JPH0925287A - Synthetic lipid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new lipid capable of forming liposomes and a microemulsion having excellent properties. SOLUTION: This synthetic lipid is expressed by formula I [X is a monosaccharide residue or an oligosaccharide reside; R<1> is CH3 , CH2 CH2 NCH (NH2 )=NH, CH2 CONH2 , CH2 COOH, CH2 SH, CH2 C6 H5 , etc.; (1) is an integer of 1-8; (m) and (n) are each an integer of 1-18; (p) is an integer of 1-5], e.g. a compound of formula II. The compound of formula II which is one of the commands of formula I can be obtained by a usual method using a compound of formula III as a starting raw material. It is preferable that (1) is an integer of 3-8, (m) and (n) are each an integer of 6-18, more preferably 10-18.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a synthetic lipid having a novel structure.

[0002]

2. Description of the Related Art In recent years, in the field of medicine, it has been attempted to include a drug in a package called a liposome to enhance the organ-localizing property or to solubilize the drug by a microsphere called a micelle or a microemulsion. However, in order to put liposomes and microemulsions into practical use, there are many points to be solved such as structural instability, control of directivity to target cells and target organs, and control of particle size. For this reason, many studies have been made in the past. For example, Japanese Unexamined Patent Publication (Kokai) No. 58-49311 discloses a technique for strengthening the structure by a liposome coated with a polysaccharide derivative, and Japanese Unexamined Patent Publication (Kokai) No. 6-329558 discloses improvement of organ directivity by a liposome using a synthetic lipid. It is disclosed.

[0003]

DISCLOSURE OF THE INVENTION In order to overcome the drawbacks of liposomes and microemulsions and to provide better properties, it is necessary to find new lipids that can form liposomes and microemulsions. It is an important means together with addition, and the advent of lipids having excellent properties is craving. The present inventors have conducted intensive studies to achieve the above object, and as a result, have found that the object can be achieved by the following means and completed the present invention.

[0004]

The present invention is a synthetic lipid represented by the general formula (I).

Embedded image [Wherein, X represents a monosaccharide residue or an oligosaccharide residue, and R ¹ is a formula

Embedded image , L represents an integer of 1 to 8, m and n represent an integer of 1 to 18, and p represents an integer of 1 to 5. ]

The monosaccharide residue in the definition of X in the general formula is not particularly limited, and includes all monosaccharides regardless of the number of carbon atoms such as 5-carbon sugar, 6-carbon sugar or the structure of aldose, ketose, etc., One of the functional groups of these monosaccharides means a residue chemically bonded to another structural portion of the lipid according to the present invention. Specific examples of monosaccharides include ribose, xylose, galactose, glucose, mannose, ribulose and fructose. Further, the oligosaccharide residue is not particularly limited, and means one in which a plurality of monosaccharides such as disaccharides and trisaccharides are bound, and one functional group of these oligosaccharides is different from that of the lipid according to the present invention. It means a residue chemically bonded to a structural part. Specific examples of oligosaccharides include cellobiose, chitobiose, gentiobiose, lactose, melibiose, sucrose, trehalose, melezitose, raffinose, stachyose and the like.

Specific examples of the compound represented by the general formula (I) include the following compounds.

Embedded image These compounds can be synthesized, for example, by the following method.

[Chemical 6]

Specific examples of R ¹ are as described above, but the portion A of the compound represented by the following structural formula according to the present invention is an amino acid residue. That is, it means an amino acid residue in which a carboxyl group and an amino group bonded to the alpha carbon of an amino acid are chemically bonded to other structural parts of the lipid according to the present invention.

Embedded image Specific examples of the amino acid residue in the A portion include alanine, arginine, aspartic acid, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, leucine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, It means residues such as tyrosine and valine.

Although p in the general formula (I) is preferably an integer of 1 to 5, it is also possible to take an integer of 6 or more. When p is an integer of 2 or more, the A portion is a diamino acid, triamino acid residue or the like. For example, in the case of diamino acid, the two amino acids may be the same or different. The same applies to the case of more than triamino acids. General formula
In the structural formula shown in (I), l represents an integer of 1 to 8, preferably 3 to 8 and m and n are 1 to 18
Is more preferable, the integer of 6 to 18 is more preferable, and the integer of 10 to 18 is further preferable. The compound according to the present invention can form liposomes having excellent properties such as stability and organ transfer.

The method for producing the compound according to the present invention is not particularly limited, and it can be synthesized from an existing compound. Next, the production method of the compound according to the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0010]

【Example】

 Example 1

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1) Synthesis of N, N-dihexadecylamine ( 2 )

Embedded image [Operation] 430 g (1.8 mol) of hexadecylamine and 258 g (0.84 mol) of hexadecyl bromide were placed in a three-necked flask, 600 ml of ethanol and 387 g (2.9 mol) of potassium carbonate were added, and the mixture was vigorously stirred with a stirrer with a vacuum stirrer. While shaking
The mixture was heated under reflux for 120 hours. The reaction mixture was filtered while hot and potassium carbonate was filtered off. The filtrate was cooled, the precipitated solid was collected by filtration, and dried under reduced pressure. This solid was filtered while hot, and then distilled under reduced pressure (195 ° C to 210 ° C / 0.03 mmHg) to obtain a white solid. [Results] Yield (Yield) 207g (53%) (Reference value ⁵⁾ 43%) Melting point 63.2-63.9 ° C (Reference value ⁵⁾ 64.8-65.9 ° C)

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2) N, N-dihexadecyl-N-tert
-Butoxycarbonyl-L-alanine amide ( 3 ) synthesis

Embedded image [Operation] Nt-butoxycarbonyl-L-alanine
(Boc-Ala-OH) 2.0 g (1.0 × 10 ^-2 mol) was added to anhydrous methylene chloride 30
It was dissolved in ml and stirred with ice for 15 minutes while maintaining the temperature at 0 ° C.
2.4g of dicyclohexylcarbodiimide (DCC)
(1.2 × 10 ^-2 mol) was added and stirred, and then 4.8 g of N, N-dihexadecylamine was added. The mixture was stirred at 0 ° C for 4 hours and at room temperature overnight. Then, the reaction mixture was filtered, and the solvent was distilled off under reduced pressure from the filtrate. 40 ml of ethyl acetate was added to the residue and left in the refrigerator overnight to remove insoluble matter by filtration.
The filtrate is 10% aqueous citric acid solution (30 ml x 3), saturated saline solution (30 ml
X3), 4% aqueous sodium hydrogen carbonate solution (30 ml x 3) and saturated saline (30 ml x 3) were washed in this order, and further dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure and cooled to obtain a white oily substance. This was purified by column chromatography [Wakogel C-300 / chloroform: methanol = 30: 1 (v / v)].

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3) N, N-dihexadecyl-N- (6
-Bromohexanoyl) -L-alanine amide ( 4 ) synthesis

Embedded image [Operation] 0.8 g (1.18 mmol) of Boc-Ala-2C ₁₆ ( 2 ) was dissolved in 20 ml of anhydrous methylene chloride, 20 g (175 mmol) of trifluoroacetic acid was added with stirring at room temperature, and the mixture was stirred at room temperature for 3 hours. I did. The solvent was distilled off under reduced pressure, methylene chloride was further added, and the solvent was distilled off under reduced pressure to obtain a pale yellow oily substance. Boc group by NMR measurement
After confirming the removal of (δ = 1.43 ppm), 2.0 g of triethylamine (2
20 mmol of anhydrous methylene chloride solution (0 mmol) was added, and the mixture was cooled to 0 ° C in an ice bath.
6-Bromohexanoic acid chloride 1.
25 ml of 0 g (4.25 mmol) anhydrous methylene chloride solution was added dropwise over 45 minutes. Then, the mixture was stirred at 0 ° C. for 3 hours and at room temperature for 2 hours.
The reaction solution was added with 4% aqueous sodium hydrogen carbonate solution, saturated saline solution,
The solution was washed with a 10% aqueous citric acid solution and a saturated saline solution (all 30 ml × 3) in this order, dehydrated with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give a brown oil. This was recrystallized with methanol to obtain a white solid.

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4) N, N-dihexadecyl-N- (6
-Hydroxyhexanoyl) -L-alanine amide ( 5 )

Embedded image [Operation] (Trial 1) Br-C ₅ -Ala-2C ₁₆ ( 4 ) 1.0 g (1.6 mm
2.5 g (86 mmol) of formamide and 0.06 (3.2 mmol) of water were added to ol) and the mixture was stirred at 150 ° C. for 3 hours. 4 ml of water was added to this, extracted with methylene chloride (10 ml × 3), the solution was dehydrated with anhydrous sodium sulfate and then distilled under reduced pressure to obtain a brown oily substance. I
Since formation of ester species was confirmed by R spectrum measurement, 3 ml of a 2 mol / dm ³ NaOH aqueous solution was also added to this and refluxed for 3 hours. Was adjusted to pH = 1 with 5 mol / dm ³ HCl,
It was extracted with chloroform (10 ml × 3), water was removed with a liquid phase separation filter paper, and the solvent was distilled off under reduced pressure to obtain a pale yellow oily substance. This was purified by gel chromatography (Toyopear 1HW-40F / methanol) to obtain a yellow solid. (Trials 2, 3 and 4) The same procedure as in trial 1 was carried out, and purification was carried out by column chromatography (Wakogel C-100 / ethyl acetate).

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5) Methyl-2,3,4,6-tetra-O
-Benzyl-α-D-galactopyranoside ( 6 ) Synthesis

Embedded image [Operation] (Trial 1) Sodium hydride (60%) (1.9 g, 48 mol) was added to anhydrous DMF (20 ml), and the mixture was stirred at 0 ° C under a nitrogen atmosphere and then tetra-n-butylammonium iodide (1.8 g, 9.6 mmol) was added.
And stir to add methyl-α-D-galactose ( 5 ).
30 ml of 1.5 g (7.2 mmol) DMF solution was added dropwise over 40 minutes. 0
After stirring at 0 ° C for 1 hour, 7.8 g (75 mmol) of benzyl bromide was added dropwise, and the mixture was further stirred at 0 ° C for 1 hour and at room temperature for 16 hours.
The solution is filtered to remove excess sodium hydride and cold water 10
0 ml was added and the mixture was extracted with ether (80 ml × 2). The solvent was distilled off under reduced pressure to obtain a pale yellow liquid. The benzyl bromide was eluted from this by column chromatography (Wakogel C-300 / Benzene), and then the solvent was changed to benzene: ethyl acetate = 95: 5 to elute the target compound. (Trial 5) Methyl-α-D galactose ( 5 ) 1.0 g (5.
1 mmol) and 1.7 g (0.4 mol) of sodium hydride (60%) were dissolved in 25 ml of benzyl chloride and stirred at 125 ° C. for 80 minutes. Purification by column chromatography (Wakogel C-300 / benzen → ethyl acetate) gave a yellow transparent oil.

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6) Methyl-2,3,4,6-tetra-O
Synthesis of -benzyl-β-D-galactopyranoside ( 6 ')

Embedded image [Reagents used] 1-Methyl-β-galactose 3.0 g (15 mmol) Sodium hydride (60%) 5.1 g (0.21 mmol) Benzyl chloride 75 ml [Procedure] Same as α-form (Trial 5), but column chromatography method In the purification by, the developing solvent was changed from n-hexane to ethyl acetate. After that, recrystallization was performed with methanol to obtain a yellow white solid. [Results] Yield (yield) 3.5 g (41%) Melting point 82.0-82.4 ° C TLC (Silica gel 70FM Plate Wako / n-hexane)

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7 ) Synthesis of 2,3,4,6-tetra-O-benzylgalactopyranoside ( 7 )

[Chemical 21] [Operation] (Trials 1 and 2) 0.8 g (1.6 mmol) of compound ( 6 ) was dissolved in 20 ml of acetic acid, 8 ml of 2N hydrochloric acid was added, and the mixture was heated with stirring at 80 ° C for 10 hours. Then, acetic acid and hydrochloric acid were distilled off under reduced pressure to obtain a white viscous substance. This is subjected to column chromatography [Wakogel C-
300 / chloroform: ethyl acetate = 30: 1 (v / v)] to obtain a white viscous substance. (Trial 3, 4) 0.5 g (0.90 mmol) of compound ( 6 ) in 10 ml of acetic acid
It was dissolved in 1.2 ml of ³ mol / dm ³ sulfuric acid and stirred at 80 ° C. for 20 minutes. Then, 30 ml of cold water was added and extracted with benzene. The organic layer was washed with a 4% aqueous sodium hydrogen carbonate solution and water in this order, and then the solvent was distilled off under reduced pressure to obtain 0.49 g of a transparent oily substance. This was purified by column chromatography [Wakogel C-300 / chloroform: ethyl acetate = 30: 1 (v / v)] to obtain a white viscous substance. (Trial 5) The same operation as in Trial 3 was performed using the β-form ( 6 ′) as a raw material.

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8) 1-methoxyacetyl-2,3,4
Synthesis of 6-tetra-O-benzyl-D-galactopyranoside ( 8 )

Embedded image [Procedure] Compound ( 7 ) 100 mg (0.18 mmol, α: β = 63: 37),
Methoxyacetic acid (30 mg, 0.26 mmol), 4-dimethylaminopyridine (10 mg, 0.08 mmol) and DCC (60 mg, 0.28 mmol) were dissolved in anhydrous methylene chloride and stirred at room temperature for 24 hours. Then, the insoluble matter was filtered off, the solvent was distilled off under reduced pressure, and the obtained oily substance was dissolved in ethyl acetate and left in a refrigerator overnight. Further, the insoluble matter was filtered and the solvent was distilled off under reduced pressure to obtain a white oily substance. This was subjected to column chromatography [Wakogel C-300 / chloroform: ethyl acetate = 20:
1 (v / v)] to obtain a pale white oily substance.

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9) Ytterbium trifluoromethanesulfonate

Embedded image 5.0 g (12.6 mmol) of ytterbium oxide was dissolved in a trifluoromethanesulfonic acid aqueous solution [50% (v / v)] and refluxed for 1 hour. The insoluble matter was filtered and the solvent was distilled off under reduced pressure to obtain a white solid. This was dried under vacuum at 180 ° C. to obtain a white solid. Further, this was reprecipitated with acetonitrile-methylene chloride to obtain a white fine powder. [Results] Yield (Yield) 7.9 g (50%)

10) N, N-dihexadecyl-N-
Synthesis of [6- (2,3,4,6-tetra-O-benzylgalactopyranosyl) hexanoyl] alaninamide ( 9 )

Embedded image [Operation] [Trial 1] ( 8 ) (α: β = 70: 30) 0.40g (0.64mm
ol) and 0.45 g (0.65 mmol) of ( 5 ) were dissolved in anhydrous methylene chloride and refluxed for 30 minutes, 0.39 g (0.64 mmol) of ytterbium trifluoromethanesulfonate was added, and the mixture was heated under reflux for 1.5 hours. The resulting yellow transparent oily product was purified by column chromatography [Wakogel C-100 / ether] to remove the catalyst, and the conditions were changed [Wakogel C-300 / chloroform], followed by gel chromatography [Sephadex LH-20 / methanol: Chloroform = 1: 1 (v / v)] to obtain a transparent oil. This was dried under reduced pressure to obtain a white solid. [Trial 2] A tube filled with molecular sieve 4A was attached between the flask and the reflux condenser, and the reflux time was set to 2 hours.

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11) N, N-dihexadecyl-N-
[6- (galactopyranosyl) hexanoyl] -L-alanine amide ( 1 )

Embedded image [Operation] Compound ( 10 ) (α: β = 70: 30) 480mg (0.40mmo
l) was dissolved in 20 ml of THF, and hydrogen gas was slowly blown in for 12 hours using palladium black as a catalyst. The obtained yellow transparent oily substance was subjected to gel chromatography [Sephadex LH-20 / methanol: chloroform = 1: 1 (v / v)] and column chromatography [Wakogel C-300 / ethyl acetate: methanol = 30: 1 → 10]. : 1 (v / v)] and dried under reduced pressure to obtain a yellow viscous solid.

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Embedded image [Result] yield (yield) 220mg (67%) α: β = 73: 27 (1 calculated from integral ratio of 1 H-NMR spectrum first proton) TLC [silica gel 70FM Plate Wako / chloroform: methanol = 7: 1 ( v / v] Rf = 0.87 (single spot) Melting point 82.4-83.0 ° C

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07K 5/06 ZNA 8517-4H C07K 5/06 ZNA 5/08 8517-4H 5/08 5/10 8517-4H 5/10 7/06 8517-4H 7/06 C11C 3/00 C11C 3/00

Claims (1)

[Claims] 1. A synthetic lipid represented by the general formula (I). Embedded image [In the formula, X represents a monosaccharide residue or an oligosaccharide residue, and R ¹ is represented by the formula: , L represents an integer of 1 to 8, m and n represent an integer of 1 to 18, and p represents an integer of 1 to 5. ]