前苏联专利SU1024011A3 Method of producing carboxymethylated beta-1,3-glucans

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专利摘要:
It has been found that water-insoluble, thermogelable beta -1,3-glucan produced by microorganisms, lower polymers obtainable upon partial hydrolysis of the glucan or, carboxymethylated derivatives of said glucan or of said lower polymers are useful for inhibiting growth of tumors in a warm-blooded animal. The inhibitory activity against various tumors of these polysaccharides is very strong and significant whenever administered to warm-blooded animals from the early to very late stage of tumor progression or even when administered prior to plantation of tumors. The carboxymethylated derivatives above-mentioned are novel water-soluble compounds which can be produced by carboxymethylation of said glucan and partial hydrolyzate thereof.
公开号:SU1024011A3
申请号:SU772543704
申请日:1977-11-17
公开日:1983-06-15
发明作者:Сасаки Такума；Сугино Юкио
申请人:Такеда Кемикал Индастриз Лтд (Фирма)；
IPC主号:

专利说明:

This invention relates to a process for the preparation of new carboxymethylated, 3 glucanroses with antitumor activity. The purpose of the invention is to obtain new compounds expanding the arsenal of means of action on a living organism. The goal is achieved according to the method of producing carboxymethylated 1 -1, 3 -glucanes of the general formula H, ROV n H OR based on the known carboxymethylation of carbohydrates tO, where at least one of R is CH, COOH, and the others are atoms hydrogen, with an average degree of polymerization (p + 2) from 2 to 1000, where p is O or an integer, or their salts of RE-1, 3-glucan of formula CHjOH. I CHjOH “JrVoU Nf-O. U- FH, OH 3vi4 TAY H (And Jn OH, where the average degree of polymerization (n + 2) and n are as above, is reacted with monochloric acetic acid and its alkali metal salt in the presence of alkali, followed by isolation of the target product in free form or in the form of salt. Used in accordance with the present invention in the original, 3-glitch (TAK-N) contains L – 1, 3-bonds in its main glycosidic units and is water insoluble, it has a moderate degree of polymerization (DP) and may gelatinize at DP 170 at 0 ° in the presence of water; I get it Weeding the growth of microorganisms, such as Agrobacterium radiobacte {iF013127 ATCC), Agrobacterium radiobacter (iFO 13126, ATCC 21679, FeRM P-1166), Agrobacterium Faecali var, myxogenes NTE-V (iFO-131 0, ATC 21680, FF, FERcali var, myxogenes NTE-V (iFO-131 0, ATC 21680, FEP), mye, IIE, VE 21680 . in accordance with the present invention, lower polymers (TAK-D), obtained by partial hydrolysis of the p-glucan {TAK-N), are also used. TAK-D is obtained by partial hydrolysis of TAK-N {for example, by acid hydrolysis, alkaline and enzymatic hydrolysis with p-1,3 glucanase). Example 1. 3 g of TAK-N (average degree of polymerization) and suspension are suspended in 80 ml of isopropyl alcohol stirred at room temperature for 30 minutes. Then 8 ml of a 30% sodium hydroxide solution are slowly added with stirring for about 69 minutes. The mixture is then stirred vigorously at room temperature for an additional 90 minutes to prevent gel formation. 3.6 g of monochloroacetic acid are added and the mixture is stirred at 60-70 ° C for 5 hours to ensure carboxymethylation. The product is isolated by filtration and thoroughly washed with a mixture of methanol and acetic acid (7: 3 by volume). The precipitate is collected by filtration, washed well with aqueous methanol, methanol and acetone in the above order and dried under reduced pressure. 4 2.9 g of carboxymethylated PJ-1, 3-glucan (CMTAK) are obtained. Carboxymethyl content, (number of carboxymethyl groups per glucose residue) 0 ,. Example 2. Isropyl alcohol is suspended with 1.5 g of TAK-N (polymerization degree 540j and the suspension is stirred at room temperature for 30 minutes. Then, with stirring, 2 ml of sodium hydroxide solution is added in four portions, i.e. 0.5 ml at intervals of 15 minutes. The mixture is then stirred at room temperature for an additional 90 minutes, after which 0.9 g of monochloroacetic acid is added in three portions, i.e., 0.3g each, at intervals of for 10 minutes Carboxymethylation is carried out with stirring for 150 minutes. The product is collected in the center by fugging, dissolved in 50 ml of water and neutralized with acetic acid. 120 ml of methanol are added to the neutral solution and the precipitate is collected by centrifugation. The precipitate is washed with a mixture of 300 ml of 80% aqueous methanol and 100 ml of ethanol and then with a mixture of 3 300 ml 80 - aqueous methanol and 200 ml of ether. The product is lyophilized, 1.7 g is recovered. Carboxyme. 0.75 is a good content. Example 3 ml of isopropyl alcohol is suspended in 1.5 g of TAK (degree of polymerization) and the suspension is stirred at room temperature for 30 min. Then, a portion of 30 ml of sodium hydroxide solution i.e. 1 ml at intervals of 15 mi, then stirred at room temperature for 90 minutes. After that, 1.8 g of monochloric acetic acid are added in three portions, i.e., 0.6 g each in intervals of 10 minutes. The mixture was stirred at 150 minutes. The product resulting from the carbon-oxymethylation is collected by centrifugation, dissolved in 0 ml of water and neutralized with acetic acid. 90 ml of methanol are added to the neutral solution and the resulting precipitate is collected by centrifugation. The precipitate is washed well with a mixture of 200 ml of BO-aqueous methanol and 100 ml of ethanol, and then with a mixture of 200 ml of BO-aqueous ethanol and 200 ml of ether. Then the product, t lyophilized, get 2, About g SMTAK. Carboxymethyl content of 1.07. Example A. In 33 ml of water, 3.2 g of TAK-N (degree of polymerization 255) are suspended and 1 g of sodium hydroxide is added with stirring at room temperature followed by the addition of 2.4 g of sodium monochloroacetate. Carboxymethylation is carried out at room temperature with constant stirring for 2 hours. After that, 1 g of sodium hydroxide and 2 g of A is added, And g of sodium monochloroacetate, and then the reaction is carried out at room temperature with stirring for 3 hours. another 1 g of sodium hydroxide is added, and 2, sodium monochloroate tata. The reaction is continued at room temperature while stirring for another 2 hours. To this reaction mixture is added 1 liter of ethanol and the resulting precipitate is well washed with ethanol on a glass filter until the filtrate ceases to give a red color with phenolphthalein 1k. Then the precipitate. dried under reduced pressure. The resulting powder (3.7 g) is dissolved in 90 ml of water and neutralized with acetic acid, followed by the addition of 210 ml of ethanol. The resulting precipitate is collected by centrifugation, washed with B0 | - ethanol and lyophilized. Get 2.6 g SMTAK. Carboxymethyl content is 0.30. EXAMPLE 5 6 g) of TAK-N (polymerization degree 255) are suspended in 66 ml of water and, under ice-cooling, tr of sodium hydroxide is added with stirring, followed by 9.6 g of sodium monochloroacetate. Carboxymethylation is carried out while the scientific research institute is cooled with ice, with stirring for 2 hours. Then, g of sodium hydroxide and 9.6 g of sodium monochloroacetate are added again and the mixture is stirred under ice cooling for 3 hours. After that, another g of sodium hydroxide and 9.6 g of sodium monochloroacetate are added and the reaction is continued while cooling with ice and with stirring over 3 to 1 L of ethanol is added to the reaction mixture and the precipitate is well washed with ethanol on a glass filter until until the filtrate ceases to give a red color with phenolphthalein, after which the product is dried at 50 ° C and reduced pressure. The resulting powder (B, 6 g) was dissolved in 172 ml of water and neutralized with acetic acid. Then, after adding 4BO of ml of ethanol, the precipitate is collected by centrifugation, washed with 80% ethanol and lyophilized. . 5.6 g SMTAK are obtained. Carboxymethyl, Content 0,36. Example b. isopropyl alcohol is suspended 1.5 g of TAK-D - the lower polymer obtained by partial hydrolysis of TAK-N, degree of polymerization 299- Carboxymethylation is carried out similarly to example 2. The product is washed as in example 2, 1.9 g of CMTAK is obtained. The carboxymethyl content of 0.59. The indicated TAK-D is suspended in Q ml of isopropyl alcohol and, as in Example 3, carboxymethylation is carried out. The reaction product is washed as in Example 3, 2.2 g CMTAK are obtained. The carboxymethyl content is 1.13. Example 7. Isopropyl alcohol is suspended with 1.5 g of TAK-D (polymerization degree 113). Analogously to Example 2, carboxymethylation is carried out. The reaction product is collected by centrifugation, dissolved in tO ml of water and neutralized with acetic acid. 90 ml of methanol is added to the neutral solution and the resulting precipitate is collected by centrifugation, and then it is washed well first with 200 ml of 80% methanol and then with 200 ml of aqueous ethanol, and then it is lyophilized. Obtain 1.4 g SMTAK. Carboxyl content of 0.51. Similarly to that described from 1.5 g of TAK-D (polymerization degree 68), 1 g of CMTAK is obtained. Carboxyl content of 0.36. Example 8, isopropyl alcohol is suspended in 1.5 g of TAK-D (polymerization degree 113). Analogously to Example 3, carboxymethylation is carried out. The reaction product is washed as in Example T to obtain 2.3 g of CMTAK (carboxyl content 1.22). Similar to that described from 1.5 g of TAK-D (degree of polymerization 68 is obtained I, g of CMTAK, carboxyl content is 0,. Example 9 1.3 g of TAK-N (DP 960) are suspended in 0 ml of isopropyl alcohol, and the suspension is stirred at at room temperature for 30 minutes. Then, with stirring, in 4 equal portions, 2 ml of 30% sodium hydroxide solution are added at intervals of 15 minutes. The mixture is then stirred at room temperature for 90 minutes. Then 0.9 g is added monochloroacetic acid in three equal portions with intevalami for 10 min. Carboxymethylation thus conducted at 50 ° C with stirring for 150 minutes, the product is collected by centrifugation and washed with 20 ml of 95% isopropyl alcohol, the residual current is re-suspended in 20 95 isopropyl alcohol and neutralized with acetic acid, the resulting precipitate is collected by centrifugation and washed well with water methanol, methanol, and acetone in this order and dried under reduced pressure to obtain 1.6 g n SMTAK. Carboxymethyl content 0.40. Example 10 B + Oml of isopropyl alcohol is suspended with 1.5 g of TAK-D (DP 15) and the suspension is stirred at room temperature for 30 minutes. Then, with stirring, add 2, ml of 30% sodium hydroxide in four equal portions fM at intervals of 15 minutes. The mixture was stirred at room temperature for 90 minutes, 1.08 g of monochloroacetic acid was added in three equal portions at intervals of 10 minutes. Carboxymethylation is carried out with stirring for 150 minutes. The product is washed as in example 7, to obtain 0.8 g SMTAK. Carboxyl content is 0.19. Example 11. A 3.7 g of water is dissolved 3 g of TAK-D (polymerization degree 2), 1 g of sodium hydroxide is added with stirring at room temperature, and then 2.9 g of sodium monochloroacetate. The carboxymethylation reaction is carried out at room temperature with constant stirring for 2 hours. Then, 1 g of sodium hydroxide and 2.9 g of sodium monochloroacetate are added again, then the reaction is carried out under the same conditions for 3 hours. Then, 1 g of sodium hydroxide and 2.9 g of sodium monochloroacetate are added. The reaction is carried out under the same conditions for another 2 hours. To the reaction mixture is added 1 liter of ethanol and the resulting precipitate is washed well with ethanol on a glass filter, dried at 50 ° C and under reduced pressure. Received; the powder (3–7 g) was added to a mL of hot {95 ° C) ethylene glycol and the mixture was stirred. The resulting precipitate was removed by filtration on a glass filter as quickly as possible. The filtrate is concentrated under reduced pressure and the residue is dried at 60 ° C. Get 2.5 g SMTAK. The carboxyl content of 0.30, The data characterizing the new compounds obtained are presented in Table 1. Example 12. In 30 ml of a solution of nitric acid (nitric acid: methanol — 1:10 in volume, 1.5 g of CMTAK are suspended in suspension. The suspension is stirred at room temperature for 3 hours and filtered through a glass filter. The solid residue on a glass filter washed with 60 ml of 90% methanol and then 301 gels of methanol.The washed solid precipitate is distilled with 300 ml of distilled iodine. Then 370 mg of calcium hydroxide is added and the mixture is stirred for k h, then filtered through filter paper. The filtrate is neutralized 0 1N hydrochloric acid 50 ml of ethanol was added to the neutral solution and the resulting precipitate was collected by centrifugation. The precipitate was washed with 120 ml of 80% ethanol and dried under reduced pressure to obtain 1.3 g of the CMTAC calcium salt. Anti-tumor activity of three types of CMTAK obtained from TAK-M and autoclaved SMTAK investigated in mice.
OOrats were dissolved in distilled water for injection (pH A / 7.0). One of these samples was sterilized in an autoclave, 25 minutes) before administration. Six million S l80 cells were transplanted subcutaneously into the right groin area of 1CP-} CL mice, whose body weight was g. The tumor nodes were removed on the 35th day after the transplantation and weighed.
The average tumor weight of the treated T group, including 5 mice, was compared with the control group C, which included 10 mice, and the degree of tumor suppression was calculated (% inhibition (C – T) (CxA10). On the 35th day, the results for several animals were also calculated with a tumor removed (full regression).
The test results are presented in table 2.
As shown in Table 2, all CMTAK samples at a dose level of 10 mg / kg significantly suppress tumor growth. This growth inhibition of SMTAK was observed at dose levels of 1 to kQ mg / kg.
These results show that CMTAK can be sterilized without losing its antitumor activity and can be used in an injectable form for treating cancer.
The antitumor i effect of TAK-N, TAK-0 and SMTAK was investigated with various administration methods.
TAK-N (degree of polymerization), TAK-0 (degree of polymerization 50), TAK-0 (degree of polymerization 16) and CMTAK of example 1 were administered 5 mg / kg intravenously, subcutaneously or intraperitoneally for 10 days to TCP-ICL mice “Some 40 cells were transplanted 2k hours before the first injection; The degree of tumor growth inhibition was calculated as described above.
The test results are presented in table 3.
As can be seen from table 3 in each case there was a significant suppression of tumor growth. When administered intravenously, TAK-N, CMTAK almost completely suppressed tumor growth, and the degree of suppression was over 85% with complete regression of the tumor in 5 out of 5 mice treated with men.
Thus, TAK-N, CMTAK and .TAK-D are effective in suppressing a tumor with any route of administration.
eleven
12 Table 2
Continued table. 3

权利要求:
Claims (1)
[1]
METHOD FOR PRODUCING CARBOXYMETYLED fb-1,3-Glucans of formula I where the average degree of polymerization (n + 2) and η have the above meanings, they are reacted with monochloroacetic acid or its alkali metal salt in the presence of alkali, followed by isolation of the desired product with free form or in the form of salt.
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