FR2531436A1 - Substituted pentasaccharides possessing biological properties, their preparation and their uses as medicaments - Google Patents

Abstract

Pentasaccharides with a (D-glucosamine)-(glucuronic acid)- (D-glucosamine)-(L-iduronic acid)-(D-glucosamine) structure possessing high anti-Xa (Yin Wessler) activity. These products may be used, in particular, as antithrombotic medicaments.

Description

"Substituted pentasaccharides having properties
biology is their preparation and their applications as
that drugs "
The invention relates to novel pentasaccharides having biological properties enabling them, in particular, to control only certain stages of blood coagulation.
It also relates to a process for the synthesis of pentasaccharide derivatives.

The invention further relates to the application of the biologically active pentasaccharides in question as active ingredients of medicaments
The inventors' research, which led to the development of the invention, relates to obtaining, by way of synthesis, oligosaccharides comprising motifs constituting heparinic chants and exhibiting anti-Xa activity (Yin-Wessler). ) higher than that of h @ parine with an overall anticoagulant activity (measured according to the USP or APTT test) lower than that of heparin.

 It is recalled that the determination of the anti-Xa activity according to the Yin-Wessler test is reported by these authors in J, La Cbim.Med. 1976, 81, 298-300.

 The USP test is described in "Pharmacopea ™ United States of America", pages 229-230 (see also USP-NFF Supplement No. 2, p. 62 and USP-NF Supplement No. 4, p.90, respectively, entitled "Drug". substances "and" Dosage forms ").

The title APTT is measured according to the method of J. Caen et al, in the hemo @ tase, French scientific expansion,
Paris, 1968, pages 1 to 3-135.

 The work done by the inventors has shown that by carrying out the condensation of substituted saccharide units according to a given strategy, it was possible to obtain pentasaccharides of particular interest with regard to their specific anticoagulant properties.

 The object of the invention is therefore to provide new pentasaccharides whose basic structure comprises constituent units of the heparin chains.

 It also relates to a process for obtaining compounds with pentasaccharide structure substituted by synthesis, which therefore allows, according to an essential advantage of the invention, to develop such compounds on an industrial scale and with a degree of purity. very high.

 The invention aims; in addition, to provide active principles @t the drugs themselves, capable of inhibiting certain coagulation factors and especially the actor Xa, when they are present in the blood, with a high degree of selectivity vis-à-vis other factors including e factor II which is related to a very low activity on global coagulation.

dans laquelle - les groupes R,identiques ou différents les uns des autres, représentent un atome d'hydrogène ou un '--radical, physiologiquement accep@able, type -R1R2, dans lequel R1 représente avantageusement un anion minéral, en particulier un groupe SO3 et R2 représente un cation métallique, notamment un metal alcalin tel que le sodium ou alcaiino-terreux, tel que le calcium ou le magnésium, ou un cation organique tel qu'un dérivé ae base organique azotée, et - les substituants A, identiques ou différents les uns des autres, représentent un atome d'hydrogène, ou encore un cation métallique ou un cation organique sels que définsi cidessus. The pentasaccharides of the invention correspond to formula (I)

in which - the groups R, identical to or different from each other, represent a physiologically acceptable hydrogen atom or a '--radical, type -R 1 R 2, in which R 1 advantageously represents a mineral anion, in particular a group SO3 and R2 represents a metal cation, in particular an alkali metal such as sodium or alkaline earth metal, such as calcium or magnesium, or an organic cation such as a nitrogen-based organic base derivative, and the identical substituents A, or different from each other, represent a hydrogen atom, or a metal cation or an organic cation salts as described above.

 In preferred pentasaccharides the R groups are identical to each other. In other pentasaccharides, all the groups A are, furthermore, idonetic with each other.

 A particularly preferred pentasaccharide of this type contains as substituents of type R, a group -SO 3 Na and as substituents A an Na group.

This pentasaccharide thus corresponds to the following formula III)

 In addition, the pentasaccharides of the invention are characterized by Yin-Wessler titers which are much higher than those of heparin. More particularly, the pentasaccharide of formula II has anti-Xa (Yin-Wessler) activity equal to or greater than 2000 u / mg, up to 2500 u / mg.

In a test using a chromogenic substrate, this activity was even 4000 units anti Xa / mg (method of
TEIEN AM and LIE modified; Thrombosis Research No 10,1977, 388-410.)
This test consists in using the factor Xa marketed by SIGMA Company in solution at 8 u / ml in physiological saline, the concentration of the substrate being 1.33 mM.

 To perform this test, you can proceed as follows.

 10 μl of solution to be assayed and 300 l of human plasma diluted with 0.02 M Tris maleate buffer, pH 5, are mixed.

 Incubate for one minute at 37 OC.

 100 μl of the aforesaid factor Xa (8 μl / ml) are added and after one minute the solution obtained is injected into the substrate.

 The overall anticoagulant activity of these products is very low, 4 u / mg in the APTT test.

 These properties allow them to contol, in a specific way, certain stages of blood coagulation.

The study of these products shows that it is
able to perform antithrombotic activity then
health and that the risk of bleeding is
practically eliminated because of their low
overall anticoagulant activity.

The pentasaccharides of the invention are, in
In addition, they are advantageously free of toxicities.

 These products are therefore particularly valuable for the development of usable drugs, especially for the treatment of thromboses.

 The invention thus relates to pharmaceutical preparations which contain said pentasaccharides with high anti-Xa activity.

 More particularly, it relates to pharmaceutical preparations lacking pyrogenic substances, containing an effective amount of active ingredients in combination with pharmaceutical excipients.

 It also relates to compositions in which the pharmaceutical carrier is suitable for oral administration. Forms of administration of the invention suitable for oral administration may advantageously be gastroresistant capsules, tablets or tablets, pills, or presented as liposomes.

 Other pharmaceutical compositions include these pentasaccharides in combination with excipients suitable for rectal administration. Corresponding dosage forms are suppositories.

 Other forms of administration of the invention are aerosols or pomades.

 The invention also relates to injectable, sterile or sterilizable pharmaceutical compositions.

for intravenous, intramuscular or subcutaneous administration.

These solutions advantageously contain 1000 to 100,000 μ (Yin-Wessler) / ml of pentasaccharides, preferably 5,000 to 50,000, for example 25,000 μ / l, when these solutions are intended for subcutaneous injection. . They may contain, for example, from 500 to 10,000, in particular 5,000 u / ml of pentasaccharides, which are intended for intravenous injection or infusion.
Advantageously, such pharmaceutical preparations are presented in the form of non-recoverable syringes, ready for use.

 The invention also relates to pharmaceutical compositions containing said pentasaccharides in combination with another active ingredient, usable in particular for prophylaxis and treatment of thrombosis, such as a venotonic agent such as dihydroergotamine, a nicotinic acid salt or an agent thrombolytic as urokinase.

 The pharmaceutical compositions of the invention are particularly suitable for the control (preventive or curative) of certain stages of the coagulation of sana in humans or animals), particularly in the case where the patient is subject to risks of hypercoagulability resulting in particular from surgical operations, atheromatous processes, development of tumors and coagulation disorders by bacterial or enzymatic activators, etc.

To illustrate the invention. we indicate, below,
an example of a dosage that can be used in humans
this dosage includes, for example, the administration
to the patient from 1000 to 25 000 u (Yin and Wecsler) per
subcutaneously, one to three times daily, according to
risk level of hypercoagulability or the condition
thrombotic patient, or 1000 to 25000 u / 2 hours,
intravenous, in discontinuous administrations
at regular intervals, or continuous by infusion, or
still 1,000 to 25,000 u (three times a week) per
intramuscular or subcutaneous (these titles are
expressed in Yin-Wessler units). These doses may be
to be naturally adjusted for each patient according to the results and blood tests carried out beforehand, the nature of the affections from which he suffers and, in general, his state of health.

 The invention also relates to the application of the pentasaccharides of the invention, to the constitution of biological reagents, usable in laboratories, especially as comparative elements for the study of other substances whose anticoagulant activity is to be tested, especially at the level of factor Xa inhibition and antithrombin III assay.

 It also applies to the application of these pentasaccharides in nuclear medicine as radiopharmaceuticals. These products are then labeled with tracers chosen from those commonly used in this field, and in particular using technetium 99 m.

 For this purpose, the technetium 99 m obtained from commercial generators, in the form of non-reactive valence 7 pertechnetate desodium, is converted into reduced technetium of valence 4 which would be the most reactive form of technetium. This transformation is carried out by means of a reducing system made from tin salts (stannous chloride), iron salts (ferrous sulphate), titanium salts (tri-titanium chloride) or other salts.

 Most of the time, this simple reduction of terhe- tium is sufficient, under given pH conditions, to achieve the fixation of technetium on the molecule in question.

 The products of the invention, which constitute a kind of support, can be used at doses of the order of 100 to 200 μ Yin-Wessler.

 For the development of these radiopharmaceutical reagents, the method of P.V. KULKARNI et al. in The Journal of Nuclear Medicine 21, No. 2 ,. p. 117-121.

 The products thus labeled are advantageously used in in vivo tests for detecting and diagnosing the extension of thromboses and thrombotic states.

avec un tétrasaccharide formé d'un enchalnement de motifs respectivement de structure (acide glucuronique)-(D-glucosamine)- (acide-L-iduroniquej-(D-glucosamine) répondant à la formule (Iv)

The novel biologically active pentasaccharides of the invention are advantageously obtained by reacting a monosaccharide with a D-glucosamine structure of formula (III)

with a tetrasaccharide formed by a chailling of motifs respectively structure (glucuronic acid) - (D-glucosamine) - (L-iduronic acid- (D-glucosamine) corresponding to the formula (Iv)

 In these formulas -B1, B2 and A1 represent blocking groups of -OH groups that can be eliminated so as to allow the desired substituents to be successively introduced into the saccharide units without the remaining blocking groups being affected, or to release groups -OH data, - Z represents a nitrogen functional group, precursor of an amino group, in particular an azido group N3 or a benzyloxycarbonylamino group, - X represents a halogen atom, especially bromine or chlorine.

It will be noted that in the starting products
(III) and (IV) all positions are blocked, except "reactive positions" ie those which must be involved in the glyco reaction
sylation.

 These reactive positions, on each of the starting materials, are then substituted by radicals capable of reacting by allowing the establishment of a glycosidic bond, and which are compatible with the blocking groups. In the products chosen, these radicals are respectively constituted by a halogen and an alcohol.

 The blocking groups are chosen from groups that are inert with respect to the glycosylation reaction.

 these are Z groups which allow the maintenance throughout the process of synthesis of a nitrogen group which can then be converted into an amine functional derivative and preferably an amine salt.

 The other blocking groups protect the -OH groups of the various saccharide units of the starting materials.

 These groups are chosen so as to introduce, in a specific manner, given substituents on the saccharide units.

 It is then advantageous to use different blocking groups, namely, B1, B2, A1, depending on the substituent to be introduced.

 The groups k, which protect OH groups intended to be salified, in particular sulphated, are advantageously constituted by acyl groups, in particular acetyl groups. The B2 blocking groups that must be removed to liberate the -OH groups at the end of the process are selected from inert groups during the sulfation reaction, and are advantageously benzyl groups.

 The A1 groups allow the blocking of the carboxyl groups in the 6-position throughout the steps used for the introduction of the various substituents and are removable to allow, in particular, to carry out a salification reaction of carboxyl groups. The use of alkyl radicals, in particular methyl, is satisfactory in this respect.

 The condensation reaction carried out in the invention is based, as indicated above, on the reaction of a halide and an alcohol.

 This reaction is advantageously carried out in the middle. solvent, especially in an organic solvent of the dichloromethane type, and in the presence of catalyst such as silver triflate and also a proton acceptor, such as sym-coliidine.

 The reaction conditions, in particular concerning the temperature, the duration, the concentration of reagents, are chosen so as to carry out the glycosylation bond without altering the structure and the substituents of the various units and will be easily established by those skilled in the art. for obtaining a given type of products.

 The pentasaccharide formed at the end of this condensation is then treated so as to eliminate in sequence the blocking groups present and for.

successively introducing the desired functional groups and releasing the -OH functions in positions.

data.

 Advantageously, in a first step, the B1 groups are removed, for example using sodium hydroxide in the case of acetyl groups; which liberates the corresponding -OH positions which will then be subjected, during a second stage, to a treatment for the purpose of sulphation of these only positions.

In order to introduce a sulphate group, a sulphating agent such as a trimethylamine / SO 3 complex is advantageously used.

 The introduction of the desired cation R.sub.2 can in particular be carried out using an ion exchange resin comprising this cation, or alternatively, after passage in acid form, by neutralization with the base of the cation.

 After the sulphation step, the OH groups are liberated from their benzyl substituents and, at the same time, the Z group is converted to anin group by hydrogenation, for example, under a stream of hydrogen, in the presence of hydrogenation catalyst, operating under conditions compatible with the maintenance of the sulphate group at the 6-position of the D-glucosamine units.

 In order to have glucosamine units containing functional nitrogen groups in position 2, the pentasaccharide obtained at the end of the preceding stage is subjected to the action of a suitable agent. The introduction of sulfate groups is thus advantageously carried out with the sulphating agent trimethylamine / SO 3 indicated above.

 It appears appropriate to carry out this reaction at basic pH, from about 9 to 10. To obtain the desired salt, an ion exchange resin containing the cation which it is desired to introduce is advantageously used, or else, after passing under acid form, neutralized with the base of the cation.

 In a further step, the protecting groups of the carboxylic groups are removed for salification of the acid functions, for example with the aid of a metal hydroxide.

 Intermediate pentasaccharides are novel products and as such are also within the scope of the invention.

 The tetrasaccharide of formula IV used in the condensation reaction mentioned above is advantageously obtained from the corresponding product, but comprising in the reactive position (namely on the carbon in position 4 of the uronic acid structure unit) a group temporary blocking. By this term is meant a group which temporarily protects an OH group intended to be engaged during the synthesis process in a glycosylation reaction and not an OH group intended to be released or functionalized.

This group must be chosen from among those compatible with the other groups present as well as with the conditions implemented in the process. of synthesis.

 An appropriate group is, for example, of the monochloroacetyl type. But it is clear that other groups meeting the above requirements may be used.

avec un disaccharide de structure (D-glucosamine)-(acide L-iduronique) répondant à la formule (VI)

According to a preferred embodiment of the process of the invention, the tetrasaccharide comprising the above temporary blocking group is advantageously obtained by reaction on the one hand of a disaccharide structure (glucuronic acid) - (D-gluco-samine) having the formula (V)

with a disaccharide of structure (D-glucosamine) - (L-iduronic acid) having the formula (VI)

In these formulas, the substituents Al, s1,
B2, Z and X have the meanings given above and B3 a temporary blocking group as defined above.

 The condensation reaction between these two disaccharides is advantageously carried out under conditions of the type used for the reaction of the tetrasaccharide of formula (IV) with the monosaccharide of formula (III).

et d'un monosaccharide à structure D-glucosamine de formule (VIII)

The disaccharide of formula (V) is in turn advantageously obtained from a monosaccharide with a uronic acid structure of formula (VII)

and a monosaccharide with a D-glucosamine structure of formula (VIII)

in which A1, B3, X and Z have the meanings given above. Preferably, both
substituents -O-B3 in the derivative of formula (VIII) form
a 1,6-anhydro bridge.

The disaccharide resulting from the condensation
monosaccharides treated and treated, according to the
usual techniques, to have a single position
reactive, namely on the anomeric carbon, and introduce
so a group X on this carbon.

The derivative of formula (VIII) is advantageously
obtained from the derivative, described in the application for
patent FR No. 82 09392 of May 28, 1982 in the name of the Applicant, which
is then treated according to conventional techniques so
to introduce the blocking group B3 and the reactive group X.

 As disaccharide of formula V-advantageously uses a product as obtained according to the process described in the patent application FR No. 82 01575 of 1.02.1982, in the name of the Applicant.

 Other features and advantages of the invention will appear in the description of the examples which follow and with reference to FIGS. 1 to 4 which represent the example of the fractional scheme corresponding to the syntheses described in the examples.

 The numerical references used in these figures for the compounds are repeated in the examples to designate the same compounds.

The abbreviations used have the following meanings
Ac represents an acetyl group,
- B a benzyl group
- Me a methyl group
- McAO a monochloroacetyl group, and
- Y a -COoB group.

EXAMPLE 1
Synthesis of disaccharide 3 or (1-bromo-3,6-di-O-acetyl-2-azido-4-O-2,3-di-O-benzyl-4-chloroacetyl-s-D-glucopyranosyl) methyl uronate / -D-glucopyranose,
This synthesis includes
A) the preparation of monosaccharide 14 or methyl (1-bromo-2,3-di-O-benzyl-4-O-chloroacetyl-α-D-glucopyranosyl) uronate;
B) - the condensation of compound 14 with monosaccharide 15 leading to disaccharide 1;
C) acetolysis of compound 1 leading to disaccharide 2; and
D) bromination of the disaccharide 2 to give the drift 3
A) Preparation of the monosaccharide 14
This synthesis is carried out starting from the monosaccharide 16 or (prop-1'-enyl 2,3-di-O-benzyl
α-glucopyranoside) methyl uronate (the synthesis of which is reported in patent application FR 8209392) by
1: chloroacetylation of the compound 16 in position d
2: unblocking of the anomeric carbon;
3: bromination of the anomeric carbon 1 -hloroacetylation of the compound 16 leading to the compound
se 17, namely (prop-1'-enyl 2,3-di-O-benzyl-4
0-chloroacetyl-α-D-glucopyranoside) uronate
Thyle
2.8 g of compound 16 are dissolved in 30 ml of pyridine (6.56 mmol). After cooling to OOC, 10 ml of a solution of 2 ml of chloroacetyl chloride in 20 ml of dichloromethane is added dropwise.

After 30 minutes, the mixture is evaporated to dryness, the residue is taken up in 200 ml of chloroform, washed with a 10% solution of KHSO 4, then with water, dried and concentrated. The syrup obtained is chromatographed on silica gel (200 g, eluent, AcOEt / hexane, 1/3, v / v). 2.7 g of pure compound 17 are thus obtained in syrup form (80% yield); [α] @ 20 = + 2 (c = 1.5, chioroform).

Elemental analysis and the NMR spectrum confirm the expected structure.

2) Anomeric carbon unblocking leading to the compound
18 or (2,3-di-O-benzyl-4-O-chloroacetyl-D-glucopyra
nose) methyl uronate
2.71 g (5.3 mmol) of the derivative 17 are dissolved in 80 ml of an acetone / water mixture (5/1, v / vi), mercuric oxide (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 residue is taken up in chloroform The chloroform phase is washed with 10% KI solution and then with water.After evaporation, the product is crystallized from an ethyl acetate / hexane mixture to give 2 g of a solid of mp 105-1070 ° C; 4.70 (eq, 1, chloroform) Elemental analysis and NMR study confirm structure (80% yield).

3) Bromination of the anomeric carbon leading to the compound
14 or (1-bromo-2,3-di-O-benzyl-4-O-chloroacetyl-α-D-
methyl glucopyranosyl) uronate
2 g (4.30 mmol) of the compound 18 are dissolved in 50 ml of dichloromethane. 4.8 ml (34.4 mmoles) of sym-collidine is added at 0 ° C. followed by bimoumethylene dimethyl ammonium bromide (17 mmol) prepared according to
HEPBURN DR and HUDSON HRJ Chem Soc. Perkin I (1976) 754-757. After 4 hours of reaction, the mixture is diluted with 100 ml of dichloromethane and then 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 14 is obtained in the form of a syrup (90% yield). = + 82.50 (c = 1.5, chloroform) .The elemental analysis and the NMR study confirm the structure.

B) Preparation of disaccharide 1 or (3, 6-acetyl-1,6-anhydro-2-azido-4-O - / (2,3-di-O-benzyl-4-chloroacetate)
methyl-ss-D-glucopyranosyl) methyl uronate / ss-D-gluco
pyranose.

 This synthesis is based on the condensation of monosaccharides 14 and 15.

 To a solution of 870 mg (3.8 mmol) of dichloromethane was added 1 g of drierite (0.5 g of 4 A molecular sieves, powder and 0.525 g of freshly prepared silver carbonate. After stirring for 2 hours, 670 mg (1.3 mmol) of compound 14 are added dropwise to OOC, the solids are removed by filtration and the syrup obtained after concentration is chromatographed on silica gel. silica (50 g, eluent: chloroform / ethyl acetate, 4/1, v / v) to give the disaccharide 1 as a foam (421 mg, 50%). [& alpha] D20 = -17 (c = 1, chloroform) Elemental analysis confirms the structure The NMR study confirms the ss configuration of the interglycosidic bond.

C) Preparation of Disaccharides of Structure 2 by Ace
tolysis of disaccharide 1
The disaccharide 2 is prepared by subjecting the disaccharide 1 to an acetolysis reaction as follows.

 300 mg of compound 1 are dissolved in a mixture of 4 ml of acetic anhydride and 0.5 ml of freshly distilled trifluoroacetic acid. The reaction mixture is stirred at 180 ° C. for 18 h, then evaporated to dryness and coevaporated with toluene. The residue is chromatographed on a column of silica gel (15 g). Elution with dichloromethane, ethyl acetate (19/1, v / v). 282 mg of a mixture of anomeric acetates of structure 2 are recovered in the form of a colorless syrup (yield 86%). The ratio of forms α in the ss forms, determined by MNR analysis, is 4/1.

NMR Spectrum (90 MHz, CDCl3) υ: 7.35 (m, 10H, 2 Ph); 6.28 (d, H 9 JI, 2 = 3.5 Hz);
5.56 (d, H19, J1.2 = 8.5 Hz);
5.50 (dd, H3ss, J2.3 = gHz, J3.4 = 8.5 Hz);
5.15 (dd, H 2, J 2, 9.3 Hz, J 3.4 = 8.5 Hz);
4.43 (d, 1H, 1H, J1.2, 8.8 Hz);
3.80 (AB system, 2H, Cl-CH 2 -CO); 3.75 (s, 3H, COOMe);
3.60 (dd, H2ss, J1.2: 8Hz, J2.39Hz);
3.57 (dd, H2 α J1.2 = 35Hz, J2.3 = 9Hz);
2.25; 2.10; 2.09 (3s, 9H, 30Ac)
D) Preparation of the disaccharide 3 by bromination of the di
saccharides 2
The mixture of acetates of structure 2 was subjected to the action of TiBr 4: A solution of 140 mg of the mixture of acetates 2 in a mixture of 3 ml of dichloromethane and 0.3 ml of acetate was stirred with stirring. of ethyl at 17-18 ° C., under a dry argon atmosphere, in the presence of 140 mg of TiBr4 (# 2 equivalents) for 20 hours.

After cooling to 00.degree. C. and diluting with 30 ml of dichloromethane, the mixture is washed with ice-water and then with a 5% aqueous solution of potassium bromide and then with water and dried over sodium sulfate. filtered and evaporated. The residue is chromatographed on a column of silica gel (10 g). Elution with dichloromethane-ethyl acetate (19: 1, v / v) is carried out in order of elution.
bromide 3 (74 mg, yield 50%) in the form of a colorless, unstable syrup (immediately engaged in the next reaction); NMR spectrum (90 MHz, CDCl 3): 7.35 (m, 10H, 2Ph); 6.38 (d, 1H, H1, J1.2 = 3.5 Hz);
5.55 (dd, 1H, H3, J2 3 = 10 Hz, J3 4 = 8.5 Hz);
5.15 (t, 1H, H4 ', J3', 4 '= J4', 5 '= 9 Hz);
4.80 (AB + ls system, 4H, 2 OCH2 Ph);
4.42 (d, 1H, H1 ', J1,', 2 '= 7 Hz); 3.79 (AB system,
2H, Cl-CH2-CO);
3.75 (s, 3H, COOMe); 2.24; 2.10 (2s, 6E, 20Ac)
a fraction (28 mg, yield 20%) corresponding to the unreacted starting material,
a fraction migrating little corresponding to products of 0-partial debenzylation.

EXAMPLE 2
synthesis of the tetrasaccharide 6
The tetrasaccharide 6 is prepared by carrying out the condensation of the disaccharides 3 and 4 in step a) by subjecting the tetrasaccharide 5 formed in step b) to a selective reaction of -O-demonochloroacetylation at the 4-position. Derivative 4 is prepared according to application FR 82 01575. a) Condensation reaction
A mixture of 64 mg (80 M) of freshly prepared bromide 3, 51 mg (60 μM) of compound 4 and 80 mg of powdered 4 A molecular sieve in 1.5 ml of anhydrous dichloroethane is stirred for a period of one hour. half an hour at room temperature, under a dry argon atmosphere, and then cooled to -200 ° C. 20 ml (150 μM of de-sym-collidine and 31 mg (120 μM) of silver triflate are successively added. The reaction mixture is stirred for 1 hour at -200 ° C. and then the temperature is allowed to rise to room temperature for 15 hours.When the mixture is diluted with 50 ml of dichloromethane, the solids are filtered off and the filtrate is washed with an ice-cold aqueous solution of acid. 1 M hydrochloric acid and then with water (twice), then dried over sodium sulfate, filtered and evaporated.

The residue is chromatographed on a column of silica gel (8 g, 230-400 mesh gel). Elution with hexane-ethyl acetate (4: 3, v / v) makes it possible to recover 37 mg of tetrasaccharide (yield 39%) in the form of a colorless glass. [α] D20 = + 56 (c 0.6, CHCl3); NMR spectrum (90 MHz, CDCl 3) δ: 7.30 (m, 30H, 6Ph); 5.41 (d.degree., 1H, H3, J2 "3" =
10 Hz, J3, 4, = 9 Hz);
5.05 (dd, 1H, H ", 4, J3", 4 "= 9.5 Hz, J4", 5 "
8.5 Hz);
5.00 (d, H1 ", J1", 2 "low, overlap with others
protons);
3.72 (AB + Is system, 5H, Cl-CH2CO and COOMe);
3.63 (s, 3P, COOMe), 3.20 (dd, 1H, H2 ", J1", 2 ").
3.5 Hz, J2 ", 3": 10 Hz)
2.20; 2.11, 2.00 (3s, 12H, 40Ac).

 Elution of the column with ethyl acetate-hexane (2: 1, v / v) yields 23 mg of starting material 4 (44% yield).

b) Reaction of -O-dechloroacetylation
A solution of 36 mg (23 M) of the tetrasaccharide S in 1.25 ml of a mixture of pyridine and 0.25 ml of absolute ethanol is heated at 1000C in the presence of 7 mg (100 M) of thiourea for 20 minutes. . After cooling and evaporation to dryness, the solid residue is taken up with 20 ml of water and extracted with chloroform (5 times 5 ml). The organic phases are washed with a 10% aqueous solution of sodium hydrogen sulphate, with water, dried over sodium sulphate, filtered and evaporated. The residue is chromatographed on a column of silica gel (3 g). Elution with ethyl acetate-hexane (3: 2, v / v) gives 27 mg of derivative 6 (80% yield) as a colorless glass; [α] D20 = + 61 (c = 0.8, chloroform); NMR spectrum (90 MHz, CDCl 3 then D 2 O): 7.30 (m, 30H, 6 Ph); 5.45 (d.degree., 1H, H3 "J2", 3 "=
10 Hz, J3 "4" = 9 Hz);
3.83 (s, 3H, COOMe); 3.67 (s, 3H, COOMe);
3.25 (dd) 1H, H2 "J1", 2 "3.5 Hz, J2", 3 ": 10 Hz);
2.75 (1H, OH, exchanged with D20, OH at 4, uronic acid unit.

2.15; 2.04 (25, 12H, 4 OAc) EXAMPLE 3
Synthesis of pentasaccharide 8 or
1) A condensation reaction is carried out between the tetrasaccharide 6 and the monosaccharide z, which leads to the pentasaccharide 8.

 A mixture of 27 mg (54 M) bromide 7 prepared according to H. PAULSEN and W. STENZEL, Chem. Ber., 111 (1978) 2334-2347, 26 mg (18 M) of the tetrasaccharide 6 and 50 mg of powdered molecular sieve 4 in 0.8 ml of dichloroethane is stirred for 1/2 hour at room temperature. under dry argon atmosphere, then cooled to -200C. 16 ml (120 μM) of svm-collidine and 26 mg (100 M) of silver triflate are added successively and the reaction mixture is stirred for 18 h allowing the temperature to rise slowly to room temperature.

 After treatment identical to that described for the preparation of 5, the residue is chromatographed on a column of silica gel (5 g, 230-400 mesh gel).

By solution with a hexane-ethyl acetate mixture (4: 3, v / v), 30 mg of pentasaccharide 3 are recovered in the form of a colorless glass (yield 90%). [α] D20 = + 670 (c 1, chloroform).

NMR spectrum (90 MHz, CDCl3).

#: 7.30 (m, 40H, 8 Ph); 5.53 (d, 1H, H1 "", J1 "", 2 "" =
3.5 Hz);
5.40 (d = 1H, H +, J211.3: 10Hz, J3 +, 4 "= 9Hz);
3.76 (s, 3H, COOMe); 3.61 (5, 34, cooMe);
3.30-3.10 (2 dd of recovery, 2H, H2 "and H'21,
J = 3.5 and 10 Hz);
2.10; 2.03; 2.00 (3s, 15H, 50Ac
EXAMPLE 4
Preparation of pentasaccharide 13
The following steps are used
a) elimination of acetyl groups (pentasaccharide 9)
b) sulfation of the -OH groups thus released (pentasaccharide 10)
c) Hydrogenation to liberate -OH groups protected by benzyl groups and for trans
form the group -N3 in group -NH2 (pentasaccharide 11)
d) Sulfation of NH 2 groups (pentasaccha ryde (12) harmful saponification groups
-COOMe in position 6 (pentasaccharide 13).

These steps are performed as follows
a) - removal of acetyl groups from
derivative 8
A solution of 28 mg pentasaccharide 8
in a mixture of 2.5 ml of 1,2-dimethoxyethane and 0.8 ml of methanol is cooled to 0 ° C. with stirring.

1 ml of a 1M solution of sodium hydroxide, dropwise, is then added.
drop by drop, in 10 minutes. The reaction mixture is stirred for 1 h at 0 ° C., then 12 h at room temperature.

After cooling to OOC, 3 ml of 1 M hydrochloric acid are added and the milk mixture is immediately extracted with chloroform (5 x 5 ml). The organic phases are washed with water, dried over sodium sulfate, filtered and evaporated. The residue is taken up in 2 ml of methanol and treated with an ethereal solution of diazomethane (excess until persistence of the yellow color) for half an hour.

After evaporation to dryness, the residue is chromatographed on a column of silica gel (2 g, 230-400 mesh gel). Elution with dichloromethanemethanol (15: 1, v / v) makes it possible to recover 18 mg of pentasaccharide 9 (yield 72%) in the form of an in-color glass. [α] D20 = + 570 (c = 1, chloroform);
NMR Spectrum (90 MHz, CDCl3 then D20): 6: 7.30 (m, 40H, 8 Ph); 5.50 (d, 1H, J1.2 = 3.5 Hz, H1
a 2-azido unit); 3.78 (s, 3H, cooMe);
3.28 (s, 3H-, COOMe); absence of OAc signals in the
zone 1.50 <5 c 2.50
b) sulfation of -OH groups
To a solution of compound 9 (22 mg) in dimethylformamide (0.5 ml) is added trimethylamine / SO 3 complex (22 mg, 2.5 eq / OH). The reaction mixture is heated at 500 ° C. for about 14 hours. The trimethylamine / SO 3 complex (10 mg) is then added again and the reaction is allowed to proceed for 24 hours. Methanol (0.5 ml) and chloroform are added to the reaction mixture. (0.5 ml). The solution is introduced at the top of an equilibrated LH20 Sephadex column in CHCl3 / CH3OH (1/1, v / v). Fractions containing the sulfated product are pooled and the solvent is evaporated. This gives a glass (30 mg).

This glass is then chromatographed on silica gel (10 g) in a solvent consisting of 3 parts of the mixture
ethyl acetate / pyridine acetic acid / water,
(6/2/0, 6/1, v / v / v / v) and 2 parts of the acetate mixture
ethyl / pyridine / acetic acid / water, (5 / b / 1/3, v / v / v / v) parts of the mixture ethyl acetate / pyridine / acetic acid / water, 5/5/1/3 , v / v / v / v.

Fractions containing the desired product are pooled and concentrated. After evaporation of the solvents, the residue obtained is dissolved in methanol added to water and then passed through a column of Dowex 50 W x 4, Na +, equilibrated in a mixture of methanol / water (50/50, v / v). .

The sodium salt is thus obtained; c) hydrogenation
The product obtained above is dissolved in methanol (3.7 ml) added with water (0.3 ml).

To this solution, the catalyst (Pd / c, 5%, 40 mg) is added and stirred under a hydrogen atmosphere for 5 days.

After removal of the catalyst by filtration, the analysis of the U.V spectrum of the solution obtained shows the complete disappearance of the absorption due to the benzyl groups. The solvent is then evaporated, leaving a residue, namely compound 11.

 d) sulfation of the -NH2 groups, followed by saponification of the carboxyl groups.

The compound is dissolved in water (4ml). The pH is then adjusted to 9.5 and then the trimethylamine / SO 3 complex (54 mg) is added to the solution. The pH is maintained at 9.5 throughout the duration of the reaction by addition of 0.1N sodium hydroxide.

After one night, a further addition of sulphating agent (27 mg) is carried out. A final addition is made after 24 hours.

After 48 hours, sodium hydroxide (3M, 34 ml) is added to the compound 12 formed, and the solution is then stirred for 3 hours at room temperature so as to hydrolyse the methyl esters of the uronic acid type units.

The reaction mixture is then neutralized and then concentrated to a volume of about 2 ml.

The solution thus obtained is deposited by a column of Sephadex G (100 ml) eluted with water.

The collected fractions were analyzed by UV absorption (206 nm) and polarometry (265 nm). The fractions having optical activity are pooled, the solvent is removed and the residue taken up in about 2 ml of water and lyophilized.

The derivative 13 is thus obtained in the form of a white powder (5.6 mg, 25% relative to the product 9).

 The RNN study confirms the expected structure.

Claims (1)

 - CLAIM  Pentasaccharides characterized in that they correspond to the following formula (i)

 in which - the groups R, identical or different from each other, represent a hydrogen atom or a physiologically acceptable radi cal, of the type -R 1 R 2, in which P * 1 advantageously represents a mineral salt, in particular a group SO 3 and R2 represents a metal cation, in particular an alkali metal such as sodium or alkaline earth metal, such as calcium or magnesium, or an organic cation such as a nitrogenous organic base derivative, and  the substituents A, which are identical or different from  each other, represents a hydrogen atom, or  still a metal cation or an organic cation  as above.