JPH04257595A - Glycerol derivative - Google Patents

Abstract

PURPOSE:To provide the subject derivatives useful as an intermediate for synthesis of isoprenoid type phospholipids capable of producing a membrane, excellent in biocompatibility and membrane flow properties with high barrier properties, and also excellent in dispersion stability and chemical stability and capable of forming an intermolecule association. CONSTITUTION:A natural diterpene, (7R,11R)-phytol of formula I is hydrogenated in the presence of platinum oxide catalyst, subsequently oxidized in the presence of ruthenium trichloride catalyst to obtain a carboxylic acid derivative. The resultant carboxylic acid derivative is then converted to its acid chloride using thionyl chloride and then made to react with 3-benzyl-sn-glycerol in the presence of diisopropylethylamine. Benzyl group is subsequently eliminated to obtain a glycerol-1,2-diester derivative represented by formula II. This derivative is then allowed to react with diphenylphosphorochloridate in the presence of diisopropylethylamine to obtain its triphosphate and the resultant triphosphate is subsequently reacted with hydrogen in the presence of platinum oxide, thus obtaining the objective glycerol derivative shown by formula III (R and R' are H or phosphoric acid-protecting group; (n) is 1-3).

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to glycerol derivatives which are important intermediates in the synthesis of novel phospholipids and which can themselves form a molecular aggregate state.

0002

BACKGROUND OF THE INVENTION It is generally known that when amphipathic molecules such as phospholipids are dispersed in water, they assume a special form of molecular assembly. Among these, liposomes are closed endoplasmic reticulum formed from a lipid bilayer membrane, and have a water layer inside. In recent years, liposomes have been used in the medical and pharmaceutical fields to hold water-soluble substances and to use them as drug carriers and diagnostic agents. Many attempts have been made to use it as a medicine (for example,
Sunamoto et al., Bioscience and Industry, Vol. 47, p. 475, 1989). Furthermore, attempts are being made to utilize liposomes in cosmetics and the like by taking advantage of their water-retaining and moisturizing effects.

[0003] When dispersing phospholipids in water and using them as liposomes or emulsions, the most important thing is whether they can be easily dispersed and a uniform dispersion can be obtained.
Another issue is whether the resulting dispersion is stable. Therefore, it goes without saying that it is extremely important to use a material with good dispersibility and stability. Taking liposomes as an example, the fluidity of the liposomal lipid bilayer membrane changes significantly depending on temperature (gel-liquid crystal phase transition). It is known that the ease of movement of molecules in a bilayer film in a gel state and in a liquid crystal state is much greater in the liquid crystal state in all cases of lateral diffusion, flip-flop, and exchange. In general, liposomes composed of hydrophobic fatty acid residues with a small number of carbon atoms or highly unsaturated lipids have high membrane fluidity, whereas liposomes that are saturated and have a relatively large number of carbon atoms have low membrane fluidity. The phase transition temperature is also generally high. Therefore, by changing the chain length and degree of unsaturation of the fatty acid residues of the lipid used, the membrane fluidity of the liposome and the closely related lipid dispersibility and membrane barrier ability can be adjusted.

[0004] For example, lipids containing unsaturated fatty acids such as egg yolk phosphatidylcholine have a low phase transition temperature, so they are in a liquid crystal state above room temperature and form a soft film. Lipids containing unsaturated fatty acids are essential for living organisms in order for biological membranes in a liquid crystal state to function as barriers, and biological membranes have acquired this property due to sudden changes in environmental factors such as temperature. This is thought to be due to the advantage that the physical properties of the film change in a buffering manner. This phenomenon is also important when liposomes are used as drug carriers; for example, when incorporating hydrophobic drugs into liposome membranes, it is better to use egg yolk phosphatidylcholine containing unsaturated fatty acids than liposomes made only of saturated fatty acids. It often has good dispersibility and high inclusion efficiency.

[0005] However, the polyunsaturated fatty acids contained in egg yolk phosphatidylcholine are susceptible to peroxidation reactions due to oxygen and have poor storage stability, which is a major drawback. Therefore, in consideration of stability, it is advantageous to use phospholipids consisting only of saturated fatty acids that are less susceptible to oxygen attack. However, even if liposomes are prepared using dimyristoyl phosphatidylcholine, a naturally occurring saturated phospholipid, as a membrane component, it is extremely difficult to retain glucose within the liposomes at temperatures above the phase transition temperature. Furthermore, it is known that even if liposomes made only of dipalmitoylphosphatidylcholine are prepared, they are unstable and readily aggregate and precipitate. In general, phospholipids containing only saturated fatty acids have a dense arrangement, especially below the phase transition temperature.
It is unrealistic to prepare liposomes using these alone because they are inflexible and tend to exclude foreign molecules and cause phase separation.

[0006] As described above, excellent biocompatibility, good membrane fluidity, and excellent dispersibility and stability are fundamentally contradictory properties, and conventionally used saturated fatty acids There has been no material that satisfies all of these requirements, whether it is a phospholipid consisting solely of phospholipids or a phospholipid containing unsaturated fatty acids.

[0007] Therefore, we conducted an investigation to find a material that satisfies all of these properties, and as a result, we found the material in the biomembranes of bacteria. Bacteria, unlike animals and plants, do not normally contain polyunsaturated fatty acids in their biological membranes. It has been 30 years since Japanese researchers first discovered that branched fatty acids (isoacids and antiisoacids) exist as the main fatty acids in bacterial lipids (S. Akashi and K.
Saito, J. Biochem. , vol. 47, p. 222, 1960). At present, more than several hundred types of bacteria are known that have branched fatty acids as the main fatty acids in biological lipids (T. Kaneda, Bacterio
l. Rev. , vol. 41, p. 391, 1977).

Using this as a model, diacylphosphatidylcholines with various branched fatty acids were recently synthesized, and their phase transition temperatures were measured. As a result, it was revealed that the phase transition temperature of lipids having branched fatty acids is lower than that of phospholipids having straight chain acids, and there is a difference of about 16 to 28°C. In other words, branched fatty acids contribute more to the fluidity of modern biological membranes than their straight-chain counterparts (
Satoshi Kaneda, Bioscience and Industry, vol. 48,
229, 1990). These branched fatty acids are considered to be desirable materials because, unlike polyunsaturated fatty acids, they are less susceptible to oxygen attack and are chemically stable. However, isoacids and antiisoacids are not universally present in nature, and since they are usually mixtures with different hydrophobic structures, it is very difficult to separate and purify them. The remaining method is chemical synthesis, but the major drawbacks are that easily available raw materials are limited and carbon chain extension from there requires too many steps, making it unsuitable for mass synthesis.

[0009] In order to solve these problems, biomembranes of archaea have recently attracted attention. In 1977, Woese et al.
This concept was proposed by comparing the base sequences of , University of Tokyo Press, 1988). All archaeal polar lipids known so far are glycerolipids with ether bonds, and their most significant feature is that their hydrocarbon chains are saturated isoprenoids with 20 or 40 carbon atoms. Saturated isoprenoids are also less susceptible to oxygen attack and are chemically stable, so if artificial lipids are designed and synthesized using these lipids as models, it is expected that materials with unique properties will be obtained.

Since chain isoprenoids are relatively easy to obtain compared to isoacids and antiisoacids, research on lipids incorporating these into the hydrophobic part has recently been reported (K. Yamauchi et al, Bi
ochim. Biophys. Acta 1003
Volume, 151 pages, 1989, K. Yamauchi
et al, J. Am. Chem. Soc. ，
Volume 112, page 3188, 1990, L. C. St
ewart et al., Chem. Phys. L
ipids vol. 54, p. 115, 1990, Yamauchi et al., Proceedings of the 1990 Spring Annual Meeting of the Chemical Society of Japan, 1793
p. 1794; Toda et al., Proceedings of the 1990 Spring Annual Meeting of the Chemical Society of Japan, p. 1793). As a result, it was found that a lipid bilayer membrane formed from isoprenoid type lipids has a low phase transition temperature and a high membrane barrier ability. However, in the molecular design of isoprenoid-type artificial lipids that have been reported so far, the method of linking the glycerol moiety and the hydrocarbon chain is an ether type, which is the same as in archaeal biomembranes, so the synthesis method is not versatile, and it is difficult to synthesize it in large quantities. A major drawback was that it was unsuitable for

[0011]

[Problems to be Solved by the Invention] As mentioned above, there has been no material that satisfies all of our requirements, neither the conventionally used phospholipids consisting only of saturated fatty acids nor the phospholipids containing unsaturated fatty acids. In particular, phospholipids with branched fatty acids such as isoacids and antiisoacids and chain isoprenoid skeletons are expected to have promising properties.
It has been extremely difficult to isolate and purify these from nature and to chemically synthesize them. Therefore, the purpose of the present invention is to provide properties that cannot be achieved with the conventionally used phospholipids consisting only of saturated fatty acids or phospholipids containing unsaturated fatty acids, that is, excellent biocompatibility, good membrane fluidity, and barrier performance of the membrane. The purpose of the present invention is to provide a versatile and important synthetic intermediate that can easily synthesize isoprenoid-type phospholipids that have high dispersibility, excellent dispersibility, and chemical stability. Another object of the present invention is to provide a useful material that can take the form of a molecular assembly by itself.

0012

[Means for Solving the Problems] The above objects have been achieved by discovering a glycerol derivative represented by the following general formula (I). General formula (I)

001
3]

[Case 2]

That is, the present invention is characterized in that the method of linking the hydrophobic part having an isoprenoid skeleton and the glycerol part is an ester bond instead of the conventionally known ether bond.

In the formula, n represents an integer from 1 to 3. That is, when n=1, the carbon skeleton of the hydrophobic part is a monoterpene, n
When = 2, it is a sesquiterpene, and when n = 3, it is a diterpene. However, when phospholipids are used in a lipid bilayer state such as liposomes, n = 3, and when phospholipids are used as micelles, it is called a diterpene. It is preferable that n=1 or 2. Furthermore, regarding the stereochemistry of the asymmetric carbon atoms present in the molecule, it may be a racemic form or an optically active form. These can be appropriately selected in consideration of the ease of obtaining the raw materials, but with regard to the stereochemistry of the branched methyl group of the isoprenoid hydrophobic part, those having the absolute configuration (R) are preferable. Such optically active isoprenoids can be produced by using naturally occurring terpenes as raw materials or by the method of Noyori et al. (J. Org. Chem., Vol. 53, 708).
Page, 1988, J. Am. Chem. Soc.
It is also possible to prepare the compound by asymmetric hydrogenation according to the method described in J.D., Volume 109, Page 1596, 1987).

R and R' represent hydrogen or a phosphoric acid protecting group. The protecting group for phosphoric acid can be selected from known groups used in the synthesis of ordinary nucleic acids and phospholipids, taking into account ease of synthesis, deprotection conditions, etc. Specifically, benzyl group, phenyl group, o-chlorophenyl group, p-chlorophenyl group, methyl group, 2,2,2-trichloroethyl group, 2,2,2-tribromoethyl group, 2-cyanoethyl group, allyl group. and cyclopropylmethyl group, among which preferred are benzyl group, phenyl group, and methyl group.

The phosphoric acid group may also form a salt with a suitable counter ion. However, it is desirable that the salt be physiologically and pharmacologically acceptable. Specific examples include ammonium salts, alkali metal salts such as sodium salts and potassium salts, and alkaline earth metal salts such as magnesium salts and calcium salts, among which sodium salts and potassium salts are particularly preferred. Conversion to such salts can be accomplished by conventional means.

The compounds of the present invention can be synthesized using known methods such as H. A review by Eibl (Chem.
Phys. Lipids, volume 26, page 405,
(1980) is effective. The synthesis method can be appropriately selected in consideration of the ease of obtaining reagents and the scale of the reaction. Specific examples of the compounds of the present invention are shown below, but the present invention is not limited thereto.

[0019]

[Chemical formula 3]

[0020] As an example, exemplified compounds I of the present invention are shown below.
A synthesis example of -1 is described below. However, the synthesis method is not limited to the one shown here, and various synthetic routes are possible. In addition, various protecting groups, solvents, and reagents are represented by commonly used abbreviations.

[0021]

[C4]

[0022]

[Example] Example 1 Synthesis of exemplified compound I-1 1)
Synthesis of Intermediate 1 The natural diterpene (7R,11R)-phytol (200 g) was dissolved in ethanol (1000 ml), platinum oxide (1 g) was added, and the reaction mixture was incubated at room temperature for 6 hours under a hydrogen atmosphere. Stirred. After the reaction, insoluble substances were removed by filtration through Celite, and the filtrate was concentrated under reduced pressure to obtain intermediate 1 ((3RS,7R,11R)-phytanol).
201 g of was obtained as an oil. IR νmax
(film) 3340 (br s), 2960
(s), 2930 (s), 2870 (s),
1465 (s), 1380 (s), 1370
(m), 1060 (s), 735 (w) cm
-1

2) Synthesis of Intermediate 2 Intermediate 1 (40 g) was mixed with carbon tetrachloride:acetonitrile:
Dissolved in a mixed solvent (700 ml) of water = 2:2:3,
Ruthenium trichloride n-hydrate (500 mg)
and sodium metaperiodate (70 g) were added, and the reaction mixture was vigorously stirred at room temperature for 4 hours. After the reaction was completed, insoluble substances were removed by filtration through Celite, the filtrate was diluted with methylene chloride, the organic layer was separated, and the aqueous layer was extracted with methylene chloride. The organic layers were combined, washed once with water, and then dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain intermediate 2 ((3RS,7R,11R)-
29 g of phytanic acid) was obtained as an oil. IR ν
max (film) 3600-2400 (br
m), 2960 (s), 2930 (s), 2
870 (s), 1715 (s), 1465 (m
), 1380 (m), 1370 (w), 13
00 (m), 940 (w) cm-1

[0024]
3) Synthesis of Intermediate 3 Thionyl chloride (18 g) was added to a solution of Intermediate 2 (30 g) in toluene (150 ml), and the reaction mixture was diluted to 40 g.
Stir for hours. After gas generation has stopped and the reaction has finished,
The solvent and excess thionyl chloride were distilled off at normal pressure. The residue was dried under reduced pressure to obtain the desired intermediate 3 ((3RS,7R,1
1R)-phytanoyl chloride) as an oil 32
I got g. IR νmax (film) 296
0 (s), 2930 (s), 2870 (s)
, 1800 (s), 1465 (s), 138
0 (s), 1370 (m), 990 (m),
825 (s) cm −1

4) Synthesis of intermediate 4 3-benzyl-sn-glycerol (5.4 g,
Literature [Synthesis 503 pages, 1985]
(prepared by the method described) and diisopropylethylamine (10 g) in methylene chloride (100 ml).
Intermediate 3 (22.1 g) in methylene chloride (50 ml
) solution was added and the reaction mixture was stirred at room temperature for 20 hours in the presence of a catalytic amount of 4-N,N-dimethylaminopyridine. The reaction mixture was washed with water and a saturated aqueous sodium chloride solution, and then dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel chromatography (eluent: hexane/ethyl acetate = 2
19 g of intermediate 4 as an oily substance
Obtained. IR νmax (film) 3030 (
w), 2960 (s), 2930 (s), 2
870 (s), 1745 (s), 1500 (w
), 1460 (s), 1380 (s), 13
70 (m), 1245 (m), 1165 (s
), 1120 (s), 1110 (sh), 7
30 (m), 695 (s) cm −1

5) Synthesis of Intermediate 5 To a solution of Intermediate 4 (10 g) in ethyl acetate (200 ml) was added 5% palladium-carbon (1 g).
The reaction mixture was stirred at room temperature for 6 hours under hydrogen atmosphere. After the reaction, insoluble substances were removed by filtration through Celite, and the filtrate was concentrated under reduced pressure to obtain 8.6 g of the desired intermediate 5 as an oil.
Obtained. IR νmax (film) 3460
(br m), 2950 (s), 2920 (s
), 2870 (s), 1745 (s), 14
65 (s), 1380 (s), 1370 (s
h), 1240 (m), 1165 (s), 1
130 (m), 1100 (w), 1045 (m
) cm −1

6) Synthesis of Intermediate 6 Intermediate 5 (5 g) and diisopropylethylamine (
To a solution of 1.15 g) in methylene chloride (50 ml) was added diphenylphosphorochloridate (2.4 g) and the reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was diluted with methylene chloride, washed with water, saturated sodium bicarbonate solution, and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain 5.2 g of Intermediate 6 as an oil. IR νmax (film) 3060 (w
), 2950 (s), 2930 (s), 28
60 (s), 1745 (s), 1595 (s
), 1490 (s), 1460 (s), 13
80 (m), 1370 (sh), 1290 (
m), 1190 (s), 1160 (m), 1
060 (m), 960 (s), 755 (s)
, 735 (m), 685 (m) cm -1

[
7) Ethyl acetate (60 ml) of synthetic intermediate 6 (5.2 g) of I-1
) Platinum oxide (100 mg) was added to the solution and the reaction mixture was stirred at room temperature under hydrogen atmosphere for 8 hours. After the reaction was completed, insoluble substances were removed by filtration through Celite, and the filtrate was concentrated under reduced pressure to obtain 4.3 g of the desired I-1 as an oil. IR
νmax (film) 3600-2000 (
br m), 2950 (s), 2920 (s)
, 2860 (s), 1745 (s), 1460
(s), 1380 (s), 1370 (sh)
, 1240 (s), 1165 (s), 106
0 (s), 1020 (s) cm-1

0029
] Next, as Example 2, a synthesis example of Compound I-2 of the present invention will be described. However, the synthesis method is not limited to that shown here. For example, there is a method in which a reaction is carried out using phosphorology (1,2,4-triazolide) as a phosphorylating agent and the active triester intermediate produced in the system is hydrolyzed (C.B. Reese et al, Tet
rahedron Lett. , 5059 pages, 1979
), and a method in which one benzyl group of the resulting phosphoric acid triester is removed by performing a reaction using dibenzyl phosphorochloridate as a phosphorylating agent (A.E. Ste.
panov and V. I. Shevets, C.
hem, Phys, Lipids, vol. 41, 21
Page, 1980), but various synthetic routes are also possible. In addition, various protecting groups, solvents, and reagents are represented by commonly used abbreviations.

[0030]

[C5]

Example 2 Synthesis of Exemplified Compound I-2 Phenylphosphorodichloridate (620 mg) was added to a solution of Intermediate 5 (2 g) described in Example 1 and triethylamine (350 mg) in methylene chloride (10 ml). was added and the reaction mixture was stirred at room temperature for 2 hours. After adding water and stirring at room temperature for 30 minutes, the mixture was diluted with methylene chloride, washed with water and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (eluent: chloroform/methanol = 9/1),
1.9 g of I-2 was obtained as an oily substance. IR ν
max (film) 3600-2400 (br
m), 3040 (w), 2950 (s), 2
920 (s), 2860 (s), 1745 (s
), 1600 (w), 1460 (s), 13
80 (s), 1370 (sh), 1240 (
s), 1160 (s), 1030 (s),
740 (s), 695 (m) cm −1

00
Example 3 Synthesis of Exemplified Compound I-3 Using farnesol, a naturally occurring sesquiterpene, as a starting material, hydrogenation, oxidation of alcohol to carboxylic acid, and acid chloride were carried out in the same manner as in Example 1. Conversion, esterification, removal of the benzyl group by hydrolysis, phosphoric acid esterification, and removal of the phenyl group by hydrolysis were performed in this order to obtain Compound I-3 as an oil. IR νmax
(film) 3600-2200 (br m),
2950 (s), 2920 (s), 2860
(s), 1745 (s), 1460 (s),
1380 (s), 1370 (sh), 1240
(s), 1165 (s), 1060 (s),
1020 (s) cm-1

Example 4
Synthesis of I-4 Using geraniol, a naturally occurring monoterpene, as a starting material, hydrogenation, oxidation of alcohol to carboxylic acid, conversion to acid chloride, esterification, and hydrogenation were carried out in the same manner as in Example 1. Removal of the benzyl group by decomposition, phosphoric acid esterification, and removal of the phenyl group by hydrolysis were performed in this order to obtain Compound I-4 as an oil. IR νmax (film) 3550-21
00 (br m), 2960 (s), 2930
(s), 2860 (s), 1745 (s), 1
460 (s), 1380 (s), 1370 (
m), 1240 (s), 1165 (s), 1
060 (s), 1020 (s) cm-1

003
4]

[Effects of the Invention] By using the compound of the present invention, the following remarkable effects can be obtained. (1) Synthesis is easy because the glycerol moiety and the hydrocarbon chain are linked by an ester bond rather than the ether type found in archaeal biomembrane lipids, which is particularly advantageous for large-scale synthesis. (2) Chain isoprenoids serving as raw materials are easier to obtain with reliable structure and stereochemistry than isoacids and antiisoacids, which is also advantageous for synthesis. (3) The compound of the present invention is an important intermediate that facilitates the synthesis of isoprenoid-type phospholipids. These phospholipids have properties that cannot be achieved with the conventionally used phospholipids consisting only of saturated fatty acids or phospholipids containing unsaturated fatty acids, namely excellent biocompatibility, good membrane fluidity, and membrane barrier ability. Furthermore, it is considered to have particularly excellent properties in terms of dispersibility and chemical stability. (4) Furthermore, the compound of the present invention is an amphipathic molecule that can itself be a component of a lipid bilayer membrane, and since it has a phosphate group, it is possible to impart a negative charge to the system. Therefore, it can be used as a material with unique properties in the fields of emulsion and biomembrane mimetic research.

Claims (1)

[Claims] 1. A glycerol derivative represented by the following general formula (I). General Formula (I) embedded image In the formula, n represents an integer of 1 to 3. R and R' represent hydrogen or a phosphoric acid protecting group. The phosphate group is intended to include a state in which it forms a salt with a suitable counter ion. Furthermore, regarding the stereochemistry of the asymmetric carbon atoms present in the molecule, it may be an optically active form or a racemic form.