DK174284B1 - Biologically active tetrasaccharides - Google Patents

Abstract

The oligosaccharides of the invention contain or consist of a tetrasaccharide chain sequence of formula: <IMAGE> in which R1 denotes an inorganic anion, R2 is identical with R1 or denotes a hydrogen atom, N1 and N2 denote an amine functional group.

Description

DK 174284 B1

Biologically active tetrasaccharides

The present invention relates to novel biologically active tetrasaccharides.

5

The present invention is a further development of the invention which is the subject of Danish Patent Application No. 143/83, published July 16, 1983. This patent application discloses a process for producing oligosaccharides consisting of or containing fragments of chains 10 of acidic natural mucopolysaccharides as well as derivatives of these oligosaccharides. It should be noted that the term "acidic mucopolysaccharides" refers to such derivatives which are also frequently referred to as glucosaminoglucuron glycans. These derivatives are formed by oligosaccharides and polysaccharides, which are in particular the chains of biologically active derivatives, such as These chains are mainly composed of alternating components: amino sugar-uronic acid or vice versa. In these components, the amino sugar species in particular exhibits a D-glucoseamine structure (referred to as a) and the uronic acid group a D-glucuronic acid structure (referred to as b) or L-iduronic acid structure ( referred to as c).

20

The high flexibility which is characteristic of the process described in the above-mentioned Danish patent application makes it possible to produce chain formations of the desired repeat groups or components according to the desired stereochemical configuration and with determined substitutions. Thus, it is particularly possible to obtain such oligosaccharides which form analogous compounds to the structure of fragments from heparin chains.

These fragments may advantageously include the octasaccharide chain structure ABCDEFGH obtained so far by the applicant by enzymatic depolymerization of heparin, as disclosed in U.S. Patent No. 4,401,662 and having the structural formula DK 174284 B1 - ΟΊΌ rt - £ jyφ & y ^^ y °: 'O so "HKS <j“ O'. 'I •' • .iÅC. Oil «ftso" OM'j SHSO, λ acd. ': - c, h \

By depolymerizing heparin, fragments of different lengths and with different compositions are initially obtained. By chromatographic fractionation, fractions containing predominantly similar compounds in structure can then be obtained.

US Patent No. 4,401,662 discloses that the antithrombin activity and the anti-10 Xa activity (measured by the so-called Yin Wessier titer) were measured for some of these fractions. When comparing the measurement results, it was concluded that the fractions would probably contain compounds comprising the sequence CDEF if the fraction should have an affinity for antithrombin (ATIII), however, although the presence of the sequences ABCDEF and 15 CDEFGH was found in the fraction. Further, fractions containing the sequence DEFGH were found to have a high anti-Xa activity, but fractions of EFGH had a low activity.

The synthetically produced oligosaccharides may also comprise only a portion of this chain structure ABCDEFGH, or may comprise this chain structure.

The letters A - H indicated in the formula, as used in the present specification, denote a structure type, the substituents being identical or different from those shown in the formula.

The synthetically prepared oligosaccharides, as described in the aforementioned patent application, constitute particularly interesting biologically reactive substances as well as substances useful as reference compounds. In addition, they possess pharmacological properties which confer upon them a utility of great importance as active ingredients in pharmaceuticals.

Some of these synthetically produced oligosaccharides have also been found to be particularly active in the blood coagulation.

Indeed, if an anti-Xa activity (as measured by the so-called Yin-Wessler titer) has been detected in a trisaccharide of the DEF structure, it is possible to demonstrate a very strong affinity for antithrombin 111 with a pentasaccharide of the structure DEFGH ( ATIII) and an anti-Xa activity (Yin-Wessler) which is very high, amounting to at least 2,000 Yin-Wessler units / mg.

The tested product has the formula and is known from DK patent application no.

15 143/83 coo "r-OSO, f" OSO ~ / ^ 0. / Kr / Γ ° \ / ~ 0Vh / ~ ° \ f \ QH XqX ^ oh y (} A ^ so3 v ^ QA ^ h ° ~ , / ίΛ ^ οι · Λ011 nhso3 OH NHSO3 OSO "VT.iS0"

An anti-Xa activity of at least 2,000 Yin-Wessler-20 units / mg has been demonstrated.

It has now surprisingly been found, while continuing with the above-mentioned research work, a sufficiently high activity in a certain group of lower oligosaccharides (since this term "lower" must be understood in relation to the penetasaccharides) to enable utilization as an active ingredient anti-thrombotic drugs.

DK 174284 B1 4

The continuation of these research works has led to the preparation of a specific group of oligosaccharides, which have proved advantageous to have a broad therapeutic spectrum.

Thus, the present invention aims to provide a novel group of tetrasaccharides, the structure of which corresponds to the structure of fragments of the chains of mucopolysaccharides.

These tetrasaccharides can be used as reactive substances within the laboratory and as active ingredients in drugs.

The tetrasaccharides of the invention which are of the kind set forth in the preamble of claim 1 are peculiar to that of the characterizing part of claim 1.

15

The studies carried out by the inventors in this field have demonstrated the importance of the sequence DEFG, which corresponds to the block designated as irregular and present in the natural heparin molecule. In view of the prior art, it is particularly surprising that this sequence confers the pharmacological properties of the tetrasaccharide itself, which can be utilized in a very wide therapeutic range.

Thus, the defined tetrasaccharides contain two repeating units D-glucosamine-D-glucuronic acid (a-b) and D-glucosamine-L-iduronic acid (a-c).

25

The components mentioned above as included in the compounds are interconnected by type 1 -4 bonds, and they include the following bonds: o a β a 1 4b, a 1 · * 4c, and b 1 ^ 4a.

5 DK 174284 B1

Surprisingly, the tetrasaccharides of the structure DEFG have themselves shown to have pharmacological properties sufficiently potent to permit the use of these products as active ingredients in drugs.

5

The economic interest in these products can be measured against the higher oligosaccharides to the extent that they only require the use of four components or repeating groups for the synthesis thereof.

A family, x, of tetrasaccharides, which is preferred because of their high anti-Xa activity and their high affinity for ATIII, are compounds of the general formula IIa set forth in claim 1, wherein R 2 represents an inorganic anion.

Tetrasaccharides of this family, in which R 2 represents a sulfate anion, are particularly preferred because of their analogy with the natural products.

The particularly interesting tetrasaccharides in this regard also contain sulfate groups for at least some of the other substitutions, or in particular for all the substitutions R 1 and / or Ni and N 2.

20

A preferred tetrasaccharide of this type corresponds to product # 12 of the following Example 3 having the formula: - - oso 2 coo "r" ° s03 NHS03 oh nhso

Other inorganic anions, such as the phosphate group, may be present in place of the sulfate groups.

25 DK 174284 B1 6

Another family γ of the tetrasaccharides, which is preferred, especially because of its fibrinolytic activity, contains a substituent R 2 which represents a hydrogen atom.

5

Tetrasaccharides of this type advantageously contain sulfate groups for at least some of the other substitutions R 1 and / or Ni and N 2. A tetrasaccharide of this group corresponds to the formula: oso ^ coo ~ pO.SO 3 Η0ο ^^ ο4ΐ ^ 01 '<ivi nhS03 oh NHSo: OSO ~ 10 3 3

As is the case in the x family, the products advantageously contain instead of one or more sulfate groups or instead of the total number of these groups other inorganic anions such as phosphate anions.

In the various types of tetrasaccharides highlighted above, the various inorganic anions and carboxylic acid groups are advantageously present in the form of salts with an inorganic cation, especially a metal cation, especially an alkaline cation such as sodium, magnesium or calcium, or a cation of led by a nitrogenous organic base such as triethylammonium.

Advantageously, the tetrasaccharides of the invention can be prepared by the synthetic procedure described below.

In accordance with the most general definition of this process, two compounds consisting of or terminated, respectively, are reacted with components having D-glucosamine structure and with D-glucuronic acid structure or L-iduronic acid structure.

One of the amino sugar or uronic acid components consists of an alcohol in which the OH group in the alcohol function occupies the position 4. The other component then possesses an activated, anomeric carbon atom, i. including a reactive group capable of establishing with the OH group in the alcohol the desired -O-glycosylation bond, in accordance with the desired stereochemical configuration to form a sequence of amino-sugar uronic acid or vice versa.

In particular, the groups located on the components used to form the tetrasaccharide chain should meet the following requirements: The reactive group in the amino sugar component or uronic acid component should be compatible with the protecting groups and / or functional groups which located on the components; the protecting groups for the OH groups and optionally the amino groups or the carboxylic acid groups should be compatible with each other and with the groups which are starting compounds for the amino groups or the carboxylic acid groups when present; the protecting groups and the starting compounds should be inert to the glycosylation reaction and to the reactive groups, allowing the subsequent substitutions to be placed in the various locations and, if desired, sequentially.

The glycosylation reaction is carried out in such a way that it does not change the structure of the components of these products or the nature of the various substituents present.

DK 174284 B1 8

The glycosylation step described above is repeated in such a way as to obtain the desired length of the chain.

In order to carry out this extension of the glucidin backbone, amino acid or uronic acid components which include temporary protecting groups, i.e. groups which are capable of selectively blocking a position in the amino sugar component or uronic acid component which is intended to form part of a new glycosylation reaction. It concerns groups which are removable in the presence of other groups present on the components during recovery of an alcohol.

After the formation of the desired glucidin skeleton, the chain thus formed is subjected to one or more chemical reactions for the purpose of introducing a given type of functional groups or successively several types of groups to form, if desired, derivatives of these functional groups. groups.

To introduce specific substituents, i.e. When certain substituents are in defined positions, it is advantageous to use starting compounds containing several types of protecting groups, namely (1) one or more semi-permanent groups and (2) one or more permanent groups.

The semi-permanent groups are removable in the first place and make it possible to introduce the desired functional groups into the positions they have occupied. The permanent groups, on the other hand, are capable of maintaining the protection of the OH groups during the introduction of functional groups into the locations of the semi-permanent groups.

In addition, the starting compounds of the amino sugar type contain in the 2-position a nitrogen-containing group which allows the presence of a nitrogen-containing functional group to be maintained during the implementation of the process according to the invention. This nitrogen-containing group preferably consists of groups such as N3 or -NHCOO-CH2-C6H5 or any other group which forms a precursor for the amino group or for an amino group derivative, especially -NHSO3 or -NH-acyl, especially -NH-COCH3 .

With respect to the carboxylic acid groups in the uronic acid component, these are blocked by groups which are inert to the reactions employed in the replacement of the protecting groups and can be removed at the end of synthesis to release the carboxylic acid groups, possibly at the end of the salt formation. These protecting groups for the carboxylic acid group are advantageously selected from alkyl groups or aryl groups.

These treatments are used to prepare the oligosaccharides according to the invention.

Preferably, in a preferred method of obtaining the sequence DEFG, a halide, more particularly the bromide, of a saccharide having the structure EF is synthesized, the synthesis of which is described in the previously mentioned patent application with an alcohol having a repeat group of the structure G.

The disaccharide EF includes a temporary group at the 4-position of component E. This group is advantageously selected from acyl groups, especially the acetyl or chloroacetyl group.

25

The condensation reaction is carried out in a solvent medium, especially an organic solvent especially of the dichloromethane or dichloroethane type.

A catalyst is advantageously employed. generally a silver salt or a mercury salt, e.g., silver trifluoromethanesulfonate, which is generally referred to as DK 174284 B1 10 silver triflate, silver carbonate, silver oxide, mercuribromide or mercuricyanide. Also used is a proton acceptor such as symcoilidin as well as a means for capturing the water which may be present and / or capturing the hydrogen halide acid formed, for example a molecular sieve 4Å.

5

An examination of the reaction conditions shows that it is appropriate to operate at ambient temperature or at a lower temperature, which can reach 0 ° C or even lower, under an atmosphere of an inert gas such as nitrogen or argon.

10

After the formation of the trisaccharidic chain with the structure EFG, the temporal group on component E is removed in accordance with classical methods for restoring the -OH group. This latter is then involved in a glycosylation reaction with the halide, especially with the bromide, of the component of structure D.

More specifically, to prepare tetrasaccharides of formula (IIa) wherein R 1 represents a sulfate group, R 2 is hydrogen atom and Ni, and N 2 is identical and represents sulfoamino groups, starting materials containing the following groups are used.

The protecting groups for the -OH groups in these various components intended to be sulfated are protected by acyl groups, especially acetyl groups, while the OH groups intended to be released at the end of the synthesis are protected with a permanent group such as the benzyl group.

The 2-positions of the amino sugar components are substituted by groups such as N3 or NH-COO-CH2-C6H5, and the 6-positions of the uronic acid components are taken by carboxylic acid groups protected by an alkyl group, especially by a methyl group.

11 DK 174284 B1

The step of functionalizing the tetrasaccharide chain formed, i.e. for the sequential introduction of specific substitutions, is now carried out analogously to the method described in the aforementioned Danish patent application 143/83.

These reaction conditions allow the functionalization step to be carried out, for example, as follows: First, the sulfate groups are first selectively introduced after removing the -O-acetyl groups used for the blocking. This reaction is carried out in such a way that it does not affect the benzyl groups and nitrogen containing groups and carboxylic acid groups present.

For this purpose, a saponification reaction is advantageously carried out by means of a strong base such as sodium hydroxide.

This reaction is preferably carried out at a temperature lower than the ambient temperature, more particularly in the vicinity of 0 ° C.

20

Hydrolysis thus obtained is subjected to hydrolysis by the action of an alkylating agent to introduce to the carboxylic acid groups the protective alkyl groups which have been removed during the hydrolysis.

By reaction with a sulfating agent, one first obtains the introduction of the sulfate groups into the positions released by the hydrolysis, which have been left free after the action of the alkylating agent.

The reaction conditions 30 satisfactory for carrying out the sulfation include the use of a sulfating agent, such as a complex of trimethylamine / SO 3.

> * DK 174284 B1 12

This reaction is advantageously carried out in a solvent such as dimethylformamide. It is preferable to operate at a temperature higher than ambient temperature, generally at 50 ° C, which corresponds to a reaction time of approx. 12 hours.

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

The removal of the benzyl groups is advantageously carried out by catalyzed hydrogenation under conditions compatible with the maintenance of the sulfate groups and with the conversion of the nitrogenous groups to functional amino groups.

Preferably, one operates under a hydrogen pressure and in the presence of a Pd / C catalyst.

This reaction is advantageously carried out in an organic solvent medium, especially in water with an alcoholic medium.

20

In order to obtain the hydrogenation of the nitrogen-containing groups acting as precursors and the removal of the groups which are protecting groups for the -OH groups, the reaction is advantageously carried out with a duration of approx.

3 - 4 days.

25

As already stated, the functional amino groups are in the form of N-acetyl or N-sulfate derivatives in the biologically active molecules considered.

DK 174284 B1 13

To form N-acetyl groups, the reaction product coming from the hydrogenation reaction is subjected to the action of an acetylating agent. In this connection, acetic anhydride is a particularly suitable agent.

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

It may also be desired to form N-sulfate groups, which may be carried out by means of a sulfating agent of the above type. pH values greater than 9, preferably of the order of 9-10, are used for the sulfation.

After the sulfation reaction, the addition of a strong base allows the carboxylic acid groups to be released.

15

The reaction products thus formed can be readily converted to salts by suitable cation exchange resins. In the natural products, the cation is especially made of baking soda. Thus, natural cation exchange resins are advantageously used.

20

Salts of potassium, lithium, magnesium or calcium can also be formed.

A proton-exchange resin is then used to neutralize the acid thus formed with the base of the cation in question.

The study of the pharmacological effects of the tetrasaccharides of the invention has made it possible to demonstrate their interest in the therapy.

In particular, they exert an activity on fibrinolysis by increasing the content of circulating activator of plasminogen and sensitizing blood clot to light.

30 DK 174284 B1 14

Studies on various experimental models have been performed according to the technique described by Vairel et al. in Ann. Pharmaceuticals Frangaises, 1983, 41, No. 4, pp. 339-353.

Thus, 15 minutes after intravenous injection, rabbits (anesthesia 20 minutes prior to administration) of 0.25 mg oligosaccharides per kg increases the content of the plasminogen activator in the circulating blood.

For example, with the tetrasaccharide DEFG of formula (III), an average increase of the brightness ranges of 17.80 is observed, while a reduction of 0.5 is observed with physiological saline solution used as a control substance.

Studies carried out with tetrasaccharides according to the invention in which the component of structure F contains an -SO 3 group at the 3-position have shown an anti-Xa activity that is clearly greater than the activity of heparin and a strong affinity for AT -III. In the case, for example, of the tetrasaccharide of formula (III) (product # 11), the anti-Xa activity measured with a homogeneous substrate is 600 units of anti-Xa / mg (method of Teien AM and Lie modification, Thrombosis Research no. 10,937,388-410)

The therapeutic efficacy of the products in question has been investigated with well-defined animal models to determine their anti-thrombosis capabilities under known pathological conditions.

25

The following results were obtained with the model for modified stasis in rabbits (see Thromb. And Hemost. 46, (1) 117, 1981 by Andersen et al.).

In rabbits, 50 µg / kg of the product dissolved in a saline solution (in an amount of 100 µg / kg) was administered intravenously 5 minutes prior to administration of a thrombogenic agent. The thrombogenic agent consisted either of rabbit serum or of agent DK 174284 B1 PCC / RW (concentrate of a complex of prothrombin and viper venom (Rup-per)). The left and right cervical veins were graded on a scale of up to 10.

5 Complete protection (rating 0) against the effects of thrombogenes introduced with rabbit serum and partial protection against the thrombogenic agent PCC / RW (rating 5) was observed, with control animals showing a rating of 10.

The tetrasaccharides of the invention, in which the component of structure F contains at the 3-position an OH group, do not appear to have activity against the Xa factor, and thus exhibit a much greater specificity than the group of oligosaccharides having an SO group at the 3-position of the component of structure F, against the fibrinolytic activity.

15

The toxicological studies on the products of the invention have shown their harmlessness, which makes them valuable in the manufacture of pharmaceuticals.

The tetrasaccharides of the invention can be used in pharmaceutical formulations, and in particular, pharmaceuticals which are free of pyrogenic substances and which contain an effective amount of the active ingredients in combination with pharmaceutical excipients. The tetrasaccharides of the invention are particularly useful in such compositions in which the pharmaceutical carrier is suitable for oral administration. Administration forms suitable for oral administration may advantageously be gastro-resistant gelatin capsules, compressed powders or tablets, pills, or the compounds may be in the form of liposomes.

30

Other pharmaceutical compositions contain these tetrasaccharides in combination with excipients suitable for administration through the rectum. Corresponding administration formulations consist of suppositories.

Other forms of administration consist of aerosols or pomades.

The tetrasaccharides of the invention can also be used in pharmaceutical preparations for injection, which are sterile or which can be sterilized, for administration, either intravenously or intramuscularly or subcutaneously. These solutions advantageously contain products of the family x in an amount of 1,000 - 100,000 units of μ (Yin-Wessler) / ml oligosaccharides, preferably 5,000-50,000, e.g. 25,000 μ / ml when these solutions are intended for subcutaneous injection. For example, they can contain from 500 to 10,000, especially 5,000 μ / ml oligosaccharides when intended for intravenous injection or perfusion.

Such pharmaceutical compositions are advantageously available in the form of disposable syringes ready for use.

Also, pharmaceutical compositions containing the aforementioned tetrasaccharides may be combined with other active ingredients.

The pharmaceutical compositions containing the tetrasaccharides of the invention are particularly suitable for the regulation (preventive or curative) of certain stages of blood coagulation in humans or animals, especially in the case where the patient is at risk of hypercoagulability, especially as a result of surgical intervention, the atheromatosis process, the development of tumors, disruption of coagulation through bacterial activators or enzymatic activators, etc.

In order to illustrate the invention, an example of posology which is useful in humans in connection with the products of the family x is given below. administration to the patient of 1,000 to 25,000 units of μ (Yin and Wessler) subcutaneously, one to three times a day, depending on the level of risk of hypercoagulation or thrombotic state in the patient, or from 1,000 to 25,000 μ / 24 hours at 5 intravenous administration, at discontinuous administration at regular intervals or continuously by perfusion, or also 1,000-25,000 μ (three times a week) by intramuscular or subcutaneous injection (these contents are indicated in Yin-Wessler units). These dosages can, of course, be regulated for each patient as a function of the results and of the prior blood tests performed, and of the nature of the patient's suffering and, in general, of the patient's health.

In the case of products of the family γ, one is administered 1-100 mg / day, depending on the patient's condition and the pharmaceutical form used.

15

In addition to the pharmaceutical compositions containing the tetrasaccharides as they exist, pharmaceutical compositions may also contain at least one oligosaccharide as defined above, which is conjugated with a covalent bond to a soluble carrier or to an insoluble carrier, advantageously by a sugary species having only one sugar. reducing group.

Preferred conjugates fixed to soluble carriers in particular consist of tetra-saccharides AT-III conjugates containing a sequence DEFG of formula (IIa), in particular a sulfate group. Such products constitute particularly interesting drugs for the prevention of thrombosis in the case of AT-III deficiency conditions.

Other preferred conjugates with soluble carriers are tetra-saccharides of the general formula (IIa), which are fixed to a carrier, such as a protein, especially polylysine or bovine serum albumin.

DK 174284 B1 18

These products are useful as immunogenic substances which in themselves are source of antibody circulation products in vivo, or as monoclonal antibodies cloned in vitro by a suitable technique.

The present tetrasaccharides are bound to insoluble carriers by other preferred conjugates. Here you will advantageously use the classic carriers. These conjugates can be used as immunosorbents, for example, in a highly specific purification of AT-III and in the determination thereof, or in the preparation of blood-compatible thrombotic polymers, by fixing the oligosaccharides on biologically compatible polymers when contains a sequence DEFG according to the family described above x.

The tetrasaccharides of the invention can also be used in the field of nuclear medicine as radiopharmaceutical products. In that case, these products are marked by a trecer, selected from those commonly used in this field, especially by means of technetium 99 m.

To this end, technetium 99 m obtained from commercially available generators in the form of non-reactive sodium pertechnetate with valence 7 is converted 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 particular molecule under the specified pH conditions.

30 DK 174284 B1 19

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

Further development of these radiopharmaceutical reaction components may 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 tests 10 in vivo for the detection and diagnosis of the extent of thrombosis and of thrombotic sites.

The present tetrasaccharides can also be used for the determination of the specificity of numerous enzyme systems involved in the metabolism of glucosaming glucagon glycans.

The following examples illustrate other advantageous characteristics associated with the compounds of the invention and the process for their preparation.

Reference is made to Figures 1 to 5 which illustrate the products used in the syntheses described.

In these figures, the numbering of formulas is used in the same way as in the examples for describing uniform products.

25 The formulas in question use the following abbreviations:

Ac - acetyl Me - methyl Bn - benzyl 30 Lev - leuvinyl MCAO - monochloroacetyl and DK 174284 B1 20 Z - benzyloxycarbonyl.

EXAMPLE 1 Preparation of trisaccharide # 3 having the structure EFG, i.e. benzyl-O- [methyl-2,3-di-O-benzyl-4-O-chloroacetyl-β-D-glucopyranosyluronate- (1-4) -O- (3,6-di-O-acetyl-2-azido -2-Deoxy-α-D-glucopyranosyl) - (1-4) -O- [methyl-2-O-acetyl-3-O-benzyl-αL-idopyranoside uronate] The synthesis of trisaccharide # 3 is carried out by condensation between a halogenide having the structure EF and an alcohol having the structure G. These reaction components are numbered 1 and 2 respectively in the specification.

This condensation step is carried out as follows:

A solution of 738 mg (0.92 mmol) of the halide 1 and 428 mg (1 mmol) of the alcohol 2 in 15 ml of anhydrous dichloroethane is stirred at 20 ° C in the presence of a powdery molecular sieve. Then 150 ml of collidine is added and then 262 mg (10 mmol) of silver triflate. The reaction mixture is diluted 20 after 1 hour at -20 ° C with dichloromethane and filtered. The organic phase is washed with 10% KHSO 4 in water, dried (Na 2 SO 4) and then concentrated to dryness. The obtained foam (1.07 g) is chromatographed over silica gel (toluene / ethyl acetate; 4/1; v / v) to give the pure trisaccharide 3 (699 mg; 63%) as a white foam.

[Cz] d: + 25 ° (c 1.4; chloroform).

EXAMPLE 2 Preparation of a tetrasaccharide # 6 having the structure DEFG, i.e. benzyl-O- (6-O-acetyl-2-azido-3,4-di-O-benzyl-2-des-oxy-α-D-glucopyranosyl) - (1-4) -O- [methyl] -2,3-di-O-benzyl-β-D-glucopyranosyluronate] - (1-4) -0- (3,6-di-O-acetyl-2-azido-2-deoxy-α-D-glucopyranosyl) - (1 - * 4) -0- (methyl 2-O-acetyl-3-O-benzyl-αL-idopyranosiduronate).

This synthesis is carried out by condensing a halide 5 with structure D and an alcohol 4 corresponding to the trisaccharide 3 in which the OH group at the 4-position of component E has been unblocked.

Thus, it is described successively: (a) obtaining the trisaccharide 4 from the trisaccharide 3 prepared as described in Example 1, (b) the condensation of 4 with the halide 5.

15

Step a: 669 mg (0.55 mmol) of the trisaccharide 3 prepared as described in Example 1 is dissolved in a mixture of 3.8 ml of lutidine and 1.25 ml of acetic acid. Then, methanol is added followed by a solution of hydrazine dithiocarbonate obtained through the method described by Boeckel and Beetz in Tetrahedron letters 24 (1983) 3775-3778. After 2 hours at ambient temperature, the solution is diluted by adding 200 ml of dichloromethane, then washed (saturated NaHCCb and water; 10% KHSO4 in water), dried (Na2SO4) and concentrated to dryness. The pure reaction product (530 mg; 83%) is obtained after chromatography over silica gel (toluene / ethyl acetate; 2/1; v / v).

[Α] D: -29 ° (c 1.29; chloroform).

Elemental Analysis for C54H11O20N3: <DK 174284 B1 22

Calculated: C 60.50 - H 5.74 N 3.92 Found: C 60.46 - H 5.74 - N 4.11.

Step b: 5

A solution of 494 mg (0.455 mmol) of the alcohol 4 in dichloromethane and of 1.096 g (2.2 mmol) of the halide 5 is treated with 632 mg of silver triflate in the presence of 360 μc symcollidine as described in the synthesis. of the compound 3. After purification over a column of silica gel (gradient 10 chloroform - * chloroform / ethyl acetate; 20/1; v / v) the pure derivative 6 (575 mg; 89%) is obtained.

[οφ: + 43 ° (1.27; chloroform) Elemental analysis for C76H84O24N6:

Calculated: C 61.61 - H 5.71 - N 5.67 Found: C 61.57 - H 5.76 - N 5.61.

EXAMPLE 3

Preparation of the tetrasaccharide # 11 having the structure DEFG, i.e. O- (2-deoxy-6-O-sulfo-2-sulfamido-α-D-glucopyranosyl) - (1-4) -25- (PD-glucopyranosyluronate) - (1- * 4) -0- (2 -desoxy-3,6-di-O-sulfo-2-sulfamido-α-D-glucopyranosyl) - (1-4) -0-2-O-sulfo-α-L-idopyranuronate, octane sodium salt.

This tetrasaccharide is obtained by sequentially blocking the OH groups and introducing the desired groups on the tetrasaccharide 6 prepared as described in Example 2.

The following successive steps are used: 1 - release of the OH groups blocked with acetyl groups; 2 - formation of the sodium salts of the sulfate groups; 3 - forming the sodium salts of the carboxylic acid groups; 4 - Release of the OH groups blocked with benzyl groups and 10 conversion of the azido groups to amino groups; 5 - esterification of the amino groups.

These steps are carried out as follows: 15 1 - Transfer of -OAc to -OH leading to the tetrasaccharide 7.

0.340 g of the derivative 6 prepared as described in Example 2 is dissolved in a mixture of 30.6 ml of methanol, 3.5 ml of chloroform and 4.25 ml of water. Then 4.25 ml of 5-N hydroxide solution is added. After 4 hours at ambient temperature, 90 ml of chloroform and then 8 ml of 8N hydrochloric acid are added. After decanting, the chloroform phase is separated. The aqueous phase is washed with chloroform (3x10 ml). The chloroform phases are collected and dried (Na 2 SO 4). The evaporation residue obtained after concentration to dryness is methylated with 25 diazomethane and then chromatographed over a silica gel column, eluting with a gradient (dichloromethane - dichloromethane / ethyl acetate; 1/1; v / v). Compound 7 is obtained (0.173 g; 57%).

[ci] d: 14 ° (c 1; chloroform).

Elemental analysis for CeOHreOaiNe, 0.5 H2O

30 DK 174284 B1 24

Calculated: C 61.68 - H 5.93 - N 6.34 Found: C 61.62 - H 5.84 - N 6.31.

5 2 - Transfer of -OH to -Q-SQsNa leading to tetrasaccharide # 8

117 mg (0.068 mmol) of Compound 7 is dissolved in 3 ml of anhydrous dimethylformamide and then sulfated overnight at 50 ° C in the presence of 123 mg of trimethylamine / SO3. The reaction mixture is then diluted by the addition of a methanol / chloroform mixture (1/1; v / v; 3 mL), then chromatographed over a column of "Sephadex LH 20" which is equilibrated in chloroform / methanol (-1/1; v / v) The reaction product thus obtained is used directly for the preparation of 9.

15 3 - Transfer of COOMe to COONa leading to tetrasaccharide No, 9

357 mg (0.207 mmol) of compound 8 is dissolved in a mixture of 8 ml of methanol and 2 ml of water, to which 1.2 ml of 5N sodium hydroxide is added. After 3 hours of reaction, the reaction mixture is passed through a 20 column of the resin “Dowex 50 - H + M which is equilibrated with a mixture of ethanol / water (8/2; v / v). The eluate is passed through a column of "Dowex 50 - Na +" which is equilibrated with the same solvent. After evaporation to dryness, the reaction product 9 is obtained in a pure state (0.267 g) after chromatography over a silica gel column (30 g; 25 ethyl acetate / pyridine / acetic acid / water; 160/77/19/42; v / v / v / v) , followed by a passage over an ion exchanger “Sephadex SPC 25” (18 g) equilibrated with the Na + form in a methanol / water mixture (8/2; v / v).

[α] D: 5.50 (1.025; methanol).

- Transfer of OBn to -OH and of -N ^ to -NH? leading to tetrasaccharide # 10

256 mg (0.147 mmol) of compound 9 is dissolved in a mixture of methanol / water (9/1; v / v; 10 ml) and then hydrogenated in the presence of a catalyst (Pd / C 10%; 130 mg ) for 5 days. The catalyst is replaced with fresh catalyst and the reaction is continued for 4 days. The UV spectrum of the product then shows the absence of benzylated derivatives. The product obtained (0.17 g) is transferred directly to the preparation of Compound No. 11 10 5 - Transfer of -NH? to -NHSCKNa leading to tetrasaccharide No. 11

Gradually, a solution of 9.17 g (0.152 mmol) of compound 10 in 10 ml of water is added 140 mg (0.75 mmol) of the pyridine / SO3 complex, maintaining pH at 9.5 by the addition of 2 M-sodium hydroxide. A renewed addition of the sulfating complex is made after one hour (70 mg) and after one night (70 mg). The reaction mixture is then placed on top of a column of "Sephadex G 50" (300 x 2.5 cm) eluted with 0.2M sodium chloride. The reaction product is then absorbed on top of a 20 column of the "Biore AG 1 x 2" resin. (1.6 x 10 cm) on the Na + form eluted with a gradient of sodium chloride (0.5M - 3M) The fractions containing the reaction product are pooled.

The salts are removed by chromatography over "Sephadex G-25" (100 mg). Finally, the reaction product is lyophilized (111 mg; 54%).

[α] D: + 46 (c 0.85; water).

Obtaining a component G containing a -OCH3 instead of an -OBn group at the anomeric carbon atom, tetrasaccharide # 12, is employed in the treatment steps described above.

The meanings of the substituents A ^ are listed in the following table. A5 of formula 5 shown in FIG. In for compounds 6-12.

Aι A2 A3 A4 A5 6 Ac Bn Na Me Bn 7 H »Bn Na Me Bn 8 SOaNa Bn N3 - Me Bn 9 SOaNa Bn Na Na + Bn

10 SO 3 Na H NH 2 Na + H

11 SOaNa H NHSOaNa Na + H

12 SOaNa H NHSOaNa Na + CH3 5 NMR 1H for the tetrasaccharide of the invention is shown in FIG. 5 in the lower part. In the upper part, for comparison, NMR spectra of the corresponding pentasaccharide containing an OSO3 group at the 3-position of component F. are indicated. The signal is attributed to 5.51 ppm observed at DEFGH 10 containing the OSO33 group, component F at the 3-position at the more proton of the repeat group glucosamine-3-O-sulfate. This signal is offset to approx. 5.45 ppm at the corresponding component F in the non-sulfated state, and it superimposes the corresponding signal from the component H.

The NMR spectral analysis characteristics of the penta-saccharide DEFGH used for comparison are as follows: 1 H NMR: in D 2 O relative to TSP internal;

Component D; 5.64 (H1) 3.30 (H-2) 3.63 (H-3) 3.57 (H-4) 3.90 (H-5) 4-4.5 (H-6.6) .

Component S: 4.62 (H-1) 3.40 (H-2) 3.87 (H-3).

Component P: 5.45 (H-1) 3.25-3.30 (H-2).

Component G: 5.24 (H-1) 4.33 (H-2) 4.20 (H-3) 4.11 (H-4) 4.81 (H-5).

Component H: 5.44 (H-1a) 4.70 (H-1 &) 3.25 (H-2a) 3.05 (H-20) 3.69 (H-3) 3.79 (H-4) ).

5

Claims (3)

1. Tetrasaccharides, characterized in that they have the structure DEFG of the general formula QRj_coo 'ORj lla i'j os, n2 i:' Aj wherein the groups R ^ which are the same or different represent an inorganic anion, especially a sulfate group. or a phosphate group, R 2 has one of the meanings specified for R 1, or represents a hydrogen atom, Ni and N 2, which are the same or different, represents -NH 2, a sulfoamino group, a phosphoamino group or -NH-COR 3, wherein R 3 represents a alkyl group, and salts thereof, preferably those for which the substituent R2 at the 3-position of component F represents an inorganic anion, especially a sulfate group, and for which one, more or all of the substitutions R2, Ni and / or N2 include a sulfate. 20 Tetrasaccharide according to claim 1 of the formula: p-OSOj coo '/ ° \ / ° \ / · / T ° \ / “* ° \ (oh / qAox / 9 ^, so3 / qA ^ S0' ^ · ° η æa : NKS03 Ofi NHS03 OS03 DK 174284 B1 29 Tetrasaccharides according to claim 1, characterized in that the substituent R 2 at the 3-position of the component (F) represents a hydrogen atom and that at least some of the other substitutions R 1, Ni and / or N 2 include a sulfate group. 5