JPH0576448B2 - - Google Patents

Description

[Detailed description of the invention]

[0001]

[Field of Industrial Application] The present invention relates to a lipid serum clarifying agent characterized by containing a sulfated polysaccharide as an active ingredient.

BACKGROUND OF THE INVENTION Hyperlipidemia often occurs in association with diseases such as malignant tumors, arteriosclerosis, diabetes, and nephrotic syndrome. In recent years, hyperlipidemia has been on the rise with the increase in the above-mentioned diseases. Currently, effective drugs for these treatments include, for example, dextran sulfate and heparin. That is, dextran sulfate has the effect of lowering dextranceride, which is a polymer of α-1,6-linked D-glucopyranose produced by microorganisms such as Leuconostoc mesenteroides. have It also has anticoagulant, antihyaluronidase, and fibrinolytic effects, and is known as an effective drug for treating thrombosis, hyperlipidemia, and arteriosclerosis. It is known that the larger the molecular weight and the higher the ion content, the stronger these physiological activities, but the higher the toxicity.

[0002]

[Case 2]■■■ Turtle shell [0006] ■■■

[0003] On the other hand, heparin, a mucopolysaccharide that exists in animal fibers, has a wide range of physiological effects such as strong blood coagulation inhibitory effects and lipid serum clarifying effects, and its activity is much higher than that of human heparinoids. Although strong,
The quality of the standard product is not constant, the structure is complex, and the isolation method is complicated. Heparin is characterized by having sulfated amino acids, such as the following units, in its molecule.

[0004]

[Chemical formula 3] ■■■ Tortoise shell [0007] ■■■ (However, in the formula, X represents H or SO ₃ ^- .)

[0005]

Problems to be Solved by the Invention and Means for Solving the Problems The present inventors have conducted intensive research with the aim of synthesizing a substance having lipid serum clarifying action, and as a result, they have found that α-1, as shown in the general formula above, We succeeded in synthesizing a novel sulfated polysaccharide that does not exist in nature, which has a 6-linked sugar chain and also contains an amino group such as heparin in its molecule, and the sulfated polysaccharide is
As shown in the Examples below, it was discovered that it has an excellent lipid serum clarifying effect, and the present invention was completed.

[0006] The present invention provides a sulfated polysaccharide consisting of the following structural units or a sulfated polysaccharide consisting of structural units and structural units (where n is the number of structural units in the sulfated polysaccharide, m is the number of structural units in the sulfated polysaccharide, and This is the number of structural units in a sulfated polysaccharide, m/n is 0 to 5, and m+n is an integer of 30 to 150) as an active ingredient.

[Case 4] ■■■ Tortoise shell [0008] ■■■

[0007] The active ingredients of the present invention will be explained below. The active ingredient of the present invention is a sulfated polysaccharide as shown in the general formula above, and can be synthesized, for example, by the following method. Previously, the present inventors used an azide sugar as a precursor of an amino sugar, which has an α'-1,6-linked sugar chain and has a high degree of polymerization that contains an amino group such as heparin in the molecule. Succeeded in synthesizing a high amino sugar polymer and its copolymer with D-glucose (Japanese Unexamined Patent Application Publication No. 1983-1999)
No. 180603, No. 57-180604, No. 57-180605, No. 180603, No. 57-180604, No. 57-180605, No.
57-180606). The process is shown below.

[0008]

[Case 5] ■■■ Tortoise shell [0009] ■■■

[0009]

[C6]

[ka]

【0010】

[C7]

[Chemical formula] (In the formula, Bz is a benzoyl group, Ms is a
A methanesulfonyl group, Bn is a benzyl group, and n and m indicate the number of each structural unit constituting the compound (10) or (11). )

[0011] That is, compound (2) can be easily obtained by treating 1,6-anhydro-β-D-glucopyranose (1) derived from β-D-glucopyranose with benzoyl chloride. [M.Cerny et al Collection
Czechoslov.Chem.Commun.Vol.26, 2542 (1961)
reference. ]. Compound (2) is dissolved in dry pyridine and cooled on ice. Add methanesulfonyl chloride dropwise while stirring.
Gradually return to room temperature. After 3 hours, the mixture is poured into a large amount of ice water and stirred to precipitate crude crystals of compound (3).
After filtration, washing with water, drying, and recrystallization from methanol, compound (3) is obtained as white crystals. Dissolve compound (3) in chloroform and cool on ice. A sodium methoxide solution prepared by dissolving metallic sodium in methanol is added dropwise to this while stirring. After the mixture was left at room temperature overnight, it was neutralized with 5% hydrochloric acid and concentrated to dryness under reduced pressure. Dilute the residue with acetone
Extract twice and concentrate the extract under reduced pressure to obtain an oil. Silica gel column chromatography (developing solvent chloroform-methanol, 100:1v/v)
Upon purification, a mixture of compounds (4-a) and (4-b) is obtained. A mixture of compounds (4-a) and (4-b) is dissolved in dry tetrahydrofuran, and sodium hydride (purity 60%) is added under ice cooling, followed by stirring for 30 minutes. Next, benzyl bromide is added and reacted at room temperature for 4 hours. Add ice-cooled saturated aqueous ammonium chloride solution, stir for 30 minutes, and then extract three times with ether.
The extract was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and the resulting oil was purified using silica gel column chromatography (developing solvent benzene-ethyl acetate 20:
1v/v), oily compound (5-a)
A mixture of and (5-b) is obtained. A mixture of compounds (5-a) and (5-b) is dissolved in a mixed solvent of ethanol and saturated ammonium chloride water, sodium azide is added, and the mixture is heated under reflux for 60 hours with stirring. After cooling, distilled water is added, ethanol is removed under reduced pressure, and the remaining aqueous solution is extracted with chloroform. The extract was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain an oil. Purification by column chromatography on silica gel (developing solvent: benzene-ether, 20:1 v/v) yields a mixture of compounds (6-a) and (6-b). A mixture of compounds (6-a) and (6-b) is dissolved in dry tetrahydrofuran and cooled on ice. Add sodium hydride (purity 60%) and stir for 30 minutes, then add benzyl bromide. After stirring at room temperature for 3 hours, an ice-cooled saturated aqueous ammonium chloride solution was added, and the mixture was stirred for an additional 30 minutes. This mixed solution is extracted with ether, and the extract is dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain an oil. Silica gel chromatography (developing solvent, hexane-ethyl acetate 5:1v/
Purification in step v) yields oily compound (7). It crystallizes if left standing and can be recrystallized from cyclohexane. Compound (7) is vacuum dried under high vacuum of 10 ⁻⁵ mmHg overnight and dissolved in methylene chloride previously dried over calcium hydride in a vacuum ampoule. This solution was mixed with phosphorus pentafluoride, benzoyl fluoride, and methylene chloride that had been reacted for 30 minutes in a polymerization tube at -60℃, and after reacting at -60℃ for 23 hours, the polymerization ampoule was opened. and pour in methanol. At this time, the polymer precipitates, so chloroform is added to it until the polymer is sufficiently dissolved, neutralized with an aqueous sodium bicarbonate solution, washed with water, and dried over anhydrous sodium sulfate. After removing the desiccant by filtration, the filtrate is concentrated, and petroleum benzine is added thereto for reprecipitation. The operations of dissolution, concentration, and reprecipitation are performed twice more, and the polymer is dissolved in benzene and freeze-dried to obtain polymer (8). Polymers having various molecular weights and degrees of polymerization can be obtained by carrying out the reaction in the same manner as above while changing the amount of compound (7) used and the polymerization time. This is reduced by the following method to obtain aminopolysaccharide (1). The reduction of polymer (8) is carried out by Birch reduction, ie with an alkali metal in liquid ammonia. Alkali metals include potassium, sodium, and lithium. It is necessary to add at least 6 atoms of alkali metal to 1 unit in the polymer.
Usually, it is desirable to use three times the amount. Polymer (8) is added after being dissolved in a suitable solvent such as 1,2-dimethoxyethane or diglyme. The reaction time is suitably about -70 to -80°C, and the reaction time is suitably about 30 minutes to 3 hours. That is, metallic sodium and liquid ammonia are mixed and kept at -78°C, and a solution of polymer (8) in 1,2-dimethoxyethane is gradually added dropwise. 1,2-
Dimethoxyethane must be thoroughly dried with metallic sodium in advance. During the reaction, it is best to stir the system to make it homogeneous. After reacting for a predetermined time, the reaction is stopped by adding ethanol or ammonium chloride, and a small amount of water is added and left overnight to evaporate ammonia.
Unreacted polymer is removed by extraction with methylene chloride. After dialysis against distilled water for 3 days and filtering out any insoluble portion, the insoluble portion is vacuum dried, and the soluble portion is concentrated and freeze-dried to obtain aminopolysaccharide (). On the other hand, compound (9) is 1,6-anhydro-β
-D-glucopyranose (1) can be easily obtained by treating with benzyl bromide or the like. The thus obtained compounds (7) and (9) are copolymerized and reduced by the following method to obtain an amino sugar copolymer () consisting of structural units and structural units.
get. The copolymerization reaction is carried out by dissolving the compounds (7) and (9) in a sufficiently dried solvent and reacting with a Lewis acid as a catalyst. Suitable solvents include methylene chloride and chloroform. . Lewis acids include phosphorus pentafluoride, antimony pentachloride,
Examples include boron trifluoride etherate, but phosphorus pentafluoride is most suitable in terms of yield and stereoregularity of the resulting copolymer. The amount of Lewis acid used is 2 based on the total number of moles of compounds (7) and (9).
~7 mol%, especially 2-3 mol% is optimal. Note that various copolymers can be obtained by changing the mixing ratio of compounds (7) and (9). The reaction temperature is suitably about -30 to -60°C, and the polymerization time is suitably about 40 to 60 hours. This polymerization reaction is preferably carried out in vacuum or in an inert gas (eg, _N2 gas) atmosphere. In this way, an azido sugar-containing polysaccharide (10) consisting of a structural unit (10) and a structural unit (10) having an azide group in the molecule is obtained, which is then subjected to Birch reduction as described above to form an amino sugar. A copolymer () is obtained. The sulfated polysaccharide, which is the active ingredient of the present invention, is obtained from the polymers (1) and () thus obtained by the following reaction. The polymer () or () is pretreated to swell and then suspended in a suitable solvent. Suitable solvents include pyridine, dimethylsulfoxide, dimethylformamide, and the like. This suspension is reacted with the sulfating agent (and the solvent). As a sulfating agent, chlorsulfonic acid, piperidine-N
- Sulfuric acid etc. are suitable. The appropriate amount of the sulfating agent to be used is 20 to 30 moles based on the amino monosaccharide residue of the starting material. The reaction temperature and reaction time vary depending on the solvent and sulfating agent, but are approximately 70-100℃,
45 to 60 minutes is appropriate. After the reaction, the reaction mixture is allowed to cool, distilled water is added to stop the reaction, and then the reaction is neutralized with an alkali such as sodium hydroxide, and ethanol or the like is added to precipitate the polymer. This is centrifuged, dissolved in distilled water, dialyzed, and then concentrated and dried to obtain the sulfated polysaccharide as the active ingredient.

[0012]

[Chemical formula 8] ■■■ Tortoise shell [0012] ■■■ Where n is the number of structural units in the copolymer (), and m is the number of structural units in the copolymer ().

[0013] As described above, the active ingredient of the present invention can be obtained, but in the case of a homopolymer (), the intrinsic viscosity is about 0.15 to 0.18 and the ionic content is about 16 to 18%, and in the case of a copolymer (), the intrinsic viscosity is about 0.07~0.10,
Suitably, a sulfur content of about 10-15% is used. The drug of the present invention can be administered by the most appropriate method of administration depending on the clinical symptoms. That is, intravenous injection, subcutaneous injection, intramuscular injection, oral administration, etc. are used, but in the case of acute symptoms, intravenous administration is still appropriate. Furthermore, the active ingredient of the present invention can be dissolved or suspended in a suitable inert solvent, such as sterile or physiological saline, or an inert carrier or filler can be added thereto.
For example, parenteral preparations such as injections; powders, granules, capsules, tablets, coatings, syrups,
It is appropriate to use it in the form of a liquid solution or other oral preparation.

[0014] Examples of the inert carrier or filler include calcium phosphate, calcium carbonate, glucose, lactose, sucrose, dextrin, sucrose ester, starch, sorbitol,
Examples include mannitrite, crystalline cellulose, talc, kaolin, synthetic aluminum silicate, carboxymethylcellulose, methylcellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, gum arabic, gum tragacanth, gelatin, powdered agar, and sierracus. The active ingredient of the present invention is usually preferably contained in an amount of 1 to 90% by weight based on the weight of the composition. These contents can be changed appropriately depending on the dosage form.

[0015] The present invention will be described in detail below using production examples, working examples, and test examples, but the present invention is not limited by these in any way. Production example 1 Polymer (-a) (Mn=3.5×10 ⁴ , DPn=95)
(100mg, _NH2 group: 0621mmol, OH group: 1.24mmol)
Grind in water (10 ml) and centrifuge. Then 3 times in ethanol (20 ml) followed by ether (20 ml)
ml) and suspended in pre-dried pyridine (8 ml). Chlorsulfonic acid (1 ml, 15 mm
ol) and dry pyridine (6 ml), immerse in a boiling water bath, and allow to react for 1 hour. After cooling, distilled water (20 ml) is added to stop the reaction, neutralized with 2.5N aqueous sodium hydroxide solution (7.5 ml), and ketanol (50 ml) is added to precipitate the polymer. This is centrifuged, dissolved in distilled water, dialyzed for 3 days, concentrated and freeze-dried to obtain sulfated aminopolysaccharide homopolymer (-a). (183.7
mg, 81.0%) [Physical substance of homopolymer (-a)] C H N S Elemental analysis value Actual value 20.16 4.18 3.75 16.14 Calculated value (C ₆ H ₉ O ₁₀ NS ₂ Na ₂ ) 19.73 2.48 3.83
17.56 Ninhydrin reaction: negative IR: 580 cm ^-1 (M), 800 cm ^-1 (M), 610 cm ^-1
(M), 1240cm ^-1 (s, broad), 1510cm ^-1
(M) ¹³ C-NMR: δ97.53ppm (1C, C-1), 75.54
(3C, C-2, C-4, C-5), 74.68 (3C,
C-2, C-4, C-5), 71.03 (3C, C-
2, C-4, C-5), 68.07 (1C, C-6),
55.07 (1C, C-3) [α] _D ²⁵ +116.9° (C = 1.0, CHCl ₃ ) [η] = 0.17 (in H ₂ O, 30°C)

Production Example 2 Polymer (-a) (DPn=144, m/n=1.04,
Dissolve 100mg) in water (2ml) and add 20ml of ethanol.
Add and precipitate. After centrifugation, the mixture was further stirred and separated in ethanol and then in ether, and then suspended in pre-dried pyridine (8 ml). Chlorsulfonic acid (1
ml, 15 mmol) and dry pyridine (6 ml),
Place in a boiling water bath and let react for 1 hour. After cooling, distilled water (20ml) was added to stop the reaction.
Neutralize with 2.5N aqueous sodium hydroxide solution (7.5ml),
Add ethanol (50 ml) to precipitate the polymer. This is centrifuged, dissolved in distilled water, dialyzed for 3 days, concentrated and lyophilized to obtain a sulfated amino sugar-containing polysaccharide [copolymer (-a)] (168 mg, 76.2%). [Physical properties of copolymer (-a)] Elemental analysis: C H N S Measured value 17.40 3.39 1.55 14.65 Calculated value C ₆ H _8.589 O _10.211 N _0.489 S _1.90 Na _1.90・3H ₂ O 17.59 3.59 1.68 14.87 (Sulfation rate : SO ₃ ^- 1.90 per sugar residue) IR: 580cm ^-1 (M), 800cm ^-1 (M), 1240cm ^-1
(S, broad), 1510cm ^-1 (W) [α] _D ²⁵ = +100.2° (C = 1.0, H ₂ O) [η] = 0.07 (H ₂ O, 30°C)

[0017] Production example 3 copolymer (-b) (DPn=100-120, m/n
=4.76, _NH2 group 0.114mmol, OH group 1.74mmol, 100
The reaction and post-treatment were carried out in the same manner as in Production Example 2, except that sulfated amino sugar-containing polysaccharide (-b) was obtained (176 mg, 72.0%). [Physical properties of copolymer (-B)] Elementary analysis: C h N S Actual value 22.76 4.67 10.82 Calculating value C ₆ H _9.115 O _8.185 S _1.07 NA _{1.07 22.16} 4.68 0.55 SO ₃ ^- 1.07 pieces per base) IR: 580cm ^-1 (M), 800cm ^-1 (M), 1240cm ^-1
(S, broad) [α] _D ²⁵ = +109.6° (C = 1.0, H ₂ O) [η] = 0.15 (H ₂ O, 30°C)

Example 1 (injection) 5 g of the sulfated polysaccharide obtained in Production Example 1, 0.2 g of sodium bicarbonate, and 0.4 g of sodium chloride were dissolved in distilled water for injection to make 100 ml, and then dissolved by a conventional method. As an injection. Example 2 (Tablet) After saturating 200 g of the sulfated polysaccharide obtained in Production Example 2 with 140 g of lactose, the tablet was passed through a US standard sieve (60 mesh). The mixture is then moistened with 40 g of alcoholic polyvinylpyrrolidone and passed through a 12 mesh sieve to form granules and dried. After passing the dry granules through a 16 mesh sieve, 50 g of talc and 20 g of starch are added and then tablets weighing 450 mg are produced. Example 3 (Granule) 200 g of the sulfated polysaccharide obtained in Production Example 3, 150 g of methyl cellulose, 80 g of cornstarch, and some flavoring are mixed and passed through a 60-mesh sieve. The mixture is moistened with 20 g of alcoholic polyvinylpyrrolidone and then granulated through a stainless steel sieve with a diameter of 0.7 mm.

[0019] Test example 1 (lipid serum clearing activity) Using Japanese white rabbits (male) weighing 2.5 to 3.0 kg, the test substance (0.5% physiological saline solution) was intravenously injected at a rate of 5 mg/Kg, and 10 minutes later, the ear vein was injected. Blood was collected from each patient, and lipid serum purification activity in plasma was measured using the Hara-Kuzutani method (Proteins, Nucleic Acids, and Oxygen, 10 1224-1229, 1965). That is, 10% sodium citrate aqueous solution in advance
Collect 2.4ml of whole blood with a syringe containing 0.1ml, and add 3000r.
Obtain plasma by centrifugation at pm for 5 minutes. Separate PH
A substrate solution is prepared by dissolving 550 mg of bovine albumin in 8.5 ml of pH 7.4 phosphate buffer and then adding 1.5 ml of emulsion Ediol (manufactured by Calbiochem) diluted 10 times with pH 7.4 phosphate buffer. Mix 1 ml of plasma and 1 ml of substrate solution, and immediately measure the absorbance (-) at a wavelength of 660 nm with a layer length of 5 mm.
logT ₁ ). After incubation for 3 hours in a constant temperature water bath at 37° C., the absorbance (−logT ₂ ) is measured in the same manner. On the other hand, the absorbance (-logT ₃ and -logT ₄ ) before and after incubation is measured using plasma before administration of the test substance as a control. Calculate the lipid serum clearing activity of the test substance using the following formula. Lipid serum clearing activity (Δ−logT) = {(−logT ₁ )−(−
logT ₂ )}−{(−logT ₃ )−(−logT ₄ )} The results are shown in Table 1.

【0020】

[Table 1] ■■■ Turtle shell [0018] ■■■

[0021] It can be seen that all of the active ingredients of the present invention have an excellent lipid serum clarifying effect. The acute toxicity (LD ₅₀ ) in mice and rats was 3 g/Kg or more and 2 g/Kg or more (intravenous injection) for all active ingredient compounds, respectively.

[0022]

Effects of the Invention As is clear from the above examples, the active ingredient of the present invention has a lipid serum clarifying effect superior to that of dextran sulfate, and also has acute toxicity (LD ₅₀ ).
It is also more than 3g/Kg (mouse) and more than 2g/Kg (rat), and is expected to be used as an extremely promising drug.

Claims (1)

[Claims] Claim 1: A sulfated polysaccharide consisting of the following structural units or a sulfated polysaccharide consisting of two structural units (where n is the number of structural units in the sulfated polysaccharide, m is the sulfated polysaccharide) It is the number of structural units in the polysaccharide, m/n is 0 to 5, and m+
n represents an integer of 30 to 150. ) as an active ingredient. [Chemical 1] ■■■ Tortoise shell [0005] ■■■