JPH0372234B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to phospholipid derivatives, and particularly to phospholipid derivatives having two polymerizable groups in one molecule. Various attempts have been made to encapsulate medicinal substances and enzymes in minute capsules and provide them as pharmaceuticals. The early method of microencapsulation was the encapsulation of polymer compounds by emulsification method
-2757 and Japanese Patent Publication No. 50-17950), and encapsulation accompanied by the production of a polymer (polyamide, etc.) by interfacial polycondensation reaction (Japanese Patent Publication No. 7395 and 7396, 1989). However, in these methods, for example, the toxicity of the polymer that is the encapsulating material and the organic solvent required in the synthesis process (e.g.
There are problems with toxicity due to the residual presence of dioxane, acetone, xylene, benzene) in the capsule, and the large particle size of the capsule (several μm to 1000 μm), which tends to cause problems such as blood clots. There were problems in using the capsules as medicines. By the way, the main purpose of encapsulating pharmaceutical substances and enzymes in microcapsules is to maintain the activity of pharmaceutical substances and enzymes that are unstable in vivo for a long time.
The goal is to maintain the effect for a long time. Therefore, especially when aiming to use microcapsules as a pharmaceutical product, the following conditions, including the problems of the prior art described above, are particularly important. (1) to make the microcapsule particle size even smaller (several μm or less), (2) to improve the stability of the capsule in vivo, and (3) to reduce the toxicity of the capsule material. Among these conditions, (1) and (2)
As a technology that satisfies this requirement, we utilize the fact that various phospholipids, which are components of biological membranes, form microscopic spherical aggregates (liposomes, particle size 0.05 to 10 μm) in water. Recently, a technique has been reported in which a lipid compound is synthesized, and the resulting liposome is polymerized while being kept intact to obtain microcapsules. For example, SL
Journal of the American by Regen et al.
Chemical Society, 104 , 791-795 (1982) (hereinafter referred to as literature), by H-H. Hub et al.
Angewante Chemie International Edition English version, 19 , 938-940 (1980) (hereinafter referred to as literature), Angewante Chemie by A. Akimoto et al.
International Edition English version, 20 , 90-91 (1981)
(hereinafter referred to as literature ()), and patent application 1983-
No. 140963 (hereinafter referred to as literature), etc. These synthetic phospholipid compounds form liposomes (or vehicles) in water. In order to microencapsulate this liposome as it is, the polymerizable groups in each compound (i.e.,
In the compound of literature (), a vinyl group, in the compound of literature (), a diacetylene group, and in the compound of literature (), a butadiene group) are polymerized. When liposomes made of phospholipid compounds having only one polymerizable functional group in one molecule as described in these documents are polymerized, crosslinking does not occur and a linear polymer is produced. Polymerized liposomes may be somewhat lacking in physical stability. Therefore, by adding a phospholipid compound that acts as a crosslinking agent to a phospholipid compound that has one polymerizable functional group in one molecule, crosslinking occurs in the resulting polymer, making the microcapsules physically more robust. It is desirable to That is, an object of the present invention is to provide a novel phospholipid derivative that can cause crosslinking to conventional phospholipid compounds. In order to achieve the above object, the present invention provides a novel phospholipid derivative having two polymerizable groups in one molecule. In this invention, a styrene nucleus is introduced as a polymerizable group because of its high photopolymerizability. That is, the present invention is based on the general formula (wherein n is a positive integer from 3 to 16). The phospholipid derivative of the present invention represented by the general formula (A) is first prepared by the general formula (however, n is as stated above) or general formula (C) It is obtained by reacting a compound represented by (where n is as described above) with L-α-glycerophosphocholine (as a cadmium chloride complex) (hereinafter abbreviated as GPC). This condensation is expressed as GPC and the formula
(B) or compound of formula (C) molar ratio 1:2 to 1:
25% and an appropriate amount of dehydrochlorination agent (pyridine,
4-(N,N-dimethyl)aminopyridine, etc.) in a suitable solvent (eg, anhydrous tetrahydrofuran, chloroform, dichloromethane, etc.) at a temperature of 0°C to 30°C. The reaction time is
Usually 10 to 30 hours. The acid chloride represented by formula (B) is described by S. Swan et al. in Organic Synthesis, 2, 276 (1950),
and Organic Synthesis, 3, by J.Cason.
169 (1955), the formula obtained by improving the method described in (However, as mentioned above, n is a methyl group,
An alkanedicarboxylic acid monoester monochloride represented by an ethyl group, a propyl group, or a butyl group is reacted with ethylbenzene bromide in the presence of anhydrous aluminum chloride to obtain the formula A compound represented by (where n and R are as described above) is obtained. This is then treated with excess alkali (e.g. sodium hydroxide, potassium hydroxide).
After heating and reacting in alcohol, the formula is acidified with hydrochloric acid etc. The compound shown is obtained. A compound represented by formula (B) is obtained by reacting this with thionyl chloride, oxalyl chloride, or the like. Further, the acid anhydride represented by formula (C) can be easily prepared by treating the carboxylic acid represented by formula (F) with 1/2 molar amount of dicyclohexylcarbodiimide in anhydrous chloroform or dichloromethane. The phosphatidylcholine-type phospholipid derivatives of the present invention can be obtained using general methods for obtaining liposomes from natural phospholipids (e.g., Biochimia et Biophysia Acta, 135, by D. Papahadjopoulos et al.
639-652 (1967), etc.), for example, by mixing with a phospholipid having one polymerizable group in one molecule described in the literature () or () to make a liposome, and then adding an appropriate amount of Polymerization techniques (e.g.
Corresponding partially crosslinked liposomes (particle size 0.02 to 1 μm) can be obtained by irradiation with ultraviolet light, etc.).
The molar ratio of the phospholipid compound having one polymerizable group in the molecule and the phospholipid derivative of the present invention represented by the general formula (A) in the liposome is usually 20:
1 to 1000:1, preferably 40:1 to
The ratio is 200:1. In addition, in general formula (A), the value of n is preferably larger because liposomes are more easily formed. Therefore, the microcapsules with a partially crosslinked structure obtained by adding the phosphatidylcholine type phospholipid of this invention have increased physical stability, and therefore can contain pharmaceutical substances, enzymes, etc. It is now possible to encapsulate more stably (by polymerization in a physiologically acceptable aqueous solution containing medicinal substances, enzymes, etc.) and is considered to be more useful as pharmaceuticals or as reagents for various tests such as clinical tests. . In addition, by encapsulating photographic photosensitizers, dyes, etc., it becomes a material for obtaining clearer photographs and printed images. Examples of this invention will be described below. Example 1 Synthesis of 1,2-bis[9-(p.vinylbenzoyl)nonanoyl]glycero-3-phosphocholine (acid chloride method) (A) Synthesis of p-(ethoxycarbonylnonanoyl)phenylethyl bromide S .Organic Synthesis by Swan et al., 2 , 276
Sebacic acid monoethyl ester monochloride synthesized according to the method described in (1950)
A mixture of 10.0 g (40.2 mmol), 6.2 g (33.5 mmol) of bromoethylbenzene, and 60 ml of dry nitrobenzene was stirred under ice cooling in an Erlenmeyer flask equipped with a calcium chloride drying tube, and 11.2 g (83.8 mmol) of anhydrous aluminum trichloride was added to the mixture. ) was added. The mixture was stirred in an ice bath for 4 hours and then at room temperature overnight. After cooling again in an ice bath, ice and water were added to the mixture, followed by the slow addition of 5.7 ml of concentrated hydrochloric acid. After adding appropriate amounts of methylene chloride and water and shaking, the methylene chloride layer was separated, and methylene chloride was added to the aqueous layer for further extraction. After combining the methylene chloride layers and dehydrating them with anhydrous sodium sulfate,
Methylene chloride and nitrobenzene were removed by evaporation under reduced pressure and fractionated with a silica gel column (diameter 4 cm, length 35 cm, eluent benzene) to obtain the desired product. Yield 6.6 grams (yield 49%). Mass spectrum. : M ⁺ = 396 (molecular weight of C ₂₀ H ₂₉ O ₃ Br ₁ 396.398) Silica gel TLC (benzene/ether = 20/
1): Rf = approx. 0.47 (ultraviolet absorption, coloration due to bromothymol blue) IR spectrum (cm ^-1 ) (sodium chloride pellet): 2940, 2860, 1730, 1680, 1610.1440,
1410, 1370, 1260, 1220, 1180, 1130,
1095, 1030 Elemental analysis value: C60.20 (60.5) H7.61 (7.4) However, the value in parentheses is the calculated value of C ₂₀ H ₂₉ O ₃ Br ₁ ¹³ C-NMR spectrum δ value: Carbon number δ (ppm) 1 33.1 2 39.0 3 143.9 4 128.4 5 128.8 6 135.8 7 199.8 8 38.5 9, 14 24.9, 24.3 10-13 29.3, 29.1 15 34.3 16 173.7 17 60 .1 18 14.3 (B) p-(chlorocarbonyl Synthesis of nonanoyl)styrene (B-1) 3.3 g (8.3 mmol) of p-(ethoxycarbonylnonanoyl) phenylethyl bromide obtained in (A) above was mixed with 40 ml of primary ethanol and 6 g of powdered potassium hydroxide. The mixture was heated under reflux for 2.5 hours and then cooled on ice. After filtering and drying the generated precipitate,
Dissolved in 100ml of water. Add 5 ml of concentrated hydrochloric acid to this solution.
was added, and the resulting precipitate was collected and dried under reduced pressure. This product was recrystallized from a mixture of 50 ml of hot toluene and 150 ml of petroleum ether to give the desired product (p-(hydroxycarbonylnonanoyl)styrene). Yield 1.1 grams (46% yield). ^13C -NMR spectrum (δ value):

【table】

[Table] (B-2) In a 100 ml eggplant-shaped flask with a calcium chloride drying tube, add the (p-(hydroxycarbonylnonanoyl)styrene) obtained in (B-1) above.
1.0 g (3.5 mmol) and dry benzene 25
ml and 1 drop of dimethylformamide were added and stirred. Add 1 ml of oxalic acid dichloride to this.
(11.8 mmol) was added dropwise, and after stirring at room temperature for 2 hours, the solvent was removed under reduced pressure. The residue was dried with an oil pump to give the desired chloride product, p-(chlorocarbonylnonanoyl).
I got styrene. IR spectrum: 1805cm ^-1 (νco(cocl)) (KBr
pellets) (C) 1,2-bis[9-(p-vinylbenzoyl)
Synthesis of [nonanoyl]glycero-3-phosphocholine L-α-glycerophosphocholine cadmium chloride chain (GPC) was synthesized by J.S. Chedra.
Chemistry and Physics of Lipids, Volume 4, 104
- Synthesized according to p. 108, 1970. 0.48 g (1 mmol) of GPC and 10 ml of glass beads were added to a 50 ml Erlenmeyer flask, and the mixture was cooled on ice and stirred. Add 20 mmol of p-(chlorocarbonylnonanoyl)styrene to this and 10 ml of dry chloroform.
1.1 ml (14 mmol) of dry pyridine was added. This mixture was stirred for 30 minutes under ice cooling, and then reacted overnight at room temperature. The reaction mixture was filtered through a Buchner funnel and the beads were washed with chloroform. After the filtrate was evaporated and the residue was dried under reduced pressure, 50 ml of dry acetone was added and centrifuged. The acetone layer was collected and the precipitate was treated again with 100 ml of dry acetone. Combine the acetone layer,
After evaporation, the residue was dissolved in a mixed solvent of chloroform/methanol/water/(5/4/1), passed through an Amberlite IR-45/IRC-50 (1/1) column, and eluted with 100 ml of the same mixed solvent. I let it happen. After removing the solvent from the effluent by evaporation (benzene bumping),
It was purified using a silica gel column, and the fraction containing the target portion was purified again using a silica gel column (chloroform/methanol/water = 65/25/4). The target portion was collected, the solvent was distilled off, water was removed by benzene azeotropy, and the residue was dried under reduced pressure over phosphorus pentoxide to obtain the desired product. Yield 0.15g. Yield 16
%. IR spectrum (cm ^-1 ): 2940, 2865, 1735,
1680, 1605, 1470, 1405, 1385, 1250,
1230, 1095, 1070, 995, 975, 915, 840 Elemental analysis value (weight%): N1.72 (1.76) However, the values in parentheses are C ₄₄ H ₆₄ N ₁ O ₁₀ P ₁ (molecular weight 797, 965 ) for the calculated value. ^13C -NMR spectrum ( _CDCl3 / _CD3CD ,
TMS): δ (ppm) Carbon number δ (ppm) 9′ 24.4 11′ 24.9 10′ 29.1−29.3 12′ 34.2, 34.1 8′ 38.6 6 54.3 4 59.3 1 62.8 3 63.6 5 61.6 2 70.4 1′ 116.7 4′ 126.3 5 ′ 128.5 2′ 135.9 6 ' 136.0 3' 142.0 13' 174.3, 173.1 7' 200.4 The Rf value of thin layer chromatography (silica gel, chloroform/methanol/water = 65/25/4) matched that of the standard product (Sigma egg yolk lecithin). Example 2 1,2-bis[9-(p-vinylbenzoyl)
Synthesis of [nonanoyl]glycero-3-phosphocholine (acid anhydride method) GPC0.44g (1mmol), p-hydroxycarbonylnonanoylstyrene 4.8g (5mmol), 4-
(N,N-dimethylaminomethyl)pyridine 0.24
(2 mmol), 150 ml of dry chloroform, and 100 ml of glass spheres (approximately 3 mm particle size), while stirring vigorously, add 1.24 g of dicyclohexylcarbodiimide.
After adding (6 mmol), nitrogen gas was blown into the reactor and the reactor was tightly stoppered. After stirring in the dark for two nights, the reaction mixture was filtered and the liquid was evaporated under reduced pressure. The residue was transferred to an ion exchange column (Amberlite MB-1) (solvent: chloroform/methanol/water = 4/5/
1) After the treatment, the target eluate was collected and evaporated to dryness under reduced pressure, and the residue was purified using a silica gel column (elution drying: chloroform/methanol = 1/0 ~
1/9). The target portion was collected, evaporated under reduced pressure, and then dried under reduced pressure to obtain 0.98 g (yield: 61%) of the target product. Analytical data were consistent with the compound synthesized in Example 1. Example 3 Synthesis of crosslinked liposomes 1,2-bis[9-(p-vinylbenzoyl)
Nonanoyl] Glycero-3-phosphocholine 0.016
g (0.02 mmol) and 1.5 g (2.0 mmol) of 1-[8-(p-vinylbenzoyl)nonanoyl]2-stearyl-glycero-3-phosphocholine. was dissolved in a mixed solvent of chloroform/methanol, the solvent was distilled off using a water jet pump in a one-bottle eggplant flask, and the solution was dried under reduced pressure using an oil pump. After adding 50 ml of N ₂ saturated water to this and shaking with a Vortex mixer, ultrasonic stirring treatment was performed in an N ₂ atmosphere.
A nearly uniform and transparent liposome dispersion was obtained. Collect 4 ml of this dispersion, transfer it to a quartz glass cell, and fill it with N ₂
After replacement, it was sealed tightly. This was irradiated with light using a 30W low-pressure mercury lamp in a 50°C bath at a distance of 5 cm from the lamp to perform a photopolymerization reaction. The ultraviolet absorption spectra of phospholipid compounds were measured over time. As the reaction progressed, the absorption in the characteristic absorption band (around λ _nax 260 nm) decreased, and the absorption disappeared after about 15 minutes, confirming that the polymerization was complete. When the shape of this polymer was measured using an electron microscope (Hitachi Model H-500), it was found to be microspherical with an average particle size of 0.03 μm. When an equal volume of ethanol was added to this polymer solution, its shape did not change. Example 4 1,2-bis[4-(p-vinylbenzoyl)
Synthesis of p-(hydroxycarbonylbutanoyl)-3-phosphocholine (A) Synthesis of p-(hydroxycarbonylbutanoyl)styrene 25 g of glutaric acid monoethyl ester and 15 ml of dry benzene were added to an eggplant-shaped flask equipped with a calcium chloride drying tube and stirred. After adding 20 ml of oxalic acid chloride and one drop of dry N,N-dimethylformamide to this, the mixture was stirred in a bath at 35°C for 5 hours.
The solvent was then removed using an evaporator.
The residue was subjected to vacuum distillation and a fraction with a boiling point of 110°C/2 mmHg was collected to obtain 23 grams of 4-ethoxycarbonylbutanoyl chloride. Infrared absorption spectrum (sodium chloride plate): 1800cm (ν _cpcl ),
1740 cm (ν _c = _p , ester). 200 ml of dry benzene, 24.1 g of ethylbenzene bromide and 27.0 g of 4-ethoxycarbonylbutanoyl chloride were added to an eggplant-shaped flask with a calcium chloride tube, cooled on ice, and stirred. To this, 38.5 g of anhydrous aluminum chloride was added in five portions, and the mixture was stirred at room temperature for 20 hours. After cooling on ice again, add 20ml of ice water and add 25ml of concentrated hydrochloric acid.
was dripped slowly. Then, methylene chloride
Extracted twice with 0.5 liters, the organic layer was collected and dehydrated with anhydrous sodium sulfate. After filtration, the filtrate was concentrated using an evaporator, and the residue was transferred to a silica gel column (diameter 4 cm, length 30 cm, elution solvent benzene ~
The target product was collected by fractionation and purification using benzene/diethyl ether (9/1) to obtain crude p-(ethoxycarbonylbutanoyl)phenylethyl bromide. Yield 80%. 10 grams of p-(ethoxycarbonylbutanoyl) phenylethyl bromide, 10 grams of powdered potassium hydroxide, 100 ml of ethanol, and 10 mg of hydroquinone were placed in an eggplant-shaped flask with a reflux tube, and the mixture was heated at 70°C.
The mixture was heated and stirred in a bath for 1.5 hours. Then cooled with ice,
The precipitate was collected by centrifugation (2000 rpm, 15 minutes) at 0°C and dried. To this was added 50 ml of water to dissolve it, and after filtering off insoluble matter, the filtrate was cooled on ice. To this, 10 ml of concentrated hydrochloric acid was added dropwise, and the resulting precipitate was collected by filtration, washed with ice-cold water, and then dried under reduced pressure. Further, it was recrystallized from 200 ml of hot benzene and 800 ml of petroleum ether to obtain 4.6 g (yield: 68%) of p-hydroxycarbonylbutanoyl)styrene. Elemental analysis value (weight%): C 71.53 (71.54) H
6.49 (6.47) (Values in parentheses are calculated values for C ₁₃ H ₁₄ O ₃ (molecular weight 218.252)) IR spectrum (cm ^-1 ) (potassium bromide pellets): 1700, 1675, 1630, 1600, 1560, 1450 ，
1430, 1410, 1375, 1340, 1310, 1280,
1230, 1180, 1115, 1070, 985, 925, 910,
870, 835 ¹³ C-NMR spectrum δ value: (in CDCl ₃ TMS standard) Carbon number δ (ppm) 1 117.0 2,16 137.2 3 143.6 4 127.4 5 129.5 7 201.1 8 38.5 9 20.7 10 34.0 11 177.0 Mass spectrum: 218 (M ⁺ ) (B) 1,2-bis[4-(p- vinyl benzoyl)
Synthesis of [butanoyl]glycero-3-phosphocholine 4.5 grams of p-(hydroxycarbonylbutanoyl)styrene synthesized in Example 4(A) and 30 ml of dry benzene were placed in an eggplant-shaped flask equipped with a calcium chloride drying tube and stirred. To this, 5 ml of oxalic acid chloride was added, and then one drop of dry N,N-dimethylformamide was added, and after reacting for 2 hours in a 35°C bath, the solvent was distilled off using an evaporator. The residue was dried using a vacuum pump to obtain p-(chlorocarbonylbutanoyl)styrene. In an Erlenmeyer flask with a calcium chloride drying tube,
GPC0.69g, glass beads (2-3mm diameter)
20 ml and 20 ml of dry methylene chloride were added and stirred. To this, a solution of the carboxylic acid chloride synthesized above in 20 ml of dry methylene chloride was added, and then 2.2 ml of dry pyridine was added, and the mixture was heated at room temperature for 24 hours.
Stir for hours. After post-treatment according to the method of Example 1(C), the product was purified using a silica gel column to obtain the desired product. Yield 11%. Thin layer chromatography (silica gel, chloroform/methanol/water = 65/25/4) The Rf value matched that of egg yolk lecithin (Sigma). Monospot (ultraviolet light, bromothymol blue indicator). ^13C -NMR spectrum δ value: (in CDCl ₃ , TMS
standard) Carbon number δ (ppm) 1 62.8 2 70.6 3 63.9 4 59.4 5 66.8 6 54.4 1' 116.9 2', 6' 135.9 3' 142.4 4' 126.5 5' 128.4 7' 199.3 8' 37.5 9' 19.3 10' 32.2, 33.4 11' 173.2, 172.9 Mass spectrum: 658 (M+1) Example 5 In Example 3, 1,2-bis[9-(p-
vinylbenzoyl)nonanoyl]glycero-3-
Instead of phosphocholine, 1,2-bis[4-(p
-vinylbenzoyl)butanoyl]glycero-3
- Exactly the same experiment was performed except that phosphocholine (0.02 momol) was used, and 1,2-bis[4-
It was confirmed that (p-vinylbenzoyl)butanoyl]glycero-3-phosphocholine functions as a crosslinking agent and can be used for producing stable crosslinked liposomes.

Claims (1)

[Claims] 1. General formula (where n is a positive integer from 3 to 16).