HK1094806B - Heparin-derived oligosaccharide mixtures, preparation thereof and pharmaceutical compositions containing said mixtures - Google Patents

Description

Oligosaccharide mixture derived from heparin, method for preparing same and pharmaceutical composition containing same

The invention relates to oligosaccharide mixtures derived from heparin and having an average molecular weight of 1800-2400 Dalton, which are characterized by a strong anti-Xa (aXa) activity and by no anti-Iia (aIIa) activity, to a process for their preparation and to pharmaceutical compositions containing them.

The heparin is a sulfated glycosaminoglycan mixture of animal origin, primarily for anticoagulation and antithrombotic use.

However, the drawbacks of heparin have limited its use. Bleeding may be caused especially due to its high anticoagulant activity (aIIa). (workshop on Thrombosis and hemostasis) (Seminirs in Thrombosis and Hemostasis), Vol.5, supplement, 3 (1999)).

Low molecular weight heparins, particularly those obtained by alkaline depolymerization of heparin esters and sold in practice, such as enoxaparin (enoxaparine), also have high aIIa activity.

Recently, some very low molecular weight heparins have been described in the prior art. For example, in the patent US 6384021, the anti-Xa activity of these products is 100-120UI/mg and the anti-IIa activity is 2-8 UI/mg. In the international applications WO 02/08295 and WO 2004/033503, the anti-Xa activity of these products is in particular 100-190UI/mg, whereas the anti-IIa activity is less than 5 UI/mg. However, any of these very low molecular weight heparins does not effectively have an anti-Xa activity higher than 190UI/mg, while the anti-IIa activity is zero or substantially zero (UI ═ international units).

An anti-IIa activity of substantially zero (in other words, substantially no anti-IIa activity) is to be understood as an activity of less than 0.2 UI/mg.

The object of the present invention is to provide oligosaccharide mixtures which have a very high selectivity for activated factor X (factor Xa) and at the same time have no or essentially no anti-IIa activity.

It is therefore an object of the present invention to provide oligosaccharide mixtures having the general structure of the polysaccharides constituting heparin and having the following characteristics:

their average molecular weight is 1800-2400 daltons, the anti-Xa activity is 190UI/mg-450UI/mg, and no or essentially no anti-IIa activity.

-constituent oligosaccharides of the mixture

Containing 2 to 16 sugar building blocks,

-having a 4, 5-unsaturated uronic acid-2-O-sulphate building block at one of the termini,

-and contains a hexasaccharide of the formula:

the hexasaccharide Δ IIa-II contained in the oligosaccharide mixtures described in the inventionsIs a sequence with a strong affinity for ATIII, characterized by an aXa activity higher than 740 UI/mg.

As the alkali metal or alkaline earth metal salt, sodium, potassium, calcium and magnesium salts are preferable.

The average molecular weight is determined by high pressure liquid chromatography using two columns in series, such as those sold under the trade names TSK G3000 XL and TSK G2000 XL. The detection is performed using a refractometer. The eluent used was lithium nitrate at a flow rate of 0.6 ml/min. The system was calibrated using standards prepared by separation of enoxaparin (IBF) by agarose-polyacrylamide gel chromatography. This preparation is accomplished according to the techniques described in Barrowcliffe et al, Thromb. Res., 12, 27-36(1977-78) or D.A. Lane et al, Thromb. Res., 12, 257-271 (1977-78). Results were calculated using GPC6 software (Perkin Elmer).

anti-Xa activity was determined by amidohydrolysis of the chromogenic substrate according to the principle described by Teien et al, Thromb. Res., 10, 399-410 (1977). Quantification was performed according to the method described in the monograph on low molecular weight heparins of the current european pharmacopoeia, but with a different replication buffer, i.e. polyethylene glycol 6000(PEG 6000) instead of albumin in the buffer Tris-nacliph 7.4.

The anti-Xa activity was determined relative to a standard very low molecular weight Heparin (HTBPM) of 140-180U/mg (dry basis). The activity of the standard HTBPM was determined relative to an international standard sample of low molecular weight heparin.

This standard HTBPM was prepared according to the method described in patent application WO 02/08295, in particular WO 2004/033503. The activity of the standard HTBPM was determined relative to an international standard sample of low molecular weight heparin.

The anti-IIa activity was determined by amidohydrolysis of chromogenic substrates according to the method described in the monograph on low molecular weight heparins of the current European pharmacopoeia. The anti-IIa activity was determined relative to a standard very low molecular weight Heparin (HTBPM), the measured activity of which was 2.1 UI/mg. The activity of the standard HTBPM was determined relative to an international standard sample of low molecular weight heparin.

According to a preferred mode, the oligosaccharide mixture according to the invention contains 20-100% hexasaccharide moieties. In particular, the mixture contains 30-60% of hexasaccharide fraction.

In addition, the mixture of the invention contains 20-70% of the hexasaccharide Δ IIa-II in the hexasaccharide fraction of the oligosaccharide mixturesIs. Particularly preferably, part of the moieties Δ IIa-II in the hexasaccharide fractionsIs as high as 25-50%.

The percentage of the hexasaccharide fraction is determined by high pressure liquid chromatography on columns of TSK G3000 XL and TSK G2000 XL or by preparative separation of the hexasaccharide fraction.

In this case, the mixture is chromatographically separated using a column packed with polyacrylamide agarose gel, the mixture is eluted with sodium bicarbonate solution, preferably the concentration of the sodium bicarbonate solution is 0.1 to 1mol/l, still more preferably the concentration of the separation is 1mol/l, determined by ultraviolet spectroscopyDetection by method (254nm) after separation, sodium bicarbonate solution of the hexasaccharide fraction is neutralized with glacial acetic acid, the solution is then concentrated under reduced pressure to a sodium acetate concentration of greater than 30% by weight, the hexasaccharide fraction is precipitated with 3-5 volumes of methanol, the hexasaccharide fraction is recovered by filtration using a No. 3 sintered glass filter, and the resulting hexasaccharide mixture can be analyzed by High Performance Liquid Chromatography (HPLC) to determine the hexasaccharide Δ IIa-IIsThe content of Is. The hexasaccharide Δ IIa-II can be isolated by preparative HPLC chromatography or affinity chromatography on an antithrombin III agarose column according to techniques employed by those skilled in the artsIs (M.hook, I.BJork, J.Hopwood and U.Lindahl, F.E B.Sletters, 656 (1976)).

The average molecular weight of the mixtures according to the invention is preferably 1900-.

According to a preferred mode, the oligosaccharide mixture of the invention is characterized in that its anti-Xa activity is 190-410UI/mg and the anti-IIa activity is zero or essentially zero. Very particular preference is given to an anti-Xa activity of 200-300 UI/mg.

The object of the invention is therefore particularly preferably a mixture having the following characteristics:

average molecular weight 1950 and 2150 daltons,

the anti-Xa activity is 190-410UI/mg, the anti-IIa activity is zero or essentially zero,

they contain 30-60% hexasaccharide fraction containing 25-55% of Δ IIa-IIsThe Is portion.

The activity of the oligosaccharide mixture of the invention is obtained by a very unique process disclosed below. It is well known to those skilled in the art that the physicochemical characteristics of the polysaccharide mixture and the activity thereof are related to the method of obtaining (J.Med Chem 33(6)1639-2093 (1990)).

The oligosaccharide mixtures of the invention can be prepared by depolymerisation of the benzyl ester quaternary ammonium salt of very low molecular weight Heparin (HTBPM), which is itself prepared according to the methods described in patent applications WO 02/08295 and WO 2004/033503, in an organic medium. Generally speaking, it concerns the depolymerization of very low molecular weight heparins, and it is obtained in particular by depolymerisation of esterified heparins in the presence of a strong base, preferably in dichloromethane, and in the presence of less than 3% of water.

In particular, the HTPBM used as starting material in the present invention can be prepared according to the methods described in patent applications WO 02/08295 and WO 2004/033503.

In particular, the HTBPM used as starting product has an aXa activity higher than 140UI/mg, an aIIa activity lower than 5UI/mg and an average molecular weight of 2000-3000 Dalton. The aXa activity was determined relative to the standard HTBPM, which measured the activity to be 158 UI/mg. Standard HTBPM activity was determined relative to a standard international sample of low molecular weight heparin.

The starting HTPBMs obtained according to the process as described above are depolymerized using a strong organic base with a pka preferably higher than 20 (e.g. r.schwesinger et al, angle.chem.int.ed. engl. 26, 1167-1169(1987) or r.schwesinger et al, angle.chem. 105, 1420(1993) define the properties of the preferred phosphazene family). The depolymerized HTBPM benzyl ester quaternary ammonium salt is then converted to the sodium salt, the residual esters are saponified and the resulting product is optionally purified. The following reaction scheme illustrates the invention:

n ═ X + Y + Z (average disaccharide Total sulfation Rate)

x is the site sulfation rate and the complementary part is represented by H

Y is site sulfation rate, the complement Z is site sulfation rate expressed by H group, and COCH is used₃Base representation supplement

Therefore, another object of the present invention is a process for the preparation of an oligosaccharide mixture as defined above, characterized in that a very low molecular weight heparin having an aXa activity higher than 140UI/mg, an aIIa activity lower than 5UI/mg and an average molecular weight of 2000-3000 Dalton is subjected to the following chemical reactions:

a) salt exchange is carried out by the action of benzethonium chloride to obtain heparin benzethonium (benzethonium),

b) esterifying the obtained heparin benzyl ethylammonium by the action of benzyl chloride, and treating with sodium acetate alcoholic solution to obtain heparin benzyl ester sodium salt with very low molecular weight,

c) the benzyl ester obtained undergoes a salt exchange to give a quaternary ammonium salt, preferably a benzethonium salt (benzethonium), cetylpyridinium or cetyltrimethylammonium salt,

d) by depolymerization with a strong organic base having a pKa preferably higher than 20, to obtain a depolymerized heparin of very low molecular weight,

e) the very low molecular weight depolymerized heparin quaternary ammonium salt is converted into sodium salt,

f) saponification and optional purification of the residual ester is carried out.

In the present invention, it is very particularly preferred that in the depolymerization step (step d), the sequence having affinity for ATIII in the oligosaccharide mixture can be unexpectedly enriched by utilizing the high selectivity of the phosphazene base, preferably the strong base/ester molar ratio is 0.2 to 5, very particularly 0.6 to 2.

For the best selectivity and maximum preservation of sequences having affinity for ATIII, treatment with 1 molar equivalent of phosphazene base, based on the HTBTM benzyl ester, benzethonium salt, is best carried out at a water content of less than 0.3%.

The phosphazene base is preferably a base of the formula:

in the formula, R₁-R₇The radicals, which may be identical or different, represent straight-chain, branched or cyclic alkyl radicals having from 1 to 6 carbon atoms, where appropriate R₃And R₄Can form a 6-membered heterocyclic ring with the group-N-P-N-carried by them. Particularly preferably, the subject of the present invention is a process as defined above, characterized in that the base used in the depolymerization step d) is 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 3, 2-diazaphospho-benzene (phosphene); formal naming: 2- [ (1, 1-Dimethylethyl) imino group]-N, N-diethyl-1, 2, 2, 2, 3, 5, 6-octahydro-1, 3-dimethyl-1, 3, 2-diazaphospho-2-amine).

The salt exchange step a) reaction is preferably carried out by reacting excess benzethonium chloride with HTBPM sodium at a temperature of about 15-25 ℃. Preferably the salt/heparin sodium molar ratio is 2.5-3.5.

The esterification of step b) is preferably carried out in a chlorinated organic solvent, such as chloroform or dichloromethane, at a temperature of 25 to 45 c, preferably 30 to 40 c. The ester is then recovered as the sodium salt by precipitation with a 10% by weight solution of sodium acetate in an alcohol such as methanol. Generally, 1 to 1.2 volumes of alcohol are used per volume of reaction medium. The amount of benzyl chloride and the reaction time are adjusted to achieve an esterification rate of 40 to 100%, preferably 70 to 90%. Preferably, 0.5 to 1.5 parts by weight of benzyl chloride are used per 1 part by weight of the heparinium benzethonium salt. It is also preferred that the reaction time is from 10 to 35 hours.

Therefore, the esterification rate of the quaternary ammonium salt of heparin benzyl ester achieved by the method of the invention is 40-100%, and preferably 70-90%.

In general, the depolymerized heparin benzyl ester quaternary ammonium salt is converted to the sodium salt by treating the reaction medium with an alcoholic solution of sodium acetate, preferably 10% (w/v) methanol solution of sodium acetate, at a temperature of 15-25 ℃.

The weight equivalent of the acetate salt added is preferably 3 times the amount of heparin benzyl ester quaternary ammonium salt subsequently used in the depolymerization reaction. The HTBPM benzyl ester quaternary ammonium salt obtained is preferably a benzethonium salt, a cetylpyridinium salt or a cetyltrimethylammonium salt.

The salt exchange reaction of step c) is carried out in an aqueous medium at a temperature of 10-25 ℃ using a quaternary ammonium chloride, preferably using benzethonium chloride, cetylpyridinium or cetyltrimethylammonium salt. The molar ratio of quaternary ammonium chloride/sodium heparin benzyl ester is preferably 2.5-3.5.

Generally, the saponification is carried out in an aqueous medium at a temperature of 0 to 20 deg.C, preferably 0 to 10 deg.C, using an alkali metal hydroxide such as sodium hydroxide, potassium hydroxide, lithium hydroxide. Generally, 1 to 5 molar equivalents of alkali metal hydroxide are used. The saponification reaction is preferably carried out in the presence of 1 to 2 molar equivalents of an alkali metal hydroxide.

The final product is optionally purified using any known method for purifying depolymerised heparin (e.g. according to the method described in EP 0037319B 1). The purification is preferably carried out in an aqueous medium at a temperature of from 10 to 50 ℃ using hydrogen peroxide. Preferably, this operation is carried out at from 20 to 40 ℃.

The inventive mixtures in the form of their sodium salts can be converted into other alkali metal or alkaline earth metal salt forms. Optionally, one salt may be converted to another salt using the method described in patent FR 7313580.

The invention makes hexasaccharide delta IIa-IIsIs greatly enriched. It is well known to those skilled in the art that when low molecular weight heparins are produced and the heparins are depolymerized, the anti-Xa activity of the resulting product decreases dramatically, even to zero. In the case of using a method capable of obtaining enoxaparin, heparine (fraxiparine), tedipine (fraagmine), tinzaparin sodium (innohep) (or lopararin), aclidinium sodium (norifflo), embolex (or Sandoparin), Fluxum (or Minidalton), reviparin (clivarine) and Hibor, if these HBPMs are depolymerized again using their original method, these HBPMs are depolymerizedThis phenomenon can be observed. This is due to the fact that these methods preserve low selectivity for the bit point ATIII.

In the present invention, if HTBPM from the phosphazene depolymerization process is used as a raw material, a reverse phenomenon thereof inevitably occurs. The activity of the oligosaccharide mixture, aXa, increased and even exceeded the activity of heparin used to prepare HTBPM. This may be due to the significant selectivity of the phosphazene base to preserve sequences having affinity for ATIII.

This characteristic of the process can also be observed by obtaining the average molecular weight of the oligosaccharide mixture. For example, when depolymerizing an HTBPM with an average molecular weight of 2400 daltons, a 2000Da average molecular weight oligosaccharide mixture is obtained. It was observed that the sequences having affinity for ATIII (hexa-and octa-saccharides) were protected from the action of phosphazene base, which resulted in other sequences being destroyed and removed, and thus, a product of average molecular weight tending toward that of the non-depolymerized constituent was obtained; i.e. hexasaccharide Δ Ha-IIsIs (1834 g/mol). It should be noted that in the step of alkaline hydrolysis of heparan with phosphazene, the average molecular weight is from about 15000Da to about 2400 Da.

In addition, the method of the present invention can increase the activity and selectivity of factor Xa, and the method can also be applied to low molecular weight heparins in general. For example, enoxaparin, freicharin, tedipin, tinzaparin sodium (or lolaparin), aclidinium sodium, Embollex (or Sandoparin), Fluxum (or Minidalton), reviparin and Hibor may be cited as examples. It is also possible to refer to certain very low molecular weight heparins having an anti-Xa activity, such as those described in US 6384021 (2000-4000Da) or WO 02/08295(1500-3000Da), which have an anti-Xa activity of less than 140UI/mg (in particular 100-140 UI/mg).

This characteristic of the process is represented by the unexpected anti-Xa activity obtained with mixtures of oligosaccharides with average molecular weight (190UI/mg < aXa < 450 UI/mg; 1800Da < PM < 2400 Da).

According to one embodiment of the invention, removal of the disaccharide and tetrasaccharide moieties, which do not specifically bind to ATIII, also increases the selectivity of the oligosaccharide mixture for factor Xa. In this case, the mixture is chromatographically separated using a column packed with polyacrylamide agarose gel or polyacrylamide gel. This mixture was eluted with sodium bicarbonate solution. Preferably, the sodium bicarbonate solution is a 0.1-1mol/l solution. It is still more preferred to carry out this separation at a concentration of 1mol/l. This detection was carried out by UV spectrometry (254nm). After removal of the disaccharide and tetrasaccharide moieties, the mixture of oligosaccharide in sodium bicarbonate solution was neutralized with glacial acetic acid. This solution is concentrated under reduced pressure to give a sodium acetate solution with a concentration of more than 20% by weight. This oligosaccharide mixture was precipitated by adding 3-5 volumes of methanol. The high affinity oligosaccharide mixture is then recovered by filtration. If necessary, purification can be carried out using appropriate column desalting. Example 6 illustrates this alternative procedure, resulting in very low molecular weight heparins with anti-Xa activity higher than 400 UI/mg.

Therefore, another object of the present invention is a process for the preparation of an oligosaccharide mixture as defined above, having an increased selectivity for factor Xa, characterized in that the disaccharide and tetrasaccharide moieties are also removed by chromatography, in particular by gel column chromatography using loaded polyacrylamide agarose gels.

The mixture of the invention can be used as a pharmaceutical.

The oligosaccharide mixtures of the invention are useful as anticoagulants, in particular they are useful in the treatment or prevention of venous and arterial thrombosis, deep vein thrombosis, pulmonary embolism, unstable angina, myocardial infarction, myocardial ischemia, peripheral arterial occlusive disease and atrial fibrillation, in the prevention and treatment of smooth muscle cell proliferation, atherosclerosis and arteriosclerosis, in the treatment and prevention of cancer by modulating angiogenesis and growth factors, and in the treatment and prevention of diabetes, such as diabetic retinopathy and diabetic nephropathy.

The invention also relates to pharmaceutical compositions containing as active principle a mixture of formula (I), optionally in combination with one or more inert excipients.

These pharmaceutical compositions are, for example, subcutaneous or intravenous solutions. Other pharmaceutical compositions of the invention may also be administered by pulmonary (inhalation) or oral routes of administration.

The dosage may vary with the age, weight and physical condition of the patient. For adults, the dosage is generally 20-100mg daily, administered by intramuscular or subcutaneous routes.

The following examples illustrate the invention without limiting it.

Preparation 1: very low molecular weight heparins are obtained, the initial aXa activity of which is equal to 158.8IU/mg

Very low molecular weight Heparin (HTBPM) used as starting product in example 1 can be prepared according to patent application WO 2004/033503 using sodium heparin, with the steps a-f as described previously, the depolymerization step being carried out in the presence of 2-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 3, 2-diazaphosphoramine, in the presence of a percentage of water lower than 0.6%.

Characteristics of the very Low molecular weight heparins obtained

The characteristics of the depolymerized heparin thus obtained are as follows:

average molecular weight: 2400 Dalton

anti-Xa activity: 158.8UI/mg

anti-IIa Activity: 3.1UI/mg

anti-Xa activity/anti-IIa activity ratio: 51

Preparation 2: very low molecular weight heparins are obtained, the initial aXa activity of which is equal to 158IU/mg

Very low molecular weight Heparins (HTBPM) used as starting products in examples 2, 3, 4, 5 can be prepared according to patent application WO 2004/033503 using sodium heparin, with the steps a-f as described previously, the depolymerization step being carried out in the presence of 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 3, 2-diazaphosphoheterobenzene, in the presence of a percentage of water lower than 0.6%.

Characteristics of the very Low molecular weight heparins obtained

The characteristics of the depolymerized heparin thus obtained are as follows:

average molecular weight: 2450 Dalton

anti-Xa activity: 158UI/mg

anti-IIa Activity: 2.1UI/mg

anti-Xa activity/anti-IIa activity ratio: 75

Preparation 3: HTBPM, benzethonium salt

Salt exchange reaction of HTBPM to benzethonium salt (corresponding to step a) of the process):

12.53g (20.7mmol) of the sodium salt of HTBPM obtained according to preparation 1 are charged into a 500ml Erlenmeyer flask A and dissolved with 85ml of water (yellow solution).

31.62g (70.5mmol) of benzethonium chloride are introduced into a 100ml Erlenmeyer flask B (colorless solution) with 250ml of water.

The contents of B were poured into A and the mixture was stirred at room temperature for about 1h then allowed to settle for about 1h then the supernatant discarded, then the same volume of water (250ml) was added and stirred for about 15min and allowed to settle for about 30min then the supernatant discarded, then the same volume of water (250ml) was added and the mixture stirred for about 15min then filtered and the cake washed 3 times with water, 200ml each time the wet beige solid was dried in a reduced pressure (6kPa) oven at 80 ℃ for about 18h after dehydration to give 35.56g of HTM BPbenzylethylammonium salt.

The yield reaches 89%.

Preparation 4:

salt exchange reaction of HTBPM to benzethonium salt (corresponding to step a) of the process):

17.93g (30.2mmol) of the sodium salt of HTBPM obtained according to preparation 2 are charged into a 1l Erlenmeyer flask A and dissolved with 120ml of water (yellow solution).

45g (0.1mol) of benzethonium chloride are introduced into a 500ml Erlenmeyer flask B (colorless solution) with 360ml of water.

The contents of B were poured into A and the mixture was stirred at room temperature for about 1h. Followed by settling for about 1h. The supernatant was discarded and then the same volume of water (500ml) was added. Stirring is continued for about 15min and settling takes place for about 30min. The supernatant was discarded, and then the same volume of water (500ml) was added. The mixture was stirred for about 15min and then filtered. The filter cake was washed 3 times with 200ml of water each time. The wet beige solid was dewatered and dried in a reduced pressure (6kPa) oven at 80 ℃ for about 48 h. 49.5g of HTBPM benzethonium salt are obtained.

The yield reaches 87%.

Example 1:

very low molecular weight Heparins (HTBPM) are obtained by the process of the invention comprising a 77% esterification step and a depolymerization step using a base derived from phosphazene in an anhydrous medium.

Esterification of HTBPM (step b of the process):

35.39g (18.3mmol) of the benzethonium salt HTBPM obtained according to preparation 3 (water content 0.20%) are dissolved in 183.3g of anhydrous dichloromethane and placed in a 500ml three-necked flask. 29.5ml (25.7mmol) of benzyl chloride are added at a temperature of 30 ℃. The esterification rate after about 23 hours of reaction at 30 ℃ was 77%. After cooling to room temperature (22. + -. 3 ℃ C.), the reaction mixture is poured into 490ml of a 10% sodium acetate in methanol. The mixture was stirred at room temperature for about 1h. Followed by settling for about 1h. The supernatant was discarded, and then the same volume of methanol (250ml) was added. Stirring is continued for about 30min and settling is carried out for about 45 min. The supernatant was discarded, and then the same volume of methanol (250ml) was added. Followed by settling for about 16 h. The supernatant was discarded, and then the same volume of methanol (350ml) was added. The mixture was stirred for about 5min, and then the suspension was filtered. The filter cake was washed 2 times with 50ml of water each time, dewatered and then dried under reduced pressure (6kPa) at 40 ℃ for about 18h. 34.48g of crude HTBPM benzyl ester sodium salt was obtained, reaching an esterification rate of 77%.

Purification of HTBPM benzyl ester sodium salt (step b) of the process):

34.48g crude HTBPM benzyl ester sodium salt dissolved in 350ml 10% NaCl aqueous solution. This solution was poured into 1.57l of methanol. The suspension was stirred for about 40min, followed by settling for about 16 h. The supernatant was discarded, and then the same volume of methanol (1.5l) was added. Stirred for about another 1h and settled for about 1.5 h. The supernatant was discarded, and then the same volume of methanol (1.2l) was added. The mixture was stirred for about 15min and then filtered. The filter cake was washed 3 times with 50ml each time of methanol. The white wet solid was dehydrated and then dried under reduced pressure (6kPa) at 40 ℃ for about 18h. 6.07g of HTBPM sodium benzyl ester was obtained.

The esterification rate reaches 50 percent.

Salt exchange reaction of HTBPM benzyl ester sodium salt to benzethonium sodium (step c) of the process):

in a 250ml Erlenmeyer flask A6 g (9.14mmol) of HTBPM benzyl ester sodium salt are dissolved with 40ml of water.

At the same time, 13.93g (31mmol) of benzethonium chloride and 110ml of water were charged into a 250ml Erlenmeyer flask B.

The contents of B were poured into A. The suspension was stirred at room temperature (22. + -. 3 ℃) for about 1h, followed by settling for about 1h. The supernatant was discarded and then the same volume of water (140ml) was added. It was stirred for about 15min and settled for 1h. The supernatant was discarded and then the same volume of water (140ml) was added. The mixture was stirred for about 15min, followed by settling for about 30min. The supernatant was discarded and replaced by the same volume of water (140 ml). Stirred for about 5min, then filtered. The filter cake was washed 3 times with 50ml of water each time and dried under reduced pressure (6kPa) at 80 ℃ for about 18h after dehydration. 17.43g of HTBPM benzyl ester phenethyl ammonium salt was obtained.

The yield reaches 100 percent.

Depolymerization of HTBPM benzyl ester phenethyl ammonium salt in anhydrous medium: the water content is not detected to be less than 0.01 percent (institute) Step d) of the method)

17.43g (9.14mmol) of HTBPM from preparation 2 was charged into a 250ml three-necked flask with 122ml of anhydrous dichloromethane. 17.4g of additiveAnd (3) a molecular sieve. Stirring was carried out at room temperature (22. + -. 3 ℃ C.) for about 18h under an argon atmosphere.

This solution was transferred to a 250ml three-necked flask and the molecular sieve was separated from the mixture. 2.64ml (9.14mmol) of 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 3, 2-diazaphosphobenzene were added and the mixture was stirred at 22. + -. 3 ℃ for 24h under an argon atmosphere.

Conversion of Quaternary ammonium salt to sodium salt (step e of the process)

At the same time, 730ml of 10% sodium acetate in methanol was prepared in a 2l Erlenmeyer flask. To this solution 8.71g of Hyflo supercel diatomaceous earth was added. This reaction mixture was poured into the methanol solution while maintaining the temperature at about 4 ℃. The suspension is stirred at this temperature for about 15min. Followed by settling at room temperature for about 45min, after which the supernatant was discarded and then the same volume of methanol (450ml) was added. Stirring is continued for about 15min and settling is carried out for 45 min. The supernatant was discarded again and the same volume of methanol (420ml) was added. Stirred for about 15min, then filtered through a No. 3 sintered glass filter. The filter cake was washed 2 times with 70ml each time with methanol and dried under reduced pressure (6kPa) at 50 ℃ for about 18h after dehydration. This gave 4.35g of crude depolymerized HTBPM (sodium salt) in diatomaceous earth (8.71 g).

The yield reaches 72.5 percent.

Saponification of the crude depolymerized HTBPM (sodium salt) (step f1 of the process)):

4.35g (6.63mmol) of crude depolymerized HTBPM (sodium salt) in diatomaceous earth was dissolved in 46ml of water and then filtered through a No. 3 sintered glass filter. The diatomaceous earth was rinsed with 2 parts of water, 30ml each. The filter cake was charged to a 500ml Erlenmeyer flask. 823. mu.1 (9.94mmol) of 35% sodium hydroxide solution were added at a temperature of about 4 ℃. Stirring was carried out at this temperature for about 3 h. The medium was neutralized by adding 1N HCl solution, followed by addition of 11.5g NaCl and 80ml methanol. After stirring for about 15min, 210ml methanol was added. The suspension was stirred for about 1h and then subsequently settled for 30min. The supernatant was discarded and the same amount of methanol (230ml) was added. Stirring was carried out for about 15min, followed by settling for 30min. The supernatant was discarded and the same amount of methanol (210ml) was added. Stirred for about 15min, then filtered. The filter cake was washed 2 times with 9ml of methanol each time, dewatered and then dried under reduced pressure (6kPa) at 50 ℃ for about 18h. 2.95g of crude depolymerized HTBPM (sodium salt) were obtained.

The yield reaches 73.7 percent.

Purification of the crude depolymerized HTBPM (sodium salt) (step f2 of the process):

1.5g of crude depolymerized HTBPM sodium salt was charged to a 50ml three-necked flask with 16ml of water. The solution was warmed to 40 ℃ in about 10min. The pH was adjusted to about 9.7 by the addition of 0.1N sodium hydroxide. The solution was filtered through a 0.45 μm membrane and then 84 μ l of 30% aqueous hydrogen peroxide was added. The mixture was stirred at room temperature for 2h while maintaining the pH at 9.7. + -. 0.1 by addition of 0.1N sodium hydroxide. The reaction mixture was then neutralized with 0.1N HCl, 2g nacl added, after stirring for about 10min, the solution was filtered with a 0.45 μm membrane, 14ml methanol was poured at a temperature of about 4 ℃, the solution was stirred at room temperature for about 15min, then 36ml methanol was added, the suspension was stirred for about 1h, at which time the stirring was stopped, followed by settling for about 30min, then the supernatant was withdrawn and discarded (40ml), 40ml methanol was added to the settled precipitate, stirring for about 10min, the precipitate was allowed to settle for about 30min, the supernatant was withdrawn and discarded (45ml), then the precipitate in suspension was filtered, the resulting white cake was washed with 2 parts methanol, 3ml methanol each, the wet solid was dehydrated and dried at a temperature of about 50 ℃ under reduced pressure (6kPa), after drying for about 18h, to yield 1.303g pure depolymerized HTBPM (sodium salt).

The yield reaches 86.8 percent.

Characterization of the depolymerized HTBPM thus obtained

Average molecular weight: 1950 Dalton

Polydispersity index: 1.1

anti-Xa activity: 283U/mg

AIIa Activity: no detection (< 0.2U/mg)

Example 2:

very low molecular weight Heparins (HTBPM) are obtained by the process of the invention comprising a 49% esterification step and a depolymerization step in anhydrous medium using a base derived from phosphazene.

Esterification of HTBPM (step b of the process)：

13.29g (7.6mmol) of the benzethonium salt HTBPM obtained according to preparation 4 are dissolved in 70.43g of anhydrous dichloromethane and introduced into a 100ml three-necked flask (the water content of the reaction medium is 0.073%). At a temperature of 30 ℃ 12.3ml (107mmol) of benzyl chloride were added. The esterification rate after about 7 hours of reaction at 30 ℃ was 49%. After cooling, the reaction mixture was poured into 160ml of 12% sodium acetate in methanol. The mixture was stirred at room temperature for 1h and then allowed to settle for about 16 h. The supernatant was discarded, and then the same volume of methanol (100ml) was added. Stirred for about 1h, then settled for about 1h. The supernatant was discarded again and the same volume of methanol (100ml) was added. Stirred for about 5min, then filtered. The filter cake was washed with 2X 40ml of methanol, dewatered and then dried in an oven at 40 ℃ under reduced pressure (6kPa) for about 18h. 3.90g of crude HTBPM benzyl ester sodium salt was obtained with an esterification rate of 49%.

Purification of HTBPM benzyl ester (49% esterification) sodium salt (step b of the process).

3.90g of crude HTBPM benzyl ester sodium salt was dissolved in 39ml of 10% NaCl aqueous solution. The solution was poured into 176ml of methanol. The suspension was stirred for about 15min and then subsequently settled for 2 h. The mixture was filtered. The filter cake was resuspended in 175ml of methanol and stirred for 10min. Filtration and washing of the filter cake with 2 parts of methanol, 10ml each. After dewatering the white wet solid, it was dried in an oven at 40 ℃ under reduced pressure (6kPa) for about 18h. 2.62g of HTBPM benzyl ester sodium salt are obtained.

The total yield of the esterification section reaches 57.3 percent.

Salt exchange reaction of HTBPM benzyl ester to benzethonium salt (step c) of the process):

2.62g (4.37mmol) of HTBPM benzyl ester sodium salt are dissolved in 20ml of water (Erlenmeyer flask A). Meanwhile, 5.92g (13.2mmol) of benzethonium chloride and 60ml of water were charged into Erlenmeyer flask B. The contents of "B" were poured into "a". The suspension was stirred at room temperature for about 1h, then subsequently settled for 1h. The supernatant was discarded and the same volume of water (70ml) was added. Stirred for about 15min, then settled for 1h. The supernatant was discarded again and the same volume of water (70ml) was added. Stirring for about 5min, and filtering. The filter cake was washed with 3 parts of water, 50ml each, dewatered and then dried in an oven at 80 ℃ under reduced pressure (6kPa) for about 18h. 6.85g of HTBPM benzyl ester phenethyl ammonium salt was obtained.

The yield reaches 99 percent. The water content in the benzethonium salt was 0.6%.

Depolymerization of HTBPM benzyl ester phenethyl ammonium salt:

6.80g (4.3mmol) of HTBPM was charged to a 100ml three-necked flask with 54ml of anhydrous dichloromethane, the mixture was warmed to 30 ℃ and then stirred until dissolution was complete the water content of the reaction mixture was estimated to be about 0.05%. 1.25ml (4.3mmol) of 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 3, 2-diazaphosphoheterobenzene was added and stirred at 30 ℃ for 24h under an inert atmosphere.

Conversion of Quaternary ammonium salt to sodium salt (step e of the process)

In parallel, 270ml of a 10% sodium acetate methanol solution was prepared in a 1l Erlenmeyer flask. The reaction mixture was poured into the methanol solution while maintaining the temperature at about 4 ℃. The suspension was stirred at room temperature for about 1h. Followed by settling for 1h. The supernatant was discarded, and then the same amount of methanol (165ml) was added. Stirred for about 1h and then settled for 1h. The supernatant was discarded again and the same amount of methanol (170ml) was added. Stir for about 15min and filter. The filter cake was washed with 3 parts of methanol, 40ml each, dewatered and then dried in an oven at 50 ℃ under reduced pressure (6kPa) for about 18h. 2.29g of crude depolymerized HTBPM sodium salt were obtained.

The yield reaches 89%.

Saponification of crude HTBPM sodium salt (step f1 of the process)):

2.29g (3.8mmol) of the crude depolymerized HTBPM sodium salt are dissolved in 23ml of water. The solution was filtered through a 0.8 μm membrane and then charged into a 100ml three-necked flask. Mu.l (5.73mmol) of 30% sodium hydroxide solution are added at a temperature of about 3 ℃. Stirring was carried out at this temperature for about 2 h.

Glacial acetic acid was added to half of the reaction mixture for neutralization, followed by addition of 367mg of solid sodium acetate and 13ml of methanol. The solution was stirred for about 15min, then 65ml methanol was added. The resulting suspension was stirred for about 30min and then subsequently settled for about 16 h. The supernatant was discarded and the same amount of methanol (36ml) was added. Stirring was continued for about 30min, followed by settling for about 30min. The supernatant was discarded and the same amount of methanol (16ml) was added. Stir about 15min and filter through a 0.22 μm membrane. The filter cake was washed 2 times with 5ml of methanol each time, dewatered and then dried in an oven at 50 ℃ under reduced pressure (6kPa) for about 18h. 563mg of crude depolymerized HTBPM (sodium salt) were obtained.

The yield reaches 52.6 percent.

Purification by AcONa precipitation of the crude depolymerized HTBPM (sodium salt) (step f2 of the process):

560mg of the crude depolymerized HTBMP (sodium salt) were charged to a 10ml three-necked flask with 5.6ml of water. The brown solution was warmed to 40 ℃ over 10min. The pH was adjusted to 9.7 by addition of 0.1N sodium hydroxide. The solution was filtered through a 0.45 μm membrane and 28. mu.l of 30% aqueous hydrogen peroxide was added. The mixture was stirred at room temperature for 2h while keeping the pH 9.5. + -. 0.1 constant by addition of 0.1N sodium hydroxide. The reaction mixture was neutralized with 0.1N HCl and 620mg NaCl was added. After stirring for 10min, the solution was filtered through a 0.45 μm membrane. Poured into 4.35ml of methanol at a temperature of about 4 ℃. The solution was stirred at room temperature for 15min. 11.2ml of methanol were added. The suspension was stirred for 1h. At this point, the stirring was stopped and the mixture was allowed to settle for 1 hour. The supernatant was then extracted and discarded (13.5 ml). 13.5ml of methanol was added to the precipitated precipitate, and the mixture was stirred for 15min. The precipitation was continued for about 30min. The supernatant was extracted and discarded (13 ml). 13ml of methanol were added, and the precipitate in suspension was filtered. The resulting white filter cake is then washed with 2 parts of methanol, 5ml each. The wet solid was dewatered and dried under reduced pressure (6kPa) at a temperature of 50 ℃. After drying for 18h, 376mg of pure depolymerized HTBPM (sodium salt) were obtained. The yield reaches 67 percent.

The depolymerized HTBPM thus obtained was characterized as follows:

anti-Xa activity: 191UI/mg

Average molecular weight: 2100Da

Example 3:

very low molecular weight Heparins (HTBPM) are obtained according to the process of the invention, which comprises a step of esterification at 73% and a step of depolymerization in an anhydrous medium using a base derived from phosphazene.

Esterification of HTBPM (step b of the process):

13.7g (7.3mmol) of the benzethonium salt of HTBPM obtained according to preparation 4 were dissolved in 73.67g of anhydrous dichloromethane and charged into a 100ml three-necked flask (determination of the water content of the reaction medium at 0.23%). 13ml (113mmol) of benzyl chloride were added at a temperature of 30 ℃ after a reaction time of about 20h at 30 ℃ until the esterification rate reached 73%. after cooling to room temperature, the reaction mixture was poured into 210ml of a 12% sodium acetate in methanol solution. The mixture was stirred at room temperature for 30min, then allowed to settle for about 1.5 h. The supernatant was discarded, and then the same volume of methanol (140ml) was added. Stir 15min and filter the suspension. The filter cake was washed with two portions of methanol, 100ml each, dewatered and then dried in an oven at 40 ℃ under reduced pressure (6kPa) for about 18h. 13.3g of crude HTBPM benzyl ester sodium salt was obtained with an esterification rate of 73%.

Purification of HTBPM benzyl ester (esterification 73%) sodium salt (step b of the process)

13.3g crude HTBPM benzyl ester sodium salt dissolved in 133ml 10% NaCl aqueous solution. The solution was poured into 600ml of methanol. The suspension was stirred for about 15min and then allowed to settle for about 1h. The supernatant was discarded, and then the same volume of methanol (400ml) was added. Stirred for about 5min, then filtered. The filter cake was washed 3 times with 100ml each time of methanol. The wet white solid was dehydrated and dried in an oven at 40 ℃ under reduced pressure (6kPa) for about 18h. 2.33g of HTBPM benzyl ester sodium salt are obtained.

The esterification rate reaches 49.6 percent.

Salt exchange reaction of HTBPM benzyl ester phenethyl ammonium salt (step c of the process):

in a 100ml Erlenmeyer flask "A" was dissolved 2.27g (3.53mmol) of HTBPM benzyl ester sodium salt using 15ml water. Meanwhile, 5.22g (11.6mmol) of benzethonium chloride and 55ml of water were charged into a 100ml Erlenmeyer flask "B".

The contents of "B" were poured into "A". The suspension was stirred at room temperature for about 1h, then subsequently settled for about 1h. The supernatant was discarded and the same volume of water (50ml) was added. Stirred for about 15min and then settled for about 1h. The supernatant was discarded and the same volume of water (50ml) was added. Stirring for another 5min, and then filtering. The filter cake was washed with 3 parts of water, 50ml each, dewatered and dried in an oven at 80 ℃ under reduced pressure (6kPa) for about 18h. 5.67g of HTBPM benzyl ester phenethyl ammonium salt was obtained.

The yield reaches 98 percent. The water content of the product obtained is 1%.

Depolymerization of HTBPM benzyl ester benzyl ethyl ammonium salt (step d of the process):

a100 ml three-necked flask with 40ml of anhydrous dichloromethane was charged with 5.45g (3.3mmol) of HTBPM. The water content of the mixture was estimated to be about 0.1%. The mixture was warmed to 30 ℃. Mu.l (3.3mmol) of 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 3, 2-diazaphosphobenzene were added and stirred at 30 ℃ for 24h under an argon atmosphere.

Conversion of quaternary ammonium salts to sodium salts (step e) of the process):

meanwhile, 200ml of a 10% sodium acetate methanol solution was prepared in a 500ml Erlenmeyer flask. The reaction mixture was poured into the methanol solution while maintaining the temperature at about 4 ℃. The suspension was stirred at room temperature for about 1h. Followed by settling for about 1h. The supernatant was discarded, and then the same amount of methanol (150ml) was added. Stirring was carried out for about 30min, followed by settling for about 30min. The supernatant was discarded again and the same amount of methanol (150ml) was added. Stirred for about 15min, then filtered. The filter cake was washed 3 times with 50ml of methanol and dried under reduced pressure (6kPa) at 50 ℃ for about 18h after dewatering. 1.40g of crude depolymerized HTBPM sodium salt was obtained. The yield reaches 65.8 percent.

Saponification of the crude depolymerized HTBPM sodium salt (step f1 of the process):

1.40g (2.18mmol) of crude depolymerized HTBPM (sodium salt) was dissolved in 14ml of water. The solution was charged to a 100ml three-necked round bottom flask. At a temperature of about 4 ℃ 351. mu.l (3.5mmol) of 30% sodium hydroxide solution were added. Stirring was carried out at this temperature for about 2 h. Glacial acetic acid (100%) was added to neutralize the solution, then 7g of solid sodium acetate and 130ml of methanol were added. The suspension was stirred for 30min and then subsequently settled for about 1h. The supernatant was discarded and the same amount of methanol (80ml) was added. Stirring was continued for about 30min, followed by settling for about 16 h. The supernatant was discarded and the same amount of methanol (80ml) was added. Stirred for about 15min, then filtered through a 0.45 μm membrane. The filter cake was washed 2 times with 10ml of methanol and dried under reduced pressure (6kPa) at 50 ℃ for about 18h after dehydration. 1.15g (yield: 89.4%) of crude depolymerized HTBPM (sodium salt) was obtained.

The yield reaches 89.4 percent.

Crude depolymerized HTBPM (sodium salt) purification (step f2 of the process):

373mg of crude depolymerized HTBPM (sodium salt) was charged to a 10ml three-necked flask with 3.7ml of water. The solution was warmed to 40 ℃ over 10min. The pH was adjusted to about 9.5 by the addition of 1N sodium hydroxide. The solution was filtered through a 0.45 μm membrane and then 18 μ l of 30% aqueous hydrogen peroxide was added. The mixture was stirred at room temperature for about 2h while maintaining the pH at 9.5. + -. 0.1 by the addition of 0.1N sodium hydroxide. The reaction mixture was neutralized with 0.1N HCl, then 430mg NaCl was added. After stirring for about 10min, the solution was filtered through a 0.45 μm membrane. 3ml of methanol are poured in at a temperature of about 4 ℃. The solution was stirred at room temperature for 15min. Then 7.7ml of methanol were added. The suspension was stirred for about 1h. At this point, the stirring was stopped and then allowed to settle for about 40 min. The supernatant was then extracted and discarded (10 ml). To the precipitated precipitate, 10ml of methanol was added and stirred for 15min. The precipitate was allowed to settle for about 30min. The supernatant was extracted and discarded (10 ml). 10ml of methanol was added, and the suspended precipitate was filtered through a 0.45 μm membrane. The resulting white filter cake was washed with 4 parts of methanol, 5ml each. The wet solid was dried after dewatering under reduced pressure (6kPa) at a temperature of about 50 ℃. After drying for about 18h, 199mg of pure depolymerized HTBPM (sodium salt) were obtained. The yield reaches 54 percent.

The characteristics of the depolymerized HTBPM thus obtained are as follows:

average molecular weight: 2000 daltons. Polydispersity index: 1.1

anti-Xa activity: 252UI/mg

Example 4:

HTBPM is obtained by the process of the invention comprising a 96% esterification step and a depolymerization step with BEMP.

Esterification of HTBPM (step b of the process)

14.45g (7.7mmol) of the benzethonium salt of HTBPM obtained according to preparation 4 were dissolved in 75.79g of anhydrous dichloromethane and charged into a 250ml three-neck flask. (the water content of the reaction medium is 0.20%). 12.4ml (108mmol) of benzyl chloride are added at 30 ℃. The esterification rate after reaction at a temperature of 30 ℃ for about 26h was 96%. After cooling to room temperature, the reaction mixture was poured into 180ml of 12% sodium acetate in methanol. The mixture was stirred at room temperature for about 30min, then subsequently settled for about 30min. The supernatant was discarded, and then the same volume of methanol (150ml) was added. Stirred for about 15min, then filtered. The filter cake is washed with 2 parts of methanol, 100ml each, and dried under reduced pressure (6kPa) at 40 ℃ for about 18h after dehydration. 3.67g of crude HTBPM benzyl ester sodium salt was obtained with an esterification rate of 96%.

Purification of HTBPM benzyl ester (96% esterified) sodium salt (step b) of the process):

3.67g crude HTBPM benzyl ester sodium salt dissolved in 37ml 10% NaCl aqueous solution (3.7g NaCl in 37ml water solution). The solution was poured into 167ml of methanol. The suspension was stirred for about 15min and then subsequently settled for about 1h. The supernatant was discarded, and then the same volume of methanol (38ml) was added. Stir for about 5min and filter. The filter cake was washed 2 times with 30ml of methanol each time. The wet white solid was dewatered and dried under reduced pressure (6kPa) at 40 ℃ for about 18h. 2.76g of HTBPM benzyl ester sodium salt are obtained.

The esterification rate reaches 54.4 percent.

Salt exchange reaction of HTBPM benzyl ester to benzethonium salt (step c) of the process):

in a 100ml Erlenmeyer flask "A" was dissolved 2.83g (4.29mmol) of HTBPM benzyl ester sodium salt using 20ml of water. Meanwhile, 6.35g (14.2mmol) of benzethonium chloride and 50ml of water were charged into a 100ml Erlenmeyer flask "B".

The suspension was stirred at room temperature for about 1h, then allowed to settle for about 1h, the supernatant discarded, the same volume of water (60ml) was added and stirred for about 15min, then allowed to settle for about 1h, the supernatant discarded, the same volume of water (60ml) was added and stirred for about 5min, then filtered, the cake was washed 4 times with 50ml of water each, dried under reduced pressure (6kPa) at 80 ℃ for about 18h after dewatering to give 7.0g HTBPM benzyl ester ethylammonium salt.

The yield was determined to be about 100%. The water content was 0.23%.

Depolymerization of HTBPM benzyl ester phenethyl ammonium salt (step d) of the process):

3.67g (2.3mmol) of HTBPM were charged to a 50ml three-necked round-bottom flask with 27ml of anhydrous dichloromethane. The mixture was warmed to 30 ℃. 676. mu.l (2.3mmol) of 2-t-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 3, 2-diazaphosphobenzene were added and stirred at 30 ℃ for 24h.

Conversion of Quaternary ammonium salt to sodium salt (step e of the process)

Meanwhile, 150ml of 10% sodium acetate in methanol was prepared in a 250ml Erlenmeyer flask. The reaction mixture was poured into its methanol solution while maintaining the temperature at about 4 ℃. The suspension was stirred at room temperature for about 1h. Followed by settling for about 1h. The supernatant was discarded, and then the same amount of methanol (100ml) was added. Stirring for 30min, followed by settling for 30min. The supernatant was discarded again and the same amount of methanol (100ml) was added. Stir 15min and filter. The filter cake was washed 3 times with 40ml of methanol each time, dewatered and dried under reduced pressure (6kPa) at 50 ℃ for about 18h. 966mg of depolymerized HTBPM sodium salt are obtained.

The yield reaches 64 percent.

Saponification of HTBPM benzyl ester sodium salt (step f1 of the process):

942mg (1.43mmol) of depolymerized HTBPM sodium salt was dissolved in 9.5ml of water. The solution was charged to a 100ml three-necked round bottom flask. 236. mu.l (2.35mmol) of 30% sodium hydroxide solution are added at a temperature of about 4 ℃. Stirring was carried out at this temperature for about 2 h. Glacial acetic acid (100%) was added to neutralize the solution. 4.5g of solid sodium acetate and 85ml of methanol are then added. The suspension was stirred for about 30min and then subsequently settled for about 1h. The supernatant was discarded and the same amount of methanol (40ml) was added. Stirring was continued for about 30min, followed by settling for about 16 h. The supernatant was discarded, and the same amount of methanol (40ml) was added. Stirred for about 30min and filtered through a 0.45 μm membrane. The filter cake was washed 2 times with 10ml of methanol and dried under reduced pressure (6kPa) at 50 ℃ for about 18h after dehydration. 776mg of crude depolymerized HTBPM (sodium salt) were obtained.

The yield reaches 91.4 percent.

Crude depolymerized HTBPM (sodium salt) purification-step f2 of the process:

758mg of crude depolymerized HTBPM (sodium salt) was charged to a 25ml three-neck flask with 7.6ml of water. The solution was warmed to 40 ℃ over 10min. The pH was adjusted to about 9.5 by the addition of 0.1N sodium hydroxide. The solution was filtered through a 0.45 μm membrane and then 38 μ l of 30% aqueous hydrogen peroxide was added. The mixture was stirred at room temperature for about 2h while the pH was kept constant at 9.5. + -. 0.1 by the addition of 0.1N sodium hydroxide. The reaction mixture was neutralized with 1N HCl and 880mg NaCl was added. After stirring for about 10min, the solution was filtered through a 0.45 μm membrane. 6.2ml of methanol are poured in at a temperature of about 4 ℃. The solution was stirred at room temperature for about 15min. 16ml of methanol are added and the suspension is stirred for about 1h. At this point, the stirring was stopped and the suspension was filtered. The filter cake was washed with 2 portions of methanol, 15ml each. The wet solid was dewatered and dried under reduced pressure (6kPa) at a temperature of about 50 ℃. After drying for about 18h, 490mg of pure depolymerized HTBPM (sodium salt) were obtained. The yield reaches 65 percent.

The depolymerized HTBPM thus obtained was characterized as follows:

average molecular weight: 2000 Dalton

Polydispersity index: 1.1

anti-Xa activity: 205UI/mg

Example 5:

HTBPM is obtained by the process of the invention comprising a 96% esterification step and a depolymerization step using tert-butylimino-tris (dimethylamino) phosphane.

Decomposition of HTBPM benzyl ester benzyl ethyl ammonium saltPoly (step d) of the process):

3.67g (2.3mmol) of HTBPM benzyl ester phenethylammonium salt (96% esterified) obtained according to example 4, with a water content of 0.23%, are charged in a 50ml three-necked flask with 30ml of anhydrous dichloromethane. The mixture was warmed to 30 ℃. 595. mu.l (2.3mmol) of tert-butylimino-tris (dimethylamino) phosphane are added and stirred at 30 ℃ for 24h.

Conversion of Quaternary ammonium salt to sodium salt (step e of the process)

Meanwhile, 160ml of 10% sodium acetate methanol solution was prepared in a 250ml Erlenmeyer flask. The reaction mixture was poured into methanol solution while maintaining the temperature at about 4 ℃. The suspension was stirred at room temperature for about 1h. Followed by settling for about 1h. The supernatant was discarded, and then the same amount of methanol (120ml) was added. Stirring was carried out for about 30min, followed by settling for 30min. The supernatant was discarded again and the same amount of methanol (125ml) was added. Stir for about 15min and filter. The filter cake was washed 3 times with 40ml of methanol each time, dewatered and dried under reduced pressure (6kPa) at a temperature of 50 ℃ for about 18h. 982mg of crude depolymerized HTBPM sodium salt were obtained. The yield reaches 65 percent.

Saponification of the crude depolymerized HTBPM sodium salt (step f1 of the process):

980mg (1.49mmol) of the crude depolymerized HTBPM sodium salt are dissolved in 10ml of water. The solution was then charged to a 100ml three-necked round bottom flask. Mu.l (2.45mmol) of 30% sodium hydroxide solution are added at a temperature of about 4 ℃. Stirring was carried out at this temperature for about 2 h. Glacial acetic acid (100%) was added to neutralize the solution. 4.9g of solid sodium acetate and 95ml of methanol are then added. The suspension was stirred for about 30min and then subsequently settled for about 1h. The supernatant was discarded and the same amount of methanol (60ml) was added. Stirring was continued for about 30min, followed by settling for about 16 h. The supernatant was discarded, and the same amount of methanol (60ml) was added. Stirred for about 30min and filtered through a 0.45 μm membrane. The filter cake was washed 2 times with 10ml of methanol and dried under reduced pressure (6kPa) at 50 ℃ for about 18h after dehydration. 809mg of crude depolymerized HTBPM (sodium salt) were obtained.

The yield reaches 91.6 percent.

Purification of the crude depolymerized HTBPM (sodium salt) -step f2 of the process:

792mg of crude depolymerized HTBPM (sodium salt) was charged to a 25ml three-neck flask with 8ml of water. The solution was warmed to 40 ℃ over 10min. The pH was adjusted to about 9.5 by the addition of 0.1N sodium hydroxide. The solution was filtered through a 0.45 μm membrane and then 39.6 μ l of 30% aqueous hydrogen peroxide was added. The mixture was stirred at room temperature for about 2h while maintaining the pH at 9.5. + -. 0.1 by the addition of 0.1N sodium hydroxide. The reaction mixture was neutralized with 1N HCl and 1.04g NaCl was added. After stirring for about 10min, the solution was filtered through a 0.45 μm membrane. 7.3ml of methanol are poured in at a temperature of about 4 ℃. The solution was stirred at room temperature for about 15min. 18.8ml of methanol are added and the suspension is stirred for about 1h. At this point, the stirring was stopped and filtration was carried out. The filter cake was washed with 3 portions of methanol, 15ml each. The wet solid was dewatered and dried under reduced pressure (6kPa) at a temperature of about 50 ℃. After drying for about 18h, 538mg of pure depolymerized HTBPM (sodium salt) were obtained. The yield reaches 67.9 percent.

The depolymerized HTBPM thus obtained was characterized as follows:

average molecular weight: 2100 Dalton

Polydispersity index: 1.1

anti-Xa activity: 209UI/mg

Example 6:

HTBPM is obtained using the method of the invention, which includes a supplemental separation step using chromatography, while removing the disaccharide and tetrasaccharide moieties.

The oligosaccharide mixture described in example 1 (286mg) was dissolved in 20ml of mobile phase (0.2mol/l aqueous sodium bicarbonate).

These chromatographic conditions were as follows:

-a mobile phase: 0.2mol/l sodium bicarbonate solution

-a stationary phase: gel biogel P6

-a column: length 1m, diameter 5 cm.

-the detection wavelength: 240 nm.

Fractions greater than or equal to hexasaccharides were collected and combined. They were further neutralized with acetic acid and then concentrated until 200g/l of sodium acetate solution was obtained. To the resulting solution was added 5 volumes of methanol with stirring. The suspension was stirred for about 18h and then filtered through a 0.45 μm membrane. The filter cake was dried under reduced pressure (6kPa) at a temperature of about 40 ℃ for about 6 h. The product obtained is precipitated again, then dissolved in the minimum amount of water and desalted using a Sephadex G10 column. After concentration of the desalted fraction, lyophilization was performed to give 109mg of product. The yield reaches 38%.

The oligosaccharide mixture thus obtained is characterized as follows:

anti-Xa activity: 403UI/mg

The percentage of oligosaccharides was as follows:

Mw(Da)	polydispersity	Di％	Tetra％	Hexa％	Octa％	Deca％	＞Deca％
								2150	1.0	0	0	53.83	32.58	10.5	3.5

Pharmacological activity of the compounds of the invention:

examples	Average molecular weight	anti-Xa Activity UI/mg	anti-IIa Activity
				1	1950	283	＜0.2
2	2100	191	0
				3	2000	252	0
4	2000	205	0
				5	2100	209	0
6	2150	403	0

Hexasaccharide Δ IIa-II in Compounds of the inventions-percentage of Is:

examples	Percentage of hexasaccharide moiety	Hexasaccharide delta IIa-II in the hexasaccharide moietyPercentage of Is
			1	31％	46％
2	30％	26％

Examples	Percentage of hexasaccharide moiety	Hexasaccharide delta IIa-II in the hexasaccharide moietyPercentage of Is
			3	33％	33.5％
4	32％	30.8％
			5	31.5％	28.7％
6	53.8％	46％

Claims (40)

1. A mixture of oligosaccharides, wherein said oligosaccharides have the general structure of the polysaccharides that constitute heparin, and further having the following characteristics:

average molecular weight 1800-,

the anti-Xa activity was 190-450IU/mg,

an anti-IIa activity of less than 0.2IU/mg,

-oligosaccharides present in the mixture:

containing 2 to 16 sugar building blocks,

-having a 4, 5-unsaturated uronic acid-2-O-sulphate building block at one of the termini,

-and contains a hexasaccharide of formula:

characterized in that the oligosaccharide mixture contains 20-100% of the hexasaccharide fraction and the hexasaccharide fraction contains 20-70% of the hexasaccharide Δ IIa-II as defined aboves-Is。

2. Oligosaccharide mixture according to claim 1, characterized in that the oligosaccharide mixture contains 30-60% hexasaccharide moieties.

3. Oligosaccharide mixture according to claim 1, characterized in that the hexasaccharide fraction contains 25-50% of the hexasaccharide Δ IIa-II as defined in claim 1s-Is。

4. Oligosaccharide mixture according to any of claims 1-3, characterized in that it has an average molecular weight of 1900 and 2200 daltons.

5. Oligosaccharide mixture according to claim 4, characterized in that it has an average molecular weight of 1950 and 2150 daltons.

6. Oligosaccharide mixture according to any of claims 1-3, characterized in that it has an anti-Xa activity of 190 IU/mg and an anti-IIa activity of less than 0.2 IU/mg.

7. Oligosaccharide mixture according to claim 6, characterized in that its anti-Xa activity is 200-300 IU/mg.

8. Oligosaccharide mixture according to any of claims 1-3, characterized in that it has the following characteristics:

average molecular weight 1950 and 2150 daltons,

the anti-Xa activity is 190 IU/mg and the anti-IIa activity is less than 0.2IU/mg,

the oligosaccharide mixture contains 30-60% of the hexasaccharide fraction, whereas the hexasaccharide fraction contains 25-55% of Δ IIa-IIs-Is。

9. Process for the preparation of the oligosaccharide mixture according to any of claims 1-8, characterized in that very low molecular weight heparins with an anti-Xa activity higher than 140IU/mg, an anti-IIa activity lower than 5IU/mg and an average molecular weight of 2000-3000 daltons are subjected to the following chemical reactions:

a) the salt exchange is carried out by the action of benzethonium chloride to obtain heparin benzethonium,

b) the obtained heparin benzyl ethylammonium is esterified and treated by the action of benzyl chloride, so as to obtain heparin benzyl ester sodium salt with very low molecular weight,

c) the obtained heparin benzyl ester sodium salt with very low molecular weight is subjected to salt exchange to obtain quaternary ammonium salt,

d) depolymerisation with a strong organic base having a pKa higher than 20, to obtain depolymerised heparin quaternary ammonium salts of very low molecular weight,

e) depolymerized very low molecular weight heparin quaternary ammonium salt is converted to sodium salt,

f) residual ester saponification is carried out and optionally purification is carried out.

10. The process according to claim 9, wherein the molar ratio of strong base/ester used in the depolymerization step d) is 0.2 to 5.

11. The process according to claim 10, wherein the molar ratio of strong base/ester used in the depolymerization step d) is 0.6 to 2.

12. The process according to claim 9, characterized in that the depolymerization step d) is carried out using a phosphazene base.

13. The process according to any one of claims 9 to 12, characterized in that the depolymerization step d) is carried out with a water content of less than 0.3%, operating with 1 molar equivalent of phosphazene base, based on the very low molecular weight heparin benzyl ester, benzethonium salt.

14. The process according to claim 12, wherein the phosphazene base used in the depolymerization step d) is a base of the formula:

in the formula, R₁-R₇The radicals, which are identical or different, represent a linear, branched or cyclic alkyl radical having from 1 to 6 carbon atoms, or R₃And R₄Form a 6-membered heterocyclic ring with the-N-P-N-group they bear.

15. The production method according to claim 12, wherein the phosphazene base used in the depolymerization step is 2-t-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 3, 2-diazaphospholbenzene.

16. The method according to claim 9, wherein the esterification rate of heparin benzyl ester sodium salt in step b) is 40-100%.

17. The method according to claim 16, wherein the esterification rate of heparin benzyl ester sodium salt in step b) is 70-90%.

18. The process according to claim 9, characterized in that the very low molecular weight heparin benzyl ester quaternary ammonium salt prepared according to the process of step b) of claim 9 is converted into very low molecular weight heparin benzyl ester sodium salt by treating the reaction medium with an alcoholic solution of sodium acetate at a temperature of 15-25 ℃.

19. The method of claim 18, wherein the solution of sodium acetate is a 10% w/v solution of sodium acetate in methanol.

20. The method of claim 18, wherein the amount of sodium acetate added in esterification step b) is 3 times the amount of quaternary ammonium salt of heparin benzyl ester used in the depolymerization reaction.

21. Process for the preparation of an oligosaccharide mixture according to claim 9, characterized in that the quaternary ammonium salt obtained in step c) is a benzethonium salt or a cetyltrimethylammonium salt.

22. The method according to claim 9, wherein the saponification in step f is carried out in an aqueous medium at a temperature of 0 to 20 ℃ using an alkali metal hydroxide.

23. The method according to claim 22, wherein the saponification is carried out at a temperature of 0 to 10 ℃.

24. The method according to claim 22, wherein the alkali metal hydroxide is sodium hydroxide, potassium hydroxide or lithium hydroxide.

25. The method according to claim 22, wherein 1 to 5 molar equivalents of the alkali metal hydroxide are used.

26. The method according to claim 25, wherein 1 to 2 molar equivalents of alkali metal hydroxide are used.

27. The method according to claim 9, wherein the disaccharide and tetrasaccharide moieties are removed by using column chromatography using polyacrylamide agarose gel-packed.

28. Method for the preparation of an oligosaccharide mixture according to any of claims 1-8 according to any of claims 9-12, characterized in that instead of very low molecular weight heparin as defined in claim 9, low molecular weight heparin with a molecular weight of 1500-3000 dalton or very low molecular weight heparin with an anti-Xa activity of 100-140UI/mg is used as starting product.

29. Method for the preparation of an oligosaccharide mixture according to any of claims 1-8 according to any of claims 9-12, characterized in that instead of very low molecular weight heparin as defined in claim 9, very low molecular weight heparin with a molecular weight of 1500-3000 dalton with an anti-Xa activity of 100-140UI/mg is used as starting product.

30. The method of claim 28, wherein the low molecular weight heparin is selected from the group consisting of enoxaparin, nadroparin, dalteparin, tinzaparin, adexaparin, sertorxaparin, parnaparin, reviparin, and bemiparin.

31. An oligosaccharide mixture as claimed in any one of claims 1 to 8 obtainable by a process as claimed in any one of claims 9 to 30.

32. A medicament comprising as a medicament the oligosaccharide mixture as claimed in any one of claims 1 to 8.

33. A medicament having anticoagulant activity, which comprises as a medicament the oligosaccharide mixture of any one of claims 1-8.

34. A medicament according to claim 32 or 33 for the treatment or prophylaxis of venous and arterial thrombosis, deep vein thrombosis, pulmonary embolism, unstable angina, myocardial infarction, myocardial ischemia, peripheral arterial occlusive conditions and atrial fibrillation, smooth muscle cell proliferation, atherosclerosis and arteriosclerosis, cancer, and diabetes.

35. The medicament of claim 34, wherein the diabetes is diabetic retinopathy and diabetic nephropathy.

36. A pharmaceutical composition comprising at least one medicament according to claim 32 and one or more pharmaceutically inert excipients or carriers or additives.

37. Pharmaceutical compositions according to claim 36, characterized in that they are subcutaneous or intravenous solutions.

38. Pharmaceutical compositions according to claim 36, characterized in that they are formulations for administration by inhalation via the pulmonary route.

39. Pharmaceutical compositions according to claim 36, characterized in that they are formulated for oral administration.

40. Method for determining the anti-Xa activity of the oligosaccharide mixture according to any of the claims 1-8, characterized in that an amide hydrolysis method with a chromogenic substrate is used, wherein the replication buffer is polyethylene glycol 6000.