JPH093093A - Cholesterol derivative - Google Patents

Abstract

PURPOSE: To obtain the subject new derivative which is a specific cholesterol derivative having an N-carbonylimidazolyl oxyalkylene chain, can be bonded to a functional substance at its end through a covalent bond and is useful as a component for forming a cytoplastic membrane such as a liposome, etc. CONSTITUTION: This cholesterol derivative of formula I (OA is a 2-4C oxyalkylnen group; (n) is an average number of addition mols of the oxyalkylene groups and is a positive number of 1-1,000; when (n) is >=2, the oxyalkylene groups may be different or same and may be added in a random state or added in a block state; R is H or methyl). The derivative can be efficiently bonded to a functional substance at the end of the (poly)oxyalkylene chain through a convalent bond and is useful as a component for forming a liposome for a liquid crystal raw material, a medicine transporting material, a diagnostic, a sensor, an immobilized catalyst, etc. The compound is obtained by reacting a cholesterol of formula II with an alkylene oxide and then with N'N- carbonylimidazole.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel and useful cholesterol derivative. More specifically, various functional liposomes or fat emulsions such as liquid crystal raw materials, drug carriers for medicines, test agents, diagnostic agents, sensors, immobilized catalysts, bioreactors, bioelectronic devices, microcapsule substitutes, cosmetic raw materials, etc. The present invention relates to a cholesterol derivative used for production of endoplasmic reticulum.

[0002]

2. Description of the Related Art It has been pointed out that when liposomes are administered in vivo, most of them are trapped in reticuloendothelial organs such as the liver and spleen, so that sufficient effects cannot be obtained (Ca.
ncer Res., 43 , 5328 (1983)). Therefore, as a method for improving the problems of being trapped in the reticuloendothelial organ and the problems of low stability such as disintegration and aggregation of the liposome itself, a polyethylene glycol chain is introduced on the surface of the liposome. Has been attempted (for example, WO90 / 3484, JP-A-1-249717,
FEBS letters, 268 , 235 (1990)). In addition, it has been clarified that liposomes modified with polyethylene glycol can maintain blood levels for a long period of time (Bi
Ochem. Biophys. Acta., 1066 , 29-36 (1991)). However, since the liposome having a polyethylene glycol chain introduced by such a method does not react with the functional substance, the functional substance cannot be immobilized on the surface of the liposome.

Further, in Japanese Patent Laid-Open No. 4-346918,
First, a liposome having a maleimide group is reacted with a protein having a thiol group (thiolated protein), and then a compound having a polyalkylene glycol (thiolated polyalkylene glycol) moiety having a thiol group added to a residual maleimide group is reacted. Due to
It has been described that drug-containing antibody-bound liposomes with improved uptake in reticuloendothelial organs are obtained. However, this liposome has a problem that the expected effect cannot be sufficiently obtained because the antibody is hidden under the polyalkylene glycol layer and the reaction with the antigen at the target site is hindered.

Further, in JP-A-5-508388, α-
A liposome preparation comprising a polyoxyethylene derivative having an anion group represented by stearyl-ω-propionic acid-polyoxyethylene is disclosed. However, since the hydrophobic portion of this polyoxyethylene derivative is a monoalkyl group, it is easily detached from the liposome membrane,
Therefore, there is a problem that liposomes containing such a polyoxyethylene derivative as a film-forming component are inferior in long-term stability.

On the other hand, as an example of introducing a polyethylene glycol chain into cholesterol, Japanese Patent Laid-Open No. 1-249798
JP-A No. 1994-242 discloses a 2,4-bis (o-methoxypolyethylene glycol) -6-cholesteryl-s-triazine derivative, and the stability of the liposome is improved by using this derivative as a membrane-forming component of the liposome. Is described. However, immobilization of functional substances and the like is not considered in this derivative.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to enable various functional substances to be immobilized on the tip of a (poly) oxyalkylene chain easily and efficiently by covalent bonding, and small functionalities such as liposomes can be obtained. It is an object of the present invention to provide a novel and useful cholesterol derivative that can be used as a cell-forming component.

[0007]

The present invention is a cholesterol derivative represented by the following general formula (1).

Embedded image [In the Formula, OA is a C2-C4 oxyalkylene group, n
Is the average number of moles of oxyalkylene groups added, which is from 1 to 100.
Represents a positive number of zero. When n is 2 or more, the oxyalkylene groups may be the same or different and may be added randomly or in a block. R
Represents a hydrogen atom or a methyl group. ]

In the present invention, "(poly) oxyalkylene" means "oxyalkylene and / or polyoxyalkylene" and "(poly) alkylene" means "alkylene and / or polyalkylene".

The oxyalkylene group represented by OA of the general formula (1) is an oxyalkylene group having 2 to 4 carbon atoms, and is an oxyethylene group, an oxypropylene group, an oxytrimethylene group or an oxy-1-ethylethylene group. , Oxy-1,2-dimethylethylene group, oxytetramethylene group and the like. These oxyalkylene groups are
Ethylene oxide, propylene oxide, oxetane,
It is a group obtained by addition-polymerizing an alkylene oxide such as 1-butene oxide, 2-butene oxide or tetrahydrofuran.

In the general formula (1), n is a positive number of 1 to 1000, preferably 10 to 300, more preferably 20 to 120. When n is 2 or more, the types of the oxyalkylene groups may be the same or different. In the latter case, they may be added randomly or in blocks.

When imparting hydrophilicity, it is preferable that ethylene oxide is added alone as OA, and in this case, n is preferably 10 or more. When different types of alkylene oxides are added, it is desirable that ethylene oxide is added in an amount of 20 mol% or more, preferably 50 mol% or more. When imparting lipophilicity to the (poly) oxyalkylene chain, the number of added moles other than ethylene oxide is increased.

The cholesterol derivative represented by the general formula (1) can be easily produced, for example, by the following two-step reaction. First of all, in the first stage reaction,
An alkylene oxide is addition-polymerized with the hydroxyl group in cholesterol to produce a (poly) oxyalkylene adduct of cholesterol (hereinafter abbreviated as Chol-OA). At this time, the charged molar ratio of cholesterol and alkylene oxide is selected according to the number of repetitions of the target (poly) oxyalkylene, and is, for example, 1: 1 to 1: 1000.
It is desirable that the ratio is 0, preferably 1:10 to 1: 300. The reaction uses metallic sodium, metallic potassium, sodium hydroxide, potassium hydroxide and the like as a catalyst, and an organic solvent such as toluene, benzene, chloroform and carbon tetrachloride as a solvent at 0 to 150 ° C. for 30 minutes to
It can be performed by stirring for 200 hours.

Next, in the second-step reaction, the terminal hydroxyl group of Chol-OA obtained in the first-step reaction is converted to N,
N'-carbonyldiimidazole or a substitution product thereof is used for N-carbonylimidazolation. At this time, Ch
The molar ratio of ol-OA and N, N'-carbonyldiimidazole or its substitution product was 1: 0.5 to 1: 1.
It is desirable that the ratio is 0, preferably 1: 0.9 to 1: 2. The reaction is solvent-free or benzene, toluene,
Using an organic solvent such as chloroform, carbon tetrachloride, acetonitrile, ethyl acetate, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, etc., -100 to +
It can be performed by stirring at 150 ° C., preferably 0 to 80 ° C., for 1 minute to 200 hours, preferably 30 minutes to 6 hours. After completion of the reaction, distillation, recrystallization, reprecipitation, adsorbent treatment, column treatment, ion exchange, gel filtration,
It can be isolated and purified by a method such as ultrafiltration or dialysis.

The reaction formula when ethylene oxide is used as the alkylene oxide is shown in the following formula (2). In the formula, n and R have the same meanings as described above.

Embedded image

The cholesterol derivative of the present invention thus obtained can be used as an endoplasmic reticulum-forming component or the like. Here, the endoplasmic reticulum means a particle having a structure in which the hydrophilic group of the cholesterol derivative of the present invention or this cholesterol derivative and another endoplasmic reticulum-forming component is oriented toward the aqueous phase at the interface. Specific examples thereof include liposomes, which are closed vesicles composed of bilayers; fat emulsions obtained by emulsifying a mixture of vegetable oil and phospholipids; micelles and the like.

Since the cholesterol derivative of the present invention has a functional group such as an amino group, a hydroxyl group or a thiol group, in particular, an oxycarbonylimidazole group having a high reactivity with a primary amino group or a substituted product thereof. , Easily reacts with a functional substance having such a functional group to form a covalent bond. Therefore, by using the cholesterol derivative of the present invention as an endoplasmic reticulum-forming component, it is possible to introduce a (poly) oxyalkylene chain into the endoplasmic reticulum,
The reactivity to the functional substance having the functional group can be imparted, and the reactive vesicle can be produced.

The functional substance having the above-mentioned functional group is not particularly limited, but substances derived in vivo such as antibodies, enzymes, nucleic acids, saccharides, chemiluminescent substances, fluorescent substances, radioisotope labeling substances, dyes, pigments, Preferred are medicines and agricultural chemicals.

[0018]

The cholesterol derivative of the present invention is novel and useful. By using the cholesterol derivative of the present invention as an endoplasmic reticulum-forming component, a (poly) oxyalkylene chain can be introduced into the endoplasmic reticulum, and at the tip of the (poly) oxyalkylene chain, various kinds of easily and efficiently can be introduced. The functional substance can be immobilized by a covalent bond.

[0019]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited thereto. Example 1 Toluene 500 ml, cholesterol 10 g (26 mmol) and sodium metal 0.6 g in an autoclave
(26 mmol) was added and the mixture was stirred for 30 minutes, 52 g (1.2 mol) of ethylene oxide was added, and the mixture was stirred at 100 ° C. for 24 hours. After completion of the reaction, the product was washed with 5% hydrochloric acid and distilled water, and then toluene was distilled off under reduced pressure to give an intermediate in which ethylene oxide was added to the hydroxyl group of cholesterol (hereinafter, referred to as Chol-PEG, a molecular weight of about 240).
0, the number of moles of ethylene oxide added was about 46) (yield 96%). Further, 5 g (2 mmol) of Chol-PEG and 0.32 g (2 mmol) of N, N'-carbonyldiimidazole were added to 10 ml of chloroform, and the mixture was stirred at room temperature for 6 hours. After completion of the reaction, the product was purified by reprecipitation in diethyl ether to obtain a white powdery cholesterol derivative represented by the following formula (3). The progress of the reaction is as follows: in the IR spectrum (KBr method), the disappearance of hydroxyl groups (OH stretching, 3430 cm ⁻¹ ) in Chol-PEG and = NC
Generation of (= O) O-bond (C = O stretching, 1760c
m ^-1 ).

[0020]

Embedded image

¹ H-N of the obtained cholesterol derivative
The MR and IR analysis results are as follows. ¹ H-NMR (270 MHz, CDCl ₃ , TMS, δ;
ppm, J; Hz) 8.14 ( a ; s, 1H) 7.43 ( b ; s, 1H) 7.08 ( c ; s, 1H) 4.67 ( d ; t, 2H, J = 2. 5) 3.66 ( e ; m, about 182H) 5.37 ( 6 ; d, 1H, J = 5.0) 4.44 ( 8 ; m, 1H) 2.33 ( 7 ; d, 2H, J = 6.9) 2.05-1.0 (derived from a sterol skeleton other than 1 to 7 ) 1.01 ( 5 ; s, 3H) 0.88 ( 3 ; d, 3H, J = 1.0) 86 ( 1 , 2 ; d, 6H, J = 5.2) 0.68 ( 4 ; s, 3H) IR (KBr method, cm ^-1 ) 1760 (C = O stretching)

Example 2 500 ml of toluene, 10 g of cholesterol (26 mmol) and 0.6 g of sodium metal in an autoclave
(26 mmol) and after stirring for 30 minutes, 3.02 g (52 mmol) of propylene oxide was added, and the mixture was added at 100 ° C. for 4 minutes.
After stirring for an hour, 52 g of ethylene oxide (1.
2 mol) was added and the mixture was stirred at 100 ° C. for 24 hours. After completion of the reaction, the product is washed with 5% hydrochloric acid and distilled water, and then toluene is distilled off under reduced pressure to give an intermediate in which propylene oxide and ethylene oxide are added in a block form to the hydroxyl group of cholesterol (hereinafter, referred to as Chol-PPG-P.
EG, molecular weight about 2500, propylene oxide addition mole number 2, ethylene oxide addition mole number about 46)
Was obtained (95% yield). Furthermore, 10 ml of chloroform
5 g (2 mmol) of the above Chol-PPG-PEG and 0.32 g of N, N′-carbonyldiimidazole
(2 mmol) was added, and the mixture was stirred at room temperature for 6 hours. After completion of the reaction, the product was purified by reprecipitation in diethyl ether to obtain a white powdery cholesterol derivative represented by the following formula (4).
The progress of the reaction was confirmed in the same manner as in Example 1.

[0023]

Embedded image

¹ H-N of the obtained cholesterol derivative
The MR and IR analysis results are as follows. ¹ H-NMR (270 MHz, CDCl ₃ , TMS, δ;
ppm, J; Hz) 8.14 ( a ; s, 1H) 7.43 ( b ; s, 1H) 7.08 ( c ; s, 1H) 4.67 ( d ; t, 2H, J = 2. 5) 3.60 ( e , g , h ; m, about 182H) 1.12 ( f ; s, about 6H) 5.37 ( 6 ; d, 1H, J = 5.0) 4.44 ( 8 ; m, 1H) 2.33 ( 7 ; d, 2H, J = 6.9) 2.05 to 1.0 (derived from sterol skeleton other than 1 to 7 ) 1.01 ( 5 ; s, 3H) 0.88 (3; d, 3H, J = 1.0) 0.86 (1, 2; d, 6H, J = 5.2) 0.68 (4; s, 3H) IR (KBr method, cm ^-1) 1760 (C = O expansion / contraction)

Reference Example 1 Egg yolk phosphatidylcholine 20 mg (26 μmol), cholesterol 3.9 mg (10 μmol) and Example 1
Put 2.4 mg (10 wt% of the above two) of the cholesterol derivative obtained in step 2 into an eggplant-shaped flask, and add 2 ml.
Was dissolved in benzene and then freeze-dried. To this, 1 ml of physiological saline was added, and multi-layer liposomes were obtained by bath-type ultrasonic irradiation and a vortex mixer. Furthermore, 3.0, 1.0, 0.2μ by Extruder
m of the polycarbonate membrane were sequentially passed through to obtain large unilamellar reactive liposomes. The particle size of the obtained reactive liposome was measured by a laser scattering particle size distribution meter (NICOM
The particle size was 181 nm (CV value 15%) as measured by NICOMP370 HPL (trademark) manufactured by P. After the liposomes were allowed to stand at 5 ° C. for 3 weeks, the particle size was measured and found to be 195 nm (CV value 23%).
It was excellent in stability.

Claims (1)

[Claims] 1. A cholesterol derivative represented by the following general formula (1): Embedded image [In the Formula, OA is a C2-C4 oxyalkylene group, n
Is the average number of moles of oxyalkylene groups added, which is from 1 to 100.
Represents a positive number of zero. When n is 2 or more, the oxyalkylene groups may be the same or different and may be added randomly or in a block. R
Represents a hydrogen atom or a methyl group. ]