JPH01258691A - Phospholipid derivative and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (m and n are 1-22). EXAMPLE:1-Octadecanoyl-2-(9-(tetrahydro-2H-pyran-2-yl)oxy) nonanoyl-3-glycero- phosphorylcholin. USE:An immune activator. PREPARATION:A 1-monoacyl-3-glycerophosphorylcholin is reacted with an aliphatic anhydride having a protecting group expressed by the formula III to provide a phospholipid derivative expressed by formula IV, which is then treated with an acid.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel pharmacologically effective phospholipid derivative and a method for producing the same.

(Prior art) There are many phospholipid compounds represented by the following structural formula: (wherein R represents an alkyl group and Rz represents an unsaturated hydrocarbon group) in the lipids of the vesicle membrane. The unsaturated hydrocarbon group R2 at the 2-position in the formula is substituted with various functional groups by an in vivo oxidation reaction, producing pharmacologically effective compounds.

(Problems to be Solved by the Invention) However, there has been no report to date on the successful production of a phospholipid compound having a functional group at the 2-position in the above formula.

An object of the present invention is to provide a novel pharmacologically effective phospholipid derivative having a functional group at the 2-position in the above formula, and a method for producing the same.

(Means for Solving the Problems) The novel pharmacologically effective phospholipid derivative of the present invention is a compound having a hydroxyl group at the 2-position as represented by the following structural formula (1).

1'' (wherein, m is 1 to 22, and n is 1 to 22).
The present invention is a compound having a specific substituent at the 2-position as represented by the following general formula: (wherein m is 1-22 and n is 1-22).

In the formulas (1) and (II) above, m is 1 to 22, and n is 1 to
22, but it lacks pharmacological effectiveness outside this range.

The phospholipid derivatives (H and (n)) of the present invention can be produced by the method described below.

That is, in the presence of a tertiary amine, 1-monoacyl-3-glycerophosphorylcholine (III) represented by the following general formula (% formula %) (where m is 1 to 22) is converted to the following general formula: (In the formula, n is 1 to 22.) By reacting with a fatty acid anhydride having a protecting group represented by the formula (II) to obtain a compound of formula (II), pyranyl The group is removed to obtain the desired phospholipid (I).

The synthetic route is shown in the figure below.

-(m) (II) (In the formula, m is 1 to 22, and n is 1 to 22.) The reaction temperature and reaction time in the first step reaction are usually 30 to
100°C for 2 to 48 hours, more preferably 40
~70°C for 2 to 8 hours. If the temperature is less than 30°C or less than 2 hours, there will be a large amount of unreacted materials, and if the temperature is more than 100°C or more than 48 hours, by-products will increase and the yield will decrease. Regarding the acid treatment in the second step, the reaction temperature and reaction time are usually 0°C to 70°C and 1 to 10 hours, more preferably 30 to 50°C and 2 to 5 hours. If the temperature is less than 0°C or less than 1 hour, there will be a large amount of unreacted substances, and if the temperature is more than 70°C or more than 10 hours, by-products will increase and the yield will decrease.

Examples of the 1-monoacyl-3-glycerophosphorylcholine represented by the general formula (I[I) include 1-propanoyl-3-glycerophosphorylcholine, 1-hexanoyl-3-glycerophosphorylcholine, and 1-nonanoyl-3
-glycerophosphorylcholine, 1-dodecanoyl-3-
Glycerophosphorylcholine, 1-tetradecanoyl-3
-Glycerophosphorylcholine, 1-hexadecanoyl-
3-glycerophosphorylcholine, 1-octadecanoyl-3-glycerophosphorylcholine, 1-eicosanoyl-3-glycerophosphorylcholine, 1-docosanoyl-
Examples include 3-glycerophosphorylcholine.

Acid anhydride (rV) can be obtained by the following method. The synthetic route is shown in the figure below.

(IV) (In the formula, k is θ~4, and n is 1~22.) The reaction temperature and reaction time in the first addition reaction are usually 0~6
0°C, 1 to 7 hours, more preferably 10 to 30 hours
°C for 1 to 3 hours. If the temperature is less than 0°C or less than 1 hour, there will be a large amount of unreacted materials, and if the temperature exceeds 60°C or more than 7 hours, the yield will decrease due to increased amount of by-products.

The alkaline hydrolysis reaction is usually carried out at 10 to 70°C and 1 to 2
4 hours, more preferably 20-50°C, 2-1
It is 0 hours. If it is less than 10°C or less than 1 hour, there will be a large amount of unreacted substances, and if it is more than 70°C or more than 24 hours, by-products will increase and the yield will decrease.

The acid anhydride reaction is usually carried out at 10 to 70°C for 1 to 24 hours, more preferably at 30 to 50°C for 3 to 12 hours. If it is less than 10℃ or less than 1 hour, there will be a lot of unreacted substances (,
When the temperature exceeds 70°C or exceeds 24 hours, the yield decreases due to increased amount of by-products.

As the acid anhydride (rV), 3-((tetrahydro-2
1(-bilan-2-yl)oxy)propionic anhydride, 6-((tetrahydro-2H-bilan-2-yl)oxy)hexanoic anhydride, 8-((tetrahydro-2H
-bilan-2-yl)oxy)octanoic anhydride, 9-
((Tetrahydro-2I]-bilan-2-yl)oxy)nonanoic anhydride, 10-((tetrahydro-2H-bilan-2-yl)oxy)decanoic anhydride, 12-((
Tetrahydro-2H-bilan-2-yl)oxy)dodecanoic anhydride, 14-((tetrahydro-2H-bilan-2-yl)oxy)tetradecanoic anhydride, 16-(
(tetrahydro-2H-bilan-2-yl)oxy)hexadecanoic anhydride, 1s-cctetrahydro-2H-
biran-2-yl)oxy)octadecanoic anhydride, 2
O-((tetrahydro-2H-bilan-2-yl)oxy)eicosanoic anhydride, 21((tetrahydro-2H
-bilan-2-yl)oxy)tocosanoic acid anhydride and the like.

Furthermore, examples of the tertiary amine used in the first step in the production method of the present invention include triethylamine, trimethylamine, pyridine, and 4-dimethylaminopyridine. Examples of acids used in the acid treatment in the second step include hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic acid, phosphoric acid, and p-toluenesulfonic acid.

After the reaction is completed, the reaction mixture is concentrated and the residue is purified by separation means such as silica gel chromatography.
Pure objects (1) and (II) can be obtained.

(Effects of the Invention) The phospholipid derivative of the present invention is a novel substance, has efficacy as an immunostimulant, and is a pharmacologically extremely useful compound.

In addition, the novel method for producing phospholipid derivatives of the present invention allows the phospholipid derivatives to be obtained in high yield and purity using a small number of steps and using easily available raw materials, and is suitable for industrial use. This is an extremely useful manufacturing method.

(Examples) Hereinafter, the present invention will be explained in more detail by giving Examples and Comparative Examples.

Example 1 9-
14.0 g of methyl hydroxynonanoate, 204 g of p-)luenesulfonic acid, 12.3 g of 3,4-dihydro-2H-bilane and 130 g of benzene were introduced, and 2
After the reaction was stirred for an hour, benzene was distilled off under reduced pressure.The reaction mixture was then subjected to silica gel column chromatography (developing solution: n-hexane: diethyl ether = 80:20).
When separated by volume ratio)), 9-((tetrahydro-
Methyl 2H-bilan-2-yl)oxy)nonanoate 12
．． 45 g (yield 61.6%) was obtained.

9-(
Methyl (tetrahydro-2H-bilan-2-yl)oxy)nonanoate 5.86 g, potassium hydroxide 2.23
After the completion of the reaction, 30 g of water and 30 g of water were introduced and stirred at room temperature for 12 hours, and the mixture was washed with diethyl ether.

The reaction solution was adjusted to pH 3 to 4 with a 2N aqueous sulfuric acid solution, extracted with diethyl ether, and concentrated. When the concentrated solution was separated using silica gel column chromatography (developing solution: n-hexane:diethyl ether-70:30 (volume ratio)), 3
4.03 g (yield 72.5%) of 9-((tetrahydro-2H-bilan-2-yl)oxy)nonanoic acid was obtained.

100mJ with stirring bar! 9 in an eggplant-shaped flask with a stopcock
-((tetrahydro-2H-bilan-2-yl)oxy)nonanoic acid 3.50 g, N,N'-dicyclohexylcarbodiimide 2.81 g, and carbon tetrachloride 60-
was introduced and allowed to react at room temperature for 15 hours. After the reaction is complete,
The precipitated white solid was removed by filtration to obtain a carbon tetrachloride solution of 9-((tetrahydro-2H-bilan-2-yl)oxy)nonanoic anhydride.

An appropriate amount of carbon tetrachloride solution was collected and carbon tetrachloride was distilled off under reduced pressure to obtain 2.48 g of 9-((tetrahydro-214-bilan-2-yl)oxy)nonanoic anhydride. To this, 1.30 g of 1-octadecanoyl-3-glycerophosphorylcholine, 0.624 g of N'-dimethylaminopyridine, and 12 g of dimethyl sulfoxide were added.
- was added and stirred at 50°C for 6 hours.

After the reaction was completed, dimethyl sulfoxide was eluted using silica gel column chromatography using acetone as a developing solution. Next, the desired 1-octadecanoyl-
2-(9-((tetrahydro-21-1-bilane-2-
1.64 g (yield: 86.5%) of yl)oxy)nonanoyl)-3-glycerophosphorylcholine was obtained.

The results of mass spectrum, infrared spectrum and elemental analysis were as follows.

Mass spectrum (m/z): 764 ((M+H
)” )1740 (C・0 stretching vibration) Elemental analysis: (calculated value): C62,91% H10,22%
N 1.83% P 4.06% (actual value): C62,12% H11,01%N
1.81% P 4.31% The group bonded to the 2nd carbon position of the phospholipid was confirmed using an enzyme that specifically decomposes the ester bond at the 2nd carbon position.

The compound 100cm synthesized by the above method was treated with snake venom phospholipase A2 (naja naja venom; S
IGMA Chew.

Corp, ) 0.3 mg, 5% CaC1z aqueous solution 0,
It was added to a mixture of 1 mg of ethyl ether and 10 mg of ethyl ether, and the mixture was stirred at room temperature overnight to react. After the reaction, the mixture was separated by silica gel thin layer chromatography (developing solution: chloroform:methanol:water=65:25:4 (volume ratio)). Seven fractions of fatty acid components with an R2 value of around 0.90 were fractionated and extracted with methanol. After methyl esterifying this fatty acid component by a conventional method, gas chromatography analysis revealed that 9-((tetrahydro-2H
-bilan-2-yl)oxy)nonanoic acid, 95.8%
Met.

From the above analysis results, 1-octadecanoyl-2-(9
It was confirmed that -((tetrahydro-2H-pyran-2-yl)oxy)nonanoyl-3-glycerophosphorylcholine was synthesized.

Example 2 10.5 g of methyl 6-hydroxyhexanoate was used instead of methyl 9-hydroxynonanoate, appropriate amounts of other reagents were used according to the molar ratio of Example 1, and the experimental method was the same as that of Example 1. However, 11.7 g of methyl 6-((tetrahydro-2H-pyran-2-yl)oxy)hexanoate (
A yield of 70.7%) was obtained.

6.37 g of methyl 6-((tetrahydro-2H-pyran-2-yl)oxy)hexanoate was used, appropriate amounts of other reagents were used according to the molar ratios of Example 1, and the experimental method was changed to Example 1.
According to the results, 4.98 g of 6-((tetrahydro-2H-pyran-2-yl)oxy)hexanoic acid (yield 83
．． 2%).

6-((Tetrahydro-2H-bilan-2-yl)oxy)hexanoic acid was converted into an acid anhydride according to Example 1.

1.10 g of 1-hexadecanoyl-3-glycerophosphorylcholine was used instead of 1-octadecanoyl-3-glycerophosphorylcholine, and the other reagents were as in Example 10.
Appropriate amounts were used according to the molar ratio. When the experimental method was carried out in the same manner as in Example 1, 1-hexadecanoyl-2-(6-(
(Tetrahydro-2H-bilan-2-yl)oxy)hexanoyl)-3-glycerophosphorylcholine 1.29
g (yield: 83.8%).

The results of mass spectrum, infrared spectrum and elemental analysis were as follows.

Mass spectrum (m/z): 694 (CM+
H)'')1740 (C=O stretching vibration) Elemental analysis: (calculated value): C60, 61% H9, 81%N
2.02% P 4.47% (actual value) i C59,71% l-110,3
1% N 1.90% P 4.19% 6-((tetrahydro-
As a result of gas chromatography analysis of 2H-bilan-2-yl)oxy)hexanoic acid according to Example 1, 9
It was 5.3%.

From the above analysis results, 1-hexadecanoyl-2-(6
It was confirmed that -((tetrahydro-2H-bilan-2-yl)oxy)hexanoyl)-3-glycerophosphorylcholine was synthesized.

Example 3 12.01 g of methyl 12-hydroxydodecanoate was used in place of methyl 9-hydroxynonanoate, and appropriate amounts of other reagents were used according to the molar ratios in Example 1.

When the experimental method was according to Example 1, 9.36 g (yield 57.1%) of methyl 12-((tetrahydro-2H-bilan-2-yl)oxy)dodecanoate was obtained. This is 7
．． 83g was used, other reagents were used in appropriate amounts according to the molar ratios in Example 1, and the experimental method was in accordance with Example 1. As a result, 12-(
6.23 g (yield: 83.3%) of (tetrahydro-2H-bilan-2-yl)oxy)dodecanoic acid was obtained. This was converted into an acid anhydride according to Example 1.

1.15 g of 1-dodecanoyl-3-glycerophosphorylcholine was used in place of 1-octadecanoyl-3-glycerophosphorylcholine, and appropriate amounts of other reagents were used according to the molar ratios in Example 10. The experimental method was carried out in the same manner as in Example 1, and 1.50 g of l-dodecanoyl-2-(12-((tetrahydro-2H-bilan-2-yl)oxy)dodecanoyl)-3-glycerophosphorylcholine was obtained.
(yield 79.4%).

The results of mass spectrum, infrared spectrum and elemental analysis were as follows.

Mass spectrum (m/z): 722 ((M+
H)” )1740 (C=0 stretching vibration) Elemental content Fr: (calculated value): C61,58% H9,99%N
1.94% P 4.30% (actual value): C61,61% B 10.51%
N 1.71% P 4.03% 12-((tetrahydro-2
As a result of analyzing H-bilan-2-yl)oxy)dodecanoic acid by gas chromatography according to Example 1, 93
．． It was 8%.

From the above analysis results, l-dodecanoyl-2-(12-
((tetrahydro-2H-bilan-2-yl)oxy)
It was confirmed that dodecanoyl-3-glycerophosphorylcholine was synthesized.

Example 4 9-(
Add 2.582 g of (tetrahydro-2H-bilan-2-yl)oxy)nonanoic anhydride, 0.647 g of 4-dimethylaminopyridine, 1.585 g of 1-octadecanoyl-3-glycerophosphorylcholine and 10 d of dimethyl sulfoxide, The reaction was carried out at 50°C for 7.5 hours.

After the reaction is complete, the reaction mixture is subjected to silica gel column chromatography (
Developing solution: acetone, followed by separation with chloroform: methanol, N, aqueous ammonia - 60:20:3 (volume ratio)), and 1-octadecanoyl-2-(9-((tetrahydro-2H-bilan-2- yl)oxy)nonanoyl)
-3-glycerophosphorylcholine 2.003g (yield 8
6.5%).

Next, 1-octadecanoyl-2-(9-((tetrahydro-
2H-bilan-2-yl)oxy)nonanoyl)-3-
0.712 g of glycerophosphorylcholine and 50% of an 85% acetic acid aqueous solution were added, and the mixture was reacted at 40°C for 3 hours.

After the reaction, the reaction mixture was concentrated and subjected to silica gel column chromatography (developing solution: chloroform:methanol:water-2:
5:1 (volume ratio)). The result l
-Octadecanoyl-2-(9-hydroxynonanoyl)-3-glycerophosphorylcholine 0.366 g (yield 57.8%) was obtained.

The results of mass spectrum, infrared spectrum and elemental analysis were as follows.

Mass spectrum (si/z): 680 ((M
+H]”) 1740 (C・0 stretching vibration) 3400 (0-H stretching vibration) Elemental analysis: (Calculated value): C61.86% 810.46%
N 2.06% P 4.57% (measured value): C62,11%)l 10.69
%N 1.98% P 4.39% The group bonded to the carbon at the 2-position was confirmed using an enzyme that specifically decomposes the ester bond at the 2-position of the 4-phospholipid.

The compound 100cm synthesized by the above method was treated with snake venom phospholipase At (naja naja venom; S
IGMA Che+w.

Corp, ) 0.3w, 5% CaClz aqueous solution 0.1
d and diethyl ether toy, and the mixture was stirred at room temperature overnight to react.

After the reaction, silica gel thin layer chromatography (developing solution;
Chloroform:methanol:acetic acid:water=so:2s:8
: 4 (volume ratio)). A spot containing fatty acid components with an R2 value of around 0.85 was fractionated and extracted with methanol. This fatty acid component was esterified by a conventional method and then analyzed by gas chromatography. As a result, the same peak as that of a compound obtained by similarly esterifying standard 9-hydroxynonanoic acid was obtained with a purity of 93.8%.

From the above analysis results, l-octadecanoyl-2-(9
It was confirmed that -hydroxynonanoyl)-3-glycerophosphorylcholine was synthesized.

Example 5 6-((tetrahydro-2H-bilan-2-yl)oxy)hexanoic anhydride 1.63 instead of 9-((tetrahydro-2H-bilan-2-yl)oxy)nonanoic anhydride Example 4, except that 0.981 g of 1-hexadecanoyl-3-glycerophosphorylcholine and 0.485 g of 4-dimethylaminopyridine were used instead of 1-octadecanoyl-3-glycerophosphorylcholine. 0.725 g (yield: 60.1%) of the target 1-hexadecanoyl-2-(6-hydroxyhexanoyl)-3-glycerophosphorylcholine was obtained by reacting under the same conditions as above and using the same operating method. .

The results of mass spectrum, infrared spectrum, and elemental analysis were as follows.

Mass spectrum (m/z): 610 ((M+
H)") 1740 (C=0 stretching vibration) 3400 (0-H stretching vibration) Elemental analysis: (calculated value): C59,11% H9,85%N
2.30% P 5.09% (actual value): C58,78% H10,03%
N 2.19% P 5.01% In addition, the group bonded to the carbon position 2 was confirmed in the same manner as in Example 4 using an enzyme that specifically decomposes the ester bond at the carbon position 2 of the phospholipid. . As a result, it was found by gas chromatography that the purity of 6-hydroxyhexanoic acid at the second position was 95.1%.

From the above analysis results, 1-hexadecanoyl-2-(6
It was confirmed that -hydroxyhexanoyl)-3-glycerophosphorylcholine was synthesized.

Example 6 1.71 g of 12-((tetrahydro-2H-bilan-2-yl)oxy)dodecanoic anhydride instead of 9-((tetrahydro-2H-bilan-2-yl)oxy)nonanoic anhydride and 1-decanoyl-3-instead of l-octadecanoyl-3-glycerophosphorylcholine.
The reaction was carried out under the same conditions as described in Example 4, except that 0.681 g of glycerophosphorylcholine and 0.353 g of 4-dimethylaminopyridine were used, and the desired 1-decanoyl-2-(12-hydroxy dodecanoyl)-3-glycerophosphorylcholine 0.435g (
A yield of 43.1%) was obtained.

The results of mass spectrum, infrared spectrum and elemental analysis were as follows.

Mass spectrum (m/z) 2610 ((M+H
)” )1740 (C・0 stretching vibration) 3400 (0-H stretching vibration) Elemental analysis: (Calculated value): C59,11% 8 9.85%
N 2.30% P 5.09% (measured value): C59.01% H9.92%N
2.50% P 4.87% When the group bonded to the carbon 2 position of the phospholipid was measured using the same method as in Example 4, the purity of 12-hydroxydodecanoic acid was 95.3%. This was confirmed by gas chromatography.

From the above analysis results, it was confirmed that 1-decanoyl-2-(12-hydroxydodecanoyl)-3-glycerophosphorylcholine was synthesized.

Comparative Example 1 A mixture of 9-hydroxynonanoic acid and N,N'-dicyclohexylcarbodiimide was used instead of 9-((tetrahydro-2H-pyran-2-yl)oxy)nonanoic anhydride. When the operation was carried out according to Example 4, 1-octadecanoyl-2-(9-hydroxynonanoyl)-3-glycerophosphorylcholine was not obtained.

Comparative Example 2 The procedure of Example 4 was followed except that 9-acetoxynonanoic anhydride was used instead of 9-((tetrahydro-2H-pyran-2-yl)oxy)nonanoic anhydride. Because it is difficult to selectively remove groups,
1-octadecanoyl-2-(9-hydroxynonanoyl)-3-glycerophosphorylcholine was not obtained.

Claims (4)

[Claims] (1) A phospholipid derivative represented by the following general formula: ▲Mathematical formula, chemical formula, table, etc. ▼(I) (In the formula, m is 1 to 22 and n is 1 to 22.) (2) A phospholipid derivative represented by the following general formula: ▲Mathematical formula, chemical formula, table, etc.▼(II) (In the formula, m is 1 to 22 and n is 1 to 22.) (3) 1-monoacyl-3-glycerophosphorylcholine represented by the following general formula ▲ Numerical formulas, chemical formulas, tables, etc. ▼ (III) (In the formula, m is 1 to 22) and the following general formula: Formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(IV) (In the formula, n is 1 to 22.) The phospholipid according to claim 2, which is reacted with a fatty acid anhydride having a protecting group represented by A method for producing a phospholipid derivative, the method comprising obtaining a derivative (II). (4) 1-monoacyl-3-glycerophosphorylcholine represented by the following general formula ▲ Numerical formulas, chemical formulas, tables, etc. ▼ (III) (In the formula, m is 1 to 22) and the following general formula: Formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(IV) (In the formula, n is 1 to 22.) The phospholipid according to claim 2, which is reacted with a fatty acid anhydride having a protecting group represented by A method for producing a phospholipid derivative, which comprises obtaining the derivative (II) and then treating it with an acid to obtain the phospholipid derivative (I) according to claim 1.