HK1145031A - Heparin-derived polysaccharide mixtures, preparation thereof and pharmaceutical compositions containing same - Google Patents

Description

heparin-derived polysaccharide mixtures, process for their preparation and pharmaceutical compositions containing them

The divisional application of PCT application PCT/FR2003/002960 entitled "polysaccharide mixtures derived from heparin, methods for their preparation and pharmaceutical compositions containing them", filed on 8/10/2003 in 2003, with a date of entry into the national phase of china on 6/4/2005 and application number 200380100992.5.

The present invention relates to mixtures of polysaccharides derived from heparin, to processes for their preparation and to pharmaceutical compositions containing them.

Heparin is a sulfated glycosaminoglycan mixture of animal origin that finds application primarily for its anticoagulant and antithrombotic properties.

However, the drawbacks of heparin itself limit its use. In particular, its high anticoagulant activity (anti-IIa activity) may cause bleeding.

Low molecular weight heparins have been proposed by alkaline depolymerization of heparin esters (EP 40144); however, these products still have a high IIa-resistance activity.

In US 6384021, very low molecular weight heparins have also been described. However, in the examples in which the Xa activity did not exceed 120IU, and the Xa/IIa resistance ratio was 15-50.

In WO 0208295, different from US 6384021, very low molecular weight heparins were prepared, which have an activity of 100-150IU and in some application examples have a very high Xa/IIa resistance ratio.

Nevertheless, there is a need to improve the anti-Xa activity of these drugs, especially to achieve an activity above 150IU/mg, and to increase the anti-Xa/anti-IIa ratio, and therefore, there is a need to develop new processes for the preparation of heparin derivatives.

It is therefore an object of the present invention to increase the ratio of the anti-Xa activity to the anti-Xa/IIa resistance by improving the processes described in the prior art, in particular by controlling the water percentage during this depolymerization step. The heparin thus obtained has excellent antithrombotic activity, an anti-Xa activity similar to that of heparin, a reduced risk of bleeding and a very low anti-IIa activity. Meanwhile, the half-life period of the product is obviously higher than that of heparin.

The object of the present invention is therefore novel polysaccharide mixtures derived from heparin which have a selective activity on activated factor X (factor Xa) and activated factor II (factor IIa) which is superior to that of heparin.

It should be understood that a mixture of polysaccharides having an average molecular weight of 1500-.

The object of the present invention is therefore a mixture of these sulfated oligosaccharides, which have the general structure of the polysaccharides that make up heparin and which also have the following characteristics:

-they have an average molecular weight of 1500-3000 daltons, an anti-Xa activity of 120-200IU/mg, an anti-IIa activity of less than 10IU/mg and a ratio of anti-Xa activity/anti-IIa activity of more than 30,

the oligosaccharides constituting these mixtures contain 2-26 saccharide units, have 4, 5-unsaturated uronic acid 2-O-sulfate units at one end, and contain the hexasaccharide Δ IIa-II of the formulas-Is：

The hexasaccharide Δ IIa-II contained in the oligosaccharide mixtures described in the inventionsIs a sequence with high affinity for ATIII, characterized by an anti-Xa activity higher than 740U/mg.

The oligosaccharide mixtures described in the present invention are in the form of alkali metal or alkaline earth metal salts.

For alkali metal or alkaline earth metal salts, sodium, potassium, calcium and magnesium salts are preferred.

The average molecular weight can be determined by high pressure liquid chromatography using a tandem double column, such as high pressure liquid chromatographs sold under the tradenames TSK G3000 XL and TSK G2000 XL. The detection was performed using a refractometer. The eluent used was lithium nitrate at a flow rate of 0.6 ml/min. This system was calibrated using standards prepared by agarose-polyacrylamide gel chromatography (IBF) fractionation of enoxaparin (AVENTIS). Prepared according to the method 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). These results were calculated using GPC6 software (Perkin Elmer).

anti-Xa activity was determined according to the chromogenic substrate amidolysis method described by Teien et al, Thromb. Res., 10, 399-410(1977), using the first international standard for low molecular weight heparin as a standard.

The anti-IIa activity was determined using the technique described in Anderson L.O. et al, Thromb. Res., 15, 531-541(1979), using the first international standard for low molecular weight heparins as a standard.

Preferably the hexasaccharide fraction is 15-25% of the oligosaccharide mixture.

Preferably, the mixture of the invention contains 8-15% of the hexasaccharide Δ IIa-II in the hexasaccharide fraction of the oligosaccharide mixtures-Is。

The percentage of the hexasaccharide fraction can be determined analytically by high pressure liquid chromatography using TSK G3000 XL and TSK G2000 XL columns, or by preparative separation of the hexasaccharide fraction. In this situationIn this case, a polyacrylamide agarose gel packed column is used, for example, commercially available under the trade mark Ultrogel ACA202^RThe mixture was chromatographed on a packed column of (Biosepra). The mixture was eluted with sodium bicarbonate solution. Preferably, this sodium bicarbonate solution is a 0.1-1mol/l solution. More preferably, this separation is carried out with a sodium bicarbonate solution having a concentration of 1 mol/l. Detection was performed by UV spectroscopy (254 nm). After fractionation, the sodium bicarbonate solution of the hexasaccharide fraction was neutralized with glacial acetic acid. This solution was then concentrated under reduced pressure to a sodium acetate concentration of above 30% by weight. The hexasaccharide fraction was then precipitated by adding 3-5 volumes of methanol. This hexose fraction was recovered by filtration using a sintered glass filter No. 3. The hexasaccharide mixture obtained can be analyzed by HPLC (high performance liquid chromatography) to determine the hexasaccharide Δ IIa-IIsThe content of Is. The hexasaccharide Δ IIa-II can be isolated using preparative HPLC chromatography or using affinity chromatography using an antithrombin III agarose column according to techniques employed by those skilled in the art (m.hook, i.bjork, j.hopwood and u.lindahl, f.e.b.sletter, vol 656(1) (1976))s-Is。

It is particularly preferred that the anti-Xa activity of the mixtures of the invention is 150-200 IU/mg.

Preferably, the mixtures according to the invention have an anti-IIa activity of less than 5IU/mg, particularly preferably 0.5 to 3.5 IU/mg. The application examples described below demonstrate that the IIa-resistance activity of the mixtures according to the invention is between 1.1 and 1.6IU/mg when using the preferred features of the method.

Preferably, these mixtures have a ratio of Xa-activity/IIa-activity resistance of more than 50, particularly preferably more than 100.

Preferably, the average molecular weight of the mixture of the invention is 2000-.

Thus, very particularly, the object of the present invention are those mixtures as defined above whose anti-Xa activity is 150-200IU/mg, anti-IIa activity is 0.5-3.5IU/mg and the average molecular weight is 2400-2650 daltons.

The oligosaccharide mixtures of the invention may be prepared by saponification and optionally purification of the residual esters, preferably using a strong organic base having a pKa of greater than 20 (preferably having properties similar to those of the phosphazenes as defined, for example, in accordance with r.schwesinger et al, angle.int.ed. engl. 26, 1167-1169(1987), r.schwesinger et al, angle.1420. 105, 1993), in an organic medium by depolymerization of the heparin benzyl ester quat followed by conversion of the depolymerised heparin benzyl ester quat to the sodium salt. The process of the present invention repeats the main steps of the process as described in WO 0208295, while adding an essential feature which enables the resulting oligosaccharide mixture of the present invention to have the physicochemical characteristics and activities as described previously.

In fact, in order to obtain the specific oligosaccharide mixtures of the invention, it is necessary to control the selectivity of this base by very precise control of the water content in such mixtures during the depolymerization step.

In fact, the process of the invention is characterized by a high selectivity of the base controlled during the depolymerization. The invention enables such heparins to be depolymerised while maximally maintaining these sequences with affinity for ATIII, e.g.the hexasaccharides delta IIa-II described in the inventionsIs. This critical step of the process ensures that the polysaccharide of the invention is obtained.

This feature of the process makes this mixture of oligosaccharides with average molecular weight unexpectedly show anti-Xa activity (activity 150IU/mg < anti-Xa < 200IU/mg at an average molecular weight of 2000-3000 daltons). This selectivity is due to the very specific physicochemical characteristics of the phosphazene base, having a pKa of greater than 20 and a very high steric hindrance and a weak nucleophilicity.

This effect is fully manifested when the reaction medium is anhydrous. Conversely, a drastic decrease in the selectivity of the depolymerization is observed as the water content in the reaction medium increases. The sequence which retains affinity for ATIII is reduced, as a result of which the anti-Xa activity is greatly reduced. In the presence of small amounts of water, the phosphazene base becomes protonated and these reaction species become quaternary ammonium hydroxides. In this case, the very high steric hindrance and the weak nucleophilicity are lost, and thus the quality of the resulting product is seriously affected. This effect was clearly observed when the depolymerization experiments were carried out with controlled water content.

The following table summarizes the effect of water content on depolymerization selectivity (this parameter is only changed in these experiments: the stoichiometry, dilution and temperature of the reagents remain unchanged according to standards known to those skilled in the art. the base used is the phosphazene base 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 2, 3-diazaphosphoricyclohexene).

Water content%	0.05％	0.1％	0.2％	0.3％	0.4％	0.57％	1.8％	2.5％
									anti-Xa IU/mg	192	177	161	132	122	120	105	99
IIa resistant IU/mg	1.3	1.5	1.4	1.4	1.3	1.4	3.1	13.4
									Anti Xa/IIa resistance	148	118	115	94	94	85.7	34	7.4

In order to obtain the best selectivity and maximum retention of affinity sequences for ATIII, this step is preferably carried out at a water content of less than 0.6%, very preferably less than 0.3%, based on 1 molar equivalent of phosphazene base, based on heparin benzyl ester, benzethonium (benzethonium) salt.

Very particularly, therefore, the object of the present invention is a depolymerization step of a quaternary ammonium salt of heparin benzyl ester obtained according to a process known to a person skilled in the art, characterized by the use of a base of the phosphazene type, particularly in a solution of dichloromethane containing less than 0.6% of water. Preferably, the percentage of water should be less than 0.3%, particularly preferably less than 0.2%.

It is preferred that the molar ratio of strong base/ester is from 0.2 to 5, preferably from 0.6 to 2, particularly preferably from 0.8 to 1.2. Thus, the use of equimolar ratios is a preferred embodiment of the invention.

Other aprotic solvents known to those skilled in the art, such as THF or DMF, may be used.

These heparin benzyl ester quaternary ammonium salts are preferably benzethonium, cetylpyridinium or cetyltrimethylammonium salts.

These phosphazene base preferably have the formula:

wherein R1-R7 are the same or different and represent alkyl.

In the foregoing formula, the alkyl group contains 1 to 6 straight or branched carbon atoms.

The object of the present invention is therefore a process for preparing an oligosaccharide mixture according to the invention, which process comprises the following steps:

a) the sodium salt of heparin is exchanged (transmethylation) through the action of benzethonium chloride,

b) the benzyl chloride is used for esterifying the benzethonium heparin salt,

c) the obtained benzyl ester salt is exchanged into quaternary ammonium salt,

d) depolymerisation of the heparin benzyl ester quat using the method defined above,

e) the quaternary ammonium salt is converted into a sodium salt,

f) optionally saponifying the heparin by the action of a base, such as sodium hydroxide,

g) in particular by the action of an oxidizing agent, such as hydrogen peroxide, optionally purified.

The following reaction scheme illustrates the invention:

n ═ X + Y + Z (mean sulfation level of disaccharide)

X is the degree of sulfation of the site, the remainder being represented by the group H

Y-the degree of sulfation of the site, the remainder being represented by the group H

Z is the degree of sulfation of the site, the remainder being formed by the group COCH₃Represents

Generally, the depolymerised 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 with a 10% (w/v) solution of sodium acetate in methanol, at a temperature of 15-25 ℃ (step e). The weight equivalent of acetate added is preferably three times the amount of heparin benzyl ester quaternary ammonium salt added to this depolymerization reaction.

In general, the saponification reaction is carried out at a temperature of 0 to 20 ℃, preferably 0 to 10 ℃, in an aqueous medium using an alkali metal hydroxide, for example sodium hydroxide, potassium hydroxide, lithium hydroxide (step f). Typically, 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.

This final product (step g) may optionally be purified using any known method for purifying depolymerised heparin (e.g. EP 0037319B 1). Preferably, this purification is carried out in an aqueous medium using hydrogen peroxide at a temperature of 10-50 ℃. Preferably, this operation is carried out at 20-40 ℃.

The heparin benzyl ester quaternary ammonium salt can be prepared according to the following reaction scheme:

a) the conversion of heparin sodium with benzethonium chloride to give the benzethonium heparin salt (salt exchange),

b) esterifying the benzethonium salt obtained above by treatment with an alcoholic solution of sodium acetate using benzyl chloride to obtain heparin benzyl ester sodium salt,

c) the sodium salt of heparin benzyl ester is exchanged for a quaternary ammonium salt, preferably benzethonium, cetylpyridinium or cetyltrimethylammonium salt.

The reaction of step a) is carried out by the action of an excess of benzethonium chloride with sodium heparin at a temperature of 15-25 ℃. Advantageously, the salt/heparin sodium molar ratio is between 3 and 4.

The starting material heparin used is preferably porcine heparin. The porcine heparin may be previously purified to reduce its dermatan sulfate content, according to the method described in patent FR 2663639.

The esterification reaction of step b) is carried out at a temperature of 25-45 ℃, preferably 30-40 ℃, preferably in a chlorine-containing organic solvent, such as chloroform or dichloromethane. The ester is then recovered as the benzethonium salt by precipitation with 10% by weight sodium acetate in an alcohol such as methanol, the sodium salt being the sodium salt. Typically 1-1.2 volumes of alcohol are used per volume of reaction medium. The amount of benzyl chloride and the reaction time are adjusted to achieve a degree of esterification of 50 to 100%, preferably 70 to 90%. Preferably, 0.5-1.5 parts by weight of benzyl chloride is used per 1 part by weight of heparin benzethonium salt. Likewise, the reaction time is preferably 10 to 35 hours.

The salt exchange of step c) is carried out in an aqueous medium at a temperature of 10-25 ℃ using a quaternary ammonium chloride, preferably benzethonium chloride, cetylpyridinium chloride or cetyltrimethylammonium chloride. Advantageously, the quaternary ammonium chloride/heparin benzyl ester sodium salt molar ratio is between 2 and 3.

The inventive mixtures in the form of the sodium salts can be converted into further alkali metal or alkaline earth metal salts. The conversion from one salt to another can optionally be achieved using the method described in patent FR 7313580.

The mixtures of the invention are non-toxic and can therefore be used as medicaments.

The oligosaccharide mixtures of the invention can be used as antithrombotic agents. In particular, they are useful for the treatment or prophylaxis of venous and arterial thrombosis, deep vein thrombosis, pulmonary vascular infarction, unstable angina, myocardial infarction, myocardial ischemia, peripheral arterial obstructive disease and atrial fibrillation. They are also useful 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 diabetic conditions such as diabetic retinopathy and nephropathy.

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

These pharmaceutical compositions are, for example, solutions which can be administered by subcutaneous or intravenous injection. They may also be administered pulmonary (inhalation) or by oral administration.

The dosage may vary with the age, weight and health of the patient. The dosage for intramuscular or subcutaneous administration to adults is usually 20-100mg per day.

The invention also relates to

1. Mixtures of sulfated oligosaccharides, having the general structure of the polysaccharides that make up heparin, also have the following characteristics:

-they have an average molecular weight of 1500-3000 daltons, an anti-Xa activity of 120-200IU/mg, an anti-IIa activity of less than 10IU/mg and a ratio of anti-Xa activity/anti-IIa activity of more than 30,

the component oligosaccharides of these mixtures:

-containing from 2 to 26 saccharide units,

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

-a hexasaccharide Δ IIa-II comprising the formulas-Is：

And these oligosaccharide mixtures are in the form of alkali metal or alkaline earth metal salts.

2. Oligosaccharide mixture according to claim 1, characterized in that the alkali or alkaline earth metal salt is selected from sodium, potassium, calcium and magnesium salts.

3. Oligosaccharide mixture according to claim 1 or 2, characterized in that it contains a hexasaccharide fraction which represents 15-25% of the oligosaccharide mixture.

4. Oligosaccharide mixture according to claim 3, characterized in that the hexose fraction of the oligosaccharide mixture comprises 8-15% of the hexoses Δ IIa-IIs-Is。

5. Oligosaccharide mixture according to any of claims 1-4, characterized in that its anti-Xa activity is 150-200 IU/mg.

6. Oligosaccharide mixture according to any of claims 1 to 5, characterized in that it has an anti-IIa activity of less than 5IU/mg, in particular 0.5-3.5 IU/mg.

7. Oligosaccharide mixture according to any of claims 1 to 6, characterized in that it has a ratio of the anti-Xa activity/anti-IIa activity of more than 50, in particular more than 100.

8. Oligosaccharide mixture according to any of claims 1 to 7, characterized in that it has an average molecular weight of 2000-.

9. Oligosaccharide mixture according to any of claims 1-8, characterized in that it has an anti-Xa activity of 150-200IU/mg, an anti-IIa activity of 0.5-3.5IU/mg and an average molecular weight of 2400-2650 daltons.

10. A process for the preparation of an oligosaccharide mixture according to any of the claims 1-9, during which process the depolymerization of heparin benzyl ester quaternary ammonium salt is carried out in an organic medium in the presence of a strong organic base having a pKa above 20, characterized in that the organic base used is a phosphazene base, in particular a dichloromethane solution containing a phosphazene base having a water percentage of less than 0.6%.

11. The process according to item 10 for the preparation of the oligosaccharide mixture of item 1, wherein the water content is less than 0.3%, preferably less than 0.2%.

12. The production method according to item 10 or 11, characterized in that the heparin benzyl ester quaternary ammonium salt is benzethonium, cetylpyridinium or cetyltrimethylammonium salt.

13. The production process according to item 10 or 11, characterized in that the phosphazene base is a base of the following formula:

in the formula R₁-R₇Are identical or different and represent a linear or branched alkyl radical having from 1 to 6 carbon atoms.

14. The production method according to item 10 or 11, characterized in that the molar ratio of strong base/ester is 0.2 to 5, preferably 0.6 to 2.

15. A method for preparing oligosaccharides according to any one of items 1 to 9 using heparin, wherein the following operations are performed:

a) the sodium salt of the heparin is exchanged under the action of benzethonium chloride,

b) the benzyl chloride is used for esterifying the benzethonium heparin salt,

c) the obtained benzyl ester salt is exchanged into quaternary ammonium salt,

d) depolymerizing the heparin benzyl ester quat using the method of item 10 or 11,

e) the quaternary ammonium salt is converted into a sodium salt,

f) optionally saponifying said heparin by the action of a base, such as sodium hydroxide,

g) in particular by the action of an oxidizing agent, such as hydrogen peroxide, optionally purified.

16. The process according to item 15, characterized in that the reaction of step a) is carried out by the action of an excess of benzethonium chloride with sodium heparin at a temperature of about 15-25 ℃ and a salt/sodium heparin molar ratio of 3-4.

17. The process according to item 15, characterized in that the esterification reaction of step b) is carried out at a temperature of 25 to 45 ℃, preferably 30 to 40 ℃, in a chlorinated organic solvent, such as chloroform or dichloromethane, and then the ester is recovered in the form of the sodium salt by precipitation with a 10% by weight solution of sodium acetate in alcohol, such as methanol, at a concentration of 1 to 1.2 volumes of alcohol per volume of reaction medium.

18. Process according to item 15 or 17, characterized in that the degree of esterification of the quaternary ammonium salt of heparin benzyl ester is between 50 and 100%, preferably between 70 and 90%.

19. The method according to item 15, 17 or 18, characterized in that 0.5-1.5 parts by weight of benzyl chloride is used per 1 part by weight of heparin benzethonium salt and the reaction time is 10-35 hours.

20. The process according to item 15, characterized in that the salt exchange of step c) is carried out in an aqueous medium at a temperature of 10-25 ℃ using quaternary ammonium chlorides, preferably benzethonium chloride, cetylpyridinium chloride or cetyltrimethylammonium chloride.

21. The process according to item 20, characterized in that the quaternary ammonium chloride/heparin benzyl ester sodium salt molar ratio is between 2 and 3.

22. Process according to item 15, characterized in that the depolymerised heparin benzyl ester quaternary ammonium salt is converted into the sodium salt by treating the reaction medium with an alcoholic sodium acetate solution, preferably with a 10% methanolic sodium acetate solution (weight/volume), at a temperature of 15-25 ℃ (step e).

23. The process according to claim 15, characterized in that the saponification is carried out (step f) in an aqueous medium at a temperature of 0-20 ℃, preferably 0-10 ℃, using an alkali metal hydroxide, such as sodium hydroxide, potassium hydroxide or lithium hydroxide.

24. The process according to item 23, characterized in that 1 to 5 molar equivalents of alkali metal hydroxide, preferably 1 to 2 molar equivalents of sodium hydroxide, are used.

25. Process according to claim 15, characterized in that the purification is carried out with hydrogen peroxide in an aqueous medium at a temperature of 10-50 ℃, preferably 20-40 ℃ (step g).

26. An oligosaccharide according to any one of claims 1-9 for use as a medicament.

27. An oligosaccharide according to any one of claims 1-9 as a medicament having antithrombotic activity.

28. The medicament according to item 26 or 27 for the treatment or prevention of venous and arterial thrombosis, deep vein thrombosis, pulmonary vascular infarction, unstable angina, myocardial infarction, myocardial ischemia, peripheral arterial obstruction disease and atrial fibrillation, smooth muscle cell proliferation, atherosclerotic diseases and atherosclerosis, for the treatment and prevention of cancer by modulation of angiogenesis and growth factors, and for the treatment and prevention of diabetic conditions such as diabetic retinopathy and nephropathy.

29. A pharmaceutical composition comprising at least one drug as defined in item 26 and one or more pharmaceutically inert excipients or carriers or additives.

30. The pharmaceutical composition according to item 29, characterized in that it is a solution administered by subcutaneous or intravenous injection.

31. The pharmaceutical composition according to item 29, characterized in that it is a formulation to be administered by the pulmonary inhalation method.

32. Pharmaceutical composition according to item 29, characterized in that it is a formulation administered by the oral method.

33. A polysaccharide mixture as defined in any of the items 1 to 9, which is obtainable by a process as defined in item 15.

The following examples illustrate the invention without limiting it.

Example A: preparation of heparin benzyl ester benzyl ethyl ammonium salt

Benzethonium heparin salt

A solution of 25g benzethonium chloride in 125ml of water is added to a solution of 10g heparin sodium salt in 100ml of water, the product is filtered, washed with water and dried.

Heparin benzyl ester (sodium salt)

16ml of benzyl chloride are added to a solution of 20g of heparin benzethonium salt in 80ml of dichloromethane. The solution was heated at a temperature of 30 ℃ for 12 hours. Then 108ml of 10% sodium acetate in methanol are added and the mixture is filtered, washed with methanol and dried. This gives 7.6g of heparin benzyl ester in the form of the sodium salt, whose degree of esterification is 77%.

Heparin benzyl ester (benzyl ethyl ammonium salt)

36g (0.0549mol) of heparin benzyl ester (sodium salt) and 540ml of distilled water were introduced into a2 liter Erlenmeyer flask A. After homogenization at a temperature of about 20 ℃, a pale yellow solution is obtained. A solution of 64.45g (0.1438mol) benzethonium chloride and 450ml of water was prepared in a 1 l Erlenmeyer flask B with magnetic stirring. The solution in Erlenmeyer flask B was poured into the solution in Erlenmeyer flask A over about 35 minutes with stirring. It was observed that a large amount of white precipitate had formed. Erlenmeyer flask B was rinsed with 200ml of distilled water and washing water was added to Erlenmeyer flask A. The stirring was then stopped and the suspension was allowed to settle for an additional 12 hours. Once this time has elapsed, the clear portion of the supernatant is removed and discarded. 560ml of water were then added to the settled precipitate (in the form of a slurry) and the mixture was stirred for 20 minutes. This precipitate was allowed to settle for about 30 minutes. The supernatant was removed and discarded (560 ml). The precipitated precipitate was washed with about 560ml of distilled water, and this washing operation was repeated twice. In the final washing operation, this precipitate was in the form of a suspension and was filtered through a No. 3 sintered glass filter. This filter cake was then washed 4 times with 200ml of distilled water each time. This white wet solid was dehydrated and dried under reduced pressure (2.7kPa) at a temperature of about 60 ℃. After drying for 12 hours, 87.5g of heparin benzyl ester were obtained as benzethonium salt. The yield thereof was 94.9%.

Example B: description of the hexasaccharide ATIII (. DELTA.IIa-IIs-Is)

At D₂Proton spectrum in O, 500MHz, T298K, δ, expressed in ppm: 1.97(3H, s), 3.18(1H, dd, 10 and 3Hz), 3.30(1H, t, 8Hz), 3.37(1H, dd, 10 and 3Hz), 3.60(2H, m), 3.65-3.85(6H, m), 3.87(2H, m), 3.95(1H, d, 8Hz), 4.03(IH, d, 8Hz), 4.05-4.13(4H, m), 4.16-4.45(8H, m), 4.52(1H, d, 8Hz), 4.67(1H, m), 5.06(1H, d, 6Hz), 5.10(1H, d, 3Hz), 5.33(1H, d, 4Hz), 5.36(1H, d, 3Hz), 5.46(1H, d, 3Hz), 5.72H, 4 Hz).

4-deoxy- α -L-threo-hexenosylruronic acid- (1 → 4) -2-deoxy-2-acetylamino-6-O-sulfo- α -D-glucopyranosyl- (1 → 4) - β -D-glucopyranosyl uronic acid- (1 → 4) -2-deoxy-2-sulfonylamino-3, 6-di-O-sulfo- α -D-glucopyranosyl) - (1 → 4) -2-O-sulfo- α -L-iduronate uronic acid- (1 → 4) -2-deoxy-2-sulfonylamino-6-O-sulfo- α -D-glucopyranosyl decasodium salt.

Examples 1-7 and 12 illustrate the effect of water content on the selectivity of the polymerization and on the anti-Xa and anti-IIa activity of the products obtained.

Examples 8-10 illustrate the influence of the number of base equivalents on the anti-Xa and anti-IIa activity of the resulting products (using a water content of 0.1%)

Example 11 illustrates the use of a phosphazene base other than 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 2, 3-diazaphosphocyclohexene:

tert-butyliminotris (pyrrolidin-1-yl) phosphine was used.

Example 1

Depolymerization and conversion to sodium salt (0.1% water):

70ml of methylene chloride was placed in Erlenmeyer flask A. 10g (0.006mol) of heparin benzyl ester obtained as described in example A (degree of esterification: 75%, benzethonium salt) are added slowly with stirring. The water content of the reaction medium is adjusted to 0.1%. The solution was heated to 40 ℃ under nitrogen. After complete dissolution, the solution was cooled to a temperature of about 20 ℃ and then 1.75ml (0.006mol) of 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 2, 3-diazaphosphocyclohexene were added. The resulting mixture was stirred at a temperature of about 20 ℃ for 24 hours. At this time, a solution of 30g of anhydrous sodium acetate in 300ml of methanol was prepared in Erlenmeyer flask B. After complete dissolution, 5g of Hyflo supercel diatomaceous earth was added to the solution. The reaction mixture in Erlenmeyer flask A was poured into a solution of sodium acetate in methanol at a temperature of about 5 ℃ over 1 minute 30 seconds with magnetic stirring. After stirring for 5 minutes, this suspension was decanted for 1 hour 30 minutes. The clear portion of the supernatant was separated and discarded (220 ml). 220ml of methanol were then added to this settled precipitate and stirred for a further 5 minutes. The precipitate was allowed to settle for an additional about 1 hour and 20 minutes. This supernatant (250ml) was separated and discarded. 250ml of methanol were then added to this settled precipitate and the mixture was stirred for 5 minutes. The precipitate in the suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was washed with 100ml of methanol. The pale yellow wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After 18 hours of drying, 2.51g of sodium salt of depolymerized heparin in kieselguhr (5g) were obtained. The yield thereof was 64%.

Saponification:

2.5g (0.0038mol) of the crude sodium salt of depolymerized heparin obtained above in diatomaceous earth (5g) and 17ml of water were placed in a 50ml Erlenmeyer flask. This suspension was filtered through a No. 3 sintered glass filter and rinsed twice with 5ml of water each time. The obtained filtrate was charged into a 150ml Erlenmeyer flask. 0.4ml (0.004mol) of 30% sodium hydroxide solution was added with magnetic stirring at a temperature of about 5 ℃. After the addition was complete, the mixture was stirred for 2 hours. The solution was neutralized with 1N HCl and 3g of sodium chloride was added. After dissolution, 21ml of methanol were added to the reaction medium. After stirring for 15 minutes, 44ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was allowed to settle at a temperature of about 5 c for 45 minutes. The supernatant (90ml) was then separated and discarded. 90ml of methanol was added to this settled precipitate and stirred for 5 minutes. This precipitate was allowed to settle for about 20 minutes. The supernatant (80ml) was separated and discarded. To the precipitated precipitate was added 80ml of methanol, followed by stirring for 5 minutes. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was washed with 50ml of methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying for 18 hours, 1.31g of crude depolymerized heparin (sodium salt) was obtained. The yield was 66%.

And (3) purification:

1.3g of the previously obtained crude depolymerized heparin and 13ml of distilled water were charged into a 50ml Erlenmeyer flask. This mixture was warmed to 40 ℃ under magnetic stirring. The pH was adjusted to 9.7. + -. 0.1 by addition of IN sodium hydroxide. The reaction medium is filtered using a 0.45 μm membrane and 0.07ml of 30% aqueous hydrogen peroxide solution are added. After stirring at a temperature of about 20 ℃ for about 2 hours, the mixture was neutralized by adding 1N HCl followed by the addition of 2g of sodium chloride. The solution is then filtered through a 0.45 μm membrane and poured into 14ml of methanol. The solution was then cooled to 10 ℃ and stirred for about 15 minutes. Then, 36ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was allowed to decant for about 15 minutes. The supernatant (50ml) was then separated and discarded. 50ml of methanol was added to this settled precipitate and stirred for 5 minutes. The precipitate was allowed to settle for about 25 minutes. The supernatant (50ml) was separated and discarded. The precipitate in the suspension was then filtered through a No. 3 sintered glass filter. The resulting white filter cake was washed with 50ml of methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying for 18 hours, 1.13g of pure depolymerized heparin (sodium salt) was obtained. The yield thereof was 87%.

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

average molecular weight: 2600 Dalton

anti-Xa activity: 177IU/mg

anti-IIa Activity: 1.5IU/mg

anti-Xa activity/anti-IIa activity ratio: 118

Example 2

Depolymerization and conversion to sodium salt (0.2% water)

70ml of methylene chloride was charged into Erlenmeyer flask A. 10g (0.006mol) of heparin benzyl ester (degree of esterification: 75%, benzethonium salt) obtained as described in example A were added slowly with stirring and under nitrogen pressure. The water content of the reaction medium is adjusted to 0.2%. After complete dissolution, 1.75ml (0.006mol) of 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 2, 3-diazaphosphocyclohexene were added. This mixture was stirred at a temperature of about 20 ℃ for 24 hours. At this time, a solution of 30g of anhydrous sodium acetate in 300ml of methanol was prepared in Erlenmeyer flask B. After complete dissolution, 5g of Hyflo supercel diatomaceous earth was added to the solution. The reaction mixture in Erlenmeyer flask A was poured into the above-mentioned sodium acetate methanol solution within 1 minute and 30 seconds, with magnetic stirring and at a temperature of about 5 ℃. After stirring for 5 minutes, this suspension was decanted for 2 hours. The clear portion (220ml) of the supernatant was separated and discarded. 220ml of methanol was added to the precipitated precipitate and stirred for 5 minutes. This precipitate was allowed to settle again by about 2 hours. The supernatant (230ml) was separated and discarded. 230ml of methanol were added to this settled precipitate. The precipitate in the suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was then washed with 150ml of methanol. The pale yellow wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After 18 hours of drying, 2.63g of crude depolymerized heparin in celite (5g) was obtained. The yield thereof was 67%.

Saponification:

2.5g (0.0038mol) of the crude sodium salt of depolymerized heparin obtained above in diatomaceous earth (5g) and 18ml of water were placed in a 50ml Erlenmeyer flask. This suspension was filtered through a No. 3 sintered glass filter and rinsed twice with 5ml of water each time. The obtained filtrate was charged into a 150ml Erlenmeyer flask. 0.4ml (0.004mol) of 30% sodium hydroxide solution was added with magnetic stirring at a temperature of about 5 ℃. After the addition was complete, the mixture was stirred for 2 hours. The solution was neutralized with 1N HCl and 2g sodium chloride was added. 14ml of methanol were added to the reaction medium. After stirring for 15 minutes, 36ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was allowed to settle at a temperature of about 5 c for 45 minutes. The supernatant (80ml) was then separated and discarded. 80ml of methanol was added to this settled precipitate and stirred for 5 minutes. To the precipitated precipitate was added 80ml of methanol, followed by stirring for 5 minutes. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was washed with 50ml of methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying for 48 hours, 2.3g of crude depolymerized heparin (sodium salt) were obtained. The yield was 65%.

And (3) purification:

1.4g of the previously obtained crude depolymerized heparin and 15ml of distilled water were charged into a 50ml Erlenmeyer flask. This mixture was warmed to 40 ℃ under magnetic stirring. The pH was adjusted to 9.7. + -. 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered using a 0.45 μm membrane and 0.07ml of 30% aqueous hydrogen peroxide solution are added. Stirring was carried out at a temperature of about 40 ℃ for about 2 hours, after which the mixture was cooled to a temperature of 20 ℃ and neutralized by addition of 1N HCl. To the reaction medium was added 2g of sodium chloride. The solution is then filtered through a 0.45 μm membrane and poured into 14ml of methanol. The solution was then cooled to 10 ℃ and stirred for about 15 minutes. At this time, 36ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was allowed to decant for about 15 minutes. The supernatant (40ml) was then separated and discarded. 40ml of methanol was added to this settled precipitate and stirred for 5 minutes. The precipitate was allowed to settle for about 20 minutes. The supernatant (50ml) was separated and discarded. The precipitate in the suspension was then filtered through a No. 3 sintered glass filter. The resulting white filter cake was washed with 50ml of methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying for 18 hours, 1.2g of pure depolymerized heparin (sodium salt) was obtained. The yield thereof was 86%.

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

average molecular weight: 2650 Dalton

anti-Xa activity: 161IU/mg

anti-IIa Activity: 1.4IU/mg

anti-Xa activity/anti-IIa activity ratio: 115

Example 3

Depolymerization and conversion to sodium salt (0.3% water)

70ml of methylene chloride was charged into Erlenmeyer flask A. 10g (0.006mol) of heparin benzyl ester (degree of esterification: 75%, benzethonium salt) obtained as described in example A were added with stirring and under nitrogen pressure. The water content of the reaction medium is adjusted to 0.3%. After complete dissolution, 1.75ml (0.006mol) of 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 2, 3-diazaphosphocyclohexene were added. This mixture was stirred at a temperature of about 20 ℃ for 24 hours. At this time, a solution of 30g of anhydrous sodium acetate in 300ml of methanol was prepared in Erlenmeyer flask B. After complete dissolution, 5g of Hyflo supercel diatomaceous earth was added to the solution. The reaction mixture in Erlenmeyer flask A was poured into the above-mentioned sodium acetate methanol solution within 1 minute and 30 seconds, with magnetic stirring and at a temperature of about 5 ℃. After stirring for 5 minutes, this suspension was decanted for 1 hour 10 minutes. The clear portion (220ml) of the supernatant was separated and discarded. 220ml of methanol was added to the precipitated precipitate and stirred for 5 minutes. The precipitate in the suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was then washed with 100ml of methanol. The wet solid was dewatered and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After 18 hours of drying, 2.57g of crude depolymerized heparin sodium salt in diatomaceous earth (5g) were obtained. The yield thereof was 66%.

Saponification:

2.5g (0.0038mol) of the crude sodium salt of depolymerized heparin obtained above in diatomaceous earth (5g) and 18ml of water were placed in a 50ml Erlenmeyer flask. This suspension was filtered through a No. 3 sintered glass filter and rinsed twice with 5ml of water each time. The obtained filtrate was charged into a 150ml Erlenmeyer flask. 0.4ml (0.004mol) of 30% sodium hydroxide solution was added with magnetic stirring at a temperature of about 5 ℃. After the addition was complete, the mixture was stirred for 2 hours. The solution was neutralized with 1N HCl and 3g of sodium chloride was added. 15ml of methanol were added to the reaction medium. After stirring for 15 minutes, 36ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was decanted for 1 hour. The supernatant (70ml) was then separated and discarded. 70ml of methanol was added to this settled precipitate and stirred for 5 minutes. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was washed with 50ml of methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of 40 ℃. After drying for 48 hours, 1.42g of crude depolymerized heparin (sodium salt) was obtained. The yield was 62%.

And (3) purification:

1.4g of the previously obtained crude depolymerized heparin and 14ml of distilled water were charged into a 50ml Erlenmeyer flask. This mixture was warmed to 40 ℃ under magnetic stirring. The pH was adjusted to 9.7. + -. 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered using a 0.45 μm membrane and 0.07ml of 30% aqueous hydrogen peroxide solution are added. After stirring at a temperature of about 20 ℃ for about 2 hours, 1N HCl was added to the mixture for neutralization, followed by addition of 2g of sodium chloride. After dissolution, the solution was filtered through a 0.45 μm membrane and poured into 14ml of methanol. The filtrate was then cooled to 10 ℃ and stirred for 15 minutes. At this time, 36ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was decanted for about 40 minutes. The supernatant (50ml) was then separated and discarded. 50ml of methanol was added to this settled precipitate (in the form of a slurry) and stirred for 5 minutes. The precipitate was allowed to settle for about 25 minutes. The precipitate in the suspension was then filtered through a No. 3 sintered glass filter. The resulting white filter cake was washed with 50ml of methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying for 18 hours, 1.24g of pure depolymerized heparin (sodium salt) was obtained. The yield thereof was 89%.

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

average molecular weight: 2400 Dalton

anti-Xa activity: 132IU/mg

anti-IIa Activity: 1.4IU/mg

anti-Xa activity/anti-IIa activity ratio: 94

Example 4

Depolymerization and conversion to sodium salt (0.4% water)

70ml of methylene chloride was charged into Erlenmeyer flask A. 10g (0.006mol) of heparin benzyl ester (degree of esterification: 75%, benzethonium salt) obtained as described in example A were added slowly with stirring. The water content of the reaction medium is adjusted to 0.4%. The solution was heated at 30 ℃ under nitrogen. After complete dissolution, the temperature was again lowered to about 20 ℃ and 1.75ml (0.006mol) of 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 2, 3-diazaphosphocyclohexene were added. This mixture was stirred at a temperature of about 20 ℃ for 24 hours. At this time, a solution of 30g of anhydrous sodium acetate in 300ml of methanol was prepared in Erlenmeyer flask B. After complete dissolution, 5g of Hyflo supercel diatomaceous earth was added to the solution. The reaction mixture in Erlenmeyer flask A was poured into the above-mentioned sodium acetate methanol solution within 1 minute and 30 seconds, with magnetic stirring and at a temperature of about 5 ℃. After stirring for 5 minutes, this suspension was decanted for 2 hours. The clear portion (80ml) of the supernatant was separated and discarded. 80ml of methanol was added to the precipitated precipitate and stirred for 5 minutes. The precipitate was allowed to settle for about 1 hour. The supernatant was separated and discarded (80 ml). 80ml of methanol was added to the precipitated precipitate and stirred for 5 minutes. The precipitate in the suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was then washed with 150ml of methanol. The pale yellow wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After 18 hours of drying, 3.25g of crude depolymerized heparin sodium salt in celite (5g) was obtained. The yield was 83%.

Saponification:

3.1g (0.0018mol) of the previously obtained crude sodium salt of depolymerized heparin in diatomaceous earth (10g) and 21ml of water were placed in a 50ml Erlenmeyer flask. This suspension was filtered through a No. 3 sintered glass filter and rinsed twice with 6ml of water each time. The obtained filtrate was charged into a 150ml Erlenmeyer flask. 0.7ml (0.007mol) of 30% sodium hydroxide solution was added with magnetic stirring at a temperature of about 5 ℃. After the addition was complete, the mixture was stirred for 2 hours. The solution was neutralized with 1N HCl and 4g of sodium chloride was added. 28ml of methanol were added to the reaction medium. After stirring for 15 minutes, 72ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was decanted for 1 hour. The supernatant (90ml) was then separated and discarded. 90ml of methanol was added to this settled precipitate and stirred for 5 minutes. The precipitate was allowed to settle for an additional 20 minutes. The supernatant (90ml) was then separated and discarded. 90ml of methanol was added to this settled precipitate and stirred for 5 minutes. The suspended precipitate was filtered through a No. 3 sintered glass filter. The resulting filter cake was washed with 50ml of methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying for 48 hours, 1.9g of crude depolymerized heparin (sodium salt) was obtained. The yield was 67%.

And (3) purification:

1.9g of the previously obtained crude depolymerized heparin and 19ml of distilled water were charged into a 50ml Erlenmeyer flask. This mixture was warmed to 40 ℃ under magnetic stirring. The pH was adjusted to 9.7. + -. 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered using a 0.45 μm membrane and 0.1ml of 30% aqueous hydrogen peroxide solution are added. After stirring at a temperature of about 20 ℃ for about 2 hours, 1N HCl was added to the mixture for neutralization, followed by addition of 2g of sodium chloride. This solution was filtered through a 0.45 μm membrane, and 14ml of methanol was poured in and stirred for 15 minutes. At this time, 36ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was decanted for 15 minutes. The supernatant (40ml) was then separated and discarded. 40ml of methanol was added to this settled precipitate and stirred for 5 minutes. The precipitate was allowed to settle for about 20 minutes. The supernatant (50ml) was separated and discarded. 500ml of methanol was added to this settled precipitate and stirred for 5 minutes. The precipitate was allowed to settle for about 20 minutes. The precipitate was then filtered through a No. 3 sintered glass filter. The resulting white filter cake was washed with 50ml of methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying for 72 hours, 1.56g of pure depolymerized heparin (sodium salt) was obtained. The yield thereof was 82%.

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

average molecular weight: 2350 Dalton

anti-Xa activity: 122IU/mg

anti-IIa Activity: 1.3IU/mg

anti-Xa activity/anti-IIa activity ratio: 94

Example 5

Depolymerization and conversion to sodium salt (0.57% water)

140ml of methylene chloride was charged into Erlenmeyer flask A. 20g (0.019mol) of heparin benzyl ester (degree of esterification: 75%, benzethonium salt) obtained as described in example A were slowly added with stirring. The water content of the reaction medium was adjusted to 0.57%. After complete dissolution, 3.5ml (0.012mol) of 2-t-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 2, 3-diazaphosphocyclohexene were added. This mixture was stirred at a temperature of about 20 ℃ for 24 hours. At this time, a solution of 60g of anhydrous sodium acetate in 600ml of methanol was prepared in Erlenmeyer flask B. After complete dissolution, 10g of Hyflo supercel diatomaceous earth was added to the solution. The reaction mixture in Erlenmeyer flask A was poured into the above-mentioned sodium acetate methanol solution within 1 minute and 30 seconds, with magnetic stirring and at a temperature of about 4 ℃. After stirring for 5 minutes, this suspension was decanted for 30 minutes. The clear portion (400ml) of the supernatant was separated and discarded. 400ml of methanol was added to the precipitated precipitate and stirred for 5 minutes. This precipitate was allowed to settle for about 1 hour. The supernatant (420ml) was separated and discarded. 420ml of methanol was added to the precipitated precipitate and stirred for 5 minutes. The suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was then washed with 200ml of methanol. The pale yellow wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After 18 hours of drying, 6.66g of crude depolymerized heparin sodium salt in celite (10g) was obtained. The yield was 85%.

Saponification:

6.66g (0.0101mol) of the previously obtained crude sodium salt of depolymerized heparin in diatomaceous earth (10g) and 47ml of water were placed in a 50ml Erlenmeyer flask. This suspension was filtered through a No. 3 sintered glass filter and rinsed twice with 15ml of water each time. The obtained filtrate was charged into a 150ml Erlenmeyer flask. 1.1ml (0.011mol) of 30% sodium hydroxide solution are added with magnetic stirring at a temperature of about 5 ℃. After the addition was complete, the mixture was stirred for 2 hours. This solution was neutralized with 1N HCl and 9.5g of sodium chloride was added. 66ml of methanol were added to the reaction medium. After stirring for 15 minutes, 171ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was allowed to settle out at a temperature of about 5 ℃ for 3/4 hours. The supernatant (160ml) was then separated and discarded. 160ml of methanol was added to this settled precipitate and stirred for 5 minutes. The precipitate was allowed to settle for an additional 20 minutes. The supernatant (180ml) was then separated and discarded. To this settled precipitate was added 180ml of methanol and stirred for 5 minutes. The suspension was filtered through a No. 3 sintered glass filter. The filter cake obtained is washed twice more with 50ml of methanol each time. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying for 18 hours, 4.53g of crude depolymerized heparin (sodium salt) were obtained. The yield was 74%.

And (3) purification:

4.53g of the previously obtained crude depolymerized heparin and 45ml of distilled water were placed in a 100ml Erlenmeyer flask. This mixture was warmed to 40 ℃ under magnetic stirring. The pH was adjusted to 9.7. + -. 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered using a 0.45 μm membrane and 0.25ml of 30% aqueous hydrogen peroxide solution are added. After stirring at a temperature of about 20 ℃ for about 2 hours, 1N HCl was added to the mixture for neutralization, followed by addition of 5.5g sodium chloride. This solution was filtered through a 0.45 μm membrane and 38ml of methanol was poured at a temperature of about 10 ℃. The solution was then raised to 20 ℃ and stirred for 15 minutes. At this point, l 00ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was decanted for 20 minutes. The supernatant (90ml) was then separated and discarded. 90ml of methanol was added to this settled precipitate and stirred for 5 minutes. The precipitate was allowed to settle for about 25 minutes. The supernatant (100ml) was separated and discarded. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting white filter cake was washed with 50ml of methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying for 18 hours, 3.7g of pure depolymerized heparin (sodium salt) are obtained. The yield thereof was 82%.

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

average molecular weight: 2200 Dalton

anti-Xa activity: 120IU/mg

anti-IIa Activity: 1.4IU/mg

anti-Xa activity/anti-IIa activity ratio: 86

Example 6

Depolymerization and conversion to sodium salt (1.8% water)

140ml of methylene chloride was charged into Erlenmeyer flask A. 20g (0.019mol) of heparin benzyl ester (degree of esterification: 75%, benzethonium salt) obtained as described in example A were slowly added with stirring. The water content of the reaction medium is adjusted to 1.8%. After complete dissolution, 3.5ml (0.012mol) of 2-t-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 2, 3-diazaphosphocyclohexene were added. This mixture was stirred at a temperature of about 20 ℃ for 24 hours. At this time, a solution of 60g of anhydrous sodium acetate in 600ml of methanol was prepared in Erlenmeyer flask B. After complete dissolution, 10g of Hyflo supercel diatomaceous earth was added to the solution. The reaction mixture in Erlenmeyer flask A was poured into the above-mentioned sodium acetate methanol solution within 1 minute and 30 seconds, with magnetic stirring and at a temperature of about 4 ℃. After stirring for 5 minutes, this suspension was decanted for 30 minutes. The clear portion (400ml) of the supernatant was separated and discarded. 400ml of methanol was added to the precipitated precipitate and stirred for 5 minutes. This precipitate was allowed to settle for about 1 hour. The supernatant (420ml) was separated and discarded. 420ml of methanol was added to the precipitated precipitate and stirred for 5 minutes. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was then washed with 200ml of methanol. The pale yellow wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 50 ℃. After 18 hours of drying, 7.54g of crude depolymerized heparin sodium salt in celite (10g) was obtained. The yield was 96%.

Saponification:

7.54g (0.0101mol) of the previously obtained crude sodium salt of depolymerized heparin in diatomaceous earth (10g) and 53ml of water were placed in a 50ml Erlenmeyer flask. This suspension was filtered through a No. 3 sintered glass filter and rinsed twice with 15ml of water each time. The obtained filtrate was charged into a 150ml Erlenmeyer flask. 1.25ml (0.012mol) of 30% sodium hydroxide solution are added with magnetic stirring at a temperature of about 4 ℃. After the addition was complete, the mixture was stirred for 2 hours. This solution was neutralized with 1N HCl and 10.5g of sodium chloride was added. 70ml of methanol were added to the reaction medium. After stirring for 15 minutes, 190ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was allowed to settle at a temperature of about 4 ℃ for 3/4 hours. The supernatant (180ml) was then separated and discarded. To this settled precipitate was added 180ml of methanol and stirred for 5 minutes. The precipitate was allowed to settle for an additional 20 minutes. The supernatant (180ml) was then separated and discarded. To this settled precipitate was added 180ml of methanol and stirred for 5 minutes. The suspended precipitate was filtered through a No. 3 sintered glass filter. The filter cake obtained is washed twice more with 50ml of methanol each time. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of 50 ℃. After drying for 18 hours, 5.53g of crude depolymerized heparin (sodium salt) were obtained. The yield was 80%.

And (3) purification:

5.53g of the previously obtained crude depolymerized heparin and 55ml of distilled water were placed in a 100ml Erlenmeyer flask. This mixture was warmed to 40 ℃ under magnetic stirring. The pH was adjusted to 9.7. + -. 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered using a 0.45 μm membrane and 0.31ml of 30% aqueous hydrogen peroxide solution are added. After stirring at a temperature of about 20 ℃ for about 2 hours, 1N HCl was added to the mixture for neutralization, followed by addition of 7g of sodium chloride. This solution is filtered through a 0.45 μm membrane and 49ml of methanol are poured at a temperature of about 10 ℃. The solution was then raised to 20 ℃ and stirred for 15 minutes. At this time, 126ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was allowed to settle for 20 minutes. The supernatant (105ml) was then separated and discarded. 105ml of methanol was added to this settled precipitate and stirred for 5 minutes. The precipitate was allowed to settle for about 25 minutes. The supernatant (110ml) was separated and discarded. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting white filter cake was washed with 50ml of methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 55 ℃. After drying for 18 hours, 4.53g of pure depolymerized heparin (sodium salt) are obtained. The yield thereof was 82%.

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

average molecular weight: 2600 Dalton

anti-Xa activity: 105IU/mg

anti-IIa Activity: 3.1IU/mg

anti-Xa activity/anti-IIa activity ratio: 34

Example 7

Depolymerization and conversion to sodium salt (2.5% water)

140ml of methylene chloride was charged into Erlenmeyer flask A. 20g (0.019mol) of heparin benzyl ester (degree of esterification: 75%, benzethonium salt) obtained as described in example A were slowly added with stirring. The water content of the reaction medium is adjusted to 2.5%. After complete dissolution, 3.5ml (0.012mol) of 2-t-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 2, 3-diazaphosphocyclohexene were added. This mixture was stirred at a temperature of about 20 ℃ for 24 hours. At this time, a solution of 60g of anhydrous sodium acetate in 600ml of methanol was prepared in Erlenmeyer flask B. After complete dissolution, 10g of Hyflo supercel diatomaceous earth was added to the solution. The reaction mixture in Erlenmeyer flask A was poured into the above-mentioned sodium acetate methanol solution within 1 minute and 30 seconds, with magnetic stirring and at a temperature of about 4 ℃. After stirring for 5 minutes, this suspension was decanted for 1 hour. The clear portion (400ml) of the supernatant was separated and discarded. 400ml of methanol was added to the precipitated precipitate and stirred for 5 minutes. This precipitate was allowed to settle for about 30 minutes. The supernatant (400ml) was separated and discarded. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was then washed with 200ml of methanol. The pale yellow wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 50 ℃. After 18 hours of drying, 7.78g of crude depolymerized heparin sodium salt in kieselguhr (10g) are obtained. The yield was 99.6%.

Saponification:

7.78g (0.0119mol) of the previously obtained crude sodium salt of depolymerized heparin in diatomaceous earth (10g) and 79ml of water were placed in a 50ml Erlenmeyer flask. This suspension was filtered through a No. 3 sintered glass filter and rinsed twice with 15ml of water each time. The obtained filtrate was charged into a 150ml Erlenmeyer flask. 1.3ml (0.012mol) of 30% sodium hydroxide solution are added with magnetic stirring at a temperature of about 4 ℃. After the addition was complete, the mixture was stirred for 2 hours. The solution was neutralized with 1N HCl and 10g of sodium chloride was added. 60ml of methanol are added to the reaction medium. After stirring for 15 minutes, 190ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was allowed to settle at a temperature of about 4 ℃ for 3/4 hours. The supernatant (180ml) was then separated and discarded. To this settled precipitate was added 180ml of methanol and stirred for 5 minutes. The precipitate was allowed to settle for an additional 20 minutes. The supernatant (180ml) was then separated and discarded. To this settled precipitate was added 180ml of methanol and stirred for 5 minutes. The suspended precipitate was filtered through a No. 3 sintered glass filter. The filter cake obtained is washed twice more with 50ml of methanol each time. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of 50 ℃. After drying for 18 hours, 5.87g of crude depolymerized heparin (sodium salt) were obtained. The yield was 82%.

And (3) purification:

5.87g of the previously obtained crude depolymerized heparin and 59ml of distilled water were placed in a 100ml Erlenmeyer flask. This mixture was warmed to 40 ℃ under magnetic stirring. The pH was adjusted to 9.7. + -. 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered using a 0.45 μm membrane and 0.34ml of 30% aqueous hydrogen peroxide solution are added. After stirring at a temperature of about 20 ℃ for about 2 hours, 1N HCl was added to the mixture for neutralization, followed by addition of 7g of sodium chloride. This solution is filtered through a 0.45 μm membrane and 49ml of methanol are poured at a temperature of about 10 ℃. The solution was then raised to 20 ℃ and stirred for 15 minutes. At this time, 126ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was allowed to settle for 20 minutes. The supernatant (105ml) was then separated and discarded. 105ml of methanol was added to this settled precipitate and stirred for 5 minutes. The precipitate was allowed to settle for about 25 minutes. The supernatant (110ml) was separated and discarded. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting white filter cake was washed with 50ml of methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 55 ℃. After drying for 18 hours, 5.21g of pure depolymerized heparin (sodium salt) are obtained. The yield thereof was 89%.

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

average molecular weight: 3550 daltons

anti-Xa activity: 99IU/mg

anti-IIa Activity: 13.4IU/mg

anti-Xa activity/anti-IIa activity ratio: 7.4

Example 8

Depolymerization and conversion to sodium salt (0.5 equiv base)

140ml of methylene chloride was charged into Erlenmeyer flask A. 20g (0.019mol) of heparin benzyl ester (degree of esterification: 75%, benzethonium salt) obtained as described in example A were slowly added with stirring. After complete dissolution at a temperature of about 30 ℃ and cooling to a temperature of about 20 ℃ 1.75ml (0.006mol) of 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 2, 3-diazaphosphocyclohexene were added. This mixture was stirred at a temperature of about 20 ℃ for 24 hours. At this time, a solution of 60g of anhydrous sodium acetate in 600ml of methanol was prepared in Erlenmeyer flask B. The reaction mixture in Erlenmeyer flask A was poured into the above sodium acetate methanol solution under magnetic stirring at a temperature of about 4 ℃. After stirring for 1 hour, this suspension was decanted for 2 hours. The clear portion of the supernatant (420ml) was separated and discarded. 420ml of methanol was added to the precipitated precipitate and stirred for 30 minutes. This precipitate was allowed to settle for a further 18 hours. The supernatant (400ml) was separated and discarded. 400ml of methanol was added to the precipitated precipitate and stirred for 30 minutes. The clear portion (400ml) of the supernatant was separated and discarded. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was then washed twice with 100ml of methanol each time. The pale yellow wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 60 ℃. After drying, 5.7g of crude depolymerized heparin sodium salt was obtained. The yield was 73%.

Saponification:

5.7g (0.0086mol) of the previously obtained crude sodium salt of depolymerized heparin and 53ml of water were placed in a 100ml Erlenmeyer flask. 0.93ml (0.009mol) of 30% sodium hydroxide solution are added with magnetic stirring at a temperature of about 4 ℃. After the addition was complete, the mixture was stirred for 2 hours. The solution was neutralized with 1N HCl and 6g sodium chloride was added. 42ml of methanol were added to the reaction medium. After stirring for 15 minutes, 108ml of methanol was added, followed by stirring for 30 minutes. At this point the stirring was stopped and the suspension was allowed to settle at a temperature of about 4 ℃ for 30 minutes. The supernatant (180ml) was then separated and discarded. To this settled precipitate was added 180ml of methanol and stirred for 5 minutes. The precipitate was allowed to settle for an additional 30 minutes. The supernatant (170ml) was then separated and discarded. 170ml of methanol was added to this settled precipitate and stirred for 30 minutes. The suspended precipitate was filtered through a No. 3 sintered glass filter. The filter cake obtained is washed twice more with 30ml of methanol each time. The wet solid is dehydrated and then dried under reduced pressure at a temperature of 60 ℃. After drying, 3.5g of crude depolymerized heparin (sodium salt) was obtained. The yield was 67.4%.

And (3) purification:

3.5g of the previously obtained crude depolymerized heparin and 35ml of distilled water were charged into a 100ml Erlenmeyer flask. This mixture was warmed to 40 ℃ under magnetic stirring. The pH was adjusted to 9.6. + -. 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered using a 0.45 μm membrane and 0.18ml of 30% aqueous hydrogen peroxide solution are added. After stirring at a temperature of about 20 ℃ for about 2 hours, 1N HCl was added to the mixture for neutralization, followed by the addition of 3.6g of sodium chloride. This solution is filtered through a 0.45 μm membrane and 27ml of methanol are poured at a temperature of about 10 ℃. The solution was then raised to 20 ℃ and stirred for 15 minutes. At this time, 65ml of methanol was added, followed by stirring for 30 minutes. At this point the stirring was stopped and the suspension was allowed to settle for 30 minutes. The supernatant (80ml) was then separated and discarded. 80ml of methanol was added to this settled precipitate and stirred for 30 minutes. The precipitate was allowed to settle for about 30 minutes. The supernatant (70ml) was separated and discarded. 70ml of methanol was added to this settled precipitate and stirred for 30 minutes. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting white filter cake was washed twice more with 30ml of methanol each time. This wet solid was dehydrated and dried under reduced pressure at a temperature of about 60 ℃. After drying, 2.8g of pure depolymerized heparin (sodium salt) was obtained. The yield thereof was 80%.

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

average molecular weight: 2900 Dalton

anti-Xa activity: 146.1IU/mg

anti-IIa Activity: 5.1IU/mg

anti-Xa activity/anti-IIa activity ratio: 28.6

Example 9

Depolymerization and conversion to sodium salt (0.6 equiv base)

280ml of dichloromethane are charged into a three-necked flask A. 40g (0.024mol) of heparin benzyl ester (degree of esterification: 75%, benzethonium salt) obtained as described in example A were slowly added with stirring. The solution was completely dissolved at about 30 ℃ and then cooled to about 20 ℃ and 4.2ml (0.014mol) of 2-t-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 2, 3-diazaphosphocyclohexene was added. This mixture was stirred at a temperature of about 20 ℃ for 24 hours. At this time, a solution of 60g of anhydrous sodium acetate in 600ml of methanol was prepared in Erlenmeyer flask B. Half of the reaction mixture in three-necked flask A was poured into the above sodium acetate in methanol solution under magnetic stirring at a temperature of about 4 ℃. After stirring for 1 hour, this suspension was decanted. The clear portion of the supernatant (310ml) was separated and discarded. 310ml of methanol was added to the precipitated precipitate and stirred for 1 hour. This precipitate was allowed to settle for a further 18 hours. The supernatant (400ml) was separated and discarded. 400ml of methanol was added to the precipitated precipitate and stirred for 1 hour. The supernatant (300ml) was separated and discarded. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was then washed twice with 100ml of methanol each time. The pale yellow wet solid was dehydrated and dried under reduced pressure at a temperature of about 60 ℃. After drying, 6g of crude depolymerized heparin sodium salt was obtained. The yield was 77%.

Saponification:

6g (0.0091mol) of the crude sodium salt of depolymerized heparin obtained above and 56ml of water were placed in a 250ml Erlenmeyer flask. 1ml (0.010mol) of 30% sodium hydroxide solution was added with magnetic stirring at a temperature of about 4 ℃. After the addition was complete, the mixture was stirred for 2 hours. The solution was neutralized with 1N HCl and 6.4g of sodium chloride was added. 45ml of methanol were added to the reaction medium. After stirring for 15 minutes, 115ml of methanol was added, followed by stirring for 30 minutes. At this point the stirring was stopped and the suspension was allowed to settle at a temperature of about 4 ℃ for 30 minutes. The supernatant (170ml) was then separated and discarded. 170ml of methanol was added to this settled precipitate and stirred for 30 minutes. The precipitate was allowed to settle for an additional 30 minutes. The supernatant (140ml) was then separated and discarded. 140ml of methanol was added to this settled precipitate and stirred for 30 minutes. The suspended precipitate was filtered through a No. 3 sintered glass filter. The filter cake obtained is washed twice more with 30ml of methanol each time. The wet solid is dehydrated and then dried under reduced pressure at a temperature of 60 ℃. After drying, 3.6g of crude depolymerized heparin (sodium salt) was obtained. The yield was 65.3%.

And (3) purification:

3.5g of the previously obtained crude depolymerized heparin and 35ml of distilled water were charged into a 100ml Erlenmeyer flask. This mixture was warmed to 40 ℃ under magnetic stirring. The pH was adjusted to 9.6. + -. 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered using a 0.45 μm membrane and 0.18ml of 30% aqueous hydrogen peroxide solution are added. After stirring at a temperature of about 20 ℃ for about 2 hours, 1N HCl was added to the mixture for neutralization, followed by the addition of 3.5g of sodium chloride. This solution is filtered through a 0.45 μm membrane and 25ml of methanol are poured at a temperature of about 10 ℃. The solution was then raised to 20 ℃ and stirred for 15 minutes. At this time, 63ml of methanol was added, followed by stirring for 30 minutes. At this point the stirring was stopped and the suspension was allowed to settle for 30 minutes. The supernatant (70ml) was then separated and discarded. 70ml of methanol was added to this settled precipitate and stirred for 30 minutes. The precipitate was allowed to settle for a few more minutes. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting white filter cake was washed twice more with 30ml of methanol each time. This wet solid was dehydrated and dried under reduced pressure at a temperature of about 60 ℃. After drying, 2.5g of pure depolymerized heparin (sodium salt) was obtained. The yield thereof was 71.4%.

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

average molecular weight: 2600 Dalton

anti-Xa activity: 150.5IU/mg

anti-IIa Activity: 3.2IU/mg

anti-Xa activity/anti-IIa activity ratio: 47

Example 10

Depolymerization and conversion to sodium salt (0.8 equiv base)

70ml of methylene chloride are introduced into a three-necked flask A. 10g (0.006mol) of heparin benzyl ester (degree of esterification: 75%, benzethonium salt) obtained as described in example A were added slowly with stirring. The solution was completely dissolved at a temperature of about 30 ℃ and then cooled to a temperature of about 20 ℃ and 1.38ml (0.004mol) of 2-t-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 2, 3-diazaphosphocyclohexene was added. This mixture was stirred at a temperature of about 20 ℃ for 24 hours. At this time, a solution of 30g of anhydrous sodium acetate in 300ml of methanol was prepared in Erlenmeyer flask B. The reaction mixture in three-necked flask A was poured into the above sodium acetate methanol solution over 1 minute and 15 seconds with magnetic stirring and at a temperature of about 4 ℃. After stirring for 5 minutes, this suspension was decanted for 1 hour. The clear portion (190ml) of the supernatant was separated and discarded. 190ml of methanol was added to the precipitated precipitate and stirred for 5 minutes. This precipitate was allowed to settle for about 30 minutes. The supernatant (190ml) was separated and discarded. 190ml of methanol was added to the precipitated precipitate, and stirred for 30 minutes. The supernatant (190ml) was separated and discarded. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was then washed with 150ml of methanol. The pale yellow wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying for 18 hours, 3.05g of crude depolymerized heparin sodium salt are obtained. The yield was 80%.

Saponification:

3.05g (0.0048mol) of the previously obtained crude depolymerized heparin sodium salt and 21ml of water were placed in a 100ml Erlenmeyer flask. The solution was filtered through a No. 3 sintered glass filter and rinsed twice with 6ml of water each time. The obtained filtrate was charged into a 150ml Erlenmeyer flask. 0.6ml (0.006mol) of 30% sodium hydroxide solution are added with magnetic stirring at a temperature of about 4 ℃. After the addition was complete, the mixture was stirred for 2 hours. The solution was neutralized with 1N HCl and 4g of sodium chloride was added. 28ml of methanol were added to the reaction medium. After stirring for 15 minutes, 72ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was allowed to settle at a temperature of about 4 ℃ for 30 minutes. The supernatant (80ml) was then separated and discarded. 80ml of methanol was added to this settled precipitate and stirred for 5 minutes. The precipitate was allowed to settle for an additional 30 minutes. The supernatant (80ml) was then separated and discarded. 80ml of methanol was added to this settled precipitate and stirred for 30 minutes. The suspended precipitate was filtered through a No. 3 sintered glass filter. The resulting filter cake was washed with 50ml of methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of 40 ℃. After drying, 1.6g of crude depolymerized heparin (sodium salt) was obtained. The yield was 57%.

And (3) purification:

1.6g of the previously obtained crude depolymerized heparin and 16ml of distilled water were charged into a 50ml Erlenmeyer flask. This mixture was warmed to 40 ℃ under magnetic stirring. The pH was adjusted to 9.6. + -. 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered using a 0.45 μm membrane and 0.08ml of 30% aqueous hydrogen peroxide solution are added. After stirring at a temperature of about 20 ℃ for about 2 hours, 1N HCl was added to the mixture for neutralization, followed by addition of 2g of sodium chloride. This solution was filtered through a 0.45 μm membrane and 14ml of methanol were poured at a temperature of about 10 ℃. The solution was then raised to 20 ℃ and stirred for 15 minutes. At this time, 36ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was allowed to settle for 30 minutes. The supernatant (50ml) was then separated and discarded. 50ml of methanol was added to this settled precipitate and stirred for 5 minutes. The precipitate was allowed to settle for about 30 minutes. The supernatant (50ml) was separated and discarded. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying, 1.25g of pure depolymerized heparin (sodium salt) was obtained. The yield thereof was 78%.

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

average molecular weight: 2400 Dalton

anti-Xa activity: 154.3IU/mg

anti-IIa Activity: 1.6IU/mg

anti-Xa activity/anti-IIa activity ratio: 96.4

Example 11

Depolymerization and conversion to the sodium salt

140ml of methylene chloride are charged into a three-necked flask A. 20g (0.019mol) of heparin benzyl ester (degree of esterification: 75%, benzethonium salt) obtained as described in example A were slowly added with stirring. The solution was completely dissolved at a temperature of about 40 ℃ and then cooled to a temperature of about 20 ℃ and 3.5ml (0.011mol) of t-butylimino-tris (pyrrolidin-1-yl) phosphine was added. This mixture was stirred at a temperature of about 20 ℃ for 24 hours. At this time, a solution of 60g of anhydrous sodium acetate in 600ml of methanol was prepared in Erlenmeyer flask B. After complete dissolution, 10g of hyflo supercel diatomaceous earth was added to the solution. The reaction mixture in three-necked flask A was poured into the above sodium acetate methanol solution over 1 minute and 30 seconds with magnetic stirring and at a temperature of about 4 ℃. After stirring for 5 minutes, this suspension was decanted for 30 minutes. The clear portion (400ml) of the supernatant was separated and discarded. 400ml of methanol was added to the precipitated precipitate and stirred for 5 minutes. This precipitate was allowed to settle for about 1 hour and 20 minutes. The supernatant (250ml) was separated and discarded. 250ml of methanol was added to the settled precipitate and stirred for 5 minutes. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was then washed with 200ml of methanol. The pale yellow wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying, 5.39g of crude depolymerized heparin sodium salt was obtained. The yield was 69%.

Saponification:

5g (0.0076mol) of the previously obtained crude depolymerized heparin (benzyl ester, sodium salt) and 35ml of water were placed in a 50ml Erlenmeyer flask. The solution was filtered through a No. 3 sintered glass filter and rinsed twice with 10ml of water each time. The obtained filtrate was charged into a 250ml Erlenmeyer flask. 1ml (0.01mol) of 30% sodium hydroxide solution was added with magnetic stirring at a temperature of about 4 ℃. After the addition was complete, the mixture was stirred for 2 hours. The solution was neutralized with 1N HCl and 6g sodium chloride was added. 42ml of methanol were added to the reaction medium. After stirring for 15 minutes, 104ml of methanol was added, followed by stirring for 15 minutes. At this point the stirring was stopped and the suspension was allowed to settle for 1 hour at a temperature of about 4 ℃. The supernatant (140ml) was then separated and discarded. 140ml of methanol was added to this settled precipitate and stirred for 5 minutes. The precipitate was allowed to settle for an additional 45 minutes. The supernatant (160ml) was then separated and discarded. 160ml of methanol was added to this settled precipitate and stirred for 5 minutes. The suspended precipitate was filtered through a No. 3 sintered glass filter. The resulting filter cake was washed with 100ml of methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying, 2.7g of crude depolymerized heparin (sodium salt) were obtained. The yield was 59%.

And (3) purification:

2.6g of the previously obtained crude depolymerized heparin and 25ml of distilled water were charged into a 50ml Erlenmeyer flask. This mixture was warmed to 40 ℃ under magnetic stirring. The pH was adjusted to 9.7. + -. 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered using a 0.45 μm membrane and 0.15ml of 30% aqueous hydrogen peroxide solution are added. After stirring at a temperature of about 20 ℃ for about 2 hours, 1N HCl was added to the mixture for neutralization, followed by the addition of 3g of sodium chloride. This solution is filtered through a 0.45 μm membrane and 21ml of methanol are poured at a temperature of about 10 ℃. The solution was then raised to 20 ℃ and stirred for 15 minutes. At this time, 54ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was allowed to settle for 20 minutes. The supernatant (50ml) was then separated and discarded. 50ml of methanol was added to this settled precipitate and stirred for 5 minutes. The precipitate was allowed to settle for about 20 minutes. The supernatant (50ml) was separated and discarded. The precipitate in suspension was then filtered through a No. 3 sintered glass filter. The resulting white filter cake was washed with 50ml of methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying for 18 hours, 2.35g of pure depolymerized heparin (sodium salt) are obtained. The yield thereof was 90%.

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

average molecular weight: 2400 Dalton

anti-Xa activity: 167.5IU/mg

anti-IIa Activity: 1.1IU/mg

anti-Xa activity/anti-IIa activity ratio: 152

Example 12

Depolymerization and conversion to sodium salt (0.05% water)

140ml of methylene chloride are charged into a three-necked flask A. 20g (0.019mol) of heparin benzyl ester (degree of esterification: 75%, benzethonium salt) obtained as described in example A were slowly added with stirring. To this reaction medium was added 20g of 4A molecular sieve, the water content was adjusted to 0.05% while stirring slowly for 48 hours. The supernatant was transferred to a three-necked flask a under an inert atmosphere. After complete dissolution, 3.5ml (0.012mol) of 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 2, 3-diazaphosphocyclohexene were added. This mixture was stirred at a temperature of about 20 ℃ for 24 hours. At this time, a solution of 60g of anhydrous sodium acetate in 600ml of methanol was prepared in Erlenmeyer flask B. After complete dissolution, 10g of Hyflo supercel diatomaceous earth was added to the solution. The reaction mixture in three-necked flask A was poured into the above sodium acetate methanol solution over 2 minutes with magnetic stirring and at a temperature of about 4 ℃. After stirring for 5 minutes, this suspension was decanted for 1 hour. The clear portion of the supernatant (420ml) was separated and discarded. 420ml of methanol was added to the precipitated precipitate and stirred for 15 minutes. This precipitate was allowed to settle for about 1 hour. The supernatant (450ml) was separated and discarded. 450ml of methanol was added to the settled precipitate and stirred for 15 minutes. The suspension was then filtered through a No. 3 sintered glass filter. The resulting filter cake was then washed with 200ml of methanol. The pale yellow wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 50 ℃. After drying for 16 hours, 5.36g of crude depolymerized heparin sodium salt in diatomaceous earth (10g) were obtained. The yield was 68.6%.

Saponification:

5.36g (0.00817mol) of the previously obtained crude sodium salt of depolymerized heparin and 50ml of water were placed in a 50ml Erlenmeyer flask. The suspension was filtered through a No. 3 sintered glass filter and rinsed four times with 15ml of water each time. The obtained filtrate was charged into a 150ml Erlenmeyer flask. 1.01ml (0.0122mol) of 35% sodium hydroxide solution are added with magnetic stirring at a temperature of about 4 ℃. After the addition was complete, the mixture was stirred for 3 hours. The solution was neutralized with 1N HCl and 11g of sodium chloride was added. 77ml of methanol are added to the reaction medium. After stirring for 15 minutes, 200ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was allowed to settle for 1 hour at a temperature of about 4 ℃. The supernatant (240ml) was then separated and discarded. 240ml of methanol was added to this settled precipitate and stirred for 10 minutes. The precipitate was allowed to settle for an additional 30 minutes. The supernatant (225ml) was then separated and discarded. 225ml of methanol was added to this settled precipitate and stirred for 10 minutes. The suspension was filtered through a No. 3 sintered glass filter. The filter cake obtained is washed twice more with 50ml of methanol each time. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 40 ℃. After drying for 18 hours, 2.65g of crude depolymerized heparin (sodium salt) were obtained. The yield was 53.7%.

And (3) purification:

2.65g of the crude depolymerized heparin obtained above and 26.5ml of distilled water were charged into a 100ml Erlenmeyer flask. This mixture was warmed to 40 ℃ under magnetic stirring. The pH was adjusted to 9.7. + -. 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered using a 0.45 μm membrane and 0.25ml of 30% aqueous hydrogen peroxide solution are added. After stirring at a temperature of about 20 ℃ for about 2 hours, 1N HCl was added to the mixture for neutralization, followed by the addition of 3g of sodium chloride. This solution is filtered through a 0.45 μm membrane and 21ml of methanol are poured at a temperature of about 10 ℃. The solution was then raised to 20 ℃ and stirred for 15 minutes. At this time, 54ml of methanol was added, followed by stirring for 1 hour. At this point the stirring was stopped and the suspension was allowed to settle for 45 minutes. The supernatant (46ml) was then separated and discarded. To this settled precipitate, 46ml of methanol was added and stirred for 5 minutes. The precipitate was allowed to settle for about 30 minutes. The supernatant (50ml) was separated and discarded. The precipitate in suspension was then filtered through a No. 4 sintered glass filter. The resulting white filter cake was washed with two more portions of 10ml methanol. This wet solid was dehydrated and dried under reduced pressure (6kPa) at a temperature of about 50 ℃. After drying for 18 hours, 2.363g of pure depolymerized heparin (sodium salt) were obtained. The yield thereof was 89.1%.

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

average molecular weight: 2500 Dalton

anti-Xa activity: 192IU/mg

anti-IIa Activity: 1.3IU/mg

anti-Xa activity/anti-IIa activity ratio: 148.

Claims (11)

1. Process for the preparation of oligosaccharide mixtures as described below using heparin, in which the following operations are carried out:

a) the sodium salt of the heparin is exchanged under the action of benzethonium chloride,

b) the benzyl chloride is used for esterifying the benzethonium heparin salt,

c) the obtained benzyl ester salt is exchanged into quaternary ammonium salt,

d) depolymerizing the heparin benzyl ester quaternary ammonium salt by the method of item 10 or 11 of the specification,

e) the quaternary ammonium salt is converted into a sodium salt,

f) optionally saponifying said heparin by the action of a base, such as sodium hydroxide,

g) in particular by the action of an oxidizing agent, such as hydrogen peroxide, optionally purified,

said mixture of sulfated oligosaccharides, which have the general structure of the polysaccharides that make up heparin, also has the following characteristics:

-they have an average molecular weight of 1500-3000 daltons, an anti-Xa activity of 120-200IU/mg, an anti-IIa activity of less than 10IU/mg and a ratio of anti-Xa activity/anti-IIa activity of more than 30,

the component oligosaccharides of these mixtures:

-containing from 2 to 26 saccharide units,

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

-hexasaccharides Δ IIa-IIs-Is comprising the following formula:

and these oligosaccharide mixtures are in the form of alkali metal or alkaline earth metal salts.

2. The process according to claim 1, characterized in that the reaction of step a) is carried out by the action of an excess of benzethonium chloride with heparin sodium at a temperature of about 15-25 ℃ and a salt/heparin sodium molar ratio of 3-4.

3. The process according to claim 1, characterized in that the esterification reaction of step b) is carried out at a temperature of 25-45 ℃, preferably 30-40 ℃, in a chlorinated organic solvent, such as chloroform or dichloromethane, and then the ester is recovered in the form of its sodium salt by precipitation with a 10% by weight solution of sodium acetate in an alcohol, such as methanol, at a concentration of 1-1.2 volumes of alcohol per volume of reaction medium.

4. A process according to claim 1 or 3, characterized in that the degree of esterification of the heparin benzyl ester quaternary ammonium salt is 50-100%, preferably 70-90%.

5. The process according to claim 1, 3 or 4, characterized in that 0.5-1.5 parts by weight of benzyl chloride are used per 1 part by weight of heparin benzethonium salt, the reaction time being 10-35 hours.

6. The process according to claim 1, characterized in that the salt exchange of step c) is carried out in an aqueous medium at a temperature of 10-25 ℃ using quaternary ammonium chlorides, preferably benzethonium chloride, cetylpyridinium chloride or cetyltrimethylammonium chloride.

7. The process according to claim 6, characterized in that the molar ratio quaternary ammonium chloride/sodium salt of heparin benzyl ester is between 2 and 3.

8. The process according to claim 1, characterized in that the depolymerised heparin benzyl ester quaternary ammonium salt is converted into the sodium salt by treating the reaction medium with an alcoholic sodium acetate solution, preferably with a 10% methanolic sodium acetate solution (w/v), at a temperature of 15-25 ℃ (step e).

9. The process according to claim 1, characterized in that the saponification (step f) is carried out in an aqueous medium at a temperature of 0-20 ℃, preferably 0-10 ℃, using an alkali metal hydroxide, such as sodium hydroxide, potassium hydroxide or lithium hydroxide.

10. The process according to claim 9, characterized in that 1-5 molar equivalents of alkali metal hydroxide, preferably 1-2 molar equivalents of sodium hydroxide, are used.

11. The method according to claim 1, characterized in that the purification (step g) is carried out in an aqueous medium at a temperature of 10-50 ℃, preferably 20-40 ℃, using hydrogen peroxide.