DE2152059A1 - Alkali salt of carboxylkylglucan and process for its preparation - Google Patents

Description

The invention relates to new polysaccharides, methods for their production and for their use. In particular, the invention relates to new polysaccharides which Have effectiveness against tumors.

It is known that some polysaccharides have physiological activity such as anti-tumor activity or anti-virus activity; However, most of these physiologically active polysaccharides are too insoluble in water, to use them as medicinal products by intravenous injection and when using them by subcutaneous,

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intradermal and intramuscular injection they tend to. to cause various side effects. Even water. soluble polysaccharides dissolve in v / ater in a LIenge of at most 0.2 and fo ~ these polysaccharides are generally insoluble after isolation and drying.

For dissolving various drugs]. · Are often surface-active Agents or the like used, "however, they cannot apply to the physiologically active polysaccharides. be used because polysaccharide conformations are higher Dimensions can be changed by the 'action of surface-active agents in such a way that they reduce their physiological activity lose.

It has now been found that by reacting water-insoluble ß- (1 -> 3) glucan and 0 - ("I * 6) glucan with & -halogen-substituted lower fatty acids or their derivatives and treating the resulting Garboxy-lower- alkyl-ß- (1 - * 3) glucan and carboxy-lower-alkyl-α - (1 - * 6) glucan for the production of their alkali salt, the alkali salt of: carboxy-lower-alkyl-ß- (1 - 3) glucan and the alkali salt of carboxy-lower-alkyl- ά- (1 4 6) glucan - which are easily soluble in water and have high physiological activity. It has also been found that when using physiologically active glucans as starting material, the physiological Activity of the starting materials is greatly increased, that physiologically active glucans are obtained from physiologically inactive glucans and that

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the toxicity of the end product obtained physiologically effective ü-lucane is very low.

The invention relates to an alkali salt of carboxy-lower-alkyl-ß- (1 - »3) glucan or of carboxy-lower alkyl- ^a - (1 - * 6) glucan, preferred compounds being the alkali salts of the carboxymethyl derivatives.

The invention also relates to anti-tumor agents, which as -active substance an alkali salt of carboxy-nider- _ · alkyl-ß- (1 4 3) glucan or an alkali salt of carboxy-lower-alkyl-a - (1 -) 6) contain glucan.

The method of the present invention for producing an alkali metal salt of carboxy-lower-alkyl-ß- (1 - ^ 3) glucan or -α - ("1 -} 6) glucan is characterized in that one ß- (1 · * 3) glucan or α - (1 4 6) glucan with a cc -halogen-substituted lower fatty acid or its derivative converts the carboxy-lower-alkyl-ß- (i - * 3) glucan thus formed or α - (1 -> 6) glucan dissolves in water, the pH of the aqueous Adjust the solution to a value between 6.5 and 8.5 and the alkali salt of carboxy-lower-alkyl-ß- (1 -) 3) or α - (1 ·> 6) glucan by adding a water soluble one organic solvent precipitates, the amount of which is not less than 2 times the volume of the aqueous solution with adjusted pH value.

The glucans used as starting materials, which he "f in

can be used properly are in water

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BATH ORIGINAL

insoluble or poorly soluble ß- (1 - * 3) glucan or α „(-j j 5) glucan. Examples of the glucan include lentinan, which is glucan with only ß- (1> 3) glucoside bonds as a glycoside bond and which is obtained by extracting the fruiting body of Lentinus edodes Sing or Flammulina veltipes Sing with hot water (Saishin - Igaku, Recent Medicine, Volume 25, page 1043, 1970), Pachymaran which can be obtained by mild oxidation, reduction and hydrolysis of Pachyman (Recent Medicine, Volume 25, page 1043, 1970), and α- (Ί - * 6) glucan obtained by extracting the fruiting body from Lentinus edodes Sing. It was assumed that pachyman, a polysaccharide obtained from the extract of Poria cocos V / 'olf, is β- (1 ) 3) glucan. Recently, however, it was found that this compound is not a ß- (1 4 3) glucan, since there are several ß- (1 4 6) glucopyranoside bonds that are linked to the main chain via side chains and internal ß- (1 - * 6) -Bonds in the ß- (1 * 3) main chain.

To a-halogen-substituted lower fatty acids and their

Derivatives that can be used for the purposes of the invention include monochloroacetic acid, uonobromoacetic acid, α -monochlorobutyric acid, α -monobromobutyric acid, alkali salts these acids and lower alkyl esters of these acids, such as methyl monochloroacetate, ethyl monochloroacetate, propyl monochloroacetate, Butyl monochloroacetate or methyl monobromoacetate.

_ 5 _ BAD ORIGINAL

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According to the invention, the starting material β- (1 - * 3) glucan and α- (1 ■ * 6) glucan is suspended in water or alcohol, the suspension is stirred, after which an aqueous solution of caustic alkali is added to the suspension with stirring.

ct-halogen-lower-fatty acid or its derivatives are added to the alkaline-made G-lucan suspension. The amount of the caustic alkali used is generally 1 to 2.5 moles per mole of the α-halogen-substituted lower fatty acid or its derivative used. Is preferably used from caustic alkali in an amount of from λ 1/5 to 1/20 of the initial volume, a 20-to 30 / o aqueous solution. The α-halogen-substituted lower fatty acid or its derivative is used in an amount of 0.8 to 2.2 mol per G-lucopyranoside ring of the G-lucan used. The reaction can be completed by heating the reaction system on a Y / water bath at from 30 to 6.0 ° C. for 2 to 10 hours.

The end product according to the invention can be obtained and purified by the methods described below. The precipitate in the reaction mixture is isolated by any suitable method, such as filtration or centrifugation, washed in an acetic acid-alcohol solution of about pH 6 and then washed with aqueous methanol at a concentration greater than 70 % . The precipitate obtained can be subjected to further purification by washing with absolute methanol and / or acetone »

The precipitate isolated from the reaction mixture can

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can also be dissolved in water, the pH of the aqueous solution being adjusted to a value between 4.0 and 8.5, preferably between 6.5 and 8.0 with an acid such as acetic acid. Then water-soluble organic solvent is added to the aqueous solution to precipitate the alkali salt of carboxy-lower-alkyl-ß- (1 -} 3) glucan or the alkali salt of carboxy-lower-alkyl-α- (1 -} 6) glucan. ß- (1 - »3) glucan or -α- (1 - * 6) glucan can preferably be carried out in the presence of not more than 1 % of an alkali salt or alkaline earth salt. Preferred examples of alkali salts are sodium acetate and sodium chloride and a preferred example of an alkaline earth salt is calcium sulfate. The amount of the water-soluble organic solvent used is not less than twice the volume of the original volume of the aqueous solution. If desired, the isolated precipitate of the The final product can be further purified by washing with the water-soluble organic solvent.

The products according to the invention are alkali salts of carboxy-lower-alkyl-β- (1 ■ * 3) glucan and alkali salts of carboxy-lower-alkyl-aO ■> 6) glucan. These compounds have physiological activity such as anti-tumor activity. If effective against tumors ß- (1 -ψ 3) glucan is used as starting material for the carboxyalkylation reaction according to the invention, the initial anti-tumor effectiveness of the ß- (1 ■> 3) glucan decreases with the

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Increase the degree of substitution until the degree of substitution is about 0.4. This degree of substitution increases the anti-tumor effectiveness of the carboxyalkyl-ß- (1 -> 3) glucans is strong up to a substitution degree of 1.5 «Ss assumed that the anti-tumor activity of the carboxyalkyl-ß- (1 · # 3) glucans does not come from the feed, but rather is newly obtained, the reasons for it are not yet known. Metabolism test results have shown that the Garboxyalkylglucan has no effect against tumors after the. Oarboxyalkyl radicals in the living Tissues have been cleaved off and it is believed that the carboxyalkyl radicals of glucan are important for play his activity.

example 1

Pachyman obtained by extracting Poria cocos Wolf with an aqueous alkaline solution was used in the manner oxidizes, reduces and then hydrolyzes, as in the reference by "Saishin Igakushi", Recent Medicine, Volume 25, page 1043 »1970, where Pachymaran was produced (Figure 1 shows the infrared absorption spectrum of the preserved pachymaran). 3 g of pachymaran were vigorously stirred in 80 ml of isopropanol at room temperature the resulting solution was 8 ml of a 30 μl sodium hydroxide solution and then 3 g of solid methyliaonochloroacetic acid were added The resulting solution was stirred on a water bath maintained at 40 to 45 ° C for 3 hours and cooled to room temperature with stirring. The reaction

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mixture was filtered with the aid of a glass frit, the resulting precipitate was dissolved in 100 ml of 70% methanol with given a content of 5 ml of glacial acetic acid, stirred for 10 minutes and washed 3 times with 100 ml of absolute methanol and ether. · The precipitate obtained was dried in vacuo, whereby

2.2 g carboxymet; hyl-ß- (1> 3) glucan was obtained in the form of a colorless powder, hereinafter referred to as product Ia will. Figure 2 shows the infrared absorption spectrum of the product Ia.

• The reaction described was carried out using "1 g. Pachymaran repeated. The precipitate isolated from the reaction mixture was dissolved in 100 ml of water containing 2 ml of glacial acetic acid, dissolved and 300 inl acetone was added to carboxymethyl-ß- (1 ■ * 3) to precipitate glucan. The precipitate was Purified by washing with absolute methanol, the product obtained was referred to as' product 1b.

1 g of the product Ib was dissolved in 50 ml of water, and the pH of the aqueous solution was raised with aqueous NaOH solution set and there were 0.5 ml of 20 $ aqueous sodium acetate solution added. The precipitate formed was isolated and referred to as product Ic.

Example 2
200 g of Pachymaran were at 380 rpm for 30 minutes in.

5.3 1 isopropanol was stirred, then 324 ml of 30 NaOH solution were added dropwise over 115 minutes to the pachymaran

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and the mixture was stirred for 40 minutes. The mixed solution was heated to 50 G, 138 g of solid ionochloroacetic acid was added and the mixture was stirred at 400 rpm for 3 hours. The reaction mixture was cooled to 17 ° C., filtered with the aid of a glass frit and the precipitate obtained was washed with 300 ml of isopropanol. The precipitate was stirred in 10 1 of 80 % MeOH for 17 hours at room temperature, isolated by filtration and washed with 300 ml of 80% aqueous methanol. The washed precipitate was dissolved in 6 l water, solid substance was removed by filtration and the pH of the filtrate was adjusted to 8.7 with acetic acid. 400 ml of the filtrate, the pH of which had been adjusted, were added 20% strength aqueous solution of sodium acetate was added, 10 l of acetone were also added and the mixture was stirred for 30 minutes. The precipitate formed was isolated by filtration, stirred for 30 minutes in 2 liters of 80% aqueous methanol and then stirred in 2 liters of absolute methanol for 30 minutes. The precipitate was isolated by filtration and dried in vacuo at 40 ° C. for 24 hours. This gave 257 g of sodium carboxymethylpachymaran (product II).

Example 3

1 g of the product I was dissolved in 10 ml of 1N NaOH solution and 50 ml of methanol was added to the solution. The precipitate formed was collected by filtration with 70% strength aqueous methanol, 90% aqueous methanol, absolute

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Methanol and then washed with acetone and dried. It ' 0.8 g of complete sodium carboxymethyl-ß- (1 -? 3,) glucan obtained, which is referred to as product III. Figure 3 shows the infrared absorption spectrum of this compound.

Example 4

3 g of pachymaran were used for 2 hours in the same way as in example 1 reacted with 3 x 6 g of methyl monochloroacetate. The resulting precipitate was neutralized in acetic acid-methanol solution, Washed and dried as in Example 1, 3.5 g of carboxymethyl-ß- (1 - * 3) glucan (product IV) in the form of a colorless powder. The infrared absorption spectrum shown in Fig. 4 and the results the titration showed that this compound is the complete sodium salt.

Example 5

1 g of Pachymaran and 1.2 g of butyl monoehloracetat were how

in Example 4, implemented, whereby sodium carboxymethyl-ß- (1 ■) 3) glucan was obtained as a colorless powder (product V). The infrared absorption spectrum of the product V is shown in FIG shown.

Example 6

2 g of pachymaran were stirred in 60 ml of isoamyl alcohol, 6 ml of 20 / S ITaOH solution were added to the resulting solution and the mixture was stirred for 3 hours at room temperature.

2 g of α-bromobutyric acid was added to the mixture and it became

Stirred for 2 otunds on an asaerbad of 50 G.

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The precipitate formed was treated as in Example 1,

where 1.6 g of a-alkylcarboxymethyl-ß- (1 -> 3) Glucan was obtained in the form of a colorless powder (Product VI).

Example 7

1 g of 13- (1 - 3) glucan, which is obtained by extracting the .fruit body from Lentinus edodes Sing with hot water, fractionating and Purification (Lentinan) was stirred for 30 minutes at room temperature in 30 ml of isopropanol. To the · Lentinan 3 ml of 25% NaOH solution and then 3 ml of isopropanol were added containing "1.2 g of monochloroacetic acid was added dropwise and the reaction mixture was 150 minutes on a stirred at 50 C held water bath. The precipitate formed was isolated from the reaction mixture by filtration, poured into 35 ml of 80% aqueous methanol, the

Contained 2 ml of glacial acetic acid, and stirred for 10 minutes. The precipitate was collected by filtration, three times with 40 ml washed with absolute methanol and dried, with 0.8 g of carboxymethyl-13- (1 -> 3) glucan (product VII) were obtained as a colorless powder.

Example 8

1 g of α - (1 · '■ · # 6) glucan obtained by extracting the fruiting body by Lentinus edodes Sing, Fractionation and Purification, was put into a mixed solution of 35 ml isopropanol and stirred in 5 ml of water, the suspension were

3 ml of 25% aqueous NaOII were added and the mixture was Stirred for 30 minutes. There were 1.2 g of Iionochloressigsaure

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BATH ORIGINAL

% \ 120.5 % - Ψ -

added and the reaction was completed by heating the reaction system to a water bath at 50 C with stirring ο The precipitate was isolated by filtration, 30 liinut'en in 35 ^m ^ 80 / & - aqueous methanol was stirred and then washed three times with absolute methanol The precipitate obtained was dried, leaving 0.7 g
Carboxy-methyl-ex - (1, -56) glucan were obtained in the form of a colorless powder (product VIII).

Example 9

Glucan with β- (1 -i 3) -glueosid bonds as a main chain was produced by extracting the fruiting body of Flammulina veltipes Sin, fractionating and purifying. 1 g of the glucan (referred to as material F) was reacted with 1.2 g of ilonochloroacetic acid in the manner described in Example 5. 1.0 g of carboxymethylglucan with β- (1 -} 3) -glucoside bonds as main chain were obtained (product IX).
The anti-tumor activity, the degree of substitution by carboxyalkyl groups, the percentage of sodium salt and
the water solubility of products I to X and the starting materials were tested. The results obtained are shown in Table 1 below.

EiAD ORIGINAL,

Table 1

sample solubility Substitutions Content of Inhibition Complete (mg / 100 ml H ₂ O): degree (', S) Na courtship (; 5) rate {', *) Regression 46.65 0 0 93 4/9 Pachymaran 7500. 0.815 75 96 8/10 Product I-a 7500: 0.829 50 31 0/10 Product 1-b 9300 0.822 100 ■ 100 10/10 Product I-e 9300 0.808 100 100 ■ 10/10 Product II , 9400 0.815 100 100 10/10 Product III 4300 0.618 100 100 ι 10/10 KX Product IV "■ 4700 0.628 Ίοο 98 8/10 <α Product V 5800 0.701 72 · 90 8/10 «A J" Product VI 38.25 . 0 0 100 10/10 *** Lentinan 7700 0.560 ■ 92 100 10/10 Product VII 8000 0 0 -85 0/10 ' o> T α - (A ^ 6) -glucan 7100 0.723 98 20th 2/10 ■ "*. -, Product '/ 11I 230 P. 0 80 5/10 Material ib ¹ 6100 0.670 94 92 8/10 Product IX

ro δ ο

ro

cn ro C =) on co

-H-

Comments:

1) · Solubility tests were carried out at 30G.

2) Swiss "" albino owls. "(Swiss" albino nice) 'were in the right groin subcu ^ aneTn jectionsof O.O5 ml ascites of Sarcoma 180 administered. 24 hours after the injection and then daily for a total of 10 days a group of 10 mice received intraperitoneal injections of the. Sample. A comparison group received no injection of the " Rehearse. After 5 weeks, the lice and the tumors were killed excised and balanced. The inhibitory effect of the samples on the tumors was determined from the treated and untreated mice and the values of the inhibition rate and the complete regression were determined.

Lethal dose of product I-a and pachymaran were tested on mice, ils was a value of more than 4 g / kg for I-a and a value of 1.5 g / kg for pachymaran established.

The anti-tumor activity of Product I-a was also tested using tumors other than Sarcoma-180. the The results obtained are in the following table listed.

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tumor

Honest- ■ i

Carcinoma

1ÄM1O2. C3H / Arinia

Ade.no carcinoma

CCLi Adeno-SV / il / iiS.

carcinoma

Table 2

dose used (ng / kg. Inhibiii-Haus χ day). ' 'onsrate

- 100 χ to

- 5 x 10

5 χ \ 0

40.0

78.9

36.4

complete regression

0/6

0/5

0/10

09g 1 7/1

BAO LOCATIONNM.

Claims (12)

Claims i.1 alkali salt of a carboxy- lower-alkyl-ß- (1 -> 3) -lilucan. 2. Alkali salt according to claim 1, characterized in that the ß- (1 - ^ 3) -glucan pachymaran or Lentinan is. 3. Alkali salt according to claim 1, characterized in that the ß- (1 - »3) -glucan a .by extracting Sing the fruiting body of Flammulina veltipes with hot water, fractionating the extract and purifying it Product is. 4. alkali salt according to claim 1 to 3, characterized in that g e k e η η draws that it contains methyl groups as alkyl groups. 5. Alkali salt of carboxymethyl-a- (1 ■> 6) -glucan. 6. alkali salt according to claim 5 »characterized in that the a- (1 V6) -glucan by extracting of the fruiting body of Lentinus edodes Sing obtained with hot water, fractionating the extract and cleaning Is glucan. - 17 -209817/1361 7. Process for the preparation of an alkali salt of carboxy-lower ~ alkyl-ß- ( 1 -> 3) glucan or a- (1 ^ 6) -glucan, thereby characterized in that ß- (1 - »3) -glucan or a- (1 - ^ 6) ~ glucan with an a-halogen-substituted lower fat acid or its derivative is reacted, the resulting carboxy-lower-alkyl-ß- (1 - * 3) -glucan or α- (1 -> 6) -Glucan is dissolved in water, adjust the pH of the aqueous solution to a value between 4 and 8.5 »preferably 6.5 and 8.5 is set by adding such an amount of a water-soluble organic Solvent not less than 2 times the volume of the pH-adjusted aqueous solution, the alkali salt of carboxy-lower-alkyl-ß- (1 -> 3) - or -a- (1- £ 6) -glucan fails. 8. The method according to claim 7, characterized in that the alkali metal salt is precipitated with the aid of a water-soluble organic solvent in the presence of not more than 1 % of an alkali metal salt or alkaline earth metal salt. 9. The method according to claim 7 or 8, characterized in that after reacting the ß- (1 -? · 3) -glucan or'a- (1 - »6) -glucans with an α-halogen-substituted lower fatty acid or those Derivative isolated the precipitate formed in the reaction mixture and washed with aqueous, more than 70% Lgem methanol and with absolute methanol. - 18 - 209817/1361 10. Anti-tumor agent, characterized in that it is an alkali salt of a carboxy-lower-alkyl-ß- (1 -> 6) -glucan or a carboxy-lower alkyl-a- (1 -> 6) contains glueans. 11. Composition according to claim 10, characterized in that it is sodium-carboxy-methyl-ß- (1 - »3) -glucan as the active ingredient contains. 12. Means according to claim 11, characterized in that that the ß- (1 - * 3) -glucan is pachymaran or lentinan. 209817/1361