JPS63139186A - Triple-chain phospholipid compound and production thereof - Google Patents

Abstract

NEW MATERIAL:The compound of formula I [R is alkyl, alkenyl or alkadienyl; X is -O- or -OCH2CH2N<+>(CH3)3]. EXAMPLE:Tris(2,4-decadienoyl)pentaerythritol phosphate.2Na salt. USE:A raw material for liposome useful as a surfactant and a drug-including and carrying material. PREPARATION:The compound of formula III can be produced by reacting triacylpentaerythritol of formula II (e.g. tridecanoylpentaerythritol) with phosphorus oxychloride in the presence of a base and reacting the reaction product with a mixed aqueous solution of sodium bicarbonate and ethylenediaminetetraacetic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a novel synthetic phospholipid compound having three fatty acid derivatives.

[Prior Art and Problems] Natural phospholipids are widely distributed in animals and plants, and a wide variety of components are known. For example, phospholipids with high content include phosphatidylcholine, phosphatidic acid, and phosphatidylethanolamine. It is common practice to isolate these by extraction and use them as natural surfactants. In addition, in recent years, it has been discovered that these phospholipids form endoplasmic reticulum consisting of a phospholipid double membrane, so-called liposomes, and various attempts have been made to encapsulate medicinal substances, enzymes, etc. into these liposomes and provide them as medicines.
It is very useful in that it can encapsulate either water-soluble or fat-soluble substances. However, liposome aqueous solutions made using these natural phospholipids still have problems such as poor storage stability or enzymatic decomposition by phospholipase etc. when administered in vivo, resulting in poor stability of liposome particles. . Furthermore, naturally occurring phospholipids have heterogeneous constituent fatty acids and must be used as a mixture, and natural phospholipids with a uniform composition must be obtained by complex synthesis (for example, HoE ibl, “Re5ea
rch Monographs in Ce1l a
ndT 1ssue physiology 7,
L iposomes: fromphysical
5structure to therapeutic
applicati-ons, editor C,G
, Kniaht ”DDl 9-47゜ELSEV
IER/NORTH-HOLLAND BIOMEDIC
AL PRESS (AMST-ERDAM) (198
1).

Therefore, an object of the present invention is to provide a novel compound that does not exist in nature but can be used as a surfactant like ordinary phospholipids and can form liposomes.

[Means for Solving the Problems] The compound of the present invention is a phospholipid type compound having three fatty acid chains that can be synthesized by a simple method using easily available pentaerythritol as a raw material. Although the compounds of the present invention are structurally different from natural glycerophospholipids, they function in exactly the same way, including the ability to form liposomes. Furthermore, in the case of containing an alkagenyl group, stabilization in terms of both physical properties and enzyme resistance is expected by polymerization in a liposome state in the presence of ultraviolet light or a radical initiator.

According to the present invention, there is provided a phospholipid type compound represented by the general formula (wherein R is an alkyl group, alkenyl group, or alcalgenyl group, and X is 10- or 100H20H2N (CH:l)3). Ru. The compound where X is 10- is a natural phosphatidic acid, and the compound where X is 10-CH
The gold compound represented by 2CH2N (CH3)3 can be said to be a synthetic phospholipid similar to natural phosphatidylcholine.

A compound similar to phosazic acid with the general formula (where R
(as described above), firstly, pentaerythritol is dissolved in a mixed solvent of lysine or pyridine and N,N-dimethylformamide.
Monotrityl pentaerythritol ((
HOCH2)E C-CH20CPh yo),
3 or more equivalents of pyridine or 4 in an aprotic organic solvent (e.g. dichloromethane, chloroform, N,N-dimethylformamide, tetrahydrofuran, acetone, etc.)
A condensation reaction is performed at room temperature for 5 to 24 hours using 3 or more equivalents of the carboxylic acid represented by R-COOH and 3 or more equivalents of N,N'-dicyclohexylcarbodiimide in the presence of -N,N-dimethylaminopyridine. Alternatively, by reacting with an acid chloride represented by 3 equivalents or more of R-COCf, a triester body represented by the general formula % (where R is as described above) is obtained. Carboxylic acid (R-COOH) used here
is a fatty acid that usually exists as a fatty acid residue component of natural phospholipids, such as saturated fatty acids having 10 to 22 carbon atoms or linoleic acid, linuric acid, having unsaturated double bonds,
In addition to using oleic acid, arachidonic acid, etc., synthetic fatty acid compounds having conjugated double bonds may also be used. All naturally occurring fatty acid derivatives are commercially available. Fatty acids with conjugated double bonds can be synthesized by various methods. For example, α,β-unsaturated aldehyde and diethylphosphonoacetic acid ethyl ester are synthesized by 1,8-diazabicyclo[5,4, 0] 2,4-Alkanedienoic acid ethyl ester obtained by condensing -7-undecene (hereinafter abbreviated as DBU) as a base at room temperature is hydrolyzed with an aqueous alkali hydroxide solution at room temperature to 50°C, and then neutralized. How to get (
route (A)) and 2,4-alkanedienoic acids obtained by condensing a saturated aldehyde and 4-diethylphosphonocrotonic acid ethyl ester at 0°C to 40°C using sodium hydride in tetrahydrofuran or dimethoxyethane as a base. A method of obtaining it by hydrolyzing and neutralizing ethyl ester (route (B)) can be used.

CH3(CH2)mCH=CH(-CF(mCT(-C
OOH route (A) CH3 (CH2) mCHO+ (Et
o) 2P-CH2CF(-CH-COOEt-1→1
)OH- CH3(CH2)mCT-1=CH-CH=CH-CO
OEt ICH3(CH2)mCH=CH-CH=CH
-COOH route (B) Detritylation of the resulting compound represented by general formula (II) is carried out by adding a methanol solution of 1 to 3 equivalents of boron trifluoride in chloroform or dichloromethane at 0°C to room temperature for 3 to 24 hours. This can be carried out by stirring to obtain an alcohol compound represented by the general formula (where R is as described above). This compound is dissolved in a solvent such as dichloromethane, trichloroethylene or chloroform, and added to a solution of 1 to 4 equivalents of triethylamine and the same amount of phosphorus oxychloride in the same solvent, stirred and reacted at room temperature for 5 to 24 hours, and then the solvent is distilled off. Then, tetrahydrofuran was added and dissolved, and a sodium hydrogen carbonate aqueous solution (0.5M) and an ethylenediaminetetraacetic acid aqueous solution (0.5M, IHlo, 5) were added.
After adding a 5:1 mixed solution of and stirring at room temperature for 3 to 10 hours, the tetrahydrofuran layer was separated, collected, and dried under reduced pressure. Add acetone to the solution extracted with chloroform and recrystallize, or chloroform:methanol:water-6
The desired compound represented by the general formula (Ia) can be obtained by purification by silica gel column chromatography using 5:25:4 as a solvent. In this case, the phosphate residue is 2
Exists as sodium salt.

On the other hand, in order to produce a phospholipid represented by the general formula (where R is as described above), which is a compound similar to phosphatidylcholine, an alcohol compound represented by the general formula (I[r) is used in a solvent such as dichloromethane, trichloroethylene or chloroform. In the presence of 1 to 4 equivalents of triethylamine, 1 to 4 equivalents of 2-chloro-2-oxo-1,3,2-
After reacting the dioxaphosphorane at room temperature for 5 to 24 hours, the solvent was distilled off and then acetonitrile or N,N-
Add and dissolve dimethylformamide, add 3 to 100 times the equivalent of trimethylamine, and incubate at 60 to 80°C in a sealed tube for 2 hours.
Allow to react for 4-48 hours. After distilling off the solvent, the residue is dissolved in methanol and purified by passing it through a column of a mixture of cation and anion exchange resin (for example, Amberlite MB-1) using methanol or a mixed solvent of methanol:chloroform:water.

The eluate was dried under reduced pressure, and either recrystallized with a mixed solvent of chloroform-acetone or subjected to silica gel column chromatography (solvent: chloroform: methanol: water = 65
: 25 : 2-4) to obtain the general formula (I
The compound b') is obtained.

Differential scanning calorimetry shows that when the triple-stranded phospholipid represented by the general formula (1) obtained in this way is dispersed in an aqueous medium in the same way as normal phospholipids, it becomes a dispersion solution with a lipid bilayer structure. (DSC). Further, the compound of the present invention can be dispersed in an aqueous medium and then vigorously shaken using a portex mixer or treated with an ultrasonic homogenizer to obtain a homogeneous solution exhibiting a transparent or pale milky white color. This solution is a solution of endoplasmic reticulum with a lipid bilayer structure, so-called liposomes. The particle size was determined by transmission electron microscopy, laser particle analyzer (Coulter Electronics (USA), Model N4), and gel filtration chromatography. This was confirmed through measurements. In other words, any of the phospholipid compounds of the present invention can be used as a drug carrier by forming liposomes in the same way as ordinary natural or synthetic diacylglycerophosphatidic acid or diacylglycerophosphatidylcholine, and by encapsulating drugs, enzymes, etc. in these liposomes. It shows. Furthermore, in the compound represented by the general formula (■), R is -CH=CH-
CH=CH(CH2)m-CH3 (where m is 4-16
A liposome-dispersed aqueous solution of a phospholipid compound (an integer of
~80°C), the polymerized liposome solution is rapidly polymerized and becomes a polymerized liposome solution. This polymerization further enhances and stabilizes the mechanical and physical strength of the liposome. In addition, the phospholipid compound of the present invention has a structure different from that of naturally occurring glycerophospholipids, and when administered in the form of liposomes, it is thought that it is unlikely to become a substrate for phospholipase, which is a phospholipid degrading enzyme in the body. It is expected to have improved in vivo stability compared to liposomes made of lipids. In view of the above, the compound of the present invention is a completely new type of phospholipid, and can be used as a surfactant to help disperse drugs in water, or as a raw material for liposomes that serve as drug-embedded carriers. be.

[Examples] Examples of the compounds of the present invention will be shown below, but these are not intended to limit the present invention. When displaying instrumental analysis values, the following abbreviations are used. Melting point: mp, Electron ionization mass spectrum: EIMS, Fast atomic bombardment mass spectrum: FABMS, Infrared absorption spectrum: ■R, Proton nuclear magnetic resonance spectrum: I
H-NMR113C-Nuclear magnetic resonance spectrum: 13
C-NMR0 Synthesis Example 1 Synthesis of 2,4-alkashenoic acid by route (A) Diethylphosphonoacetic acid ethyl ester 36.32g (0
, 162 mol) in anhydrous acetonitrile (0.4 Jl
20.55 g (0,135 mol) of 1,8-diazabicyclo[5,4,0]-7-undecene and 87 g (0,162 mol) of lithium chloride e were added to the mixture and stirred at room temperature for 10 minutes. trans-2-octenal 170 (0,
After reacting at room temperature for 16 hours, 0.61 mol of 5% hydrochloric acid was added, extracted with ether (300 mf!,
After washing with 00m), it was dried with anhydrous magnesium sulfate.

The solvent was distilled off under reduced pressure, and the remaining yellow oil was purified by silica gel column chromatography (silica gel 60 (Nia O~230 mesh manufactured by Merck, solvent n-hexane:ethyl ether-15:1) to obtain ethyl 2.4-decanedienoate. The ester was obtained in a yield of 11.0 (3 g (42% yield)).

TLC<silica gel 60SRf = 0.47 (petroleum ether: ethyl ether = 9:1)). 10.5o (53.5 mmol) of the obtained ethyl ester was dissolved in a mixed solution of 5.29 (l) of potassium hydroxide in an aqueous solution (50m) and ethanol (100d) for 12 hours at room temperature and then reacted for 1 hour at 50°C. , neutralized with concentrated hydrochloric acid to pH 2.0, cooled to 4°C, collected the precipitated crystals, washed with water and vacuum dried to obtain 7.77Q of 2.4-decanedienoic acid (yield: 8
6%).

mp, 39.0-42.0℃ E IMS: M! 168 (C Ko H2S 02
) I R (KBr): C1680 (C=O
), 1630 oJ: and 1610 cm-” (C
= C).

IH-NMR (CDCjh): δ 0.89 (3H.

t, -C, LL), 2.18 (2H, m, -〇upper 10 H=CH-) 5.7-7.5p11+11
(4H,m)13C-NMR (CDCf3): δ
13,98 (C-10>, 22.49 (C-
9), 28.34 (C-7), 31.42 (
C-8'), 33.05 (C-6), 118.3
5 (C-2), 128.23 (C-4), 146.
26 (C-5), 147.57 (C-3),
173.11 (C-1).

However, the number in parentheses is the carbon number.

By a similar method, trans-2-decenal 21.43
0 (0,139 mol) to 2,4-dodecanedienoic acid e
, ig' (total yield 23%) obtained mp, 48.5 ~
50.0℃ E IMS: M word 196 (C12H2o 02) I
R (KBr): L/1680 (C=O),
1630 length 1610cm-engineering (C=C).

IH-NMR (CDCJ13): δ0.88(3)-1
゜t, -CH:l), 1.28 (10H, s, -
(History ratio L) s-), 2.20 (2H, m, -C ratio LCH=CH-), 5.78 (I H, d, J
=15.1Hz, -CH=CL-COOH), 5.
9 to 6.4 (21-1, m, -C, L=C) (-C
H=CH-COOH), 7.2-7.5 (1H, m, -
CH=CH-CH=CH-C00H), 11.76
(I H,,brs,-COO ratio),"3C-NM
R(CD(,Ca): δ14.07 (C-12)
, 22.67 (C-11) , 28.67 (C
-,7'), 29.13, . 29.17 (C-8,
9>, 31.80 (C-10) 33.08 (
C-6), 118.38 (C-2), 1
28.27 (C-4), 146.20 (
C-5), 147.51 (C-3),
173.08 (C-1).

However, the number in parentheses is the carbon number.

Synthesis Example 2 Synthesis of 2,4-alkanedienoic acid by route (B) 60% sodium hydride 12.0Q (0.3 mol)
Ethyl 4-diethylphosphonocrotonate (manufactured by Aldrich) in a tetrahydrofuran solution (0.5 Jl) under cooling to -20°C. 75. After dropping Oa (0.3 mol), the mixture was stirred for 1 hour. Tetradecyl aldehyde 63,7(]
(0.3 mol of tetrahydrofuran solution (0.2 Jl)
) was added at -20°C, returned to room temperature, reacted for 1 hour, and then poured with cold water at 21°C.
Pour into ethyl ether-n-hexane (0.6 Jl: 0
．． 6J) and extracted with n-hexane (1Jl), and the combined organic layers were washed with saturated brine (1fx2) and dried over anhydrous sodium sulfate. The residual oil obtained by distilling off the solvent was subjected to silica gel chromatography (silica gel 3.0K
g, n-hexane:ethyl ether-20:1)
24.11 g (yield 26%) of 2,4-octadecanedienoic acid ethyl ester was obtained. It is a waxy solid with a low melting point.

TLC (silica gel 60, Rf = 0.74 with n-hexane:ethyl ether = 4 = 1) shows a single product.

Then the obtained ethyl ester 22Q (0,071
mol) in a mixed solution of 10% potassium hydroxide aqueous solution (60 m) and 100 d of ethanol and heated to 60°C.
After reacting for an hour, the mixture was neutralized to pl-12.5 with concentrated hydrochloric acid, cooled to 4°C, and the precipitated crystals were collected and air-dried.

Recrystallization from n-hexane and a small amount of dichloromethane yields colorless needle-like crystals of 2,4-octadecanedienoic acid 16.
．． 5 g (yield 83%) was obtained.

EIMS: M! 280 (Cte) + 3202 > Elemental analysis value (%): Calculated value as Cl8H3202: C
77,09, H11,50, Actual value: C77,20,
Hll, 45° I H-NMR and 13 C-NMR are as in Reference Example 1, 2.
, 4-dodecanedienoic acid.

Similarly, 2,4-hexadecadienoic acid was obtained from dodecylaldehyde in a yield of 24%.

mp, 60.0-61.0°C E IMS: M: 252 (Ct 6 H2a 0
2) Elemental analysis value (%): Calculated value as C engineering H2802: C76,14, Hll, 18, Actual value: C7
2,4-tetradecadienoic acid was obtained in a yield of 20% from decyl aldehyde at 6,08, Hll, and 59°.

mp, 53.5-54.5℃ E IMS: M! 224 (C14H2402) Elemental analysis value (%) Calculated value as Cs 4 H2402:
C74,95, Hlo, 78, actual value; C74,6
5, Hlo, 93° Synthesis Example 3 Synthesis of tritylpentaerythritol Pentaerythritol 500 (0,367 mol) with pyridine (70d) and N,N-dimethylformamide (200
The mixed solution of d) was heated to 90°C and trityl chloride 25
(1 (0,090 mol) was added in 4 parts over about 1 hour, and the reaction was carried out at 100°C for 3 hours.
Pour the water into the container, remove the aqueous layer by decantation, and remove the water (0,
After washing with 5.1×2), remove the residual viscous oil with chloroform 0.4. / and the insoluble portion was removed by filtration. The filtrate was dried, and 0.31% of benzene was added and dissolved in n-hexane o, i.
After adding Jl and leaving it at 4°C for 3 hours, the precipitated crystals were collected by filtration, washed with a mixed solvent of benzene:n-hexane = 2:1, and dried under vacuum to obtain tritylpentaerythritol 18.
．． 5i;l (yield 55%) was obtained. mp, 155~
The temperature was 156.5°C.

Elemental analysis value: Calculated value (%) 0 as C24th 2604
76.17.86.92, Actual value (%) C76.08
, H7,1g Example 1 Production of compound represented by general formula (II) (a)
8.13 g (33 mmol) of myristoyl chloride was added to a dichloromethane solution (100 m) of trityl pentaerythritol 3.78 T (10 mmol) and pyridine 2.69 T (34 mmol) at room temperature.
Allowed time to react. Add 200 d of chloroform and add 1 d of water.
00dX2), after washing with saturated saline (100t+f),
It was dried with anhydrous magnesium sulfate. After distilling off the solvent under reduced pressure, the residue was recrystallized from a mixed solvent of methanol and ethyl ether to obtain 9.33 g (yield: 89%) of trimyristoyl monotrityl pentaerythritol (compound numbered 3 in Table 1).

mp, 47.0-48.0°C.

IR (KBr or CHCjh): C17
30 Oyohi 106106O1 (Ester C=0 and C
-O>IH-NMR (CDCJ13): δ 0,89
(9H.

S, -ChL3), 2.19 (6H,t, -CH2
CLLcoo->, 3.14 (2H, S, -C-
C, LLOCPh5), 4.11 (6H,s, -C
OOC length 3C-CH20CPh 3 ) and 7.
1 to 7.5 ppm (15H, m, proton of trityl group) 3C-NMR (CDCjh): δ86.7.1
27.1127.7,128,7,143.5 (trityl group) and 173.2 (-history 00 CH2+ding-〇
).

Compounds numbered 1 to 4 in Table 1 were synthesized in a similar manner. The final oily compound was purified by silica gel column chromatography (solvent: benzene). IR,I H-N
MR and 13C-NMR were in good agreement with those of trimyristoylmonotritylpentaerythritol.

(b) Tritylpentaerythritol 14,32 (1
(37,8 mol), 2,4-decadienoic acid 21,0 (]
(125 mmol), 1.53 g (12.5 mmol) of 4-N,N-dimethylaminopyridine in dichloromethane (300 mmol) was added 30.9 g (1<150 mmol) of N,N'-dicyclohexylcarbodiimide. The reaction was stirred at room temperature for 24 hours. The precipitated N,N'-dicyclohexylurea was filtered off, the filtrate was concentrated under reduced pressure, and the resulting crude oil was subjected to silica gel column chromatography (silica gel 60.1 kg).

benzene) and the resulting oil was dried in vacuo.

26.250 (yield: 84%) of tri(2,4-decajenoyl)tritylpentaerythritol (compound No. 5 in Table 1) was obtained as a pale yellow oil.

IR (CHCjh): 1700 (varnish y/1zc
−o) and 1635.1610 cm −1 (C= C
).

'H-NMR (CDCJla): 60.90
(9H, t, -C Dannie), 1.34 (18H,
m, Chh+CH2 Sting CH2), 2.20 (6H
, m, -CH2COOCH2'-), 3.22 (2
)-1,S,-C=CI-1200Ph3),4
．． 25 (6H,S, -1cOOcH2juctoC->
, 5.67 (3H,d, J=15.1Hz
, -〇H=CH-COOCH2-), 5.9-6.
3(6H, m, -CH= CL-CH-CH-C00-
), 6.9-7.5(18)-1,m, -CH=C)-1-
13C-NMR (CDCf3): 643.06 (erythritol quaternary carbon), 60.
67 (ΣC-History H20CPh 3 ), 62.63
(-Coo main H2 dimension C->, 86.70 (-
0-stop Pha), 166.7 (-shi○0CH2,000) Compounds numbered 6 to 9 in Table 1 were synthesized in the same manner. IR, l H-
NMR, and 13c NMR were very similar to those of tri(2,4-decajenoyl)tritylpentaerythritol.

Example 2 Preparation of a compound represented by the general formula (I [
7.0 g (0,0147 mol) in dichloromethane 3
A methanolic solution of 14% boron trifluoride (7,2me (0,0149 mol)) was added to the solution, stirred at room temperature for 24 hours, washed with cold water (200meX twice), separated into layers, and dried over anhydrous magnesium sulfate. . After distilling off the solvent, the residue was purified by silica gel column chromatography (solvent: chloroform:methanol = 40:1). Furthermore, by recrystallizing from ethanol:diethyl ether, tristearoylpentaerythritol (Table 2
9.30 (1 (yield: 68%) of compound No. 4) was obtained.

IR (KBr): 3460 cm-1 (0-H).

Compounds shown in Table 2 were synthesized in a similar manner.

Example 3 Preparation of a compound represented by the general formula (A) 1.0 g (1.3 mmol) of trimyristoylpentaerythritol obtained in Example 2 was mixed with 0.30 g of phosphorus oxychloride.
g (1,95 mmol) and triethylamine 0.200
(2.0 mmol) in dichloromethane (30 ml) and stirred at room temperature for 12 hours. 100 d of benzene was added thereto and precipitated triethylamine hydrochloride was filtered off and the solution was concentrated under reduced pressure.

70ml of tetrahydrofuran for the remaining oily substance! Dissolved in 0.5M sodium bicarbonate aqueous solution 50- and 0.25-
M-ethylenediaminetetraacetic acid aqueous solution (pH 10,5)
20 mL of the mixed solution was added, and the mixture was stirred and reacted at room temperature for 12 hours. 50 d of saturated brine was added and the separated tetrahydrofuran layer was collected and dried under reduced pressure. The remaining solid was dissolved in a mixed solution of chloroform:methanol:water=50d:50d:50m, and the chloroform layer was collected and dried under reduced pressure.

Dissolve in chloroform (30 m2) again, filter out the insoluble portion, reprecipitate in 70 d of acetone, and centrifuge (2800 r).
pm, 4℃.

The precipitate was collected (for 30 minutes), washed with acetone, and then dried under vacuum in the presence of phosphorus pentoxide. 0.97 (1 (yield: 85%)) of colorless crystals of trimyristoyl pentaerythritol phosphate-2Na salt (compound represented by general formula (Ia) (R is CH3(CH2) 12-)) was obtained.I
R (KBr): l/3400°1260, 11
60.1100 (phosphate), 1730cm-
'(Ester C=○). ” H-NMR (CDC, C3
): δ0.88 (9H, t, -Cl1L>, 2
．． 30 (61-1, brs, -CLLCOO-),
3.70 (2) -1°brs, -CLLOP (0)
-), 4.18 (6H.

brs, total-cooc ratio Esuding). 13 (, -NMR(CDC1
3): δ43.03 (-History H20P (0)-),
63.27 (-〇〇CH2-), 173.96
(-COOCH2-). FABMS: [M+1]!
891゜[M+Na]”913(C47HsgPtO
tONa2 = 890). Elemental analysis value: C47l
-(s B Pt○1uNa2 ・Calculated value (%) as 2H20; C60,89, HIo, 11, analytical value (%)
): C61,02,1-(10,52.

Similarly, 0.577 (1 (0.98 mmol)) of tri(2,4-decajenoyl)pentaerythritol was reacted with 0.3070 (2 mmol) of phosphorus oxychloride and 0.2020 (2 mmol) of triethylamine for post-treatment. The crude product obtained was purified by silica gel column chromatography (silica gel 60.50, solvent chloroform:methanol:water-65:25:4), and the eluate was shaken with a saturated aqueous sodium bicarbonate solution to separate the layers. Tris(2,4-decajenoyl)pentaerythritol phosphate 2Na salt (general formula (Ia)
(R is CH3(CH2>40 days-C)-1-C1-1=
The yield of the compound represented by C1-1-) was 0.51111
, with a yield of 73%. I R (K B r ): l
/3380,1250.1130,1090 (Hoss 7m
-t), 1700 (ester c=o), 1640.1
610cm” (C=C). FAB MS: [
M+1] + 711, [M 10Na]
+ 733 (However, C3sHssP10to
Na2=710).

” H-NMR (CDCj!, + +CD3 0D
): δ0.90 (9H,t, -Ctani->1.35 (18H.

S, -(CLLjT), 2.40 (6H,m, -
Cdan” CH=CH-), 4.20 (2H, brs,
-CL20P (ONa)2, 4.37
(6H,s, -C0OCH2-), 5.78
(3H, d, J=15.4Hz;-CH
=CH--Coo-), 6.0-6.4 (6H.

m, -Q old-= C, [-C) (= CH-COO
-), 7.1 to 7.5 (3H, m, -CH=CH-
C00-).

13C-NMR (CDCf3 +CD3 0D)
: δ14.07 (C-13), 22.67
(C-12), 28.58 (C-10), 31
．． 62 (C-11>, 33.26 (C-9),
43.35 (C-2), 64.0 (C-1 and C-3
), 118.50 (C-5), 128.44 (C-
7), 146.02 (C-8), 146.
46 (C-6) 167.76 (C-4). However, the number in parentheses indicates the carbon number.

Tridecanoylpentaerythritol 4 obtained in Preparation Example 2
．． 00 (6,68 mmol) and triethylamine 1.
39 m (10 mmol) and 0.041 (] (0,334 mmol) of 4-N,N-dimethylaminopyridine in dichloromethane solution at room temperature.
Oxo-1,3,2-dioxaphosphorane 1.10++
(10 mmol) was added, and after stirring reaction for 12 hours,
Additionally, 1.39 d of triethylamine and 2-chloro-2
-oxo-1,3,2-dioxaphosphorane 1.10 (
] and reacted for a total of 18 hours. The completion of the reaction was confirmed by silica gel TLC using a Nisilica gel 60 plate (manufactured by Merck), a benzene:ether-5=1 solvent, and coloration due to iodine adsorption, and the Rf value of the raw alcohol was 0.
45. The Rf value of the reaction product phosphate is 0.2
It was 7.

Ether 50d was added to the reaction solution, the precipitated triethylamine hydrochloride was filtered off, and the filtrate was concentrated under reduced pressure to obtain a brown oily residue. This was dissolved in 30°C of anhydrous acetonitrile, cooled in a dry ice-acetonitrile bath, 8ml of trimethylamine was added, and the tube was sealed in a stainless steel reaction tube. Bath temperature 70
After heating and stirring at 'C for 20 hours, the mixture was cooled to room temperature, opened, and acetonitrile was distilled off under reduced pressure. Dissolve the remaining wax-like solid in methanol 100ne and add ion exchange resin Amberlite MB- which has been previously washed with methanol.
1 column (2.5 x 30 cm) and eluted with 0.44 of a mixed solvent of chloroform:methanol-1=2. The eluate was dried under reduced pressure to obtain a colorless solid. Art7734.
100! ], solvent chloroform:methanol:water-6
The colorless solid obtained by purifying the colorless solid by 5:25:4) was vacuum-dried over phosphorus pentoxide to obtain a compound of general formula (Ib) in which R is CH3-(CH2)8- (number in Table 3). Compound 1) was obtained. Yield 2.28Q, yield 45%
. The melting point was unclear. I R (KBr): ν
3390 (0-H of water), 1725 (ester C=O)
, 1240 (ester C-0) 1170.1080,1
040,980c#I-1 (phosphocholine).

IH-NMR (CDCfyo): 60.88 (
9H.

t, CHa CH2-), 1.26 (36H
,S,CH3(CH,=)s CH2-),
1.57 (6H,m, -CH2CLLCH2
COO-), 2.29 (6H.

t, J -7,4Hz, -CH2C,LLCoo-)
, 3.39 (91-1,s, -N (C
H''-)3), 3.86-CH20LLN (CH:I)3), 4
．． 14 (6H.

S, -CH2C00Cdan"-1), 4.24 (H
,brs.

-0CH2CH214(CHs)s),”
C-NMR (CDCJla): 614,1
3 (C-13), 22.70 (IC-12
), 24.95 (C-6), 29.34~
29.49 (Q-7 to C-10), 31.8
8 (C-11) 34.19 (C-5), 4
2.54 (C-2>, 54.38 (C-γ)
, 59.41 (C-α) , 62.95 (
C-3) 64.10 ((, -1) , 66.46
(C-β). However, the number in parentheses indicates the carbon number.

(CR3CH2CH2CH2CH2CH2CH2CH2
CH2CoocH2suj3j2si'109 a
y 6 s 4 3 From 2.800 (4.18 mmol) of tri(2,4-dodecagenoyl)pentaerythritol obtained in Example 2 in a similar manner, R in general formula (Ib) is CH:l ( CH2)s
Compounds represented by CH=CI-1-CH=CH- (Table 3
Collect the compound number 6 in as a colorless waxy solid.
2.09G (2.50 mmol) was obtained in a yield of 60%. Although the melting point was unclear, the color gradually changed to reddish brown when the temperature exceeded about 150°C. IR (KBr): ν340
0 (water 0-H), 1710 (ester C=O>, 164
0.1615 (C' = C), 1245 (ester C-0), 1140,1085,1060,970
(J-” (phosphocholine).

IH-NMR (CDCf3): δ0,88 (91-(,t, C ratio 1cH2-), 1
．． 28 (30, H, S, CH3 (CHLfT), 2
．． 17 (6H, brs, -CH2--CH=CH-
), 36-3.35 (9H,S,N (CH”-)3),
3.75 (2H, brs, -CH2CLL-N (
CI-1a1co) 3.95 (2H, brs, -C
-CLLOP (0)-), 4,27(81-1,br
s, -GOOC ratio L- and -〇Cdan''-CH2N
(CH3)3), 5.74 (31-1
゜d, J=15.1l-(z, -CH=Cl-1-
COO-), 5.8-6.3 (6H, m, -CL-〇L
-CH=CI-1-COO->, 7.00-7.3
5 (3H, m, -CH=CH-Coo-).

”3C-NMR (CDCfa +CD:l
OD): δ14.10 (C-15), 22
．． 73 (C-14), 28.78 (C-1
0), 29.19 and 29.25 (C-11 and C-12), 31.88 (C-13), 33
．． 17 (C-9), 43.02 (C-2)
, 54.38 (C-γ) , 59.13 (C
-α), 63.07 (C-3) 64.40
<C-1), 66.43 (C-β), 11
B, 26 (C-5), 128.27 (C-7
), 145.94 (C-8), 146.2
6 (C-6), 167.26 (C-4)
However, the number in parentheses indicates the carbon number.

(CH3CH2CH2CH2CH2CH2CH2CH2CH=
CH-CH=CH2Cα)CH,7i1s ji11
3 1211 +09 8 7 6 5 4
3 Compounds shown in Table 3 were synthesized by the same method.

Experimental example Confirmation experiment for liposome formation 100 columns of compounds represented by the general formula (Ib >
! The mixture was placed in an eggplant flask and dissolved in chloroform 2-, and the chloroform was evaporated to form a thin film on the vessel wall. Further, the solvent was completely removed by vacuum drying at room temperature for 5 hours, and then 10 d of distilled water was added and shaken. The obtained emulsion solution was subjected to ultrasonic treatment (Ultrasonic homogenizer US-600 (Nippon Seiki Seisakusho)) while slowly blowing nitrogen gas at an output of 150 to 300 μA for 10 to 2 minutes.
When this was carried out intermittently for 0 minutes, a homogeneous solution with almost no turbidity was obtained. In addition, the compound numbered 3.4.8°9 in Table 4 was slightly cloudy. The reason for this turbidity is thought to be that large multilayered liposomes are mainly formed.

Solutions prepared from compounds numbered 1 to 4 in Table 4 were used as they were in the following confirmation analysis of liposome formation.
Solutions from compounds numbered 5 to 9 were further polymerized. Oxygen was removed from the aqueous solution obtained by ultrasonication by blowing nitrogen gas (at a rate of 100 to 307 per minute), and the solution was then transferred to a quartz test tube under a nitrogen gas atmosphere and sealed tightly.

Polymerization was carried out by irradiation with ultraviolet light (a photoreaction device (manufactured by Riko Kagaku Sangyo ■) equipped with a low-pressure mercury lamp (32W)) in a water bath at a bath temperature of 40 to 50°C to obtain an aqueous solution of polymeric liposomes. Completion of polymerization was confirmed by the disappearance of the ultraviolet absorption spectrum (λmax 250 nm) based on the conjugated diene moiety. It is believed that polymerization occurs according to the following reaction mode.

(CH, (CH2)m-CH-cH-CH-CH-CO
OCH2+3C-CH2OP-OCH2CH2N (C
6↓The time required to complete the ultraviolet light irradiation polymerization was within about 1 hour.

Moreover, there was no change in the solution state before and after polymerization.

The formation of liposomes in the aqueous dispersion solution thus obtained was confirmed by a method already established for liposomes made of ordinary natural phospholipids. Differential thermal analysis (DSC) shows that the phospholipid has a bilayer structure.

(manufactured by Seiko), and the gel liquid crystal phase transition temperature (Tc
)It was determined. In addition, transmission electron microscopy observation by negative staining method using 4% uranyl acetate aqueous solution (newly manufactured by Hitachi Seisakusho), submicron particle size measuring device Coulter N4
D (manufactured by Coulter Electronics) and TSK
G5000PW and G6000PW column set (
Gel filtration chromatography (GFC) using Toyo Soda Kogyo Co., Ltd. (solvent: 0.1M phosphate buffer (pH 7,0) and calibration curve using Sigma standard proteins (thyroglobin, apoferritin, albumin) ) The liposome shape and liposome diameter were determined by combining the ,
The results in Table 4 do not indicate fixed diameter values.

[Effects of the Invention] As described above, the present invention provides a phospholipid compound that can be used as a surface-active substance like natural phospholipids and can form liposomes, and a method for producing the same.

Applicant's representative Patent attorney Takehiko Suzue Written amendment 1, Indication of the case Patent Application No. 1984-284610 2, Name of the invention Double-stranded phospholipid compound and its manufacturing method 3, Relationship with the person making the amendment Patent Applicant: 1) Shun Hide 4, Agent UBE Building 7, 3-7-2 Kasumigaseki, Chiyoda-ku, Tokyo;
Contents of the amendment (1) Add "Patent application pursuant to the proviso to Article 38 of the Patent Act" to the title of the application.

(2) Next to the "Title of Invention" column in the application, a new column will be created for "Number of inventions stated in one claim" and the number of inventions "3" will be added.

= 2-

Claims (5)

[Claims] (1) General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (Here, R is an alkyl group, alkenyl group, or alkadienyl group, and X is -O- or -OCH_2CH_2^
A phospholipid compound represented by +(CH_3)_3). (2) The phospholipid compound according to claim 1, wherein the substituent R is -(CH_2)-_nCH_3 (where n is an integer of 8 to 20). (3) Substituent R is -CH=CH-CH=CH-(CH_2)-_mCH_
3 (here, m is an integer of 4 to 16). (4) General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (Here, R is an alkyl group, an alkenyl group, or an alkadienyl group) After reacting triacylpentaerythritol with phosphorus oxychloride in the presence of a base, , a phospholipid represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (where R is an alkyl group, alkenyl group, or alkadienyl group), which is characterized by reacting with a mixed aqueous solution of sodium hydrogen carbonate and ethylenediaminetetraacetic acid. Method of manufacturing the compound. (5) General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (Here, R is an alkyl group, an alkenyl group, or an alkadienyl group) -1,3,
A general formula characterized by reaction with 2-dioxaphosphorane and then trimethylamine ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (Here, R is an alkyl group, an alkenyl group, or an alkadienyl group). Method for producing phospholipid compounds.