JPH0635478B2 - Cationic amphiphile and method for producing the same - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention has an affinity (amphipathicity) for both an aqueous solvent and an organic solvent. Therefore, in water, molecules are bound to each other to form a closed endoplasmic reticulum ( The present invention relates to a chaotic amphipathic substance composed of an oligopeptide derivative which forms a liposome) and acts as an emulsifier in a two-component system of water and oil, and a method for producing the same. Industrial applications of this invention include foods, cosmetics, dyes and other emulsifiers, stabilizers,
It is suitable for use as a dispersant, a wetting agent, and as a drug carrier material for encapsulating a water-soluble drug in the endoplasmic reticulum.

Conventional Technology As a conventional technology, phospholipids, which are the basic skeleton of cell membranes, and particularly phosphatidylcholine (generally called lecithin), which is the most abundant phospholipid in nature, are known (eg, Yasuo Kagawa, “Ecology”). Membrane "(June 27, 1978), Iwanami Shoten, p39). However, most of these lecithins are made from soybean or egg yolk, and are commercially available as a mixture of various phospholipids having different structures in terms of purity, and are used industrially. Therefore, the physical and chemical stability of the endoplasmic reticulum is poor, which hinders its use as a drug carrier. In addition, it is very difficult to obtain highly pure lipids by separation and purification, because phospholipids having slightly different alkyl chain lengths and structures are mixed, which is very difficult.

DISCLOSURE OF THE INVENTION Under the circumstances, the present invention has a dramatically higher purity as compared with a natural product, and has the same amphipathic characteristics as a natural product, and is stable for a long time. The present invention has been made for the purpose of providing a novel amphipathic substance having excellent endoplasmic reticulum formation ability.

Means for Solving the Problems As a result of intensive studies for developing a novel amphipathic substance, the present inventors have found that kaothin, which is an oligopeptide derivative derived from an imino acid having one carboxyl group, It was found that a sex amphiphile can meet the purpose, and the present invention has been completed based on this finding.

That is, the present invention has the general formula (Where X is a halogen atom, A is an oligopeptide residue derived from an imino acid having one carboxyl, R is
Provides a cationic amphipathic substance composed of an oligopeptide derivative represented by a long-chain alkyl group having 12 to 22 carbon atoms. X in the general formula (I) is a halogen atom, and examples thereof include a chlorine atom and a bromine atom. Also, A1, A2,
, An is an imino acid residue having one carboxyl group (hereinafter also referred to as imino acid residue), and examples thereof include a sarcosine residue, a proline residue and the like. R is a long-chain alkyl group having 12 to 22 carbon atoms, and examples thereof include dodecyl group, tetradecyl group, hexadecyl group, octadecyl group, eicosyl group and docosyl group.

The compounds represented by the general formula (I) are all novel compounds which have not been published in the literature. For example, general formula P-AOH (II) [wherein A is an imino acid having one carboxyl group] A compound represented by the general formula: (Wherein X represents a halogen atom and R represents a long-chain alkyl group having 12 to 22 carbon atoms) and a glutamic acid diamide salt are coupled with diethylphosphorusocyanidate or diphenylphosphoryl azide to protect them. It can be produced by removing the group.

An oligopeptide having the N-terminal protected in the general formula (II) used as a starting compound in this case, for example, P-A1-A
2-A3-OH [A1, A2 and A3 in the formula are imino acid residues having one carboxyl group (hereinafter also referred to as imino acid residues), and they may be the same or different. First, an imino acid having one carboxyl with an imino group protected (hereinafter also referred to as inomiic acid), -A2
-OH, an imino acid in which a carboxyl group is protected, H-A3
By reacting with-to form a dipeptide, and then removing the imino-protecting group, and then reacting this with an imino-protected imino acid, -A1-OH to form a tripeptide. Obtained by removing the end protecting group. As the imino protecting group, the carboxyl protecting group, and the coupling agent in synthesizing the oligopeptide, reagents and methods used in ordinary peptide synthesis can be used in an appropriate combination. The peptides, which are production intermediates, can be easily isolated and purified by washing with acid and alkali, recrystallization and reprecipitation.

In addition, the long-chain alkyl derivative of the general formula (III) is, for example, glutamic acid with an amino group protected is reacted with hydroxysuccinimide to give a bifunctional active ester, which is then reacted with a long-chain monoalkylamine. Obtained by removing the amino protecting group.

The coupling of the compound of the general formula (II) and the compound of the general formula (III) is carried out by dissolving both components in an organic solvent at low temperature with sufficient stirring, while adding diethylphosphorocyanidate (DEPC) thereto. Alternatively, diphenylphosphoryl azide (DPPA) dissolved in a very small amount of organic solvent was added, and then triethylamine (TEA) dissolved in a very small amount of organic solvent was added, and the mixture was added at low temperature for several hours. Stir overnight at room temperature. Then the acid,
The desired product can be obtained by washing with alkali.

The molar ratio in the reaction is (II) :( III): DEPC
(Or DPPA): TEA can be prepared by reacting 1: 1: 1: 2, but (III), DEPC (or DPPA) and TEA are preferably used in 0.1 to 0.2 equivalent excess. There is no problem if the reaction temperature is 0 ° C. or lower, but 0 to −5 ° C. is suitable for promoting the reaction. As the solvent, dimethylformamide, methylene chloride, chloroform, tetrahydrofuran, etc. are used from the viewpoint of solubility and reaction rate. Of these, dimethylformamide, which dissolves peptides well, is desirable. In particular, it is desirable to completely dissolve the compounds of the general formulas (II) and (III) in order to increase the reaction yield. As the reaction time, 2 to 6 hours at a low temperature and half a day at room temperature are suitable.

In order to purify and isolate the target compound from the reaction mixture thus obtained, an organic solvent immiscible with water such as chloroform is added to the reaction mixture, and the organic layer is subjected to thin layer chromatography to obtain a single product of the target compound. Wash with weak acid aqueous solution, weak alkaline aqueous solution, and saturated saline until spots are obtained. As the weak acid aqueous solution, 10% citric acid aqueous solution, 2% dilute hydrochloric acid, and as the weak alkaline aqueous solution, 4% sodium hydrogen carbonate aqueous solution is used. From the thus washed organic layer, a trace amount of water is removed with a suitable desiccant such as anhydrous sodium sulfate, and the solvent is removed under reduced pressure to obtain an oily substance. This can be obtained as a solid or amorphous substance by treating with water or a mixed solvent of water / chloroform / methanol.

Finally, to remove the N-terminal protecting group, 25% hydrogen bromide /
Acetic acid, or with 3-4M hydrogen chloride / organic solvent at room temperature for 1-
After treating for several hours and removing the solvent under reduced pressure, water or a mixed solvent of water / chloroform / methanol can be added to the residue to obtain a solid compound of the present invention.

In the compound of the present invention, the actually measured elemental analysis value agrees with the calculated value within an error range, and the infrared absorption spectrum shows that it is 1640 to
1660 cm ^-1 represent a characteristic absorption derived from an amide carbonyl group, in the ¹³ C-NMR, [delta] value is 14.1 ppm, 2
2.7 ppm, 29.3 to 29.7 ppm, 32.6 ppm, 3
At 9.8 to 40.2 ppm, a signal attributed to each methylene or methyl group carbon of the long-chain alkyl group, and further a signal of carbon contained in each imino acid residue and amino acid residue can be observed. The product can be identified.

EFFECTS OF THE INVENTION The compound of the present invention can obtain vesicles having a size of several nanometers to several microns by adding distilled water to these and shaking them by hand or performing ultrasonic treatment. By pre-dissolving a water-soluble drug in distilled water, the molecular assembly containing the drug in the aqueous solution region of the endoplasmic reticulum is mixed with a hydrophobic substance. A molecular assembly in which the compounds coexist is obtained.

Next, the present invention will be described in more detail with reference to examples. As the Rf value of thin layer chromatography, a value using a chloroform / methanol (5/1, volume ratio) mixed solvent as a developing solvent is Rf1, and chloroform / methanol / acetic acid (9
5/5/1, volume ratio) Rf2, n-butanol / acetic acid / water (4/1/2, volume ratio) mixed solvent was used as the developing solvent. It was set to Rf3.

Reference Example 1 Preparation of (A) t-butyloxycarbonyl-L-prolyl-L-prolyl-L-prolyl-L-proline 1.50 g (0.0048 mol) of t-butyloxycarbonyl-L-prolyl-L-proline ) And N-methylmorpholine 0.53 ml (0.0048 mol) in chloroform 1
After dissolving in 5 ml and cooling to -20 ° C, 0.63 ml of isobutyl chloroformate (0.00
48 mol) was added. 15 minutes stirring, L-prolyl-L-proline benzyl ester hydrochloride 1.63 g
(0.0048 mol) and then 0.53 ml (0.0048 mol) of N-methylmorpholine were added, and the mixture was reacted by stirring at -20 ° C for 6 hours and further at room temperature overnight. The chloroform solution containing the reaction mixture was washed repeatedly with 4% aqueous sodium hydrogen carbonate solution, 10% aqueous citric acid solution and saturated saline solution until the thin layer chromatogram gave a single spot. Then, it was washed with distilled water and dried over anhydrous sodium sulfate. Then, the solvent was removed under reduced pressure to obtain 2.56 g (yield 89%) of a colorless transparent syrup. This t-butyloxycarbonyl-L-prolyl-L
2.56 g of -prolyl-L-prolyl-L-proline benzyl ester was dissolved in 40 ml of t-butyl alcohol, and catalytic hydrogen reduction was carried out for 4 hours using 5% palladium / carbon as a catalyst. The catalyst was filtered off, the solvent was removed under reduced pressure, and the colorless transparent oil was solidified with petroleum ether and recrystallized from ethyl acetate / petroleum ether to give 1.75 g of the desired compound having a melting point of 95-97 ° C (yield 81 %) Was obtained. The physical properties of this product are as follows.

Rf value of thin layer chromatography Rf1 = 0.13, Rf2 = 0 Elemental analysis value (as C ₂₅ H ₃₈ O ₇ N ₄ .H ₂ O) C H N calculated value (%) 57.24 7.69 10. 68 Measured value (%) 57.45 7.78 10.30 (B) Instead of the proline residue in (A) of Reference Example 1, each corresponding imino acid residue was used, and the following scanning was conducted. The compound shown in was obtained.

t-Butyloxycarbonyl-sarcosyl-sarcosyl-sarcosyl-sarcosine (semisolid) t-butyloxycarbonyl-sarcosyl-sarcosyl-sarcosine (semisolid) (C) L Production of glutamic acid didodecylamide hydrobromide Benzyloxycarbonyl -L-glutamic acid 5g
(0.0178 mol) and 4.09 g (0.0356 mol) of N-hydroxysuccinimide were dissolved in 30 ml of dimethylformamide, and 8.07 g (0.039 mol) of dicyclohexylcarbodiimide was dissolved while stirring at 0 ° C. 10 ml of dimethylformamide solution were added. The mixture was stirred overnight at 0 ° C., insoluble by-products were filtered, and the filtrate was removed under reduced pressure to give a colorless transparent foam solidified with ether. 1 g (0.0021 mol) of hydroxysuccinimide ester derivative of glutamic acid (melting point 73 to 74 ° C.) obtained by recrystallization from ethyl acetate / isopropanol and 0.78 g (0.0042 mol) of dodecylamine
Was dissolved in 10 ml of chloroform and left at room temperature for 2 days. The reaction solution was washed with 4% aqueous sodium hydrogen carbonate solution and distilled water, and the solvent was removed under reduced pressure to give a white solid.
Wash with ether and methanol, melting point 138-139 ° C
1.17 g (yield 91%) of the compound of was obtained. 25% hydrogen bromide / 0.83 g (0.00135 mol) of this compound
4.2 ml of acetic acid solution was reacted for 2 hours, the formed precipitate was dissolved in ether, and the solvent-free residue was recrystallized from a water / chloroform / methanol mixed solvent to give a melting point of 118-.
The target compound at 122 ° C. was obtained. The physical properties of this product are as follows.

Rf value RF1 = 0.49 in thin layer chromatography, RF2 = 0.03 Elemental analysis _{(C 29} H ₆₀ O as a _{2 N} 3 Br) C H _N calc (%) 61.90 10.75 7.47 Actual value (%) 61.57 10.69 7.61 (D) Instead of dodecylamine in (C) of Reference Example 1,
By using octadecylamine in exactly the same manner,
L-Glutamic acid dioctadecylamide hydrobromide (melting point 125-127 ° C) was obtained.

Example 1 Preparation of L-prolyl-L-prolyl-L-prolyl-L-prolyl-L-glutamic acid didodecylamide hydrochloride t-Butyloxycarbonyl-L-prolyl-L-prolyl-L-prolyl-L-proline 0.53g (0.001
04 mol) and 0.70 g (0.00124 mol) of L-glutamic acid didodecylamide hydrobromide were dissolved in 50 ml of dimethylformamide, and 0.27 ml of diphenylphosphoryl azide (0 ml) was added while stirring at 0 ° C. ．
5m of dimethylformamide solution containing 10024 mol)
l, followed by the addition of 5 ml of a dimethylformamide solution containing 0.32 ml of triethylamine. After stirring at 0 ° C. for 6 hours, the mixture was stirred at room temperature overnight. Chloroform (100 ml) was added to the reaction solution, which was washed twice with 10% aqueous citric acid solution, 4% aqueous sodium hydrogen carbonate solution, saturated saline solution and distilled water, and the chloroform layer was dried over anhydrous sodium sulfate, and then the solvent was removed. The residual oil was purified by silica gel column chromatography. 0.65 g of the obtained pale yellow oil (Rf1 = 0.77) was dispersed in ethyl acetate and reacted with 10 ml of 3.5N hydrogen chloride / ethyl acetate for 1 hour at room temperature. The light yellow oil obtained by removing the solvent was recrystallized from water / methanol / chloroform,
400 mg of the target compound as a pale yellow solid having a melting point of 76-78 ° C.
(Yield 77%) was obtained. The gel-liquid crystal phase transition temperature (Tc) of this product was a doublet of 51 ° C. and 55 ° C. (determined by differential scanning calorimetry), and the critical aggregate formation concentration was 3.
It was 1 × 10 ⁻⁵ mole / 1 (determined by the surface tension method). Other physical properties of this are as follows.

Specific rotation ▲ [α] ²⁵ _D ▼ = −81.58 ° (c1.07, ethanol) Rf value of thin layer chromatography RF1 = 0.16, RF2 = 0 Elemental analysis value (C ₄₉ H ₈₈ O ₆ N ₇ Cl · H ₂ O · CH
₃ OH) CHN calculated value (%) 62.77 9.90 10.25 measured value (%) 63.02 9.94 9.42 The ¹³ C-NMR spectrum of this product is shown in FIG. 1.

Example 2 Preparation of sarcosyl-sarcosyl-sarcosyl-L-glutamic acid didodecylamide hydrochloride 0.77 g (0.00232 mol) of t-butyloxycarbonyl-sarcosyl-sarcosyl-sarcosine and L-
Glutamic acid didodecylamide hydrobromide 1.57 g
70 ml of dimethylformamide (0.00279 mol)
2 ml of a dimethylformamide solution containing 0.45 g of diethylphosphorus cyanidate while stirring at 0 ° C., followed by triethylamine
2 ml of a dimethylformamide solution containing 72 ml was added.
Then, the same treatment as in Example 1 was carried out to obtain t-butyloxycarbonyl-sarcosyl-sarcosyl-sarcosine-L-.
2.00 g of glutamic acid didodecylamide (yield 100
%) Was obtained. This product (1.84 g, 0.00231 mol) was dispersed in ethyl acetate and treated with hydrogen chloride in the same manner as in Example 1 to obtain 260 mg (yield 15%) of a white solid having a melting point of 163 to 164 ° C. This compound had a gel-liquid crystal phase transition temperature (Tc) of 47 ° C. (determined by differential scanning calorimetry) and had a critical aggregate formation concentration of 2.8 × 10 ⁻⁵ mole / 1.
(Determined by the surface tension method). Other physical properties of this are as follows.

Specific rotation ▲ [α] ²⁵ _D ▼ = −7.85 ° (c1.07, ethanol) Rf value of thin layer chromatography RF1 = 0.04, RF3 = 0 Elemental analysis value (C ₃₈ H ₇₅ O ₅ N ₆ Cl / 1 / 2H ₂ O
C H N calculated value (%) 61.63 10.34 11.35 Measured value (%) 61.45 10.38 11.12 The ¹³ C-NMR spectrum of this product is shown in FIG.

Example 3 Sarcosyl-sarcosyl-sarcosyl-sarcosyl-L
-Preparation of glutamic acid dioctadecyl amide hydrochloride t-butyloxycarbonyl-sarcosyl-sarcosyl-sarcosyl-sarcosine 0.40 g (0.00099)
4 mol) and 0.87 g (0.00119 mol) of L-glutamic acid dioctadecylamide hydrobromide were dispersed in 100 ml of dimethylformamide, and 0.19 g of diethylphosphorusanidate was added while stirring at 0 ° C. 5 ml of a dimethylformamide solution containing 0.31 ml of triethylamine was added, followed by 5 ml of a dimethylformamide solution containing 0.31 ml of triethylamine. Then, the same treatment as in Example 1 was carried out to obtain the target compound 4 as a white solid having a melting point of 181 to 184 ° C.
00 mg (yield 74%) was obtained. The physical properties of this product are as follows.

Rf value RF1 = 0.07 in thin layer chromatography, RF2 = 0 Elemental analysis _{(C 53 H 104 O 6 N} 7 as _{Cl · H 2 O) C H} N calc (%) 64.37 10.80 9 .91 measured value (%) 64.10 10.75 9.75

[Brief description of drawings]

FIG. 1 shows ¹³ C-N of Example 1 among the compounds of the present invention.
MR spectrum diagram (in deuterated chloroform, 25 ° C), second
The figure is the ¹³ C-NMR spectrum diagram of Example 2 (25 ° C. in deuterated methanol).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display area B01J 13/02 C07K 1/06 1/10 7/06 Z 8318-4H // A23L 1/03 8214 -4B 1/035 8214-4B A61K 7/00 C 7252-4C N 7252-4C T 7252-4C C07K 99:00

Claims (2)

[Claims] 1. A general formula (Wherein X is a halogen atom, A is an oligopeptide residue derived from an imino acid having one carboxyl,
R represents a long-chain alkyl group having 12 to 22 carbon atoms), and is a cationic amphipathic substance consisting of an oligopeptide derivative. 2. A compound represented by the general formula P-A-OH [wherein A represents an oligopeptide residue derived from an imino acid having one carboxyl group, and P represents an N-terminal protecting group]. And the general formula (Wherein X represents a halogen atom and R represents a long-chain alkyl group having 12 to 22 carbon atoms) and a glutamic acid diamide salt represented by the formula (3) is coupled with diethylphosphorocyanidate or diphenylphosphoryl azide. Characteristic general formula (X, A and R in the formula have the same meanings as described above) A method for producing a cationic amphipathic substance comprising an oligopeptide derivative represented by the formula: