JPS636079B2 - - Google Patents

Description

Detailed Description of the Invention The present invention provides choline cytidine diphosphate (hereinafter referred to as
Abbreviated as CDP-choline. ).

CDP-choline is an important drug used for recovery from impaired consciousness and functional impairment caused by brain trauma, and various methods for its production are known. for example,
(1) A method of condensing cytidine-5'-phosphate (hereinafter abbreviated as 5'-CMP) and phosphorylcholine.
(Tokuko Sho 35-13024, Sho 46-37594, Sho 46
−18990, etc. )(2) A method of reacting an activated derivative of 5′-CMP with phosphorylcholine. (Tokuko Showa 42-
1384, Special Publication Showa 46-2101, etc. )(3) A method of reacting an activated derivative of phosphorylcholine with 5'-CMP.
(Special Publication No. 45-4747, No. 52-16115, etc.) (4) Method via cytidine-5'-diphosphate ethanolamine. (Special Publication No. 39-6541, No. 47-11476,
etc. ) etc. are known. However, these methods have disadvantages in the use of expensive reagents, reaction, operation, yield, or both, and are not necessarily industrially advantageous.

The present inventors have solved the drawbacks of such conventional methods, and have developed CDP-CDP, which is inexpensive, easy to react and operate, and is industrially advantageous.
As a result of intensive research aimed at establishing a method for producing choline, we discovered a new general formula that is easy to handle, inexpensive, and highly reactive. (In the formula, N(R) ₂ represents a morpholino group or a lower dialkylamino group.) Intermediate represented by:
Phosphorylcholine diamidates were discovered.
Conventionally, only monosubstituted amides of phosphorylcholine were known (for example, Japanese Patent Publication No. 45-27561, Japanese Patent Publication No. 47-38929, West German Patent Publication No. 2359245). Difficult to synthesize because the positively charged quaternary ammonium nitrogen in the molecule easily forms salts within the molecule, and only one of the two hydroxyl groups in the phosphoric acid residue is esterified or amidated. It was said to be. As a result of intensive research, the present inventors have found that phosphorylcholine diamides, which have not been described in any literature, can be easily produced using inexpensive industrial chemicals, and the present inventors can easily produce diamides of phosphorylcholine, which have not been described in literature, by combining the present compound and 5'-CMP in the presence or absence of a solvent. Furthermore, by conducting the reaction in the presence or absence of acids and bases, we discovered a method to eliminate the drawbacks of conventional methods and obtain CDP-choline in a short period of time and in good yields, leading to the completion of this research. Ivy. That is, according to the present invention, the phosphorylcholine diamidates of the general formula [] and 5'-
By reacting with CMP, CDP-choline can be obtained in good yield.

As shown in detail later, phosphorylcholine diamidates, which are one of the starting materials used in the present invention, can be obtained easily and in good yield by, for example, reacting β-chloroethylphosphoryl diamidates with trimethylamine. You can get it.

Any solvent may be used in the present invention as long as it does not inhibit the reaction, and there is no particular need to make it anhydrous. Preferably, dimethylformamide, dimethylacetamide, etc. are used, but it can also be carried out without a solvent. The reaction temperature is from room temperature to the boiling point of the solvent, preferably from 80° to 140°C. reaction time is 30
It is carried out from 1 minute to 24 hours, but preferably 1
~4 hours is advantageous. Phosphorylcholine diamidates can be used in an amount equal to or more than 5'-CMP.

The acid to be present in the reaction system is preferably an organic acid such as trichloroacetic acid, but may also be an inorganic acid such as sulfuric acid or hydrogen chloride. The amount of acid used is preferably about 0.01 to 1 mole of the phosphorylcholine diamidate used.

The target product can be isolated from the reaction mixture by a conventional method. For example, after the reaction, the solvent is distilled off, the supernatant is decanted, or a solvent is added to precipitate, the resulting residue is dissolved in water, and the target product is adsorbed by an anion exchange resin. , followed by elution with dilute aqueous formic acid. eluted
The CDP-choline fraction is collected, concentrated and dried to obtain the target product, CDP-choline.

A feature of the present invention is that the compound of the general formula [], which has a very high activity, is utilized for the synthesis of CDP-choline. That is, the compound of general formula [] is 5'-
Once reacted with CMP, the following general formula [], (In the formula, N(R) ₂ represents a morpholino group or a lower dialkylamino group) is provided as a reaction intermediate, but under the reaction conditions, it is hydrolyzed without isolation, almost quantitatively and High purity
The point is to obtain CDP-choline. Furthermore, the general formula []
By selecting an appropriate solvent (e.g. dimethylacetamide, etc.),
5'-CMP is first converted into an organic amine salt such as tri-n-butylamine salt, as in a known example (Japanese Patent Publication No. 45-4747) in which tri-n-butylamine salt of 5'-CMP is reacted with phosphorylcholine monoamidate in an orthochlorophenol solvent. The reaction can be carried out with 5'-CMP itself without the need for further reaction. In addition, the reaction time for the reaction between the general formula [] and 5'-CMP is extremely short at 100°C for 3 hours, compared to 18 hours at 100°C in the case of the example cited in Japanese Patent Publication No. 45-4747. The subsequent operation is also an extremely industrially advantageous method in which the supernatant can be removed by decanting without the need to specifically distill off the solvent.

Phosphorylcholine diamidates, which are compounds of the general formula [] in the present invention, can be produced by the three methods described below, and an appropriate method can be selected depending on the individual compound, reaction conditions, etc. Details of these methods are as follows: I will explain later.

Manufacturing method (1) (In the formula, N(R) ₂ represents a morpholino group or a lower dialkylamino group.) via chloroethylphosphoryl diamidates (general formula []). β-chloroethylphosphoryl dichloride (Reference example, J.Amer.Chem.Soc., 51 ,
953 (1929)) with morpholine or lower dialkylamine in an anhydrous organic solvent to obtain a compound of general formula []. Then, the compound of general formula [] is converted into general formula [] by reacting it with trimethylamine in a sealed tube in a high yield. Any reaction solvent may be used as long as it does not inhibit the reaction, but examples include aromatic hydrocarbons such as benzene, hydrogen halides such as chloroform, ethers such as tetrahydrofuran, ketones such as acetone, and acetonitrile. Mention may be made of nitriles and amides such as dimethylformamide, but advantageously nitriles such as acetonitrile are preferred. The reaction temperature is between room temperature and the boiling point of the solvent, preferably between 50° and 100°C. Reaction times range from several hours to several days. The reaction product is obtained as crystals or oil by concentrating the reaction solution.

This production method is industrially superior in that it allows the compound of general formula [] to be produced easily and at low cost.

Manufacturing method (2) (In the formula, N(R) ₂ represents a morpholino group and a lower dialkylamino group.) Choline chloride and the known chlorophosphoryl diamidate (general formula []) (Reference examples, J.Chem.
Soc., 1958 , 1963) in the presence of a base to obtain phosphorylcholine diamidate chloride [] represented by the general formula []. As the reaction solvent, polar organic solvents such as
Dimethylformamide, dimethylsulfoxide, etc. can be mentioned. As the base, an inorganic base such as sodium hydride, an organic base such as 2,6-lutidine, pyridine, etc. are used. The reaction temperature is between room temperature and the boiling point of the solvent,
Desirably, it is carried out at room temperature to 150°C. The reaction time ranges from several hours to several days, but is preferably around 8 hours. The reaction product is obtained as crystals or oil of phosphorylcholine diamidate chloride by filtering the precipitate and concentrating the filtrate under reduced pressure.

This production method is an excellent method in that it allows the compound of general formula [] to be produced with good yield.

Manufacturing method (3) (In the formula, N(R) ₂ represents a morpholino group or a lower dialkylamino group.) Known phosphorylcholine dichloridate (Literature example, Compt. Rend., 257 , 183, (1963), Japanese Patent Publication No. 47
-32970) with morpholine or lower alkylamine, phosphorylcholinediamidate chloride represented by the general formula [] is obtained. As the reaction solvent, polar solvents such as dimethylformamide, dimethylsulfoxide, acetonitrile, etc. are used, but the reaction can also be carried out without a solvent. The reaction temperature is carried out between below freezing and the boiling point of the solvent, preferably between -5°C and room temperature. The reaction time ranges from several hours to several days, and advantageously several hours. The reaction product is obtained as crystals or oil of phosphorylcholine diamidate chloride by filtering the precipitate and concentrating the filtrate under reduced pressure. This production is excellent in that it allows the compound of the general formula [] to be produced easily and in a short time.

Examples of the present invention will be described in detail below, but the present invention is not limited to these examples.

Example 1 1.2 g of phosphorylcholine dimorpholito chloride (general formula [ ], N(R) ₂ = morpholino group)
1 g of 5'-CMP was suspended in 4.5 ml of dimethylacetamide and reacted at 100°C for 3 hours with stirring. After the reaction, the supernatant dimethylacetamide was decanted, the residue was dissolved in water, and the pH was adjusted to 9.5 with aqueous ammonia.
Adsorb to 120 ml of Diaion SA11B (formic acid type) and elute with 0.01-0.03M formic acid. CDP-choline fractions were collected, concentrated, and dried to obtain 1.44 g of CDP-choline. This compound was analyzed by paper chromatography (ethanol, 0.5M ammonium acetate 5:2), high pressure filter paper electrophoresis (0.2M phosphate buffer PH7.4, 1500V,
2 hours), nuclear magnetic resonance absorption spectrum (in heavy water),
Infrared absorption spectrum (potassium bromide disk),
The ultraviolet absorption spectrum (PH11, PH7.0, PH1.5) completely matched that of standard CDP-choline.

Example 2 Phosphorylcholine dimolholitochloride
1.2 g and 1.0 g of 5'-CMP were mixed and reacted by heating at 100°C for 4 hours with stirring. After the reaction, the reaction mixture was dissolved in water, purified using an ion exchange resin in the same manner as in Example 1, and the CDP-choline fraction was collected, concentrated, and dried to obtain 1.28 g of pure CDP-choline. Obtained. The thus obtained CDP-choline was compared and identified with the standard CDP-choline in the same manner as in Example 1, and it was found to be completely identical.

Example 3 Phosphorylcholine dimorpholitochloride
1.2g and 1.0g of 5′-CMP with 4.5g of dimethylacetamide
ml and reacted at 100°C for 3 hours with stirring.
After the reaction, the supernatant dimethylacetamide was decanted, the residue was dissolved in water, and purified using an ion exchange resin in the same manner as in Example 1. The CDP-choline fraction was collected, concentrated, and dried. ,
1.38 g of pure CDP-choline was obtained. The thus obtained CDP-choline was prepared in the same way as in Example 1.
When compared and identified with CDP-choline, they were completely identical.

Example 4 Phosphorylcholine-N・N'-diethyldiamidate chloride (general formula [], R=ethyl)
1.98 g and 0.96 g of 5'-CMP were added to 3 ml of dimethylacetamide and reacted at 120°C for 3 hours with stirring.
After the reaction, the same treatment as in Example 1 was carried out to obtain 0.88 g of CDP-choline. The thus obtained CDP-choline was compared and identified with the standard CDP-choline in the same manner as in Example 1, and it was found to be completely identical.

Example 5 20 ml of benzene is added to 10 g of β-chloroethylphosphoryl dichloride, and a solution of 17.7 g of morpholine dissolved in 60 ml of benzene is gradually added dropwise while stirring under ice cooling. After the addition was completed, the mixture was stirred at room temperature for 1 hour, and the precipitated morpholine hydrochloride was collected by filtration. If the filtrate is concentrated to dryness and left at 10°C or lower, crystals will precipitate.
When the crystals are washed with ether and dried, the melting point is 53° ~
13.1 g of white crystals of β-chloroethylphosphoryl dimorpholinate (general formula [], N(R) ₂ =morpholino group) at 58° C. were obtained.

Elemental analysis (as C ₁₀ H ₂₀ ClN ₂ O ₄ P) Theoretical value C: 40.21%, H: 6.75%, N: 9.37% Analytical value C: 40.14%, H: 6.83%, N: 9.38% Next, this β - Add 20 g of a 30% acetonitrile solution of trimethylamine to 5 g of chloroethylphosphoryl dimorpholidate and react in a sealed tube at 70° C. for 48 hours. After the reaction, the precipitate is collected by filtration, the filtrate is concentrated, a small amount of dioxane is added, and when it is left to stand at below 10°C, it crystallizes to form phosphorylcholine dimorpholidite chloride (general formula []N
5.4 g of white crystals of (R) ₂ =morpholino group) were obtained.

Elemental analysis (as C ₁₃ H ₂₉ ClN ₃ O ₄ P・H ₂ O) Theoretical value C: 41.73%, H: 8.36%, N: 11.22% Analytical value C: 41.85%, H: 8.41%, N: 11.39% Using the obtained phosphorylcholine dimorpholithochloride, the same procedure as in Examples 1 to 3 was carried out to obtain choline cytidine diphosphate.

Example 6 20 ml of benzene is added to 10 g of β-chloroethylphosphorodichloride, and a solution of 15.6 g of diethylamine dissolved in 60 ml of benzene is added dropwise while stirring under ice cooling. After the addition was completed, the mixture was stirred at room temperature for 1 hour, the precipitate was collected by filtration, and the filtrate was concentrated. By distilling the residue under reduced pressure, β-chloroethylphosphoro-N.
N'-diethyldiamidate (general formula []R=
ethyl) was obtained.

Next, this β-chloroethylphosphoro-N・
20 g of a 30% acetonitrile solution of trimethylamine was added to 5 g of N'-diethyldiamidate, and the mixture was allowed to react in a sealed tube at 70° C. for 48 hours. After the reaction, the reaction solution is concentrated, dioxane is added, and insoluble matter is filtered off. When the filtrate is concentrated to dryness, phosphorylcholine-
3.65 g of N.N'-diethyldiamidate chloride (general formula []R=ethyl) was obtained. This is a paper chromatography monospot (ethanol: 0.5M - ammonium acetate,
10:1, detected with HI reagent), gas, and silica gel thin layer chromatography (chloroform:methanol, 1:2, HI reagent,
Detected with iodine). nuclear magnetic resonance absorption spectrum,
δ1.17 (12H, t, J=7Hz), δ3.1 (8H, m,),
δ3.27 (9H, s), δ3.72 (2H, m), δ4.35 (2H,
m), [solvent methanol- _d4 , internal standard TMS,
Varian T-60] Using the obtained phosphorylcholine-N·N'-diethyldiamidate chloride, the same procedure as in Example 4 was carried out to obtain choline cytidine diphosphate.

Example 7 1.4 g of choline chloride and 2.55 g of chlorophosphoryl dimorpholidate were mixed with 5 ml of dimethyl sulfoxide.
0.24 g of sodium hydride while stirring.
was added and reacted at room temperature for 8 hours with stirring. After the reaction, a small amount of precipitate is collected by filtration, and the filtrate is concentrated to dryness under reduced pressure. Add a small amount of acetonitrile to the residue,
Filter, concentrate the filtrate, and leave it at below 10°C.
White crystalline phosphorylcholine dimorpholitochloride (general formula []N(R) ₂ = morpholino group)
2.05g of was obtained. This product completely matched the crystals obtained in Example 5 in melting point and paper chromatography (ethanol:0.5M ammonium acetate, 10:1, detected with HI reagent).

Using the obtained phosphorylcholine dimorpholithochloride, the same procedure as in Examples 1 to 3 was carried out to obtain choline cytidine diphosphate.

Example 8 1 g of phosphorylcholine dichloridate is added to 4 ml of acetonitrile, and 1.36 g of morpholine is added dropwise while stirring and cooling. After the addition was completed, the mixture was stirred at room temperature for 3 hours, 10 ml of benzene was added, and the mixture was further stirred at room temperature for 30 minutes, and the precipitated white crystals were collected by filtration. The filtrate is concentrated to dryness, a small amount of acetonitrile is added to the residue, it is filtered, and the filtrate is left at 10°C or lower to produce white crystals of phosphorylcholine dimorpholitochloride.
0.56g was obtained. This crystal completely matched the crystal obtained in Example 5 in melting point, paper chromatography, and nuclear magnetic resonance absorption spectrum.

Using the obtained phosphorylcholine dimorpholithochloride, the same procedure as in Examples 1 to 3 was carried out to obtain choline cytidine diphosphate.

Claims (1)

[Claims] 1. General formula [] (In the formula, N(R) ₂ represents a morpholino group or a lower dialkylamino group.) Production of choline cytidine diphosphate, which is characterized by reacting a phosphorylcholine diamidate represented by the formula with cytidine-5'-monophosphoric acid. Method. 2 General formula [] (In the formula, N(R) ₂ represents a morpholino group or a lower dialkylamino group) is reacted with trimethylamine to obtain phosphorylcholine diamidates represented by the general formula []. , and then reacting with cytidine-5'-monophosphoric acid. 3 General formula [] (In the formula, N(R) ₂ represents a morpholino group or a lower dialkylamino group) is reacted with choline chloride to obtain phosphorylcholine diamidates represented by the general formula [ ], A method for producing choline cytidine diphosphate, which comprises then reacting it with cytidine-5'-monophosphoric acid. 4. A method for producing choline cytidine diphosphate, which comprises reacting phosphorylcholine dichloridate with morpholine or lower dialkylamine to obtain phosphorylcholine diamidates represented by the general formula [], and then reacting with cytidine-5'-monophosphoric acid. .