Branched degradable polyethylene glycol derivative

文档序号:1865928 发布日期:2021-11-19 浏览:27次 中文

阅读说明:本技术 分支型分解性聚乙二醇衍生物 (Branched degradable polyethylene glycol derivative ) 是由 吉冈宏树 大坂间顺规 羽村美华 稻叶高德 西山伸宏 松井诚 武元宏泰 野本贵大 孙小 于 2020-03-26 设计创作,主要内容包括:本发明提供一种用于对下式(1)所示的生物相关物质进行修饰的用途的在细胞内进行分解的分支型分解性聚乙二醇衍生物。(式中,各符号如本说明书中所定义)。(The present invention provides a branched degradable polyethylene glycol derivative which is used for modification of a biologically relevant substance represented by the following formula (1) and is degraded in a cell.)

1. A degradable polyethylene glycol derivative represented by the following formula (1):

wherein n is 45 to 950, W is an oligopeptide of 5 to 47 residues having a symmetric structure with glutamic acid as the center, a is 2 to 8, X is a functional group capable of reacting with a biologically relevant substance, and L1And L2Each independently is a 2-valent spacer group.

2. The degradable polyethylene glycol derivative according to claim 1, wherein the oligopeptide of W having a symmetrical structure centering on glutamic acid is an oligopeptide having the following structure of W1, W2 or W3:

wherein Glu is a glutamic acid residue, and Z is a degradable oligopeptide of 2 to 5 residues consisting of neutral amino acids excluding cysteine.

3. The degradable polyethylene glycol derivative according to claim 2, wherein the degradable oligopeptide of Z is an oligopeptide having glycine as a C-terminal amino acid.

4. The degradable polyethylene glycol derivative according to claim 2 or 3, wherein the degradable oligopeptide of Z is an oligopeptide having at least 1 hydrophobic neutral amino acids with a hydropathic index of 2.5 or more.

5. The degradable polyethylene glycol derivative according to any one of claims 1 to 4, wherein the total molecular weight is 20,000 or more.

6. The degradable polyethylene glycol derivative according to any one of claims 1 to 5, wherein L is1Is a carbonyl, urethane, amide, ether, thioether, secondary amino, or urea linkage; or alkylene groups with or without these bonds and/or groups.

7. The degradable polyethylene glycol derivative according to any one of claims 1 to 6, wherein L is2Is an alkylene group; or an alkylene group containing at least one bond and/or group selected from a carbonyl group, a urethane bond, an amide bond, an ether bond, a thioether bond, a secondary amino group, and a urea bond.

8. The degradable polyethylene glycol derivative according to any one of claims 1 to 7, wherein X is selected from the group consisting of an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxide group, a maleimide group, a substituted maleimide group, a vinylsulfonyl group, an acrylate group, a substituted sulfonate group, a sulfonyloxy group, a carboxyl group, a mercapto group, a dithiopyridyl group, an α -haloacetyl group, an alkylcarbonyl group, an iodoacetamido group, an alkenyl group, an alkynyl group, a substituted alkynyl group, an amino group, an oxyamino group, a hydrazide group and an azide group.

Technical Field

The present invention relates to a branched degradable polyethylene glycol derivative which is degraded in a cell and is used for modifying a biologically relevant substance.

Background

In general, when a drug using a biologically relevant substance such as a hormone, a cytokine, an antibody, or an enzyme is administered into a living body, the drug is rapidly excreted from the living body by glomerular filtration in the kidney or by uptake by macrophages in the liver, spleen, or the like. Therefore, the blood half-life is shortened, and it is often difficult to obtain a sufficient pharmacological effect. In order to solve this problem, attempts have been made to chemically modify biologically relevant substances with a hydrophilic polymer such as a sugar chain or polyethylene glycol, or albumin. As a result, the blood half-life of the biologically-relevant substance can be prolonged due to the increase in molecular weight, the formation of a hydrated layer, and the like. It is also known that modification with polyethylene glycol can reduce the toxicity or antigenicity of biologically relevant substances and improve the solubility of poorly water-soluble drugs.

Since the bio-related substance modified with polyethylene glycol is covered with a hydrated layer formed by hydrogen bonding of ether bond of polyethylene glycol and water molecule, the size of the molecule is increased, so that glomerular filtration in kidney can be avoided. Further, it is known that the interaction between opsonin and the cell surface constituting each tissue is reduced, and migration into each tissue is reduced. Polyethylene glycol is known to be an excellent material capable of extending the blood half-life of a biologically-relevant substance, and its property is that the larger the molecular weight, the higher the effect. Studies on bio-related substances modified with polyethylene glycol having a high molecular weight of 4 ten thousand or more have been conducted so far, and the blood half-life thereof can be remarkably prolonged.

Polyethylene glycol is used as the best standard among modifying agents for the improvement of the properties of bio-related substances, and a plurality of polyethylene glycol modifying agents are now on the market and used in medical sites. On the other hand, the European Medicines Agency (EMA) of 2012 reported that when a biologically-relevant substance modified with polyethylene glycol having a molecular weight of 4 ten thousand or more was administered to animals for a long period of time at a predetermined administration amount or more, cavitation occurred in cells of a part of the tissue (non-patent document 1). Currently, considering that there is no report that the generation of cavitation itself adversely affects the human body, and that the dose used in the above-mentioned report of EMA is extremely high compared with the dose generally used in the medical field, it can be said that there is no problem in the safety of the therapeutic preparation modified with polyethylene glycol having a molecular weight of 4 ten thousand or more which is currently manufactured and sold. However, treatment regimens employing high amounts and prolonged administration of polyethylene glycol modified formulations to patients in the treatment of very specific diseases (e.g., dwarfism, etc.) can also be envisioned. Therefore, there is expected to be a potential need for the development of polyethylene glycol-modified preparations that do not produce vacuoles in cells, and that can be applied in such special situations.

In non-patent document 2, when polyethylene glycol in an amount larger than the amount of a conventional polyethylene glycol-modified preparation is administered alone to an animal for a long period of time, no cavitation was observed at a molecular weight of 2 ten thousand, and the occurrence of cavitation was observed at a molecular weight of 4 ten thousand. As one means for suppressing cavitation, reduction of the molecular weight of polyethylene glycol is conceivable, but there is a problem that the blood half-life of a bio-related substance cannot be sufficiently improved if the molecular weight is reduced.

There are reported examples of techniques for promoting the decomposition of high molecular weight polyethylene glycol into low molecular weight polyethylene glycol in vivo and excretion from the kidney. Patent document 1 describes a polyethylene glycol derivative having a thioether bond or a peptide bond site that can be cleaved in vivo. It is described that the polyethylene glycol derivative is decomposed in vivo to a molecular weight suitable for excretion from the kidney. However, no data on specific breakdown was shown at all, nor was it shown that excretion from the kidney promoted such data. Further, there is no description about vacuoles of cells.

Patent document 2 describes a polyethylene glycol derivative having an acetal moiety that can be hydrolyzed in a low pH environment in vivo. It is described that the polyethylene glycol derivative is decomposed in vivo to a molecular weight suitable for excretion from the kidney. However, there is no data specifically promoting excretion from the kidney, and there is no description about vacuole of cells. Further, it is known that these acetal moieties capable of hydrolysis are also slowly decomposed in blood, and it is not expected that the half-life of the modified bio-related substance in blood will be sufficiently improved.

On the other hand, there are reported examples of polyethylene glycol derivatives into which degradable oligopeptides are introduced for effective drug release, hydrogels that decompose in vivo, and the like.

Non-patent document 3 describes polyethylene glycol derivatives having an oligopeptide portion decomposed by an enzyme. Among them, it is reported that an oligopeptide is introduced as a linker (linker) between an anticancer agent and polyethylene glycol, and the oligopeptide is decomposed by an enzyme specifically expressed around a tumor, thereby efficiently releasing the anticancer agent. The purpose of this is to release the anticancer agent, and not to impart degradability to polyethylene glycol for the purpose of suppressing cell vacuoles.

Non-patent document 4 describes a hydrogel using a cross-linking molecule having an oligopeptide portion decomposed by an enzyme and a multibranched polyethylene glycol derivative. Among them, oligopeptides are used as crosslinking molecules for linking the multibranched polyethylene glycol derivatives, and further, the hydrogel can be imparted with degradability by enzymes. The purpose of this is to prepare a degradable hydrogel, and not to impart degradability to polyethylene glycol for the purpose of suppressing cell vacuolation.

Patent document 3 describes a branched polyethylene glycol derivative having an oligopeptide as a skeleton. Wherein the oligopeptide is used as a basic skeleton of the polyethylene glycol derivative, rather than imparting degradability through an enzyme. The oligopeptide is characterized by containing amino acids having an amino group or a carboxyl group in a side chain, such as lysine and aspartic acid, and is intended to synthesize a branched polyethylene glycol derivative using these amino acids for the reaction. Polyethylene glycol derivatives that are not intended to inhibit vacuolation of cells.

Further, polyethylene glycol derivatives used for modification of biologically relevant substances generally include straight-chain type and branched type, and non-patent document 5 describes that branched type significantly extends the blood half-life of biologically relevant substances as compared with straight-chain type. In recent years, polyethylene glycol-modified preparations on the market are mostly branched. However, to date, there have been no reports in the art of branched polyethylene glycol derivatives that inhibit cellular vacuoles.

As described above, there is a need for a branched high molecular weight polyethylene glycol derivative which is stable in blood, improves the blood half-life of a modified bio-related substance, specifically decomposes intracellularly when taken into cells, and inhibits the production of vacuoles in cells.

Documents of the prior art

Patent document

Patent document 1: japanese Kokai publication No. 2009-527581

Patent document 2: international publication No. 2005/108463

Patent document 3: international publication No. 2006/088248

Non-patent document

Non-patent document 1: EMA/CHMP/SWP/647258/2012

Non-patent document 2: daniel G.Rudmann, et al., Toxicol. Pathol.,41,970-

Non-patent document 3: france sco M Veronese, et al, Bioconjugate chem, 16,775-784(2005)

Non-patent document 4: jiyuan Yang, et al, Marcomol biosci, 10(4),445-

Non-patent document 5: yulia Vugmeysterang, et al, Bioconjugate chem.,23,1452-

Disclosure of Invention

Problems to be solved by the invention

The problem of the present invention is to provide a branched polyethylene glycol derivative having a high molecular weight which does not cause cavitation of cells. More specifically, it is possible to provide a branched degradable polyethylene glycol derivative which can be used effectively for modification of a biologically relevant substance and which is stable in blood in vivo and is degraded intracellularly by an industrial production method.

Means for solving the problems

The present inventors have conducted intensive studies to solve the above problems, and as a result, have invented a branched degradable polyethylene glycol derivative having an oligopeptide that degrades inside cells.

That is, the present invention is as follows.

[1] A degradable polyethylene glycol derivative represented by the following formula (1):

[ solution 1]

(wherein n is 45 to 950, W is an oligopeptide of 5 to 47 residues having a symmetrical structure with glutamic acid as the center, a is 2 to 8, X is a functional group capable of reacting with a biologically relevant substance, and L1And L2Each independently a 2-valent spacer group).

[2] The degradable polyethylene glycol derivative according to [1], wherein the oligopeptide of W having a symmetrical structure centering on glutamic acid is an oligopeptide having the structure of W1, W2 or W3 below.

[ solution 2]

[ solution 3]

[ solution 4]

(wherein Glu is a glutamic acid residue, and Z is a degradable oligopeptide of 2 to 5 residues comprising a neutral amino acid excluding cysteine)

[3] The degradable polyethylene glycol derivative according to [2], wherein the degradable oligopeptide of Z is an oligopeptide having glycine as a C-terminal amino acid.

[4] The degradable polyethylene glycol derivative according to any one of [2] or [3], wherein the degradable oligopeptide of Z is an oligopeptide having at least 1 hydrophobic neutral amino acid having a hydropathic index of 2.5 or more.

[5] The degradable polyethylene glycol derivative according to any one of [1] to [4], wherein the total molecular weight is 20,000 or more.

[6]According to [1]~[5]The degradable polyethylene glycol derivative of any one of the above, wherein L is1Is a carbonyl, urethane, amide, ether, thioether, secondary amino, or urea linkage; or an alkylene group that may contain such bonds and/or groups.

[7]According to [1]~[6]The degradable polyethylene glycol derivative of any one of the above, wherein L is2Is an alkylene group; or an alkylene group containing at least one bond and/or group selected from a carbonyl group, a urethane bond, an amide bond, an ether bond, a thioether bond, a secondary amino group, and a urea bond.

[8] The degradable polyethylene glycol derivative according to any one of [1] to [7], wherein X is selected from the group consisting of an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxide group, a maleimide group, a substituted maleimide group, a vinylsulfonyl group, an acrylate group, a substituted sulfonate group, a sulfonyloxy group, a carboxyl group, a mercapto group, a dithiopyridyl group, an α -haloacetyl group, an alkylcarbonyl group, an iodoacetamido group, an alkenyl group, an alkynyl group, a substituted alkynyl group, an amino group, an oxyamino group, a hydrazide group and an azide group.

Effects of the invention

The branched degradable polyethylene glycol derivative of the present invention is stable in blood in vivo and has an oligopeptide in its structure which is degraded by an intracellular enzyme. Therefore, the degradable polyethylene glycol derivative is stable in blood, and can impart a blood half-life equivalent to that of a conventional non-degradable polyethylene glycol derivative to a biologically-relevant substance. Further, the degradable polyethylene glycol derivative is rapidly degraded at the oligopeptide site when taken into the cell, thereby suppressing the generation of vacuoles in the cell. The oligopeptide constituting the degradable polyethylene glycol derivative has a symmetrical structure with glutamic acid as the center, and the same degradable oligopeptide Z is bonded to the ends of all polyethylene glycol chains. Therefore, polyethylene glycol decomposition products generated during intracellular decomposition have the same molecular weight and the same structure, and have the characteristic of uniform discharge from tissues and cells.

Since vacuolation of cells by polyethylene glycol is more likely to occur as the molecular weight of polyethylene glycol increases, a desired molecular design of degradable polyethylene glycol is to decompose into molecules having a smaller molecular weight in cells. However, when a high molecular weight degradable polyethylene glycol is produced by sequentially connecting polyethylene glycols having a small molecular weight via degradable oligopeptides, the number of steps increases. In addition, polyethylene glycol having two different functional groups is required as a raw material, and the by-produced impurities are complicated, so that it is not suitable for industrial production. On the other hand, the branched degradable polyethylene glycol of the present invention is prepared by bonding a degradable oligopeptide with a cheap and easily available methoxypolyethylene glycol derivative as a raw material, and then introducing 2 polyethylene glycol chains into the structure at a time in the reaction with a glutamic acid derivative, and thus the number of steps can be greatly reduced in the production thereof. Furthermore, by using glycine as the amino acid at the C-terminal end of the oligopeptide, impurities generated in the production process can be reduced, and the branched degradable polyethylene glycol derivative of the present invention can be industrially produced.

Drawings

FIG. 1 shows Compound (p3) (I) of example 1 2 2NH-E(FG-200ME)) GPC analysis result (2).

FIG. 2 shows the compound (p3) (recovered from the cells in the cell lysis test in example 8 2 2NH-E(FG-200ME)) GPC analysis result (2).

FIG. 3 shows the compound of example 5 (p13) 2 2 2(NH-E{E(FG-100ME)}) GPC analysis result (2).

FIG. 4 shows the compound (p13) (recovered from the cells in the cell lysis test in example 8 2 2 2NH-E{E(FG-100ME)}) GPC analysis result (2).

FIG. 5 shows images of sections of the choroid plexus of mice chronically dosed with methoxy PEG (polyethylene glycol) amine 40kDa of example 9 (arrows indicate vacuoles).

FIG. 6 shows the chronic administration of Compound (p3) of example 9 2 2NH-E(FG-200ME)) Images of sections of mouse brain choroid plexus.

FIG. 7 shows the chronic administration of PBS, methoxy PEG amine 40kDa, methoxy PEG amine 20kDa, Compound (p3) ((p 3)) 2 2NH-E(FG-200ME)) Images of sections of mouse brain choroid plexus (stained portions show accumulation of PEG).

FIG. 8 shows radioisotope-labeled NH in example 112-E(FG-200ME)22-branched PEG amine 40kDa and 2-branched PEG amine 20 kDa.

Detailed Description

The present invention is described in detail below.

The degradable polyethylene glycol derivative according to the present invention is represented by the following formula (1).

[ solution 5]

(in the formulaN is 45 to 950, W is an oligopeptide of 5 to 47 residues with a symmetrical structure taking glutamic acid as the center, a is 2 to 8, X is a functional group capable of reacting with biologically related substances, and L1And L2Each independently is a spacer group having a valence of 2)

The total molecular weight of the polyethylene glycol derivative of formula (1) of the present invention is usually 4,000 to 160,000, preferably 10,000 to 120,000, and more preferably 20,000 to 80,000. In a preferred embodiment of the present invention, the polyethylene glycol derivative of formula (1) of the present invention has a total molecular weight of 20,000 or more. The molecular weight referred to herein means the number average molecular weight (Mn).

N in formula (1) is the number of repeating units of polyethylene glycol, and is usually 45 to 950, preferably 110 to 690, and more preferably 220 to 460.

In formula (1), a is the number of polyethylene glycol chains bonded to the oligopeptide, and is usually 2 to 8, preferably 2 or 4 or 8, and more preferably 2 or 4.

L in the formula (1)1And L2Each independently represents a 2-valent spacer, and these spacers are not particularly limited as long as they are groups capable of forming a covalent bond, and L is1Preferably an amide bond, an ether bond, a thioether bond, a urethane bond, a secondary amino group, a carbonyl group or a urea bond; or an alkylene group that may contain such bonds and/or groups.

In addition, L2Preferably an alkylene group; or an alkylene group containing at least one bond and/or group selected from an amide bond, an ether bond, a thioether bond, a urethane bond, a secondary amino group, a carbonyl group, and a urea bond. L is2Preferably, the carbon atoms are bonded to the repeating units of the polyethylene glycol.

L1And L2Particularly preferred modes of (b) are shown in the following group (I). Furthermore, 2 to 5 spacer groups of group (I) may also be combined. As the 2-valent spacer, an ester bond and a carbonate bond are not suitable because they are slowly decomposed in blood in vivo.

Group (I):

[ solution 6]

In the formulae (z1) to (z11), s represents an integer of 0 to 10, preferably an integer of 0 to 6, and more preferably an integer of 0 to 3. In addition, in the formulae (z2) to (z11), s may be the same or different. L is1In the case of an asymmetric 2-valent spacer, the bonding position with another adjacent group is not particularly limited, and if the right side of the spacer represented by the above formula in the group (I) represents the bonding position with W and the left side represents the bonding position with X, two bonding positions can be adopted in the case where the left side represents the bonding position with W and the right side represents the bonding position with X. Likewise, L2In the case of asymmetric 2-valent spacers, if the spacer of the above group (I) is represented by the formula2CH2When the bonding position of (3) and the left side represent the bonding position with W, the left side may be represented by OCH2CH2And the right side shows two bonding positions at the bonding position with W.

As L in formula (1)1The group (I) preferably has a group represented by (z3), (z4), (z6), (z7), (z8), (z9) or (z10), and more preferably has a group represented by (z3), (z6), (z9) or (z 10).

As L in formula (1)2Preferred are groups of group (I) represented by (z1), (z2), (z3), (z4), (z5), (z6), (z7), (z8) or (z11), and more preferred are groups of group (z3), (z5) or (z 11).

W in formula (1) is not particularly limited as long as it is an oligopeptide of 5 to 47 residues having a symmetrical structure with glutamic acid as the center, which is stable in blood in vivo and is decomposed by intracellular enzymes, and the amino acids constituting the oligopeptide are preferably neutral amino acids excluding cysteine, except glutamic acid constituting the center. The oligopeptide having a symmetric structure with glutamic acid as the center is a compound in which the same peptide is bonded to the carboxyl group at the α -position and the carboxyl group at the γ -position of glutamic acid, and the peptide paired with glutamic acid is an oligopeptide having a symmetric structure. The oligopeptide generally has a composition ratio of the number of neutral amino acids to the number of glutamic acids (number of neutral amino acids/number of glutamic acids) of 2 to 10, preferably 2 to 8, and more preferably 2 to 6. The amino acids constituting W are substantially in the L form.

Particularly preferred modes of W are shown in the following group (II).

Group (II):

[ solution 7]

[ solution 8]

[ solution 9]

(wherein Glu is a glutamic acid residue, and Z is a decomposable oligopeptide of 2 to 5 residues consisting of neutral amino acids excluding cysteine.)

Z in (w1) to (w3) is preferably an amino acid having an amino group or a carboxyl group in a side chain, specifically an oligopeptide composed of a neutral amino acid not containing lysine, aspartic acid, or glutamic acid. In the synthesis of the branched degradable polyethylene glycol derivative of formula (1) of the present invention, when the polyethylene glycol derivative and the oligopeptide as raw materials are bonded by reaction, the carboxyl group at the C-terminal end of the oligopeptide is used in the condensation reaction with the polyethylene glycol derivative. However, when the oligopeptide has an amino acid having an amino group or a carboxyl group in a side chain, a side reaction between oligopeptides by a condensation reaction occurs, and the polyethylene glycol derivative is not introduced into a carboxyl group at the C-terminal end as a target, but is also introduced into an impurity of the carboxyl group in the side chain.

Since such impurities are difficult to remove by a purification step such as ordinary extraction or crystallization, it is desirable to use oligopeptides composed of amino acids having no amino group or carboxyl group in the side chain in order to obtain a target product with good purity. The amino acids constituting Z are alpha-amino acids and are furthermore substantially in the L-form.

Since cysteine as a neutral amino acid has a thiol group and forms a disulfide bond with another thiol group, (w1) to (w3) Z is preferably an oligopeptide composed of a neutral amino acid containing no cysteine.

In addition, Z in (w1) to (w3) is preferably an oligopeptide having glycine as a C-terminal amino acid. When the carboxyl group at the C-terminal is reacted with a polyethylene glycol derivative, it is basically necessary to activate the carboxyl group at the C-terminal with a condensing agent or the like. It is known that amino acids other than glycine are easily epimerized in the activation step, and that stereoisomers are produced as by-products. By using achiral glycine as the C-terminal amino acid of the oligopeptide, a high-purity target product free from a by-product of a stereoisomer can be obtained.

Further, Z in (w1) to (w3) is a hydrophobic neutral amino acid having a hydropathic index of 2.5 or more, specifically preferably an oligopeptide having at least 1 of phenylalanine, leucine, valine and isoleucine, and more preferably an oligopeptide having phenylalanine. The quantitation achieved by Kate (Kyte) and Doolittle (Doolittle) indicates the hydropathic index (hydropathicity index) of the hydrophobicity of amino acids, with larger values indicating more hydrophobic amino acids (Kyte J & Doolittle RF,1982, J Mol Biol,157: 105-.

Z in (w1) to (w3) is not particularly limited as long as it is stable in blood in vivo and has the ability to be decomposed by intracellular enzymes, and is an oligopeptide of 2 to 5 residues comprising neutral amino acids excluding cysteine, and specific examples thereof include glycine-phenylalanine-leucine-glycine, glycine-phenylalanine-glycine, glycine-leucine-glycine, valine-citrulline-glycine, valine-alanine-glycine, phenylalanine-glycine and the like, and preferably glycine-phenylalanine-leucine-glycine, glycine-glycine, phenylalanine-glycine, glycine-serine-glycine, cysteine-glycine, and the like, Glycine-phenylalanine-glycine, valine-citrulline-glycine, valine-alanine-glycine or phenylalanine-glycine, more preferably glycine-phenylalanine-leucine-glycine, glycine-phenylalanine-glycine, valine-citrulline-glycine or phenylalanine-glycine, and still more preferably glycine-phenylalanine-leucine-glycine or phenylalanine-glycine.

X in formula (1) is not particularly limited as long as it is a functional group that reacts with a functional group present in a biologically relevant substance such as a physiologically active protein, peptide, antibody, or nucleic acid to be chemically modified to form a covalent bond. Examples thereof include "Harris, J.M. Poly (Ethylene Glycol) Chemistry; plenum Press, New York,1992(J.M. Harris, polyethylene glycol chemistry; Proelainan Press: New York, 1992), "Hermanson, G.T. bioconjugate Techniques,2nd ed.; academic Press: San Diego, CA,2008(G.T. Hermanson, Biocoupling technology, second edition; Academic Press: San Diego, Calif.; 2008) "and" PEGylated Protein Drugs: Basic Science and Clinical Applications; veronese, f.m., ed.; birkhauser: basel, Switzerland, 2009 (Pegylated protein drugs: basic science and clinical applications, F.M. Werlensted, Bukhaus Press: Basel, Switzerland, 2009) "and the like.

The "functional group reactive with a biologically-relevant substance" represented by X in formula (1) is not particularly limited as long as it is a functional group capable of chemically bonding to a functional group such as an amino group, a thiol group, an aldehyde group, a carboxyl group, an unsaturated bond, or an azide group of the biologically-relevant substance.

Specific examples thereof include an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxide group, a carboxyl group, a mercapto group, a maleimide group, a substituted maleimide group, a hydrazide group, a dithiopyridyl group, a substituted sulfonate group, a vinylsulfonyl group, an amino group and an oxyamino group (H)2N-O-group), an iodoacetamido group, an alkylcarbonyl group, an alkenyl group (e.g., allyl group or vinyl group), an alkynyl group, a substituted alkynyl group (e.g., an alkynyl group substituted with a hydrocarbon group having 1 to 5 carbon atoms described later), an azido group, an acrylic group, a sulfonyloxy group (e.g., an alkylsulfonyloxy group), an α -haloacetyl group, etc., preferably an active ester group, an active carbonate group, an aldehyde group, an isoacetyl groupThe isocyanate group, isothiocyanate group, epoxide group, maleimide group, substituted maleimide group, vinylsulfonyl group, acrylic group, sulfonyloxy group (e.g., alkyl-sulfonyloxy group having 1 to 5 carbon atoms), substituted sulfonate group, carboxyl group, mercapto group, dithiopyridyl group, α -haloacetyl group, alkynyl group, substituted alkynyl group (e.g., alkynyl group having 2 to 5 carbon atoms substituted with hydrocarbon group having 1 to 5 carbon atoms described later), allyl group, vinyl group, amino group, oxyamino group, hydrazide group and azide group, more preferably active ester group, active carbonate group, aldehyde group, maleimide group, oxyamino group and amino group, and particularly preferably aldehyde group, maleimide group and oxyamino group.

In other suitable embodiments, the functional group X can be classified into group (III), group (IV), group (V), group (VI), group (VII), and group (VIII) described below.

Group (III): functional group capable of reacting with amino group of bio-related substance

Examples thereof include groups represented by the following (a), (b), (c), (d), (e), (f), (g), (j) or (k).

Group (IV): functional group capable of reacting with thiol group of bio-related substance

Examples thereof include groups represented by the following (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k) or (l).

Group (V): functional group capable of reacting with aldehyde group of bio-related substance

Examples thereof include groups represented by the following (h), (m), (n) or (p).

Group (VI): functional group capable of reacting with carboxyl group of bio-related substance

Examples thereof include groups represented by the following (h), (m), (n) or (p).

Group (VII): functional group capable of reacting with unsaturated bond of bio-related substance

Examples thereof include groups represented by the following (h), (m) or (o).

Group (VIII): functional group capable of reacting with azide group of bio-related substance

Examples thereof include those shown in the following (l).

[ solution 10]

In the functional group (j), W in the formula1Represents a halogen atom such as a chlorine atom (Cl), a bromine atom (Br), or an iodine atom (I), and is preferably Br or I, more preferably I.

In addition, in the functional group (e) and the functional group (l), Y in the formula1And Y3Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms, preferably a hydrocarbon group having 1 to 5 carbon atoms. The hydrocarbon group having 1 to 5 carbon atoms includes, specifically, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, and the like, and preferably methyl or ethyl.

In addition, in the functional group (k), Y in the formula2Specifically, the alkyl group may include methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, hexyl, nonyl, vinyl, phenyl, benzyl, 4-methylphenyl, trifluoromethyl, 2,2, 2-trifluoroethyl, 4- (trifluoromethoxy) phenyl, and the like, and preferably methyl, vinyl, 4-methylphenyl, or 2,2, 2-trifluoroethyl.

The active ester group means an ester group having an alkoxy group with a high leaving ability. Examples of the alkoxy group having a high leaving ability include alkoxy groups derived from nitrophenol, N-hydroxysuccinimide, pentafluorophenol and the like. The active ester group is preferably an ester group having an alkoxy group derived from N-hydroxysuccinimide.

The activated carbonate group means a carbonate group having an alkoxy group with a high leaving ability. Examples of the alkoxy group having a high leaving ability include alkoxy groups derived from nitrophenol, N-hydroxysuccinimide, pentafluorophenol and the like. The active carbonate group is preferably a carbonate group having an alkoxy group derived from nitrophenol or N-hydroxysuccinimide.

The substituted maleimide group is a maleimide group in which a hydrocarbon group is bonded to a carbon atom of one of the double bonds of the maleimide group. Specific examples of the hydrocarbon group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, and a tert-butyl group, and a methyl group or an ethyl group is preferable.

The substituted sulfonate group means a sulfonate group in which a hydrocarbon group which may contain a fluorine atom is bonded to a sulfur atom of the sulfonate group. Specific examples of the hydrocarbon group which may contain a fluorine atom include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a tert-butyl group, a hexyl group, a nonyl group, a vinyl group, a phenyl group, a benzyl group, a 4-methylphenyl group, a trifluoromethyl group, a 2,2, 2-trifluoroethyl group, a 4- (trifluoromethoxy) phenyl group and the like, and a methyl group, a vinyl group, a 4-methylphenyl group or a 2,2, 2-trifluoroethyl group is preferable.

One of the preferable embodiments of formula (1) is a 2-branched degradable polyethylene glycol derivative represented by the following formula (2) wherein W is W1 and a is 2.

[ solution 11]

(wherein Glu, Z, n, X, L1And L2Same as above)

One of the preferred embodiments of formula (1) is a 4-branched degradable polyethylene glycol derivative represented by the following formula (3) wherein W is W2 and a is 4.

[ solution 12]

(wherein Glu, Z, n, X, L1And L2Same as above)

One of the preferable embodiments of formula (1) is an 8-branched degradable polyethylene glycol derivative represented by the following formula (4) wherein W is W3 and a is 8.

[ solution 13]

(wherein Glu, Z, n, X, L1And L2Same as above)

The branched degradable polyethylene glycol derivative of the present invention can be produced, for example, through the following steps.

[ solution 14]

(in the step (A), PEG is a polyethylene glycol chain, peptide is an oligopeptide, Pro is a protecting group, and L3Is a spacer group with a valence of 2.)

In the step, PEG is a polyethylene glycol chain, and the molecular weight is in the range of 2000 to 42000, as defined by n, which is the number of repeating units of the above-mentioned polyethylene glycol, that is, n is 45 to 950.

The peptide in the step (a) is an oligopeptide having the same meaning as Z. In this step, an oligopeptide in which the amino group at the N-terminal is protected with a protecting group is used.

Pro in the step is a protecting group, and the protecting group is a component which prevents or inhibits the reaction of a functional group capable of undergoing a specific chemical reaction in a molecule under a certain reaction condition. Protecting groups vary depending on the type of functional group that is protected to be capable of chemical reaction, the conditions used, and the presence of other functional groups or protecting groups in the molecule. Specific examples of protecting groups may be found in many common books, for example, those described in "Wuts, p.g.m.; greene, T.W.protective Groups in Organic Synthesis,4th ed.; Wiley-Interscience, New York,2007(P.G.M. Wus; T.W. Green, fourth edition in organic Synthesis; Wiley-Interscience, New York, 2007) ". Further, the functional group protected by a protecting group can regenerate its original functional group by deprotection, that is, chemical reaction, using reaction conditions suitable for the respective protecting groups. Typical deprotection conditions for protecting groups are as described in the above mentioned documents.

L in the process3Is related to the above-mentioned L1And L2Spacer group with 2 valences of the same meaning.

Reaction a is a step of obtaining a polyethylene glycol derivative (1) by bonding a carboxyl group of an oligopeptide having an N-terminal amino group protected by a protecting group to an amino group of a polyethylene glycol derivative having a methoxy group at one terminal through a condensation reaction.

The protective group for the amino group at the N-terminal of the oligopeptide is not particularly limited, and examples thereof include an acyl-based protective group and a carbamate-based protective group, and specifically include a trifluoroacetyl group, a 9-fluorenylmethoxycarbonyl (Fmoc) group, a tert-butoxycarbonyl group, and the like.

The condensation reaction is not particularly limited, but is preferably a reaction using a condensing agent. As the condensing agent, a carbodiimide-based condensing agent such as Dicyclohexylcarbodiimide (DCC) or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) may be used alone, or a reagent such as N-hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBt), or 1-hydroxy-7-azabenzotriazole (HOAt) may be used together. Furthermore, condensing agents such as HATU, HBTU, TATU, TBTU, COMU, and 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholine hydrochloride n-hydrate (DMT-MM) having higher reactivity may also be used. In addition, a base such as triethylamine or dimethylaminopyridine may be used to accelerate the reaction.

Impurities generated by the side reaction in the reaction, remaining oligopeptides and condensing agents which are not consumed in the reaction, and the like are preferably purified and removed. The purification is not particularly limited, and purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[ solution 15]

Deprotection B is a step of deprotecting the protecting group of polyethylene glycol derivative (1) obtained in reaction a to obtain polyethylene glycol derivative (2). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use oligopeptide and L3Without decomposition of the spacer group having a valence of 2. The present step may be carried out as a part of the step of reaction a.

Impurities and the like generated by side reactions in the deprotection reaction are preferably purified and removed. The purification is not particularly limited, and purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[ solution 16]

Reaction C is a step of bonding the amino group of the polyethylene glycol derivative (2) obtained by deprotection B to two carboxyl groups of the glutamic acid derivative whose amino group is protected by a protecting group by a condensation reaction to obtain a branched polyethylene glycol derivative (3) having a structure in which 2 degradable polyethylene glycol chains are linked by a glutamic acid residue.

As in the case of the above reaction A, a reaction using a condensing agent is preferable, and a base such as triethylamine or dimethylaminopyridine may be used to accelerate the reaction.

The protecting group of the amino group of glutamic acid is not particularly limited, and examples thereof include an acyl-based protecting group and a carbamate-based protecting group, and specifically include trifluoroacetyl group, 9-fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl, and the like.

Impurities produced by the side reaction in the reaction, polyethylene glycol derivatives remaining without being consumed in the reaction, and the like are preferably purified and removed. The purification is not particularly limited, and purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[ solution 17]

Deprotection D is a step of deprotecting the protecting group of the polyethylene glycol derivative (3) obtained in reaction C to obtain a polyethylene glycol derivative (4). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use oligopeptide and L3Without decomposition of the spacer group having a valence of 2. The present step may be performed as a part of the step of reaction C.

Impurities and the like generated by side reactions in the deprotection reaction are preferably purified and removed. The purification is not particularly limited, and purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[ solution 18]

Reaction E is a step of bonding the amino group of the polyethylene glycol derivative (4) obtained by deprotection D to two carboxyl groups of a glutamic acid derivative whose amino group is protected by a protecting group by a condensation reaction to obtain a branched polyethylene glycol derivative (5) having a structure in which 4 degradable polyethylene glycol chains are linked by a glutamic acid residue.

It can be reacted and purified under the same conditions as in the above reaction C.

As a method for removing polyethylene glycol impurities having different molecular weights or functional groups from the polyethylene glycol derivative (5), purification techniques described in Japanese patent laid-open Nos. 2014-208786 and 2011-79934 can be used.

[ solution 19]

Deprotection F is a step of deprotecting the protecting group of the polyethylene glycol derivative (5) obtained in reaction E to obtain a polyethylene glycol derivative (6). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use oligopeptide and L3Without decomposition of the spacer group having a valence of 2. Can be reacted and purified under the same conditions as the above deprotection D. The present step may be carried out as a part of the step of the reaction E.

[ solution 20]

Reaction G is a step of bonding the amino group of the polyethylene glycol derivative (6) obtained by deprotection F to two carboxyl groups of a glutamic acid derivative whose amino group is protected by a protecting group by a condensation reaction to obtain a branched polyethylene glycol derivative (7) having a structure in which 8 degradable polyethylene glycol chains are linked by a glutamic acid residue.

It can be reacted and purified under the same conditions as in the above reaction C.

[ solution 21]

Deprotection H is a step of deprotecting the protecting group of the polyethylene glycol derivative (7) obtained in reaction G to obtain a polyethylene glycol derivative (8). Can be reacted and purified under the same conditions as the above-mentioned deprotection F. The present step may be carried out as a part of the step of the reaction G.

By carrying out the above-mentioned reaction A, deprotection B, reaction C and deprotection D, a 2-branched degradable polyethylene glycol derivative (4) can be obtained. The 4-branched degradable polyethylene glycol derivative (6) can be obtained by continuing the reaction E and deprotection F using the 2-branched degradable polyethylene glycol derivative (4) as a starting material. Further, by continuing the reaction G and deprotection H, 8-branched degradable polyethylene glycol derivative (8) can be obtained.

The polyethylene glycol derivatives (4), (6) and (8) obtained in deprotection D, deprotection F and deprotection H each have an amino group, and can be converted into various functional groups by using the amino group.

The step of converting the terminal amino group of the polyethylene glycol derivative into another functional group is not particularly limited, and basically, it can be easily converted into various functional groups by using a compound having an active ester group capable of reacting with an amino group, a general reaction reagent such as an acid anhydride or an acid chloride.

For example, when it is desired to convert an amino group at the terminal of a polyethylene glycol derivative into a maleimide group, a target substance can be obtained by reacting the maleimide group with the following reagent.

[ solution 22]

For example, when it is desired to convert an amino group at the terminal of a polyethylene glycol derivative into a carboxyl group, the target compound can be obtained by reacting the carboxyl group with succinic anhydride or glutaric anhydride.

For example, when it is desired to convert an amino group at the terminal of a polyethylene glycol derivative into a hydroacid group, the target compound is obtained by a condensation reaction with a ring-opened product of a cyclic ester such as caprolactone.

These reagents are low molecular weight reagents, and have a large solubility difference from polyethylene glycol derivatives of high molecular weight polymers, and therefore can be easily removed by a common purification method such as extraction or crystallization.

The degradable polyethylene glycol obtained through the above-mentioned steps is required to be stable in blood and to have a property of being degraded only in cells. In order to appropriately evaluate the performance, for example, the stability of degradable polyethylene glycol in blood and the degradability in cells can be evaluated by the following tests.

In these evaluations, all the evaluation samples were tested in a lump for polyethylene glycol derivatives having one amino group, taking into account the influence of the kind of functional group of the polyethylene glycol derivative.

The test method for evaluating the stability of the degradable polyethylene glycol derivative in blood is not particularly limited, and examples thereof include a test using serum of a mouse, a rat, a human, and the like. Specifically, the degradation rate can be evaluated by dissolving a polyethylene glycol derivative in serum to a concentration of 1 to 10mg/mL, incubating the resulting solution at 37 ℃ for 96 hours, collecting the polyethylene glycol derivative contained in the serum, and measuring GPC. The decomposition rate was calculated based on the% peak area of the GPC main fraction (main fraction) of the polyethylene glycol derivative before the stability test and the% peak area of the GPC main fraction of the polyethylene glycol derivative after the stability test. Specifically, the following formula is used.

Decomposition rate ═ peak area before test% peak area after test ÷ peak area before test ÷ peak area% before test x 100

For example, if the peak area% of the GPC main fraction of the degradable polyethylene glycol derivative before the stability test is 95% and the peak area% of the GPC main fraction after the test is 90%, the degradation rate is calculated as follows.

Decomposition rate (95-90) ÷ 95 × 100 ═ 5.26 (%)

Since the target blood half-life cannot be obtained when the degradable polyethylene glycol derivative is degraded in blood, the degradation rate after 96 hours in the stability test is preferably 10% or less, and more preferably 5% or less.

The test method for evaluating the intracellular degradability of the degradable polyethylene glycol derivative is not particularly limited, and examples thereof include a test in which cells are cultured using a medium containing the degradable polyethylene glycol derivative. The cells or the medium used herein are not particularly limited, and specifically, the decomposition rate can be evaluated by dissolving the polyethylene glycol derivative in the RPMI-1640 medium to a concentration of 1 to 20mg/mL, culturing the macrophage RAW264.7 at 37 ℃ for 96 hours using the medium, recovering the polyethylene glycol derivative in the cells, and measuring GPC. The decomposition rate can be calculated using the% peak area of the GPC main fraction of the polyethylene glycol derivative before and after the test, similarly to the stability test.

For example, if the peak area% of the GPC main fraction using the decomposable polyethylene glycol derivative before the decomposition test of cells is 95%, and the peak area% of the GPC main fraction after the test is 5%, the decomposition rate is calculated as follows.

Decomposition rate (95-5) ÷ 95 × 100 ═ 94.7 (%)

Since vacuoles of target cells cannot be suppressed if the degradable polyethylene glycol derivative cannot be efficiently degraded in cells, the degradation rate after 96 hours in the degradability test is preferably 90% or more, and more preferably 95% or more.

The test method for evaluating the blood half-life and in vivo distribution of the degradable polyethylene glycol derivative is not particularly limited, and examples thereof include a test in which the degradable polyethylene glycol derivative is labeled with a radioisotope or a fluorescent substance, administered to a mouse or a rat, and monitored.

It is considered that the lytic peptide introduced into the polyethylene glycol derivative imparts degradability to polyethylene glycol in a cell, but the pharmacokinetics of polyethylene glycol may be changed due to the peptide structure. Therefore, in order to confirm the influence of the introduced peptide structure on pharmacokinetics, it is necessary to compare the half-life in blood and the distribution in vivo thereof with polyethylene glycol derivatives having the same molecular weight and not having degradability. Specifically, a polyethylene glycol derivative labeled with a radioisotope and not degradable and a degradable polyethylene glycol derivative are administered to mice, and the radiation dose of blood and organs is measured at a plurality of time points to perform quantitative measurement.

The test method for evaluating the vacuolation inhibition of cells by a degradable polyethylene glycol derivative is not particularly limited, and examples thereof include a test in which the drug is administered to a mouse or a rat for a long period of time, at a high frequency, and at a high dose, and a slice image of an organ or organ that is thought to be likely to generate vacuolation is confirmed, as described in non-patent document 2.

Specifically, the polyethylene glycol derivative is dissolved in physiological saline to a concentration of 10 to 250mg/mL, and the administration is continued 3 times a week, 4 weeks or more and 20 to 100 μ L per week through the mouse tail vein, whereby paraffin sections of the brain choroid plexus, spleen or the like of organs considered to be likely to cause cavitation are prepared and stained, and then the section images are confirmed by a pathological method to evaluate the cavitation inhibition.

In addition, in this evaluation, the amount of polyethylene glycol to be administered is required to be excessively larger than the amount of polyethylene glycol generally used in the art.

Non-patent document 2 describes that vacuolation of cells by high molecular weight polyethylene glycol is related to accumulation of polyethylene glycol in tissues. The test method for evaluating the accumulation property in the cells of the degradable polyethylene glycol derivative is not particularly limited, and the evaluation can be performed by using a slice image prepared in the same manner as the above-described vacuole evaluation. The evaluation of the accumulation of polyethylene glycol can be carried out by confirming stained slice images of the brain choroid plexus, spleen, etc. of organs thought to be susceptible to vacuolation by a pathological method.

In addition, in this evaluation, the amount of polyethylene glycol to be administered is required to be excessively larger than the amount of polyethylene glycol generally used in the art.

Examples

Obtained in the examples below1H-NMR was obtained from JNM-ECP400 or JNM-ECA600, manufactured by JEOLDATUM, Inc. Used in the assaySample tube, use of D in deuterated solvents2O or CDCl containing Tetramethylsilane (TMS) as internal standard3And d6-DMSO. The molecular weight and amine purity of the obtained polyethylene glycol derivative were calculated using liquid chromatography (GPC and HPLC). The liquid chromatography system used was "HLC-8320 GPC EcoSEC" manufactured by Tosoh corporation and "ALLIANCE" manufactured by WATERS corporation for GPC. Analytical conditions for GPC and HPLC are shown below.

GPC analysis (molecular weight measurement)

Standard polymer: molecular weight determination by GPC analysis was performed using polyethylene glycols having molecular weights of 8,000, 20,000, 50,000, and 100,000 as standard polymers.

A detector: differential refractometer

A chromatographic column: ultrahydrogel 500 and 250 (manufactured by WATERS Co., Ltd.)

Mobile phase: 100mM Acetate buffer (Acetate buffer) + 0.02% NaN3(pH5.2)

Flow rate: 0.5mL/min

Sample amount: 5mg/mL, 20. mu.L

Temperature of the column: 30 deg.C

HPLC analysis (determination of amine purity)

A detector: differential refractometer

A chromatographic column: TSKgel SP-5PW (manufactured by Tosoh corporation)

Mobile phase: 1mM Sodium phosphate buffer (Sodium phosphate buffer) (pH6.5)

Flow rate: 0.5mL/min

Injection amount: 5mg/mL, 20. mu.L

Temperature of the column: 40 deg.C

[ example 1]

Compound (p3) (p) 2 2NH-E(FG-200ME)) Synthesis of (2)

[ solution 23]

[ example 1-1]

[ solution 24]

L-phenylalanyl-glycine (Fmoc-Phe-Gly) protected to the N-terminus by 9-fluorenylmethoxycarbonyl (Fmoc group) (0.267g, 6.0X 10-4mol, manufactured by Dubian chemical Co., Ltd.) and methoxy PEG having propylamino group at the terminal (6.0g, 2.8X 10)-4To "SUNBRIGHT MEPA-20T" manufactured by Nichikoku K.K., 21,120 mol, was added dehydrated N, N' -dimethylformamide (60g), and the mixture was dissolved at 30 ℃ with heating. Then, diisopropylethylamine (192. mu.L, 1.2X 10) was added-3mol, manufactured by Kanto chemical Co., Ltd.) and (1-cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylamino-morpholin-carbenium hexafluorophosphate (COMU) (0.321g, 7.5X 10)-4mol, manufactured by Sigma-Aldrich corporation), and allowed to react at room temperature under a nitrogen atmosphere for 1 hour. After completion of the reaction, the reaction mixture was diluted with chloroform (600g), and a saturated aqueous sodium bicarbonate solution (240g) was added thereto, followed by washing with stirring at room temperature for 15 minutes. After separating the aqueous layer and the organic layer, a saturated aqueous sodium bicarbonate solution (240g) was added to the organic layer again, and the mixture was stirred at room temperature for 15 minutes to wash the mixture, thereby recovering the organic layer. To the obtained organic layer (chloroform solution), magnesium sulfate (2.4g) was added, and the mixture was stirred for 30 minutes to dehydrate, followed by suction filtration using an aleurite funnel on which Oplite was spread on a 5A filter paper. The filtrate obtained was concentrated at 40 ℃, ethyl acetate (240g) was added to the concentrate, and the mixture was stirred until uniform, followed by addition of hexane (120g), and stirring was carried out at room temperature for 15 minutes to precipitate a product.After the precipitate was recovered by suction filtration using a 5A filter paper, it was dissolved in ethyl acetate (240g) again, and hexane (120g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (120g), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p1) ((p 1))ME-200GF-Fmoc). The yield was 5.1 g.

1H-NMR(d6-DMSO):1.62ppm(m,2H,-CO-NH-CH2- 2CH-CH2-O-(CH2-CH2-O)n-CH3),2.80ppm(m,1H,-NH-CO-CH- 2CH-C6H5),3.04ppm(m,1H,-NH-CO-CH- 2CH-C6H5),3.10ppm(m,2H,-CO-NH- 2CH-CH2-CH2-O-(CH2-CH2-O)n-CH3),3.24ppm(s,3H,-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- 3CH) 3.48ppm (m, about 1,900H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3),4.20ppm(m,4H),7.33ppm(m,9H),7.66ppm(m,4H,Ar),7.88ppm(d,2H,Ar),8.27ppm(t,1H)

[ examples 1-2]

[ solution 25]

ME-200GF-Fmoc (4.9g, 2.3X 10) obtained in example 1-1 was added-4mol) was added to N, N' -dimethylformamide (29.4g), and the mixture was dissolved at 30 ℃ with heating. Piperidine (1.55g, 1.8X 10) was added-2mol, Wako pure chemical industries, Ltd.) was reacted at room temperature under a nitrogen atmosphere for 2 hours. After completion of the reaction, ethyl acetate (300g) was added thereto and stirred until uniform, and hexane (150g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. After the precipitate was recovered by suction filtration using 5A filter paper, it was dissolved in ethyl acetate (300g) again, hexane (150g) was added thereto, and the mixture was stirred at room temperature for 15 minutes to obtain a productAnd (4) precipitating. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (150g), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p2) (R) 2ME-200GF-NH). The yield was 3.9 g.

1H-NMR(d6-DMSO):1.62ppm(m,2H,-CO-NH-CH2- 2CH-CH2-O-(CH2-CH2-O)n-CH3) 1.64ppm (width, 1H), 2.59ppm (dd,1H, -NH-CO-CH- 2CH-C6H5),2.98ppm(dd,1H,-NH-CO-CH- 2CH-C6H5),3.10ppm(q,2H,-CO-NH- 2CH-CH2-CH2-O-(CH2-CH2-O)n-CH3),3.24ppm(s,3H,-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- 3CH) 3.48ppm (m, about 1,900H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3),7.24ppm(m,6H,-NH-CO-CH-CH2- 6 5CH-NH-), 7.73ppm (t,1H), 8.12ppm (Wide, 1H)

[ examples 1 to 3]

[ solution 26]

L-glutamic acid (Fmoc-Glu-OH) protected by Fmoc group to the N-terminus (16.0mg, 4.3X 10)-5mol manufactured by Du chemical industries Co., Ltd.) and ME-200GF-NH obtained in example 1-22(2.0g,1.0×10-4mol) was added with dehydrated N, N' -dimethylformamide (10g), and the mixture was dissolved by heating at 30 ℃. Then, diisopropylethylamine (19.2. mu.L, 1.1X 10) was added-4mol, manufactured by Kanto chemical Co., Ltd.) and 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholine hydrochloride N hydrate (DMT-MM) (39.0mg, 1.1X 10)-4mol, Wako pure chemical industries, Ltd.) was reacted at room temperature under a nitrogen atmosphere for 1 hour. Then, piperidine (0.5g, 5.9X 10) was added-3mol, andmanufactured by Wako pure chemical industries, Ltd.) was reacted at room temperature under a nitrogen atmosphere for 2 hours. After completion of the reaction, the reaction mixture was diluted with toluene (80g), and hexane (40g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. After the precipitate was recovered by suction filtration using a 5A filter paper, it was dissolved in toluene (80g) again, and hexane (40g) was added thereto, followed by stirring at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (40g), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p3) (b) 2 2NH-E(FG-200ME)). The yield was 1.6 g. The molecular weights are shown in table 1. HPLC: the amine purity was 92%.

1H-NMR(d6-DMSO):1.54ppm(m,2H,-NH-CO-CH(NH2)- 2CH-CH2-),1.62ppm(m,4H,-CO-NH-CH2- 2CH-CH2-),1.97ppm(m,2H,-NH-CO-CH(NH2)-CH2- 2CH-),2.74ppm(dd,1H,-CO-NH-CH- 2CH-C6H5),2.81ppm(dd,1H,-CO-NH-CH- 2CH-C6H5),3.11ppm(m,11H),3.24ppm(s,6H,-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- 3CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3),4.49ppm(m,1H,-CO-NH-CH-CH2-C6H5),4.57ppm(m,1H,-CO-NH-CH-CH2-C6H5),7.25ppm(m,10H,-CO-NH-CH-CH2- 6 5CH),7.74ppm(m,2H),8.44ppm(m,2H),8.61ppm(m,2H)

[ example 2]

Compound (p4) (p) 2MA-E(FG-200ME)) Synthesis of (2)

[ solution 27]

The compound (p3) (200mg, 5.0X 10) obtained in example 1 was added-6mol) was dissolved in acetonitrile (160mg) and toluene (1.0 g). Then, N-methylmorpholine (10mg, 1.0X 10) was added-5mol, manufactured by Kanto chemical Co., Ltd.) and 3-Maleimidopropionic acid N-hydroxysuccinimide ester (8.0mg, 3.0X 10)-5mol, manufactured by osaka synthetic organic chemistry research institute corporation), and reacted at room temperature under a nitrogen atmosphere and under light shielding for 6 hours. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (50g) containing 2, 6-di-t-butyl-p-cresol (BHT) (10mg), and hexane (25g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (25g) containing BHT (5mg), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p4) ((25 g)) 2MA-E(FG-200ME)). The yield was 137 mg. The molecular weights are shown in table 1. The purity of maleimide is 90%, (1H-NMR)。

1H-NMR(d6-DMSO):1.62ppm(m,6H),1.99ppm(m,2H,-NH-CO-CH(NH2)-CH2- 2CH-),2.34ppm(m,2H,-NH-CO- 2CH-CH2-maleimide), 2.75ppm (dd,1H, -CO-NH-CH- 2CH-C6H5),2.82ppm(dd,1H,-CO-NH-CH- 2CH-C6H5),3.11ppm(m,11H),3.24ppm(s,6H,-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- 3CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3),4.04ppm(m,2H,-NH-CO-CH2- 2CHMaleimide), 4.49ppm (m,2H, -CO-NH-CH-CH2-C6H5),6.98ppm(s,2H,-CO-CH-CH-CO-),7.25ppm(m,10H,-CO-NH-CH-CH2- 6 5CH),7.69ppm(dt,2H),8.04ppm(d,1H),8.29ppm(dd,2H),8.41ppm(dt,2H)

[ example 3]

Compound (p8) (p) 2AL-E(FG-200ME)) Synthesis of (2)

[ solution 28]

[ example 3-1]

Compound (p5) (p) 2HO-E(FG-200ME)) Synthesis of (2)

[ solution 29]

Mixing epsilon-caprolactone (114mg, 1.0X 10)-3mol, manufactured by Tokyo chemical industry Co., Ltd.) was dissolved in 1N NaOH (0.8mL, 8.0X 10-4mol, manufactured by Kanto chemical Co., Ltd.) was reacted for 2 hours to prepare an aqueous solution of 6-hydroxycaproic acid (0.88M). Further, the compound (p3) (2.0g, 5.0X 10) obtained in example 1 was added-5mol) was dissolved in acetonitrile (8.0 g). Then, the above 6-hydroxycaproic acid aqueous solution (114. mu.L, 1.0X 10)-4mol), diisopropylethylamine (20. mu.L, 1.2X 10)- 4mol, manufactured by Kanto chemical Co., Ltd.) and DMT-MM (21mg, 6.0X 10)-5mol, Wako pure chemical industries, Ltd.) was added to the acetonitrile solution of (p3), and reacted for 1 hour at room temperature under a nitrogen atmosphere. After completion of the reaction, the reaction mixture was concentrated at 40 ℃ and chloroform (24g) was added to the obtained concentrate to dissolve it. Saturated aqueous sodium bicarbonate (10g) was added thereto, and the mixture was stirred at room temperature for 15 minutes to wash the mixture. After separating the aqueous layer and the organic layer, a saturated aqueous sodium bicarbonate solution (10g) was added to the organic layer again, and the mixture was stirred at room temperature for 15 minutes to wash the mixture, thereby recovering the organic layer. To the obtained organic layer (chloroform solution), magnesium sulfate (1.2g) was added, and the mixture was stirred for 30 minutes to dehydrate, followed by suction filtration using an aleurite funnel in which Oplite was spread on a 5A filter paper. The obtained filtrate was concentrated at 40 ℃, toluene (50g) was added to the concentrate and stirred until uniform, then hexane (25g) was added thereto, and stirred at room temperature for 15 minutes to precipitate a product. The precipitate was collected by suction filtration using a 5A filter paper, and then dissolved in toluene (50g) again, followed by addition of hexane(25g) The mixture was stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (10g) containing BHT (2mg), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p5) (10g) 2HO-E(FG-200ME)). The yield was 1.5 g.

1H-NMR(CDCl3):1.37ppm(m,2H,HO-CH2-CH2- 2CH-CH2-CH2-CO-NH-),1.55ppm(m,4H,HO-CH2- 2CH-CH2- 2CH-CH2-CO-NH-),1.77ppm(m,4H,-CO-NH-CH2- 2CH-CH2-O-(CH2-CH2-O)n-CH3),1.85ppm(m,1H),2.01ppm(m,2H,HO-CH2-CH2-CH2-CH2- 2CH-CO-NH-),3.01ppm(m,1H),3.24ppm(m,8H),3.38ppm(s,6H,-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- 3CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3),4.03ppm(m,4H),4.14ppm(m,1H),4.48ppm(m,2H,-CO-NH-CH-CH2-C6H5) 6.95ppm (Wide, 1H), 7.00ppm (Wide, 1H), 7.26ppm (m,10H, -CO-NH-CH-CH)2- 6 5CH) 7.66ppm (Wide, 1H), 8.29ppm (Wide, 1H)

[ examples 3-2]

Compound (p6) (p) 2SC-E(FG-200ME)) Synthesis of (2)

[ solution 30]

The compound (p5) (500mg, 1.3X 10) obtained in example 3-1 was added-5mol) was dissolved in dichloromethane (3.5 g). Then, N' -disuccinimidyl carbonate (51mg, 2.0X 10) was added-4mol, manufactured by Tokyo chemical industry Co., Ltd.) and pyridine (24. mu.L, 3.0X 10)-4molManufactured by kanto chemical corporation) at room temperature under a nitrogen atmosphere for 8 hours. After completion of the reaction, the reaction solution was washed with 5% saline, magnesium sulfate (0.1g) was added thereto, and the mixture was stirred at 25 ℃ for 30 minutes, followed by suction filtration using a Fukusan funnel in which Oplite was spread on a 5A filter paper. The obtained filtrate was concentrated, and toluene (50g) was added to the concentrate to dissolve it, followed by addition of hexane (25g) and stirring at room temperature for 15 minutes to precipitate a product. After the precipitate was recovered by suction filtration using a 5A filter paper, it was dissolved in toluene (50g) again, and hexane (25g) was added thereto, followed by stirring at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (25g) containing BHT (5mg), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p6) ((25 g)) 2SC-E(FG-200ME)). The yield was 286 mg. Purity of activated carbonate 92% ((1H-NMR)。

1H-NMR(CDCl3) 1.38ppm (m,2H, succinimide-OCO-CH)2-CH2- 2CH-CH2-CH2-CO-NH-), 1.59ppm (m,2H, succinimide-OCO-CH)2-CH2-CH2- 2CH-CH2-CO-NH-), 1.75ppm (m,6H), 1.85ppm (m,1H), 2.13ppm (m,2H, succinimide-OCO-CH)2-CH2-CH2-CH2- 2CH-CO-NH-),2.83ppm(s,4H,-CO- 2 2CH-CH-CO-),3.01ppm(m,1H),3.19ppm(m,6H),3.38ppm(s,6H,-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- 3CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3) 4.03ppm (m,3H), 4.18ppm (m,1H), 4.31ppm (t,2H, succinimide-OCO- 2CH-CH2-CH2-CH2-CH2-CO-NH-),4.50ppm(m,2H,-CO-NH-CH-CH2-C6H5) 6.98ppm (Wide, 1H), 7.15ppm (Wide, 1H), 7.26ppm (m,10H, -CO-NH-CH-CH)2- 6 5CH) 7.81ppm (Wide, 1H), 8.37ppm (Wide, 1H)

[ examples 3 to 3]

Compound (p7) (p) 2DE-E(FG-200ME)) Synthesis of (2)

[ solution 31]

The compound (p6) (250mg, 6.3X 10) obtained in example 3-2 was added-6mol) in chloroform (2 g). Then, 1-amino-3, 3-diethoxypropane (10. mu.L, 6.3X 10) was added-5mol, manufactured by ACROS ORGANICS corporation), at room temperature under a nitrogen atmosphere for 3 hours. After completion of the reaction, the reaction mixture was diluted with toluene (25g), and hexane (12.5g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using a 5A filter paper to collect the precipitate, followed by washing with hexane (12.5g) containing BHT (2.5mg), suction filtration was performed using a 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p47) ((p 47)) 2DE-E(FG-200ME)). The yield was 185 mg.

1H-NMR(CDCl3):1.20ppm(t,6H,( 3CH-CH2-O)2-CH-),1.32ppm(m,2H,(CH3-CH2-O)2-CH-CH2-CH2-NH-COO-CH2-CH2- 2CH-CH2-CH2-CO-NH-),1.58ppm(m,2H,(CH3-CH2-O)2-CH-CH2-CH2-NH-COO-CH2-CH2-CH2 2-CH-CH2-CO-NH-),1.76ppm(m,4H,-CO-NH-CH2- 2CH-CH2-O-(CH2-CH2-O)n-CH3),1.82ppm(m,2H,(CH3-CH2-O)2-CH- 2CH-CH2-NH-COO-CH2-CH2-CH2-CH2-CH2-CO-NH-),2.11ppm(m,2H,(CH3-CH2-O)2-CH-CH2-CH2-NH-COO-CH2- 2CH-CH2-CH2-CH2-CO-NH-),2.16ppm(m,1H),2.70ppm(m,1H),3.06ppm(m,2H),3.25ppm(m,11H),3.38ppm(s,6H,-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- 3CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3),4.02ppm(m,8H),4.17ppm(m,1H),4.51ppm(m,2H,-CO-NH-CH-CH2-C6H5),4.55ppm(t,1H,(CH3-CH2-O)2-CH-), 5.36ppm (Width, 1H), 6.47ppm (Width, 1H), 6.98ppm (Width, 2H), 7.26ppm (m,10H, -CO-NH-CH-CH)2- 6 5CH) 7.81ppm (Wide, 1H), 8.36ppm (Wide, 1H)

[ examples 3 to 4]

Compound (p8) (p) 2AL-E(FG-200ME)) Synthesis of (2)

[ solution 32]

The compound (p7) (150mg, 3.8X 10) obtained in example 3-3 was added-6mol) was dissolved in a phosphate buffer (2.25g) adjusted to pH 1.90 and reacted at room temperature under a nitrogen atmosphere for 3 hours. After the reaction, 0.1N aqueous sodium hydroxide (0.89g) was added to adjust the pH to 6.40, and then sodium chloride (0.56g) was added to dissolve the mixture. To the obtained solution, 0.1N aqueous sodium hydroxide solution (0.60g) was added dropwise to adjust the pH to 7.06, followed by addition of chloroform (3g) containing BHT (0.6mg), stirring at room temperature for 20 minutes, and the product was extracted into an organic layer. After the organic layer and the aqueous layer were separated and the organic layer was collected, chloroform (3g) containing BHT (0.6mg) was added again to the aqueous layer, and the mixture was stirred at room temperature for 20 minutes to extract the product into the organic layer. The organic layers obtained in the 1 st extraction and the 2nd extraction were combined and concentrated at 40 ℃, and the obtained concentrate was diluted with toluene (30g), and hexane (15g) was added and stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using a 5A filter paper, and the precipitate was collected, followed by washing with hexane (15g) containing BHT (3.0mg), suction filtration was performed using a 5A filter paper, and vacuum drying was performed to obtain the aboveThe compound (p8) (p) 2AL-E(FG-200ME)). The yield was 84 mg. The molecular weights are shown in table 1. The aldehyde purity is 92%, (1H-NMR)。

1H-NMR(CDCl3):1.32ppm(m,2H,CHO-CH2-CH2-NH-COO-CH2-CH2- 2CH-CH2-CH2-CO-NH-),1.57ppm(m,2H,CHO-CH2-CH2-NH-COO-CH2-CH2-CH2- 2CH-CH2-CO-NH-),1.76ppm(m,4H,-CO-NH-CH2- 2CH-CH2-O-(CH2-CH2-O)n-CH3),1.82ppm(m,1H),2.10ppm(m,2H,CHO-CH2-CH2-NH-COO-CH2- 2CH-CH2-CH2-CH2-CO-NH-),2.16ppm(m,1H),2.71ppm(m,2H,CHO- 2CH-CH2-NH-COO-CH2-CH2-CH2-CH2-CH2-CO-NH-),3.02ppm(m,1H),3.26ppm(m,8H),3.38ppm(s,6H,-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- 3CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3),4.01ppm(m,4H),4.16ppm(m,1H),4.49ppm(m,2H,-CO-NH-CH-CH2-C6H5) 5.59ppm (Wide, 1H), 6.36ppm (Wide, 1H), 6.93ppm (Wide, 2H), 7.08ppm (Wide, 1H), 7.26ppm (m,10H, -CO-NH-CH-CH)2- 6 5CH) 7.80ppm (width, 1H), 8.37ppm (width, 1H), 9.79ppm (s,1H,CHO-CH2-CH2-NH-COO-)

[ example 4]

Compound (p9) (p) 2 2NHO-E(FG-200ME)) Synthesis of (2)

[ solution 33]

The compound obtained in example 3-1 was converted intoCompound (p5) (300mg, 7.5X 10-6mol) was dissolved in toluene (2.4g) at 30 ℃ with heating and azeotropically dehydrated under reduced pressure. Then, the concentrate was dissolved in chloroform (2.4g), and N-hydroxyphthalimide (7.3mg, 4.5X 10) was added-5mol, Wako pure chemical industries, Ltd.), triphenylphosphine (35mg, 1.4X 10- 4mol, manufactured by Kanto chemical Co., Ltd.) and diisopropyl azodicarboxylate (22. mu.L, 1.1X 10)-4mol, manufactured by ACROS ORGANICS corporation), and reacted at room temperature under a nitrogen atmosphere for 4 hours. After completion of the reaction, methanol (9.1. mu.L) was added to the reaction solution, stirred at 25 ℃ for 30 minutes, and concentrated at 40 ℃. The concentrate was diluted with toluene (3.0g) and azeotroped, and then the concentrate was dissolved in toluene (1.5g), and ethylenediamine monohydrate (24. mu.L, 3.0X 10)-4mol, manufactured by Kanto chemical Co., Ltd.), and reacted at room temperature under a nitrogen atmosphere for 1 hour. After completion of the reaction, the reaction mixture was diluted with toluene (50g), and hexane (25g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (20g), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p9) ((p 9)) 2NHO- 2E(FG-200ME)). The yield was 156 mg. The molecular weights are shown in table 1. HPLC: the purity of the amine oxide is 91%.

1H-NMR(CDCl3):1.32ppm(m,2H,H2N-O-CH2-CH2- 2CH-CH2-CH2-CO-NH-),1.56ppm(m,4H,H2N-O-CH2- 2CH-CH2- 2CH-CH2-CO-NH-),1.76ppm(m,4H,-CO-NH-CH2- 2CH-CH2-O-(CH2-CH2-O)n-CH3),1.85ppm(m,1H),2.10ppm(m,2H,H2N-O-CH2-CH2-CH2-CH2- 2CH-CO-NH-),2.17ppm(m,1H),3.01ppm(m,1H),3.24ppm(m,8H),3.38ppm(s,6H,-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- 3CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3),4.03ppm(m,2H),4.17ppm(m,1H),4.49ppm(m,2H,-CO-NH-CH-CH2-C6H5) 5.37ppm (Width, 2H), 6.40ppm (Width, 1H), 6.95ppm (Width, 2H), 7.12ppm (Width, 1H), 7.26ppm (m,10H, -CO-NH-CH-CH)2- 6 5CH) 7.74ppm (Wide, 1H), 8.31ppm (Wide, 1H)

[ example 5]

Compound (p13) (p) 2 2 2NH-E{E(FG-100ME)}) Synthesis of (2)

[ chemical 34]

[ example 5-1]

Compound (p10) (p)ME-100GF-Fmoc) Synthesis of (2)

[ solution 35]

L-phenylalanyl-glycine (Fmoc-Phe-Gly) (533mg, 1.2X 10) having its N-terminal protected by Fmoc group was used in the same manner as in example 1-1-3mol, manufactured by Dubian chemical Co., Ltd.) and methoxy PEG having propylamino group at the terminal (9.9g, 1.0X 10-3mol, number average molecular weight 9,896, "sunberght MEPA-10T" manufactured by japan oil corporation, as a raw material, to obtain the above compound (p10) ((p 10))ME-100GF-Fmoc). The yield was 9.2 g.

1H-NMR(d6-DMSO):1.62ppm(m,2H,-CO-NH-CH2- 2CH-CH2-O-(CH2-CH2-O)n-CH3),2.80ppm(m,1H,-NH-CO-CH- 2CH-C6H5),3.04ppm(m,1H,-NH-CO-CH- 2CH-C6H5),3.10ppm(m,2H,-CO-NH- 2CH-CH2-CH2-O-(CH2-CH2-O)n-CH3),3.24ppm(s,3H,-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- 3CH) 3.48ppm (m, about 900H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3),4.20ppm(m,4H),7.33ppm(m,9H),7.66ppm(m,4H,Ar),7.88ppm(d,2H,Ar),8.27ppm(t,1H)

[ examples 5-2]

Compound (p11) (p) 2ME-100GF-NH) Synthesis of (2)

[ solution 36]

The compound (p10) (9.2g, 4.6X 10) obtained in example 5-1 was used in the same manner as in example 1-2-4mol) to obtain the above compound (p11), (p11) 2ME-100GF-NH). The yield was 8.7 g.

1H-NMR(d6-DMSO):1.62ppm(m,2H,-CO-NH-CH2-CH 2 -CH2-O-(CH2-CH2-O)n-CH3) 1.64ppm (width, 1H), 2.59ppm (dd,1H, -NH-CO-CH- 2CH-C6H5),2.98ppm(dd,1H,-NH-CO-CH- 2CH-C6H5),3.10ppm(q,2H,-CO-NH- 2CH-CH2-CH2-O-(CH2-CH2-O)n-CH3),3.24ppm(s,3H,-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- 3CH) 3.48ppm (m, about 900H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3),7.24ppm(m,6H,-NH-CO-CH-CH2- 6 5CH-NH-), 7.73ppm (t,1H), 8.12ppm (Wide, 1H)

[ examples 5 to 3]

Compound (p12) (p) 2 2NH-E(FG-100ME)) Synthesis of (2)

[ solution 37]

L-glutamic acid (Fmoc-Glu-OH) (135mg, 3.7X 10) having its N-terminus protected by Fmoc group was used in the same manner as in example 1-3-4mol, manufactured by Biffman chemical Co., Ltd.) and the compound (p11) (8.5g, 8.5X 10) obtained in example 5-2-4mol) as a starting material, successively subjected to a reaction and deprotection to obtain the above-mentioned compound (p12) ((p 12) 2NH- 2E(FG-100ME)). The yield was 6.6 g. HPLC: the amine purity was 95%.

1H-NMR(d6-DMSO):1.54ppm(m,2H,-NH-CO-CH(NH2)- 2CH-CH2-),1.62ppm(m,4H,-CO-NH-CH2- 2CH-CH2-),1.97ppm(m,2H,-NH-CO-CH(NH2)-CH2- 2CH-),2.74ppm(dd,1H,-CO-NH-CH- 2CH-C6H5),2.81ppm(dd,1H,-CO-NH-CH- 2CH-C6H5),3.11ppm(m,11H),3.24ppm(s,6H,-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- 3CH) 3.64ppm (m, about 1,800H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3),4.49ppm(m,1H,-CO-NH-CH-CH2-C6H5),4.57ppm(m,1H,-CO-NH-CH-CH2-C6H5),7.25ppm(m,10H,-CO-NH-CH-CH2- 6 5CH),7.74ppm(m,2H),8.44ppm(m,2H),8.61ppm(m,2H)

[ examples 5 to 4]

Compound (p13) (p) 2 2 2NH-E{E(FG-100ME)}) Synthesis of (2)

[ solution 38]

L-glutamic acid (Fmoc-Glu-OH) (15.2mg, 4.1X 10) having its N-terminus protected by Fmoc group was used in the same manner as in example 1-3-5mol, manufactured by Biffman chemical Co., Ltd.) and the compound (p12) (2.0g, 1.0X 10) obtained in example 5-3-4mol) as a starting material, successively subjected to a reaction and deprotection to obtain the above-mentioned compound (p13) ((p 13) 2NH- 2 2E{E(FG-100ME)}). The yield was 1.2 g. The molecular weights are shown in table 1. HPLC: the amine purity was 94%.

1H-NMR(d6-DMSO):1.62ppm(m,14H),2.00ppm(m,6H,-NH-CO-CH(NH2)-CH2- 2CH-),2.78ppm(m,4H),3.11ppm(m,14H),3.24ppm(s,16H,-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- 3CH) 3.64ppm (m, about 3,600H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3),4.19ppm(m,2H),4.51ppm(m,4H),7.25ppm(m,20H,-CO-NH-CH-CH2- 6 5CH),7.71ppm(m,4H),7.89ppm(m,1H),8.45ppm(m,9H)

[ example 6]

Compound (p16) (p) 2 2NH-E(GFLG-200ME)) Synthesis of (2)

[ solution 39]

[ example 6-1]

Compound (p14) (p)ME-200GLFG-Fmoc) Synthesis of (2)

[ solution 40]

Using L-glycyl-phenylalanyl-leucyl-glycyl protected at the N-terminus by Fmoc group, the preparation was carried out in the same manner as in example 1-1Amino acid (Fmoc-Gly-Phe-Leu-Gly) (66mg, 1.1X 10-4mol, manufactured by Dubian chemical industries Co., Ltd.) and methoxy PEG having propylamino group at the terminal (1.5g, 7.1X 10)-5mol, number average molecular weight 21,120, "sunberg MEPA-20T" manufactured by japan oil co., ltd., as a raw material, to obtain the above compound (p14) ((mME-200GLFG- Fmoc). The yield was 1.2 g.

1H-NMR(CDCl3):0.89ppm(d,3H,-NH-CO-CH-CH2-CH( 3CH)2)、0.91ppm(d,3H,-NH-CO-CH-CH2-CH( 3CH)2),1.53ppm(m,2H,-NH-CO-CH- 2CH-CH(CH3)2),1,70ppm(m,1H,-NH-CO-CH-CH2-CH(CH3)2),1.80ppm(m,2H,-CO-NH-CH2- 2CH-CH2-O-(CH2-CH2-O)n-CH3),3.10ppm(dd,1H,-NH-CO-CH- 2CH-C6H5),3.18ppm(dd,1H,-NH-CO-CH- 2CH-C6H5) 3.33ppm (m,7H), 3.74ppm (m, about 1,900H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3) 4.31ppm (width, 1H), 4.55ppm (t,1H, -NH-CO-CH-CH2-C6H5) 6.91ppm (Wide, 1H), 7.00ppm (Wide, 1H), 7.28ppm (m,5H, -NH-CO-CH-CH)2- 6 5CH) 7.33ppm (t,2H, Ar), 7.41ppm (m,3H, Ar), 7.73ppm (m,3H, Ar), 7.89ppm (d,2H, Ar), 7.98ppm (Width, 1H)

[ example 6-2]

Compound (p15) (p) 2ME-200GLFG-NH) Synthesis of (2)

[ solution 41]

The compound (p14) (1.2g, 5.7X 10) obtained in example 6-1 was used in the same manner as in example 1-2-5mol) to obtain the above compound (p15), (p15) 2ME-200GLFG-NH). The yield was 1.0 g.

1H-NMR(CDCl3):0.89ppm(d,3H,-NH-CO-CH-CH2-CH( 3CH)2)、0.91ppm(d,3H,-NH-CO-CH-CH2-CH( 3CH)2),1.53ppm(m,2H,-NH-CO-CH- 2CH-CH(CH3)2),1,70ppm(m,1H,-NH-CO-CH-CH2-CH(CH3)2),1.80ppm(m,2H,-CO-NH-CH2- 2CH-CH2-O-(CH2-CH2-O)n-CH3),3.10ppm(dd,1H,-NH-CO-CH- 2CH-C6H5),3.18ppm(dd,1H,-NH-CO-CH- 2CH-C6H5) 3.33ppm (m,7H), 3.74ppm (m, about 1,900H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3) 4.31ppm (width, 1H), 4.55ppm (t,1H, -NH-CO-CH-CH2-C6H5) 6.91ppm (Wide, 1H), 7.00ppm (Wide, 1H), 7.28ppm (m,5H, -NH-CO-CH-CH)2- 6 5CH) 7.98ppm (Wide, 1H)

[ examples 6 to 3]

Compound (p16) (p) 2 2NH-E(GFLG-200ME)) Synthesis of (2)

[ solution 42]

L-glutamic acid (Fmoc-Glu-OH) (8.3mg, 2.3X 10) having its N-terminus protected by Fmoc group was prepared in the same manner as in example 1-3-5mol, manufactured by Biffman chemical Co., Ltd.) and the compound (p15) (1.0g, 4.8X 10) obtained in example 6-2-5mol) as a starting material, successively subjected to a reaction and deprotection to obtain the above-mentioned compound (p16) ((p 16) 2NH-E 2(GFLG-200ME)). The yield was 0.5 g. The molecular weights are shown in table 1. HPLC: the amine purity was 90%.

1H-NMR(CDCl3):0.89ppm(d,6H,-NH-CO-CH-CH2-CH( 3CH)2)、0.91ppm(d,6H,-NH-CO-CH-CH2-CH( 3CH)2),1.53ppm(m,4H,-NH-CO-CH- 2CH-CH(CH3)2),1,70ppm(m,2H,-NH-CO-CH-CH2-CH(CH3)2),1.77ppm(m,4H,-CO-NH-CH2- 2CH-CH2-O-(CH2-CH2-O)n-CH3),1.85ppm(m,1H),3.01ppm(m,1H),3.24ppm(m,8H),3.38ppm(s,6H,-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- 3CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)2-CH2-CH2-O-( 2 2CH-CH-O)n-CH3),4.03ppm(m,4H),4.14ppm(m,1H),4.48ppm(m,2H,-CO-NH-CH-CH2-C6H5) 6.95ppm (Wide, 1H), 7.00ppm (Wide, 1H), 7.26ppm (m,10H, -CO-NH-CH-CH)2- 6 5CH) 7.66ppm (Wide, 2H), 8.29ppm (Wide, 2H)

Comparative example 1

Compound (p18) (p) 2LY-400NH) Synthesis of (2)

[ solution 43]

Comparative examples 1 to 1

Compound (p17) (p)LY-400BO) Synthesis of (2)

[ solution 44]

2-branched polyethylene glycol activated ester of lysine skeleton (3.0g, 7.5X 10) used in polyethylene glycol modifier on the market-5mol, "SUNBRIGHT LY-400 NS" manufactured by Nichikoku K.K., having a number average molecular weight of 39,700 was dissolved in toluene (15g) at 40 ℃ under heating, and N- (tert-butoxycarbonyl) -1, 2-diaminoethane (48. mu. mu.M) was addedL,3.0×10-4mol, manufactured by Tokyo chemical industry Co., Ltd.) was reacted at 40 ℃ for 1 hour under a nitrogen atmosphere. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (12g), and hexane (14g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. After the precipitate was recovered by suction filtration using a 5A filter paper, it was dissolved in ethyl acetate (27g) again, and hexane (14g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (30g), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p17) (b)LY-400BO). The yield was 2.7 g.

1H-NMR(CDCl3):1.37ppm(m,2H),1.43ppm(s,9H,-CH2-NH-CO2-C-( 3CH)3),1.51ppm(m,2H),3.15ppm(m,2H),3.38ppm(s,6H,-O-(CH2-CH2-O)n- 3CH) 3.65ppm (m, about 3,650H, -O- (R-) - (R)) 2 2CH-CH-O)n-CH3),4.21ppm(m,4H)

Comparative examples 1 and 2

Compound (p18) (p) 2LY-400NH) Synthesis of (2)

[ solution 45]

The compound (p17) (1.0g, 2.5X 10) obtained in comparative example 1-1-6mol) was dissolved in ion-exchanged water (4.0g), and methanesulfonic acid (57. mu.L, 8.8X 10) was added-4mol, manufactured by Kanto chemical Co., Ltd.), at 40 ℃ under a nitrogen atmosphere for 6 hours. After the reaction, the reaction mixture was diluted with ion-exchanged water (6.0g), a 1N aqueous solution (0.9g) of sodium hydroxide was added to adjust the pH to 12, and then sodium chloride (2.5g) was added to dissolve the mixture. Chloroform (10g) containing BHT (1.0mg) was added to the obtained solution, and the mixture was stirred at room temperature for 20 minutes to extract the product into an organic layer. The organic layer and the aqueous layer were separated, the organic layer was recovered, and then concentrated at 40 ℃, and the obtained concentrate was diluted with toluene (30g), hexane (15g) was added and stirred at room temperature for 15 minutes to allowThe product was precipitated. Suction filtration was performed using a 5A filter paper to collect the precipitate, followed by washing with hexane (15g) containing BHT (3.0mg), suction filtration was performed using a 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p18), (p18) 2LY-400NH). The yield was 0.7 g. The molecular weights are shown in table 1. HPLC: the amine purity was 97%.

1H-NMR(CDCl3):1.37ppm(m,2H),1.51ppm(m,2H),3.15ppm(m,2H),3.38ppm(s,6H,-O-(CH2-CH2-O)n- 3CH) 3.65ppm (m, about 3,650H, -O- (R-) - (R)) 2 2CH-CH-O)n-CH3),4.21ppm(m,4H)

[ Table 1]

Sample name Molecular weight (M)n)
Example 1 Compound (p3) 42,417
Example 2 Compound (p4) 42,534
Example 3 Compound (p8) 42,334
Example 4 Compound (p9) 42,190
Example 5 Compound (p13) 38,234
Example 6 Compound (p16) 42,398
Comparative example 1 Compound (p18) 39,654

[ example 7]

Stability test in serum

To a 1.5mL microcentrifuge tube, 1mL of mouse or human serum was added, and each polyethylene glycol derivative was added to a concentration of 5.0 mg/mL. After incubation at 37 ℃ for 96 hours, 200. mu.L of the sample was taken, acetonitrile was added thereto, and the mixture was stirred with a vortex mixer for 1 minute to precipitate proteins in serum, and after centrifugation, the supernatant was collected. Next, hexane was added to the recovered solution to remove hydrophobic substances such as fatty acids, and the mixture was stirred with a vortex mixer for 1 minute, centrifuged, and the lower layer was recovered. The solution was concentrated under vacuum, and the polyethylene glycol derivative was recovered from the serum. Then, GPC analysis was performed to calculate the decomposition rate of the decomposable polyethylene glycol derivative.

The decomposition rate was calculated as follows:

decomposition rate ═ peak area of 40kDa before assay% — peak area of 40kDa after assay%/(% peak area of 40kDa before assay) × 100 ×

The results are shown in table 2 below.

[ Table 2]

According to Table 2, no decomposition was observed in the serum of the compounds (p3), (p13) and (p16) which are decomposable polyethylene glycol derivatives, as in the case of the compound (p18) which is a non-decomposable polyethylene glycol derivative and methoxy PEG amine 40 kDa. That is, the degradable polyethylene glycol derivative is stable in blood.

[ example 8]

Disintegration test Using cells

Using 10mL of the culture medium RPMI-1640 (10% FBS Pn/St), 10X 10 was inoculated into a 100mm dish6The cells were cultured at 37 ℃ for 24 hours in RAW264.7, then replaced with a medium containing various polyethylene glycol derivatives dissolved therein to a concentration of 10mg/mL, and cultured at 37 ℃ for 96 hours. After the culture, the cells were dissolved in a 1% SDS solution, diluted with Phosphate Buffered Saline (PBS), acetonitrile was added thereto, and the mixture was stirred with a vortex mixer for 1 minute to precipitate the protein in the cell lysate, and after centrifugation, the supernatant was collected. Next, hexane was added to the recovered solution to remove hydrophobic substances such as fatty acids, and the mixture was stirred with a vortex mixer for 1 minute, centrifuged, and the lower layer was recovered. The solution was concentrated under vacuum, and the polyethylene glycol derivative was recovered from the cells.

In addition, in order to confirm the decomposition in the culture medium for cell culture, only in the dissolved various polyethylene glycol derivatives to the concentration of 10mg/mL culture medium at 37 degrees C were cultured for 96 hours, according to the same operation of polyethylene glycol derivatives recovery.

Then, GPC analysis of each of the collected polyethylene glycol derivatives was performed, and the decomposition rate of the decomposable polyethylene glycol derivative was calculated according to the same calculation formula as in example 7.

The results are shown in table 3 below. Furthermore, GPC spectra of the compounds (p3) and (p13) before and after the cell experiment are shown in fig. 1 and fig. 2, and fig. 3 and fig. 4, respectively.

[ Table 3]

From Table 3, it was confirmed that the compounds (p3) and (p16) which are degradable polyethylene glycol derivatives are efficiently degraded in cells (degradation rate 99%) and degraded from 4 to 2 million in molecular weight. In addition, in the compound (p13), decomposition from 4 to 1 million in molecular weight was observed at a decomposition rate of 99%. Since these degradable polyethylene glycol derivatives do not decompose in the medium used for cell culture, they were confirmed to be specifically degraded in the cells. On the other hand, no intracellular degradation was observed in both the compound (p18) which is a non-degradable polyethylene glycol derivative and methoxy PEG amine 40 kDa.

[ example 9]

Vacuole formation evaluation test by animal test

Compound (p3) NH which is a decomposable polyethylene glycol derivative having an amino group at the end and a molecular weight of 4 ten thousand2-E(FG-200ME)2And a non-decomposable methoxy PEG amine of 40kDa, and subjected to blank cannon formation evaluation by an animal experiment. The mouse breed is Balb/c (8 weeks old, male), polyethylene glycol solution using physiological saline to prepare polyethylene glycol derivatives to a concentration of 100mg/mL, via the tail vein of the mouse 20 u L. Continuous administration was continued 3 times a week for 4 weeks, and after completion of administration, mice were fixed by perfusion with 4% paraformaldehyde aqueous solution to prepare paraffin sections. Vacuole formation in brain choroid plexus epithelial cells was assessed by HE staining and immunostaining with anti-PEG antibodies. The immunostaining was carried out using an immunostaining Kit (Bond reference Polymer Detection Kit, manufactured by Leica) and an anti-PEG antibody (B-47 antibody, manufactured by Abcam). Images of choroid plexus brain sections subjected to immunostaining with anti-PEG antibody are shown in FIG. 5 (methoxy PEG amine 40kDa) and FIG. 6 (NH)2-E(FG-200ME)2) In (1).

As a result, NH as a decomposable polyethylene glycol2-E(FG-200ME)2Vacuole formation was significantly inhibited compared to methoxy PEG amine 40 kDa.

The amount of polyethylene glycol administered in this example was finally estimated to be an optimized amount for evaluating vacuolation, and was very large compared with the amount of polyethylene glycol generally administered in this technical field.

[ example 10]

Evaluation test of accumulation Property of polyethylene glycol Using animal experiment

Compound (p3) NH which is a decomposable polyethylene glycol derivative having an amino group at the end and a molecular weight of 4 ten thousand2-E(FG-200ME)2And non-degradable methoxy PEG amine 20kDa, methoxy PEG amine 40kDa and PBS as control, and the accumulation of polyethylene glycol was evaluated by animal experiments. The mouse breed is Balb/c (8 weeks old, male), polyethylene glycol solution using physiological saline to prepare polyethylene glycol derivatives to a concentration of 62.5mg/mL, via mouse tail vein administration of 100 u L. Continuous administration was continued 3 times a week for 4 weeks, and after completion of administration, mice were fixed by perfusion with 4% paraformaldehyde aqueous solution to prepare paraffin sections. Immunostaining with anti-PEG antibody was performed to evaluate the accumulation in the brain choroid plexus epithelial cells. Images of each section of the brain choroid plexus after immunostaining are shown in fig. 7.

According to FIG. 7, the mouse choroid plexus section administered with PBS containing no polyethylene glycol was not stained, whereas the section was confirmed to be extensively stained brown in 40kDa, which is a nondegradable methoxy PEG amine. The stained portion indicates accumulation of PEG. On the other hand, in NH which is a decomposable polyethylene glycol2-E(FG-200ME)2The sections of (a) were less stained brown, showing the same accumulation as 20kDa, methoxy PEG amine with a molecular weight of half of that. As a result, the degradable polyethylene glycol significantly inhibited the accumulation of polyethylene glycol in the tissue due to its degradability, compared to the non-degradable methoxy PEG amine 40kDa, which was the same molecular weight.

The amount of polyethylene glycol administered in this example was finally optimized for evaluation of the accumulation property, and was very large compared with the amount of polyethylene glycol generally administered in this technical field.

[ example 11]

Pharmacokinetic experiments with animal experiments (radioisotopes)

NH which was a decomposable polyethylene glycol derivative having an amino group at the terminal and a molecular weight of 4 ten thousand2-E(FG-200ME)240kDa (average molecular weight: about 42,000, SUNBRIGHT GL2-400PA manufactured by Nichikoku K.K.) and 20kDa (average molecular weight: about 20,000, SUNBRIGHT GL2-200PA manufactured by Nichikoku K.K.) as nondegradable 2-branched PEG amines were dissolved in 50mM aqueous sodium bicarbonate solution at a concentration of 10mg/mL, and Bolton-Hunter (N-hydroxysuccinimide hydroxyphenylpropionate) reagent (0.4625MBq) was added thereto, followed by stirring with a vortex stirrer and then allowed to react at room temperature overnight. The reaction solution was separated by a PD-10 column, and the content of the reaction solution was confirmed by using a polyethylene glycol color developing agent (ammonium thiocyanate and cobalt nitrate) and a gamma counter for each fraction125And (3) the fraction I is recovered.

The obtained radioisotope-labeled polyethylene glycol derivative was used to evaluate pharmacokinetics in animal experiments. The mouse strain was Balb/c (8 weeks old, male), the polyethylene glycol solution was prepared using physiological saline to an unlabelled polyethylene glycol derivative concentration of 10mg/mL, and a small amount of a radioisotope-labeled polyethylene glycol derivative was added thereto, and 100. mu.L was administered via the tail vein of the mouse. Then, at 1,3, 6, 24, 48, and 72 hours, blood and organs were taken out from the mice, and the retention of the labeled polyethylene glycol derivative was measured using a γ counter.

NH as radioisotope-labeled degradable polyethylene glycol derivative2-E(FG-200ME)2The results of the pharmacokinetic experiments of 40kDa and 20kDa 2-branched PEG amines which are non-degradable polyethylene glycol derivatives are shown in FIG. 8.

According to FIG. 8, NH2-E(FG-200ME)2Compared to non-degradable 2-branched PEG amine 40kDa, which is the same molecular weight, showed the same degree of blood half-life. On the other hand, NH2-E(FG-200ME)2Compared with the non-degradable 2-branched PEG amine 20kDa with the molecular weight of 20kDa, the molecular weight of the PEG amine is obviously improvedShowing a significantly long blood half-life.

Industrial applicability

The degradable polyethylene glycol derivative of the present invention is a high molecular weight polyethylene glycol derivative that does not cause cavitation of cells, and can be effectively used for modification of biologically relevant substances, and is stable in blood in vivo and degraded in cells.

The present application is based on Japanese patent application No. 2019-069449 (application date: 2019, 3, 29), the contents of which are incorporated herein in their entirety.

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