JPH0699501B2 - Polymer arabinofuranosylcytosine derivative and method for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel polymeric arabinofuranosylcytosine derivative having low toxicity and excellent carcinostatic activity, and a method for producing the same.

[0002]

2. Description of the Related Art Some of nucleic acid derivatives have excellent anticancer activity, and arabinofuranosylcytosine is one of them, which shows activity against leukemia and is clinically used. . However, since this arabinofuranosylcytosine is toxic to bone marrow cells and other normal cells, it is necessary to give sufficient consideration to side effects during its administration, and its application range is inevitably limited. It was

[0003]

The present invention overcomes the drawbacks of the conventional arabinofuranosylcytosine derivatives and reduces the toxicity without impairing the anticancer activity of the novel arabinofuranosylcytosine derivatives. It was made for the purpose of providing an arabinofuranosylcytosine derivative.

[0004]

The present inventors have conducted various studies on reducing the toxicity of an arabinofuranosylcytosine derivative, and as a result, have found that a certain spacer was added to a divinyl ether-maleic anhydride copolymer. It was found that a polymer compound in which an arabinofuranosylcytosine derivative residue is introduced via the compound shows a decrease in toxicity while maintaining the anticancer action originally possessed by the arabinofuranosylcytosine derivative. The present invention has been completed based on the above.

That is, the present invention has the general formula

[Chemical 3] (In the formula, a hydrogen atom or an arabinofuranosyl cytosine residue, n is an integer of 1 to 11), and 10 to 500 of the repeating units, and at least 10 mol% thereof are The present invention provides a polymeric arabinofuranosylcytosine derivative and a salt thereof, each of which is composed of a unit in which R in the formula is an arabinofuranosylcytosine residue.

The high molecular weight arabinofuranosyl cytosine derivative and its salt are novel compounds which have not been published in the literature, and are represented by the general formula in an inert organic solvent.

[Chemical 4] In the divinyl ether-maleic anhydride copolymer represented by the formula (m is an integer of 10 to 500), the general formula H2N- (CH2) n-COOH (where n in the formula is 1 to
It can be produced by reacting an aminocarboxylic acid represented by the formula (which is an integer of 11) with arabinofuranosylcytosine, and converting the product into a salt if desired.

General formula (II) used as a raw material in this case
The divinyl ether-maleic anhydride copolymer is obtained by radical copolymerization of divinyl ether and maleic anhydride according to a known method [Macromolecular synthesis].
r Synthesis, "Vol. 8, pages 89-94 (1982)].

This divinyl ether-maleic anhydride copolymer itself exhibits excellent anticancer activity against various solid cancers, and by introducing an arabinofuranosylcytosine derivative residue, a higher anticancer activity can be obtained. Exerts a cancer effect.

With regard to this copolymer, those having a molecular weight of 100,000 or less have low toxicity and are particularly suitable in the present invention.

An aminocarboxylic acid having a carbon number of 2 to 12 is used as a spacer when introducing an arabinofuranosylcytosine residue into the divinyl ether-maleic anhydride copolymer. Examples of such aminocarboxylic acids include glycine, 3-
Aminopropionic acid, 4-aminobutyric acid, 5-aminovaleric acid, 8-aminocaprylic acid and the like can be mentioned.

The reaction between the divinyl ether-maleic anhydride copolymer and the aminocarboxylic acid is carried out by reacting an inert organic solvent such as N-methylpyrrolidone, dimethylformamide, diethylacetamide, dimethylsulfoxide, and acetone with divinylether-maleic anhydride. It is carried out by dissolving the copolymer, adding an excess amount of aminocarboxylic acid thereto, and reacting with stirring under slightly alkaline conditions. The reaction time at this time is usually 1 to 5 hours.

The reaction product thus obtained has the structural formula

[Chemical 5] The arabinofuranosyl cytosine represented by the following formula is used in an amount of 5 to 100 based on the divinyl ether-maleic anhydride copolymer.
Add and react at a mol% ratio. The reaction time at this time is usually about 1 to 10 hours.

After completion of the reaction, the desired arabinofuranosylcytosine derivative is obtained by removing unreacted substances by, for example, ultrafiltration and lyophilizing. This can then be replaced, if desired, with pharmacologically acceptable salts such as sodium, potassium, calcium, magnesium salts. Further, at this time, by adding a hydroxide or soluble salt of sodium, potassium, calcium, magnesium or the like to the reaction mixture to form a corresponding salt, and then performing ultrafiltration and lyophilization. It is also possible to obtain salts of arabinofuranosylcytosine derivatives.

Generally, a polymer obtained by radical polymerization, such as a divinyl ether-maleic anhydride copolymer, has a wide molecular weight distribution. Therefore, gel filtration or ultrafiltration should be repeated at the stage of binding an aminocarboxylic acid. Therefore, it is preferable to narrow the molecular weight distribution.

The polymer arabinofuranosylcytosine derivative of the present invention has the general formula (III)

[Chemical 6] (N in the formula has the same meaning as described above) or is composed of only the repeating unit or a compound represented by the general formula (IV)

[Chemical 7] (N in the formula has the same meaning as described above) and a repeating unit. In the latter case, the general formula (II
It is necessary that the repeating unit of I) is contained in a proportion of at least 10 mol%. General formula (III)
When the number of repeating units of is small, the activity against leukemia derived from arabinofuranosylcytosine is reduced.

The content of arabinofuranosylcytosine in the polymeric arabinofuranosylcytosine derivative of the present invention based on the weight is in the range of 5 to 36% by weight, and is composed of only the repeating unit represented by the general formula (III). 3 if given
6% by weight.

[0017]

The polymer arabinofuranosylcytosine derivative of the present invention gradually releases the anticancer active substance arabinofuranosylcytosine under physiological conditions and exerts an anticancer effect. For example, in the case of the compound of n = 5 in the general formula (I), 0.05M phosphate buffer solution (pH 7.2) at 37 ° C
In, 80% of the bound arabinofuranosylcytosine is released in 3.5 days.

Further, the high molecular weight arabinofuranosyl cytosine derivative of the present invention was administered once to the CD2F1 mouse in which P388 leukemia cells were intraperitoneally transplanted, and the life prolonging effect was examined 150 days after the transplantation. %Met. In contrast, the survival rate of arabinofuranosylcytosine tested in the same manner was only 22%. In addition, the incidence of side effects was far lower in the polymeric arabinofuranosylcytosine derivative of the present invention.

[0019]

EXAMPLES Next, the present invention will be described in more detail by way of examples.

Example 1 800 mg of a divinyl ether-maleic anhydride copolymer having an average molecular weight of 30,000 (corresponding to n = 112) was dissolved in 5 ml of N-methylpyrrolidone anhydride, and 3.2 g of 6-aminohexanoic acid was added thereto. Was dissolved in 5 ml of distilled water and added while stirring.

While the pH of the reaction solution was adjusted to 9.0 with a 2N aqueous sodium hydroxide solution, the reaction was carried out for 2 hours. After completion of the reaction, the mixture was diluted to 500 ml with distilled water and adjusted to pH 4.0 with 6N hydrochloric acid, and then PTHK membrane (manufactured by Millipore, molecular weight cutoff 10
Of the PM30 membrane (Amicon, cut molecular weight 30,000) was collected, desalted and washed with distilled water, and freeze-dried to obtain 900 mg of a dried product. 108 mg of this divinyl ether-maleic anhydride copolymer having 6-aminohexanoic acid as a spacer is dissolved in 5 ml of distilled water, and 243 mg of arabinofuranosylcytosine and 288 mg of water-soluble carbodiimide are added with stirring at room temperature. After reacting for 3 hours, the product was washed with distilled water using a PCAC ultrafiltration membrane (Millipore, cut-off molecular weight 1000) to remove unreacted arabinofuranosylcytosine, and then the pore size was 0.01 μm. It was sterilized by a Millipore filter and lyophilized. The product thus obtained (181 mg) was a white fluffy solid and was easily soluble in water and physiological saline. The ultraviolet absorption spectrum of this product in distilled water (Fig. 1) has an absorption characteristic of arabinofuranosylcytosine at 272 nm, and the arabinofuranosylcytosine content calculated from the calibration curve is 23.2% by weight.
Met.

Example 2 An aqueous solution of 120 mg of the polymer arabinofuranosylcytosine derivative obtained in Example 1 was adjusted to pH 7.6 with 1N sodium hydroxide, and then PCAC manufactured by Millipore Corporation.
Demineralize and wash distilled water with an ultrafiltration membrane to 0.01
It was sterilized by a Millipore filter having a pore size of μm and freeze-dried. In this way, 131 mg of a sodium salt-type polymer arabinofuranosylcytosine derivative was obtained,
This was more soluble in water and saline than the product obtained in Example 1. The ultraviolet absorption spectrum in the aqueous solution was almost the same as in FIG.

Example 3 As in Example 1, divinyl ether-maleic anhydride copolymer having an average molecular weight of 30,000 (corresponding to n = 112) 900
mg was dissolved in 5 ml of anhydrous acetone, and 1.3 g of 3-aminopropionic acid was added to a solution of 5 ml of distilled water with stirring. At the time of addition, the pH of the solution was adjusted to 9.0 with a 2N sodium hydroxide solution, and the mixture was reacted for 2 hours. The reaction solution was diluted to 500 ml with distilled water, and the pH was adjusted to 4 with 6N hydrochloric acid.
Adjust to 0 and pass the Amicon YM100 ultrafiltration membrane (molecular weight cutoff 100,000) and the PM30 membrane made by the same company (30,000) and collect the fractions, desalting and washing with distilled water. After freeze-drying, 585 mg of a dried product was obtained. Copolymer 120 having 3-aminopropionic acid as a spacer
mg in the same manner as in Example 1 in 5 ml of distilled water, 243 mg of arabinofuranosylcytosine and water-soluble carbodiimide 2
88 mg was added and reacted, and 202 mg of white flocculent solid product was obtained using the same processing method. From the absorbance of this product in distilled water at 272 nm ultraviolet, it was found that the arabinofuranosylcytosine content was 21.6% by weight.

Example 4 As in Example 1, divinyl ether-maleic anhydride copolymer having an average molecular weight of 30,000 (corresponding to n = 112) 600
Dissolve mg in 5 ml of anhydrous N-methylpyrrolidone,
-Aminododecanoic acid was added to a suspension of 2.8 g in 10 ml of distilled water with stirring. When the pH of the reaction solution is adjusted to 11 with a 2N sodium hydroxide aqueous solution, a uniform solution is formed and the reaction proceeds. After reacting for 2 hours, diluted to 300 ml with distilled water, adjusted to pH 6.0 with 6N hydrochloric acid, and desalted and washed with distilled water using a PM30 ultrafiltration membrane (molecular weight cut off of 30,000) manufactured by Amicon. Lyophilization gave 646 mg of dried product. 52 mg of this copolymer having 12-aminododecanoic acid as a spacer was dissolved in 5 ml of distilled water, and
In the same manner as above, 122 mg of arabinofuranosylcytosine and 144 mg of water-soluble carbodiimide were added and reacted, and 34 mg of a white fluffy solid was obtained using the same treatment method. From the absorbance at 272 nm of ultraviolet absorption of this product in distilled water, it was found that the arabinofuranosylcytosine content was 32.5% by weight.

Example 5 As in Example 1, a divinyl ether-maleic anhydride copolymer having an average molecular weight of 5000 (equivalent to n = 19) was prepared.
5 g was dissolved in 5 ml of anhydrous N-methylpyrrolidone to give 6-
Aminohexanoic acid (3.6 g) was added to a solution of distilled water (10 ml) with stirring. The pH of the reaction solution was adjusted to 9.0 with a 2N aqueous sodium hydroxide solution and allowed to react for 2 hours. After completion of the reaction, the solution was diluted to 500 ml with distilled water, adjusted to pH 4.0 with 6N hydrochloric acid, desalted and washed with Amicon PM10 ultrafiltration membrane (molecular cutoff of 10,000) using distilled water, and freeze-dried. Then, 1.0 g of a dried product was obtained. 81 mg of this copolymer having 6-aminohexanoic acid as a spacer was treated with 5 ml of distilled water in the same manner as in Example 1 to obtain 182 mg of arabinofuranosylcytosine and water-soluble carbodiimide 21.
6 mg was added and reacted, and 56 mg of white flocculent solid was obtained using the same processing method. From the absorbance of this product in distilled water at ultraviolet 272 nm, it was found that the arabinofuranosylcytosine content was 22.1% by weight.

Example 6 As in Example 1, a divinyl ether-maleic anhydride copolymer having an average molecular weight of 120,000 (equivalent to n = 451)
0 g was dissolved in 5 ml of anhydrous N-methylpyrrolidone to give 6-
5.8 g of aminohexanoic acid was added to a solution prepared by dissolving 10 ml of distilled water with stirring. The pH of the reaction solution was adjusted to 9.0 with a 2N aqueous sodium hydroxide solution and allowed to react for 2 hours. After the reaction was completed, the solution was diluted to 500 ml with distilled water, adjusted to pH 4.0 with 6N hydrochloric acid, desalted / washed with distilled water using an Amicon YM100 ultrafiltration membrane (molecular cutoff of 100,000), and freeze-dried. did. Copolymer having 6-aminohexanoic acid thus obtained as a spacer 108 mg
In the same manner as in Example 1 with 243 mg of arabinofuranosylcytosine and water-soluble carbodiimide 2 in 5 ml of distilled water.
88 mg was added and reacted, and 66 mg of a white fluffy solid was obtained by the same treatment method. From the absorbance of this product in distilled water at ultraviolet 272 nm, it was found that the arabinofuranosylcytosine content was 25.0% by weight.

Reference Example 1 Anticancer activity of high molecular weight arabinofuranosylcytosine derivative Four-week-old female CD2F1 mice were intraperitoneally treated with 1 × 10 6 P.
388 leukemia cells were transplanted, and 24 hours after the transplantation, the drug was administered once intraperitoneally. Same sex in control and experimental groups,
Six mice of the same age group were used, and the mice were bred under SPF conditions. The anticancer effect was determined by the center of the survival days of the mice, and the survival rate was compared with the survival time of the control group mice represented by the following formula.

[0028]

[Equation 1]

Table 1 shows the survival days and the survival rate of the control group and the treatment group. As is clear from this table, control mice die at 9.0 days, whereas arabinofuranosylcytosine has 11.0 days, high molecular weight arabinofuranosylcytosine has 21.0 days, and arabinofuranosylcytosine has 21.0 days. The anticancer activity against mouse P388 leukemia was remarkably improved by using nofuranosyl cytosine as a polymer derivative.

[0030]

[Table 1]

[0031]

INDUSTRIAL APPLICABILITY The high molecular weight arabinofuranosylcytosine derivative of the present invention is a novel compound of low toxicity which realizes sustained release of arabinofuranosylcytosine, which is an anticancer active substance, and further, arabinofuranosyl. It has the advantage of showing superior anti-cancer activity to cytosine alone.

[Brief description of drawings]

FIG. 1 is an ultraviolet absorption spectrum diagram of a polymeric arabinofuranosylcytosine derivative in Example 1 and arabinofuranosylcytosine for comparison in distilled water.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI Technical indication location A61K 31/765 AGB 9454-4C C08F 222/06 MLT (C08F 222/06 216: 16)

Claims (2)

[Claims] 1. A general formula: (Wherein R is a hydrogen atom or an arabinofuranosylcytosine residue, n is an integer of 1 to 11), and 10 to 500 repeating units are included, and at least 10 mol% thereof is A polymeric arabinofuranosylcytosine derivative and a salt thereof, each of which is composed of a unit in which R in the above formula is an arabinofuranosylcytosine residue. 2. A compound of the general formula: ## STR2 ## in an inert organic solvent. In the divinyl ether-maleic anhydride copolymer represented by the formula (m is an integer of 10 to 500), the general formula H2N- (CH2) n-COOH (where n in the formula is 1 to
The polymeric arabi of claim 1 which is characterized by reacting an aminocarboxylic acid represented by the formula 11) with arabinofuranosylcytosine, and converting the product into a salt if desired. A method for producing a nofuranosyl cytosine derivative or a salt thereof.