RU2319707C1 - Method for preparing sulfated derivatives of arabinogalactan possessing anticoagulating and hypolipidemic activity - Google Patents

Abstract

FIELD: chemical-pharmaceutical industry.

SUBSTANCE: invention relates to a method for synthesis of arabinogalactan-base sulfated biopolymers wherein arabinogalactan is the main polysaccharide from Siberian larch. Invention describes a method for preparing sulfated derivatives of arabinogalactan that involves interaction of arabinogalactant in dimethylsulfoxide medium in the ratio 1:3 with a sulfating agent representing complex SO₃ - dimethylformamide in the concentration of SO₃ ≥18%. The process is carried out at continuous stirring, at temperature 20^°C for 30 min followed by isolation of product both in acid and saline forms. The substitution degree of biopolymer macromolecule is 8.1-12.65% as measured for the quantitative content of sulfur. Synthesized sulfated derivatives of arabinogalactan retain the structural organization, water-solubility and membranotropicity of natural polysaccharide, and elicit high physiological (anticoagulating and hypolipidemic) activity also.

EFFECT: improved method of synthesis.

2 cl, 3 dwg, 3 ex

Description

The invention relates to the pharmaceutical industry, medicine, pharmacology, and for the production of sulfated biopolymers based on arabinogalactan with anticoagulant and hypolipidemic activity. Arabinogalactan, the main polysaccharide of Siberian larch, is a non-toxic product and is recommended for use in the food industry (A.B. Mulin. Development of technology for the production of flour confectionery using arabinogalactan. (Abstract of dissertation for the degree of candidate of technical sciences M., 1998)) . The multifunctionality and water solubility of arabinogalactan allows us to consider it as a promising synthon for the synthesis of various compounds, including pharmaceutical and medical preparations.

A method is described for producing sulfated derivatives of arabinogalactan isolated from Siberian larch by reacting it with a sulfating mixture in the presence of freshly distilled dimethyl sulfoxide (DMSO) for 30 minutes at a temperature of 20 ° C with its further neutralization or evaporation and precipitation in ethyl alcohol. The sulphating mixture is a SO ₃ -dimethylformamide complex obtained by distilling gaseous sulfur trioxide (SO ₃ ) from oleum to freshly distilled dimethylformamide (DMF). The content of SO ₃ in DMF is at least 18%.

Described sulfated arabinogalactan derivatives of the formula AG-SO ₃ R,

where AG is arabinogalactan,

SO ₃ R - sulfate group,

R = H is the acid form of the sulfated derivative AG,

R = Me is the salt form, where Me are metals of groups I and II.

The degree of substitution of the biopolymer macromolecule in terms of the quantitative sulfur content is 8.1-12.65%.

These compounds are water-soluble, exhibit membranotropic properties, that is, they are able to deliver drugs to the cell (Arabinogalactan derivatives and uses thereof. C. Jun, P. Enriques, S. Palmacci, LJ Josephson, JMLewis. US Patent 5478576) and have anticoagulant and lipid-lowering activity.

Cardiovascular disease is the most common cause of death in industrialized countries and comes second after mortality from infectious diseases. Their pathogenesis is based on atherosclerotic changes in blood vessels resulting from a violation of the hemostatic system, a deterioration in the rheological properties of the blood, and a violation of lipid metabolism in the body. A possible approach to solving the problem of prevention and treatment of atherosclerosis, as well as coronary heart disease is the use of drugs with antiatherogenic activity, i.e. reducing or preventing atherosclerotic damage to blood vessels. One of these drugs is heparin, but its widespread use as an anti-atherogenic agent is significantly limited by the short half-life in the body, as well as deep and prolonged hypocoagulation (risk of bleeding). In addition, the high cost of production of heparin (to obtain 1 kg of heparin, it is necessary to process up to 10 tons of cattle lungs) has led to the search for its synthetic and natural analogues. To date, it is known that various sulfated polysaccharides of both natural (chondroitin sulfates and other sulfated glycosaminoglycans) and synthetic (dextran sulfates, chitosan sulfates, etc.) origin have to a greater or lesser extent anti-atherogenic activity. Since they contain, like heparin, O-sulfate groups, the leading role of which in the manifestation of anti-atherogenic activity is proved to date (Rozkin M.Ya., Levina M.N., Efimov VS, Usov A.I. / / Pharmacol. And toxicol. - 1991. - T. 54. - No. 5. - P.40-42. And Berngtsson G., Olivercona T., Hook M. et al. // Biochem. J. - 1980. - Vol. 189. - No. 3. - P. 625-633.).

A known method of producing a sulfated derivative of AG in both acidic and salt forms (Chemical Abstracts 1968, vol. 69, No. 11, 44182q). The complexes SO ₃ -pyridine (1) and SO ₃ -triethylamine (2) are used as sulfating mixtures. At the same time, sulfation of AG with the help of complex (1) occurs within 3 hours at 70 ° C, and with the help of complex (2) within 3 hours at 50-55 ° C, then 2 hours at 20 ° C. This method has several disadvantages.

1. Preparation of the SO ₃ -pyridine complex is carried out by adding chlorosulfonic acid to an excess of pyridine. In addition to the sulfating complex, a by-product of pyridinium chloride is formed, the presence of which is difficult to eliminate.

2. The presence in the reaction mixture of pyridinium chloride leads to the production of sulfated polysaccharide derivatives of reduced physiological activity.

3. SO ₃ -pyridine complex is obtained directly in the sulfation reaction, which does not allow its standardization.

4. The sulfation reaction by this complex itself is carried out at high t = 70 ° C for a long time (3 hours), which leads to degradation of the polysaccharide molecule.

5. When using 1 and 2 sulfating mixtures, the problem arises of eliminating the persistent unpleasant odor of the final products, moreover, the SO ₃ -triethylamine complex is very toxic.

Another method known in the literature for producing a sulfated derivative of AG (Kamitakahara H., Mikawa Y., Hori M., Tsujihata S., Minato K., Nakatsubo F. Syntheses, characterization, and biological activities of sulfated polysaccharides // 10 ^th Int. Symp . on Wood Pulp Chem. Yokohama, Japan. 1999. Vol.2. P. 238-241) consists in sulfating AG with the SO ₃ -pyridine complex in the presence of DMF at t = 20 ° C for several days. Then the reaction mixture is neutralized with 2N NaOH, the obtained GPC product is purified (by gel permeation chromatography) on P-6 bio gel (eluent is water) or by dialysis against distilled water for several days.

This method also has several disadvantages.

1. Dimethylformamide is used as a solvent for both the sulfating mixture and AG itself. AG does not dissolve in dimethylformamide; therefore, a heterogeneous synthesis system is formed, which reduces the efficiency of the reaction.

2. Sulfation at t = 20 ° C, but for a long time leads to degradation of the AG molecule.

The aim of the invention is the synthesis of sulfated derivatives of arabinogalactan in acid and salt forms with anticoagulant and lipid-lowering activity.

The technical result of this invention is that to obtain sulfated derivatives, polysaccharide AG, isolated from Siberian larch, was used, its multifunctionality, water solubility and membrane-activeness are clear advantages compared with other synthons (cellulose, chitin, chitosan, etc.) used for synthesis of biologically active substances.

For the synthesis, arabinogalactan was used, which was obtained in laboratory conditions from an aqueous extract of Siberian larch wood and purified by reprecipitation in alcohol and a sulfating mixture in the form of a SO ₃ -DMF complex with an SO ₃ concentration _of ≥18%.

The arabinogalactan molecule isolated from Siberian larch wood has a branched structure and a molecular weight of about 15-20 kD. The main chain of the biopolymer macromolecule is represented by the galactan core, and the D-galactopyranose units are linked by β-1,3-bonds, most of the galactose units have side branches at C-6. The side chains contain 3,6-di-O- and 6-O-substituted β-D-galactopyranose residues and 3-O-substituted β-L-arabinofuranose residues. The terminal non-reducing residues are β-D-galactopyranose, β-D-arabinofuranose and β-L-arabinopyranose. The ratio of galactose to arabinose is 7.5: 1, two-thirds of arabinose residues are present in furanose form, one third in pyranose form. Uronic acids in arabinogalactan of Siberian larch were not found.

Arabinogalactans are present in almost all coniferous species of wood, however, they differ from each other in structure, namely, the type of binding of monosaccharide units, their ratio and, as a result, physico-chemical properties: solubility in water, molecular weight, degree of polydispersity, polyfunctionality.

The distinctive features of arabinogalactan isolated from Siberian larch wood are the multifunctionality, water solubility and membrane activity of the biopolymer macromolecule, which serves as clear advantages compared to other synthons used for the synthesis of biologically active substances. In addition, in larch wood, Siberian arabinogalactan is present in the maximum amount of 10-25% for coniferous wood, and therefore Siberian larch serves as a reliable source of its production.

This goal is achieved in that to the solution of AG in DMSO in the ratio (1: 3) a SO ₃ -DMF complex with a SO ₃ concentration _of ≥18% was added with stirring. Sulfation was carried out for 30 minutes at t = 20 ° C with continuous stirring. After that, the reaction mixture was neutralized with a 10% aqueous alkali solution (KOH, NaOH, CaOH, BaOH, etc.) according to a universal indicator and planted in ethyl alcohol.

The resulting salt of the sulfated arabinogalactan derivative was purified by dialysis against distilled water. Then the dialyzed solution was evaporated to a minimum volume and precipitated in ethyl alcohol.

The sulfated derivative AG in acid form was prepared without neutralizing the reaction mixture. For this, it was evaporated to a minimum volume and precipitated in ethyl alcohol, the precipitate was washed with ethyl alcohol on a No. 40 glass porous filter from the residues of the sulfating mixture and DMSO.

The resulting preparations are water-soluble and exhibit membrane-tropic properties, like natural AG. MM of the obtained derivatives was determined by GPC on Sephadex G 100; 0.3% aqueous NaCl solution served as eluent.

The content of sulfate groups in the obtained preparations was determined in terms of the quantitative sulfur content by the titrimetric method (bariummetry) using specific reagents. A quantitative analysis of the preparations obtained by the claimed method showed that, depending on the form (acidic or saline) of the sulfated arabinogalactan derivative, the degree of substitution of the biopolymer macromolecule in terms of the quantitative sulfur content is 8.1-12.65%. The presence of the sulfate group and the chemical treatment performed do not cause degradation of the polymer matrix. (MM _{acid form} = 22.8 kD, MM _{salt form} = 15.7-22.1 kD)

The proposed method is characterized by the following advantages:

- the synthesis is carried out in a homogeneous medium (AG is dissolved in DMSO),

- at a low reaction temperature (20 ° C) and a short duration of exposure to the sulfating complex on AG (30 minutes),

- the SO ₃ -DMF complex is used as a sulfating mixture; its advantages are:

- storage stability (from 0 to + 4 ° C), after 6 months of storage, the concentration of SO ₃ in the complex is reduced by 1%;

- the possibility of standardization and constant monitoring of the content of SO ₃ in the complex both during receipt and during storage;

- high reactivity due to the fact that DMF is a very weak base (weaker than pyridine);

- ease of use (SO ₃ -DMF complex is a liquid from yellow to orange, depending on the content of SO ₃ , which can be taken with an ordinary pipette).

The developed synthesis method allows to obtain sulfated derivatives of AG in both acidic and salt forms with a different set of cations. Moreover, the molecular weight of the derivatives obtained does not significantly differ from the natural matrix (MM _AG = 15-20 kD, MM _{acid form} = 22.8 kD, MM _{salt form} = 15.7-22.1 kD).

The obtained sulfated derivatives of AG retain the structural organization, water solubility and membrane activity of the natural polysaccharide, and have anticoagulant and hypolipidemic activity.

The following examples illustrate the invention.

Example 1

1 g of AG was dissolved with stirring in 3 ml of DMSO, then 10 ml of the SO ₃ -DMF complex with an SO ₃ concentration _of ≥18% was added. Sulfation was carried out with vigorous stirring at t = 20 ° C for 30 minutes. After that, the reaction mixture was neutralized with 26 ml of 10% aqueous KOH solution and planted in 500 ml of ethyl alcohol. The resulting precipitate was filtered on a No. 40 glass porous filter, dissolved in 50 ml of distilled water, and dialyzed against distilled water for 6 days. The dialyzed solution was evaporated to 30 ml and planted in 150 ml of ethyl alcohol. The yield of the obtained product was 95%, the degree of substitution of the biopolymer macromolecule in terms of quantitative sulfur content was 10.3%. MM = 15.7 kD.

Example 2

1 g of AG was dissolved with stirring in 3 ml of DMSO, then 10 ml of the SO ₃ -DMF complex with an SO ₃ concentration _of ≥18% was added. Sulfation was carried out with vigorous stirring at t = 20 ° C for 30 minutes. After that, the reaction mixture was evaporated to a minimum volume and planted in 500 ml of ethyl alcohol. The resulting precipitate was filtered off and washed on a No. 40 glass porous filter with ethyl alcohol. The yield of the obtained product was 90%, and the degree of substitution of the biopolymer macromolecule in terms of the quantitative sulfur content was 8.1%. MM = 22.8 kD.

Example 3

1 g of AG was dissolved with stirring in 3 ml of DMSO, then 10 ml of the SO ₃ -DMF complex with an SO ₃ concentration _of ≥18% was added. Sulfation was carried out with vigorous stirring at t = 20 ° C for 30 minutes. After this, the reaction mixture was neutralized with 17 ml of a 10% aqueous solution of Ca (OH) ₂ and precipitated in 500 ml of ethyl alcohol. The resulting precipitate was filtered on a No. 40 glass porous filter, dissolved in 50 ml of distilled water, and dialyzed against distilled water for 6 days. The dialyzed solution was evaporated to 30 ml and planted in 150 ml of ethyl alcohol. The yield of the obtained product was 98%, the degree of substitution of the macromolecule - hypocholesterolemic effect (figure 2), atherogenic lipoproteins (β-lipoproteins) - anti-atherogenic activity (figure 3).

Figure 1 shows the anticoagulant activity of sulfated arabinogalactan.

As can be seen from the data presented in figure 2, the potassium salt of sulfated arabinogalactan reduces the level of total serum cholesterol (cholesterol total) by 4.9%, sulfated arabinogalactan in acid form by 4.2%, while heparin reduces this figure is only 0.8%. The hypolipidemic effect of sulfated arabinogalactan is also manifested in a decrease in the level of atherogenic lipoproteins (β-LP) (Fig. 3), so the acid form reduces this indicator by 4.2%, while potassium salt - by 8.3%, heparin reduces atherogenic lipoproteins by 2.1%.

Thus, the salt form of sulfated arabinogalactan (potassium salt) has a pronounced anticoagulant and lipid-lowering activity comparable to that of heparin, the acid form of sulfated arabinogalactan exhibits lipid-lowering activity in the absence of a significant effect on blood coagulation.

Claims (2)

1. The method of producing sulfated derivatives of arabinogalactan isolated from Siberian larch wood by reacting arabinogalactan in a solvent with a sulfating agent with continuous stirring and at a constant temperature of 20 ° C for 30 minutes with further isolation of the product, characterized in that the solution is subjected to interaction with arabinogalactan in dimethyl sulfoxide at a ratio of 1: 3, and as a sulfating mixture, a SO ₃ complex, dimethylformamide with a SO ₃ concentration _of ≥18%, is used. 2. The method according to claim 1, characterized in that the selection of the product is carried out by neutralization with metal hydroxides of groups I and II or by evaporation of the reaction mixture, and then planting in ethyl alcohol.

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