JPS634558B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the general formula (In the formula, R is an acyl group having 2 to 4 carbon atoms, and X is a hydrogen atom, an alkali metal atom, or an ammonium group. ⁾
This invention relates to O ² '-diacyl-adenosine-3',5'-cyclic phosphoric acid, its production method, and an anticancer agent containing it as an active ingredient. The novel compounds of the present invention are, for example, as follows. 2-fluoro-N ⁶ , O ² ′-diacetyl-adenosine-3′,5′-cyclic phosphoric acid, 2-fluoro-
N ⁶ , O ² ′-dipropylyl-adenosine-3′,5′-
Cyclic phosphoric acid, 2-fluoro- ^N6 , ^O2' -dibutyryl-adenosine-3',5'-cyclic phosphoric acid and alkali metal salts thereof, such as potassium or sodium salts, ammonium salts, preferably organic ammonium salts, For example, triethylammonium salt,
Tributylammonium salt etc. The novel compound of the formula is useful as an anticancer agent because it has extremely low toxicity and excellent anticancer activity. A compound of the formula has the general formula ( ^wherein R and Carboxylic acid represented by and general formula It is produced by reacting with an acid anhydride represented by (in these formulas, R has the above-mentioned meaning). A compound of the formula has the general formula 2-fluoro-adenosine-3',5'-cyclic phosphoric acid or its salt represented by the general formula It can be produced by reacting with an acid anhydride represented by the formula (in which R has the above meaning) (see JP-A-56-29599). Among the starting compounds of the formula, 2-fluoro-adenosine-3',5'-cyclic phosphoric acid (X=H, a)
For example, by reacting 2-fluoro-adenosine-5'-phosphate () with dicyclohexycarbodiimide to close the phosphoric acid moiety of the compound () at the 3' position (Thomas et al., The Journal of・Biological Chemistry, Volume 251, 6757
~6766 p. 1976), or by reacting 2-amino-adenosine-3',5'-cyclic phosphoric acid (2) with borofluoric acid and a nitrous acid compound to perform the Schiemann reaction (Yamatsugu et al. Author, Chemical and Pharmaceutical Bulletin, Volume 26, 2391-2395
p. 1978). Salts of 2-fluoro-adenosine-3',5'-cyclic phosphoric acid (a) in the phosphoric acid moiety can likewise be used as starting compounds for the process of the invention; It can be obtained by reacting with a metal compound such as sodium hydroxide or potassium hydroxide, or a tertiary amine such as triethylamine or tributylamine. To prepare a compound of the formula, the following procedure can be performed, for example. 2-Fluoro-adenosine-cyclic phosphoric acid or its salt () in an organic solvent,
For example, dissolved in amines such as pyridine, picoline, and lutidine, alcohols such as methanol, ethanol, and glycerin, ethers such as dioxane, dimethyl sulfoxide, etc., or mixtures thereof,
This formula

An acid anhydride of the formula is added, and the mixture is reacted, usually at about 30 to 110°C, preferably 50 to 70°C, for about 20 minutes to 24 hours, with stirring if necessary. As the acid anhydride, for example, butyric anhydride, propionic anhydride, acetic anhydride, etc. are used, and the amount added is usually about 1 to 50 times the molar amount of the compound (). Then, the thus obtained 2-fluoro-O ² ′-
Monoacyl-adenosine-3',5'-cyclic phosphoric acid or its salt () is isolated in the form of a reaction mixture or preferably by conventional methods and then treated with a carboxylic acid of formula ROH in the presence of an inorganic acid, preferably sulfuric acid. and expression

The reaction is carried out with the acid anhydride of the formula, usually at about 80 to 120°C, preferably 90 to 110°C, with stirring if necessary, for about 5 to 30 minutes. As the carboxylic acid, for example, butyric acid, propionic acid, acetic acid, etc. are used, and as the acid anhydride, butyric anhydride, propionic anhydride, acetic anhydride, etc. are used, and each of these is used in an amount of about 1 to 300 times the molar amount of the compound of the formula. It is preferable to add. Isolation and purification of the target compound () are carried out by conventional methods.
For example, this can be carried out by using an appropriate combination of column chromatography using activated carbon, ion exchange resin, alumina, silica gel, etc., or organic solvent precipitation using an organic solvent such as ether, chloroform, hexane, etc. X is H
Compounds of formula I which are can be led to the corresponding salts in the cyclic phosphate moiety by the action of alkali metal compounds such as sodium hydroxide, potassium hydroxide, etc. or with amines, preferably tertiary amines, and these salts By reacting with an acid, a free cyclic phosphoric acid of the formula in which X is H is obtained. Further, a compound of the formula in which X is an ammonium group, for example, a triethylammonium group, can be converted into its alkali metal salt by treatment with an alkali metal type cation exchange resin. According to the method of the invention, a compound of formula
% in a good yield. The novel compound of the formula shows excellent anticancer activity with significantly low toxicity and is therefore useful as an anticancer agent. Therefore, the present invention further provides an anticancer agent containing the compound of the formula as an active ingredient. This anticancer drug is also excellent in that it has almost no side effects. The active ingredients of the formula may be used alone or as a mixture of two or more, but they are usually mixed with excipients, diluents, or auxiliaries and formulated in a conventional manner. Excipients, diluents and adjuvants include common solid or liquid substances that do not affect the active ingredients, such as water, vegetable oils, floatants, surfactants, gelatin, cocoa butter, lauric fat, glycerin, macros. Galls, sugars, magnesium stearate, preservatives, etc. are used. The anticancer agent is preferably applied by intravenous injection, oral administration, mucosal application, etc., and formulations suitable for these administration methods include injections, capsules, suppositories, and the like. The dosage of this anticancer drug is appropriately selected depending on the symptoms to be treated, age, administration form, etc., but it is usually 1 to 300 mg/Kg of the active ingredient per day for adults, preferably 10 to 60 mg/Kg.
It is administered at once or in several doses. In order to determine the anticancer effect and acute toxicity of the compound of formula, the following animal experiment was conducted. Experimental Example 1 (Anticancer effect on mice) Animals used: ICR mouse (female), 5 weeks old, weight 20-23 g,
6 animals per group. Experimental method: ICR mice were injected with Sarcoma 180 (S-
180) Ascites cancer was transplanted, and ascites was removed with a syringe on the 7th day after transplantation. 0.1 ml of this ascites fluid per animal (number of cancer cells: 1×10 ⁶ cells) was intraperitoneally transplanted into each experimental animal (6 animals per group). From the second day of transplantation, the active ingredient 2-fluoro- ^N6 , ^O2' -dibutyryl-adenosine-3',5'-cyclic sodium phosphate was administered once a day for 5 days to treat ascites. It was administered intraperitoneally to cancer-grafted mice. The active ingredient was suspended in physiological saline to which 0.5% carboxymethyl cellulose was added and administered. Seven days after transplantation, ascitic fluid was removed from each mouse, the ratio of the ascites weight to the cancer cell weight was determined, and this ratio was multiplied by the ascites weight to calculate the total cancer cell weight. In addition, the tumor formation rate (%) was determined from the following formula: total cancer cell weight/total cancer cell weight of control x 100, and anticancer activity was determined as follows. Anticancer activity Tumor production rate + 65-41% 40-11% 10-0% As is clear from the results shown in the following table, the compounds of the present invention exhibit extremely strong anticancer effects on ascites cancer cells.

[Table] Experimental Example 2 (Acute Toxicity) Animals used: ICR mouse (female), 5 weeks old, weight 20-23 g,
6 animals per group. Experimental method: Test compound in 1% (w/v) saline solution 0.1
~0.5 ml was administered as a bolus to the animals intraperitoneally. The LD ₅₀ value is determined by the Richfield-Wilcoxon method.
It was calculated from the number of animals alive and dead on the second day after administration. The LD ₅₀ of 2-fluoro-N ⁶ , O ² '-dibutyl-adenosine cyclic phosphate sodium salt was 193 mg/Kg. Preparation of a compound of formula: Example A Add a small amount of triethylamine to 500 mg of 2-fluoro-adenosine-3',5'-cyclic phosphoric acid and prepare 2-
Prepare fluoro-adenosine-3',5'-cyclic phosphate triethylammonium salt, dissolve this in 10 ml of N,N-dimethylformamide, and add 10 ml of pyridine.
and 10 ml of butyric anhydride, and reacted at 50 to 70°C for 5 hours. After the reaction is complete, 500 ml of ether is added to the reaction mixture, and the mixture is left at 5°C. The precipitated crystals were filtered, washed with ether, and dried to yield 607 mg of crude crystals of 2-fluoro-O ² '-monobutyryl-adenosine-3',5'-cyclic triethylammonium phosphate.
is obtained. The obtained crude crystals were mixed with 0.2 g of silica gel, adhered to the silica gel, dried, and placed on a silica gel column (1.7 cm in diameter, 12 cm in length).
Elution with chloroform/methanol (volume ratio 80:20) and evaporation of the fraction containing the target substance to dryness yielded purified 2-fluoro-O ^2' -monobutyl-adenosine-3',5'-cyclic triethylammonium phosphate. Salt is obtained. Melting point: 166-168℃ (decomposition) Ultraviolet absorption spectrum: λPH ^7.0 _nax (nm) 261.5, 269, 0 (shoulder) Infrared absorption spectrum (KBr method): 1020, 1090, 1250, 1375, 1493, 1590, 1610,
Absorption maxima are shown at 1655, 1743, 2495, and 2675 cm ^-1 . Mass spectrum [trimethylsilylation method (see Lawson et al., Journal of the American Chemical Society, Vol. 93, p. 1014, 1971)]: Molecular ion peak: M (TMS) ₂ 561 m/e NMR spectrum (D ₂ O ): δ (ppm) 0.68 (t), 1.32 (m), 2.26 (t), 4.1
6
(s) 4.71 (m), 5.36 (d), 5.48 (s), 7.89
(s) Paper chromatography: Toyo Paper No. 51A used Developing solvent: ethanol/0.5M-ammonium acetate (volume ratio 5:2) Rf value = 0.70 Thin layer chromatography: DEAE cellulose used Developing solvent: chloroform/ Methanol (volume ratio 1:1) Rf value = 0.46 Example B 500 mg of 2-fluoro-adenosine-3',5'-cyclic phosphoric acid was suspended in 8 ml of water, and a small amount of triethylamine was added dropwise to dissolve it. 2-fluoro-adenosine-
A 3′,5′-cyclic triethylammonium phosphate salt is obtained. Dissolve 601 mg of this salt in 10 ml of N,N-dimethylformamide, add 10 ml of pyridine, and further add 10 ml of butyric anhydride, and react at 50 to 70°C for 5 hours. After the reaction is completed, 500 ml of ether is added to the reaction mixture, and the mixture is allowed to stand at 5°C, and the precipitated crystals are filtered, washed with ether, and dried. 200 mg of the obtained crude crystals of 2-fluoro-O ² ′-monobutyryl-adenosine-3′,5′-cyclic phosphate triethylammonium salt
Dissolve in 2 ml of water and add weakly acidic ion exchange resin IRC-
50 (manufactured by Rohm and Haas) (diameter 1.7 cm, length 25 cm), elute with water, and concentrate the fraction containing the target compound under reduced pressure. The obtained concentrate was mixed with 0.2 g of silica gel to adhere to the silica gel, and after drying, it was placed on a silica gel column (diameter 1.7 cm, length 12 cm), and chloroform/methanol (volume ratio 80:
20) and the fraction containing the target substance is evaporated to dryness to yield 2-fluoro-O ² '-monobutyryl-
Adenosine-3′,5′-cyclic phosphate sodium salt
167mg is obtained. Melting point: 205-207℃ (decomposition) Ultraviolet absorption spectrum: λPH ^7.0 _nax (nm) 261.5, 269.0 (shoulder) Infrared absorption spectrum (KBr method): 1020, 1090, 1250, 1375, 1493, 1590, 1610,
Absorption maximum is shown at 1655 and 1743 cm ^-1 . Mass spectrum (trimethylsilylation method): Molecular ion peak: M (TMS) ₂ 561 m/e Elemental analysis (as C ₁₄ H ₁₆ O ₇ N ₅ PFNa・1.5H ₂ O): C H N Theoretical value (%) 36.06 4.11 15.02 Actual value (%) 35.98 4.16 14.57 Paper chromatography: Paper and developing solvent are the same as Example A Rf value = 0.70 Thin layer chromatography: Adsorbent and developing solvent are the same as Example A Rf value = 0.46 Example 1 300 mg of 2-fluoro-O ² ′-monobutyryl-adenosine-3′,5′-cyclic phosphate triethylammonium salt obtained in Example A was mixed with 10 ml of butyric anhydride and n
- Dissolve in 10ml of butyric vinegar, add 2-3 doses of concentrated sulfuric acid (97%), and react at 95-105℃ for 15 minutes. After the reaction was completed, 300 ml of ether was added to the reaction mixture, and the mixture was then allowed to stand at 5°C. The precipitated crystals were filtered, washed with ether, and dried to give 2-fluoro-N ⁶ ,O ² ′-dibutyryl-adenosine-3. ′、
419 mg of crude crystals of 5′-cyclic triethylammonium phosphate salt are obtained. Next, the crystals were mixed with 0.2 g of silica gel to adhere to the silica gel, dried, and then transferred to a silica gel column (diameter 1.7 cm,
12 cm in length) and eluted with chloroform/methanol (volume ratio 80:20), and the fraction containing the target substance was evaporated to dryness to yield purified 2-fluoro-N ⁶ ,O ² '-dibutyryl. -adenosine-3′,
A 5′-cyclic triethylammonium phosphate salt is obtained. Melting point: 137-139℃ (decomposition) Ultraviolet absorption spectrum: λpH ^7.0 _nax (nm) 242.0 (shoulder), 248.5, 278.0, 287.0
(Shoulder) Mass spectrum (trimethylsilylation method): Molecular ion peak: M (TMS) ₁ 559 m/e Infrared absorption spectrum (KBr method): 1016, 1098, 1252, 1382, 1477, 1617, 1732,
Maximum absorption is shown at 2495 and 2675 cm ^-1 . NMR spectrum (D ₂ O): δ (ppm) 0.89 (2 t), 1.67 (m), 2.47
(m), 4.29 (s), 4.93 (m), 5.59 (d),
6.17 (s), 8.35 (s) Paper chromatography: Paper and developing solvent are the same as Example A Rf value = 0.85 Thin layer chromatography: Adsorbent and developing solvent are the same as Example A Rf value = 0.64 Example 2 2-fluoro-N ⁶ ,O ² ′- obtained in Example 1
Add 200 mg of crude crystals of dibutyryl-adenosine-3′,5′-cyclic triethylammonium phosphate to 2 ml of water.
Dissolve in IRC-50 column (diameter 1.7 cm, length
25 cm), elute with water, and concentrate the fraction containing the target compound under reduced pressure. The obtained concentrate was mixed with 0.2 g of silica gel to adhere to the silica gel, dried, and then placed on a silica gel column (diameter 1.7 cm, length 12 cm) and chloroform/methanol (volume ratio 80:20). When the fraction containing the target substance is evaporated to dryness, 2-fluoro-N ⁶ , O ² ′-dibutyryl-adenosine-3′,
112 mg of 5'-cyclic phosphate sodium salt are obtained. Melting point: 165-167℃ (decomposition) Ultraviolet absorption spectrum: λpH ^7.0 _nax (nm) 242.0 (shoulder), 248.5, 278.0, 287.0
(Shoulder) Infrared absorption spectrum (KBr method) 1016, 1098, 1252, 1382, 1477, 1617, 1732cm
^-1 indicates absorption maximum. Mass spectrum (trimethylsilylation method): Molecular ion peak: M (TMS) ₁ 559 m/e Elemental analysis (as C ₁₈ H ₂₂ O ₈ N ₅ PFNa・1.5H ₂ O): C H N Theoretical value (%) 40.31 4.70 13.06 Actual value (%) 40.19 4.78 12.56 Paper chromatography: Paper and developing solvent are the same as Example A Rf value = 0.85 Thin layer chromatography: Adsorbent and developing solvent are the same as Example A Rf value = 0.64 Example 3 (Injection) Add 4.1 g of common salt and distilled water for injection to 5 g of 2-fluoro-N ⁶ , O ² '-dibutyryl-adenosine-3',5'-cyclic sodium phosphate to make a total volume of 500 ml. 2 ml of this solution is dispensed into ampoules, which are melted and sealed (each ampoule contains 20 mg of active ingredient), and then sterilized with high-pressure steam to form an injection. Example 4 (Suppositories) Macrogol 1000 150g and Macrogol 4000
Dissolve 45g of 2-fluoro-
Add 5 g of N ⁶ , O ² ′-dibutyryl-adenosine-3′,5′-cyclic sodium phosphate salt, stir until uniformly distributed, and inject into 100 suppository molds.
Cool and solidify to make suppositories. Example 5 (Capsule) 2-fluoro-N ⁶ , O ² '-dibutyryl-adenosine-3',5'-cyclic sodium phosphate 3 g, crystalline cellulose 1 g, magnesium stearate 50
mg, 2 g of lactose, and 2.5 g of potato starch are mixed well, and the mixture is filled into 100 capsules to make capsules.

Claims (1)

[Claims] 1. General formula 2-fluoro-N ⁶ , O ² '- represented by (in the formula, R is an acyl group having 2 to 4 carbon atoms, and X is a hydrogen atom, an alkali metal atom, or an ammonium group)
Diacyl-adenosine-3′,5′-cyclic phosphoric acid or its salt. 2 General formula (In ^the formula, R and Carboxylic acid represented by ROH and general formula A general formula characterized by reacting with an acid anhydride represented by (R in these formulas has the meaning below) 2-fluoro-N ⁶ , O ² '- represented by (in the formula, R is an acyl group having 2 to 4 carbon atoms, and X is a hydrogen atom, an alkali metal atom, or an ammonium group)
Process for producing diacyl-adenosine-3′,5′-cyclic phosphoric acid or its salt. 3 General formula 2-fluoro-adenosine-3',5'-cyclic phosphoric acid or its salt represented by the general formula The general formula obtained by reacting with an acid anhydride represented by (in the formula, R has the meaning below) (In ^the formula, R and Carboxylic acid represented by ROH and general formula A general formula characterized by reacting with an acid anhydride represented by (R in these formulas has the meaning below) 2-fluoro-N ⁶ , O ² '- represented by (in the formula, R is an acyl group having 2 to 4 carbon atoms, and X is a hydrogen atom, an alkali metal atom, or an ammonium group)
Process for producing diacyl-adenosine-3′,5′-cyclic phosphoric acid or its salt. 4 General formula 2-fluoro-N ⁶ , O ² '- represented by (in the formula, R is an acyl group having 2 to 4 carbon atoms, and X is a hydrogen atom, an alkali metal atom, or an ammonium group)
An anticancer agent containing diacyl-adenosine-3',5'-cyclic phosphoric acid or its salt as an active ingredient.