JPS61130302A - Ribofuranan sulfate having anticoagulant activity and its production - Google Patents

Abstract

PURPOSE:To obtain ribofuranan sulfate having an anticoagulant activity, by sulfating ribofuranan. CONSTITUTION:Ribofuranan sulfate of formula I (wherein n is 10-500 and R is H or SO3Na) is obtained by sulfating ribofuranan with a sulfating agent. When n in formula I is smaller than 10, the anticoagulant activity is decreased, while when it is larger than 500, the synthesis of this compound is difficult. Examples of the sulfating agents which can be used include sulfuric anhydride/ trimethylamine complex, chlorosulfonic acid and piperidine N-sulfate. Ribofuranan sulfate of formula I has an anticoagulant activity in the medical field and therefore a substance containing it as an effective component can be used as a medical polymeric material which must prevent coagulation of the blood, such as a material for artificial hearts and artificial blood vessels. Ribofuranan which is a starting material for ribofuranan sulfate can be obtained by, for example, a process shown in formula II (wherein Bn is CH2C6H5).

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a sulfated ribofuranan having anticoagulant activity in the medical field and a method for producing the same.

(Prior Art) Thrombosis or hyperlipidemia often occurs in conjunction with diseases such as malignant tumors, arteriosclerosis, urinary tract disease, 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. Texturan sulfate can be used against microorganisms such as Leuconostocmesente.
It is a sulfate ester of Textran, which is a polymer of α-1,6-linked fc and D-glucobylanose produced by roides), and has an anticoagulant effect and is used as an effective drug for the treatment of thrombosis. Are known.

Heparin, a mucopolysaccharide that exists in animal tissues,
It has a wide range of physiological effects, including strong anticoagulant effects and lipid serum clarifying effects, and its activity is much stronger than that of artificial hevarinoids, but the quality of the standard products is inconsistent and its structure is complex, making it difficult to isolate. Heparin, which requires a complicated process, is characterized by having a sulfated subamino sugar in its molecule. The active site in heparin is represented by the following formula.

G B H (graphite-
benzalkonium chloride-he
The 0GBH method, which starts with the parin) method, uses a surfactant (tetramethylammonium chloride) to adsorb penzaiconium chloride, a surfactant, on the surface of a graphite coating, and binds heparin to it. ) has also been used to ionically bond heparin using the TDMAO method. TDMAO method is G
Compared to the BH method, this method is unique in that it can be applied to flexible general-purpose polymers such as polyethylene and polyvinyl chloride. Heparin ionically bound to the surface of such a material is released into the blood and is used clinically as an antithrombotic material for short-term use (within 24 hours), which exhibits its anticoagulant activity. For example, a material in which heparin is bonded to the surface of polyvinyl chloride using the T DMAC method is used as a bypass blood vessel during surgery. However, it is not suitable for long-term use because the elution rate of heparin into the blood is fast.

In addition to these methods, there is a method in which heparin is bound inside the polymer matrix, and the heparin eluted from the surface of the material into the blood is replenished by being released from within the material to maintain antithrombotic properties.

Some of the polymers used in this method have been confirmed to exhibit antithrombotic properties for 4 weeks or more in in vivo tests.

(Problem to be solved by the invention) However, these methods have two problems.

Since Hepari/ is extracted from animal tissue, the quality of the specimen is not constant, the structure is complex, and the isolation method is complicated. Furthermore, the method in which heparin is leached into the blood is not suitable for long-term use.

(Ninth Means for Solving the Problems) In order to solve these problems, the present invention attempts to chemically synthesize hevarinoids having high anticoagulant activity, such as heparin.

For this purpose, the present invention has an α-1,5-linked sugar chain and a sulfate group such as that molecule or peheparin. A novel heparinoid sulfated polysaccharide containing sulfated polysaccharides was synthesized.

The present invention is based on the following formula (where n is an integer from 10 to 500, and R=H is 5
o8Na) is a sulfated ribofuranan. n in the formula is 10-500. If n is smaller than 10, the anticoagulant activity will be low, and if n is larger than 500, it will be difficult to synthesize this compound.

The present invention also provides a method for producing sulfated ribofuranan by sulfating ribofuranan. As the sulfur recognition agent, anhydrous trimethylamine sulfate complex, chlorosulfonic acid, piperidine N-sulfuric acid, etc. can be used.

Furthermore, the present invention resides in a blood coagulation inhibitor containing this sulfated ribo-7-ranan as an active ingredient.

Since sulfated ribofuranan has anticoagulant activity, substances containing it as an active ingredient are used as medical polymer materials that need to inhibit blood coagulation, such as in artificial hearts and blood vessels.

Ribofuranan, which is a raw material for sulfated ribofuranan, can be obtained, for example, by the following process.

Bn-OH206H.

Hereinafter, the present invention will be explained in detail based on examples.
.

The following compound 1.4-anhydro-α-D-ribopyranose (2) obtained by the above procedure (see 19.3) is used.

Without water, 10.9'i of sodium hydride was suspended in 100 #t/dry dimethylformamide (DMF), and 10.9 of the compound (2) dissolved in 100-Dll[F was added dropwise with stirring. . After reacting for 1 hour, the benzyl chloride of 31d dissolved in 5 (11/1) DMF K was added dropwise and stirred at room temperature for about 20 hours. The reaction mixture was poured into a large amount of ice water, extracted with chloroform, and then concentrated. Then, 20 g (yield: 84 g) of crystals of compound 1,4-anhydro-2,8-di-0-benzyl-α-D-lipopyranose (8) are obtained from n-butyl chloride. The physical properties are as follows.

m, p,: 65.0-66.5°C Specific rotation degree: [α)p 35-9° (C1, chloroform] 18G-NMR: δ128.21-138.4
0 (aromatic).

100.47 (1 (3, G-1), 82.87 (10
, C-3).

80.22 (10, G-2), 78.57 (No,
0-4).

73,. 30, 73.01 (20, (q) I, 06
H5J, 64.91 (10.

The obtained compound <3) (C7I!i!, 11.9 mmol) was vacuum dried under a high vacuum of 10 Hg overnight.
Methylene chloride, previously dried with calcium hydride, is dissolved in a vacuum angle to 15 Tlt/J.

After the polymerization tube was cooled with liquid nitrogen and the monomer solution was sufficiently frozen, boron trifluoride etherate (30 μl,
0.24 mmol) is introduced. The polymerization tube was separated from the vacuum line and placed in an ethanol bath at -40"C for 1 to 2 hours.
Shake vigorously for a minute. -40"C for 30 minutes, open the polymerization ampoule, and pour methanol to stop the reaction. At this time, the polymer precipitates, so add chloroform until the polymer is sufficiently dissolved, and add bicarbonate. Neutralize with sodium water bath, wash with water, and add anhydrous sulfuric acid)
Dry with Jum. After separately removing the desiccant t-F, the F solution is concentrated, and petroleum benzine is added to reprecipitate it. Dissolution,
The operations of concentration and reprecipitation were performed two more times, followed by bath dissolution in benzene and freeze-drying to obtain the polymer 2,3-di-0-benzyl-(1→6)-α-D-ribofuranan (4)a, s
The physical properties of the polymer (4) are as follows.

Specific light intensity: [α)D+15.3.4° (C], chloroform] MH = 1.28 x 105 (DPn =
410) The resulting polymer (4J a,o g) was dissolved in 1,2-dimethoxyethane (120+11/) previously dried with metallic sodium. /) and metallic sodium (5,8I) into a solution under a nitrogen atmosphere. The reaction system is stirred at -78°C, and after 1.5 hours, ammonium chloride is added until the blue color of the reaction system disappears. Prepare water (lQ+a/) and evaporate ammonia at room temperature. Prepare water (20 m), wash 5 times with methylene chloride, and dialyze the aqueous layer for 8 days. The water bath solution is concentrated and freeze-dried. , polymeric (1→5)-α-D-ribofuranan (5
) 1.35,! A VC yield of 53% is obtained. The physical properties of polymer (5) are as follows.

Specific light intensity: [α) D+ 164.1° (01, water)・1
80-NMR:δ]02.9a<1(3,0-1
), 8L56 (lG, G-4), 71.47
(ICj, 0-2).

70.37 (10, 0-3J, 68.59 (1
0.

(3-5).

Polymer (5) (L2 g was dissolved in pre-dried dimethyl sulfoxide (1801/), piperidiy-N-sulfuric acid (3,01i) CM was added, and 80 g was added with stirring.
Incubate at ℃ for 1 hour. ] Neutralize with 0% excess L5N sodium hydroxide aqueous solution and concentrate.

Add methanol and dissolve the precipitated polymer in water,
Dialysis, concentration, freeze-drying and polymer sulfation (1→
5) Obtain 0.1 fy of -α-D-ribofuranan. The physical properties of this polymer are as follows.

Metachromatic reaction: + ) l[n = 8.19 bovine plasma was used instead).

The test substance is dissolved in physiological saline and adjusted to 160r/l/# degree. Mayu standard heparin (160IU/)] O
r/, 1 saline t- Prepare.

The test substance solution and standard heparin solution were each 0.8°0,
F, 0.6, OJ, 0.4, 0.8.0.
2, 0.1 and 0.06- each in glass test tubes (13
X10511), add physiological saline and mix so that the total volume of the trap is 0.8 m.

Each trial lol! Bovine plasma in the tube 'I 1 w! / 2 It / each and then add 2% aqueous calcium chloride solution Q, 2 It / each, and immediately mix by gently inverting the test tube.

After 7-10 minutes, record the coagulation status of each test tube by dividing it into classes of fcO, 0.25°0.5, 0.75, ], θ, and compare the test substance and standard when the coagulation status is 0.5. The potency of the test substance was determined from the amount of heparin. The results are shown in Table 1.

Bovine plasma: Prepare 40n of 10% sodium citrate (Na8C6H,O,-2H20) aqueous solution in a blood collection container in advance.
df and put fresh cow blood in this container 960 #l/
After mixing, centrifuge at AOOORPM for LO minutes to collect plasma.

Table 1 The sulfated (1→5)-α-D-ribofuranan of the present invention was tested for anticoagulant activity of about 11% of heparin, and was found to have about 2.7 times the activity of the control dextran sulfate. known.

In addition, rapid injection poisoning using mice! I1. (LD6o) is
I Ji' /1 cg or more (intravenous injection).

(Effects of the invention) The sulfated (1→5 no α-D-ribofuranan) of the present invention,
Table 2 shows the anticoagulant activity of sulfated (1→6)-α-D-xylofuranan and texturan sulfate as comparative examples.

Comparing sulfated polysaccharides with approximately the same molecular weight, sulfated ribofuranan has approximately three times the activity of dextran sulfate, but is slightly less active than sulfated xylofuranan. However, critical differences occur during the synthesis routes of ribofuranan and xylofuranane, particularly in the heptadmer synthesis.

That is, anhydrous xylose, which is the raw material for xylofuranan, is in the form of a syrup, so it is acetylated and purified by silica gel column chromatography, deacetylated, benzylated, and purified by column chromatography. be synthesized. On the other hand, anhydrous ribose, which is the raw material for ribofuranan, has good crystallinity, so it can be purified by recrystallization, and the benzylated product also has good crystallinity. Therefore, the monomer combination is much easier in the case of ribose than in the case of xylose.

Furthermore, the present invention not only makes it possible to chemically synthesize heparinoid having anticoagulant activity, but also allows the heparinoid to have a constant structure and control its physiological activity to a certain extent because it is a purely chemically synthesized heparinoid. Furthermore, it is also possible to incorporate heparinoids into the polymer backbone by covalently bonding them with synthetic polymers such as segmented polyurethanes to synthesize composite materials with sustained antithrombotic properties.

Furthermore, the present invention can be applied to medical materials that require antithrombotic properties, such as artificial hearts and artificial blood vessels.

Claims (1)

[Claims] 1. General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ Sulfated ribofuranan represented by (n in the formula is an integer from 10 to 500, and R=H or SO_3Na) . 2. General formula: ▲ There are mathematical formulas, chemical formulas, tables, etc. A method for producing sulfated ribofuranan by sulfating it with a sulfating agent. 3. General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ Contains sulfated ribofuranan as an active ingredient (n in the formula is an integer from 10 to 500, and R=H or SO_3Na) Blood clotting inhibitor.