JP2665630B2 - Glycerol derivatives - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a glycerol derivative which is an important intermediate in the synthesis of a novel phospholipid and which can form a molecular assembly by itself.

[0002]

2. Description of the Related Art It is generally known that when an amphipathic molecule such as a phospholipid is dispersed in water, it takes a certain special form of molecular assembly. Among them, liposomes are closed vesicles formed from lipid bilayer membranes and have an aqueous layer inside, so in recent years, in the fields of medicine and pharmacy, these liposomes hold water-soluble substances to make drug carriers and diagnostics. There have been many attempts to use it as a drug (eg,
Sunamoto et al., Bioscience and Industry, Vol. 47,
475, 1989). Further, utilization of the liposome in cosmetics and the like utilizing the water retention and moisture retention effects of liposomes has been attempted.

The most important factor in dispersing phospholipids in water and using them as liposomes or emulsions is whether they can be easily dispersed to obtain a uniform dispersion.
It also means whether the obtained dispersion is stable. Therefore, it is undoubtedly important to use a material having good dispersibility and stability. For example, taking a liposome as an example, the fluidity of a liposome lipid bilayer changes greatly with temperature (gel-liquid crystal phase transition). It is known that the mobility of molecules in the bilayer in the gel state and the liquid crystal state is much larger in the liquid crystal state in any of the lateral diffusion, flip-flop, and exchange. In general, liposomes composed of a hydrophobic fatty acid residue having a small number of carbon atoms or a lipid having a high degree of unsaturation have a high membrane fluidity, while a saturated liposome having a relatively large number of carbon atoms has a low membrane fluidity, The phase transition temperature is also generally high. Therefore, by changing the chain length and the degree of unsaturation of the fatty acid residues of the lipid used, the membrane fluidity of the liposome, the lipid dispersibility closely related thereto, and the 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 and are in a liquid crystal state at room temperature or higher, forming a soft film. Lipids containing unsaturated fatty acids are indispensable for living organisms in order for biological membranes in the liquid crystal state to function as barriers.The biological membranes have acquired such properties because of sudden changes in environmental factors such as temperature. On the other hand, it is considered that there was an advantage that the physical properties of the film changed in a buffer manner. Such a phenomenon is also important when liposomes are used as a drug carrier.For example, when a hydrophobic drug is incorporated into a liposome membrane, egg yolk phosphatidylcholine containing unsaturated fatty acids is better than liposomes consisting only of saturated fatty acids. Dispersibility is good and encapsulation efficiency is high in many cases.

[0005] However, polyunsaturated fatty acids contained in egg yolk phosphatidylcholine are susceptible to a peroxidation reaction by oxygen, and are disadvantageous in that they have poor storage stability. Therefore, considering stability, it is advantageous to use a phospholipid consisting only of a saturated fatty acid which is not easily attacked by oxygen.
However, even if a liposome is prepared using, for example, dimyristoyl phosphatidylcholine, which is a naturally occurring saturated phospholipid, as a membrane component, it is extremely difficult to retain glucose in the liposome at a temperature higher than the phase transition temperature. It is also known that liposomes composed of dipalmitoyl phosphatidylcholine alone are unstable and aggregate and precipitate immediately. In general, the arrangement of phospholipids containing only saturated fatty acids is dense especially at or below the phase transition temperature,
It is not practical to prepare liposomes using only these substances because of their inflexibility and the tendency to exclude and phase separate foreign molecules.

[0006] The properties of excellent biocompatibility, good membrane fluidity, and excellent dispersibility and stability are fundamentally contradictory properties. Neither phospholipids consisting of solely phospholipids nor phospholipids containing unsaturated fatty acids satisfy any of these requirements.

[0007] Therefore, as a result of studying to find a material that satisfies all of these properties, we sought the material for a biological membrane of bacteria. Bacteria, unlike animals and plants, do not normally contain polyunsaturated fatty acids in biological membranes. It has been 30 years since Japanese researchers first discovered that branched fatty acids (iso- and anti-iso-acids) exist as major fatty acids in bacterial lipids (S. Akashi and K. Saito, J. Bioch).
em., 47, 222, 1960). At present, several hundred types of bacteria having branched fatty acids as major fatty acids of biological lipids are known (T. Kaneda, Bacteriol. Rev., Vol. 41, 39).
1 page, 1977).

Accordingly, using this as a model, diacylphosphatidylcholines having various branched fatty acids have recently been synthesized, and their phase transition temperatures have been measured. As a result, it was revealed that the lipid having a branched fatty acid had a lower phase transition temperature than the phospholipid having a linear acid and had a difference of about 16 to 28 ° C. In other words, branched fatty acids contribute to increasing the fluidity of biological membranes of bacteria over the corresponding linear acids (Satoshi Kaneda, Bioscience and Industry, 48
Vol. 229, 1990). These branched fatty acids are unlikely to be attacked by oxygen unlike polyunsaturated fatty acids, and are considered to be desirable materials because they are chemically stable.
However, iso-acids and anti-iso-acids are not universally present in the natural world, and since they are usually mixtures having different structures of hydrophobic parts, separation and purification are very difficult. The remaining means is chemical synthesis, but easily available raw materials are limited, and the elongation of the carbon chain therefrom takes too many steps.

In order to solve such a problem, biological membranes of archaebacteria have recently attracted attention. Archaea was Wo in 1977
This concept was proposed by ese et al. by comparing the base sequences of 16s rRNA of many organisms, and three groups of highly halophilic bacteria, sulfur-dependent highly thermophilic bacteria, and methanogens are known at present. As a book, Yoga Koga, Archaea, University of Tokyo Press, 1988). Archaeal polar lipids are all glycerolipids having an ether bond as far as known so far, and the most significant feature is that the hydrocarbon chain is a saturated isoprenoid having 20 or 40 carbon atoms. Saturated isoprenoids are also unlikely to be attacked by oxygen and are chemically stable. Therefore, it is expected that a material having unique properties can be obtained by designing and synthesizing an artificial lipid using such a lipid as a model.

Since chain isoprenoids are relatively easily available as compared with isoacids and antiisoacids, studies on lipids incorporating these in the hydrophobic part have recently been reported (K. Yamauchi et al, Biochim. Biophys. Acta
1003, 151, 1989, K. Yamauchi et al, J. Am. C
hem. Soc., 112, 3188, 1990, LC Stewartet a
l, Chem. Phys. Lipids 54, 115 pages, 1990, Yamauchi et al., Proceedings of the 1990 Chemical Society Spring Meeting, 1793
P. 1794, Toda et al., Proceedings of the Chemical Society of Japan Spring Meeting, 1990, p. 1793). As a result, it was found that the lipid bimolecular membrane formed from the isoprenoid-type lipid has a low phase transition temperature and a high membrane barrier ability. However, in the molecular design of isoprenoid-type artificial lipids reported to date, the method of linking the glycerol moiety and the hydrocarbon chain is the same ether type as that of the archaeal biomembrane, so that the synthesis method is poor in versatility and mass synthesis. It was a big drawback that it was not suitable for.

[0011]

As described above, there has been no material that satisfies all of our requirements, regardless of the conventional phospholipids composed of only saturated fatty acids or phospholipids containing unsaturated fatty acids. In particular, isoacids, phospholipids having a branched fatty acid skeleton such as antiisoic acid and a chain isoprenoid skeleton are expected to have promising properties,
These were very difficult to isolate and purify from nature and to chemically synthesize. Therefore, an object of the present invention is to achieve a property which cannot be achieved by a conventionally used phospholipid composed of only a saturated fatty acid or a phospholipid having an unsaturated fatty acid, that is, excellent biocompatibility, good membrane fluidity, and barrier property of the membrane. Another object of the present invention is to provide a versatile and important synthetic intermediate which can easily synthesize an isoprenoid-type phospholipid having high properties and excellent dispersibility and chemical stability. Another object of the present invention is to provide a useful material which can take a molecular assembly state by itself.

[0012]

The above object has been achieved by finding a glycerol derivative represented by the following general formula (I). General formula (I)

[0013]

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That is, the present invention is characterized in that the method of connecting the hydrophobic part having an isoprenoid skeleton and the glycerol part is not a conventionally known ether bond but an ester bond.

In the formula, n represents an integer of 1 to 3. That is, when n = 1, the carbon skeleton of the hydrophobic part is monoterpene,
The case of n = 2 is sesquiterpene, and the case of n = 3 is diterpene. When phospholipid is used in a lipid bilayer state such as liposome, n = 3, and when phospholipid is used as micelle. It is preferable that n = 1 or 2. Regarding the stereochemistry of the asymmetric carbon atom present in the molecule, it may be racemic or optically active.
These can be appropriately selected in consideration of the availability of the raw materials, but the stereochemistry of the branched methyl group of the hydrophobic portion of the isoprenoid is preferably of the absolute configuration (R). Such optically active isoprenoids can be prepared by using a naturally occurring terpene as a raw material or by the method of Noyori et al. (J. Org. Chem., 53, 708, 1988, J. Am. C.
hem. Soc., Vol. 109, p. 1596, 1987).

R and R 'are hydrogen or benzyl, phenyl, o-chlorophenyl, p-chlorophenyl, methyl, 2,2,2-trichloroethyl, 2-
Represents a phosphoric acid protecting group selected from a cyanoethyl group, an allyl group and a cyclopropylmethyl group. The stereochemistry of the asymmetric carbon atom present in the molecule may be optically active or racemic.

The phosphate group may form a salt with an appropriate counter ion. However, it is desirable that the salt is 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. Of these, sodium salts and potassium salts are particularly preferred. Such conversion to a salt can be carried out by conventional means.

The method for synthesizing the compounds of the present invention is described in a known manner, for example, as reviewed by H. Eibl (Chem. Phys. Lipid).
s, 26, 405, 1980) is effective. The synthesis method can be appropriately selected in consideration of the availability of reagents and the scale of the reaction. Hereinafter, specific examples of the compound of the present invention are shown, but the present invention is not limited thereto.

[0019]

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In the following, the exemplified compounds I of the present invention will be described by way of examples.
The synthesis example of -1 is described below. However, the synthesis method is not limited to the method shown here, and various other synthesis routes are possible. Various protecting groups, solvents and reagents are represented by commonly used abbreviations.

[0021]

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[0022]

EXAMPLES Example 1 Synthesis of Exemplified Compound I-1 1) Synthesis of Intermediate 1 Natural diterpene (7R, 11R) -phytol (200 g)
Is dissolved in ethanol (1000 ml) and platinum oxide (1 g)
Was added, and the reaction mixture was stirred at room temperature for 6 hours under a hydrogen atmosphere. After completion of the reaction, insoluble substances were removed by filtration through Celite, and the filtrate was concentrated under reduced pressure to obtain 201 g of Intermediate 1 ((3RS, 7R, 11R) -phytanol) as an oil. IR νmax (fil
m) 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 converted to carbon tetrachloride: acetonitrile: water =
It was dissolved in a 2: 2: 3 mixed solvent (700 ml), and ruthenium trichloride n-hydrate (500 mg) and sodium metaperiodate (70 g) were added thereto. The reaction mixture was vigorously stirred at room temperature for 4 hours. Stirred. After completion of the reaction, 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 dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to give intermediate 2
29 g of ((3RS, 7R, 11R) -phytanic acid) were obtained as an oil. IR νmax (film) 3600-2400 (br m), 2960 (s), 2
930 (s), 2870 (s), 1715 (s), 1465 (m), 1380 (m), 13
70 (w), 1300 (m), 940 (w) cm ^-1

3) Synthesis of Intermediate 3 To a solution of Intermediate 2 (30 g) in toluene (150 ml) was added thionyl chloride (18 g), and the reaction mixture was stirred for 40 hours. After the gas generation stopped and the reaction was completed, the solvent and excess thionyl chloride were distilled off at normal pressure. The residue was dried under reduced pressure to obtain 32 g of the desired intermediate 3 ((3RS, 7R, 11R) -phytanoyl chloride) as an oil. IR νmax (film) 2960
(s), 2930 (s), 2870 (s), 1800 (s), 1465 (s), 1380
(s), 1370 (m), 990 (m), 825 (s) cm ^-1

4) Synthesis of intermediate 4 3-benzyl-sn-glycerol (5.4 g, literature [Synthesi
s p. 503, 1985]) and diisopropylethylamine (10 g) in methylene chloride (100 ml).
To the solution was added intermediate 3 (22.1 g) in methylene chloride (50 ml)
The 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 solution of sodium chloride 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 purified by silica gel chromatography (eluent hexane / ethyl acetate = 20/1),
Intermediate 4 was obtained as an oil, 19 g. IR νmax (film)
3030 (w), 2960 (s), 2930 (s), 2870 (s), 1745 (s),
1500 (w), 1460 (s), 1380 (s), 1370 (m), 1245 (m),
1165 (s), 1120 (s), 1110 (sh), 730 (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), and the reaction mixture was stirred at room temperature under a hydrogen atmosphere for 6 hours. After completion of 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. IR νmax (film) 3
460 (br m), 2950 (s), 2920 (s), 2870 (s), 1745
(s), 1465 (s), 1380 (s), 1370 (sh), 1240 (m), 1165
(s), 1130 (m), 1100 (w), 1045 (m) cm ^-1

6) Synthesis of Intermediate 6 Intermediate 5 (5 g) and diisopropylethylamine (1.15)
To a solution of g) in methylene chloride (50 ml) was added diphenyl phosphorochloridate (2.4 g) and the reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, dilute with methylene chloride,
The extract was washed with water, a saturated sodium hydrogen carbonate solution and a saturated 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 to obtain 5.2 g of Intermediate 6 as an oil. IR νmax (film) 3060
(w), 2950 (s), 2930 (s), 2860 (s), 1745 (s), 1595
(s), 1490 (s), 1460 (s), 1380 (m), 1370 (sh), 1290
(m), 1190 (s), 1160 (m), 1060 (m), 960 (s), 755
(s), 735 (m), 685 (m) cm ^-1

7) Synthesis of I-1 Platinum oxide (100 mg) was added to a solution of Intermediate 6 (5.2 g) in ethyl acetate (60 ml), and the reaction mixture was stirred at room temperature for 8 hours under a hydrogen atmosphere. After completion of the reaction, 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), 146
0 (s), 1380 (s), 1370 (sh), 1240 (s), 1165 (s), 10
60 (s), 1020 (s) cm ^-1

Next, as Example 2, exemplified compound I of the present invention
-2 is described below. However, the synthesis method is not limited to the method shown here. For example, a method in which a reaction is performed using phosphorodi (1,2,4-triazolide) as a phosphorylating agent to hydrolyze an active triester intermediate formed in the system (CB Reese et al, Tetrahedron Lett., 50
59, 1979) and a method in which a reaction is carried out using dibenzyl phosphorochloridate as a phosphorylating agent to remove one benzyl group of the resulting phosphoric acid triester (AE S
tepanov and VI Shevets, Chem, Phys, Lipids, 41
Vol. 21, p. 1980), but various other synthetic routes are also possible. Various protecting groups, solvents and reagents are represented by commonly used abbreviations.

[0030]

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Example 2 Synthesis of Exemplified Compound I-2 Phenylphosphorodichloridate (620 mg) was added to a solution of the 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 a 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 subjected to silica gel chromatography (eluent: chloroform /
Purification was performed using methanol = 9/1) to obtain 1.9 g of I-2 as an oily substance. IR νmax (film) 3600-2400 (br m), 3
040 (w), 2950 (s), 2920 (s), 2860 (s), 1745 (s), 16
00 (w), 1460 (s), 1380 (s), 1370 (sh), 1240 (s), 1
160 (s), 1030 (s), 740 (s), 695 (m) cm ^-1

Example 3 Synthesis of Exemplified Compound I-3 Using farnesol, which is a naturally occurring sesquiterpene, as a starting material, hydrogenation was carried out in the same manner as in Example 1.
Oxidation of alcohols to carboxylic acids, conversion to acid chlorides,
Esterification, removal of a benzyl group by hydrogenolysis, phosphoric acid esterification, and removal of a phenyl group by hydrogenolysis were performed in this order to obtain Compound I-3 as an oil. IR ν
max (film) 3600-2200 (br m), 2950 (s), 2920 (s), 2
860 (s), 1745 (s), 1460 (s), 1380 (s), 1370 (sh), 1
240 (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, and conversion to acid chloride were performed in the same manner as in Example 1. Then, benzyl group removal by esterification and hydrogenolysis, phenyl group removal by phosphoric acid esterification and hydrogenolysis were performed in this order to obtain Compound I-4 as an oil. IR νmax
(film) 3550-2100 (br m), 2960 (s), 2930 (s), 2860
(s), 1745 (s), 1460 (s), 1380 (s), 1370 (m), 1240
(s), 1165 (s), 1060 (s), 1020 (s) cm ^-1

[0034]

The following remarkable effects can be obtained by using the compound of the present invention. (1) The method of linking the glycerol moiety and the hydrocarbon chain is not the ether type found in archaeal biomembrane lipids, but is an ester bond, which facilitates synthesis, and is particularly advantageous for mass synthesis. (2) Since a chain isoprenoid as a raw material can be easily obtained with a reliable structure and stereochemistry compared with isoacids and antiisoacids, this is advantageous for synthesis. (3) The compound of the present invention is an important intermediate capable of easily synthesizing isoprenoid type phospholipid. Such phospholipids cannot be achieved with the conventional phospholipids consisting of only saturated fatty acids or phospholipids having unsaturated fatty acids, that is, they have excellent biocompatibility, good membrane fluidity, and high barrier properties of the membrane. It is considered to have high properties, and particularly excellent properties in dispersibility and chemical stability. (4) Further, the compound of the present invention is an amphipathic molecule which can itself be a component of a lipid bimolecular membrane, and can impart a negative charge to the system because it has a phosphate group. Therefore, it can be applied as a material having unique properties in the field of emulsion and biological membrane mimetics research.

Claims (1)

(57) [Claims] 1. A glycerol derivative represented by the following general formula (I). General formula (I) In the formula, n represents an integer of 1 to 3. R and R ′ are hydrogen or a benzyl group, a phenyl group, an o-chlorophenyl group,
represents a phosphoric acid protecting group selected from a p-chlorophenyl group, a methyl group, a 2,2,2-trichloroethyl group, a 2-cyanoethyl group, an allyl group and a cyclopropylmethyl group. Phosphate groups shall include the state of forming a salt with an appropriate counter ion. The stereochemistry of the asymmetric carbon atom present in the molecule may be optically active or racemic.