JPH078879B2 - Polymerizable phospholipid compound - Google Patents

Description

TECHNICAL FIELD The present invention relates to a phospholipid compound, and more particularly to a phospholipid compound having a polymerizable group (glycerophosphocholine compound).

[Prior Art] At present, various attempts have been made to provide a drug by encapsulating a drug substance, an enzyme, or the like in a minute capsule. The initial method of such microencapsulation is encapsulation of a polymer compound by an emulsification method (Japanese Patent Publication No. 45-2757 and Japanese Patent Publication No. 50-17950) and formation of a polymer (polyamide, etc.) by an interfacial polycondensation reaction. Encapsulation with
-7395 and Japanese Patent Publication No. 53-7396). However, these methods have a large particle size (several μm to 1000 μm).
m) It has a drawback that only capsules can be formed, and there is a problem in using the obtained capsules, for example, as a medicine.

By the way, the main purpose of encapsulating a drug substance or an enzyme in a microcapsule is to retain the activity of the drug substance or enzyme which is unstable in a living body for a long time, and to maintain its effect for a long time. As a technique to satisfy such conditions to a considerable extent, it is utilized that various phospholipids, which are components of biological membranes, form minute spherical aggregates (liposomes, particle size 0.02 to 10 μm) in water. Recently, a technique for synthesizing an amphipathic lipid compound having a polymerizable group and polymerizing the liposome formed by maintaining the state (shape) of the liposome to obtain stable microcapsules has recently been reported. . For example, Journal of the American Chemical Society, 10 by SLRegen et al.
4,791-795 (1982) (hereinafter referred to as Reference (I)), H-H.Hub and others
Hemy International Edition (Angewa
ndte Chemie International Edition) English version, 19,938-
940 (1980) (hereinafter referred to as Reference (II)), and Angewandte Chemie International Edition by Akimoto et al.
Edition) English version, 20,90-91 (1981) (Reference (II
I)).

In addition, these polymerizable lipid compounds are simultaneously used as one of methods for surface modification of various materials. That is, a polymerizable functional group (diacetylene group, diene group,
A stable film is obtained by using an amphipathic lipid compound having a vinyl group, etc.), forming it as a monolayer film or a multilayer film on the surface of the material, and then crosslinking it by an appropriate polymerization method (for example, irradiation with ultraviolet rays). A method of forming the is being investigated.
For example, S. SL Regen et al., Macromolecules, 16 volumes, 355-338
Page, 1983 (hereinafter referred to as reference (IV)) and the like.

In these known compounds, a diyne group, a diene group, a vinyl group (acrylic acid, methacrylic acid, etc.) is used as the polymerizable functional group. To obtain stable microcapsules or crosslinked membranes by polymerizing liposomes (vesicles) or monolayer (multilayer) membranes composed of these compounds,
There is a method in which an appropriate compound (for example, an azo compound such as azobisisobutyronitrile, an organic peroxide such as benzoyl peroxide, potassium persulfate, etc.) is used as a polymerization initiator, or a direct ultraviolet ray is irradiated. However, in the method of adding a polymerization initiator, it is necessary to embed a component (polymerization initiator) other than the polymerizable lipid in the membrane, the operation is complicated, and further, the regulation sequence of the intramolecular molecules is disturbed. There are drawbacks such as impairing the stability of the resulting polymerized film, and as the polymerization method, a method of directly irradiating ultraviolet rays or the like is preferable. The polymerizable groups that are easily polymerized by irradiation with ultraviolet rays are a diyne group and a diene group.

[Problems to be Solved by the Invention] In any case, by irradiation of light, polymerization is more easily carried out,
There is an increasing demand for developing polymerizable lipid compounds that form microcapsules by their polymerization.

An object of the present invention is to have a photopolymerizable diene residue,
It is an object of the present invention to provide a novel polymerizable phospholipid compound which forms microcapsules or a crosslinked polymer film by polymerization.

[Means for Solving Problems] According to the present invention, the general formula (N is an integer of 6 to 12).

The phospholipid compound of the present invention represented by the formula (A) is produced, for example, by the following method.

First, the general formula (Where n is as described above) is prepared. This compound uses triethyl 4-phosphonocrotonate and alkyl aldehydes, and is disclosed in the literature (K.Dorn et al, J.
ournal of the American Chemical Society, 106, 1627
Page, 1984), for example, after reacting with anhydrous tetrahydrofuran as a solvent, Rica gel column (elution solvent: benzene / ether (20 /
1)) was used to isolate the desired trans-2, trans-4-alkanadienic acid ethyl ester (C), and then 1 to 20 equivalent moles of this compound (C) sodium hydroxide, potassium hydroxide, etc. In a water-containing solvent containing a base of (for example, alcohol (methanol or ethanol) -water mixture), 30 to
After reacting at 60 ° C. for 3 hours to 24 hours, an acid such as concentrated hydrochloric acid is excessively added to acidify the compound of the formula (B).

For example, 2 to 30 times mol of oxalyl chloride is added to the fatty acid represented by the formula (B), and the mixture is reacted at 50 to 70 ° C. for 1 to 3 hours to quantitatively obtain the corresponding acid chloride (D).

(N is already described) Compound (D) is condensed with glycerol to give a compound represented by the general formula (N is as described above) is obtained.

In this condensation reaction, glycerol and the compound of formula (D) are mixed in a suitable anhydrous solvent (eg, dichloromethane, chloroform, tetrahydrofuran, etc.) containing a dehydrochlorinating agent (quinoline, pyridine, etc.) in a molar ratio of 1: 2-1: 2. It can be synthesized by reacting 3 at 0 to 30 ° C (preferably 10 to 15 ° C) for 3 to 24 hours.

The condensation product of formula (E) is then converted by Von R. Hirt et al. Into Pharmacentica Acta Helv., 33,349 (1958).
Or by NS Chandrakumar et al., Tetrahedron Letters 23,1043 (1982).
The target phospholipid compound of formula (A) is obtained by reacting in the following steps according to the method described in (1).

Or The phosphocholine-type phospholipid compound of the present invention can be obtained by a general method for obtaining liposomes from natural phospholipids (for example, D. Papahadjopoulo).
s) et al., Biochimica et Biophysica Acta, 135, 639-652.
(See (1967)) and the like, liposomes having a particle size of 0.02 to 10 μm can be formed in water by treating one kind or two or more kinds. In forming the liposome, a non-polymerizable phospholipid capable of forming a liposome together with the phospholipid compound of the present invention (eg, egg yolk lecithin, dipalmitoyl lecithin, dimyristoyl lecithin, distearoyl lecithin, phosphatidyl ethanolamine, phosphatidyl serine). , Phosphatidic acid,
Dicetyl phosphate, etc.) and / or cholesterol may be used.

Further, in the compound of formula (A), the larger the value of n, the easier the formation of stable liposomes. The liposome-dispersed aqueous solution thus obtained is subjected to UV all-light or UV monochromatic light (for example, 240 n
Irradiation with (m, 310 nm) rapidly advances the polymerization reaction and produces stable microcapsules. At the time of thermal polymerization, polymerization may be carried out using various general polymerization initiators.

In addition, the phosphocholine-type phospholipid compound of the present invention can be prepared by a general method (for example, SLRege (SLRege).
n) and others, Macromolecules, No. 1
Vol. 6, pp. 335-338 (see 1983), and after arranging them in a monomolecular form on the surface of the material to be modified (for example, plastic material), induce a polymerization reaction by a method such as UV irradiation,
A stable polymer film can be formed on the surface of the material to be modified.

The microcapsules obtained from the phosphocholine-type phospholipid compound of the present invention can be used as a drug by encapsulating a drug substance, an enzyme, etc. (by polymerization in a physiologically acceptable aqueous solution containing the drug substance, enzyme, etc.). ,
Alternatively, it can be expected as a reagent for various tests such as clinical tests.
Further, by encapsulating a photographic sensitizer or the like, a dye or the like, it also serves as a material for obtaining a clearer photograph or printed image. It can also be used as a surface modifier for photoresist materials and various materials.

[Examples] Examples will be described below.

Synthesis Examples A to D Synthesis of 2,4-alkanadienoic acid (general formula (B)) (Synthesis Example A) Sodium hydride (60% in oil, 710 mmol) and anhydrous tetrahydrofuran were placed in a silica gel blue drying tube flask. 1 was charged and cooled to 0 ° C. With stirring, a solution of 177.5 g (710 mmol) of triethylphosphonocrotonate dissolved in anhydrous tetrahydrofuran 0.1 was added dropwise over 1 hour, and then the mixture was stirred at 0 ° C. for 1 hour. A solution of tetradecyl aldehyde (947 mmol) in anhydrous tetrahydrofuran 0.5 was added dropwise to this over 1 hour, and then 0
The mixture was stirred at 0 ° C for 1 hour and further at 30 ° C for 1 hour. After recooling to 0 ° C., water 2 and saturated saline solution 2 were added. The aqueous layer was extracted with ether / hexane (1.2 / 1.2) and then with hexane (2). The organic layers were combined and dehydrated with anhydrous sodium sulfate. After filtration, the solvent was distilled off, and the residue was purified with a silica gel column (eluent: n-hexane / ether (15/1)), and the target part (thin layer chromatography, silica gel) n-hexane / ether = 20 / 1, Rf
= 0.23). 59.2 g (yield 19%) of trans-2, trans-4-octadecadienoic acid ethyl ester was obtained. IR (n
eat): 1720 (υ _{C = O} , ester), 1615,1640cm
^-1 (υ _{C = C} , diene).

Ethanol 0.25, potassium hydroxide (17.5g) / water 0.15 were added to the ester 53.4gr obtained above, and the mixture was mixed in a bath at 50 ° C for 5
Stir for hours.

Water 0.8 was added to the residue obtained by distilling off the solvent with a water pump. Furthermore, after adding 20 ml of concentrated hydrochloric acid, the mixture was thoroughly stirred. The formed precipitate was collected by filtration, washed thoroughly with water, and then dried under reduced pressure. This was recrystallized from n-hexane 0.7 to obtain the desired trans-2, trans-4-octadecadienoic acid 40.6.
g (84% yield) was obtained. mp.68 ℃; IR (KBr): 1980
(Υ _{C = O} , −COOH), 1610,1635mm ⁻¹ (υ _{C = C} , diene); E
I mass spectrum: 280 (M ⁺⁾ (MW280 of _{C 18 H 32 O 2);} 13 C
-NMR spectrum (CDCl _3, tetramethylsilane): Carbon number δ (ppm) 1 10.9 2 19.5 12 25.5 4 to 11 26.4 to 26.0 3 28.7 13 29.9 17 115.0 15 125.0 14,16 143.1,144.4 18 169.5 (Synthesis examples B to D) Tetradecyl aldehyde was prepared using the same method. Instead of dodecyl aldehyde, decyl aldehyde or octyl aldehyde, the corresponding trans-2, trans-4-alkanadienic acid was obtained. The analysis results are shown in the table.

Each compound showed almost the same ¹³ C-NMR spectrum and IR spectrum as 2,4-octadecadienoic acid (Synthesis Example A).

Synthesis Examples 1 to 4 Synthesis of 1,3-bis (2,4-arnacadienoyl) glycerol (general formula (E)) (Synthesis Example 1) 40.6 g (145 mmol) of 2,4-octadecadienoic acid The mixture was placed in a flask with a calcium chloride tube, 290 ml (381 mmol) of oxalic acid chloride was added, and the mixture was stirred in a 60 ° C bath for 2.5 hours. This was concentrated under reduced pressure, and the residual oil was dried with a vacuum pump.

Separately, 6.67 g (72.5 mmol) of anhydrous glycerol and 36.3 ml of dehydrated quinoline were dissolved in 15 ml of anhydrous chloroform in a flask with a calcium chloride tube, and while cooling in a cold bath (13 to 15 ° C), the above 2 A solution of 4,4-octadecadienoic acid chloride in 70 ml of anhydrous chloroform was slowly added dropwise over 1 hour. After the dropping was completed, the mixture was stirred overnight at room temperature in a dark place, 2.5 liters of ether was added, and this was washed with water (8
(00ml x 1 time). The ether layer was collected. Water in the water layer 0.3
After adding, the mixture was extracted with chloroform (0.5 × 1 time). The organic layers were combined and dried over anhydrous magnesium sulfate.
The filtered filtrate was concentrated under reduced pressure, and the residue was purified using a silica gel column (7 liters). As the eluent, n-
Elution fractions having a Rf = 0.27 component were collected by thin layer chromatography (silica gel, hexane / ether = 1/1) using a hexane / diethyl ether (3/1) mixture. The residue obtained by evaporating the solvent under reduced pressure with an evaporator was recrystallized from n-hexane 0.2 to obtain 16.5 g of the desired product (1,3-bis (2,4-octadecadienoyl) glycerol).
(Yield 37%) was obtained. mp.69-70 ℃; EI mass spectrum: 6
16: (M ⁺⁾ (MW616 of _{C 39 H 68 O 5);} IR (KBr): 3450
(Υ _OH ), 1710 (υ _{C = O} , −COOH), 1610,1635 cm ⁻¹ (−C
H = CHCH = CH-); Elemental analysis value (wt%): C76.07 (75.9
2) H11.79 (11.11) (However, the value in parentheses is C ₃₉ H ₆₈ O
₅ (calculated for MW616.963); ¹³ C-NMR spectrum (CDCl
₃ , tetramethylsilane): Carbon number δ (ppm) 1 14.1 2 22.7 12 28.7 4 to 11 29.7 to 29.2 13 33.1 3 31.9 a, b 65.2,68.5 17 118.2 15 128.2 14,16 145.6,146.2 18 167.3 (Synthesis examples 2 to 4) Similar method Instead of 2,4-octadecadienoic acid, 2,4-hexadecadienoic acid, 2,4-tetradecadienoic acid, and 2,4-dodecadienoic acid were used, respectively, to give the corresponding 1,3-bis (2,4-Arcanadienoyl) glycerol was obtained.
The results are shown in the table below.

In addition, each compound exhibited a ¹³ C-NMR spectrum (CDCl ₃ , tetramethylsilane) which was almost the same as 1,3-bis (2,4-octadecadienoyl) glycerol (Synthesis Example 1).

Examples 1 to 4, 1,3-bis (2,4-alcanadienoyl) -glycero-2
-Synthesis of phosphocholine (general formula (A)) (Example 1) 16.3 g (26.5) of 1,3-diacylglycerol obtained in Synthesis Example 1
eggplant type with calcium chloride tube and dropping funnel
Collect in a flask, 380 ml of anhydrous benzene, dry triethyl
Ruamine (3.21 g, 32 mmol) was added and dissolved. 10 in the cold bath
After cooling to 0 ° C, the mixture was stirred. To this, 2-chloro-2-
Oxo-1,3,2-dioxaphosphorane 4.56 g (32 mmol)
Was dissolved in 20 ml of anhydrous benzene and added dropwise over 30 minutes.
After the N₂Gas was introduced and the inside of the flask was replaced with nitrogen gas.
After that, the mixture was stirred overnight at room temperature in the dark. Insoluble matter is filtered off and filtered.
The residue obtained by concentrating the solution under reduced pressure was dried with a vacuum pump for 2 hours.
Dried This is anhydrous benzene / anhydrous acetonitrile
Dissolve in (150ml / 150ml), then put in stainless pressure tube
I see. To this, 100 ml of dry trimethylamine was added
Then, it was sealed. After reacting in a 60 ℃ bath for 4.5 hours, ice
Chilled Pour this into dry acetone 2 and stir well
After that, the precipitate was collected and dried once under reduced pressure. This
Add this to 0.2 of a methanol / chloroform (1/1) mixture.
After dissolution, mixed ionic resin (Bio Rad Laboratories, A
G Passed through column with 501-X8 (D)) 0.5. Mosquito
After washing the rum with the same solvent 1.5, the eluate was concentrated under reduced pressure.
Contracted. The residue was once dried under reduced pressure, and then dried with anhydrous water.
Freeze-drying from 0.15 to obtain the desired 1,3
-Bis (2,4-octadecadienoyl) -glycero-3
13.2 g (64% yield) of phosphocholine were obtained.

Thin layer chromatography (silica gel, chloroform /
Rf values for methanol / water (64/25/4), I ₂ and UV)
It was in agreement with the value of the commercial preparation 1,3-dipalmitoyl-glycero-2-phosphocholine (Larodan Fine Chemicals AB.).

FAB mass spectrum: 782 [M + 1] ⁺ (C ₄₄ H ₈₀ N ₁ O ₈ P ₁ M
W781); IR (KBr): 1700 (υ _{C = O} , ester), 1610,163
5cm ^-1 (υ _{C = C} , diene); Elemental analysis value (% by weight): N1.69
(1.79) (However, the values in parentheses are C ₄₄ H ₈₀ N ₁ O ₈ P ₁ (MW782.
107) represents the calculated value); ¹³ C-NMR spectrum (CDCl ₃ , tetramethylsilane); Carbon number δ (ppm) 1 14.1 2 22.7 12 28.8 4 to 11 29.7 to 29.4 3 31.9 13 33.1 f 54.3 d 59.5 a, c 63.3 e 66.4 b 70.5 17 118.6 15 128.2 14,16 145.9,145.4 18 166.8 (Example 2) ~ 4) Using the same method, in place of 1,3-bis (2,4-octadecadienoyl) glycerol,
The corresponding 1,3-diacyl-glycero-2-phosphocholine was obtained using 3-diacylglycerol. The results are shown in the table below.

In addition, the ¹³ C-NMR spectrum (CDCl ₃ , tetramethylsilane) of each compound almost agrees with the spectrum of 1,3-bis (2,4-octadecadienoyl) glycero-3-phosphocholine (Example 1). did.

[Effects of the Invention] As described above, according to the present invention, a polymerizable phospholipid compound having a diene residue having high photopolymerizability and forming a microcapsule or a crosslinked polymer film by polymerization is provided. Since this compound has a good molecular symmetry and the arrangement of the polymer chains in the double-layer film is uniform, there is no difference in the polymerization reactivity of the diene groups and efficient polymerization occurs, and the crosslinking reaction is easy and a more stable film is formed. It has the advantage that it can be formed.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12N 11/02

Claims (1)

[Claims] 1. A general formula (Here, n is an integer of 6 to 12) A polymerizable phospholipid compound represented by: