JP2015086319A - Branched polyethylene glycol modified lipid having two polyethylene glycol chains having different molecular weight and manufacturing method therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a branched polyethylene glycol modified lipid having two polyethylene glycol chains having different molecular weight increasing a hydrated layer on a liposome surface and easily being industrially manufactured and a manufacturing method therefor.SOLUTION: There is provided a branched polyethylene glycol modified lipid having two polyethylene glycol chins having different molecular weight represented by the formula (1), where each symbol are as defined in the specification.

Description

  The present invention relates to a branched polyethylene glycol-modified lipid containing two polyethylene glycol chains having different molecular weights, and a method for producing the same.

In recent years, in the field of drug delivery systems, research on liposomes as a transport carrier has been widely performed. Liposomes are closed vesicles having a diameter of several tens to several hundreds of nanometers made of molecules having a hydrophilic part and a hydrophobic part on one molecule, imitating a lipid bilayer of a cell membrane. Drugs such as anticancer agents can be stably encapsulated therein. However, since the liposome preparation is trapped by reticuloendothelial system (RES) in blood, the stability and retention in blood are low, and sufficient pharmacological effects cannot be obtained.
In order to solve this problem, attempts have been made to improve the in vivo behavior with PEG liposomes whose surface is modified with polyethylene glycol (PEG). PEG liposomes have high blood retention due to steric hindrance between the hydration layer formed by the surface PEG chain and the PEG chain itself, which suppresses interactions with serum proteins such as opsonin and cells belonging to the mononuclear cell system. Can be realized.
There is a correlation between the hydrated layer of the liposome and the blood retention, and it is known that the blood retention increases when the hydrated layer is thickened. Therefore, for the purpose of increasing the hydration layer, an increase in the addition amount of the PEG lipid derivative, which is a constituent component of the liposome, and an increase in the molecular weight of the PEG chain have been studied. However, as the molecular weight of the PEG chain increases, the structure of the PEG chain on the liposome surface changes from a vertically stretched brush structure to a mushroom structure with a three-dimensional lateral extension, so the length of the PEG chain There was a problem that the hydration layer did not increase more than expected.

  In order to solve this problem, in Non-Patent Document 1, a liposome modified by mixing two kinds of PEG lipid derivatives having different molecular weights of PEG chains increases the thickness of the hydrated layer, thereby increasing the retention of blood in the liposome. It has been reported to improve. This is thought to be due to the fact that the brush structure of the short chain PEG changes the mushroom structure of the long chain PEG to the brush structure, so that the thickness of the hydration layer is increased. However, since long-chain PEG is more easily detached from the liposome surface than short-chain PEG, this mixed modified liposome has a problem that the thickness of the hydrated layer decreases with time in blood and tumor.

  In addition, PEG phospholipid derivatives in which two types of PEG with different molecular weights are introduced in one molecule are known, and liposomes modified by this form a hydrated layer with the same thickness as mixed modified liposomes. And can be maintained. This PEG phospholipid derivative uses phosphatidylserine in which phospholipid and amino acid serine are bound as an intermediate. However, in the synthesis of phosphatidylserine, phosphatidic acid is hydrolyzed by the hydrolysis activity of phospholipase D during the reaction in an aqueous system, even though serine and phospholipase D, which is an enzyme catalyst, are only soluble in water as disclosed in Patent Document 1. As a by-product, there is a problem that the purity of phosphatidylserine is low. In addition, the bond formation reaction between phosphatidylserine and the activated PEG reagent also requires a reaction in a mixed system of an organic solvent and water, so that the activated PEG reagent is decomposed during the reaction and the purity further decreases. Invite. Therefore, since the PEG phospholipid derivative, which is the final product, requires purification by column chromatography, it causes a decrease in yield, an increase in the amount of solvent used, and a prolonged production time, which is unsuitable for industrial production.

  Thus, the appearance of a polyethylene glycol-modified lipid that effectively increases the hydration layer on the surface of the liposome and is industrially easy to produce is awaited.

JP 2004-215528 A

Pharmaceutical Journal, 2005, 125 (1), p. 149-157

  An object of the present invention is to provide a branched polyethylene glycol-modified lipid containing two polyethylene glycol chains having different molecular weights, which can increase the hydration layer on the liposome surface and can be easily produced industrially, and a method for producing the same. is there.

  As a result of intensive studies to solve the above problems, the present inventors have found that a branched polyethylene glycol-modified lipid having a novel structure and two polyethylene glycol chains having different molecular weights, a method for producing the same, and a polyethylene glycol-modified liposome And the present invention was completed.

That is, the present invention is as follows.
[1] The following formula (1)

(Wherein R ¹ and R ² are each independently a hydrocarbon group having 1 to 7 carbon atoms, and R ³ CO is an acyl group having 4 to 24 carbon atoms. M and n are each independently , An average addition mole number of oxyethylene groups, an integer between 10 and 125, and an integer satisfying n ≦ 25 and 2n ≦ m, or m ≦ 25 and 2m ≦ n X ¹ and X ² Are independently selected from the groups represented by the following formulas (3) to (7), and X ³ is selected from a carbonate bond, a carbamate bond and an ether bond.)

(In the formula, r is an integer of 1 to 5, and s is an integer of 2 to 5.)
A branched polyethylene glycol-modified lipid containing two polyethylene glycol chains having different molecular weights represented by the formula:
[2] In the above [1], X ¹ is a group represented by the above formula (7), X ² is a group represented by the above formula (5), and X ³ is a carbamate bond represented by the following formula (8). A branched polyethylene glycol-modified lipid containing two polyethylene glycol chains having different molecular weights.

(Wherein R ¹ and R ² are each independently a hydrocarbon group having 1 to 7 carbon atoms, and R ³ CO is an acyl group having 4 to 24 carbon atoms. M and n are each independently , An average addition mole number of oxyethylene groups, an integer between 10 and 125, an integer satisfying n ≦ 25 and 2n ≦ m, or m ≦ 25 and 2m ≦ n, and s is 2 to 5 Is an integer.)
[3] A branch containing two polyethylene glycol chains having different molecular weights according to the above [2], which comprises the following steps (A), (B), (C) and (D) in order. For producing a modified polyethylene glycol-modified lipid.
Step (A): 1,3-diamino-2-propanol is reacted with a polyethylene glycol derivative represented by the following formula (9) (hereinafter also referred to as compound (9)), and one of the amino groups has a polyethylene glycol chain. Then, another amino group is protected with a chemically inert protective group to obtain a compound represented by the following formula (10) (hereinafter also referred to as compound (10)).

(In the formula, R ¹ is a hydrocarbon group having 1 to 7 carbon atoms. M is an average added mole number of an oxyethylene group, and is an integer between 10 and 125, and L ¹ is N-succin. (It is an imidyloxy group or a p-nitrophenoxy group, and P is an amino-protecting group.)
Step (B): After converting the hydroxyl group of the compound represented by the formula (10) to an active group, a polyethylene glycol derivative represented by the following formula (11) (hereinafter also referred to as the compound (11)) is reacted. A step of obtaining a compound represented by the following formula (12) into which a second polyethylene glycol chain is introduced (hereinafter also referred to as compound (12)).

(In the formula, R ¹ and R ² are each independently a hydrocarbon group having 1 to 7 carbon atoms. M and n are each independently an average addition mole number of an oxyethylene group, 10 Is an integer satisfying n ≦ 25 and 2n ≦ m, or m ≦ 25 and 2m ≦ n, s is an integer of 2 to 5, and P is an amino-protecting group. is there.)
Step (C): The protecting group of the amino group of the compound represented by the above formula (12) obtained in the step (B) is deprotected, and the compound represented by the following formula (13) (hereinafter, also referred to as the compound (13)) Step).

(In the formula, R ¹ and R ² are each independently a hydrocarbon group having 1 to 7 carbon atoms. M and n are each independently an average addition mole number of an oxyethylene group, 10 And an integer satisfying n ≦ 25 and 2n ≦ m, or m ≦ 25 and 2m ≦ n, and s is an integer of 2 to 5.)
Step (D): The compound represented by the above formula (13) obtained in the step (C) is reacted with a diacylglycerol derivative represented by the following formula (14) (hereinafter also referred to as the compound (14)) to form the following formula: A step of obtaining a branched polyethylene glycol-modified lipid represented by (8).

Wherein R ¹ and R ² are each independently a hydrocarbon group having 1 to 7 carbon atoms, R ³ CO is an acyl group having 4 to 24 carbon atoms, and L ³ is N-succinimidyl. An oxy group or a p-nitrophenoxy group, wherein m and n are each independently the average number of moles added of the oxyethylene group, an integer between 10 and 125, where n ≦ 25 and 2n ≦ m. Or an integer satisfying m ≦ 25 and 2m ≦ n, and s is an integer of 2 to 5.)

  The novel branched polyethylene glycol-modified lipid (1) containing two polyethylene glycol chains having different molecular weights according to the present invention has a diacylglycerol structure as a lipid moiety, and a non-amino acid is present in the lipid moiety and the linker moiety of the PEG chain. It has a structure which is a certain glycerin derivative. Therefore, the reaction in an organic solvent is possible in all the reaction steps, and the reduction of the reaction rate due to hydrolysis of the active group of the PEGylation reagent can be prevented, so that the purity is high without using purification such as column chromatography. It can be manufactured efficiently. Furthermore, since the polyethylene glycol-modified lipid (1) of the present invention effectively increases the hydration layer on the surface of the liposome, it is useful as a DDS excellent in blood retention.

As used herein, “C _1-7 alkyl” includes, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl unless otherwise specified. , Heptyl and the like. As the _{"C 1-4} alkyl" in the present specification, among the above-mentioned _{"C 1-7} alkyl" _include the _{C 1-4.}

As used herein, “C _2-7 alkenyl” is, for example, vinyl, propenyl, isopropenyl, 2-buten-1-yl, 4-penten-1-yl, 5-hexene unless otherwise specified. -1-yl and the like can be mentioned.

As used herein, “C _2-7 alkynyl” includes, for example, 2-butyn-1-yl, 4-pentyn-1-yl, 5-hexyn-1-yl and the like, unless otherwise specified. It is done.

As used herein, “C _3-7 cycloalkyl” includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, unless otherwise specified. As the _{"C 3-6} cycloalkyl" as used herein, of the above-mentioned _{"C 3-7} cycloalkyl" _include the _{C 3-6.}

As used herein, “C _3-7 cycloalkenyl” is, for example, cyclopropenyl (eg, 2-cyclopropen-1-yl), cyclobutenyl (eg, 2-cyclobuten-1-yl) unless otherwise specified. ), Cyclopentenyl (eg, 1-cyclopenten-1-yl, 2-cyclopenten-1-yl, 3-cyclopenten-1-yl), cyclohexenyl (eg, 1-cyclohexen-1-yl, 2-cyclohexene-1) -Yl, 3-cyclohexen-1-yl) and the like.

R ¹ and R ² in the formula (1) of the present invention each independently represent a hydrocarbon group having 1 to 7 carbon atoms. Examples of the “C _1-7 hydrocarbon group” represented by R ¹ and R ² include a C _1-7 alkyl group, a C _2-7 alkenyl group, a C _2-7 alkynyl group, and a C _3-7 cycloalkyl group. A C _3-7 cycloalkenyl group, a C _3-6 cycloalkyl-C _1-4 alkyl group, a C _3-6 cycloalkenyl-C _1-4 alkyl group, a phenyl group, and a benzyl group. Preferably it is a _C1-7 alkyl group, More preferably, they are a methyl group and an ethyl group, More preferably, it is a methyl group.

R ³ CO represents an acyl group derived from a fatty acid having 4 to 24 carbon atoms (preferably 12 to 24 carbon atoms). Specific examples of R ³ CO include butyric acid, isobutyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, Mention may be made of acyl groups derived from saturated and unsaturated linear or branched fatty acids such as arachidic acid, behenic acid, erucic acid and lignoceric acid. When the number of carbon atoms in R ³ CO exceeds 24, there is a problem in forming liposomes because of poor dispersion in the aqueous phase. In addition, when the number of carbon atoms of R ³ CO is less than 4, the hydrophobicity is low, and thus the interaction with the liposome membrane is weak, causing problems when forming liposomes.

  m and n are each independently the average number of moles added of the oxyethylene group, each independently an integer between 10 and 125, and n ≦ 25 and 2n ≦ m, or m ≦ 25 and 2m. It is an integer that satisfies ≦ n. When n ≦ 25 and 2n ≦ m, or m ≦ 25 and 2m ≦ n are not satisfied, there is no difference in conformation derived from the chain length of the two polyethylene glycols when the liposome is prepared. The layer thickness may not be formed. When m or n is smaller than 10, the blood retention effect caused by the polyethylene glycol chain is reduced. In addition, when m or n is larger than 125, there is a problem that the compound (1) itself is easily detached from the liposome when the liposome is prepared.

X ¹ and X ² are each independently a linker between the polyethylene glycol chain and the glycerol derivative, and X ³ is a linker between the glycerol derivative and the lipid derivative. X ¹ and X ² are each independently selected from the groups represented by the following formulas (3) to (7), and X ³ is selected from a carbonate bond, a carbamate bond, and an ether bond.

(In the formula, r is an integer of 1 to 5, and s is an integer of 2 to 5.)
The carbamate bond is —NH—COO— or —OCO—NH—.
X ^1, X ² and X ^3, the stability of the surface of the ready availability and chemical bonding glycerin derivative as a raw material, X ¹ and X ² are the equations forming the carbamate bond (5) and ( A group selected from the group represented by 7) and the group represented by the above formula (6) constituting an amide bond is preferable, and X ³ is preferably a carbamate bond. More preferably, X ¹ and X ² are a group represented by the above formula (5) or a group represented by the formula (7), and X ³ is —NH—COO—. Particularly preferably, X ¹ is a group represented by the above formula (7), X ² is a group represented by the above formula (5), and X ³ is —NH—COO—.

(Production method)
The branched polyethylene glycol-modified lipid (8) of the present invention can be produced, for example, as follows.
After reacting 1,3-diamino-2-propanol with a polyethylene glycol derivative represented by the following formula (9) to introduce a polyethylene glycol chain into one of the amino groups, the other amino group is chemically inactivated. Protection with an active protecting group yields a compound represented by the following formula (10) (step (A)). Next, after converting the hydroxyl group of the compound represented by the following formula (10) to an active group, the polyethylene glycol derivative represented by the following formula (11) is reacted to introduce the second polyethylene glycol chain: 12) is obtained (step (B)). Subsequently, the protecting group of the amino group of the compound represented by the formula (12) obtained in the step (B) is deprotected to obtain a compound represented by the formula (13) (step (C)). Finally, the branched polyethylene glycol-modified lipid represented by the formula (8) can be obtained by reacting the compound represented by the formula (13) with the diacylglycerol derivative represented by the formula (14) (step (D)). ).

  The reaction pathway of the branched polyethylene glycol modified lipid (8) is shown below.

(Each symbol in the formula is as defined above)

  A detailed description of each step will be given below.

Although the manufacturing method in particular of a compound (10) is not restrict | limited, Preferably it can manufacture by passing through the following processes (A1) and then a process (A2).
Step (A1): A step of reacting one of the amino groups of 1,3-diamino-2-propanol with the polyethylene glycol derivative represented by the formula (9) to introduce a first polyethylene glycol chain.
Step (A2): a step of obtaining a compound represented by the formula (10) by protecting another unreacted amino group of the compound obtained in the step (A1) with a chemically inert protecting group.

In the compound (9), L ¹ is an N-succinimidyloxy group or a p-nitrophenoxy group. The reaction is carried out in an aprotic solvent such as toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, t-butyl-methyl ether, tetrahydrofuran, chloroform, methylene chloride, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, etc. Can do. In the absence of a solvent, a large amount of by-products generated by reacting two molecules of polyethylene glycol derivative and two amino groups of 1,3-diamino-2-propanol may be produced, which may reduce the yield. The amount of 1,3-diamino-2-propanol used is preferably 10 molar equivalents or more based on the compound (9). In the case of 10 molar equivalents or less, a large amount of side reaction products reacting with two molecules of polyethylene glycol derivative and two amino groups of 1,3-diamino-2-propanol tend to be produced, and the yield tends to decrease. As a mixing method of the compound (9) and 1,3-diamino-2-propanol, it is preferable to add a solution containing the compound (9) dropwise to a solution containing 1,3-diamino-2-propanol. The reaction temperature is usually 0 to 100 ° C., preferably 20 to 50 ° C. The reaction time is usually 10 minutes to 24 hours, preferably 30 minutes to 6 hours. The produced compound may be purified by a purification means such as extraction, recrystallization, adsorption treatment, reprecipitation, supercritical extraction or the like.

  In the compound (10), P is an amino-protecting group. The protecting group is not particularly limited, and is described in, for example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (author: Peter GM WUTS, THEODORA W. GREENE, 4th edition, 2006, publisher: John Wiley & Sons, Inc.). In the introduction of the protecting group, a known protecting reaction can be used. Specific protecting groups include methoxycarbonyl group, ethoxycarbonyl group, tert-butoxycarbonyl group, trimethylsilylethoxycarbonyl group, adamantyloxycarbonyl group, acetyl group, trifluoroacetyl group, benzoyl group, trifluoroacetyl group, p- Examples thereof include a toluenesulfonyl group and a 2-nitrobenzenesulfonyl group, and a tert-butoxycarbonyl group is preferable. When P is a tert-butoxycarbonyl group, the reaction can be performed by reacting the compound obtained in step (A1) with di-tert-butyl dicarbonate in the above-mentioned aprotic solvent or in the absence of a solvent. . The amount of tert-butyl dicarbonate used is not particularly limited, but is preferably equimolar or more with respect to the compound obtained in the step (A1). The reaction temperature is usually 0 to 100 ° C., preferably 20 to 80 ° C. The reaction time is usually 10 minutes to 48 hours, preferably 30 minutes to 24 hours. The produced compound may be purified by a purification means such as extraction, recrystallization, adsorption treatment, reprecipitation, supercritical extraction or the like.

Although the manufacturing method in particular of a compound (12) is not restrict | limited, Preferably it can manufacture by passing through the following processes (B1) and then a process (B2).
Step (B1): A step of converting the hydroxyl group of the compound (10) into an activating group.
Step (B2): The compound obtained by the step (B1) is reacted with the polyethylene glycol amine compound represented by the formula (11) to introduce a compound represented by the formula (12) into which the second polyethylene glycol chain is introduced. Obtaining step.

In the step (B1), the activated group is not particularly limited as long as it is an activated carbonate group that reacts with an amino group, and examples thereof include an N-succinimidyl carbonate group and a p-nitrophenyl carbonate group. From the viewpoint of properties, it is preferably an N-succinimidyl carbonate group. In the compound obtained in the step (B1), L ² is an N-succinimidyloxy group, a p-nitrophenoxy group, or the like, and preferably an N-succinimidyloxy group for the reasons described above. When the activating group is an N-succinimidyl carbonate group, the reaction is carried out in an aprotic solvent or in the absence of a solvent, compound (10), an organic base such as triethylamine, pyridine, 4-dimethylaminopyridine, Or it can carry out by adding inorganic bases, such as sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium carbonate, potassium hydroxide, and dicarbonate (N-succinimidyl). Further, the organic base and inorganic base need not be used. The amount of the organic base or inorganic base used is not particularly limited, but is preferably equimolar or more with respect to the hydroxyl group of the compound (10). An organic base may be used as a solvent. The amount of di (N-succinimidyl) carbonate used is preferably equimolar or more, more preferably equimolar to 5 molar relative to the hydroxyl group of compound (10). The reaction temperature is usually 0 to 100 ° C., preferably 20 to 80 ° C. The reaction time is usually 10 minutes to 48 hours, preferably 30 minutes to 12 hours. The produced compound may be purified by a purification means such as extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction or the like.

  The reaction in the step (B2) can be performed in the above-mentioned aprotic solvent or in the absence of a solvent. Although the usage-amount of a compound (11) does not have a restriction | limiting in particular, An equimolar or more is preferable with respect to the compound obtained at the process (B1), More preferably, it is equimolar-5 mol. The reaction temperature is usually 0 to 100 ° C., preferably 20 to 80 ° C. The reaction time is usually 10 minutes to 48 hours, preferably 30 minutes to 24 hours. The produced compound may be purified by a purification means such as extraction, recrystallization, adsorption treatment, reprecipitation, supercritical extraction or the like.

Although the manufacturing method in particular of a compound (13) is not restrict | limited, Preferably it can manufacture in the following processes (C).
Step (C): a step of deprotecting the protecting group introduced in step (A2) to obtain a compound represented by formula (13).

  The deprotection reaction is carried out according to the type of protecting group introduced in step (A2). For example, when P of the compound (12) is a tert-butoxycarbonyl group, it can be carried out by adding an acid catalyst in the above-mentioned aprotic solvent or a protic solvent such as water, methanol or ethanol. Examples of the acid catalyst include organic acids and inorganic acids. Examples of the organic acid include trifluoroacetic acid, p-toluenesulfonic acid, benzenesulfonic acid, and methanesulfonic acid. Examples of the inorganic acid include sulfuric acid and hydrochloric acid. Preferably, it is methanesulfonic acid. Since methanesulfonic acid has a slightly lower acidity than the above-mentioned other acids, there is an advantage that there is little degradation of the polyethylene glycol chain. The number of moles of the acid catalyst used for deprotection is 1 to 100 times, preferably 2 to 40 times, more preferably 4 to 20 times the number of moles of the compound (12). When the number of moles of the acid catalyst is more than 100 times, it becomes difficult to remove excess acid, and the handling of the work becomes worse. If it is less than 1 time, the deprotection reaction may not proceed sufficiently.

Although the manufacturing method in particular of a compound (8) is not restrict | limited, Preferably it can manufacture in the following processes (D).
Step (D): A step of reacting the compound (13) with the diacylglycerol derivative represented by the formula (14).

L ^{3 in the} compound (14) is a p-nitrophenoxy group or an N-succinimidyloxy group, and preferably an N-succinimidyloxy group from the viewpoint of reactivity. The reaction can be performed in the presence of a base catalyst in the aprotic solvent property described above. Examples of the base catalyst include organic bases such as triethylamine, pyridine and 4-dimethylaminopyridine, or inorganic bases such as sodium carbonate, sodium hydroxide, sodium hydrogen carbonate, sodium acetate, potassium carbonate and potassium hydroxide. The reaction temperature is usually 10 to 70 ° C, preferably 15 to 55 ° C. If the reaction temperature is lower than 10 ° C, the reaction may not proceed sufficiently. When the reaction temperature is higher than 70 ° C., the lipid portion of the diacylglycerol derivative may be decomposed due to thermal history, and a monoacyl derivative may be by-produced. The reaction time is usually 10 minutes to 48 hours, preferably 30 minutes to 24 hours. The produced compound may be purified by a purification means such as extraction, recrystallization, adsorption treatment, reprecipitation, supercritical extraction or the like.

Hereinafter, the present invention will be described more specifically based on examples. Incidentally, ¹ H-NMR, GPC and TOF-MS were used for the analysis and identification of the compounds in the examples.
^<1 analytical methods H-NMR> In of ^{1 H-NMR} analysis, manufactured by JEOL Datum (Ltd.) JNM-ECP400, using JNM-ECP600. The integrated value in the NMR measurement value is a theoretical value.
<GPC analysis method> In GPC analysis, SHODEX GPC SYSTEM-11 was used as a GPC system, and measurement was performed under the following conditions.
Developing solvent: Tetrahydrofuran Flow rate: 1 mL / min Column: SHODEX KF-801, KF-803, KF-804 (ID 8 mm x 30 cm) Column temperature: 40 ° C Detector: RI x 8 Sample amount: 1 mg / g,
Triethylene glycol, PEG4000, and PEG20000 were used for the 100 μL reference. The GPC measurement value shows the analysis value of the main peak obtained by removing high molecular weight impurities and low molecular weight impurities from the inflection point of the elution curve by vertically cutting the baseline.
Mn represents the number average molecular weight, Mw represents the weight average molecular weight, Mw / Mn represents the polydispersity, and Mp represents the peak top molecular weight.
<Analysis method of TOF-MS> In TOF-MS analysis, autoflex III made by Bruker was used. The sample matrix was 1,8,9-Anthracenetriol, the reference was Angiotensin II [M + H] 1 ⁺ Mono molecular weight 1046.5418, and Somatostain 28 [M + H] ¹⁺ Mono molecular weight 3147.4710.

Example 1
1-([2,3-distearoyl] propoxycarbonylamino) -2- (methylpolyoxyethylene-oxypropylaminocarbonyloxy) -3- (methylpolyoxyethylene-oxycarbonylamino) -propane (compound (I) ) (M ≒ 45, n ≒ 11 (when PEG molecular weight is about 2,000, 500))

(Example 1-1)
Methoxypolyethylene glycol-p-nitrophenyl dissolved in 50 g of dichloromethane was charged with 4.5 g (50 mmol) of 1,3-diamino-2-propanol and 80 g of dichloromethane in a 100 mL round bottom flask equipped with a thermometer, nitrogen blowing tube and stirrer. 10 g (5 mmol) of carbonate (SUNBRIGHT MENP-20H, weight average molecular weight 2,000, manufactured by NOF Corporation) was added dropwise over 1 hour under a nitrogen atmosphere. Reaction was performed at 25 degreeC after dripping for 3 hours. After completion of the reaction, the solution was filtered, washed 3 times with 25% brine, and the organic layer was concentrated under reduced pressure using an evaporator. The remaining concentrate was dissolved in 80 g of toluene, 3 g of sodium sulfate was added, and the mixture was stirred at 25 ° C. for 1 hour for dehydration. Then, the solution was filtered and the process by KW2000 (made by Kyowa Chemical Industry Co., Ltd.) was performed twice. 70 g of hexane was added to the treatment solution, and the precipitated crystals were separated by filtration. The obtained crystals were added with 100 g of ethyl acetate, dissolved by heating at 40 ° C., cooled to 15 ° C., and added with hexane for crystallization. The crystals separated by filtration were washed with hexane, and then the crystals were collected by filtration and dried to obtain 8.5 g of compound (II) shown below. (4.3 mmol; 85% yield)
¹ H-NMR (CDCl ₃ , internal standard TMS) δ (ppm): 2.62 (1H, dd), 2.81 (1H, dd), 3.13 (1H, m), 3.35 (1H, m), 3.38 (3H, s ), 3.52-3.78 (180H, m), 4.22 (2H, m), 5.45 (1H, br)

(Example 1-2)
Compound (II) 7.5 g (3.75 mmol) and toluene 22.5 g were added to a 50 mL round bottom flask equipped with a thermometer, a nitrogen blowing tube, a stirrer, and a cooling tube, and stirred at 40 ° C. under a nitrogen atmosphere. ) Was dissolved. Di-tert-butyl dicarbonate 1.03 g (4.7 mmol) was added, and the mixture was stirred at 40 ° C. for 3 hours. After completion of the reaction, crystallization was repeated twice with ethyl acetate and hexane, and then washed with hexane. The crystals were collected by filtration and dried to obtain 6.4 g of compound (III) shown below. (3.2mmol; 85% yield)
¹ H-NMR (CDCl ₃ , internal standard TMS) δ (ppm): 1.44 (9H, s), 3.14-3.33 (4H, m), 3.38 (3H, s), 3.52-3.77 (180H, m), 4.21 -4.24 (2H, m), 5.43 (1H, s), 5.61 (1H, s)
GPCMn: 2,195, Mw2,245, Mw / Mn: 1.011, Mp2,236

(Example 1-3)
To a 50 mL round bottom flask equipped with a thermometer, a nitrogen blowing tube and a stirrer, 6.1 g of compound (III) and 15.5 g of dichloromethane were added and stirred at 25 ° C. in a nitrogen atmosphere to dissolve the compound (III). 1.59 g (6.2 mmol) of di (N-succinimidyl) carbonate and then 0.94 g (9.3 mmol) of triethylamine were added and stirred at 25 ° C. for 4 hours. After completion of the reaction, the reaction solution was filtered and concentrated using an evaporator, and then 40 g of toluene was added and concentrated again. After the precipitate was filtered, KW700 (manufactured by Kyowa Chemical Industry Co., Ltd.) was added to the filtrate and purification was performed twice. Hexane was added to the treatment solution, and the precipitated crystals were separated by filtration. The obtained crystals were dissolved by heating at 40 ° C. with addition of ethyl acetate, cooled to 15 ° C., and added with hexane for crystallization. The crystals separated by filtration were washed with hexane, and then the crystals were collected by filtration and dried to obtain 5.5 g of compound (IV) shown below. (2.8 mmol; yield 90%)
¹ H-NMR (CDCl ₃ , internal standard TMS) δ (ppm): 1.44 (9H, s), 2.84 (4H, s), 3.33-3.34 (1H, m), 3.38 (3H, s), 3.52-3.77 (182H, m), 4.23 (2H, t), 4.85 (1H, t), 5.17 (1H, m), 5.63 (1H, s)

(Example 1-4)
To a 50 mL round bottom flask equipped with a thermometer, a nitrogen blowing tube, and a stirrer, 5.5 g (2.75 mmol) of compound (IV) and 25 g of toluene were added and stirred at 40 ° C. in a nitrogen atmosphere to dissolve the compound (IV). 1.7 g (3.4 mmol) of methoxypolyethyleneglycolpropylamine (SUNBRIGHT MEPA-05H, weight average molecular weight 500, manufactured by NOF Corporation) dissolved in 5 g of toluene was added and stirred at 40 ° C. for 3 hours. After completion of the reaction, the reaction mixture was washed twice with ion exchange water and then extracted twice with chloroform. The chloroform layer was concentrated, dehydrated with magnesium sulfate, and filtered. The treated solution was concentrated under reduced pressure using an evaporator, and crystallized with ethyl acetate and hexane at 15 ° C. The crystals were filtered off, hexane was added, and the crystals were washed to obtain 4.2 g of compound (V) shown below. (1.7mmol; 76% yield)
¹ H-NMR (CDCl ₃ , internal standard TMS) δ (ppm): 1.43 (9H, s), 1.77 (2H, t), 3.33-3.34 (4H, m), 3.38 (6H, s), 3.52-3.77 (183H, m), 4.21 (2H, d), 4.73 (1H, t), 5.21 (1H, s), 5.52 (1H, s), 5.60 (1H, s)
GPCMn: 2,721, Mw2,780, Mw / Mn: 1.022, Mp2,787

(Example 1-5)
Compound (V) (3.5 g, 1.4 mmol) and dichloromethane (35 g) were added to a 50 mL round bottom flask equipped with a thermometer, a nitrogen blowing tube, and a stirrer, and stirred at 25 ° C. in a nitrogen atmosphere to dissolve the compound (V). Methanesulfonic acid 2.0g (21mmol, 15eq) was added and stirred at 25 ° C for 3 hours. After completion of the reaction, the reaction mixture was neutralized with saturated aqueous sodium hydrogen carbonate solution and extracted with chloroform. The chloroform layer was concentrated and dehydrated, and then concentrated under reduced pressure with an evaporator, and crystallization was performed at 15 ° C. using 35 g of ethyl acetate and 35 g of hexane. After the crystals were filtered off, 35 g of hexane was added to wash the crystals to obtain 2.8 g (yield 79%) of the following compound (VI).
¹ H-NMR (CDCl ₃ , internal standard TMS) δ (ppm): 1.78 (2H, t), 2.79-2.87 (2H, m), 3.26-3.45 (3H, m), 3.38 (6H, s), 3.52 -3.77 (224H, m), 4.21 (2H, t), 4.68 (1H, s), 5.46 (1H, s)

(Example 1-6)
Add 2.71 g (1.08 mmol) of compound (VI), 0.28 g of sodium carbonate and 13.8 g of toluene to a 50 mL round bottom flask equipped with a thermometer, nitrogen blowing tube, and stirrer, and dissolve compound (VI) at 40 ° C. under a nitrogen atmosphere. I let you. Next, 1.01 g (1.32 mmol) of N-succinimidyloxycarbonyloxy-distearoyl glyceryl ether was added, and the mixture was stirred at 40 ° C. for 3 hours. After completion of the reaction, treatment with KW2000 (manufactured by Kyowa Chemical Industry Co., Ltd.) was performed 4 times to remove N-hydroxysuccinimide released by the reaction. Next, the toluene solution was concentrated, and lipid-based impurities were removed by repeatedly washing the concentrate with acetonitrile / hexane. Finally, the acetonitrile layer was concentrated under reduced pressure using an evaporator and dried to obtain 2.8 g (yield 79%) of the following compound (I).
The obtained compound (I) was developed by TLC (thin layer chromatography: chloroform / methanol = 85/15, 90/10) and then confirmed by iodine color development. The purity was 94% or more ( Use a terminal methoxy polyethylene glycol with a molecular weight of 2,000 as a reference). The analysis of residual fatty acid was developed by TLC (thin layer chromatography: chloroform / methanol = 99/5) and analyzed by color development of copper phosphate sulfate. As a result, no residual stearic acid was found.
¹ H-NMR (CDCl ₃ , internal standard TMS) δ (ppm): 0.88 (6H, t), 1.25-1.31 (56H, m), 1.60 (4H, quint), 1.78 (2H, t), 2.31 (4H , t), 3.26-3.36 (5H, m), 3.38 (6H, s), 3.52-3.77 (224H, m), 4.11-4.30 (6H, m), 4.74 (1H, br), 5.24 (1H, br ), 5.53-5.65 (3H, m)
Molecular weight (TOF-MS): 3,387

(Example 2)
Synthesis of compound (VII) (m ≒ 22, n ≒ 11 (when PEG molecular weight is about 1,000, 500))
As polyethylene glycol derivatives, methoxypolyethylene glycol-p-nitrophenyl carbonate (SUNBRIGHT MENP-10H, weight average molecular weight 1,000, manufactured by NOF Corporation) and methoxypolyethylene glycol propylamine (SUNBRIGHT MEPA-05H, weight average molecular weight 500, day) The oil was synthesized in the same manner as in Examples 1-1 to 1-6.
The obtained compound (VII) was developed by TLC (thin layer chromatography: chloroform / methanol = 85/15, 90/10) and then confirmed by iodine color development. The purity was 95% or more ( Use a terminal methoxy polyethylene glycol with a molecular weight of 1,000 as a reference). The residual fatty acid was analyzed by TLC (thin layer chromatography: chloroform / methanol = 99/5) and analyzed by color development of copper phosphate sulfate. As a result, residual stearic acid was not detected.
Molecular weight (TOF-MS): 2,370

(Example 3)
Synthesis of compound (VIII): (m≈45, n≈22 (when PEG molecular weight is about 2,000, 1,000))
As polyethylene glycol derivatives, methoxypolyethylene glycol-p-nitrophenyl carbonate (SUNBRIGHT MENP-20H, weight average molecular weight 2,000, manufactured by NOF Corporation) and methoxypolyethylene glycol propylamine (SUNBRIGHT MEPA-10H, weight average molecular weight 1,000, day) The oil was synthesized in the same manner as in Examples 1-1 to 1-6.
The obtained compound (VIII) was developed by TLC (thin layer chromatography: chloroform / methanol = 85/15, 90/10) and then confirmed by iodine color development. The purity was 97% or more ( Use a terminal methoxy polyethylene glycol with a molecular weight of 2,000 as a reference). The residual fatty acid was analyzed by TLC (thin layer chromatography: chloroform / methanol = 99/5) and analyzed by color development of copper phosphate sulfate. As a result, residual stearic acid was not detected.
Molecular weight (TOF-MS): 4,074

(Example 4) Preparation of liposome L-α-distearoylphosphatidylcholine, cholesterol, L-α-distearoylphosphatidyl-DL-glycerol sodium, and compound (I) were weighed so as to be 100: 100: 60: 15 μmol After dissolution in a chloroform / methanol mixture (4/1 (v / v)), the solvent was distilled off using an evaporator to form a lipid thin film. Furthermore, the organic solvent was completely removed by leaving still overnight in a desiccator. Thereafter, the mixture was rehydrated with 9.0% sucrose 10 mM lactate buffer, and size controlled by the extrusion method to obtain a liposome suspension. In addition, liposomes were similarly prepared for Compound (VII) and Compound (VIII).

(Examples 5 to 7) Calculation of hydrated layer thickness (FALT (Fixed aqueous layer thickness)) Prepared in Example 4 in a mixed solution of NaCl solutions of different concentrations and 9.0% sucrose 10 mM lactate buffer. Compound (I) liposomes were suspended and the zeta potential was measured. The corresponding zeta potential was plotted against the parameter κ calculated from the NaCl concentration, and the absolute value of the obtained slope was taken as the thickness of the hydrated layer. For measuring the zeta potential, Zetasizer Nano ZS (manufactured by Malvern Instruments) was used. Moreover, FALT was similarly calculated about the liposome of compound (VII) and compound (VIII), and it was set as Example 6 and Example 7, respectively.

(Comparative Example 1)
Preparation of liposomes in the same manner as in Examples 4 and 5 using lipid PEG (SUNBRIGHT GS-020, weight average molecular weight 2,000, manufactured by NOF Corporation) having one PEG chain represented by the following formula (IX) And FALT were calculated.

  The results of calculation of FALT in Examples 5 to 7 and Comparative Example 1 are summarized in Table 1.

  As shown in Table 1, the liposome using the branched polyethylene glycol-modified lipid of the present invention has a larger hydration layer than Comparative Example 1 which is a polyethylene glycol-modified lipid having one PEG chain of the same length. It was shown to form. This is probably because the short-chain PEG pushes the mushroom structure of the long-chain PEG into a brush structure.

  According to the present invention, a branched polyethylene glycol-modified lipid having a novel structure and containing two polyethylene glycol chains having different molecular weights, a method for producing the same, and a polyethylene glycol-modified liposome can be provided. The polyethylene glycol-modified lipid of the present invention is industrially easy to produce and effectively increases the hydration layer on the surface of the liposome, so that it is useful as a DDS having excellent blood retention.

Claims (3)

Following formula (1)

(Wherein R ¹ and R ² are each independently a hydrocarbon group having 1 to 7 carbon atoms, and R ³ CO is an acyl group having 4 to 24 carbon atoms. M and n are each independently , An average addition mole number of oxyethylene groups, an integer between 10 and 125, and an integer satisfying n ≦ 25 and 2n ≦ m, or m ≦ 25 and 2m ≦ n X ¹ and X ² Are independently selected from the groups represented by the following formulas (3) to (7), and X ³ is selected from any of a carbonate bond, a carbamate bond, and an ether bond.

(In the formula, r is an integer of 1 to 5, and s is an integer of 2 to 5.)
A branched polyethylene glycol-modified lipid containing two polyethylene glycol chains having different molecular weights represented by the formula: The molecular weight represented by the following formula (8), wherein X ¹ is a group represented by the above formula (7), X ² is a group represented by the above formula (5), and X ³ is a carbamate bond. A branched polyethylene glycol-modified lipid containing two different polyethylene glycol chains.

(Wherein R ¹ and R ² are each independently a hydrocarbon group having 1 to 7 carbon atoms, and R ³ CO is an acyl group having 4 to 24 carbon atoms. M and n are each independently , An average addition mole number of oxyethylene groups, an integer between 10 and 125, an integer satisfying n ≦ 25 and 2n ≦ m, or m ≦ 25 and 2m ≦ n, and s is 2 to 5 Is an integer.) The branched polyethylene glycol modification comprising two polyethylene glycol chains having different molecular weights according to claim 2, characterized in that it comprises all of the following steps (A), (B), (C) and (D) in order. A method for producing lipids.
Step (A): 1,3-diamino-2-propanol and a polyethylene glycol derivative represented by the following formula (9) are reacted, and after introducing a polyethylene glycol chain into one of the amino groups, another amino group Is protected with a chemically inert protecting group to obtain a compound represented by the following formula (10).

(In the formula, R ¹ is a hydrocarbon group having 1 to 7 carbon atoms. M is an average added mole number of an oxyethylene group, and is an integer between 10 and 125, and L ¹ is N-succin. (It is an imidyloxy group or a p-nitrophenoxy group, and P is an amino-protecting group.)
Step (B): After converting the hydroxyl group of the compound represented by the above formula (10) into an active group, a polyethylene glycol derivative represented by the following formula (11) was reacted to introduce a second polyethylene glycol chain. The process of obtaining the compound shown by following formula (12).

(In the formula, R ¹ and R ² are each independently a hydrocarbon group having 1 to 7 carbon atoms. M and n are each independently an average addition mole number of an oxyethylene group, 10 Is an integer satisfying n ≦ 25 and 2n ≦ m, or m ≦ 25 and 2m ≦ n, s is an integer of 2 to 5, and P is an amino-protecting group. is there.)
Step (C): a step of deprotecting the amino-protecting group of the compound represented by the above formula (12) obtained in the step (B) to obtain a compound represented by the following formula (13).

(In the formula, R ¹ and R ² are each independently a hydrocarbon group having 1 to 7 carbon atoms. M and n are each independently an average addition mole number of an oxyethylene group, 10 And an integer satisfying n ≦ 25 and 2n ≦ m, or m ≦ 25 and 2m ≦ n, and s is an integer of 2 to 5.)
Step (D): branched polyethylene glycol represented by the following formula (8) by reacting the compound represented by the above formula (13) obtained in the step (C) with a diacylglycerol derivative represented by the following formula (14): Obtaining a modified lipid;

Wherein R ¹ and R ² are each independently a hydrocarbon group having 1 to 7 carbon atoms, R ³ CO is an acyl group having 4 to 24 carbon atoms, and L ³ is N-succinimidyl. An oxy group or a p-nitrophenoxy group, wherein m and n are each independently the average number of moles added of the oxyethylene group, an integer between 10 and 125, where n ≦ 25 and 2n ≦ m. Or an integer satisfying m ≦ 25 and 2m ≦ n, and s is an integer of 2 to 5.)