JPH0425959B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to phospholipid compounds, and particularly to phosphatidylcholine type phospholipid compounds (glycerophosphocholine compounds) having a polymerizable group. Currently, various attempts are being made to encapsulate medicinal substances, enzymes, etc. into minute capsules and provide them as pharmaceuticals. The early method of such microencapsulation was the encapsulation of polymer compounds by emulsification method (Japanese Patent Publication No. 45-2757 and Patent Publication No. 17950-1983).
Encapsulation accompanied by the formation of polymers (polyamide, etc.) through interfacial polycondensation reactions
7395 and Special Publication No. 53-7396). However, with these methods, the particle size is large (several μm).
It has the disadvantage that only capsules (~1000 μm) can be formed, which poses a problem in using the obtained capsules, for example, as pharmaceuticals. By the way, the main purpose of encapsulating pharmaceutical substances and enzymes in microcapsules is to retain the activity of pharmaceutical substances and enzymes that are unstable in vivo for a long period of time, and to maintain their effects for a long period of time. As a technology that satisfies these conditions to a considerable degree, we utilize the fact that various phospholipids, which are components of biological membranes, form microscopic spherical aggregates (liposomes, particle size 0.03 to 10 μm) in water. Recently, a technique has been reported in which a phospholipid compound having a polymerizable group is synthesized, and the resulting liposome is polymerized while being kept intact to obtain microcapsules. For example, the Journal of the
American Chemical Society, 104 , 791-795
(1982) (hereinafter referred to as literature), Angewante Chemie International by H-H. Hub et al.
Edition English version, 19 , 938-940 (1980) (hereinafter referred to as literature), and by A. Akimoto et al.
Angewate.Chemie International Edition English version 20 , 90-91 (1981) (hereinafter referred to as literature)
There is. The synthetic phospholipid compounds described in these documents form liposomes (or vehicles) in water. In order to microencapsulate this liposome as it is, the polymerizable groups in each compound (i.e., methacrylic group for the compound in literature (), diacetylene group in the compound in literature (), and butadiene group in the compound in literature ()) are required. group) needs to be polymerized. In order to initiate the polymerization of the compound described in the literature (), an appropriate polymerization initiator (for example, an azo initiator such as azobisisobutyronitrile, an organic peroxide initiator such as benzoyl peroxide, Polymerization can also be carried out by adding an initiator (such as potassium sulfate) or by irradiation with ultraviolet light, but in this case high energy is required and light with a shorter wavelength is required. It is said that the compounds described in documents () and () are relatively easily polymerized by irradiation with ultraviolet light. An object of the present invention is to provide a novel polymerizable phospholipid compound that has a highly photopolymerizable styrene core and forms microcapsules through polymerization. According to this invention, the general formula (Here, n is an integer from 1 to 10, and l is 13
A phosphatidylcholine-type polymerizable phospholipid compound having a styrene core represented by the formula (an integer from 21 to 21) is provided. In order to produce the phospholipid compound of the present invention represented by formula (A), first, the general formula (where n is as above), the acid chloride represented by the general formula (Here, l is as above) by condensation reaction with a glycerol derivative represented by the general formula The compound shown is obtained. In this condensation reaction, the compound of formula (B) and the compound of formula (C) are used in a molar ratio of 0.5:1 to 3:1, and an appropriate amount (for example, compound (C)
at 0°C in a suitable solvent (e.g., anhydrous tetrahydrofuran, chloroform, dichloromethane, etc.) containing a dehydrochlorination agent (e.g., pyridine) in an equimolar amount.
Do this at a temperature of 30℃ to 30℃. The reaction time is usually 1 hour to 24 hours. Next, the condensation compound of formula (D) is prepared by the method described by Von R. Hirt et al. in Pharm. Acta Helv., 33 , 349 (1958) or by the method described by NSChandrakumar et al. in Tetrehadron Letters, 23 , 1043. (1982)
The desired phospholipid compound of formula (A) can be obtained by reacting in the following steps according to the method described in . or The acid chloride represented by formula (B) is first prepared by S.Swann
Organic Synthesis, 2 , 276, by et al.
(1950) is a general formula obtained by improving the method described by L. Ruzicka et al. in Helv.Chim.Acta, 10 , 691 (1927). (Here, n is as described above, and R is a methyl group, ethyl group, propyl group, or butyl group). Condensation reaction with p-aminostyrene below gives the formula (where n and R are as described above) is obtained. This is then treated with excess alkali (e.g. sodium hydroxide, potassium hydroxide).
was hydrolyzed in an alcohol-water mixed solvent, and then acidified with hydrochloric acid etc. to obtain the formula The compound shown is obtained. When this compound is reacted with thionyl chloride or oxalyl chloride,
An acid chloride represented by formula (B) is obtained. The glycerol derivative represented by general formula (C) is D.
Arnold et al., Lieb.Ann.Chem., 703 , 234
~239 (1967), by improving the method described in
It is obtained by reacting 2-phenyl-5-m-dioxanol with an alkyl bromide or an alkyl iodide, followed by hydrolysis in ethanol with sulfuric acid. In addition, among the glycerol derivatives represented by general formula (C), l is 13, 15, 17,
Alkyl bromide, which is a raw material for synthesizing 19 or 21, is conveniently available as a commercial product at low cost. The phosphatidylcholine-type phospholipid compounds of the present invention can be prepared using common methods for obtaining liposomes from natural phospholipids (e.g., Biochimia et Biophysia Acta, 135 , by D. Papahadjopoulos et al.,
639-652 (1967), etc.), the particle size can be reduced to 0.03 in water by treating one or more of them.
In forming liposomes of ~1 μm, in addition to the phospholipid compound of the present invention, non-polymerizable phospholipids having liposome-forming ability (e.g., egg yolk lecithin, dipalmitoyl lecithin, dimyristoyl lecithin, distearoyl lecithin, etc.) and/or ) Cholesterol may also be used. In general formula (A), the larger the value of n, the easier it is to form liposomes. The thus obtained liposome dispersion aqueous solution is exposed to total ultraviolet light or ultraviolet monochromatic light (for example, 240n).
The polymerization reaction proceeds rapidly by irradiating the nanoparticles with light (m, 310 nm), producing microcapsules.
There is no need to add additives such as a polymerization initiator during this polymerization. Furthermore, the polymerizable phospholipid compound of the present invention is not decomposed by ultraviolet irradiation, so it is convenient for polymerization treatment. The microcapsules obtained from the phosphatidylcholine type phospholipid compound of this invention can be encapsulated with a pharmaceutical substance, enzyme, etc. (by polymerization in a physiologically acceptable aqueous solution containing the pharmaceutical substance, enzyme, etc.). It can be expected to be used as a medicine or as a reagent 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 (A) sn-glycerol-stearyl ether-2
Synthesis of (n=17 in general formula (C)) H. Hibbert et al., J. Amer. Chem. Soc., 62 ,
2 obtained according to the method described in 1601-1613 (1929)
-Phenyl-5-m-dioxanol ([Formula]) in Jus.Lied.Ann.Chem. by D.Arnold et al.
709, 234-239 (1967) to synthesize 2-stearyloxy-1,3-propanediol. Elemental analysis value: H12.84 (12.8) (wt%, same below) _C73.75 ( _73.3 ) However, the value in parentheses is the calculated value of _C21H44O313C -NMR spectrum δ (ppm ⁾ value : _CDCl3 ,
Tetramethylsilane (TMS) IR spectrum (KBr pellet) (cm ^-1 ): 3350,
2940, 2870, 1470, 1120, 1080, 980 (B) Synthesis of N-(p-vinylphenyl)-9-ethoxycarbonylnonanamide (n=8, R=ethyl in general formula (G)) With silica gel drying tube A flask was charged with 6.0 g (50 mmol) of p-aminostyrene, 4.8 g (60 mmol) of dry pyridine, and 50 ml of anhydrous tetrahydrofuran, and cooled on ice. While stirring this, a solution of 15.0 g (60 mmol) of 9-ethoxycarbonylnonanoic acid chloride {ClCO(CH ₂ ) ₈ COOCH ₂ CH ₃ } dissolved in 50 ml of anhydrous tetrahydrofuran was slowly lowered from the dropping funnel. . After the harvesting was completed, the ice bath was removed and the mixture was stirred at room temperature overnight. The solvent was distilled off from the filtrate obtained by filtering this reaction mixture using a water jet pump. Dissolve the residue in 200ml of chloroform, then add 2N hydrochloric acid, water, 5%
The mixture was washed with an aqueous sodium carbonate solution and then with water, and then the chloroform layer was dehydrated with anhydrous sodium sulfate. After filtration, the filtrate was evaporated, the residue was dried under reduced pressure, and then applied to a silica gel column (chloroform).
It was purified by Yield 7.0 grams. Yield 42%. Elemental analysis values (weight%): C72.34 (72.47) H8.93 (8.82) N4.12 (4.23) However, the values in parentheses are calculated values for C ₂₀ H ₂₉ NO ₃ (molecular weight 331.454). ^13C -NMR spectrum ( _CDCl3 , TMS) [Table] Mass spectrum: 331 (M ⁺ ) IR spectrum (KBr pellet): 1730 (ν _c=p , ester), 3320, 1665 cm ^-1 (amide) (C) N-(p-vinylphenyl)-9-hydroxy Carbonylnonanamide (in general formula (H),
Synthesis of n=8) Add 2N sodium hydroxide to 6.0 g (18 mmol) of the carboxylic acid ester synthesized in (B) above.
90 ml and 180 ml of ethanol were added, and the mixture was stirred in a bath at 30°C for 3 hours. After removing the solvent using a water pump, the residue was dissolved in 400 ml of water and cooled on ice. To this, 15 ml of concentrated hydrochloric acid was added, and the resulting precipitate was collected, washed with ice-cold water, and then dried under reduced pressure over phosphorus pentoxide. This was recrystallized from hot ethanol/hexane to obtain the desired compound. Yield 4.6 grams. Elemental analysis value (weight%): C70.85 (71.26) H8.37 (8.30) N4.58 (4.62) However, the values in parentheses are C ₁₈ H ₂₅ NO ₃ (molecular weight
303.401) calculated value. IR spectrum (KBr pellet): 1690 (ν _c=p ,
COOH), 3320, 1665 cm ^-1 (amide) Mass spectrum: 303 (M ⁺ ) ¹³ C-NMR spectrum (d ⁶ -dimethyl sulfoxide, TMS) [Table] (D) Synthesis of a compound of general formula (D) with n=8 and l=17 1.5 g (5 mmol) of the carboxylic acid derivative obtained in (C) above in an eggplant-shaped flask with a calcium chloride drying tube After adding 40 ml of anhydrous carbon tetrachloride to this, 1.5 ml of oxalic acid chloride and dry N,
One drop of N-dimethylformamide was added, and the mixture was stirred in a 35°C bath for 2 hours. After distilling off the solvent using a water pump, drying under reduced pressure using an oil pump,
The residue was dissolved in 25 ml of anhydrous chloroform. On the other hand, 1.72 g (5 mmol) of 2-stearyloxy-1,3-propanediol synthesized in (A) above was dissolved in 40 ml of dry chloroform and 0.7 ml of anhydrous pyridine. The solution prepared above was slowly dropped into this solution from the dropping funnel, and then stirred at room temperature overnight. The reaction solution was washed with 3N hydrochloric acid, a saturated aqueous sodium chloride solution, a 5% aqueous sodium carbonate solution, and a saturated aqueous sodium chloride solution in this order, and then the chloroform layer was collected and dehydrated with anhydrous sodium sulfate. This was filtered, the filtrate was subjected to evaporation treatment, and the residue was purified with a silica gel column (benzene/diethyl ether = 5/1) to obtain the desired glycerin derivative. Yield 0.21g. Yield 7%. Thin layer chromatography (TLC) (silica gel, benzene/diethyl ether = 3/1): Rf
= 0.12 (UV, Bromthymol Blue reagent) Elemental analysis value (wt%): C74.56 (74.36) H10.92 (10.72) N2.25 (2.22) However, the values in parentheses are C ₃₉ H ₆₇ NO ₅ Calculated value of (molecular weight 629.962). Mass spectrum: 630 {(M+H) ⁺ } IR spectrum (KBr pellet): 3340 (ν _OH ),
3320, 1665 (amide), 1735cm ^-1 (ester) ¹³ C-NMR spectrum (CDCl ₃ TMS): [Table] [Table] [Table] (E) Phosphocholination reaction (in general formula (A), n
= 8, l = 17) 0.1 g (0.16 mmol) of the glycerin derivative synthesized in (D) above was placed in a flask with a calcium chloride tube, and triethylamine was added to the flask.
0.04 ml, 3 mg of 4-dimethylaminopyridine, 6 ml of anhydrous benzene and 4 ml of anhydrous dichloromethane were added, followed by stirring and cooling on ice. To this, 2-
After adding 0.04 ml of chloro-2-oxo-1,3,2-dioxaphosphorane, the mixture was stirred at room temperature overnight. After filtration, the solvent was distilled off using a water jet pump, and 4 ml of anhydrous acetonitrile was added to the residue.
This solution was placed in a pressure-resistant glass tube, 0.5 ml of triethylamine was added, the tube was tightly stoppered, and the tube was reacted in a bath at 60° C. for 7.5 hours. Then, the solvent was distilled off,
The residue was purified using a silica column (chloroform/methanol/water = 65/25/4) to obtain the desired compound. Yield 46 mg. Yield 35%. TLC (chloroform/methanol/water = 65/
25/4) is the same as the standard (purified egg yolk lecithin)
The Rf value is shown. Elemental analysis value (weight%); N3.47 (3.52) However, the value in parentheses is the calculated value of C ₄₄ H ₇₉ N ₂ O ₈ P (molecular weight 795.090). Mass spectrum: 795 (M+H) IR spectrum (KBr pellet): 1730 (ν _c=p , ester), 3320, 1670 cm ^-1 (amide) ¹³ C-NMR spectrum (CDCl ₃ , TMS): [Table] [Table] [Table] Experimental Example 1 The phospholipid compound of the present invention obtained in Example 1 was added to water at a concentration of 3.5×10 ⁻⁵ M. This was subjected to ultrasonication under nitrogen gas (50 W, 50°C for 15 minutes), then transferred to a quartz glass cell, replaced with nitrogen gas, and sealed tightly. After leaving this in a bath at 50℃ for 1 hour, it was heated using a 30W low-pressure mercury lamp (Riko Kagaku Sangyo Co., Ltd.).
Using UVL-30), the polymerization reaction was carried out by irradiating light at a location with an optical path length of 5 cm in a bath at 50°C. The ultraviolet absorption spectra of phospholipid compounds were measured over time. As the reaction progressed, absorption in a characteristic absorption band (λ _nax 271 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. Even after storing this polymer solution at room temperature for one month, the shape of the microcapsules did not change.
It was found that the polymerized microcapsules were extremely stable compared to before polymerization. Example 2 (A) Synthesis of 2-myristyloxy-1,3-propanediol According to (A) of Example 1, 2-phenyl-5-m
-Dioxanol was synthesized by reacting it with myristyl bromide. Yield 29%. Melting point 55.5-56℃ Elemental analysis value (wt%): H12.37 (12.58) C70.56 (70.78) However, the value in parentheses is the calculated value of C ₁₇ H ₃₆ O ₃ ¹³ C−
NMR spectral δ (ppm) value (CDCl ₃ ,
TMS) [Table] Mass spectrum: M ⁺ +1 = 289 (molecular weight 288) (B) Synthesis of a compound with n = 8, l = 13 in general formula (D) The carboxylic acid derivative obtained in Example 1 (C), Using 2-myristyloxypropanediol obtained in Example 2(A), a condensation reaction was carried out according to the method of Example 1(D) to obtain the desired compound. Yield 8
%. Elemental analysis value (weight%): C73.14 (73.26) H10.20 (10.36) N2.56 (2.44) However, the value in parentheses is the calculated value of C ₃₅ H ₅₉ NO ₅ (molecular weight 573.854). Mass spectrum: 574 (M+H) IR spectrum (KBr pellet): 3340 (ν _OH ),
3320, 1665 (amide), 1750cm ^-1 (ester) ¹³ C-NMR spectrum (CDCl ₃ , TMS): [Table] [Table] (C) Phosphocholination reaction (in general formula (A), n
Synthesis of compound with =8 and l=13 According to the method of Example 1(E), the glycerin derivative obtained in Example 2(B) was phosphocholinelated. yield
40%. TLC (silica gel, chloroform/methanol/water = 65/25/4) showed the same Rf value as the standard (purified egg yolk lecithin). Elemental analysis value (weight%): N2.01 (1.90) However, the value in parentheses is the calculated value of C ₄₀ H ₇₁ N ₂ O ₈ P (molecular weight 738.982) Mass spectrum: 739 (M + H) ¹³ C-NMR spectrum ( _CDCl3 , TMS): [Table] [Table] Experimental Example 2 Using the phospholipid compound obtained in Example 2, a polymerization reaction was carried out in the same manner as in Experimental Example 1, and it was confirmed that polymerized liposomes could be obtained. Note that the obtained polymer solution remained stable and did not generate any precipitate even after being stored at room temperature for one month. Example 3 (A) N-(p-vinylphenyl)-5-ethoxycarbonylpentanamide (n in general formula (G)
=4,R=ethyl) Synthesis of N-(p -vinylphenyl)-5-ethoxycarbonylpentanamide was obtained. Yield 73%. Elemental analysis values (wt%): C69.97 (69.79) H7.71 (7.69) N4.92 (5.09) However, the values in parentheses are calculated values for C ₁₆ H ₂₁ NO ₃ (molecular weight 275.347). IR spectrum (KBr pellet): 3380,
1700cm ^-1 (amide), 1735cm ^-1 (ester C=O) Mass spectrum: 275 (M ⁺ ) ¹³ C-NMR spectrum (CDCl ₃ , TMS) [Table] (B) Synthesis of N-(p-vinylphenyl)-5-hydroxycarbonylpentanamide (n = 4 in general formula (H)) In Example 1 (C), Example 3 was used as the raw material carboxylic acid ester The desired compound (n=4 in general formula (H)) was obtained using exactly the same method except that the compound obtained in (A) (18 mol) was used.
Yield 50%. IR spectrum (KBr pellet): 3310, 1660
cm ^-1 (amide), 1690 cm ^-1 (carboxylic acid C=O) Mass spectrum: 247 (M ⁺ ) ¹ H-NMR spectrum (CDCl ₃ , TMS): 1.57
(m, 4H, [formula]) 2.25 (m, 4H, [formula]) , 5.4 (q, 2H, CH ₂ =), 6.6 (q, 1H, C=CH
-), 7.5 (q, 4H, [formula] ), 10.0 (s, 1H, -COOH), 12.0 (s, 1H, -
CONH−). Elemental analysis value (wt%): C67.22 (68.00) H6.37 (6.93) N5.29 (5.66) However, the value in parentheses is the calculated value of C ₁₄ H ₁₇ NO ₃ (molecular weight 247.293). (C) Synthesis of a compound with n=4, l=17 in general formula (D) In Example 1(D), the compound obtained in Example 3(B) (5 mmol) was used as the carboxylic acid derivative. Except for this, the desired glycerin derivative was obtained using exactly the same method. Yield 8%. Elemental analysis value (weight%): C73.01 (73.26) H10.25 (10.36) N2.28 (2.44) However, the value in parentheses is the calculated value of C ₃₅ H ₅₉ NO ₅ (molecular weight 573.855). IR spectrum (KBr pellet): 3340 cm ^-1 (ν _OH ,
3330, 1670 cm ^-1 (amide), 1735 cm ^-1 (ester C=O) Mass spectrum: 573 ((M+H) ⁺ ) ¹³ C-NMR spectrum (CDCl ₃ , TMS): [Table] (D) Phosphocholination reaction (in general formula (A), n
Synthesis of compound with = 4, l = 17) In Example 1 (E), the desired compound was synthesized using exactly the same method except that the compound obtained in Example 3 (C) (0.16 mmol) was used as the glycerin derivative. (In general formula (A), n=4, l=17 compound) was obtained. Yield 47%. TLC (chloroform/methanol/water = 65/
25/4) is the same as the standard (purified egg yolk lecithin)
The Rf value is shown. Elemental analysis value (weight%): N3.70 (3.79) However, the value in parentheses is the calculated value of C ₄₀ H ₇₁ N ₂ O ₈ P (molecular weight 738.983). Mass spectrum 739 (M+H) IR spectrum: 1730 cm ^-1 (ester C=O),
3330, 1665cm ^-1 (amide) ¹³ C-NMR spectrum (CDCl ₃ , TMS): [Table] [Table] Experimental Example 3 Using the phospholipid compound obtained in Example 3 (compound with n=4 and l=17 in general formula (A)), the same method as in Experimental Example 1 was used to stabilize the compound. It was confirmed that microcapsules can be produced.

Claims (1)

[Claims] 1. General formula (Here, n is an integer from 1 to 10, and l is 13
A phosphatidylcholine type polymerizable phospholipid compound represented by an integer from 1 to 21). The phospholipid compound according to claim 1, wherein 2l is 13, 15, 17, 19 or 21.