Polyose blend that is heparin derivative, their production and pharmaceutical composition containing such blend

FIELD: chemistry.

SUBSTANCE: invention relates to the mixture of sulfated oligosaccharide having common structure of polyose that is included in heparin composition with average molecular mass ranging from 1500 to 3000 Da and proportion of anti-Xa/anti-IIa more than 30, to the method of their production and antithrombotic pharmaceutical compositions containing them.

EFFECT: production of the pharmaceutical compositions containing sulfated oligosaccharide that has antithrombotic activity.

31 cl, 12 ex

 

The present invention relates to mixtures of polysaccharides derived heparin, method for their production and pharmaceutical compositions containing them.

Heparin is a mixture of sulfated mucopolysaccharides of animal origin, and its application is based, in particular, on its anticoagulante and antithrombotic properties.

However, heparin has drawbacks that limit its enforcement. In particular, its high anticoagulant activity (anti-IIa) can cause bleeding.

Were offered low molecular weight heparins obtained by alkaline depolymerization of esters of heparin (EP 40144), but these products also have a high anticoagulant activity (anti-IIa).

The heparins with a very low molecular weight were also described in US 6384021. However, obtained in the above examples, the values of the activity of anti-Ha does not exceed 120 Honey and the ratio of anti-XA/anti-IIa is from 15 to 50.

In WO-0208295 heparins with very low molecular weights were obtained in any other way than in the US 6384021, and had an activity of from 100 to 150 Honey, some examples of the application of value relations anti-XA/anti-IIa were also very high.

In a specified class of drugs there is a continuing need to increase the activity of anti-Ha, h is particular to the values exceeding 150 Miu/mg, as well as in improving relations anti-XA/anti-IIa, and, consequently, the development of new generations of derivative heparins.

One of the purposes of the invention, therefore, is to improve the activity of anti-Ha and relations anti-XA/anti-IIa by modification of the methods described in the prior art, in particular, by controlling the percentage water content at the stage of depolymerization. Thus obtained heparins also have excellent antimicrobial activity and the activity of AXA close to the same activity of heparin, thus reducing the risk of bleeding due to the very low activity of the aIIa. Furthermore, the products according to the invention have a significantly greater duration half-life than heparin.

Thus the object of the invention is a new mixture of polysaccharides derived heparin, which has a higher selectivity for activated factor X (factor XA) and activated factor II (factor IIa)than heparin.

A mixture of polysaccharides, medium molecular weight is from 1500 to 3000 Da is considered the oligosaccharides.

Therefore, an object of the invention is a mixture of sulfated oligosaccharides having the General structure of the polysaccharides included heparin, and possessing the following is relevant features:

their medium molecular weight is from 1500 to 3000 Da, the activity of anti-Ha ranges from 120 to 200 Miu/mg activity, anti-IIa below 10 Miu/mg and the ratio of anti-XA/anti-IIa above 30,

- oligosaccharides included in the mixtures contain from 2 to 26 sharidny links, one of their end groups is link 2-O-sulfate unsaturated 4,5-uronic acid and contain hexasaccharide ΔIIa-IIs-Is of the formula

Hexasaccharide ΔIIa-IIs-Is contained in the mixture of oligosaccharides described in the present invention, is a sequence very similar to ATIII and different activity AXA, which is above 740 Honey/mg.

The mixture of oligosaccharides described in this invention is a salt of alkali or alkaline earth metal.

The preferred salts are alkali or alkaline earth metal can be called salts of sodium, potassium, calcium and magnesium.

Medium molecular weight determined by high-performance liquid chromatography using two columns connected in series, for example, the brand TSK G3000 XL and TSK G2000 XL. The definition is realized by means of refractometry. As eluent using lithium nitrate, the flow rate is 0.6 ml/min, the System is calibrated using standards obtained by fractionation Enoksaparina (AVENTIS) by chromatogr is the philosophy using aerosolgenerating gel (IBF). This method of obtaining described Barrowcliffe et al., Thromb. Res., 12, 27-36 (1977-78) or D.A.Lane et al., Thromb. Res., 12, 257-271 (1977-78). To calculate the results using the program GPC6 (Perkin Elmer).

The activity of anti-Ha measure amylolyticus method on auxochromes substrate described Teien et al., Thromb. Res., 10, 399-410 (1977), using as reference the first international standard low molecular weight heparins.

Activity, anti-IIa measured by the method described L.O. Anderson et al., Tromb. Res., 15, 531-541 (1979), using as reference the first international standard low molecular weight heparins.

Hexasaccharide fraction is preferably from 15 to 25% of a mixture of oligosaccharides.

Preferred mixtures according to the invention contain from 8 to 15% of hexasaccharide ΔIIa-IIs-Is hexasaccharide fraction of the mixture of oligosaccharides.

Content hexasaccharide fraction can be determined by analytical method by high performance liquid chromatography high-pressure columns TSK G3000 XL and TSK G2000 XL or by preparative separation hexasaccharides faction. In this case, the mixture is subjected to chromatography on columns, gel filled type polyacrylamide, agarose, for example, the brand Ultrogel ACA 202® (Biosepra). The mixture elute solution of sodium bicarbonate. Preferably as sodium hydrogen carbonate solution used solution, the molar concentration which is ranged from 0.1 mol/l to 1 mol/L. More preferably, the separation is carried out at a molar concentration of 1 mol/L. Detentio carry out UV-spectrometry (254 nm). After fractionation, the fraction of hexasaccharide in a solution of sodium bicarbonate to neutralize glacial acetic acid. Then the solution is concentrated under reduced pressure to obtain the concentration of sodium acetate is more than 30 wt.% Hexasaccharide fraction precipitated by adding 3 to 5 volumes of methanol. Hexasaccharide fraction allocate by filtration through a porous glass No. 3. The mixture of hexasaccharides can be subjected to analysis by HPLC (high performance liquid chromatography) to determine the content of hexasaccharide ΔIIa-IIs-Is. Hexasaccharide ΔIIa-IIs-Is possible to allocate preparative HPLC or affinity chromatography on a column of antithrombin III-sepharose methods used by the specialist (M.Hook, I.Bjork, J.Hopwood and U.Lindahl, F.E.B.S letters, vol656(1) (1976)).

More specifically, the mixture according to the invention have the activity of anti-Ha from 150 Miu/mg to 200 Med/mg.

Preferably the activity of anti-IIa mixtures according to the invention is less than 5 Miu/mg and more preferably from 0.5 to 3.5 Miu/mg the following application examples show the values from 1.1 to 1.6 Miu/mg, obtained using the preferred features of the invention.

Preferably agains the activity of anti-XA activity to anti-IIa mixtures according to the invention is more than 50, and more specifically more than 100.

Preferred mixtures according to the invention have a medium molecular weight of from 2000 to 3000 Da, and more specifically medium molecular weight of from 2400 to 2650 Yes.

Thus the object of the present invention are mixtures of the above with the activity of anti-Ha from 150 Miu/mg to 200 Miu/mg activity, anti-IIa from 0.5 to 3.5 Miu/mg and a medium molecular weight of from 2400 to 2650 Yes.

A mixture of oligosaccharides according to the invention can be obtained by depolymerization salt of the Quaternary ammonium complex benzyl ester of heparin in an organic environment with a strong organic base, the pKa of which is preferably above 20 (properties, mainly inherent to the family of phosphazenes described, for example, R.Schwesinger et al., Angew. Chem. Int. Ed. Engl. 26, 1167-1169 (1987), R.Schwesinger et al., Angew. Chem. 105, 1420(1993)), transfer salts of Quaternary ammonium complex benzyl ether depolimerizovannogo heparin sodium salt, by saponification of the residual esters and possible cleaning. In the method according to the invention using the main stages of the method described in WO 0208295, adding to its essential feature, which allows to obtain a mixture of oligosaccharides according to the invention, having physico-chemical properties and activities described above.

Indeed, for the specific mixture of oligosaccharides according to the invention is required to control the selectivity of the base, in order to accurately adjust the water content at the stage of depolymerization.

The method according to the invention has control of high selectivity Foundation in the process of depolymerization. It allows to carry out the depolymerization of heparin, the maximum while protecting a sequence similar to ATIII, such as hexasaccharide ΔIIa-IIs-Is described according to the present invention. This critical stage of the method ensures polysaccharides according to the invention.

The above description of the method is expressed in aktivnosta AXA, unexpected in relation to medium molecular weight mixtures of oligosaccharides (150 Miu/mg <Aha<200 Miu/mg for a medium molecular weight of from 2000 to 3000 Da). Such selectivity is associated with a very specific physico-chemical characteristics of the bases of phosphazene, namely pKa higher than 20, very high steric obstruction and low nucleophilicity.

This effect is fully manifested in anhydrous reaction medium. On the contrary, if the water content of the reaction medium increases, a sharp decrease of the selectivity of depolymerization. Protection sequences, similar to ATI II, is reduced, resulting in a significant reduction in the activity of AXA. In the presence of a small amount of water phosphazene base is protonated and the reaction is indosposeable particle becomes a Quaternary ammonium hydroxide. In this case, the properties of high steric hindrance and low nucleophilicity lost and significantly affect the quality of the resulting product. When conducting experiments on depolymerization in terms of measuring and controlling the water content can be observed the full manifestation of this effect.

The following table shows the effect of water content on the selectivity of depolymerization (when pilot testing only change this setting: indicators stoichiometry of the reagents, dilution, temperature remain constant, depending on the criteria used by the specialist. As the base used phosphazene base: 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,2,3-datafactory).

Table.
Water content, %0,05%0,1%0,2%0,3%0,4%0,57%1,8%2,5%
AXA Honey/mg19217716113212212010599
AIIa Honey/mg1,31,51,41,41δ31,43,1the 13.4
aXa/aIIa 148118115949485,7347,4

For optimum selectivity and maximum protection sequences similar to ATIII, it is preferable to use a water content, below 0.6% and preferably less than 0.3%when using 1 molar equivalent pospisilova base in relation to complex the benzyl ester of heparin salt benzene.

Thus a more specific object of the invention is phase depolymerization salt of the Quaternary ammonium complex benzyl ester of heparin obtained by methods known to the expert, characterized in that use the base of the family of phosphazenes, in particular in solution in dichloromethane with a water content of less than 0.6%. Preferably this percentage water content should be less than 0.3% and more preferably less than 0.2%.

Mainly molar ratio of the strong base/ester constitutes from 0.2 to 5, preferably from 0.6 to 2, and more preferably from 0.8 to 1.2. Use equimolecular relations refers to the preferred methods of carrying out the invention.

You can use other well-known specialist aprotic solvents such as THF and DMF.

Salt of the Quaternary ammonium complex benzyl E. the Ira heparin is preferably salt benzene, cetylpyridinium or cetyltrimethylammonium.

The Foundation of the family phosphazenes preferably have the formula

in which the radicals R1-R7, identical or different, denote alkyl radicals.

More specifically the invention relates to method described above, characterized in that the base (stage d) depolymerization) using 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,3,2-datafactory (1,2,3-datafactory-2-amine, 2-[(1,1-dimethylethyl)imino]-N,N-diethyl-1,2,2,2,3,5,6-octahydro-1,3-dimethyl):

or tert-butylamine(pyrrolidino)fastran.

In the above formulas, the alkyl radicals contain from 1 to 6 carbon atoms in straight or branched chain.

Thus the object of the invention is a method for oligosaccharide mixtures according to the invention, comprising the following stages:

a) translation in salt, sodium heparin by the action of chloride benzene,

b) etherification of heparinate benzene under benzylchloride,

c) the translation of the received complex benzyl ether salt of Quaternary ammonium,

d) depolymerization salt of the Quaternary ammonium complex benzyl ester of heparin as described above

e) transfer salts of Quaternary ammonium sodium salt,

f) possible Emile is their heparin under the action of a base, such as sodium hydroxide,

g) possible treatment, in particular, with an oxidizer such as hydrogen peroxide.

The following reaction scheme illustrates the present invention:

N=X+Y+Z (the total degree of sulfation of the middle disaccharide),

X = the degree of sulfation on the website, addition is indicated by radical N

Y = the degree of sulfation on the website, addition is indicated by radical N

Z = the degree of sulfation on the website, addition is indicated by radical PINES3,

The translation of the Quaternary ammonium complex benzyl ether depolimerizovannogo heparin sodium salt (stage e) is usually carried out by treating the reaction medium of an alcoholic solution of sodium acetate and preferably a 10% solution of sodium acetate in methanol (weight/volume) at a temperature of from 15 to 25°C. the Mass equivalent of the entered acetate preferably 3 times the weight of the salt of the Quaternary ammonium complex benzyl ester of heparin, which came in the depolymerization reaction.

Saponification (stage f) is generally carried out using an alkali metal hydroxide such as sodium hydroxide, potassium, lithium hydroxide in aqueous medium at a temperature of from 0 to 20°and preferably from 0 to 10°C. Typically use 1 to 5 molar equivalents of valenton hydroxide of an alkali metal. Preferably, the saponification is carried out in the presence of from 1 to 2 molar equivalents of alkali metal hydroxide.

The final product is clear (stage g) any known purification method depolimerizovannogo heparins (for example, EP 0037319 B1). Preferably this operation is carried out at a temperature of from 20 to 40°C.

Salt of the Quaternary ammonium complex benzyl ester of heparin can be obtained according to the following reaction scheme:

a) transfer of sodium heparin by the action of chloride benzene in heparinate benzene (translation to the formation of a salt),

b) etherification obtained in the previous phase of salt benzene under benzylchloride, then processing of an alcoholic solution of sodium acetate to obtain the sodium salt complex benzyl ester of heparin,

c) transfer of sodium salt complex benzyl ether salt of Quaternary ammonium, and preferably in salt benzene, terpinene or cetyltrimethylammonium.

At the stage of (a) the reaction is carried out under the action of excess chloride benzene at a temperature of from about 15 to 25°C. Mainly molar ratio salt/sodium heparin ranges from 3 to 4.

As a source of heparin preferably use heparin pigs. This heparin may be pre-treated to reduce the content of derm is consultate way described in the patent FR 2663639.

At the stage b) the esterification is preferably carried out in a chlorinated organic solution (e.g., chloroform, methylene chloride) at temperatures from 25 to 45°C. ester in the form of a salt benzene then get in the form of sodium salt by precipitation with 10 wt.% sodium acetate in alcohol, such as methanol. Usually use 1 to 1.2 volume fraction of alcohol on the volume fraction of the reaction medium. The number of benzylchloride and duration of the reaction allows to obtain 50-100% degree of esterification and preferably 70-90%. It is preferable to use from 0.5 to 1.5 mass parts of benzylchloride 1 mass part of the salt benzene heparin. The duration of the reaction is preferably from 10 to 35 hours.

On stage with) translation of the salt is carried out using chloride Quaternary ammonium and preferably using chloride benzene, chloride of cetylpyridinium or chloride of cetyltrimethylammonium in the aquatic environment at a temperature of from 10 to 25°C. Mainly molar ratio of chloride Quaternary ammonium/sodium salt complex benzyl ester of heparin ranges from 2 to 3.

According to the invention the mixture in the form of sodium salts can be converted into another salt of alkali or alkaline earth metal. Possible transfer from one salt to another exercise SPO is obom, described in the patent FR 7313580.

The mixture according to the invention are not toxic and, therefore, they can be used as medicines.

A mixture of oligosaccharides according to the present invention can be used as antithrombotic agents. In particular, they are suitable for the treatment or prophylaxis of venous and arterial thrombosis, deep venous thrombosis, pulmonary embolism, unstable angina, myocardial infarction, ischemia of the heart, occlusion of peripheral arteries and atrial fibrillation. They can also be used for treatment and prevention of proliferation of smooth muscle cells, atherosclerosis and arteriosclerosis, for the treatment and prevention of cancer through modulation of angiogenesis and growth factors, and for the treatment and prevention of diabetic disorders such as diabetic retinopathy and diabetic nephropathy.

The present invention relates also to pharmaceutical compositions containing as an active substance mixture of the formula (1), possibly in Association with one or more inert excipients.

As pharmaceutical compositions can, for example, to call solutions for subcutaneous or intravenous injection. They also apply through the lungs (inhalation), or oral.

The dosage depends on age, weight and condition of the section is cited patient. For an adult, it usually ranges from 20 to 100 mg / day intramuscular or subcutaneous injection.

The following examples illustrate the invention but are not restrictive.

Example

Getting benzylpiperidine, salt benzene

Heparinate benzene

In a solution of 10 g of heparin sodium in 100 ml of water is introduced a solution of 25 g of chloride benzene in 125 ml of water. The product is filtered, washed with water and dried.

Complex benzyl ester of heparin (sodium salt)

In a solution of 20 g of heparinate benzene in 80 ml of methylene chloride is injected 16 ml benzylchloride. The solution is heated to 30°C for 12 hours. Then enter 108 ml of 10% sodium acetate solution in methanol, filtered, washed with methanol and dried. Obtain 7.6 g of complex benzyl ester of heparin in the form of a sodium salt with a degree of esterification of 77%.

Complex benzyl ester of heparin (salt benzene)

In the conical flask And 2-liter injected 36 g (0,0549 mol) complex benzyl ester of heparin (sodium salt) and 540 ml of distilled water. After homogenization at a temperature of about 20°To obtain a pale yellow solution, In terms of mixing with a magnetic stirrer in a conical flask In 1 liter receive a solution 64,45 g (0,1438 mol) of chloride benzene and 450 ml of water. From the conical flask In a solution poured over 35 the minutes in a conical flask And mixing conditions. Produces abundant white precipitate. The conical flask and washed In 200 ml of distilled water and the wash water is introduced into the conical flask A. the stop Stirring and leave for 12 hours to precipitate suspension. After this time select and remove the transparent part of the distilled liquid. In the sludge (having a pasty kind) enter 560 ml of water and stirred for 20 minutes. Leave to re-deposition of approximately 30 minutes. The pooled liquid is selected and removed (560 ml). The formed precipitate is washed twice with about 560 ml of distilled water. When making the last wash the residue left in suspension and filtered through a porous glass 3. Then the precipitate washed four times with 200 ml of distilled water. Wet a white solid dried, then dried under reduced pressure (2.7 kPa) at a temperature of about 60°C. Dried for 12 hours and get to 87.5 g of benzylpiperidine, salt benzene. The output is 94,9%.

The example In

Description hexasaccharide ATIII (ΔIIa-IIs-Is)

Proton spectrum in the D2O, 500 MHz, T=298K, δ million-1: of 1.97 (3H, s)3,18 (1H, DD, 10 to 3 Hz), 3,30 (1H, t, 8 Hz), 3,37 (1H, DD, 10 to 3 Hz), 3,60 (2H, m), from the 3.65 to 3.85 (6N, m), a 3.87 (2H, m), 3,95 (1H, d, 8 Hz), a 4.03 (1H, d, 8 Hz), 4,05 up of 4.13 (4H, m), 4,16 to 4,45 (8H, m)to 4.52 (1H, d, 8 Hz), of 4.67 (1H, m), 506 (1H, d, 6 Hz), 5,10 (1H, d, 3 Hz), 5,33 (1H, d, 4 Hz), are 5.36 (1H, d, 3 Hz), 5,46 (1H, d, 3 Hz), 5,72 (1H, d, 4 Hz).

Dementieva salt (4-deoxy-α-L-threo-hexopyranoside(1→4)-2-deoxy-2-acetamido-6-O-sulfo-α-D-glyukopiranozil-(1→4)-acid-(β-D-glucopyranosyloxy-(1→4)-2-deoxy-2-sulfamido-3, 6-di-O-sulfo-α-D-glyukopiranozil-(1→4))-acid-(2-O-sulfo-α-L-iodophenethylamine)acid-(1→4)-2-deoxy-2-sulfamido-6-O-sulfo-α-D-glucopyranose.

Examples 1 through 7 and 12 illustrate the influence of water content on the selectivity of the polymerization reaction and the activity of AXA and aIIa obtained products.

Examples 8 through 10 illustrate the effect of the number of equivalents of base on the activity of AXA and aIIa obtained product with a water content of 0.1%).

Example 11 illustrates the use of another pospisilova grounds, except 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,2,3-diazaphospholidine:

Using tert-butylamine(pyrrolidin)phosphorane.

Example 1

Depolymerization and conversion to the sodium salt (0.1% of water)

In the conical flask And injected with 70 ml of dichloromethane. In terms of mixing slowly introducing 10 g (0,006 mol) complex benzyl ester of heparin (degree of esterification: 75%, salt benzene), obtained as described in example A. the water Content in the reaction medium set equal to 0.1%. The solution is heated is up to 40° C in nitrogen atmosphere. After complete dissolution temperature is reduced to a temperature of about 20°With, then add 1.75 ml (0,006 mole) of 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,2,3-diazaphospholidine. Stirred at a temperature of about 20°C for 24 hours. During this time in the conical flask To prepare a solution of 30 g of anhydrous sodium acetate in 300 ml of methanol. After complete dissolution the solution is injected 5 g celite Hyflo supercel. Under stirring with a magnetic stirrer, the reaction mixture from the conical flask And poured for 1 min 30 s in a methanol solution of sodium acetate at a temperature of about 5°C. Stirred for 5 minutes and leave the suspension to precipitate for 1 h 30 minutes the Clear part of distilled liquid is selected and removed (220 ml). In the sediment injected 220 ml of methanol and stirred for 5 minutes Leave to re-deposition of approximately 1 hour and 20 minutes the Pooled liquid is selected and removed (250 ml). In the sediment give 250 ml of methanol and stirred for 5 minutes, the Sediment in suspension is filtered through a porous glass 3. The precipitate is washed with about 100 ml of methanol. The wet solid light yellow, the substance is dried, then dried under reduced pressure (6 kPa) at a temperature of about 40°C. Dried for 18 h and gain of 2.51 g depolymerize the data heparin, sodium salt in celite (5 g). Output is 64%.

Saponification

In a conical flask of 50 ml volume injected 2.5 g (0,0038 mole) depolimerizovannogo heparin sodium salt in celite (5 g)obtained in the previous phase, and 17 ml of water. The suspension is filtered through a porous glass 3 and twice washed with 5 ml of water. The obtained filtrate is placed in a conical flask with a volume of 150 ml Under stirring with a magnetic stirrer enter 0/4 ml (0,004 mol) of 30% sodium liquor at a temperature of about 5°C. thereafter, the mixture shaken for 2 hours to Neutralize the solution by adding 1N HCl and injected 3 g of sodium chloride. After dilution of the reaction medium was added 21 ml of methanol. Stirred for 15 min and add 44 ml of methanol, and then stirred for 1 h Stirring is stopped and the suspension is left to settle for 45 minutes at a temperature of about 5°C. the Pooled liquid is selected and removed (90 ml). In the sediment injected 90 ml of methanol and stirred for 5 minutes Leave to re-deposition on 20 minutes the Pooled liquid is selected and removed (80 ml). In the sediment enter 80 ml of methanol and stirred for 5 minutes, the Sediment in suspension is filtered through a porous glass 3. The precipitate is washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure is (6 kPa) at a temperature of about 40° C. Dried for 18 h and obtain 1.31 g of the crude depolimerizovannogo heparin (sodium salt). The output is 66%.

Clean

In a conical flask of 50 ml volume injected 1.3 g of the crude depolimerizovannogo heparin obtained in the previous phase, and 13 ml of distilled water. The mixture is heated to 40°under stirring with a magnetic stirrer. the pH was adjusted to 9.7±0,1 by adding 1N sodium hydroxide. The reaction medium is filtered through a membrane with a hole size of 0.45 μm, and then enter 0,07 ml of 30% aqueous hydrogen peroxide solution. Shaken for 2 h at a temperature of about 20°C, after which the mixture is neutralized by adding 1N HCl and injected with 2 g of sodium chloride. Then the solution is filtered through a membrane with a hole size of 0.45 μm, and then pour in 14 ml of methanol. Then the solution is cooled to 10°C and stirred for about 15 minutes Add 36 ml of methanol and stirred for one hour. The stirring is stopped and the suspension is left to settle for about 15 minutes the Pooled liquid is selected and removed (50 ml). In the sediment injected 50 ml of methanol and stirred for 5 minutes Leave to re-suspension of sediment by about 25 minutes the Pooled liquid is selected and removed (50 ml). The sediment in suspension is filtered through a porous glass 3. Polucen the th white precipitate washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) at a temperature of about 40°C. Dried for 18 h and obtain 1.13 g of pure depolimerizovannogo heparin (sodium salt). The yield is 87%.

Properties depolimerizovannogo heparin obtained in this way.

Average molecular weight: 2600 Yes

The activity of anti-Ha: 177 Honey/mg

Activity, anti-IIa: 1.5 Miu/mg

The ratio of the activity of anti-XA activity to anti-IIa: 118

Example 2

Depolymerization and conversion to the sodium salt (0.2 per cent)

In the conical flask And injected with 70 ml of dichloromethane. With stirring and under nitrogen pressure, slowly introducing 10 g (0,006 mol) complex braillovega ester of heparin (degree of esterification: 75%, salt benzene), obtained as described in example A. the water Content in the reaction medium set equal to 0.2%. After complete dissolution of the added 1.75 ml (0,006 mole) of 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,2,3-diazaphospholidine. Stirred at a temperature of about 20°within 24 hours during this time in the conical flask To prepare a solution of 30 g of anhydrous sodium acetate in 300 ml of methanol. After complete dissolution the solution is injected 5 g celite Hyflo supercel. Under stirring with a magnetic stirrer, the reaction mixture from the conical flask And poured for 1 min 30 s in a methanol solution of sodium acetate at t is mperature about 5° C. Stirred for 5 min and left to precipitate for 2 hours the Clear part of distilled liquid is selected and removed (220 ml). In the sediment injected 220 ml of methanol and stirred for 5 minutes Leave to re-deposition of about 2 hours the Pooled liquid is selected and removed (230 ml). In the sediment impose 230 ml of methanol. The sediment in suspension is filtered through a porous glass 3. The precipitate is washed with about 150 ml of methanol. The wet solid light yellow, the substance is dried, then dried under reduced pressure (6 kPa) at a temperature of about 40°C. Dried for 18 hours and get 2,63 g of the crude depolimerizovannogo heparin in celite (5 g). The output is 67%.

Saponification

In a conical flask of 50 ml volume injected 2.5 g (0,0038 mole) depolimerizovannogo heparin sodium salt in celite (5 g)obtained in the previous phase, and 18 ml of water. The suspension is filtered through a porous glass 3 and twice washed with 5 ml of water. The obtained filtrate is placed in a conical flask with a volume of 150 ml Under stirring with a magnetic stirrer injected with 0.4 ml of 0.004 mole) of 30% sodium liquor at a temperature of about 5°C. thereafter, the mixture shaken for 2 hours the Solution is neutralized by adding 1N HCl and injected with 2 g of sodium chloride. In the reaction medium was added 14 ml of methanol. Peremeci is up for 15 minutes and add 36 ml of methanol, then stirred for one hour. The stirring is stopped and the suspension is left to settle for 45 minutes at a temperature of about 5°C. the Pooled liquid is selected and removed (80 ml). In the sediment enter 80 ml of methanol and stirred for 5 minutes, the Sediment in suspension is filtered through a porous glass 3. The precipitate is washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) at a temperature of about 40°C. Dried for 48 h and obtain 2.3 g of the crude depolimerizovannogo heparin (sodium salt). The yield is 65%.

Clean

In a conical flask of 50 ml volume injected 1.4 g of the crude depolimerizovannogo heparin obtained in the previous phase, and 15 ml of distilled water. The mixture is heated to 40°under stirring with a magnetic stirrer. the pH was adjusted to 9.7±0,1 by adding 1N sodium hydroxide. The reaction medium is filtered through a membrane with a hole size of 0.45 μm and type of 0.07 ml of 30% aqueous hydrogen peroxide solution. Shaken for 2 h at a temperature of about 40°C, after which the mixture is cooled to about 20°C, then neutralized by adding 1N HCl. In the reaction mixture is administered 2 g of sodium chloride. Then the solution is filtered through a membrane with a hole size of 0.45 μm, and then pour in 14 ml of methanol is. Then the solution is cooled to 10°C and stirred for 15 minutes Add 36 ml of methanol and stirred for one hour. The stirring is stopped and the suspension is left to settle for about 15 minutes the Pooled liquid is selected and removed (40 ml). In the sediment (paste type) enter 40 ml of methanol and stirred for 5 minutes Leave to re-suspension of sediment by about 20 minutes the Pooled liquid is selected and removed (50 ml). The sediment in suspension is filtered through a porous glass 3. The obtained white precipitate was washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) at a temperature of about 40°C. Dried for 18 h and obtain 1.2 g of pure depolimerizovannogo heparin (sodium salt). Yield : 86%.

Properties depolimerizovannogo heparin obtained in this way.

Average molecular weight: 2650 Yes

The activity of anti-Ha: 161 Honey/mg

Activity, anti-IIa: 1.4 Miu/mg

The ratio of the activity of anti-XA activity to anti-IIa: 115

Example 3

Depolymerization and conversion to the sodium salt (0.3% of water)

In the conical flask And injected with 70 ml of dichloromethane. With stirring and under nitrogen pressure injected 10 g (0,006 mol) complex benzyl ester of heparin (degree of esterification: 75%, salt benzene), obtained as opisanoj example A. The water content in the reaction medium set equal to 0.3%. After complete dissolution of the added 1.75 ml (0,006 mole) of 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,2,3-diazaphospholidine. Stirred at a temperature of about 20°within 24 hours during this time in the conical flask To prepare a solution of 30 g of anhydrous sodium acetate in 300 ml of methanol. After complete dissolution the solution is injected 5 g celite Hyflo supercel. Under stirring with a magnetic stirrer, the reaction mixture from the conical flask And poured for 1 min 30 s in a methanol solution of sodium acetate at a temperature of about 5°C. Stirred for 5 min and left to precipitate for 1 h 10 min Transparent part of the settled liquid is selected and removed (220 ml). In the sediment injected 220 ml of methanol and stirred for 5 minutes, the Sediment in suspension is filtered through a porous glass 3. The precipitate is washed with 100 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) at a temperature of about 40°C. Dried for 18 hours and get to 2.57 g of the crude depolimerizovannogo heparin sodium salt in celite (5 g). The output is 66%.

Saponification

In a conical flask of 50 ml volume injected 2.5 g (0/0038 mole) of the crude depolimerizovannogo heparin sodium salt in celite (5 g)obtained nepedyday stage, and 18 ml of water. The suspension is filtered through a porous glass 3 and twice washed with 5 ml of water. The obtained filtrate is placed in a conical flask with a volume of 150 ml Under stirring with a magnetic stirrer injected with 0.4 ml of 0.004 mole) of 30% sodium liquor at a temperature of about 5°C. thereafter, the mixture shaken for 2 hours the Solution is neutralized by adding 1N HCl and injected 3 g of sodium chloride. In the reaction medium was added 15 ml of methanol. Stirred for 15 min and add 36 ml of methanol, and then stirred for 1 h Stirring is stopped and the suspension is left to precipitate for 1 hour. The pooled liquid is selected and removed (70 ml). In the sediment injected with 70 ml of methanol and stirred for 5 minutes, the Sediment in suspension is filtered through a porous glass 3. The precipitate is washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) at a temperature of about 40°C. Dried for 48 h and obtain 1.42 g of the crude depolimerizovannogo heparin (sodium salt). The output is 62%.

Clean

In a conical flask of 50 ml volume injected 1.4 g of the crude depolimerizovannogo heparin obtained in the previous phase, and 14 ml of distilled water. The mixture is heated to 40°under stirring with a magnetic stirrer. the pH was adjusted to 9.7±0,1 by adding 1N is hydroxide sodium. The reaction medium is filtered through a membrane with a hole size of 0.45 μm and type of 0.07 ml of 30% aqueous hydrogen peroxide solution. Shaken for 2 h at a temperature of about 20°C, then the mixture is neutralized by adding 1N HCl and injected with 2 g of sodium chloride. After dissolving, the solution is filtered through a membrane with a hole size of 0.45 μm, and then pour in 14 ml of methanol. Then the filtrate is cooled to 10°C and stirred for 15 minutes Add 36 ml of methanol and stirred for about an hour. The stirring is stopped and the slurry for deposition on 40 minutes the Pooled liquid is selected and removed (50 ml). In the sediment (paste type) injected 50 ml of methanol and stirred for 5 minutes Leave to re-suspension of sediment for approximately 25 minutes, the Sediment in suspension is filtered through a porous glass 3. The obtained white precipitate was washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) at a temperature of about 40°C. Dried for 18 h and obtain 1.24 g of pure depolimerizovannogo heparin (sodium salt). The output is 89%.

Properties depolimerizovannogo heparin obtained in this way.

Average molecular weight: 2400 Yes

The activity of anti-Ha: 132 Honey/mg

Activity, anti-IIa: 1.4 Miu/mg

Attitude is aktivnosti anti-XA activity to anti-IIa: 94

Example 4

Depolymerization and conversion to the sodium salt (0.4% of water)

In the conical flask And injected with 70 ml of dichloromethane. While mixing, slowly inject 10 g (0,006 mol) complex benzyl ester of heparin (degree of esterification: 75%, salt benzene), obtained as described in example A. the water Content in the reaction medium set equal to 0.4%. The solution is heated to 30°C in nitrogen atmosphere. After complete dissolution is cooled to a temperature of about 20°With, then add 1.75 ml (0,006 mole) of 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,2,3-diazaphospholidine. Stirred at a temperature of about 20°within 24 hours during this time in the conical flask To prepare a solution of 30 g of anhydrous sodium acetate in 300 ml of methanol. After complete dissolution the solution is injected 5 g celite Hyflo supercel. Under stirring with a magnetic stirrer, the reaction mixture from the conical flask And poured for 1 min 30 s in a methanol solution of sodium acetate at a temperature of about 5°C. Stirred for 5 min and leave the suspension to precipitate for 2 hours the Clear part of distilled liquid is selected and removed (80 ml). In the sediment enter 80 ml of methanol and stirred for 5 minutes Leave to re-deposition within the hour. The pooled liquid is selected and removed (80 ml). In the precipitate enter 80 ml methane is a and stirred for 5 minutes The sediment in suspension is filtered through a porous glass 3. The precipitate is washed with 150 ml of methanol. The wet solid light yellow, the substance is dried, then dried under reduced pressure (6 kPa) at a temperature of about 40°C. Dried for 18 h and obtain 3.25 g of the crude depolimerizovannogo heparin sodium salt in celite (5 g). The yield is 83%.

Saponification

In a conical flask of 50 ml volume injected 3.1 g (0,0018 mole) of the crude depolimerizovannogo heparin sodium salt in celite (10 g)obtained in the previous phase, and 21 ml of water. The suspension is filtered through a porous glass 3 and twice washed with 6 ml of water. The obtained filtrate is placed in a conical flask with a volume of 150 ml Under stirring with a magnetic stirrer enter 0.7 ml (to 0.007 mol) of 30% sodium liquor at a temperature of about 5°C. thereafter, the mixture shaken for 2 hours the Solution is neutralized by adding 1N HCl and injected with 4 g of sodium chloride. In the reaction medium was added 28 ml of methanol. Stirred for 15 min and added 72 ml of methanol, and then stirred for one hour. The stirring is stopped and the slurry for deposition on the hour. The pooled liquid is selected and removed (90 ml). In the sediment injected 90 ml of methanol and stirred for 5 minutes, Leave for 20 minutes to re-deposition. Otstyvshy the I liquid is selected and removed (90 ml). In the sediment injected 90 ml of methanol and stirred for 5 minutes, the Sediment in suspension is filtered through a porous glass 3. The precipitate is washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) and at a temperature of about 40°C. Dried for 48 h and obtain 1.9 g of the crude depolimerizovannogo heparin (sodium salt). The output is 67%.

Clean

In a conical flask of 50 ml volume injected of 1.9 g of the crude depolimerizovannogo heparin obtained in the previous phase, and 19 ml of distilled water. The mixture is heated to 40°under stirring with a magnetic stirrer. the pH was adjusted to 9.7±0,1 by adding 1N sodium hydroxide. The reaction medium is filtered through a membrane with a hole size of 0.45 μm and injected with 0.1 ml of 30% aqueous hydrogen peroxide solution. Shaken for 2 h at a temperature of about 20°C, then the mixture is neutralized by adding 1N HCl and injected with 2 g of sodium chloride. Then the solution is filtered through a membrane with a hole size of 0.45 μm, and then pour in 14 ml of methanol and shaken for 15 minutes Add 36 ml of methanol and stirred for one hour. The stirring is stopped and the suspension is left to precipitate for 15 minutes the Pooled liquid is selected and removed (40 ml). In the sediment introduced 40 ml of methane is La and stirred for 5 minutes Leave to re-suspension of sediment by about 20 minutes the Pooled liquid is selected and removed (50 ml). In the sediment introduced 500 ml of methanol and stirred for 20 minutes, the Sediment in suspension is filtered through a porous glass 3. The obtained white precipitate was washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) at a temperature of about 40°C. Dried for 72 h and obtain 1.56 g of pure depolimerizovannogo heparin (sodium salt). The yield is 82%.

Properties depolimerizovannogo heparin obtained in this way.

Average molecular weight: 2350 Yes

The activity of anti-Ha: 122 Honey/mg

Activity, anti-IIa: 1.3 Miu/mg

The ratio of the activity of anti-XA activity to anti-IIa: 94

Example 5

Depolymerization and conversion to the sodium salt (0,57% water)

In the conical flask And injected with 140 ml of dichloromethane. While mixing, slowly introduce 20 g is 0.019 mole) of a compound benzyl ester of heparin (degree of esterification: 75%, salt benzene), obtained as described in example A. the water content of the reaction medium is set to 0.57 percent. After complete dissolution of the added 3.5 ml (0,012 mol) of 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,2,3-diazaphospholidine. Stirred at a temperature of about 20°within 24 hours during this time in conic the tion flask To prepare a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol. After complete dissolution the solution is injected 10 g celite Hyflo supercel. Under stirring with a magnetic stirrer, the reaction mixture from the conical flask And poured for 1 min 30 s in a methanol solution of sodium acetate at a temperature of about 4°C. Stirred for 5 min and leave the suspension to precipitate for 30 minutes Transparent part of the settled liquid 31 is selected and removed (400 ml). In the sediment introduced 400 ml of methanol and stirred for 5 minutes Leave to re-deposition within the hour. The pooled liquid is selected and removed (420 ml). In the resulting sludge is injected 420 ml of methanol and stirred for 5 minutes and Then the suspension is filtered through a porous glass 3. The precipitate is washed with 200 ml of methanol. The wet solid light yellow, the substance is dried, then dried under reduced pressure (6 kPa) at a temperature of about 50°C. Dried for 18 hours and get of 6.66 g of the crude depolimerizovannogo heparin sodium salt in celite (10 g). The yield is 85%.

Saponification

In a conical flask of 50 ml volume injected of 6.66 g (0,0101 mole) of the crude depolimerizovannogo heparin sodium salt in celite (10 g)obtained in the previous phase, and 47 ml of water. The suspension is filtered through a porous glass 3 and twice washed with 15 ml of water. The obtained filtrate is placed in a conical flask with a volume of 150 ml Under stirring with a magnetic stirrer impose 1.1 ml (to 0.011 mole) of 30% sodium liquor at a temperature of about 5° C. thereafter, the mixture shaken for 2 hours the Solution is neutralized by adding 1N HCl and injected 9.5 g of sodium chloride. In the reaction medium was added 66 ml of methanol. Stirred for 15 min and add 171 ml of methanol, and then stirred for 1 h Stirring is stopped and the slurry for deposition on 3/4 hours at a temperature of about 5°C. the Pooled liquid is selected and removed (160 ml). In the sediment injected 160 ml of methanol and stirred for 5 minutes, Leave for 20 minutes to re-deposition. The pooled liquid is selected and removed (180 ml). In the sediment enter 180 ml of methanol and stirred for 5 minutes, the Sediment in suspension is filtered through a porous glass 3. The precipitate twice washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) and at a temperature of about 40°C. Dried for 18 hours and get a 4.53 g of the crude depolimerizovannogo heparin (sodium salt). The output is 74%.

Clean

In a conical flask 100 ml impose a 4.53 g of the crude depolimerizovannogo heparin obtained at the previous stage, and 45 ml of distilled water. The mixture is heated to 40°under stirring with a magnetic stirrer. the pH was adjusted to 9.7±0,1 by adding 1N sodium hydroxide. The reaction credability through the membrane hole size of 0.45 μm and injected with 0.25 ml of 30% aqueous hydrogen peroxide solution. Shaken for 2 h at a temperature of about 20°C, then the mixture is neutralized by adding 1N HCl and injected 5.5 g of sodium chloride. Then the solution is filtered through a membrane with a hole size of 0.45 μm, and then pour in 38 ml of methanol at a temperature of about 10°C. Then the solution is heated to about 20°and shaken for 15 minutes then add 100 ml of methanol and stirred for one hour. The stirring is stopped and the suspension is left to settle for 20 minutes the Pooled liquid is selected and removed (90 ml). In the sediment injected 90 ml of methanol and stirred for 5 minutes Leave to re-suspension of sediment by about 25 minutes the Pooled liquid is selected and removed (100 ml). The sediment in suspension is filtered through a porous glass 3. The obtained white precipitate was washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) at a temperature of about 50°C. Dried for 18 h and obtain 3.7 g of pure depolimerizovannogo heparin (sodium salt). The yield is 82%.

Properties depolimerizovannogo heparin obtained in this way.

Average molecular weight: 2200 Yes

The activity of anti-Ha: 120 Miu/mg

Activity, anti-IIa: 1.4 Miu/mg

The ratio of the activity of anti-XA activity to anti-IIa: 86

Example 6

Depolymerizes the I and translation in sodium salt (1.8% of water)

In the conical flask And injected with 140 ml of dichloromethane. While mixing, slowly introduce 20 g is 0.019 mole) of a compound benzyl ester of heparin (degree of esterification: 75%, salt benzene), obtained as described in example A. the water content of the reaction medium is set to be 1.8%. After complete dissolution of the added 3.5 ml (0,012 mol) of 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,2,3-diazaphospholidine. Stirred at a temperature of about 20°within 24 hours during this time in the conical flask To prepare a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol. After complete dissolution the solution is injected 10 g celite Hyflo supercel. Under stirring with a magnetic stirrer, the reaction mixture from the conical flask And poured for 1 min 30 s in a methanol solution of sodium acetate at a temperature of about 4°C. Stirred for 5 min and leave the suspension to precipitate for 30 minutes the Clear part of distilled liquid is selected and removed (400 ml). In the sediment introduced 400 ml of methanol and stirred for 5 minutes Leave to re-deposition within the hour. The pooled liquid is selected and removed (420 ml). In the resulting sludge is injected 420 ml of methanol and stirred for 5 minutes, the Sediment in suspension is filtered through a porous glass 3. The precipitate is washed with 200 ml of methanol. lanoe solid light yellow, the substance is dried up, then dried under reduced pressure (6 kPa) at a temperature of about 50°C. Dried for 18 h and receive a rate of 7.54 g of the crude depolimerizovannogo heparin sodium salt in celite (10 g). The yield is 96%.

Saponification

In a conical flask of 50 ml volume injected rate of 7.54 g (0,0101 mole) of the crude depolimerizovannogo heparin sodium salt in celite (10 g)obtained in the previous phase, and 53 ml of water. The suspension is filtered through a porous glass 3 and twice washed with 15 ml of water. The obtained filtrate is placed in a conical flask with a volume of 150 ml Under stirring with a magnetic stirrer enter 1.25 ml (0,012 mol) of 30% sodium liquor at a temperature of about 4°C. thereafter, the mixture shaken for 2 hours the Solution is neutralized by adding 1N HCl and type of 10.5 g of sodium chloride. In the reaction medium was added 70 ml of methanol. Stirred for 15 min and add 190 ml of methanol, and then stirred for one hour. The stirring is stopped and the slurry for deposition on 3/4 hours at a temperature of about 4°C. the Pooled liquid is selected and removed (180 ml). In the sediment enter 180 ml of methanol and stirred for 5 minutes, Leave for 20 minutes to re-deposition. The pooled liquid is selected and removed (180 ml). In the sediment enter 180 ml of methanol and stirred for 5 minutes in suspended Sediment is able filtered through a porous glass 3. The precipitate twice washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) and at a temperature of about 50°C. Dried for 18 hours and get 5.53 g of the crude depolimerizovannogo heparin (sodium salt). The yield is 80%.

Clean

In a conical flask 100 ml injected 5.53 g of the crude depolimerizovannogo heparin obtained at the previous stage, and 55 ml of distilled water. The mixture is heated to 40°under stirring with a magnetic stirrer. the pH was adjusted to 9.7±0,1 by adding 1N sodium hydroxide. The reaction medium is filtered through a membrane with a hole size of 0.45 μm and injected at 0.31 ml of 30% aqueous hydrogen peroxide solution. Shaken for 2 h at a temperature of about 20°C, then the mixture is neutralized by adding 1N HCl and injected with 7 g of sodium chloride. After that, the solution is filtered through a membrane with a hole size of 0.45 μm, and then pour in 49 ml of methanol at a temperature of about 10°C. Then the solution is heated to about 20°and shaken for 15 minutes then add 126 ml of methanol and stirred for one hour. The stirring is stopped and the suspension is left to settle for 20 minutes the Pooled liquid is selected and removed (105 ml). In the sediment injected 105 ml of methanol and stirred for 5 minutes the tute for re-suspension of sediment approximately 25 minutes The pooled liquid is selected and removed (110 ml). The sediment in suspension is filtered through a porous glass 3. The obtained white precipitate was washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) at a temperature of about 55°C. Dried for 18 hours and get a 4.53 g of pure depolimerizovannogo heparin (sodium salt). The yield is 82%.

Properties depolimerizovannogo heparin obtained in this way.

Average molecular weight: 2600 Yes

The activity of anti-Ha: 105 Honey/mg

Activity, anti-IIa: 3,1 Honey/mg

The ratio of the activity of anti-XA activity to anti-IIa: 34

Example 7

Depolymerization and conversion to the sodium salt (2.5% of water)

In the conical flask And injected with 140 ml of dichloromethane. While mixing, slowly introduce 20 g is 0.019 mole) of a compound benzyl ester of heparin (degree of esterification: 75%, salt benzene), obtained as described in example A. the water Content in the reaction medium set equal to 2.5%. After complete dissolution of the added 3.5 ml (0,012 mol) of 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,2,3-diazaphospholidine. Stirred at a temperature of about 20°within 24 hours during this time in the conical flask To prepare a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol. After complete dissolution the solution is injected 10 g celite Hyflo supercel. PR is stirring with a magnetic stirrer, the reaction mixture from the conical flask And poured for 1 min 30 s in a methanol solution of sodium acetate at a temperature of about 4° C. Stirred for 5 min and leave the suspension to precipitate for 1 h Transparent part of the distilled liquid is selected and removed (400 ml). In the sediment introduced 400 ml of methanol and stirred for 5 minutes Leave to re-deposition within about 30 minutes the Clear part of distilled liquid is selected and removed (400 ml). The sediment in suspension is filtered through a porous glass 3. The precipitate is washed with 200 ml of methanol. The wet solid light yellow, the substance is dried, then dried under reduced pressure (6 kPa) at a temperature of about 50°C. Dried for 18 hours and get 7.78 g of the crude depolimerizovannogo heparin sodium salt in celite (10 g). The output is 99.6%.

Saponification

In a conical flask of 50 ml volume injected 7.78 g (0,0119 mole) of the crude depolimerizovannogo heparin sodium salt in celite (10 g)obtained in the previous phase, and 79 ml of water. The suspension is filtered through a porous glass 3 and twice washed with 15 ml of water. The obtained filtrate is placed in a conical flask with a volume of 150 ml Under stirring with a magnetic stirrer injected 1.3 ml (0,012 mol) of 30% sodium liquor at a temperature of about 4°C. thereafter, the mixture shaken for 2 hours the Solution is neutralized by adding 1N HCl and injected with 10 g of sodium chloride. In the reaction medium was added 60 ml of methane is as. Stirred for 15 min and add 190 ml of methanol, and then stirred for one hour. The stirring is stopped and the slurry for deposition on 3/4 hours at a temperature of about 4°C. the Pooled liquid is selected and removed (180 ml). In the sediment enter 180 ml of methanol and stirred for 5 minutes, Leave for 20 minutes to re-deposition. The pooled liquid is selected and removed (180 ml). In the sediment enter 180 ml of methanol and stirred for 5 minutes, the Sediment in suspension is filtered through a porous glass 3. The precipitate twice washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) and at a temperature of about 50°C. Dried for 18 hours and get by 5.87 g of the crude depolimerizovannogo heparin (sodium salt). The yield is 82%.

Clean

In a conical flask 100 ml injected by 5.87 g of the crude depolimerizovannogo heparin obtained at the previous stage, and 59 ml of distilled water. The mixture is heated to 40°under stirring with a magnetic stirrer. the pH was adjusted to 9.7±0,1 by adding 1N sodium hydroxide. The reaction medium is filtered through a membrane with a hole size of 0.45 μm and type of 0.34 ml of 30% aqueous hydrogen peroxide solution. Shaken for 2 h at a temperature of about 20 C, then the mixture is neutralized by adding 1N HCl and injected with 7 g of sodium chloride. After that, the solution is filtered through a membrane with a hole size of 0.45 μm, and then pour in 49 ml of methanol at a temperature of about 10°C. Then the solution is heated to about 20°and shaken for 15 minutes then add 126 ml of methanol and stirred for one hour. The stirring is stopped and the suspension is left to settle for 20 minutes the Pooled liquid is selected and removed (105 ml). In the sediment injected 105 ml of methanol and stirred for 5 minutes Leave to re-suspension of sediment by about 25 minutes the Pooled liquid is selected and removed (110 ml). The sediment in suspension is filtered through a porous glass 3. The obtained white precipitate was washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) at a temperature of about 55°C. Dried for 18 hours and get to 5.21 g of pure depolimerizovannogo heparin (sodium salt). The output is 89%.

Properties depolymerizing heparin obtained in this way.

Average molecular weight: 3550 Yes

The activity of anti-Ha: 99 Honey/mg

Activity, anti-IIa: 13,4 Honey/mg

The ratio of the activity of anti-XA activity to anti-IIa: 7,4

Example 8

Depolymerization and conversion to the sodium salt (0.5 equivalent OS is Finance)

In the conical flask And injected with 140 ml of dichloromethane. While mixing, slowly introduce 20 g is 0.019 mole) of a compound benzyl ester of heparin (degree of esterification: 75%, salt benzene), obtained as described in example A. After complete dissolution at a temperature of about 30°and cooling to about 20°add 1.75 ml (0,006 mole) of 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,2,3-diazaphospholidine. Stirred at a temperature of about 20°within 24 hours during this time in the conical flask To prepare a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol. Under stirring with a magnetic stirrer, the reaction mixture from the conical flask And poured into a methanol solution of sodium acetate at a temperature of about 4°C. is Stirred for one hour and leave the suspension to precipitate for 2 hours the Clear part of distilled liquid is selected and removed (420 ml). In the sediment injected 420 ml of methanol and stirred for 30 minutes Left to re-deposition for approximately 18 h of the Transparent part of the distilled liquid is selected and removed (400 ml). In the sediment introduced 400 ml of methanol and stirred for 30 minutes the Pooled liquid is selected and removed (400 ml). The sediment in suspension is filtered through a porous glass 3. The formed precipitate is washed twice with 100 ml of meth is Nola. The wet solid light yellow, the substance is dried, then dried under reduced pressure (6 kPa) at a temperature of about 60°C. After drying obtain 5.7 g of the crude depolimerizovannogo heparin, sodium salt. The output is 73%.

Saponification

In a conical flask 100 ml injected 5.7 g (0,0086 mole) of the crude depolimerizovannogo heparin, sodium salt obtained in the previous stage, and 53 ml of water. Under stirring with a magnetic stirrer injected with 0.93 ml (0,009 mole) of 30% sodium liquor at a temperature of about 4°C. thereafter, the mixture shaken for 2 hours the Solution is neutralized by adding 1N HCl and injected 6 g of sodium chloride. In the reaction medium was added 42 ml of methanol. Stirred for 15 min and add 108 ml of methanol, and then stirred for 30 minutes, the Stirring is stopped and the suspension is left to precipitate for 30 min at a temperature of about 4°C. the Pooled liquid is selected and removed (180 ml). In the sediment enter 180 ml of methanol and stirred for 5 minutes, Leave for about 30 min to re-deposition. The pooled liquid is selected and removed (170 ml). In the sediment impose 170 ml of methanol and stirred for 30 minutes the Precipitate in suspension is filtered through a porous glass 3. The precipitate washed twice with 30 ml of methanol. The wet solid is emesto drain, then dried under reduced pressure and at a temperature of about 60°C. After drying obtain 3.5 g of the crude depolimerizovannogo heparin (sodium salt). The output is 67,4%.

Clean

In a conical flask 100 ml injected 3.5 g of the crude depolimerizovannogo heparin obtained in the previous phase, and 35 ml of distilled water. The mixture is heated to 40°under stirring with a magnetic stirrer. the pH was adjusted to 9.6±0,1 by adding 1N sodium hydroxide. The reaction medium is filtered through a membrane with a hole size of 0.45 μm and type of 0.18 ml of 30% aqueous hydrogen peroxide solution. Shaken for 2 h at a temperature of about 20°C, then the mixture is neutralized by adding 1N HCl and injected 3.6 g of sodium chloride. After that, the solution is filtered through a membrane with a hole size of 0.45 μm, and then pour in 27 ml of methanol at a temperature of about 10°C. Then the solution is heated to 20°and shaken for 15 minutes then add 65 ml of methanol and stirred for 30 minutes, the Stirring is stopped and the suspension is left to precipitate for 30 minutes the Pooled liquid is selected and removed (80 ml). In the sediment enter 80 ml of methanol and stirred for 30 minutes Left for re-suspension of sediment by about 30 minutes the Pooled liquid is selected and removed (70 ml). the precipitate formed is injected 70 ml of methanol and stirred for 30 minutes The sediment in suspension is filtered through a porous glass 3. The obtained white precipitate washed twice with 30 ml of methanol. The wet solid is dried, then dried under reduced pressure and at a temperature of about 60°C. After drying obtain 2.8 g of pure depolimerizovannogo heparin (sodium salt). The yield is 80%.

Properties depolimerizovannogo heparin obtained in this way.

Average molecular weight: 2900 Yes

The activity of anti-Ha: 146,1 Honey/mg

Activity, anti-IIa: 5.1 Honey/mg

The ratio of the activity of anti-XA activity to anti-IIa: 28,6

Example 9

Depolymerization and conversion to the sodium salt (0.6 equivalent of base)

In a three-neck flask And injected 280 ml of dichloromethane. While mixing, slowly inject 40 grams (0,024 mol) complex benzyl ester of heparin (degree of esterification: 75%, salt benzene), obtained as described in example A. After complete dissolution at a temperature of about 30°and cooling to about 20°add to 4.2 ml of 0.014 mol) of 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,2,3-diazaphospholidine. Stirred at a temperature of about 20°within 24 hours during this time in the conical flask To prepare a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol. Under stirring with a magnetic stirrer half of the reaction mixture from the three-neck flask And pour in manoly solution of sodium acetate at a temperature of about 4° C. is Stirred for one hour and leave the suspension to precipitate. Transparent part of the settled liquid is selected and removed (310 ml). In the sediment injected 310 ml of methanol and stirred for one hour. Leave to re-deposition for approximately 18 h of the Transparent part of the distilled liquid is selected and removed (400 ml). In the sediment introduced 400 ml of methanol and stirred for one hour. The pooled liquid is selected and removed (300 ml). The sediment in suspension is filtered through a porous glass 3. The formed precipitate is washed twice with 100 ml of methanol. The wet solid light yellow, the substance is dried, then dried under reduced pressure and at a temperature of about 60°C. After drying obtain 6 g of the crude depolimerizovannogo heparin, sodium salt. The output is 77%.

Saponification

In a conical flask of 250 ml is administered 6 g (0,0091 mole) of the crude depolimerizovannogo heparin (sodium salt)obtained in the previous phase, and 56 ml of water. Under stirring with a magnetic stirrer inject 1 ml (0,010 mol) of 30% sodium liquor at a temperature of about 4°C. thereafter, the mixture shaken for 2 hours the Solution is neutralized by adding 1N HCl, and type of 6.4 g of sodium chloride. In the reaction medium was added 45 ml of methanol. Stirred for 15 min and add 115 ml meta is Ola, then stirred for 30 minutes, the Stirring is stopped and the suspension is left to precipitate for 30 min at a temperature of about 4°C. the Pooled liquid is selected and removed (170 ml). In the sediment impose 170 ml of methanol and stirred for 30 minutes Left for about 30 min to re-deposition. The pooled liquid is selected and removed (140 ml). In the sediment injected 140 ml of methanol and stirred for 30 minutes the Precipitate in suspension is filtered through a porous glass 3. The precipitate washed twice with 30 ml of methanol. The wet solid is dried, then dried under reduced pressure and at a temperature of about 60°C. After drying obtain 3.6 g of the crude depolimerizovannogo heparin (sodium salt). The output is 65,3%.

Clean

In a conical flask 100 ml injected 3.5 g of the crude depolimerizovannogo heparin obtained in the previous phase, and 35 ml of distilled water. The mixture is heated to 40°under stirring with a magnetic stirrer. the pH was adjusted to 9.6±0,1 by adding 1N sodium hydroxide. The reaction medium is filtered through a membrane with a hole size of 0.45 μm and type of 0.18 ml of 30% aqueous hydrogen peroxide solution. Shaken for 2 h at a temperature of about 20°C, then the mixture is neutralized by adding 1N HCl and enter 3,5g of sodium chloride. After that, the solution is filtered through a membrane with a hole size of 0.45 μm, and then pour in 25 ml of methanol at a temperature of about 10°C. Then the solution is heated to about 20°and shaken for 15 minutes then add 63 ml of methanol and stirred for 30 minutes, the Stirring is stopped and the suspension is left to precipitate for 30 minutes the Pooled liquid is selected and removed (70 ml). In the sediment injected with 70 ml of methanol and stirred for 30 minutes Left for re-suspension of sediment for a few minutes. The sediment in suspension is filtered through a porous glass 3. The obtained white precipitate washed twice with 30 ml of methanol. The wet solid is dried, then dried under reduced pressure and at a temperature of about 60°C. After drying obtain 2.5 g of pure depolimerizovannogo heparin (sodium salt). Output accounts for 71.4%.

Properties depolimerizovannogo heparin obtained in this way.

Average molecular weight: 2600 Yes

The activity of anti-Ha: 150,5 Honey/mg

Activity, anti-IIa: 3.2 Miu/mg

The ratio of the activity of anti-XA activity to anti-IIa: 47

Example 10

Depolymerization and conversion to the sodium salt (0.8 equivalent of base)

In the conical flask And injected with 70 ml of dichloromethane. While mixing, slowly inject 10 g (0,006 mol) complex benilov the th ester of heparin (degree of esterification: 75%, salt benzene), obtained as described in example A. After complete dissolution at a temperature of about 30°and cooling to about 20°add to 1.38 ml (0,004 mol) of 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,2,3-diazaphospholidine. Stirred at a temperature of about 20°within 24 hours during this time in the conical flask To prepare a solution of 30 g of anhydrous sodium acetate in 300 ml of methanol. Under stirring with a magnetic stirrer, the reaction mixture from the conical flask And poured within 1 min 15 s in a methanol solution of sodium acetate at a temperature of about 4°C. Stirred for 5 min and leave the suspension to precipitate an hour. Transparent part of the settled liquid is selected and removed (190 ml). In the sediment injected 190 ml of methanol and stirred for 5 minutes Leave to re-deposition of about 30 minutes the Clear part of distilled liquid is selected and removed (190 ml). In the sediment injected 190 ml of methanol and stirred for 30 minutes the Pooled liquid is selected and removed (190 ml). The sediment in suspension is filtered through a porous glass 3. The precipitate is washed with 150 ml of methanol. The wet solid light yellow, the substance is dried, then dried under reduced pressure (6 kPa) and at a temperature of about O°C. Dried for 18 h and the floor is up 3,05 g of the crude depolimerizovannogo heparin (sodium salt). The yield is 80%.

Saponification

In a conical flask 100 ml injected 3,05 g (0,0048 mole) of the crude depolimerizovannogo heparin (sodium salt)obtained in the previous phase, and 21 ml of water. The solution is filtered through a porous glass 3 and twice washed with 6 ml of water. The obtained filtrate is placed in a conical flask with a volume of 150 ml Under stirring with a magnetic stirrer injected with 0.6 ml (0,006 mole) of 30% sodium liquor at a temperature of about 4°C. thereafter, the mixture shaken for 2 hours the Solution is neutralized by adding 1N HCl and injected with 4 g of sodium chloride. In the reaction medium was added 28 ml of methanol. Stirred for 15 min and added 72 ml of methanol, and then stirred for 1 h Stirring is stopped and the suspension is left to precipitate for 30 min at a temperature of about 4°C. the Pooled liquid is selected and removed (80 ml). In the sediment enter 80 ml of methanol and stirred for 5 minutes, Leave for about 30 min to re-deposition. The pooled liquid is selected and removed (80 ml). In the sediment enter 80 ml of methanol and stirred for 30 minutes the Precipitate in suspension is filtered through a porous glass 3. The precipitate is washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) and the temperature is e 40° C. After drying obtain 1.6 g of the crude depolimerizovannogo heparin (sodium salt). The output is 57%.

Clean

In a conical flask of 50 ml volume injected 1.6 g of the crude depolimerizovannogo heparin/ received in the previous phase, and 16 ml of distilled water. The mixture is heated to 40°under stirring with a magnetic stirrer. the pH was adjusted to 9.6±0,1 by adding 1N sodium hydroxide. The reaction medium is filtered through a membrane with a hole size of 0.45 μm and type of 0.08 ml of 30% aqueous hydrogen peroxide solution. Shaken for 2 h at a temperature of about 20°C, then the mixture is neutralized by adding 1N HCl and injected with 2 g of sodium chloride. After that, the solution is filtered through a membrane with a hole size of 0.45 μm, and then pour in 14 ml of methanol at a temperature of about 10°C. Then the solution is heated to 20°and shaken for 15 minutes then add 36 ml of methanol and stirred for one hour. The stirring is stopped and the suspension is left to precipitate for 30 minutes the Pooled liquid is selected and removed (50 ml). In the sediment injected 50 ml of methanol and stirred for 5 minutes. Leave to re-suspension of sediment by about 30 minutes the Pooled liquid is selected and removed (50 ml). The sediment in suspension is filtered through the porous stack is about 3. The obtained wet solid is dried, then dried under reduced pressure (6 kPa) and at a temperature of about 40°C. After drying earn 1.25 g of pure depolimerizovannogo heparin (sodium salt). The yield is 78%.

Properties depolimerizovannogo heparin obtained in this way.

Average molecular weight: 2400 Yes

The activity of anti-Ha: 154,3 Honey/mg

Activity, anti-IIa: 1.6 Miu/mg

The ratio of the activity of anti-XA activity to anti-IIa: 96,4

Example 11

Depolymerization and conversion to the sodium salt

In the conical flask And injected with 140 ml of dichloromethane. While mixing, slowly introduce 20 g is 0.019 mole) of a compound benzyl ester of heparin (degree of esterification: 75%, salt benzene), obtained as described in example A. After complete dissolution at a temperature of about 40°and cooling down to 20°add 3.5 ml (to 0.011 mol) of tert-butylamine(pyrrolidin)phosphorane. Stirred at a temperature of about 20°within 24 hours during this time in the conical flask To prepare a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol. After complete dissolution the solution is injected 10 g celite Hyflo supercel. Under stirring with a magnetic stirrer, the reaction mixture from the conical flask And poured for 1 min 30 s in a methanol solution of sodium acetate at a temperature of about 4°C. Stirred for 5 min and the supply of the suspension to precipitate for 30 minutes Transparent part of the settled liquid is selected and removed (400 ml). In the sediment introduced 400 ml of methanol and stirred for 5 minutes Leave to re-deposition within approximately 1 hour 20 minutes the Pooled liquid is selected and removed (250 ml). In the sediment give 250 ml of methanol and stirred for 5 minutes, the Sediment in suspension is filtered through a porous glass 3. The precipitate is washed with 200 ml of methanol. The wet solid light yellow, the substance is dried, then dried under reduced pressure (6 kPa) at a temperature of about 40°C. after drying receive 5,39 g depolimerizovannogo heparin (complex benzyl ester, sodium salt). The output is 69%.

Saponification

In a conical flask of 50 ml volume injected 5 g (0,0076 mole) depolimerizovannogo heparin (complex benzyl ester, sodium salt)obtained in the previous phase, and 35 ml of water. The solution is filtered through a porous glass 3 and twice washed with 10 ml of water. The obtained filtrate is placed in a conical flask of 250 ml Under stirring with a magnetic stirrer inject 1 ml (0.01 mole) of 30% sodium liquor at a temperature of about 4°C. thereafter, the mixture shaken for 2 hours the Solution is neutralized by adding 1N HCl and injected 6 g of sodium chloride. In the reaction medium was added 42 ml of methanol. Stirred for 15 minutes and add 104 ml of methanol, then stirred for 15 minutes, the Stirring is stopped and the suspension is left to settle for one hour at a temperature of about 4°C. the Pooled liquid is selected and removed (140 ml). In the sediment injected 140 ml of methanol and stirred for 5 minutes Leave for 45 minutes to re-deposition. The pooled liquid is selected and removed (160 ml). In the sediment injected 160 ml of methanol and stirred for 5 minutes, the Sediment in suspension is filtered through a porous glass 3. The precipitate twice washed with 100 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) and at a temperature of about 40°C. Dried for 48 h and obtain 2.7 g of the crude depolimerizovannogo heparin (sodium salt). The output is 59%.

Clean

In a conical flask of 50 ml volume injected 2.6 g of the crude depolimerizovannogo heparin obtained in the previous phase, and 25 ml of distilled water. The mixture is heated to 40°under stirring with a magnetic stirrer. the pH was adjusted to 9.7±0,1 by adding 1N sodium hydroxide. The reaction medium is filtered through a membrane with a hole size of 0.45 μm and injected with 0.15 ml of 30% aqueous hydrogen peroxide solution. Shaken for 2 h at a temperature of about 20°C, then the mixture is neutralized by adding 1N HCl and injected g of sodium chloride. After that, the solution is filtered through a membrane with a hole size of 0.45 μm, and then pour in 21 ml of methanol at a temperature of about 10°C. Then the solution is heated to about 20°and shaken for 15 minutes then add 54 ml of methanol and stirred for 1 h Stirring is stopped and the suspension is left to settle for 20 minutes the Pooled liquid is selected and removed (50 ml). In the sediment injected 50 ml of methanol and stirred for 5 minutes Leave to re-deposition of about 20 minutes the Pooled liquid is selected and removed (50 ml). The sediment in suspension is filtered through a porous glass 3. The obtained white precipitate was washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) at a temperature of about 40°C. Dried for 18 hours and get to 2.35 g of pure depolimerizovannogo heparin (sodium salt). The yield is 90%.

Properties depolimerizovannogo heparin obtained in this way.

Average molecular weight: 2400 Yes

The activity of anti-Ha: 167,5 Honey/mg

Activity, anti-IIa: 1.1 Miu/mg

The ratio of the activity of anti-XA activity to anti-IIa: 152

Example 12

Depolymerization and conversion to the sodium salt (0.05% of water)

In the conical flask enter 140 ml of dichloromethane. While mixing, slowly introduce 20 g is 0.019 mole) of a compound is ensilage ester of heparin (degree of esterification: 75%, salt benzene), obtained as described in example A. the reaction medium introduced 20 g of zeolite 4 a and the water content was adjusted to 0.05% with slow stirring for 48 hours the Pooled liquid move in conditions inert atmosphere in a conical flask A. After complete dissolution of the added 3.5 ml (0,012 mol) of 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,2,3-diazaphospholidine. Stirred at a temperature of about 20°within 24 hours during this time in the conical flask To prepare a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol. After complete dissolution the solution is injected 10 g celite Hyflo suprecel. Under stirring with a magnetic stirrer, the reaction mixture from the conical flask And poured for about 2 minutes in a methanol solution of sodium acetate at a temperature of about 4°C. Stirred for 15 min and leave the suspension to precipitate an hour. Transparent part of the settled liquid is selected and removed (420 ml). In the sediment injected 420 ml of methanol and stirred for 15 minutes Leave to re-deposition for about an hour. The pooled liquid is selected and removed (450 ml). In the sediment injected 450 ml of methanol and stirred for 15 minutes and Then the precipitate is filtered through a porous glass 3. The precipitate is washed with 200 ml of methanol. The wet solid with whom Elo-yellow dry substance, then dried under reduced pressure (6 kPa) at a temperature of about 50°C. Dried for 16 h and get are 5.36 g of the crude depolimerizovannogo heparin sodium salt in celite (10g). The output is 68,6%.

Saponification

In a conical flask of 50 ml volume injected are 5.36 g (0,00817 mole) of the crude depolimerizovannogo heparin obtained in the previous phase, and 50 ml of water. The suspension is filtered through a porous glass 3 and four times washed with 15 ml of water. The obtained filtrate is placed in a conical flask with a volume of 150 ml Under stirring with a magnetic stirrer enter a 1.01 ml (0,0122 mol) of 35% sodium liquor at a temperature of about 4°C. afterwards the mixture is shaken for 3 hours the Solution is neutralized by adding 1N HCl and injected 11 g of sodium chloride. In reaction medium add 77 ml of methanol. Stirred for 15 min and add 200 ml of methanol, and then stirred for one hour. The stirring is stopped and the suspension is left to settle for one hour at a temperature of about 4°C. the Pooled liquid is selected and removed (240 ml). In the sediment administered with 240 ml of methanol and stirred for 10 min. Left for 30 min to re-deposition. The pooled liquid is selected and removed (225 ml). In the sediment injected 225 ml of methanol and stirred for 10 minutes, the Suspension is filtered through a porous glass is 3. The precipitate twice washed with 50 ml of methanol. The wet solid is dried, then dried under reduced pressure (6 kPa) and at a temperature of about O°C. Dried for 18 h and obtain 2.65 g of the crude depolimerizovannogo heparin (sodium salt). The output is 53.7%.

Clean

In a conical flask 100 ml injected 2.65 g of the crude depolimerizovannogo heparin obtained at the previous stage, and 26.5 ml of distilled water. The mixture is heated to 40°under stirring with a magnetic stirrer. the pH was adjusted to 9.7±0,1 by adding 1N sodium hydroxide. The reaction medium is filtered through a membrane with a hole size of 0.45 μm and injected with 0.25 ml of 30% aqueous hydrogen peroxide solution. Shaken for 2 h at a temperature of about 20°C, then the mixture is neutralized by adding 1N HCl and injected 3 g of sodium chloride. After that, the solution is filtered through a membrane with a hole size of 0.45 μm, and then pour in 21 ml of methanol at a temperature of about 10°C. Then the solution is heated to about 20°C and stirred for 15 minutes then add 54 ml of methanol and stirred for one hour. The stirring is stopped and the suspension is left to settle for 45 minutes. The pooled liquid is selected and removed (46 ml). In the sediment enter 46 ml of methanol and stirred for 5 is in. Leave to re-suspension of sediment by about 30 minutes the Pooled liquid is selected and removed (50 ml). Add 50 ml of methanol and the precipitate in suspension is filtered through a porous glass 4. The obtained white precipitate was washed with two portions of 10 ml of methanol each. The wet solid is dried, then dried under reduced pressure (6 kPa) at a temperature of about 50°C. Dried for 18 hours and get 2,363 Mr. clean depolimerizovannogo heparin (sodium salt). The output is of 89.1%.

Properties depolimerizovannogo heparin obtained in this way.

Average molecular weight: 2500 Yes

The activity of anti-Ha: 192 Honey/mg

Activity, anti-IIa: 1.3 Miu/mg

The ratio of the activity of anti-XA activity to anti-IIa: 148

1. A mixture of sulfated oligosaccharides having the General structure of the polysaccharides included heparin, having the following features: medium molecular weight is from 1500 to 3000 Da, the activity of anti-Ha ranges from 150 to 200 Miu/mg activity, anti-IIa below 10 Miu/mg and the ratio of anti-XA/anti-IIa above 30, oligosaccharides included in the mixtures contain from 2 to 26 sharidny links, one of their end groups is a link 2-0-sulfate unsaturated 4,5-uronic acid, contain hexasaccharide fraction comprising from 15 to 25% mixtures of oligosaccharides contain hexa is charidee fractions from 8 to 15% of hexasaccharide Δ IIa-IIs-Is

and have the form of salts of alkaline or alkaline earth metal.

2. The mixture of oligosaccharides according to claim 1, characterized in that the salt of an alkaline or alkaline-earth metal selected from salts of sodium, potassium, calcium and magnesium.

3. The mixture of oligosaccharides according to any one of claims 1 and 2, characterized in that activity, anti-IIa below 5 Miu/mg and in particular ranges from 0.5 to 3.5 Miu/mg)

4. A mixture of oligosaccharides according to any one of claims 1 to 3, characterized in that they relate the activity of anti-XA activity to anti-IIa above 50 and in particular above 100.

5. A mixture of oligosaccharides according to any of claims 1 to 4, characterized in that their medium molecular weight ranges from 2000 to 3000 and in particular from 2400 to 2650 Yes.

6. The mixture of oligosaccharides according to any one of claims 1 to 5, characterized in that the activity of anti-Ha ranges from 150 to 200 Miu/mg activity, anti-IIa is from 0.5 to 3.5 Miu/mg and medium molecular weight 2400 to 2650 Yes.

7. A method of producing mixtures of oligosaccharides according to any one of claims 1 to 6, in which carry out the depolymerization salt of the Quaternary ammonium complex benzyl ester of heparin in an organic medium in the presence of a strong organic base, pKa greater than 20, characterized in that the strong organic base belongs to the family of phosphazenes, in particular, R is the target dichloromethane, containing less than 0.3% of water.

8. The method of receiving according to claim 7 mixtures of oligosaccharides according to claim 1, characterized in that the water content is less than 0.2%.

9. The method of receiving according to claim 7 or 8, characterized in that the salt of the Quaternary ammonium complex benzyl ester of heparin salt is benzene, cetylpyridinium or cetyltrimethylammonium.

10. The method of receiving according to claim 7 or 8, characterized in that the base of the family of phosphazenes have the formula

in which the radicals R1-R7, identical or different, denote a linear or branched alkyl radicals containing from 1 to 6 carbon atoms.

11. The method of obtaining of claim 10, wherein the base of the family of phosphazenes used at the stage of depolymerization, is a 2-tert-Butylimino-2-diethylamino-1,3-dimethylpyridine-1,3,2-datafactory or tert-butylamine(pyrrolidino)fastran.

12. The method of receiving according to claim 7 or 8, characterized in that the molar ratio of the strong base/ester constitutes from 0.2 to 5 and preferably from 0.6 to 2.

13. The method of producing oligosaccharides according to any one of claims 1 to 6 of heparins, which includes the following stages:

a) translation in salt, sodium heparin by the action of chloride benzene,

b) etherification of the resulting heparinate benzene under the action of gasoline is chloride,

c) the translation of the received complex benzyl ether salt of Quaternary ammonium,

d) depolymerization salt of the Quaternary ammonium complex benzyl ester of heparin by the method described in claim 10 or 11,

e) transfer salts of Quaternary ammonium sodium salt,

f) saponification of heparin under the action of a base such as sodium hydroxide,

g) possible treatment, in particular, with an oxidizer such as hydrogen peroxide.

14. The method according to item 13, wherein the reaction in stage a) is performed by the action of excess chloride baseline sodium heparin at a temperature of from about 15 to 25°and the molar ratio salt/sodium heparin from 3 to 4.

15. The method according to item 13, wherein in stage b) the esterification is carried out in a chlorinated organic solution, such as chloroform or methylene chloride at a temperature of from 25 to 45°C, preferably from 30 to 40°and then ester in the form of sodium salt obtained by precipitation with 10 wt.% sodium acetate in alcohol, such as methanol, at the rate of 1 to 1.2 volume fraction of alcohol on the volume fraction of the reaction medium.

16. The method according to item 13 or 15, characterized in that the degree of esterification salt of the Quaternary ammonium complex benzyl ester of heparin ranges from 50 to 100% and predpochtitel is about 70 to 90%.

17. The method according to PP, 15 or 16, characterized in that use from 0.5 to 1.5 wt. including benzylchloride 1 wt. including salt benzene heparin during the duration of the reaction is from 10 to 35 p.m.

18. The method according to item 13, wherein the translation in salt at the stage C) is carried out using chloride Quaternary ammonium and preferably using chloride benzene, chloride of cetylpyridinium or chloride of cetyltrimethylammonium in the aquatic environment at a temperature of from 10 to 25°C.

19. The method according to p, characterized in that the molar ratio of chloride Quaternary ammonium/sodium salt of a complex benzyl ester of heparin ranges from 2 to 3.

20. The method according to item 13, wherein the translation of the sodium salt of the Quaternary ammonium complex benzyl ether depolimerizovannogo heparin (stage e) is carried out by treatment of the reaction medium of an alcoholic solution of sodium acetate and preferably 10%solution of sodium acetate in methanol (weight/volume) at a temperature of from 15 to 25°C.

21. The method according to item 13, wherein the saponification (stage f) is carried out using alkali metal hydroxide such as sodium hydroxide, potassium, lithium hydroxide, in an aqueous medium at a temperature of from 0 to 20°and preferably from 0 to 10°C.

22. The method according to item 21, wherein use from 1 to 5 molar equivalents of hydroxide melocheville, preferably from 1 to 2 molar equivalents of sodium hydroxide.

23. The method according to item 13, characterized in that the clearance (stage g) is carried out using hydrogen peroxide in an aqueous medium at a temperature of from 10 to 50°C, preferably from 20 to 40°C.

24. The oligosaccharides according to any one of claims 1 to 6 as a medicinal product has antimicrobial activity.

25. The use of oligosaccharides according to any one of claims 1 to 6 to obtain drugs having antithrombotic activity.

26. Use A.25 to obtain drugs for treatment or prophylaxis of venous and arterial thrombosis, deep venous thrombosis, pulmonary embolism, unstable angina, myocardial infarction, ischemia of the heart, occlusion of peripheral arteries, atrial fibrillation, proliferation of smooth muscle cells, atherosclerosis and arteriosclerosis, cancer through modulation of angiogenesis and growth factors and diabetic disorders such as diabetic retinopathy and diabetic nephropathy.

27. Antithrombotic pharmaceutical composition comprising the oligosaccharide according to any one of claims 1 to 6 and one or more excipients or fillers, or additives, inert pharmaceutical.

28. The pharmaceutical composition according to item 27, wherein they are R what sites for subcutaneous or intravenous injection.

29. Pharmaceutical compositions for p, characterized in that they are the preparations for inhalation, intended for insertion through the lungs.

30. Pharmaceutical compositions for p, characterized in that they are drugs intended for oral administration.

31. A mixture of oligosaccharides according to any one of claims 1 to 6, which can be obtained by the method according to item 13.



 

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12 cl, 1 tbl, 171 ex

Aminoalcohol // 2332212

FIELD: medicine; pharmacology.

SUBSTANCE: invention concerns pharmaceutical composition for prevention and treatment of diseases caused by abnormal lymphocyte quantity in peripheral blood, or for prevention and treatment of diseases, the symptoms of which can be alleviated by lymphocyte quantity reduction in peripheral blood. The composition includes a compound of the general formula where each of R1, R2 and R3 denotes unit described in the claim, its pharmaceutically acceptable salt or ether in a quantity sufficient for active component dosage from 0.0001 mg/kg/day to 1 mg/kg/day.

EFFECT: low toxicity and excellent physical and chemical properties.

16 cl, 30 ex

FIELD: medicine.

SUBSTANCE: invention concerns lipid content normalisation of thrombocyte membranes during metabolic syndrome (MS). For that purpose a patient undergoes 16-week therapy involving individual hypocalorific diet, graduated physical activity, administration of 40 mg lovastatin 2 times per day and of 10 mg lisinopril once in the morning.

EFFECT: reduced thrombus formation risk for the given patient group due to efficient and stable lipid content normalisation of thrombocyte membranes.

2 ex

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