JPS6115836A - Antitumor agent - Google Patents

Abstract

PURPOSE:An antitumor agent containing a specific water-soluble polysaccharide having physiological activity as an active constituent. CONSTITUTION:An antitumor agent containing a water-soluble branched polysaccharide having branched beta-D-glucose bonded to a backbone consisting substantially of beta-1,4-glucan and >=20 average number of branched bonds based on 100 anhydroglucose units in the backbone as an active constituent. The above- mentioned water-soluble branched polysaccharide is obtained by activating cellulose acetate having free hydroxyl groups [cellulose acetate having 1.7-2.4 degree of substitution (DS)], reacting the resultant activated cellulose acetate with ortho ester of acetyl glucose, and deacetylating the reaction product. Cellulose diacetate is obtained by aging and saponifying cellulose triacetate, but the above- mentioned step under mild conditions provides the polysaccharide having a relatively high polymerization degree.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antitumor agent containing a physiologically active water-soluble polysaccharide as an active ingredient. Specifically, after activating cellulose acetate having free hydroxyl groups, it is reacted with an orthoester of acetyl glucose, and the reaction product is deacetylated. Cellulose having β-(1→6) glucose branches is used as the active ingredient. The present invention relates to an antitumor agent.

It has been recognized that some branched polysaccharides with very short branch lengths have physiological activity. For example, lentinan is a branched polysaccharide with antitumor properties, and the polysaccharide portion of Krestin is also a branched polysaccharide.

Several attempts at branched polysaccharide synthesis have also been reported.

Kochetko'V et al. saponified a reaction product obtained by reacting cellulose acetate with an orthoester of triacetylglucose to obtain a branched polysaccharide with a degree of polymerization of about 30-35. (0arbohydr, Res, ”-9,1
(1971)), this method was aimed at producing β-(1→6) glucose branches, but the products mainly contain branches to secondary alcohols, such as β-(1→2) glucose branches. It was something that I had. Furthermore, the molecular weight of the product was approximately 7,000 or less due to significant branching during the reaction.

Pf annemUl fl 11! +rr et al. reacted an O-phenylcarbamoyl derivative of a linear polysaccharide with an orthoester of tetraacetylglucose bromide or triacetylglucose, then removed the phenylcarbamoyl group and acetyl group by saponification to obtain β-(1
→6) A branched polysaccharide having branched glucose or -(1→6) glucose is obtained.

(0arbohydr, Res, 43151 (19
75), 4763 (1976)) 56139 (1977
)).

In this method, the synthesis of the intermediate O-phenylcarbamoyl derivative is very troublesome. In addition, if the main chain is cleaved during the reaction and the product has a low weight, a water-soluble polysaccharide will be obtained, but if the degree of polymerization does not significantly decrease, a water-swellable polysaccharide will be obtained. ing. Kochet above
Even in the studies of kov and Phannenmul Ier, nothing has been reported regarding the physiological activity of the obtained branched polysaccharide. Therefore, the effects of the degree of polymerization and degree of branching of branched polysaccharides on their physiological activity have not been reported.

The present inventors have conducted extensive research on the idea that branched polysaccharides, which have a high degree of polymerization and are water-soluble, have high physiological activity and low side effects.

As a result, by activating a linear polysaccharide or an acetyl ester of a linear polysaccharide by an appropriate method and then performing a glucosylation reaction, a water-soluble branched polysaccharide can be obtained without a decrease in the molecular weight of the linear polysaccharide. Recognizing this, a special application was filed in 1982-20.
821.8 and filed the application.

The present inventors synthesized branched cellulose using the above method using cellulose acetate having free hydroxyl groups as a starting material, and found that the degree of branching (main chain anhydroglucose unit 100
They obtained a water-soluble branched polysaccharide with an average number of branched bonds of 20 or more and a molecular weight of 200,000 or more as measured by GPO, and found that this polysaccharide has high antitumor properties.

The obtained water-soluble branched polysaccharide is in the form of an aqueous solution or
It can be administered orally in the form of powders, tablets, etc. For example, to make tablets, it may be mixed with excipients such as microcrystalline cellulose and lactose, and then formed into tablets.

That is, the present invention provides a water-soluble branched polysaccharide in which β-D-glucose is branched and bonded to a main chain consisting essentially of β-1,4-glucan, the main chain comprising 10 anhydroglucose units.
The present invention relates to an antitumor agent containing as an active ingredient a water-soluble branched polysaccharide having an average number of branched bonds per zero of 20 or more.

The water-soluble branched polysaccharide used in the present invention is obtained by activating cellulose acetate having free hydroxyl groups, reacting it with orthoester of acetyl glucose, and deacetylating the reaction product.

The above cellulose acetate having a free hydroxyl group has a DS of 1
It is a so-called cellulose diacetate having a molecular weight ranging from 7 to 24. When such cellulose diacetate is glucosylated by the method shown in the Examples, the condensation reaction of glucose occurs preferentially at the 6-position hydroxyl group in the main chain anhydroglucose residue. As the degree of condensation reaction of glucose, ie, the degree of branching of the branched polysaccharide produced, increases, it also occurs to the hydroxyl group at the 2- or 3-position. Therefore, if the degree of branching is high, the 6th position (!: 2nd, 6th and 3rd positions, etc.) of a single anhydroglucose residue, where multiple glucoses are bonded, is also formed. Branching at this 6th position is preferential. This is thought to be related to antitumor properties.

Cellulose diacetate is obtained by aging and saponifying cellulose triacetate, but under the conditions normally used in industry, the main chain of cellulose is cleaved at this stage, resulting in a low degree of polymerization. On the other hand, when aging saponification is performed under mild conditions, cellulose diacetate with a relatively high degree of polymerization can be obtained.

By using cellulose diacetate, which has a relatively high degree of polymerization, it is possible to obtain a water-soluble branched polysaccharide with a relatively high degree of polymerization.
It seems that particularly high antitumor properties are exhibited when the molecular weight measured by PO is 200,000 or more and 500,000 or less. That is, the molecular weight is 20
The tumor inhibition rate was less than 55% when the molecular weight was 20,000 or less.
In the case of 10,000 or less, the deterrence rate was 75 or higher. Same molecular weight 5
00,000 or more has not been obtained in experiments, and no direct knowledge regarding antitumor properties has been obtained. However, as the molecular weight increases, the solubility in water tends to decrease. Polysaccharides are considered to be the most preferred for practical purposes.

The glycosylation reagent used to synthesize the water-soluble branched polysaccharide is synthesized by the following method.

That is, D (+) glucose is peracetylated using acetic anhydride and perchloric acid as a catalyst, and then the carbon at the 1st position is brominated using bromine and phosphorus to obtain 2.3,4.6-
Tetra-O-acetyl-a-D-glucobylanose bromide is synthesized. This material was heated under reflux with anhydrous ethanol in ethyl acetate to give 3,4.6-tri〇-acetyl-(1,2-0-ethylorthoacetyl) -1Z-
D-glucobylanose is obtained and used as a glucosylation reagent.

The above reaction is expressed as follows.

The curcosylation reaction and its deacetylation process will be described in the Examples, but the reaction can be expressed by the following formula.

The above formula is an example of a case where the branching bond is 1→6 bonds, and it is clear from the examples that in actual reactions, 1→2 and 1→3 bonds also occur, although fewer than 1→6 bonds. As shown.

The present invention will be described below with reference to examples, but the present invention is not based on these examples.

Example 1 4f of commercially available cellulose triacetate (substitution degree 290) powder was dissolved in methylene chloride to give a solution with a weight ratio of 41, and acetic acid/
Water-2,6/1. (When expressed as a ratio below, it is a capacity ratio)
The mixed solution was measured and the water concentration in the system was adjusted to 20.3 volumes, and then kept at 50°C for 48 hours for hydrolysis. Next, acetic acid/water-1/1 solution was added forcefully to raise the water concentration in the system to 25.0 volume, and then the water concentration in the system was raised to 258 volume. °C
The hydrolysis reaction was continued for 48 hours. After methylene chloride was distilled off from the reaction solution (160 hours in total), it was poured into water to precipitate the produced cellulose diacetate. Obtained 2
The degree of substitution of cellulose acetate was 2.30.

This cellulose diacetate was activated as follows.

First, it was immersed in a 1/1 water/ethanol mixed medium at room temperature for 48 hours, then in 1/3 water/ethanol for 24 hours, and then in ethanol for 3 hours, and then immersed in ether until the ethanol was completely removed. Washed and dried.

The activated cellulose diacetate was glucosylated by the following method. Activated cellulose diacetate 1.2F and 2.3
．． 4-) So0-acetyl-1,2-0-ethylorthoacetyl-α-D-glucopyra/-y, 2.211, was dissolved in chlorhensen (b, p, 132°C) 2Srrl. By heating under normal pressure and distilling off 1 to 2 ml of chlorobenzene, trace amounts of water and alcohol in the system were completely removed.

Next, 10 mN of 2,4-lutidine verchlorate as a reaction catalyst was added, and the mixture was heated under reflux for 80 minutes to cause a reaction.

After the reaction was completed, the reaction solution was poured into methanol to precipitate the product, and the precipitate was washed with ether and dried.

The glycosylation reaction was repeated once using the above product, which was similarly precipitated in methanol, washed and dried. What is obtained is acetylated branched cellulose. The molecular weight of this product was measured using GPO (stationary phase: crosslinked polystyrene, mobile phase: tetrahydrofuran) and found to be 350,000. Acetylated branched cellulose 0
．． Add 5 ml of 0.5N NaOH aqueous solution to III and
The mixture was allowed to stand for an hour to allow deacetylation. At this time, the system is a homogeneous solution. Add 0.1N HOl aqueous solution to neutralize this, make it slightly acidic, dilute with water,
Dialysis was carried out using Cheap, and impurities were removed, followed by precipitation with acetone, washing with ether, and drying under reduced pressure. The obtained product has a main chain consisting of β-1,4-glucan,
It is a water-soluble polysaccharide in which β-D-glucose is branched. (100% water dissemination) The tumor suppressing effect of the obtained water-soluble branched polysaccharide was investigated. Sarcoma Sarcoma 180 IRO-
Transplant 〈106 cells/mouse〉 into the subcutaneous axilla of a JOL female mouse, and 24 hours after implantation, add a solution of branched polysaccharide dissolved in sterile physiological saline at a ratio of 0.5μ branched polysaccharide per 1 kg of mouse body weight. After being reared for 25 days with intraperitoneal administration every other day, the sarcoma was removed and its weight was measured.

The tumor inhibition rate is given by the following formula.

However, C: weight of sarcoma in mice (10 mice) to which branched polysaccharide was not administered T: weight of sarcoma in mice (10 mice) to which branched polysaccharide was administered The inhibition rate of branched polysaccharide in this example was 89.5%.

The obtained branched polysaccharide was then subjected to methyl analysis as follows. The sample was treated with the Hakomori method three times to achieve complete methylation, followed by hydrolysis by heating with an aqueous solution of trifluoroacetic acid, and then reduction with aqueous sodium boronate to convert it into the corresponding partially methylated alditol acetate. This product was fixed and fixed by both gas chromatography and mass spectroscopy. The fixed partially methylated monosaccharides are as follows.

1.4.5-)di-0-acetyl-2,3,6-)di-
0-Methyl-D-glucitol (abbreviated as 2,3,6-TM, hereinafter the same) 1.4,5.6-tetra-0-acetyl-2,3-di-0-methyl-D-glucitol (2,3,6-TM) ,3-DM) 1.3,4.5-tetra-0-acetyl-2,6-di-0-methyl-D-glucitol (2,6-DM) 1.2,4.5-tetra-0- Acetyl-3,6-di-0-methyl-D-glucitol (3,6-DM) 1.3,4,5.6-penta-0-acetyl-2-0-
Methyl-D-glucitol (2-IVIM) 1.2,4
,5.6-penta-0-acetyl-3-〇-methyl-D
- Glucitol (3-MM) Among these, the 6th and 2nd positions of glucose residues in the main chain.

The relative proportion of units with branching at position 3 is shown (2, 3
-DM):'(3,6-DM):(2,6-DM) ratio was 77.5:14.1:8.4.

That is, more than 70% of the units having one branch had a branch at the 6th position.

Also, it corresponds to the unit south of the two branches (2-M
M), (3-MM) is present in a very small amount (6-MM), i.e., 1,2,3,4°5-penta-O-acetyl-6-〇-methyl- showing branching to the 2- and 3-positions. D-glucitol was not detected.

Further, the total degree of branching of the branched polysaccharide can be calculated using the following formula, and the result was 31.3%.

The degree of branching determined from the acetylation analysis was 26.5%, which was in close agreement with the value determined from the methylation analysis.

In addition, when measuring the 0-NMR spectrum of the dimethyl sulfoxide solution of the water-soluble branched polysaccharide of this example, β-
A signal indicating 01 was observed at 102 to 103 ppm, while a signal corresponding to the a-(1→6) bond (signal around 100.2 ppm observed in dextran) was not observed, indicating that the branched glucose bond was It is determined to be type β.

Example 2 Cellulose diacetate, which was the same as that used in Example 1, was hydrolyzed using the same method but with a longer hydrolysis reaction time than in Example 1 to obtain cellulose diacetate with a degree of substitution of 1.69. .

This cellulose diacetate was activated in the same manner as in Example 1. Cellulose diacetate after activation treatment 1.779
3,4.6-tri〇-acetyl-112-0
-Ethyl orthoacetyl-a-D-glucobylanose 2
．． The glucosylation reaction in which 859 was reacted according to the method of Example 1 was repeated twice to obtain acetylated branched cellulose.

The molecular weight of the obtained n-acetylated branched cellulose measured by GPO was 290,000.

This acetylated branched cellulose was saponified in the same manner as in Example 1. Dialysis, concentration, precipitation with acetone, and washing with ether were performed in sequence, followed by drying under reduced pressure to obtain a water-soluble branched polysaccharide. (
When the tumor inhibition rate of the obtained branched polysaccharide (water-soluble portion: 90%) was measured in the same manner as in Example 1, the inhibition rate was 77.8%.

The degree of branching of the above-mentioned branched sugar was determined by the methylation method in the same manner as in Example 1. As a result, the total degree of branching was 46.7%.

The degree of branching determined from the degree of acetylation of the acetylated branched polysaccharide was 13%, which was lower than the above value, but this is thought to be due to deacetylation occurring during the glucosylation reaction.

Furthermore, the relative ratio of branches at the 6th, 2nd, and 3rd positions of the main chain branching unit was 64.5:22.7:12.8.

Example 3 Degree of acetyl substitution 2.09, molecular weight measured by GPO 25
Examples 1 and 2 using cellulose acetate as a starting material
Glucosylation was performed in the same manner as above to obtain an acetylated branched polysaccharide. The molecular cap of this product measured by GPO was 290,000, and the total degree of branching was 47%.

The above acetylated branched polysaccharide was deacetylated to obtain a water-soluble branched polysaccharide (100% water-soluble). When the tumor inhibition rate was measured in the same manner as in Example 1.2, a value of 94.2% was obtained.

Comparative Example 1 Cellulose 3 acetate was hydrolyzed at a higher temperature than in Example 1, cellulose 2 acetate having a lower molecular weight than that used in Example 1 (degree of substitution 2.06) was used as a starting material, and the same procedure as in Example 1 was carried out. Glucosylated, branching degree 30%, molecule by GPC,
An acetylated branched polysaccharide of t125,000 was obtained. When this product was deacetylated and the tumor inhibition rate of the resulting water-soluble polysaccharide (100% water-soluble) was measured, it was found to be 53.7%.

Claims (1)

[Scope of Claims] 1. In the main chain consisting essentially of β-1,4-glucan, β
- Antitumor agent 2 whose active ingredient is a water-soluble branched polysaccharide in which D-glucose is branched and the average number of branched bonds is 20 or more per 100 anhydroglucose units in the main chain. , the water-soluble branched polysaccharide has a molecular weight of 20 as measured by GPC.
In the antitumor agent 3 according to claim 1, the water-soluble branched polysaccharide has a total molecular weight of 60,000 to 500,000, and the total number of 1→6 bonds among the total branched bonds is 60.
% or more, the antitumor agent according to claim 1