of the trimethyl amine / SO3 complex (22 mg, 2.5 equivalents / OH) is added. The reaction mixture is heated to 50 ° C for approx. 14 hours. Then the trimethylamine / SO3 complex (10 mg) is added again and the reaction allowed to develop for 24 hours. Then methanol (0.5 ml) and chloroform (0.5 ml) are added to the reaction mixture. The solution is inserted on top of a column of “Sephadex.

Which is equilibrated with a mixture of CHCl 3 / CH 3 OH (1/1, v / v). The 59 DK 174348 B1 fractions containing the sulfated reaction product are collected and the solvent is evaporated. This glass is then chromatographed over silica gel (10 g) in a solvent consisting of three parts of a mixture of ethylene acetate / pyridine / acetic acid / water (6/2 / 0.6 / 1, 5 v / v / v / v ) and two parts of a mixture of ethyl acetate / pyridine / acetic acid / water (5/5/1 / 3, v / v / v / v).

The fractions containing the desired reaction product are collected and concentrated. The evaporation residue thus obtained is dissolved after evaporation of the solvents in methanol added water and passed through a column of "Dowex 50 W x 4, Na +" which is equilibrated with a mixture of methanol / water (50/50 , v / v). In this way, the sodium salt is obtained (compound 47).

(C) Hydrooenerina

The reaction product obtained above is dissolved in methanol (3.7 ml), to which is added water (0.3 ml). The catalyst (Pd / C, 5%, 40 mg) is added to this solution and stirred for 5 days under a hydrogenate starch. After removal of the catalyst by filtration, UV spectral analysis of the solution thus obtained shows the total disappearance of the absorption due to the benzyl groups. The solvent is then evaporated to obtain an evaporation residue which is the compound 48.

25 fdi Sulfaterino of the NH? Groups and subsequent saponification of the carboxyl groups

The compound 48 is dissolved in water (4 ml). The pH is then adjusted to 9.5, then the trimethylamine / SO3 complex (54 mg) is added to the solution. The pH was maintained at 9.5 for the duration of the reaction by addition of 0.1 N sodium hydroxide.

After one night, further addition of the sulfating agent (27 mg) is continued. After 24 hours, a final portion is added.

60 DK 174348 B1

After 48 hours, sodium hydroxide (3 M, 34 ml) is added to the compound 49, whereupon the solution is stirred for 3 hours at ambient temperature to hydrolyze the methyl esters in the components of the uronic acid type. The reaction mixture is then neutralized and concentrated to a volume of ca. The solution thus obtained is added to a column of "Sephadex 25" (100 ml) which is eluted with water. The collected fractions are analyzed by UV absorption (206 nm) and by polarometry (265 nm). are optically active, are collected, the solvent is removed, and the residue is dissolved in about 2 ml of water and lyophilized.

In this way, the derivative 50 is obtained in the form of a white powder (5.6 mg, 25% relative to the starting compound 45).

15 NMR examination confirms the expected structure. In particular, for the anomeric protons in the glucosarnine units, shifts (δ, TMS) of 5.36, 5.45 and 5.52 ppm are found for the protons belonging to H, F and D.

EXAMPLE 10 (Intermediate of Formula I)

Synthesis of the disaccharide 92 of formula 25 COOMe fr X1 · - ^

BnO in I

OB π N, 30 3

Referring to FIG. 19th

The starting compound 91 (1 g) in solution in dichloromethane (50 ml) is stirred in the presence of drierite (6 g) and freshly prepared silver carbonate (4.5 g) for 1 hour under an argon atmosphere. Then the halide 90 (2.8 g) dissolved in dichloromethane (10 ml) is added. After 1.5 hours, 2.8 g of halide 90 is added again. After one night, the solids are removed by filtration and the residue obtained after evaporation of the solvents is purified over a silica column with the solvent ethyl acetate / chloroform (1 / 30; v / v).

In this way, the product is obtained 92 (866 mg; yield 42%). It is recrystallized from a mixture of hexane and ethyl acetate. Melting point: 104 - 106 ° C; [α] 20 D = O * (c = 1; chloroform).

Elemental analysis and the NMR spectrum are consistent with the desired structure.

EXAMPLE 11 (Intermediate of Formula I)

Synthesis of derivative 94 of formula 20

C OOM c _O

BnO I I

oc I 'ZO Oftn N3

Refer to Figures 19 and 20.

The derivative 92 (1.5 g) is dissolved in a mixture of chloroform and methanol (1/1; 30 v / v). Then 2 ml of sodium methanolate (2 M in methanol) is added. After 20 minutes, the solution is neutralized by the addition of the resin "Dowex 50" which leads to the derivative 93 which is not isolated. After filtration and evaporation, the classical carboxylic acid fraction liberated may be reesterified by evaporation. 62 B1 evaporation residue with a mixture of pyridine (20 ml) and acetic anhydride (2 ml) overnight After evaporation, the residue is recrystallized from ethyl acetate and hexane to give the product 94 (1.125 g; yield 81.6%).

5

Melting point: 103 - 105 ° C; [α] 20ο = β 5.2 (c - 1; chloroform).

Elemental analysis and the NMR spectrum are consistent with the structure sought.

10

As a variant, derivative 94 can be prepared by proceeding as described above, however, using derivative 95 instead of derivative 91.

EXAMPLE 12 (Intermediate of Formula I)

Synthesis of Derivative No. 97 (see Fig. 20) 20 In an initial step, an opening of the anhydro bridge is performed, and in a subsequent step a bromination reaction is carried out.

1: Opening of the 1,6-anhydro bridge Compound 94 (1 g) is dissolved in acetic anhydride (10 ml) and then cooled to -20 ° C under an argon atmosphere. To the cold solution is added concentrated sulfuric acid (100 µl). After 30 minutes, the reaction mixture is diluted with chloroform (150 ml) and poured onto an aqueous sodium hydrogen carbonate solution (26.5 g, 400 ml). At the end of the gas release, the chlorofomine phase is washed twice with saturated NaCl solution, then dried and concentrated. After chromatography over silica (50 mg) in a mixture of ethyl acetate and chloroform (1/20, v / v), the compound is obtained 96 (995 mg; yield 86.7%).

63 DK 174348 B1

This compound is in the form of a white foam.

Spectrum and elemental analysis confirm that the desired structure has been obtained.

2: Bromination

A solution of the derivative 96 (0.2 g) in dichloromethane / ethyl acetate (9/1; v / v, 4 10 ml) is added to titanium tetrabromide (213 mg). After stirring overnight with stirring, followed by dilution with dichloromethane, pour over an ice-water mixture (50 ml) and wash twice with 50 ml of ice-water. After drying and evaporation, the obtained silica syrup is chromatographed in the solvent ethyl acetate / chloroform (1/20; v / v). In this way, the derivative 97 is obtained with a yield of 25 - 50%.

NMR Spectrum: (ppm, CDCl3) 2.04 2.11 (1 singlet of 3 protons 2-Oac) 3.7 (1 singlet of 3 protons COOMe) 6.33 (1 doublet of 1 proton Hi, J 2 = 3.5 Hz) 25

Claims (22)

1. Pentasaccharides of the formula; 5 r'0ip co0 ^ ρΟίρ hi i 0? CXp Ni, where M is hydrogen or alkyl, e.g. methyl; Sp and P are identical or different and are selected from acetyl, benzoyl, aryl, benzyl, hydrogen and sulfate, with Sp and P not being benzoyl simultaneously, Ni, Nj, and N3 being independently selected from NH2 -NH-COO-benzyl , -NH-COO-acetyl, -NH-SO3 'and -NH-acetyl; T is hydrogen, benzyl, acetyl, monochloroacetyl or trichloroacetyl, or p-methoxybenzoyl; and R is selected from acetyl, alkyl, e.g. methyl, and benzyl; or -OR is halogen, e.g. Br, or imidoyl; 20 and their salts, where M is an alkali metal. Pentasaccharides according to claim 1; where M is hydrogen, or alkyl, e.g. methyl, Sp and P are identical or different and are selected from acetyl, hydrogen and sulfate; Ni, N2 and N3 are independently selected from -NH-COO-benzyl, -NH-SO3 'and -NH-acetyl; T is hydrogen, acetyl, monochloroacetyl or trichloroacetyl, p-methoxybenzoyl, and 30. is selected from acetyl, alkyl, e.g. methyl, benzyl; or -OR is halogen, e.g. Br or imidoyl; and their salts where M is an alkali metal. DK 174348 B1 Pentasaccharides according to claim 1, wherein M is alkyl, Ni, N 2 and N 3 are independently selected from NH 2 and -NH-COO-benzyl, T is hydrogen or benzyl, 5. is hydrogen or benzyl, Sp is acetyl, hydrogen or sulfate and R is benzyl. 4. Pentasaccharides of the formula: 10 fasv cocr ros <> y j-osos- <xm 1 15 nhso; ° p nhso3-oso; nhso; wherein P is selected from acetyl, benzoyl, aryl, benzyl, hydrogen and sulfate, -NHSO 3 'can be replaced by -NH-acetyl, and -OH can be replaced by alkoxy, e.g. methoxy. Rt is benzyl or acetyl, R er is benzyl, or R Ri and R together form the divalent radical -CH₂-O-. N, and N2 are independently selected from -NH-COO-benzyl, -NH-COO-acetyl, NH2 or -NH-SO3, and T is hydrogen, benzyl, acetyl, monochloroacetyl. or trichloroacetyl, or p-methoxybenzoyl, and R is selected from acetyl, alkyl, e.g. methyl, and benzyl; or OR is halogen, e.g. Br, or imidoyl, and their salts wherein M is an alkali metal. The pentasaccharides of claim 4, wherein P is hydrogen and NHSO3 · can be replaced by -NH-acetyl. 25 6. Tetrasaccharides of the formula: coot ^ 1 pOsp p o ~ v Of o5p ^ 2_ wherein M is hydrogen or alkyl, e.g. methyl, Sp and P are identical or different and are selected from acetyl, benzoyl, aryl, benzyl, hydrogen and sulfate, since Sp and P cannot be benzoyl simultaneously; The tetrasaccharide of claim 6, wherein M is alkyl; 8. Intermediates in the form of pentasaccharides of the formula: m--, COONi .. 0; p ni Γ? I ^ i P N i °? N1_ Οί? N y where M is hydrogen or alkyl, e.g. methyl; Sp and P are identical or different and are selected from acetylbenzoyl, aryl, benzyl, hydrogen and sulfate, with Sp and P not being benzoyl simultaneously; DK 174348 B1 Ni, N2, and N3 are independently selected from NH2, -NH-COO-benzyl, azido, -NH-COO-acetyl, -NH-SO3 'and -NH-acetyl; at least one of the groups N, N 2 and / or N 3 is azido, T is hydrogen, benzyl, acetyl, monochloroacetyl or trichloroacetyl, or p-methoxybenzoyl; and R is selected from acetyl, alkyl, e.g. methyl, and benzyl; or -OR is halogen, e.g. Br, or imidoyl; and their salts, where M is an alkali metal. The intermediates of claim 8, wherein M is alkyl, Ni, N2 and N3 are independently selected from NHh, azido and -NH-COO-benzyl, wherein at least one of the groups Ni, N2, and / or N3 is azido, T is hydrogen or benzyl, 15. is hydrogen or benzyl, Sp is acetyl, hydrogen or sulfate, and R is benzyl. 10 BnO \ J_ / Br N3 (44) Ni and N2 independently selected from NH2 -NH-COO-benzyl, azido, -NH-COO-acetyl, -NH-acetyl, NH2 or -NH-SO3, at least one of the groups Ni and / or N2 being azido and T is hydrogen, benzyl, acetyl, monochloroacetyl or trichloroacetyl, or p-methoxybenzoyl, and 15. is selected from acetyl, alkyl, for example methyl, and benzyl; or OR is halogen, e.g. Br, or imidoyl, and their salts wherein M is an alkali metal. Intermediates according to claim 8, wherein 20 M is hydrogen, or alkyl, e.g. methyl; Sp and P are identical or different and are selected from acetyl, hydrogen and sulfate; Ni, N2 and N3 are independently selected from -NH-COO-benzyl, azido, -NH-SO3 "and -NH-acetyl, at least one of the groups N, N2, and / or N3 being azido, 25. is hydrogen, acetyl, monochloroacetyl or trichloroacetyl, p-methoxybenzoyl, and R is selected from acetyl, alkyl, for example methyl, benzyl; or -OR is halogen, for example Br or imidoyl; and their salts where M is an alkali, metal 30 11. Intermediates in the form of tetrasaccharides of the formula: DK 174348 B1 COO ^ r-CKp pOsv y'o / ^ / c 00 \ o / 'VoQ- (χχ 111} y ^ / ° T ^ / J \ T_ / Op \ YES Os p 5 where M is hydrogen or alkyl, for example methyl, Sp and P are identical or different and are selected from acetylbenzoyl, aryl, benzyl, hydrogen and sulfate, since Sp and P cannot be benzoyl simultaneously, The intermediate of claim 11, wherein 20 M is alkyl; Ni and N2 are independently selected from azido, -NH-CO-O-benzyl, -NH-acetyl and NH2, with at least one of the groups N, and / or N2 being azido, Sp being acetyl, hydrogen or sulfate, P being benzyl or hydrogen, 25. is hydrogen, monochloroacetyl or benzyl 13. Intermediates in the form of disaccharides of the formula; COOH _OR, 30 I-o \ -o / ° n? / or, OR (I) T0 \ i-J N-OR, N DK 174348 B1 where M is hydrogen or alkyl, e.g. methyl, 5. is selected from -NH-COO-benzyl, azido, -NH-COO-acetyl, -NH-SO3 'and -NH-acetyl, T is hydrogen, benzyl, acetyl, monochloroacetyl or trichloroacetyl, or p-methoxybenzoyl ; and R is selected from acetyl, alkyl, e.g. methyl, and benzyl, or OR is halogen, e.g. 8r, or imidoyl, R1 is acetyl, benzyl, hydrogen or sulfate, or R and Rt together form the divalent radical -CH2-O-, and their salts where M is an alkali metal. The intermediates of claim 13. wherein M is Na + or alkyl, T is monochloroacetyl, hydrogen, benzyl or acetyl, R is alkyl, or -OR is bromine, R 1 is acetyl, benzyl, hydrogen or sulfate, or R 1 and R 1 together forms the divalent radical -CH2-O-. N is azido or acetylamino. 15. Intermediates in the form of disaccharides of the formula: 25.-0 / v I <VIn 'o / µ n 30 wherein T, Ri, Μ, N and R are defined as in claim 13. DK 174348 B1 Ni and N2 are independently selected from -NH-COO-benzyl, -NH-COO-acetyl and NH2, Sp is acetyl, hydrogen or sulfate, P is benzyl or hydrogen, T is hydrogen, monochloroacetyl or benzyl. The intermediates of claim 15, wherein M is alkyl or hydrogen, N is -NH-COO-benzyl or azido, T is hydrogen, monochloroacetyl or benzyl, A process for preparing a pentasaccharide of formula XXXII according to claim 4 or 6, wherein P is hydrogen, comprising a compound of formula XXIV according to claim 1 wherein Sp is acetyl, P is benzyl, Ni and N 2 is azido, N 3 is -NH-CO-O-benzyl, M is alkyl, and T is benzyl, ten is made. subject to the following reactions: 15. removal of the O-acyl blocking groups, b. sulfation of the liberated OH groups using a dimethylamine / SO3 complex as a sulfating agent, c. liberating the OH groups that are protected by benzyl groups, and conversion of the nitrogen-containing groups to functional amino groups by catalytic hydrogenation, and d. sulfation of free OH groups and of functional amino groups using a trimethylamine / SO3 complex as a sulfating agent. The process of claim 17, wherein the sulfation is carried out in a solvent, e.g. dimethylformamide, at a temperature higher than room temperature and close to 50 ° C. The process of claim 17 or claim 18, wherein the catalytic hydrogenation is carried out under hydrogen pressure in the presence of a Pd / D type catalyst in an organic solvent. DK 174348 B1 A process according to any one of claims 17-19, characterized in that the compound of formula XXIV used is prepared by reacting a compound of formula XXIII according to claim 6, wherein Sp is acetyl, P is benzyl, T is Η, M is alkyl, Ni is azido, and N2 is -NHCOO-5 benzyl having a monosaccharide of the formula: r "O Ac · A f \ OBn / Process according to claim 20, characterized in that the compound of formula XXIII used is prepared by a condensation reaction between the following disaccharides: COOM * -OAC j-O-MCAO-1 Nf 3t HO 2 -f I ODn in ,,. * 3 r0 (20) (41) Οβη 25 where MCA represents monochloroacetyl and subsequently -O-dechloroacetylation. Method according to claim 20, characterized in that the compound of formula XXIII used is prepared by a condensation reaction between the following disaccharides: DK 174348 B1 COOMe __ - Oftc r—— OAc && + 08 (1 M3 OAc NH C = 0 <f) B π (97) (41)