JPH0478617B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides 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, and m is the sulfated polysaccharide is the number of structural units in the body, m/n is 0 to 5, m+n
indicates an integer between 30 and 150. ) as an active ingredient. Thrombosis often occurs in association with diseases such as malignant tumors, arteriosclerosis, diabetes, and nephrotic syndrome. In recent years, with the increase in the above-mentioned diseases, thrombosis and hyperlipidemia have been on the rise. Currently, effective drugs for these treatments include, for example, dextran sulfate and heparin. That is, dextran sulfate is a sulfate ester of dextran, which is a polymer of α-1,6-linked D-glucopyranose produced by microorganisms such as Leuconostoc mesenteroides, and is It has a serum clarifying effect and has the effect of lowering blood cholesterol and triglycerides.
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 sulfur content, the stronger these physiological activities, but the higher the toxicity. On the other hand, heparin, a mucopolysaccharide that exists in animal tissues, has a wide range of physiological effects, including strong blood coagulation inhibitory effects and lipid serum clarifying effects, and its activity is much stronger than that of artificial heparinoids. 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 sugars, such as the following units, in its molecule. (However, in _the formula ^, We succeeded in synthesizing a novel sulfated polysaccharide that does not exist in nature, which has an α-1,6-linked sugar chain and also contains an amino group such as heparin in its molecule. but,
As shown in the Examples described later, it was discovered that it has an excellent blood coagulation inhibiting effect, and the present invention was completed. 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 azido sugar as a precursor of an amino sugar, thereby creating a highly polymerized product having an α-1,6-linked sugar chain and containing an amino group such as heparin in its molecule. Succeeded in synthesizing an amino sugar polymer and its copolymer with D-glucose (Japanese Unexamined Patent Application Publication No. 1983-1999)
−180603, No. 57-180604, No. 57-180605,
(See Publication No. 57-180606). (However, in the formula, Bz is a benzoyl group, Ms is
A methanesulfonyl group, Bn is a benzyl group, and n and m indicate the number of each structural unit constituting the compound (10) or (). ) That is, compound (2) is 1,6-anhydro-β-D derived from β-D-glucopyranose.
- Glucopyranose (1) can be easily obtained by treating with penzoyl chloride [M. Cerny etal Collection Czechoslov, Chem.
See Commun. Vol. 26, 2542 (1961). ]. Compound (2) is dissolved in dry pyridine and cooled on ice.
Add methanesulfonyl chloride dropwise while stirring and 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). Separately, after washing with water and drying, recrystallization from methanol yields compound (3) 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. The residue was extracted five times with acetone, and the extract was concentrated 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 analyzed using silica gel column chromatography (developing solvent benzene-ethyl acetate).
20:1v/v), an oily compound (5
A mixture of -a) and (5-b) is obtained. A mixture of compounds (5-a) and (5-b) was dissolved in a mixed solvent of ethanol and saturated ammonium chloride water, and sodium azide was added thereto for 60 minutes with stirring.
Heat to reflux for an hour. 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 ammonium chloride aqueous solution was added, and the mixture was stirred for an additional 30 hours.
Stir for 1 minute. This mixed solution was extracted with ether,
The extract was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain an oil. Silica gel chromatography (developing solvent, hexane-ethyl acetate 5:
1v/v) to obtain 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, which 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 separately removing the desiccant, the liquid is concentrated and petroleum benzine is added to reprecipitate it. 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 (). The reduction of polymer (8) is carried out by Perch reduction, ie with an alkali metal in liquid ammonia. Alkali metals include potassium, sodium, and lithium. Alkali metal is 1 in polymer
It is necessary to add six or more atoms per unit, and it is usually desirable to add three times that amount. Polymer (8) is added after being dissolved in a suitable solvent such as 1,2-dimethoxyethane or diglyme. The reaction temperature 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℃, and the 1,2-
Gradually add the dimethoxyethane solution 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 carrying out the reaction for a predetermined period of time, the reaction is stopped by adding ethanol or ammonium chloride, a small amount of water is added, and the mixture is left overnight to evaporate the ammonium. Unreacted polymer is removed by extraction with methylene chloride. After dialysis against distilled water for 3 days and separating any insoluble parts, the insoluble parts are vacuum dried, and the soluble parts are 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. The copolymerization reaction is carried out by dissolving compounds (7) and (9) in a sufficiently dry solvent and reacting them with a Lewis acid as a catalyst. Suitable catalysts include methylene chloride and chloroform. Examples of the Lewis acid include phosphorus pentafluoride, antimony pentafluoride, and boron trifluoride etherate, but phosphorus pentafluoride is most suitable from the viewpoint of yield and stereoregularity of the resulting copolymer. The optimum amount of Lewis acid to be used is 2 to 7 mol%, particularly 2 to 3 mol%, based on the total number of moles of compounds (7) and (9). In addition, compound (7),
By changing the mixing ratio of (9), various copolymers can be obtained. 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 in the same manner as described above to form an amino sugar. Copolymer ()
is obtained. The sulfated polysaccharide, which is the active ingredient of the present invention, is obtained from the polymers () 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 to 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, concentrated and dried to obtain the sulfated polysaccharide as the active ingredient. However, n is the number of structural units in the copolymer (), and m is the number of structural units in the copolymer (). As mentioned above, the active ingredient of the present invention can be obtained, but in the case of a homopolymer (), the intrinsic viscosity is approximately
In the case of a copolymer (), it is appropriate to use one with an intrinsic viscosity of about 0.07 to 0.10 and a sulfur content of about 10 to 15%. The drug of the present invention can be administered by the most appropriate method of administration depending on the clinical symptoms. That is,
Although intravenous injection, subcutaneous injection, intramuscular injection, or oral administration can be used, intravenous administration is still appropriate in the case of acute symptoms. In addition, the active ingredient of the present invention can be used in a suitable inert solvent,
For example, by dissolving or suspending in sterile water, physiological saline, etc., or adding an inert carrier or filler, parenteral preparations such as injections: powders, granules, capsules, tablets, coatings, syrups, etc. It is appropriate to use it in the form of a liquid solution or other oral preparation. Examples of the inert carrier or filler include calcium phosphate, calcium carbonate, glucose, lactose, sucrose, dextrin, sucrose ester, starch, sorbitol, mannitol, crystalline cellulose, talc, kaolin, synthetic aluminum silicate, carboxymethylcellulose. , methylcellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, gum arabic, gum tragacanth, gelatin, powdered agar, and silica. 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. The present invention will be explained 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) (n=3.5×10 ⁴ , n=
95) (100mg, _NH2 group: 0.621mmol, OH group: 1.24
mmol) in water (10 ml) and centrifuged. It is then centrifuged with stirring three times in ethanol (20 ml), followed by three times in ether (20 ml) and suspended in pre-dried pyridine (8 ml). This suspension is added to chlorosulfonic acid (1 ml, 15 mmol) previously reacted at 0° C. and dry pyridine (6 ml), immersed in a boiling water bath and allowed 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 ethanol (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 properties of homopolymer (-a)] Elemental analysis value (C ₆ H ₉ O ₁₀ NS ₂ Na ₂ ) C H NS Actual value 20.16 4.18 3.75 16.14 Measured value 19.73 2.48 3.83 17.56 Ninhydrin reaction: Negative IR: 580cm ^-1 (M) 800cm ^-1 (M) 610cm ^-1 (M) 1240cm ^-1 (s, broad) 1510cm ^-1 (M) ¹³ C-NMR: δ97.53ppm (1C, C -1) 75.54 74.68 71.03 (3C, C-2, C-4, C-5) 68.07 (1C, C-6) 55.07 (1C, C-3) [α] ²⁵ _D +116.9° (C=1.0 , CHCI ₃ ) [η] = 0.17 (in H ₂ O, 30°C) Production example 2 Polymer (-a) (n = 144, m/n =
1.04 (100 mg) in water (2 ml) and precipitated by adding 20 ml of ethanol. After centrifugation, the mixture was further stirred and separated in ethanol and then in ether, and then suspended in pre-dried pyridine (8 ml). This suspension is added to chlorosulfonic acid (1 ml, 15 mmol) previously reacted at 0° C. and dry pyridine (6 ml), immersed in a boiling water bath and allowed 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 ethanol (50 ml) is added to precipitate the polymer. This was centrifuged, dissolved in distilled water, dialyzed for 3 days, concentrated and lyophilized to produce a sulfated amino sugar-containing polysaccharide [copolymer (-
a)] (168 mg, 76.2%). [Physical properties of copolymer (-a)] Elemental analysis: C ₆ H _8.589 O _10.211 N _0.489 S _1.90・3H ₂ O C H N S Measured value 17.40 3.39 1.55 14.65 Measured value 17.59 3.59 1.68 14.87 (Sulfation rate: Sugar - SO ₃ ^- 1.90 per residue) IR: 580cm ^-1 (M) 800cm ^-1 (M) 1240cm ^-1 (s, broad) 1510cm ^-1 (W) [α] ²⁵ _D = +100.2° (C = 1.0, H ₂ O) [η] = 0.07 (H ₂ O, 30°C) Production example 3 Copolymer (-b) (n = 100-120, m/
n=4.76, _NH2 group 0.114 mmol, OH group 1.74 mmol,
The reaction was carried out in the same manner as in Production Example 2, except that 100 mg) was used.
After treatment, sulfated amino sugar-containing polysaccharides (-
b) was obtained (176 mg, 72.0%). [Copolimer ( -B) physical properties] element analysis: C ₆ H _9.115 O _8.025 N _0.185 S _1.07 NA _1.07 C HnS Active value 22.76 0.67 10.82 Measured value 22.16 4.68 0.55 (Sugar -remaining -remaining SO ₃ ^- 1.07 pieces per group) IR: 580cm ^-1 (M) 800cm ^-1 (M) 1240cm ^-1 (s, broad) [α] ²⁵ _D = +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. After dipping to 100ml, make an injection by regular use. Example 2 (Tablet) 200 g of the sulfated polysaccharide obtained in Production Example 2 and 140 g of lactose are mixed and then 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 agent 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. Test Example 1 (Anticoagulant activity) Potency assay method for heparin in the United States Pharmacopeia of the United States
America.XIX, pp. 229-230) (however, bovine plasma was used instead of sheep plasma). Dissolve the test substance in physiological saline to a concentration of 160γ/ml. Also, standard heparin (160IU/mg)
Prepare a 10γ/ml saline solution. Test substance solution and standard heparin solution each at 0.8,
Transfer 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, and 0.05 ml into glass test tubes (13 x 105 mm), add physiological saline to a total volume of 0.8 ml, and mix. Add 1 ml of bovine plasma* to each tube and mix, then add 0.2 ml of 2% calcium chloride aqueous solution, immediately mix by gently inverting the tube, and keep in water at 37°C. After 30 minutes, the coagulation status of each tube was changed to 0, 0.25, 0.5,
Records are divided into classes of 0.75 and 1.0, and blood clotting status is determined.
From the amount of test substance and standard heparin at 0.5,
The potency of the test substance was determined. The results are shown in Table 1. *Bovine plasma: Add 40ml of 10% sodium citrate (Na ₃ C ₆ H ₅ O ₇・2H ₂ O) aqueous solution to a blood collection container in advance.
Pour 960 ml of fresh bovine blood into this container, mix and centrifuge at 3000 rpm for 10 minutes to collect plasma.

[Table] It is known that the drug of the present invention has an anticoagulant activity about 10% that of heparin, and about 2.5 times that of the control dextran sulfate. Test Example 2 (Whole blood anticoagulant effect) Using Japanese white rabbits (male) weighing 2.5 to 3.0 kg,
The test substance (2.5% physiological saline solution) was intravenously injected at 25 mg/Kg, and blood was collected from the ear vein 10 minutes later.
Whole blood coagulation time was measured by White's modified method (Izumi Kanai, Masamitsu Kanai, eds., Clinical Test Methods Summary, Kanehara Publishing Co., Ltd., 1971, 28th edition). That is, two dry glass test tubes (10 x 120
mm) in a constant-temperature water bath at 37°C, and immediately after blood collection, remove the syringe needle and add 1 ml at a time, taking care not to create bubbles. After 5 minutes of standing, the first test tube is tilted every 30 seconds to observe the presence or absence of blood clots. Once this has solidified, observe the second test tube in the same way. The whole blood clotting time is the time from when the blood enters the syringe from the vein until it clots in the second test tube. The results are shown in Table 2.

[Consideration]

As is clear from the above examples, the active ingredient of the present invention has anticoagulant activity superior to that of dextran sulfate, and its acute toxicity (LD ₅₀ ) is 3 g/Kg or more (mouse) and 2 g/Kg or more (mice). (rats) and is expected to be used as an extremely promising drug.

Claims (1)

[Scope of Claims] 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 number of structural units in the sulfated polysaccharide, and m is the number of structural units in the sulfated polysaccharide, It is the number of structural units in the sulfated polysaccharide, m/n is 0 to 5, and m+n is 30 to
Indicates an integer of 150. ) as an active ingredient.