Multi-arm degradable polyethylene glycol derivatives

Abstract

To provide a high-molecular-weight degradable multi-arm polyethylene glycol derivative that does not cause cell vacuoles.SOLUTION: A degradable polyethylene glycol derivative is represented by the following formula (1): (In the formula, n1 and n2 are each independently 45-950; W1 and W2 are each independently an oligopeptide having 2-47 residues; a1 and a2 are each independently 1-8; Q is a C2-C12 hydrocarbon chain which may contain an oxygen atom and / or a nitrogen atom; X1 and X2 are each independently a functional group capable of reacting with a biological substance; and L1, L2, L3, L4, L5, and L6 are each independently a divalent spacer).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a multi-arm degradable polyethylene glycol derivative which is used for modifying biological substances and which is degraded within cells. [Background technology]

[0002] When drugs using bio-related substances such as hormones, cytokines, antibodies, and enzymes are administered to the body, they are usually quickly excreted from the body by glomerular filtration in the kidneys or uptake by macrophages in the liver and spleen. Therefore, their half-life in the blood is short, and it is often difficult to obtain sufficient pharmacological effects. To solve this problem, attempts have been made to chemically modify bio-related substances with hydrophilic polymers such as sugar chains and polyethylene glycol, albumin, etc. As a result, it is possible to extend the half-life in the blood of bio-related substances by increasing the molecular weight and forming a hydration layer. It is also well known that modification with polyethylene glycol can reduce the toxicity and antigenicity of bio-related substances and improve the solubility of poorly water-soluble drugs.

[0003] Biomaterials modified with polyethylene glycol are covered with a hydration layer formed by hydrogen bonds between the ether bonds of polyethylene glycol and water molecules, and the molecular size increases, allowing them to avoid glomerular filtration in the kidney. It is also known that the interaction with opsonins and the cell surfaces that make up each tissue is reduced, reducing migration to each tissue. Polyethylene glycol is an excellent material for extending the half-life of biomaterials in the blood, and it is known that its performance is more effective the larger the molecular weight. Many studies have been conducted on biomaterials modified with high molecular weight polyethylene glycol with a molecular weight of 40,000 or more, and the results show that the blood half-life can be significantly extended.

[0004] Polyethylene glycol is considered the gold standard among modifiers used to improve the performance of biomaterials, and currently, several polyethylene glycol-modified preparations are on the market and used in medical practice. On the other hand, in 2012, the European Medicines Agency (EMA) reported that when a biomaterial modified with a high molecular weight polyethylene glycol having a molecular weight of 40,000 or more is administered to an animal for a long period of time at a certain dose or more, vacuoles are generated in the cells of some tissues (Non-Patent Document 1). At present, there are no reports that the generation of vacuoles itself has a negative effect on the human body, and considering that the dose used in the previous EMA report is extremely high compared to the dose generally applied in medical practice, it can be said that the safety of therapeutic preparations modified with polyethylene glycol having a molecular weight of 40,000 or more that are currently manufactured and sold is not a problem. However, in the treatment of very special diseases (e.g., dwarfism, etc.), it can be assumed that a treatment protocol in which a polyethylene glycol-modified preparation is administered to a patient in a high dose for a long period of time will be adopted. Therefore, it is expected that there is a potential demand for the development of a polyethylene glycol-modified preparation that is applicable even in such special situations and does not generate vacuoles in cells.

[0005] In Non-Patent Document 2, when a large excess amount of polyethylene glycol was administered alone to animals over a long period of time compared with the dosage of a normal polyethylene glycol-modified preparation, no vacuoles were observed at a molecular weight of 20,000, but the occurrence of vacuoles was confirmed at a molecular weight of 40,000. One possible means of suppressing vacuoles is to reduce the molecular weight of polyethylene glycol, but reducing the molecular weight causes the problem that the half-life in the blood of biologically relevant substances cannot be sufficiently improved.

[0006] There are reports of a technique for decomposing high molecular weight polyethylene glycol into low molecular weight polyethylene glycol in the body and promoting excretion from the kidney. Patent Document 1 describes a polyethylene glycol derivative having a sulfide bond or peptide bond site that is cleaved in the body. It describes that the polyethylene glycol derivative is decomposed in the body to a molecular weight suitable for excretion from the kidney. However, no data on specific decomposition is shown, and there is no data on promotion of excretion from the kidney. Furthermore, there is no description of cell vacuoles.

[0007] Patent Document 2 describes a polyethylene glycol derivative having an acetal moiety that can be hydrolyzed in a low pH environment in the body. It is described that the polyethylene glycol derivative is decomposed in the body to a molecular weight suitable for excretion from the kidney. However, there is no data that specifically shows that excretion from the kidney is promoted, and there is no description of cell vacuoles. In addition, it is known that these hydrolyzable acetal moieties are gradually decomposed in the blood, and it is expected that the blood half-life of the modified biological substance cannot be sufficiently improved.

[0008] On the other hand, there have been reports of polyethylene glycol derivatives incorporating degradable oligopeptides for effective drug release, and hydrogels that degrade within the body.

[0009] Non-Patent Document 3 describes a polyethylene glycol derivative having an oligopeptide moiety that is degraded by an enzyme. Here, the oligopeptide is introduced as a linker between the anticancer drug and the polyethylene glycol, and it is reported that the oligopeptide is degraded by an enzyme specifically expressed around the tumor, efficiently releasing the anticancer drug. The purpose is the release of the anticancer drug, and the degradability is not imparted to the polyethylene glycol for the purpose of suppressing cell vacuoles.

[0010] Non-Patent Document 4 describes a hydrogel using a crosslinking molecule having an oligopeptide moiety that is degraded by an enzyme and a multi-branched polyethylene glycol derivative. Here, the oligopeptide is used as a crosslinking molecule that connects the multi-branched polyethylene glycol derivative, and can further impart enzymatic degradability to the hydrogel. The purpose is to prepare a degradable hydrogel, not to impart degradability to polyethylene glycol for the purpose of suppressing cell vacuoles.

[0011] Patent Document 3 describes a branched polyethylene glycol derivative having an oligopeptide skeleton. Here, the oligopeptide is used as the basic skeleton of the polyethylene glycol derivative, and is not used to impart enzymatic decomposition. The oligopeptide is characterized by containing amino acids having amino or carboxyl groups in the side chain, such as lysine and aspartic acid, and the purpose is to synthesize a branched polyethylene glycol derivative using these in the reaction. It is not a polyethylene glycol derivative intended to suppress cell vacuoles.

[0012] Furthermore, among the polyethylene glycol derivatives used for modifying biologically relevant substances, there are multi-arm types, and as described in Non-Patent Documents 5 and 6, multiple biologically relevant substances can be bound to one molecule of a multi-arm polyethylene glycol derivative. It is reported that this significantly improves the solubility of biologically relevant substances and extends their half-life in the blood. Multi-arm polyethylene glycol derivatives are known to be able to increase the activity of drugs because they can carry many drugs, and have been used in many studies, but there have been no reports of multi-arm polyethylene glycol derivatives that suppress cell vacuoles.

[0013] As described above, there is a demand for multifunctional, multi-arm, high-molecular-weight polyethylene glycol derivatives that are stable in blood, improve the blood half-life of modified biological substances, and are specifically degraded within cells when taken up into the cells, thereby suppressing the occurrence of cellular vacuoles. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] Special Publication No. 2009-527581 [Patent Document 2] WO2005 / 108463 [Patent Document 3] WO2006 / 088248 [Non-patent literature]

[0015] [Non-Patent Document 1] EMA / CHMP / SWP / 647258 / 2012 [Non-Patent Document 2] Daniel G. Rudmann, et al., Toxicol. Pathol., 41, 970-983(2013) [Non-Patent Document 3] Francesco M Veronese, et al., Bioconjugate Chem., 16, 775-784(2005) [Non-Patent Document 4] Jiyuan Yang, et al., Marcomol. Biosci., 10(4), 445-454(2010) [Non-Patent Document 5] Lin Dai, et al., Scientific Reports, 29 July (2014) [Non-Patent Document 6] M. Eugenia Giorgi, et al., Glycobiology, 22(10), 1363-1373(2012) Summary of the Invention [Problem to be solved by the invention]

[0016] The object of the present invention is to provide a high molecular weight multi-arm polyethylene glycol derivative that does not cause cellular vacuoles. More specifically, the object of the present invention is to provide a multi-arm degradable polyethylene glycol derivative that can be effectively used for modifying biological substances, is stable in the blood of a living body, and is degraded within cells, by an industrially producible method. [Means for solving the problem]

[0017] Means for Solving the Problems The present inventors have conducted extensive research to solve the above problems and have invented a multi-arm degradable polyethylene glycol derivative having an oligopeptide that is degraded within cells. That is, the present invention is as follows. [1] Below formula (1):

[0018] [ka]

[0019] (In the formula, n1 and n2 each independently represent 45 to 950; W 1 and W 2 are each independently an oligopeptide having 2 to 47 residues, a1 and a2 are each independently an oligopeptide having 1 to 8, Q is a hydrocarbon chain having 2 to 12 carbon atoms which may contain an oxygen atom and / or a nitrogen atom, and X 1 and X 2 are each independently functional groups capable of reacting with biologically relevant substances, and L 1 and L 2 and L 3 and L 4 and L 5 and L 6 Each of the groups independently represents a divalent spacer. [2] W 1 and W 2 The degradable polyethylene glycol derivative according to [1], wherein the oligopeptide is an oligopeptide having glycine as the C-terminal amino acid. [3] W 1and W 2 The degradable polyethylene glycol derivative according to any one of [1] to [2], wherein the oligopeptide is an oligopeptide having at least one hydrophobic neutral amino acid having a hydropathic index of 2.5 or more. [4] Below formula (2):

[0020] [ka]

[0021] (In the formula, n3 and n4 each independently represent 110 to 950; W 3 and W 4 are each independently an oligopeptide having 2 to 5 residues, Q is a hydrocarbon chain having 2 to 12 carbon atoms which may contain an oxygen atom and / or a nitrogen atom, and X 1 and X 2 are each independently functional groups capable of reacting with biologically relevant substances, and L 1 and L 2 and L 3 and L 4 and L 5 and L 6 Each of the groups independently represents a divalent spacer. [5] W 3 and W 4 The degradable polyethylene glycol derivative according to [4], wherein the oligopeptide is composed only of neutral amino acids and has glycine as the C-terminal amino acid. [6] W 3 and W 4 The degradable polyethylene glycol derivative according to any one of [4] to [5], wherein the oligopeptide is an oligopeptide having at least one hydrophobic neutral amino acid having a hydropathic index of 2.5 or more. [7] Below formula (3):

[0022] [ka]

[0023] (In the formula, n1 and n2 each independently represent 45 to 950; W 5 and W 6 are each independently an oligopeptide having a symmetric structure with glutamic acid as the center and consisting of 5 to 47 residues, b1 and b2 are each independently an oligopeptide having a length of 2 to 8, Q is a hydrocarbon chain having 2 to 12 carbon atoms which may contain an oxygen atom and / or a nitrogen atom, and X 1 and X 2 are each independently functional groups capable of reacting with biologically relevant substances, and L 1 and L 2 and L 3 and L 4 and L 5 and L 6 Each of the groups independently represents a divalent spacer. [8] W 5 and W 6 The degradable polyethylene glycol derivative according to [7], wherein the oligopeptide having a symmetric structure centered on glutamic acid is an oligopeptide having the following structure v1, v2 or v3.

[0024] [ka]

[0025] [ka]

[0026] [ka]

[0027] (In the formula, Glu is a glutamic acid residue, and Z is a degradable oligopeptide of 2 to 5 residues consisting of neutral amino acids excluding cysteine.) [9] The degradable polyethylene glycol derivative according to [8], wherein the degradable oligopeptide of Z is an oligopeptide having glycine as the C-terminal amino acid.

[10] The degradable polyethylene glycol derivative according to any one of [7] to [9], wherein the degradable oligopeptide Z is an oligopeptide having at least one hydrophobic neutral amino acid having a hydropathic index of 2.5 or more.

[11] The degradable polyethylene glycol derivative according to any one of [1] to

[10] , which has a total molecular weight of 20,000 or more.

[12] X 1 and X 2 are each independently 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 vinylsulfonyl group, an acryl group, a sulfonyloxy group, a carboxyl group, a thiol group, a dithiopyridyl group, an α-haloacetyl group, an alkynyl group, an allyl group, a vinyl group, an amino group, an oxyamino group, a hydrazide group, and an azide group.

[13] Below formula (4):

[0028] [ka]

[0029] (In the formula, n1 and n2 each independently represent 45 to 950; W 1 and W 2 are each independently an oligopeptide having 2 to 47 residues, a1 and a2 are each independently an oligopeptide having 1 to 8, Q is a hydrocarbon chain having 2 to 12 carbon atoms which may contain an oxygen atom or a nitrogen atom, D 1 and D. 2 are each independently biologically relevant substances, and L 3 and L 4 and L 5 and L 6 and L 11 and L 12 Each of the groups independently represents a divalent spacer. A bio-relevant substance to which a degradable polyethylene glycol derivative represented by the formula:

[14] L 11 and L 12each independently represents a urethane bond, an amide bond, an ether bond, a thioether bond, a secondary amino group, a carbonyl group, a urea bond, a triazolyl group, a maleimide and mercapto bond, an oxime bond, or an alkylene group which may contain these bonds and groups.

[15] The biological substance according to any one of

[13] to

[14] , wherein D is a hormone, a cytokine, an antibody, an aptamer or an enzyme. Effect of the Invention

[0030] The multi-arm degradable polyethylene glycol derivative of the present invention is stable in the blood of a living body and has an oligopeptide in its structure that is degraded by an intracellular enzyme. Therefore, the degradable polyethylene glycol derivative is stable in the blood and can impart a blood half-life equivalent to that of conventional polyethylene glycol derivatives that do not have degradability to a biologically relevant substance. Furthermore, when the degradable polyethylene glycol derivative is taken up into a cell, the oligopeptide site is rapidly degraded, so that the generation of cellular vacuoles, which has been a problem until now, can be suppressed. In addition, since the polyethylene glycol derivative has multiple functional groups, multiple biologically relevant substances can be introduced into one molecule, and the polyethylene glycol derivative has the characteristic of being able to enhance its pharmacological activity. In addition, by using glycine as the amino acid at the C-terminus of the oligopeptide, impurities generated during the manufacturing process can be reduced, and thus the multi-arm degradable polyethylene glycol derivative of the present invention can be industrially manufactured. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present invention will be described in detail below. The degradable polyethylene glycol derivative according to the present invention is represented by the following formula (1).

[0032] [ka]

[0033] (In the formula, n1 and n2 each independently represent 45 to 950; W 1 and W 2 are each independently an oligopeptide having 2 to 47 residues, a1 and a2 are each independently an oligopeptide having 1 to 8, Q is a hydrocarbon chain having 2 to 12 carbon atoms which may contain an oxygen atom or a nitrogen atom, and X 1 and X 2 are each independently functional groups capable of reacting with biologically relevant substances, and L 1 and L 2 and L 3 and L 4 and L 5 and L 6 are each independently a divalent spacer.

[0034] The total molecular weight of the polyethylene glycol derivative of the 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 one preferred embodiment of the present invention, the total molecular weight of the polyethylene glycol derivative of the formula (1) of the present invention is 20,000 or more. The molecular weight referred to here is the number average molecular weight (Mn).

[0035] In formula (1), n1 and n2 each represent the number of repeating units of polyethylene glycol, and are usually each independently 45 to 950, preferably each independently 110 to 690, and more preferably each independently 220 to 460.

[0036] In formula (1), a1 and a2 are W 1 and W 2 and usually each independently is 1 to 8, preferably each independently is 1 or 2 or 4 or 8, and more preferably each independently is 1 or 2 or 4.

[0037] W in Equation (1) 1 and W 2are each independently an oligopeptide of 2 to 47 residues, preferably 2 to 23 residues, more preferably 2 to 19 residues, and are not particularly limited as long as they are stable in the blood of the living body and are degraded by intracellular enzymes, but it is preferable to combine a degradable peptide with an ionic amino acid (glutamic acid, aspartic acid, lysine) or a dendrimer-like oligopeptide having a branched backbone. When introducing a polyethylene glycol chain into an oligopeptide, if amino groups, carboxyl groups, thiol groups, hydroxyl groups, etc. derived from the amino acids constituting the oligopeptide are mixed, the reaction cannot be controlled, so it is preferable to use an oligopeptide that can protect either the amino group or the carboxyl group and does not have cysteine ​​or serine.

[0038] Q in formula (1) is not particularly limited as long as it is a hydrocarbon chain having 2 to 12, preferably 2 to 8, more preferably 2 to 4 carbon atoms, which may contain an oxygen atom and / or a nitrogen atom, but is preferably an alkylene group which may contain an ether bond. As the alkylene group, an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, a heptylene group, or an octylene group is preferred, and an ethylene group, a propylene group, or a butylene group is more preferred.

[0039] Particularly preferred embodiments of Q are those shown in group (I) below.

[0040] Group (I):

[0041] [ka]

[0042] In (q1) to (q3), f in the formulas is an integer of 2 to 12, preferably an integer of 2 to 8, and more preferably an integer of 2 to 4. In (q2) to (q3), f in the formulas may be the same or different.

[0043] L in formula (1) 1 and L 2 and L 3and L 4 and L 5 and L 6 are each independently a divalent spacer, and there are no particular limitations on these spacers as long as they are groups capable of forming a covalent bond. L 1 and L 2 is a spacer connecting the polyethylene glycol chain and the functional group, and is preferably an amide bond, an ether bond, a thioether bond, a urethane bond, a secondary amino group, a carbonyl group, a urea bond, or an alkylene group which may contain these bonds and / or groups. L 3 and L 4 is a spacer connecting the polyethylene glycol chain and the oligopeptide, and is preferably 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, or a urea bond. 3 and L 4 is preferably bonded to the repeating unit of polyethylene glycol via a carbon atom. L 5 and L 6 is a spacer connecting the hydrocarbon chain Q and the oligopeptide, and is preferably an amide bond, an ether bond, a thioether bond, a urethane bond, a secondary amino group, a carbonyl group, a urea bond, or an alkylene group which may contain these bonds and / or groups. L 1 and L 2 and L 3 and L 4 and L 5 and L 6 Particularly preferred embodiments are those shown in the following group (II). Two to four spacers of group (II) may be combined. As a divalent spacer, an ester bond and a carbonate bond are not suitable because they are gradually decomposed in the blood of the living body.

[0044] Group (II):

[0045] [ka]

[0046] In (z1) to (z11), s in the formulae represents an integer of 0 to 10, preferably an integer of 0 to 6, and more preferably an integer of 0 to 3. In (z2) to (z11), s in the formulae may be the same or different.

[0047] L in formula (1) 1 and L 2 is preferably (z2), (z3), (z4), (z6), (z7), (z8), (z9), (z10) or a combination of (z2) and (z4) in group (II), and more preferably (z3), (z6), (z9), (z10) or a combination of (z2) and (z4).

[0048] L in formula (1) 3 and L 4 is preferably a group represented by (z1), (z2), (z3), (z4), (z5), (z6), (z7), (z8) or (z11) in group (II), and more preferably a group represented by (z3), (z5) or (z11).

[0049] L in formula (1) 5 and L 6 is preferably a group represented by (z3), (z4), (z6), (z7), (z8), (z9) or (z10) in group (II), and more preferably a group represented by (z3), (z6), (z9) or (z10).

[0050] X in formula (1) 1 and X 2is 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, nucleic acid, or anticancer drug, to be chemically modified to form a covalent bond. Examples of the functional group include those described in "Harris, JM Poly(Ethylene Glycol) Chemistry; Plenum Press: New York, 1992", "Hermanson, GT Bioconjugate Techniques, 2nd ed.; Academic Press: San Diego, CA, 2008", and "PEGylated Protein Drugs: Basic Science and Clinical Applications; Veronese, FM, Ed.; Birkhauser: Basel, Switzerland, 2009".

[0051] X in formula (1) 1 and X 2 The "functional group capable of reacting with a biologically relevant substance" represented by the formula (I) is not particularly limited as long as it is a functional group capable of chemically bonding with a functional group, such as an amino group, a mercapto group, an aldehyde group, a carboxyl group, an unsaturated bond, or an azide group, possessed by the biologically relevant substance. Specific examples of the alkyl group 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, an oxyamino group (HN-O- group), an iodoacetamide group, an alkylcarbonyl group, an alkenyl group (e.g., an allyl group, a 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, as described below), an azide group, an acryl group, a sulfonyloxy group (e.g., an alkylsulfonyloxy group), and an α-haloacetyl group. Preferred are an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an aryl group, an alkyl ... Examples of the alkyl group include an aryl group, an isothiocyanate group, an epoxide group, a maleimide group, a substituted maleimide group, a vinylsulfonyl group, an acryl group, a sulfonyloxy group (e.g., an alkylsulfonyloxy group having 1 to 5 carbon atoms), a substituted sulfonate group, a carboxyl group, a mercapto group, a pyridyldithio group, an α-haloacetyl group, an alkynyl group, a substituted alkynyl group (e.g., an alkynyl group having 2 to 5 carbon atoms substituted with a hydrocarbon group having 1 to 5 carbon atoms described below), an allyl group, a vinyl group, an amino group, an oxyamino group, a hydrazide group, and an azide group, more preferably an active ester group, an active carbonate group, an aldehyde group, a maleimide group, a carboxyl group, an oxyamino group, and an amino group, and particularly preferably an aldehyde group, a maleimide group, a carboxyl group, and an oxyamino group.

[0052] In another preferred embodiment, such functional group X 1 and X 2 can be classified into the following groups (III), (IV), (V), (VI), (VII) and (VIII).

[0053] Group (III): Functional groups capable of reacting with amino groups in biologically relevant substances Examples of the groups include those represented by the following (a), (b), (c), (d), (e), (f), (g), (j), and (k).

[0054] Group (IV): Functional groups capable of reacting with mercapto groups in biologically relevant substances Examples of the groups include those represented by the following (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), and (l).

[0055] Group (V): Functional groups capable of reacting with aldehyde groups in biologically relevant substances Examples of the groups include those represented by the following (h), (m), (n) and (p).

[0056] Group (VI): Functional groups capable of reacting with carboxyl groups of biologically relevant substances Examples of the groups include those represented by the following (h), (m), (n) and (p).

[0057] Group (VII): Functional groups capable of reacting with unsaturated bonds in biologically relevant substances Examples of the groups include those represented by the following (h), (m), and (o).

[0058] Group (VIII): Functional groups capable of reacting with azide groups in biologically relevant substances Examples include the group represented by (l) below.

[0059] [ka]

[0060] In the functional group (j), U1 in the formula represents a halogen atom such as a chlorine atom (Cl), a bromine atom (Br) or an iodine atom (I), and is preferably Br or I, and more preferably I.

[0061] In addition, in the functional group (e) and the functional group (l), Y 1 , Y 3each 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. Specific examples of the hydrocarbon group having 1 to 5 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, and a tertiary butyl group, and preferably a methyl group or an ethyl group.

[0062] In addition, in the functional group (k), Y in the formula 2 represents a hydrocarbon group having 1 to 10 carbon atoms which may contain a fluorine atom, and specific examples thereof 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, and a 4-(trifluoromethoxy)phenyl group, and is preferably a methyl group, a vinyl group, a 4-methylphenyl group, or a 2,2,2-trifluoroethyl group.

[0063] The active ester group is an ester group having an alkoxy group with high leaving ability. Examples of the alkoxy group with high leaving ability include alkoxy groups derived from nitrophenol, N-hydroxysuccinimide, pentafluorophenol, etc. The active ester group is preferably an ester group having an alkoxy group derived from N-hydroxysuccinimide.

[0064] The active carbonate group is a carbonate group having an alkoxy group with high leaving ability. Examples of the alkoxy group with high leaving ability include alkoxy groups derived from nitrophenol, N-hydroxysuccinimide, pentafluorophenol, etc. The active carbonate group is preferably a carbonate group having an alkoxy group derived from nitrophenol or N-hydroxysuccinimide.

[0065] The substituted maleimide group is a maleimide group having a hydrocarbon group bonded to one of the carbon atoms of the double bond 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 tertiary butyl group, and preferably a methyl group or an ethyl group.

[0066] The substituted sulfonate group is a sulfonate group in which a hydrocarbon group that may contain a fluorine atom is bonded to the sulfur atom of the sulfonate group.Specific examples of the hydrocarbon group that may contain a fluorine atom include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a tertiary 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, and a 4-(trifluoromethoxy)phenyl group, and preferably a methyl group, a vinyl group, a 4-methylphenyl group, or a 2,2,2-trifluoroethyl group.

[0067] Hereinafter, a degradable polyethylene glycol derivative showing a preferred embodiment of the present invention is represented by the following formula (2).

[0068] Below formula (2): [ka]

[0069] (In the formula, n3 and n4 each independently represent 110 to 950; W 3 and W 4 are each independently an oligopeptide of 2 to 5 residues, and Q and X 1 and X 2 , L 1 and L 2 and L 3 and L 4 and L 5 and L 6 has the same meaning as above.)

[0070] The total molecular weight of the polyethylene glycol derivative of the formula (2) 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 one preferred embodiment of the present invention, the total molecular weight of the polyethylene glycol derivative of the formula (2) of the present invention is 20,000 or more. The molecular weight here refers to the number average molecular weight (Mn).

[0071] In formula (2), n3 and n4 each represent the number of repeating units of polyethylene glycol, and are usually each independently 110 to 950, preferably each independently 220 to 690, and more preferably each independently 220 to 460.

[0072] W in Equation (2) 3 and W 4 are each independently an oligopeptide of 2 to 5 residues, and are not particularly limited as long as they are stable in the blood of the living body and degraded by intracellular enzymes, but are preferably an oligopeptide composed of an amino acid having an amino group or a carboxyl group in the side chain, specifically, a neutral amino acid not containing lysine, aspartic acid, or glutamic acid. In the synthesis of the degradable polyethylene glycol derivative of formula (2) of the present invention, when the raw material polyethylene glycol derivative and the oligopeptide are bonded by reaction, the carboxyl group at the C-terminus of the oligopeptide is used in the condensation reaction with the polyethylene glycol derivative. However, if the oligopeptide has an amino acid having an amino group or a carboxyl group in the side chain, the condensation reaction may cause a side reaction between the oligopeptides, or impurities may be introduced into the carboxyl group of the side chain, rather than the carboxyl group at the C-terminus, which is the intended polyethylene glycol derivative. Since it is difficult to remove these impurities through conventional purification processes such as extraction and crystallization, it is desirable to use oligopeptides made of amino acids that do not have amino or carboxyl groups in their side chains in order to obtain the target product with high purity. 3 and W 4 The amino acids that make up the peptide are α-amino acids and are basically L-type.

[0073] Cysteine, a neutral amino acid, has a sulfhydryl group and forms disulfide bonds with other sulfhydryl groups. 3 and W 4 is preferably an oligopeptide consisting of neutral amino acids not containing cysteine.

[0074] In addition, W 3 and W 4 is preferably an oligopeptide having glycine as the C-terminal amino acid. When reacting the C-terminal carboxyl group with a polyethylene glycol derivative, it is basically necessary to activate the C-terminal carboxyl group with a condensing agent or the like. It is known that epimerization is likely to occur with amino acids other than glycine during this activation process, resulting in the by-product of stereoisomers. By making the C-terminal amino acid of the oligopeptide achiral glycine, it is possible to obtain a highly pure target product without the by-product of stereoisomers.

[0075] In addition, W 3 and W 4 is preferably an oligopeptide having at least one hydrophobic neutral amino acid with a hydropathic index of 2.5 or more, specifically, phenylalanine, leucine, valine, or isoleucine, and more preferably an oligopeptide having phenylalanine. The hydropathy index, which was created by Kyte and Doolittle to quantitatively indicate the hydrophobicity of amino acids, indicates that the higher the value, the more hydrophobic the amino acid is (Kyte J & Doolittle RF, 1982, J Mol Biol, 157:105-132.).

[0076] W 3 and W 4is not particularly limited as long as it is stable in the blood in the living body and has the ability to be decomposed by intracellular enzymes, and is an oligopeptide of 2 to 5 residues consisting of neutral amino acids excluding cysteine. Specific examples include glycine-phenylalanine-leucine-glycine, glycine-glycine-phenylalanine-glycine, glycine-phenylalanine-glycine, glycine-leucine-glycine, valine-citrulline-glycine, valine-alanine-glycine, phenylalanine-glycine, etc., and preferably glycine-phenylalanine-leucine. Preferably, the amino acid sequence is glycine-glycine, glycine-glycine-phenylalanine-glycine, 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 even more preferably glycine-phenylalanine-leucine-glycine, or phenylalanine-glycine.

[0077] Hereinafter, a degradable polyethylene glycol derivative showing a preferred embodiment of the present invention is represented by the following formula (3).

[0078] Below formula (3): [ka]

[0079] (In the formula, W 5 and W 6 are each independently an oligopeptide having a symmetric structure of 5 to 47 residues with glutamic acid at the center, b1 and b2 are each independently an oligopeptide having a symmetric structure of 2 to 8, n1 and n2, Q, X 1 and X 2 , L 1 and L 2 and L 3 and L 4 and L 5 and L 6 has the same meaning as above.)

[0080] b1 and b2 in formula (3) are W 5 and W 6 and usually each independently is 2 to 8, preferably each independently is 2, 4, or 8, and more preferably each independently is 2 or 4.

[0081] W in Equation (3) 5 and W 6 are each independently an oligopeptide of 5 to 47 residues, preferably 5 to 23 residues, more preferably 5 to 19 residues, with glutamic acid as the center, and are not particularly limited as long as they are stable in the blood of the living body and decomposed by intracellular enzymes, but the amino acids constituting the oligopeptide are preferably composed of neutral amino acids other than cysteine, except for glutamic acid constituting the central part. The oligopeptide of symmetric structure with glutamic acid as the center means a compound in which the same peptide is bound to the α-position carboxyl group and the γ-position carboxyl group of glutamic acid, and is an oligopeptide in which the pair of peptides has a symmetric structure with glutamic acid as the center. The composition ratio of the number of neutral amino acids and glutamic acids in the oligopeptide (number of neutral amino acids / number of glutamic acids) is usually 2 to 10, preferably 2 to 8, and more preferably 2 to 6. W 5 and W 6 The amino acids that make up the protein are basically L-type.

[0082] W 5 and W 6 Particularly preferred embodiments are those shown in group (IX) below.

[0083] Group (IX):

[0084] [ka]

[0085] [ka]

[0086] [ka]

[0087] (In the formula, Glu is a glutamic acid residue, and Z is a degradable oligopeptide of 2 to 5 residues consisting of neutral amino acids except cysteine.)

[0088] Z in (v1) to (v3) is preferably an oligopeptide composed of an amino acid having an amino group or a carboxyl group in the side chain, specifically, a neutral amino acid not containing lysine, aspartic acid, or glutamic acid. In the synthesis of the multi-arm degradable polyethylene glycol derivative of formula (3) of the present invention, when the raw material polyethylene glycol derivative and the oligopeptide are bonded by reaction, the carboxyl group at the C-terminus of the oligopeptide is used in a condensation reaction with the polyethylene glycol derivative. However, if the oligopeptide has an amino acid having an amino group or a carboxyl group in the side chain, a side reaction between the oligopeptides occurs in the condensation reaction, and impurities are introduced not only to the C-terminal carboxyl group, which is the target of the polyethylene glycol derivative, but also to the carboxyl group in the side chain. Since it is difficult to remove these impurities by conventional purification processes such as extraction and crystallization, it is desirable to use oligopeptides composed of amino acids that do not have amino or carboxyl groups in their side chains in order to obtain the target product with high purity. The amino acids that make up Z are α-amino acids and are basically L-type.

[0089] Cysteine, a neutral amino acid, has a mercapto group and forms a disulfide bond with another mercapto group. Therefore, Z in (v1) to (v3) is preferably an oligopeptide composed of neutral amino acids that do not contain cysteine.

[0090] In addition, Z in (v1) to (v3) is preferably an oligopeptide having glycine as the C-terminal amino acid. When reacting the C-terminal carboxyl group with a polyethylene glycol derivative, it is basically necessary to activate the C-terminal carboxyl group with a condensing agent or the like. It is known that epimerization is likely to occur with amino acids other than glycine during this activation process, resulting in the by-production of stereoisomers. By making the C-terminal amino acid of the oligopeptide achiral glycine, it is possible to obtain a highly pure target product without the by-production of stereoisomers.

[0091] Furthermore, Z in (v1) to (v3) is preferably a hydrophobic neutral amino acid having a hydropathic index of 2.5 or more, specifically, an oligopeptide having at least one of phenylalanine, leucine, valine, and isoleucine, and more preferably an oligopeptide having phenylalanine. The hydropathy index, which quantitatively indicates the hydrophobicity of amino acids and was created by Kyte and Doolittle, indicates that the higher the value, the more hydrophobic the amino acid is (Kyte J & Doolittle RF, 1982, J Mol Biol, 157:105-132.).

[0092] Z in (v1) to (v3) is not particularly limited as long as it is stable in the blood of a living body and has the ability to be decomposed by an intracellular enzyme, and is an oligopeptide of 2 to 5 residues consisting of neutral amino acids excluding cysteine. Specific examples include glycine-phenylalanine-leucine-glycine, glycine-glycine-phenylalanine-glycine, glycine-phenylalanine-glycine, glycine-leucine-glycine, valine-citrulline-glycine, valine-alanine-glycine, phenylalanine-glycine, etc., and preferably glycine-phenylalanine. alanine-leucine-glycine, glycine-glycine-phenylalanine-glycine, 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 even more preferably glycine-phenylalanine-leucine-glycine, or phenylalanine-glycine.

[0093] A preferred embodiment of formula (3) is W 5 and W 6 is a 4-arm degradable polyethylene glycol derivative represented by the following formula (5), in which v1 is v1, b1=2, and b2=2.

[0094] [ka]

[0095] (Wherein, Glu, Z, n1 and n2, Q, X 1 and X 2 , L 1 and L 2 and L 3 and L 4 and L 5 and L 6 has the same meaning as above.)

[0096] A preferred embodiment of formula (3) is W 5 and W6 is v2, b1=4, and b2=4, and is an 8-arm degradable polyethylene glycol derivative represented by the following formula (6).

[0097] [ka]

[0098] (Wherein, Glu, Z, n1 and n2, Q, X 1 and X 2 , L 1 and L 2 and L 3 and L 4 and L 5 and L 6 has the same meaning as above.)

[0099] A preferred embodiment of formula (3) is W 5 and W 6 is v3, and b1=8, b2=8, and it is a 16-arm degradable polyethylene glycol derivative represented by the following formula (7).

[0100] [ka]

[0101] (Wherein, Glu, Z, n1 and n2, Q, X 1 and X 2 , L 1 and L 2 and L 3 and L 4 and L 5 and L 6 has the same meaning as above.)

[0102] Hereinafter, a bio-related substance having a degradable polyethylene glycol derivative bonded thereto, which represents a preferred embodiment of the present invention, is represented by the following formula (4).

[0103] Below formula (4): [ka]

[0104] (In the formula, L 11 and L 12 are each independently a divalent spacer, D 1 and D. 2 are each independently a biologically relevant substance, n1 and n2, a1 and a2, Q, W 1 and W 2 , L 3 and L 4 and L 5 and L 6 has the same meaning as above.)

[0105] L in formula (4) 11 and L 12 are each independently a divalent spacer, and these spacers are not particularly limited as long as they are groups capable of forming a covalent bond, but are preferably an amide bond, an ether bond, a thioether bond, a urethane bond, a secondary amino group, a carbonyl group, a urea bond, a triazolyl group, a maleimide and mercapto bond, an oxime bond, or an alkylene group which may contain these bonds and / or groups. L 11 and L 12 Particularly preferred embodiments are those shown in the following group (X). Two to five spacers of group (X) may be combined. As a divalent spacer, an ester bond and a carbonate bond are not suitable because they are gradually decomposed in the blood of the living body.

[0106] Group(X):

[0107] [ka]

[0108] In (z1) to (z20), s in the formulae represents an integer of 0 to 10, preferably an integer of 0 to 6, and more preferably an integer of 0 to 3. In (z2) to (z20), s in the formulae may be the same or different.

[0109] L in formula (4) 11 and L12 As the group (I), the groups represented by (z3), (z6), (z7) to (z20) are preferable, the groups represented by (z6), (z9), (z10), (z12), (z14), (z16), (z18) or (z20) are more preferable, and the groups represented by (z10), (z12), (z16) or (z20) are further preferable.

[0110] D in formula (4) is a biological substance, and is not particularly limited, but is a substance involved in the diagnosis, cure, mitigation, treatment or prevention of a disease in humans or other animals. Specifically, it includes proteins, peptides, nucleic acids, cells, viruses, etc., and suitable proteins or peptides include hormones, cytokines, antibodies, aptamers, enzymes, etc. More specifically, cytokines include interferon type I, type II, and type III, which regulate immunity, interleukins, tumor necrosis factors, and their receptor antagonists. Growth factors include erythropoietin, which is a hematopoietic factor, and granulocyte colony-stimulating factor (GCSF), which is a stimulating factor. Blood coagulation factors include factor V, factor VII, factor VIII, factor IX, factor X, and factor XII. Hormones include calcitonin, insulin, its analogs, exenatide, GLP-1, somatostatin, and human growth hormone. Antibodies include full-length antibodies, and antibody fragments such as Fab and svFV. Aptamers include DNA aptamers and RNA aptamers. Enzymes include superoxide dismutase and uricase. These proteins whose amino acid sequences have been modified by genetic engineering are also included. The above-mentioned proteins have low stability in blood, and it is desirable to modify them with polyethylene glycol to extend their half-life in blood. Suitable proteins include interferon, interleukin, erythropoietin, GCSF, factor VIII, factor IX, human growth hormone, antibody fragments, etc., more preferably human growth hormone, interferon, GCSF, erythropoietin, or antibody fragments (particularly Fab), and even more preferably human growth hormone or GCSF. Suitable peptides include insulin, bivalirudin, teriparatide, exenatide, enfuvirtide, degarelix, mifamurtide, nesiritide, goserelin, glatiramer, octreotide, lanreotide, icatibant, zicotinide, pramlintide, romiplostim, calcitonin, oxytocin, leuprorelin, and glucagon, and more preferably, insulin, exenatide, and calcitonin (especially salmon calcitonin).

[0111] A preferred embodiment of the bio-related substance represented by formula (4) is a bio-related substance represented by the following formula (8).

[0112] [ka]

[0113] (In the formula, D 1 and D. 2 , n3 and n4, Q, W 3 and W 4 , L 11 and L 12 and L 3 and L 4 and L 5 and L 6 has the same meaning as above.)

[0114] Another preferred embodiment of the bio-related substance represented by formula (4) is a bio-related substance represented by the following formula (9).

[0115] [ka]

[0116] (In the formula, D 1 and D. 2 , n1 and n2, b1 and b2, Q, W 5 and W 6 , L 11 and L 12 and L 3 and L 4 and L 5 and L 6 has the same meaning as above.)

[0117] One preferred embodiment of formula (9) is W 5 and W 6 is a bio-relevant substance to which a four-arm degradable polyethylene glycol derivative represented by the following formula (10) in which v1 is v1, b1=2, and b2=2 is bonded.

[0118] [ka]

[0119] (Wherein, Glu, Z, n1 and n2, Q, D 1 and D. 2 , L 11 and L 12 and L 3 and L 4 and L 5 and L 6 has the same meaning as above.)

[0120] One preferred embodiment of formula (9) is W 5 and W 6 is v2, and b1=4, b2=4, to which an 8-arm degradable polyethylene glycol derivative represented by the following formula (11) is bonded.

[0121] [ka]

[0122] (Wherein, Glu, Z, n1 and n2, Q, D 1 and D. 2 , L 11and L 12 and L 3 and L 4 and L 5 and L 6 has the same meaning as above.)

[0123] One preferred embodiment of formula (9) is W 5 and W 6 is v3, and b1=8, b2=8, to which a 16-arm degradable polyethylene glycol derivative represented by the following formula (12) is bonded.

[0124] [ka]

[0125] (Wherein, Glu, Z, n1 and n2, Q, D 1 and D. 2 , L 11 and L 12 and L 3 and L 4 and L 5 and L 6 has the same meaning as above.)

[0126] Suitable examples of the degradable polyethylene glycol derivative of the formula (1) of the present invention include the following degradable polyethylene glycol derivatives. [Degradable polyethylene glycol derivatives (1-1)] n1 and n2 each independently represent 220 to 460; W 1 and W 2 are each independently an oligopeptide having 2 to 9 residues (e.g., phenylalanine-glycine, glycine-leucine-phenylalanine-glycine, glycine-phenylalanine-glutamic acid-phenylalanine-glycine, glycine-leucine-phenylalanine-glycine-glutamic acid-glycine-phenylalanine-leucine-glycine); a1 and a2 are each independently 1, 2 or 4; Q is an alkylene group (e.g., an ethylene group, a propylene group); X 1 and X 2 are each independently selected from the group consisting of an active carbonate group (e.g., an N-succinimidyl carbonate group), a maleimide group, a carboxyl group, and an amino group; L 1 and L 2 are each independently an alkylene group (e.g., a methylene group, an ethylene group, a propylene group) which may contain an ether bond, or an amide bond or a urethane bond; L 3 and L 4 is an alkylene group containing a secondary amino group (e.g., a propylene group); L 5 and L 6 is a carbonyl group; A degradable polyethylene glycol derivative of formula (1).

[0127] Suitable examples of the degradable polyethylene glycol derivative of the formula (2) of the present invention include the following degradable polyethylene glycol derivatives. [Degradable polyethylene glycol derivatives (2-1)] n3 and n4 are each independently 220 to 460; W 3 and W 4 are each independently an oligopeptide of 2 to 5 residues (e.g., phenylalanine-glycine, glycine-leucine-phenylalanine-glycine); Q is an alkylene group (e.g., an ethylene group, a propylene group); X 1 and X 2 are each independently selected from the group consisting of an activated carbonate group (e.g., an N-succinimidyl carbonate group) and a maleimide group; L 1 and L 2 each independently represents an alkylene group which may contain an ether bond or an amide bond (e.g., an ethylene group, a propylene group); L 3 and L 4is an alkylene group containing a secondary amino group (e.g., a propylene group); L 5 and L 6 is a carbonyl group; A degradable polyethylene glycol derivative of formula (2).

[0128] Suitable examples of the degradable polyethylene glycol derivative of the formula (3) of the present invention include the following degradable polyethylene glycol derivatives. [Degradable polyethylene glycol derivatives (3-1)] n1 and n2 each independently represent 220 to 460; W 5 and W 6 are each independently an oligopeptide having a symmetric structure of 5 to 9 residues with glutamic acid at the center (e.g., glycine-phenylalanine-glutamic acid-phenylalanine-glycine, glycine-leucine-phenylalanine-glycine-glutamic acid-glycine-phenylalanine-leucine-glycine); b1 and b2 are each independently 2 or 4 or 8; Q is an alkylene group (e.g., a propylene group); X 1 and X 2 are each independently selected from the group consisting of a carboxyl group, an amino group, and an oxyamino group; L 1 and L 2 each independently represents an alkylene group (e.g., a methylene group, an ethylene group) which may contain an ether bond or a urethane bond; L 3 and L 4 is an alkylene group containing a secondary amino group (e.g., a propylene group); L 5 and L 6 is a carbonyl group; A degradable polyethylene glycol derivative of formula (3).

[0129] The multi-arm degradable polyethylene glycol derivative of the present invention can be produced, for example, via the following steps.

[0130] Reaction A

[0131] [ka]

[0132] (In the process, PEG is a polyethylene glycol chain, Peptide is an oligopeptide, and Pro 1 and Pro 2 is a protecting group, L 7 is a divalent spacer, and L 1 and X 1 has the same meaning as above.)

[0133] The PEG in the process is a polyethylene glycol chain, and the molecular weight is as defined by n1 and n2, which are the numbers of repeating units of polyethylene glycol described above. In other words, since n1 and n2 are each independently 45 to 950, the molecular weight range is 2,000 to 42,000.

[0134] The peptide in the process is 3 and W 4 In this process, an oligopeptide in which the N-terminal amino group is protected with a protecting group is used.

[0135] Pro in process 1 and Pro 2is a protecting group, where a protecting group is a moiety that prevents or blocks reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. Protecting groups vary depending on the type of chemically reactive functional group being protected, the conditions used, and the presence of other functional groups or protecting groups in the molecule. Specific examples of protecting groups can be found in many general textbooks, for example, "Wuts, PGM; Greene, TW Protective Groups in Organic Synthesis, 4th ed.; Wiley-Interscience: New York, 2007". In addition, functional groups protected with protecting groups can be deprotected, i.e., chemically reacted, using reaction conditions appropriate for each protecting group to regenerate the original functional group. Representative deprotection conditions for protecting groups are described in the above-mentioned literature.

[0136] L during the process 7 The L 3 and L 4 is a divalent spacer having the same meaning as above.

[0137] In reaction A, the amino group at the N-terminus is protected by the Pro 1 The carboxyl group of the oligopeptide protected with and the functional group X 1 is a protecting group Pro 2 This is a process in which the amino group of the polyethylene glycol derivative protected by the formula (1) is bonded by a condensation reaction to obtain the polyethylene glycol derivative (1). Protecting group Pro for the amino group at the N-terminus of an oligopeptide 1 is not particularly limited, and examples thereof include acyl-based protecting groups and carbamate-based protecting groups, specifically, trifluoroacetyl group, 9-fluorenylmethyloxycarbonyl group (Fmoc), tert-butyloxycarbonyl group, and the like. Functional group X of polyethylene glycol derivative 1 The protecting group Pro 2 X 1When is an amino group, examples of the protecting group include a trifluoroacetyl group, an Fmoc group, and a tert-butyloxycarbonyl group. When is a hydroxyl group, examples of the protecting group include a tetrahydropyranyl group, a tert-butyl group, and a benzyl group. When is a carboxyl group, examples of the protecting group include a methyl group, a tert-butyl group, and a benzyl group. 1 and Pro 2 are different protecting groups. The condensation reaction is not particularly limited, but a reaction using a condensing agent is preferable. 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 may be used in combination with a reagent such as N-hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBt), or 1-hydroxy-7-azabenzotriazole (HOAt). In addition, a more reactive condensing agent such as HATU, HBTU, TATU, TBTU, COMU, or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM) may be used. In order to promote the reaction, a base such as triethylamine or dimethylaminopyridine may be used. It is preferable to remove impurities produced as by-products in the reaction, or oligopeptides and condensing agents remaining unconsumed in the reaction, by purification, which is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0138] Deprotection B

[0139] [ka]

[0140] Deprotection B is the reaction of removing the protecting group Pro of the polyethylene glycol derivative (1) obtained in reaction A. 1 This is a process for obtaining a polyethylene glycol derivative (2) by deprotecting the protecting group Pro.2 , oligopeptides and L 1 and L 7 It is necessary to use conditions that do not decompose the divalent spacer. This step can also be carried out as part of the reaction A. It is preferable to remove impurities generated as by-products in the deprotection reaction by purification, which is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0141] Reaction C

[0142] [ka]

[0143] Reaction C is a process in which the amino group of the polyethylene glycol derivative (2) obtained in deprotection B is bonded to the two carboxyl groups of glutaric acid via a condensation reaction to obtain a two-arm polyethylene glycol derivative (3) in which two degradable polyethylene glycol chains are linked by glutaric acid. The glutaric acid used as the core molecule here may be replaced with other dibasic acids such as succinic acid, adipic acid, and sebacic acid, or may be a compound in which two hydroxyl groups of ethylene glycol, propylene glycol, butanediol, pentanediol, hexanediol, heptanediol, octanediol, nonanediol, decanediol, etc. are activated with nitrophenyl carbonate or succinimidyl carbonate. As in the above reaction A, the reaction is preferably carried out using a condensing agent, and a base such as triethylamine or dimethylaminopyridine may be used to promote the reaction. It is preferable to remove impurities produced as by-products in the reaction, or polyethylene glycol derivatives remaining unconsumed in the reaction, by purification. The purification method is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0144] Deprotection D

[0145] [ka]

[0146] Deprotection D is a step of removing 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. 1 and L 7 It is necessary to use conditions under which the divalent spacer of is not decomposed. This step can also be carried out as part of the step of reaction C. It is preferable to remove impurities generated as by-products in the deprotection reaction by purification, which is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like. Through the above steps, the functional group X with glutaric acid as the core molecule is obtained. 1 A two-arm polyethylene glycol derivative (4) having two groups can be obtained.

[0147] Reaction E

[0148] [ka]

[0149] (Pro in process 3 is a protecting group, L 8 is a divalent spacer, and can be used with PEG, Peptide, or Pro 1 , L 2 and X 2 has the same meaning as above.)

[0150] Pro in process 3 Pro 1 and Pro 2 is a protecting group having the same meaning as above.

[0151] L during the process 8 The L 3 and L4 is a divalent spacer having the same meaning as above.

[0152] In reaction E, the amino group at the N-terminus is protected by the Pro 1 The carboxyl group of the oligopeptide protected with and the functional group X 2 is a protecting group Pro 3 This is a process in which the amino group of the polyethylene glycol derivative protected by the formula (5) is bonded by a condensation reaction to obtain the polyethylene glycol derivative (5). As in the above reaction A, the reaction is preferably carried out using a condensing agent, and impurities produced as by-products in the reaction, or polyethylene glycol derivatives remaining unconsumed in the reaction, are preferably removed by purification.

[0153] Deprotection F

[0154] [ka]

[0155] Deprotection F is the removal of the protecting group Pro of the polyethylene glycol derivative (5) obtained in reaction E. 1 This is a process for obtaining a polyethylene glycol derivative (6) by deprotecting the protecting group Pro. 3 , oligopeptides and L 2 and L 8 It is necessary to use conditions that do not decompose the divalent spacer. This step can also be carried out as part of the reaction E. It is preferable to remove impurities generated as by-products in the deprotection reaction by purification, which is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0156] Reaction G

[0157] [ka]

[0158] In reaction G, the polyethylene glycol derivative (6) obtained in deprotection F is reacted with glutaric anhydride to obtain a polyethylene glycol derivative (7) having a carboxyl group. In addition, the amino group of the polyethylene glycol derivative (2) obtained in deprotection B is bonded to the carboxyl group of (7) through a condensation reaction to obtain a two-arm polyethylene glycol derivative (8) in which two different types of degradable polyethylene glycol chains are linked by glutaric acid. In reaction G, it is preferable to use an anhydride of a dibasic acid such as glutaric anhydride or succinic anhydride, but it is also possible to use a compound in which one carboxyl group of a dibasic acid such as succinic acid, glutaric acid, adipic acid, or sebacic acid is protected with a methyl group, a tert-butyl group, a benzyl group, etc. It is also possible to use a compound in which one of two hydroxyl groups of ethylene glycol, propylene glycol, butanediol, pentanediol, hexanediol, heptanediol, octanediol, nonanediol, decanediol, etc. is activated with nitrophenyl carbonate, succinimidyl carbonate, etc., and the other hydroxyl group is protected with a tetrahydropyranyl group, a tert-butyl group, a benzyl group, etc. In the reaction with (2), a condensing agent is preferably used, as in the above reaction A, and a base such as triethylamine or dimethylaminopyridine may be used to promote the reaction. It is preferable to remove impurities produced as by-products in the reaction, or polyethylene glycol derivatives remaining unconsumed in the reaction, by purification. The purification method is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0159] Deprotection H

[0160] [ka]

[0161] Deprotection H is the deprotection of the protecting group Pro of the polyethylene glycol derivative (8) obtained in reaction G. 2 and Pro3 This is the process of deprotecting the above to obtain the polyethylene glycol derivative (9). The deprotection reaction can be carried out by a conventional method. 1 and L 2 and L 7 and L 8 It is necessary to use conditions that do not decompose the divalent spacer. 2 and Pro 3 may be deprotected under the same conditions, or may be deprotected in separate steps. It is preferable to remove impurities generated as by-products in the deprotection reaction by purification, which is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like. Through the above process, two different functional groups X with glutaric acid as the core molecule are obtained. 1 and X 2 A two-arm polyethylene glycol derivative (9) having the following structure can be obtained.

[0162] Reaction I

[0163] [ka]

[0164] (L during the process 9 and L 10 is a divalent spacer, and can be used with PEG, Peptide, or Pro 1 has the same meaning as above.)

[0165] L during the process 9 The L 3 and L 4 is a divalent spacer having the same meaning as 10 The L 1 and L 2 is a divalent spacer having the same meaning as above.

[0166] In reaction I, the amino group at the N-terminus is protected by the Pro 1This is a process in which the carboxyl group of the oligopeptide protected by the above formula (1) is bonded to the amino group of a polyethylene glycol derivative having a hydroxyl group at one end by a condensation reaction to obtain a polyethylene glycol derivative (10). As in the above reaction A, a reaction using a condensing agent is preferable, and in particular, a reaction using a condensing agent such as DMT-MM that selectively promotes the condensation of amino groups and carboxyl groups is preferable. It is preferable to remove impurities produced as by-products in the reaction, or polyethylene glycol derivatives remaining unconsumed in the reaction, by purification.

[0167] Deprotection J

[0168] [ka]

[0169] Deprotection J is the deprotection of the protecting group Pro of the polyethylene glycol derivative (10) obtained in reaction I. 1 This is the process of deprotecting the above to obtain the polyethylene glycol derivative (11). The deprotection reaction can be carried out by a conventional method. 9 and L 10 It is necessary to use conditions that do not decompose the divalent spacer of the formula (1). This step can also be carried out as part of the step of Reaction I. It is preferable to remove impurities generated as by-products in the deprotection reaction by purification, which is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0170] Reaction K

[0171] [ka]

[0172] Reaction K is a process in which the amino group of the polyethylene glycol derivative (11) obtained in deprotection J is bonded to two carboxyl groups of adipic acid via a condensation reaction to obtain a two-arm polyethylene glycol derivative (12) in which two degradable polyethylene glycol chains are linked by adipic acid. The adipic acid used as the core molecule here may be replaced with other dibasic acids such as succinic acid, glutaric acid, and sebacic acid, or may be a compound in which two hydroxyl groups of ethylene glycol, propylene glycol, butanediol, pentanediol, hexanediol, heptanediol, octanediol, nonanediol, decanediol, etc. are activated with nitrophenyl carbonate or succinimidyl carbonate. As in the above reaction A, a reaction using a condensing agent is preferred, and a reaction using a condensing agent such as DMT-MM that selectively promotes the condensation of amino groups and carboxyl groups is particularly preferred. It is preferable to remove impurities produced as by-products in the reaction, or polyethylene glycol derivatives remaining unconsumed in the reaction, by purification. The purification method is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0173] Reaction L

[0174] [ka]

[0175] In reaction L, the two hydroxyl groups of the polyethylene glycol derivative (12) obtained in reaction K are converted to functional groups X 1 to obtain the polyethylene glycol derivative (13). As the reaction for converting a hydroxyl group into another functional group, a conventionally known method can be used. For example, a hydroxyl group can be converted into various functional groups by using the methods described in "Harris, JM Poly(Ethylene Glycol) Chemistry; Plenum Press: New York, 1992", "Hermanson, GT Bioconjugate Techniques, 2nd ed.; Academic Press: San Diego, CA, 2008", and "PEGylated Protein Drugs: Basic Science and Clinical Applications; Veronese, FM, Ed.; Birkhauser: Basel, Switzerland, 2009". For example, the hydroxyl group of (12) can be converted to an activated carbonate group by reacting a reaction reagent such as para-nitrophenyl chloroformate or disuccinimidyl carbonate with a base such as triethylamine. In addition, the hydroxyl group of (12) can be converted to an amino group or oxyamino group by the method described in Japanese Patent No. 5418360. The reaction reagent used in reaction L is a low molecular weight reagent, and has a significantly different solubility from polyethylene glycol derivatives, which are high molecular weight polymers, and therefore can be easily removed by common purification methods such as extraction and crystallization. Purification is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, etc. Through the above steps, functional group X with adipic acid as the core molecule is obtained. 1 A two-arm polyethylene glycol derivative (13) having two groups can be obtained.

[0176] Reaction M

[0177] [ka]

[0178] Reaction M is a process in which the amino group of the polyethylene glycol derivative (2) obtained in deprotection B is bonded by a condensation reaction to two carboxyl groups of a glutamic acid derivative whose amino group is protected with a protecting group, thereby obtaining a branched polyethylene glycol derivative (14) in which two degradable polyethylene glycol chains are linked by a glutamic acid residue. As in the above reaction A, the reaction is preferably carried out using a condensing agent, and a base such as triethylamine or dimethylaminopyridine may be used to promote the reaction. The protecting group for the amino group of glutamic acid is not particularly limited, and examples thereof include acyl-based protecting groups and carbamate-based protecting groups, specifically, trifluoroacetyl group, 9-fluorenylmethyloxycarbonyl group (Fmoc), tert-butyloxycarbonyl group, etc. It is preferable to remove impurities produced as by-products in the reaction, or polyethylene glycol derivatives remaining unconsumed in the reaction, by purification. The purification method is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0179] Deprotection N

[0180] [ka]

[0181] Deprotection N is a step in which the protecting group of the polyethylene glycol derivative (14) obtained in reaction M is deprotected to obtain a polyethylene glycol derivative (15). As the deprotection reaction, a conventionally known method can be used. 1 and L 7 It is necessary to use conditions that do not decompose the divalent spacer of the above. This step can also be carried out as part of the step of reaction M. It is preferable to remove impurities generated as by-products in the deprotection reaction by purification, which is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0182] Reaction O

[0183] [ka]

[0184] Reaction O is a process in which the amino group of the polyethylene glycol derivative (15) obtained by deprotection N is bonded to the two carboxyl groups of glutaric acid via a condensation reaction to obtain a four-arm polyethylene glycol derivative (16) in which four degradable polyethylene glycol chains are linked by glutaric acid. The glutaric acid used as the core molecule here may be replaced with other dibasic acids such as succinic acid, adipic acid, and sebacic acid, or may be a compound in which two hydroxyl groups of ethylene glycol, propylene glycol, butanediol, pentanediol, hexanediol, heptanediol, octanediol, nonanediol, decanediol, etc. are activated with nitrophenyl carbonate or succinimidyl carbonate. As in the above reaction A, the reaction is preferably carried out using a condensing agent, and a base such as triethylamine or dimethylaminopyridine may be used to promote the reaction. It is preferable to remove impurities produced as by-products in the reaction, or polyethylene glycol derivatives remaining unconsumed in the reaction, by purification. The purification method is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0185] Deprotection P

[0186] [ka]

[0187] Deprotection P is a step in which the protecting group of the polyethylene glycol derivative (16) obtained in reaction O is removed to obtain a polyethylene glycol derivative (17). As the deprotection reaction, a conventionally known method can be used.1 and L 7 It is necessary to use conditions that do not decompose the divalent spacer of the above. This step can also be carried out as part of the step O. It is preferable to remove impurities generated as by-products in the deprotection reaction by purification, which is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like. Through the above steps, the functional group X with glutaric acid as the core molecule is obtained. 1 A four-arm polyethylene glycol derivative (17) having four groups can be obtained.

[0188] Reaction Q

[0189] [ka]

[0190] Reaction Q is a process in which the amino group of the polyethylene glycol derivative (15) obtained by deprotection N is bonded by a condensation reaction to two carboxyl groups of a glutamic acid derivative whose amino group is protected with a protecting group, thereby obtaining a branched polyethylene glycol derivative (18) in which four degradable polyethylene glycol chains are linked by glutamic acid residues. The reaction and purification can be carried out under the same conditions as in Reaction M above. As a method for removing polyethylene glycol impurities having different molecular weights or functional groups from the polyethylene glycol derivative (18), the purification techniques described in JP 2014-208786 A or JP 2011-79934 A can be used.

[0191] Deprotection R

[0192] [ka]

[0193] Deprotection R is a step of removing the protecting group of the polyethylene glycol derivative (18) obtained in reaction Q to obtain a polyethylene glycol derivative (19). As the deprotection reaction, a conventionally known method can be used. 1 and L 7 It is necessary to use conditions under which the divalent spacer of is not decomposed. The reaction and purification can be carried out under the same conditions as for the above-mentioned deprotection N. This step can also be carried out as a part of the step of reaction Q.

[0194] Reaction S

[0195] [ka]

[0196] Reaction S is a process in which the amino group of the polyethylene glycol derivative (19) obtained by deprotection R is bonded to two carboxyl groups of glutaric acid via a condensation reaction to obtain an 8-arm polyethylene glycol derivative (20) in which eight degradable polyethylene glycol chains are linked by glutaric acid. The glutaric acid used as the core molecule here may be replaced with other dibasic acids such as succinic acid, adipic acid, and sebacic acid, or may be a compound in which two hydroxyl groups of ethylene glycol, propylene glycol, butanediol, pentanediol, hexanediol, heptanediol, octanediol, nonanediol, decanediol, etc. are activated with nitrophenyl carbonate or succinimidyl carbonate. As in the above reaction A, the reaction is preferably carried out using a condensing agent, and a base such as triethylamine or dimethylaminopyridine may be used to promote the reaction. It is preferable to remove impurities produced as by-products in the reaction, or polyethylene glycol derivatives remaining unconsumed in the reaction, by purification. The purification method is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0197] Deprotection T

[0198] [ka]

[0199] Deprotection T is a step of removing the protecting group of the polyethylene glycol derivative (20) obtained in reaction S to obtain a polyethylene glycol derivative (21). As the deprotection reaction, a conventionally known method can be used. 1 and L 7 It is necessary to use conditions that do not decompose the divalent spacer. This step can also be carried out as part of the step S. It is preferable to remove impurities generated as by-products in the deprotection reaction by purification, which is not particularly limited, and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like. Through the above steps, the functional group X with glutaric acid as the core molecule is obtained. 1 An 8-arm polyethylene glycol derivative (21) having eight groups can be obtained.

[0200] The polyethylene glycol derivatives (4), (9), (17) and (21) obtained by deprotection D, deprotection H, deprotection P and deprotection T have the functional group X 1 and X 2 These functional groups can be used to convert them into various functional groups.

[0201] For example, functional group X 1 and X 2 When is an amino group, there is no particular limitation, but 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, or a general reaction reagent such as an acid anhydride or an acid chloride.

[0202] Specifically, when it is desired to convert an amino group to a maleimide group, the target compound can be obtained by reacting it with a reagent such as the following.

[0203] [ka]

[0204] For example, when it is desired to convert the terminal amino group of a polyethylene glycol derivative to a carboxyl group, the target compound can be obtained by reacting it with succinic anhydride or glutaric anhydride.

[0205] For example, when it is desired to convert the terminal amino group of a polyethylene glycol derivative to a hydroxyl group, the target compound can be obtained by a condensation reaction with a ring-opening product of a cyclic ester such as caprolactone.

[0206] These reaction reagents are low molecular weight reagents and have a significantly different solubility from polyethylene glycol derivatives, which are high molecular weight polymers, and therefore can be easily removed by common purification methods such as extraction and crystallization.

[0207] The degradable polyethylene glycol obtained through the above-mentioned processes is required to have the ability to be stable in blood and decompose only within cells. In order to appropriately evaluate the performance, for example, the following tests can be carried out to evaluate the stability of the degradable polyethylene glycol in blood and its decomposition within cells. In addition, in consideration of the influence of the type of functional group possessed by the polyethylene glycol derivative in these evaluations, all evaluation samples were standardized to polyethylene glycol derivatives having one amino group and the tests were carried out.

[0208] There are no particular limitations on the test method for evaluating the stability of degradable polyethylene glycol derivatives in blood, and examples include tests using serum from mice, rats, humans, etc. Specifically, the polyethylene glycol derivative is dissolved in serum to a concentration of 1 to 10 mg / mL, incubated at 37°C for 96 hours, and the polyethylene glycol derivative contained in the serum is collected and measured by GPC to evaluate the degradation rate. The degradation rate is calculated from the peak area % of the GPC 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 × 100 For example, if the peak area % of the GPC main fraction of a 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 decomposition rate is calculated as follows. Decomposition rate = (95-90)÷95×100 = 5.26(%) If a degradable polyethylene glycol derivative is degraded in the blood, the desired half-life in the blood cannot be obtained. Therefore, in a stability test, the decomposition rate after 96 hours is preferably 10% or less, and more preferably 5% or less.

[0209] There are no particular limitations on the test method for evaluating the degradability of degradable polyethylene glycol derivatives in cells, and examples of such tests include a test in which cells are cultured using a medium containing a degradable polyethylene glycol derivative. There are no particular limitations on the cells and medium used here, but specifically, the polyethylene glycol derivative is dissolved in RPMI-1640 medium to a concentration of 1 to 20 mg / mL, and macrophage cells RAW264.7 are cultured using this medium at 37°C for 96 hours, after which the polyethylene glycol derivative in the cells is recovered and the degradation rate is evaluated by measuring GPC. The degradation rate is calculated using the peak area % of the GPC main fraction of the polyethylene glycol derivative before and after the test, as in the stability test. For example, if the peak area % of the GPC main fraction of a degradable polyethylene glycol derivative before the cell-based degradation test is 95% and the peak area % of the GPC main fraction after the test is 5%, the degradation rate is calculated as follows. Decomposition rate = (95-5)÷95×100 = 94.7(%) Unless a degradable polyethylene glycol derivative is efficiently degraded within a cell, it will not be able to inhibit vacuoles in the target cells. Therefore, in a degradability test, the decomposition rate after 96 hours is preferably 90% or more, and more preferably 95% or more.

[0210] There are no particular limitations on the test method for evaluating the blood half-life and biodistribution of degradable polyethylene glycol derivatives, but examples include tests in which the derivatives are labeled with a radioisotope or fluorescent substance, administered to mice or rats, and monitored. The degradable peptide introduced into the polyethylene glycol derivative gives polyethylene glycol intracellular degradability, but the peptide structure may change the pharmacokinetics of polyethylene glycol. Therefore, in order to confirm the effect of the introduced peptide structure on the pharmacokinetics, it is necessary to compare the blood half-life and distribution in the body with a non-degradable polyethylene glycol derivative of the same molecular weight. Specifically, a non-degradable polyethylene glycol derivative labeled with a radioisotope and a degradable polyethylene glycol derivative can be administered to mice, and the radiation doses in the blood and each organ can be measured at multiple time points, allowing quantitative measurements to be performed.

[0211] There are no particular limitations on the test method for evaluating the inhibition of cellular vacuoles by degradable polyethylene glycol derivatives, but examples of such methods include a test in which the derivative is administered to mice or rats for a long period of time, frequently, and in high doses, as described in Non-Patent Document 2, and images of slices of organs that are known to be prone to the development of vacuoles are examined. Specifically, a polyethylene glycol derivative is dissolved in physiological saline to a concentration of 10 to 250 mg / mL, and 20 to 100 μL is administered into the tail vein of a mouse three times a week for at least four weeks. Paraffin sections of organs known to be prone to the development of vacuoles, such as the cerebral choroid plexus and spleen, are prepared and stained, and images of the sections are examined using pathological techniques to evaluate the inhibition of vacuoles. In this evaluation, the amount of polyethylene glycol administered must be in large excess compared with the general amount of polyethylene glycol administered in the art.

[0212] Non-Patent Document 2 states that cellular vacuolation caused by high molecular weight polyethylene glycol is related to the accumulation of polyethylene glycol in tissues. There are no particular limitations on the test method for evaluating the cellular accumulation of degradable polyethylene glycol derivatives, but the evaluation can be performed from section images prepared in the same manner as the evaluation of vacuoles described above. The accumulation of polyethylene glycol can be evaluated by confirming stained section images of organs such as the cerebral choroid plexus and spleen, which are said to be prone to polyethylene glycol accumulation, using a pathological method. In this evaluation, the amount of polyethylene glycol administered must be in large excess compared with the general amount of polyethylene glycol administered in the art. EXAMPLES

[0213] The following examples were obtained: 1 H-NMR was obtained from JNM-ECP400 or JNM-ECA600 manufactured by JEOL Datum Co., Ltd. A φ5 mm tube was used for the measurement, and D2O or CDCl3 and d6-DMSO containing tetramethylsilane (TMS) as an internal standard were used as deuterated solvents. The molecular weight and amine purity of the obtained polyethylene glycol derivatives were calculated using liquid chromatography (GPC and HPLC). The liquid chromatography systems used for GPC were "HLC-8320GPC EcoSEC" manufactured by Tosoh Corporation, and for HPLC, "ALLIANCE" manufactured by WATERS was used. The analytical conditions for GPC and HPLC are shown below. GPC analysis (molecular weight measurement) Detector: Differential refractometer Column: Ultrahydrogel 500 and Ultrahydrogel 250 (WATERS) Mobile phase: 100mM Acetate buffer+0.02%NaN3(pH5.2) Flow rate: 0.5mL / min Sample volume: 5mg / mL, 20μL Column temperature: 30℃ HPLC analysis (amine purity measurement) Detector: Differential refractometer Column: TSKgel SP-5PW (Tosoh Corporation) Mobile phase: 1mM Sodium phosphate buffer (pH6.5) Flow rate: 0.5mL / min Injection volume: 5mg / mL, 20μL Column temperature: 40℃

[0214] [Example 1] Synthesis of compound (p3)

[0215] [ka]

[0216] [Example 1-1] Synthesis of compound (p1)

[0217] [ka]

[0218] L-phenylalanyl-glycine (Fmoc-Phe-Gly) (400 mg) with the N-terminus protected by a 9-fluorenylmethyloxycarbonyl group (Fmoc group) and NOF Corp.'s "SUNBRIGHT HO-200PA" (15 g) with an average molecular weight of 20,000 were dissolved in acetonitrile (60 g). Then, diisopropylethylamine (233 mg) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM) (311 mg) were added and reacted at room temperature under a nitrogen atmosphere for 1 hour. Then, piperidine (639 mg) was added and reacted at room temperature under a nitrogen atmosphere for 2 hours. After the reaction was completed, the reaction solution was diluted with toluene (500 g), hexane (300 g) was added, and the mixture was stirred at room temperature for 30 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, and then dissolved again in toluene (300 g), and hexane (150 g) was added and stirred at room temperature for 30 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, and then washed with hexane (100 g) containing 2,6-di-tert-butyl-p-cresol (BHT) (20 mg), and then suction filtration using 5A filter paper and vacuum dried to obtain the above compound (p1). Yield: 13 g. 1 H-NMR(d6-DMSO): 1.73ppm(m, 2H, -CO-NH-CH2- CH 2-CH2-(O-CH2-CH2)n-OH), 2.59ppm(dd, 1H, -NH-CO-CH- CH 2-C6H5), 2.98ppm(dd, 1H, -NH-CO-CH- CH 2-C6H5), 3.10ppm(q, 2H, -CO-NH- CH 2-CH2-CH2-(O-CH2-CH2)n-OH), 3.48ppm(m, approx. 1,900H, -CO-NH-CH2-CH2-CH2-(O- CH 2 -CH 2)n-OH), 7.24ppm(m, 5H, -NH-CO-CH-CH2- C 6 H 5), 7.73ppm (t, 1H), 8.12ppm (broad, 1H)

[0219] [Example 1-2] Synthesis of compound (p2)

[0220] [ka]

[0221] The compound (p1) (1.0 g) obtained in Example 1-1 and glutaric acid (3.2 mg) were dissolved in acetonitrile (4.0 g), and then diisopropylethylamine (8.4 mg) and DMT-MM (22 mg) were added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 3 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate (60 g), and then hexane (40 g) was added and stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using 5A filter paper and collecting the precipitate, the mixture was dissolved again in ethyl acetate (60 g), hexane (30 g) was added, and the mixture was stirred at room temperature for 30 minutes to precipitate the product. After suction filtration using 5A filter paper and collecting the precipitate, the mixture was washed with hexane (30 g) containing BHT (6 mg), suction filtration using 5A filter paper, and vacuum dried to obtain the above compound (p2). Yield: 734 mg. 1 H-NMR(d6-DMSO): 1.73ppm(m, 4H, -CO-NH-CH2- CH 2-CH2-(O-CH2-CH2)n-OH), 2.05ppm(m, 6H, -NH-CO- CH 2 CH 2 CH 2-CO-NH-), 2.59ppm(dd, 2H, -NH-CO-CH- CH 2-C6H5), 2.98ppm(dd, 2H, -NH-CO-CH- CH 2-C6H5), 3.10ppm(q, 4H, -CO-NH- CH 2-CH2-CH2-(O-CH2-CH2)n-OH), 3.48ppm(m, approx. 3,800H, -CO-NH-CH2-CH2-CH2-(O- CH 2 -CH 2)n-OH), 7.24ppm(m, 10H, -NH-CO-CH-CH2- C 6 H5), 7.73ppm (t, 2H), 8.12ppm (broad, 2H)

[0222] [Examples 1-3] Synthesis of compound (p3)

[0223] [ka]

[0224] The compound (p2) (500 mg) obtained in Example 1-2 was dissolved in dichloromethane (3.5 g), and then di(N-succinimidyl) carbonate (26 mg) and pyridine (10 mg) were added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 8 hours. After the reaction was completed, the reaction solution was washed with 5% saline (5 g), magnesium sulfate (100 mg) was added, and the mixture was stirred at room temperature for 30 minutes, and then suction filtration was performed using 5A filter paper. The obtained filtrate was concentrated, and ethyl acetate (30 g) containing BHT (6 mg) was added to the concentrate to dissolve it, and then hexane (15 g) was added and stirred at room temperature for 30 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (30 g) containing BHT (6 mg), suction filtration using 5A filter paper, and vacuum dried to obtain the above compound (p3). Yield: 378 mg. The succinimidyl carbonate conversion rate was 94% ( 1 H-NMR). 1 H-NMR(d6-DMSO): 1.73ppm(m, 4H, -CO-NH-CH2- CH 2-CH2-O-(CH2-CH2-O)n-OCO-Succinimide), 2.05ppm(m, 6H, -NH-CO- CH 2 -CH 2 -CH 2-CO-NH-), 2.59ppm(dd, 2H, -NH-CO-CH- CH 2-C6H5), 2.83ppm(s, 8H, -CO- CH 2 -CH 2-CO-), 2.98ppm(dd, 2H, -NH-CO-CH- CH 2-C6H5), 3.10ppm(q, 4H, -CO-NH- CH2-CH2-CH2-(O-CH2-CH2)n-OCO-Succinimide), 3.48ppm(m, about 3,800H, -CO-NH-CH2-CH2-CH2-(O- CH 2 -CH 2)n-OCO-Succinimide), 7.24ppm (m, 10H, -NH-CO-CH-CH2- C 6 H 5), 7.73ppm (t, 2H), 8.12ppm (broad, 2H)

[0225] [Example 2] Synthesis of compound (p8)

[0226]

change

[0227] [Example 2-1] Synthesis of compound (p4)

[0228]

change

[0229] "SUNBRIGHT DE-200PA" (20 g) manufactured by NOF Corp., with an average molecular weight of 20,000, was dissolved in toluene (80 g), di-tert-butyl dicarbonate (107 mg) was added, and the mixture was reacted at 40°C under a nitrogen atmosphere for 3 hours. After the reaction was completed, toluene (100 g) was added, and the mixture was stirred to make the mixture uniform. Then, hexane (100 g) was added and the mixture was stirred at room temperature for 30 minutes to precipitate the product, which was then suction filtered using 5A filter paper and vacuum dried. The product was then purified by ion exchange chromatography, and chloroform (500 g) was added to the recovered aqueous solution, which was stirred at room temperature for 30 minutes to extract the product in the organic layer. Sodium sulfate (10 g) was added to the obtained organic layer, which was then dehydrated by stirring at room temperature for 30 minutes, and then suction filtered using 5A filter paper. The obtained filtrate was concentrated, redissolved in toluene (200 g), hexane (100 g) was added, and the mixture was stirred at room temperature for 30 minutes to precipitate crystals. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (100 g), suction filtration using 5A filter paper, and dried in vacuum to obtain the above compound (p4). Yield: 9.1 g. HPLC: Amine purity was 98%. 1 H-NMR(d6-DMSO): 1.44ppm(s, 9H, -CH2-CH2-CH2-NH-CO-OC (CH 3 ) 3), 1.64ppm(m, 1H), 1.73ppm(m, 4H), 2.68ppm(t, 2H, -(CH2-CH2-O)n-CH2-CH2- CH 2-NH2), 3.18ppm(t, 2H, -CH2-CH2- CH 2-NH-CO-OC(CH3)3), 3.35ppm(m, 4H), 3.64ppm(m, approx. 1,900H, -NH-CH2-CH2-CH2-O-( CH 2 -CH 2-O)n-), 6.76 ppm (broad, 1H)

[0230] [Example 2-2] Synthesis of compound (p5)

[0231] [ka]

[0232] Acetonitrile (34 g) was added to L-glycyl-leucyl-phenylalanyl-glycine (Fmoc-Gly-Leu-Phe-Gly) (313 mg) whose N-terminus was protected with a 9-fluorenylmethyloxycarbonyl group (Fmoc group) and the compound (p4) (8.5 g) obtained in Example 2-1, and dissolved. Then, diisopropylethylamine (132 mg) and DMT-MM (353 mg) were added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 1 hour. Then, piperidine (361 mg) was added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 2 hours. After the reaction was completed, the reaction solution was diluted with toluene (400 g), hexane (250 g) was added, and the mixture was stirred at room temperature for 30 minutes to precipitate the product. The mixture was filtered by suction using 5A filter paper, and the precipitate was collected, and then dissolved again in toluene (400 g), hexane (200 g) was added, and the mixture was stirred at room temperature for 30 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (200 g), suction filtration using 5A filter paper, and vacuum dried to obtain the above compound (p5). Yield: 7.6 g. 1 H-NMR(d6-DMSO): 0.89ppm(d, 3H, -NH-CO-CH-CH2-CH( CH 3)2), 0.91ppm(d, 3H, -NH-CO-CH-CH2-CH( CH 3)2), 1.44ppm(s, 9H, -CH2-CH2-CH2-NH-CO-OC (CH 3 ) 3), 1.48 ppm (m, 1H, -NH-CO- CH -CH2-CH(CH3)2), 1.73ppm(m, 6H), 2.68ppm(t, 2H, -(CH2-CH2-O)n-CH2-CH2- CH 2-NH2), 3.18ppm(t, 2H, -CH2-CH2- CH 2-NH-CO-OC(CH3)3), 3.35ppm(m, 4H), 3.64ppm(m, approx. 1,900H, -NH-CH2-CH2-CH2-O-( CH 2 -CH 2-O)n-), 4.09ppm(s, 2H, -CH2-CH2-CH2-NH-CO- CH2-NH-), 4.44ppm(m, 1H), 4.92ppm(m, 1H), 6.76ppm(broad, 1H), 7.20ppm(d, 2H, -NH-CO-CH-CH2- C 6 H 5), 7.32ppm(m, 3H, -NH-CO-CH-CH2- C 6 H 5), 8.01ppm (broad, 1H), 8.32ppm (broad, 2H), 8.70ppm (broad, 2H), 9.04ppm (broad, 1H)

[0233] [Example 2-3] Synthesis of compound (p6)

[0234] [ka]

[0235] The compound (p5) (1.3 g) obtained in Example 2-2 and succinic acid (3.8 mg) were dissolved in acetonitrile (5.2 g), and then diisopropylethylamine (11 mg) and DMT-MM (29 mg) were added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 3 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate (100 g), and then hexane (60 g) was added and stirred at room temperature for 15 minutes to precipitate the product. The mixture was filtered by suction using 5A filter paper, the precipitate was collected, and then dissolved in ethyl acetate (100 g) containing BHT (20 mg), and hexane (50 g) was added and stirred at room temperature for 30 minutes to precipitate the product. The mixture was filtered by suction using 5A filter paper, the precipitate was collected, and then washed with hexane (50 g) containing BHT (10 mg), filtered by suction using 5A filter paper, and dried in vacuum to obtain the above compound (p6). Yield: 889 mg. 1 H-NMR(d6-DMSO): 0.89ppm(d, 6H, -NH-CO-CH-CH2-CH( CH 3)2), 0.91ppm(d, 6H, -NH-CO-CH-CH2-CH( CH 3)2), 1.44ppm(s, 18H, -CH2-CH2-CH2-NH-CO-OC (CH 3 )3)、1.48 ppm (m, 2H, -NH-CO- CH -CH2-CH(CH3)2), 1.73 ppm (m, 12H), 2.34 ppm (m, 4H), 2.68 ppm (t, 4H, -(CH2-CH2-O)n-CH2-CH2- CH 2-), 3.18 ppm (t, 4H, -CH2-CH2- CH 2-NH-CO-O-C(CH3)3), 3.35 ppm (m, 8H), 3.64 ppm (m, approximately 3,800H, -NH-CH2-CH2-CH2-O-( CH 2 -CH 2-O)n-), 4.09 ppm (s, 12H), 4.44 ppm (m, 2H), 4.92 ppm (m, 2H), 6.76 ppm (broad, 1H), 7.20 ppm (d, 4H, -NH-CO-CH-CH2- C 6 H 5), 7.32 ppm (m, 6H, -NH-CO-CH-CH2- C 6 H 5), 8.01 ppm (broad, 2H), 8.32 ppm (broad, 4H), 9.04 ppm (broad, 4H)

[0236] [Example 2-4] Synthesis of compound (p7)

[0237] [Chemical formula]

[0238] The compound (p6) (800 mg) obtained in Example 2-3 was dissolved in ion-exchanged water (3.3 g), 6N hydrochloric acid (0.7 g) was added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 3 hours. After the reaction was completed, 1N aqueous sodium hydroxide solution was added, the pH was adjusted to 6.5, and then sodium chloride (1.0 g) was added and dissolved. 1N aqueous sodium hydroxide solution was added to the obtained solution, the pH was adjusted to 7.10, and then chloroform (10 g) containing BHT (2 mg) was added and stirred at room temperature for 20 minutes, and the product was extracted into the organic layer. The organic layer and the aqueous layer were separated, the organic layer was recovered, and then chloroform (10 g) containing BHT (2 mg) was added again to the aqueous layer and stirred at room temperature for 20 minutes, and the product was extracted into the organic layer. The organic layers obtained in the first and second extractions were combined and concentrated at 40° C., and the obtained concentrate was dissolved in toluene (50 g), sodium sulfate (1.0 g) was added, and the mixture was dehydrated by stirring at room temperature for 30 minutes. Then, the mixture was filtered by suction using 5A filter paper, and hexane (30 g) was added to the filtrate and stirred at room temperature for 30 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (30 g) containing BHT (6 mg), filtered by suction using 5A filter paper, and dried in vacuum to obtain the above compound (p7). Yield: 675 mg. HPLC: Amine purity was 91%. 1 H-NMR(d6-DMSO):0.89ppm(d,6H, -NH-CO-CH-CH2-CH( CH 3)2), 0.91ppm(d, 6H, -NH-CO-CH-CH2-CH( CH 3)2), 1.48ppm(m, 2H, -NH-CO- CH -CH2-CH(CH3)2), 1.73ppm(m, 12H), 2.34ppm(m, 4H), 2.68ppm(t, 4H, -(CH2-CH2-O)n-CH2-CH2- CH 2-), 3.18ppm(t, 4H, -CH2-CH2- CH 2-NH-CO-OC(CH3)3), 3.35ppm(m, 8H), 3.64ppm(m, approx. 3,800H, -NH-CH2-CH2-CH2-(O- CH 2 -CH2)n-), 4.09ppm(s, 12H), 4.44ppm(m, 2H), 4.92ppm(m, 2H), 6.76ppm(broad, 1H), 7.20ppm(d, 4H, -NH-CO-CH-CH2- C 6 H 5), 7.32ppm(m, 6H, -NH-CO-CH-CH2- C 6 H 5), 8.01ppm (broad, 2H), 8.32ppm (broad, 4H), 9.04ppm (broad, 4H)

[0239] [Example 2-5] Synthesis of compound (p8)

[0240] [ka]

[0241] The compound (p7) (400 mg) obtained in Example 2-4 was dissolved in acetonitrile (320 mg) and toluene (2.1 g). Then, N-methylmorpholine (10 mg) and N-succinimidyl 3-maleimidopropionate (27 mg) were added, and the mixture was reacted at room temperature under a nitrogen atmosphere and in the dark for 6 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate (50 g) containing BHT (10 mg), and then hexane (30 g) was added and stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (30 g) containing BHT (6 mg), suction filtration using 5A filter paper, and vacuum dried to obtain the above compound (p8). Yield: 227 mg. The maleimide conversion rate was 96% ( 1 H-NMR). 1 H-NMR(d6-DMSO):0.89ppm(d,6H, -NH-CO-CH-CH2-CH( CH 3)2), 0.91ppm(d, 6H, -NH-CO-CH-CH2-CH( CH 3)2), 1.48ppm(m, 2H, -NH-CO- CH-CH2-CH(CH3)2), 1.73ppm(m, 12H), 2.34ppm(m, 4H), 2.68ppm(t, 4H, -(CH2-CH2-O)n-CH2-CH2- CH 2-), 3.18ppm(t, 4H, -CH2-CH2- CH 2-NH-CO-OC(CH3)3), 3.35ppm(m, 8H), 3.64ppm(m, about 3,800H, -NH-CH2-CH2-CH2-O-( CH 2 -CH 2-O)n-), 4.09ppm (s, 12H), 4.44ppm (m, 2H), 4.76ppm (m, 4H, -NH-CO-CH2- CH 2-Maleimide), 4.92ppm (m, 2H), 6.76ppm (broad, 1H), 6.68ppm (s, 4H, -CH2-CH2-CH2-NH-CO-CH2-CH2- C 4 NO 2 H 2), 7.20ppm(d, 4H, -NH-CO-CH-CH2- C 6 H 5), 7.32ppm(m, 6H, -NH-CO-CH-CH2- C 6 H 5), 8.01ppm (broad, 2H), 8.32ppm (broad, 4H), 9.04ppm (broad, 4H)

[0242] [Example 3] Synthesis of compound (p13)

[0243]

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[0244] [Example 3-1] Synthesis of compound (p9)

[0245]

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[0246] The compound (p1) (2.0 g) obtained in Example 1-1, sodium acetate (82 mg), and glutaric anhydride (132 mg) were dissolved in toluene (6.0 g) and reacted at 40° C. for 7 hours under a nitrogen atmosphere. After the reaction was completed, the mixture was diluted with ethyl acetate (100 g), suction filtered using 5A filter paper, hexane (50 g) was added to the obtained filtrate, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The mixture was suction filtered using 5A filter paper, and the precipitate was collected. The obtained precipitate was dissolved in ethyl acetate (100 g), hexane (50 g) was added, and the mixture was stirred at room temperature for 30 minutes to precipitate the product. The mixture was suction filtered using 5A filter paper, and the precipitate was collected, washed with hexane (50 g), suction filtered using 5A filter paper, and vacuum dried to obtain the above compound (p9). Yield: 1.8 g. 1 H-NMR(d6-DMSO): 1.73ppm(m, 2H, -CO-NH-CH2- CH 2-CH2-(O-CH2-CH2)n-OH), 2.05ppm(m, 4H, -NH-CO- CH 2 -CH 2-CH2-COOH), 2.30ppm(t, 2H, -NH-CO-CH2-CH2- CH 2-COOH), 2.59ppm(dd, 1H, -NH-CO-CH- CH 2-C6H5), 2.98ppm(dd, 1H, -NH-CO-CH- CH 2-C6H5), 3.10ppm(q, 2H, -CO-NH- CH 2-CH2-CH2-(O-CH2-CH2)n-OH), 3.48ppm(m, approx. 1,900H, -CO-NH-CH2-CH2-CH2-(O- CH 2 -CH 2)n-OH), 7.24ppm(m, 5H, -NH-CO-CH-CH2- C 6 H 5), 8.12ppm (broad, 1H), 9.04ppm (broad, 1H)

[0247] [Example 3-2] Synthesis of compound (p10)

[0248] [ka]

[0249] Compound (p9) (1.6 g) obtained in Example 3-1 and compound (p5) (1.6 g) obtained in Example 2-2 were dissolved in acetonitrile (10 g), and then diisopropylethylamine (25 mg) and DMT-MM (66 mg) were added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 3 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate (150 g), and then hexane (80 g) was added and stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using 5A filter paper and recovery of the precipitate, the mixture was dissolved again in ethyl acetate (100 g), hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using 5A filter paper and recovery of the precipitate, the mixture was washed with hexane (80 g) containing BHT (16 mg), suction filtration using 5A filter paper, and vacuum drying to obtain the above compound (p10). Yield 2.6 g. 1 H-NMR(d6-DMSO): 0.89ppm(d, 3H, -NH-CO-CH2-CH2-CH( CH 3)2), 0.91ppm(d, 3H, -NH-CO-CH2-CH2-CH( CH 3)2), 1.44ppm(s, 9H, -CH2-CH2-CH2-NH-CO-OC (CH 3 ) 3), 1.48 ppm (m, 1H, -NH-CO- CH -CH2-CH(CH3)2), 1.73ppm(m, 8H), 2.05ppm(m, 4H, -NH-CO- CH 2 -CH 2-CH2-CO-NH-), 2.30ppm(t, 2H, -NH-CO-CH2-CH2- CH 2-CO-NH-), 2.59ppm(dd, 1H, -NH-CO-CH- CH 2-C6H5), 2.98ppm(dd, 1H, -NH-CO-CH- CH 2-C6H5), 3.10ppm(m, 10H), 3.48ppm(m, approx. 3,800H, -CO-NH-CH2-CH2-CH2-(O- CH 2 -CH2)n-), 4.09ppm(s, 6H), 6.76ppm(broad, 1H), 7.24ppm(m, 10H, -NH-CO-CH-CH2- C 6 H 5), 8.12ppm (broad, 2H), 8.32ppm (broad, 3H), 9.04ppm (broad, 3H)

[0250] [Example 3-3] Synthesis of compound (p11)

[0251] [ka]

[0252] The compound (p10) (2.3 g) obtained in Example 3-2 was dissolved in ion-exchanged water (9.4 g), 6N hydrochloric acid (2.1 g) was added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 3 hours. After the reaction was completed, 1N aqueous sodium hydroxide solution was added, the pH was adjusted to 6.5, and then sodium chloride (2.5 g) was added and dissolved. 1N aqueous sodium hydroxide solution was added to the obtained solution, the pH was adjusted to 7.10, and then chloroform (20 g) containing BHT (4 mg) was added and stirred at room temperature for 20 minutes, and the product was extracted into the organic layer. The organic layer and the aqueous layer were separated, and the organic layer was recovered, and then chloroform (20 g) containing BHT (4 mg) was added again to the aqueous layer, and the mixture was stirred at room temperature for 20 minutes, and the product was extracted into the organic layer. The organic layers obtained from the first and second extractions were combined and concentrated at 40°C. The resulting concentrate was dissolved in toluene (150g), sodium sulfate (5.0g) was added, the mixture was stirred at room temperature for 30 minutes, and suction filtered using 5A filter paper. Hexane (80g) was added to the filtrate and stirred at room temperature for 30 minutes to precipitate the product. After suction filtration using 5A filter paper and recovery of the precipitate, it was washed with hexane (50g) containing BHT (10mg), suction filtration using 5A filter paper, and vacuum dried to obtain the above compound (p11). Yield: 1.7g. HPLC: Amine purity was 90%. 1 H-NMR(d6-DMSO): 0.89ppm(d, 3H, -NH-CO-CH2-CH2-CH( CH3)2), 0.91 ppm (d, 3H, -NH-CO-CH2-CH2-CH( CH 3)2), 1.48 ppm (m, 1H, -NH-CO- CH -CH2-CH(CH3)2), 1.73 ppm (m, 8H), 2.05 ppm (m, 4H, -NH-CO- CH 2 -CH 2-CH2-CO-NH-), 2.30 ppm (t, 2H, -NH-CO-CH2-CH2- CH 2-CO-NH-), 2.59 ppm (dd, 2H, -NH-CO-CH- CH 2-C6H5), 2.68 ppm (t, 2H, NH2- CH 2-CH2-CH2-(O-CH2-CH2)n-), 2.98 ppm (dd, 2H, -NH-CO-CH- CH 2-C6H5), 3.48 ppm (m, about 3,800H, -CO-NH-CH2-CH2-CH2-(O- CH 2 -CH 2)n-), 4.09 ppm (s, 6H), 7.24 ppm (m, 10H, -NH-CO-CH-CH2- C 6 H 5), 8.12 ppm (broad, 2H), 8.32 ppm (broad, 3H), 9.04 ppm (broad, 3H)

[0253] [Example 3-4] Synthesis of compound (p12)

[0254] [Chemical formula]

[0255] The compound (p11) (1.5 g) obtained in Example 3-3 was dissolved in acetonitrile (1.2 g) and toluene (7.8 g). Then, N-methylmorpholine (19 mg) and N-succinimidyl 3-maleimidopropionate (21 mg) were added, and the mixture was reacted at room temperature under a nitrogen atmosphere and in the dark for 3 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate (50 g) containing BHT (10 mg), and then hexane (30 g) was added and stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (20 g) containing BHT (4 mg), suction filtration using 5A filter paper, and vacuum dried to obtain the above compound (p12). Yield: 1.3 g. The maleimide conversion rate was 92% ( 1 H-NMR). 1 H-NMR(d6-DMSO): 0.89ppm(d, 3H, -NH-CO-CH2-CH2-CH( CH 3)2), 0.91ppm(d, 3H, -NH-CO-CH2-CH2-CH( CH 3)2), 1.48ppm(m, 1H, -NH-CO- CH -CH2-CH(CH3)2), 1.73ppm(m, 8H), 2.05ppm(m, 4H, -NH-CO- CH 2 -CH 2-CH2-CO-NH-), 2.30ppm(t, 2H, -NH-CO-CH2-CH2- CH 2-CO-NH-), 2.59ppm(dd, 2H, -NH-CO-CH- CH 2-C6H5), 2.68ppm(t, 2H, C4NO2H2-CH2-CH2-CO-NH- CH 2-CH2-CH2-O-(CH2-CH2-O)n-), 2.98ppm(dd, 2H, -NH-CO-CH- CH 2-C6H5), 3.48ppm(m, approx. 3,800H, -CO-NH-CH2-CH2-CH2-O-( CH 2 -CH 2-O)n-), 4.09ppm(s, 6H), 6.68ppm(s, 2H, -NH-CO-CH2-CH2- C 4 NO 2 H2), 7.24ppm(m, 10H, -NH-CO-CH-CH2- C 6 H 5), 8.12ppm(broad, 3H), 8.32ppm(broad, 3H), 9.04ppm(broad, 3H)

[0256] [Examples 3-5] Synthesis of compound (p13)

[0257] [ka]

[0258] The compound (p12) (800 mg) obtained in Example 3-4 was dissolved in dichloromethane (5.6 g), and then di(N-succinimidyl) carbonate (20 mg) and pyridine (8 mg) were added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 8 hours. After the reaction was completed, the reaction solution was washed with 5% saline (5 g), magnesium sulfate (100 mg) was added, and the mixture was stirred at 25°C for 30 minutes, and then suction filtration was performed using 5A filter paper. The obtained filtrate was concentrated, and toluene (100 g) was added to the concentrate to dissolve it, and then hexane (50 g) was added and stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (50 g) containing BHT (10 mg), suction filtration using 5A filter paper, and vacuum dried to obtain the above compound (p13). Yield: 653 mg. The succinimidyl carbonate conversion rate was 93% ( 1 H-NMR). 1 H-NMR(d6-DMSO): 0.89ppm(d, 3H, -NH-CO-CH2-CH2-CH( CH 3)2), 0.91ppm(d, 3H, -NH-CO-CH2-CH2-CH( CH 3)2), 1.48ppm(m, 1H, -NH-CO- CH -CH2-CH(CH3)2), 1.73ppm(m, 8H), 2.05ppm(m, 4H, -NH-CO- CH 2 -CH 2-CH2-CO-NH-), 2.30ppm(t, 2H, -NH-CO-CH2-CH2- CH 2-CO-NH-), 2.59ppm(dd, 2H, -NH-CO-CH- CH 2-C6H5), 2.68ppm(t, 2H, C4NO2H2-CH2-CH2-CO-NH- CH 2-CH2-CH2-O-(CH2-CH2-O)n-), 2.83ppm(s, 4H, -CO- CH 2 -CH 2-CO-), 2.98ppm(dd, 2H, -NH-CO-CH- CH 2-C6H5), 3.48ppm(m, about 3,800H, -CO-NH-CH2-CH2-CH2-O-( CH 2 -CH 2-O)n-), 4.09ppm (s, 6H), 6.68ppm (s, 2H, -NH-CO-CH2-CH2- C 4 NO 2 H 2), 7.24ppm(m, 10H, -NH-CO-CH-CH2- C 6 H 5), 8.12ppm (broad, 3H), 8.32ppm (broad, 3H), 9.04ppm (broad, 3H)

[0259] [Example 4] Synthesis of compound (p17)

[0260]

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[0261] [Example 4-1] Synthesis of compound (p14)

[0262]

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[0263] The above compound (p14) was obtained by the same method as in Example 1-1, using L-phenylalanyl-glycine (Fmoc-Phe-Gly) (400 mg) whose N-terminus was protected with a 9-fluorenylmethyloxycarbonyl group (Fmoc group) and NOF Corp.'s "SUNBRIGHT HO-100PA" (7.5 g) with an average molecular weight of 10,000 as raw materials. Yield: 6.5 g. HPLC: Amine purity was 98%. 1 H-NMR(d6-DMSO): 1.73ppm(m, 2H, -CO-NH-CH2- CH 2-CH2-(O-CH2-CH2)n-OH), 2.59ppm(dd, 1H, -NH-CO-CH- CH 2-C6H5), 2.98ppm(dd, 1H, -NH-CO-CH- CH 2-C6H5), 3.10ppm(q, 2H, -CO-NH- CH 2-CH2-CH2-(O-CH2-CH2)n-OH), 3.48ppm(m, approx. 950H, -CO-NH-CH2-CH2-CH2-(O- CH 2 -CH 2)n-OH), 7.24ppm(m, 5H, -NH-CO-CH-CH2- C 6 H 5), 7.73ppm (t, 1H), 8.12ppm (broad, 1H)

[0264] [Example 4-2] Synthesis of compound (p15)

[0265] [ka]

[0266] Acetonitrile (24 g) was added to L-glutamic acid (Fmoc-Glu-OH) (93 mg) whose N-terminus was protected with a 9-fluorenylmethyloxycarbonyl group (Fmoc group) and the compound (p14) (5.5 g) obtained in Example 4-1, and the mixture was dissolved by heating at 30°C. Then, diisopropylethylamine (86 mg) and DMT-MM (232 mg) were added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 1 hour. Then, piperidine (1.1 g) was added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 2 hours. After the reaction was completed, the reaction solution was diluted with toluene (150 g), hexane (80 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The mixture was filtered by suction using 5A filter paper, and the precipitate was collected, and then dissolved again in toluene (150 g), hexane (80 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (50 g) containing BHT (10 mg), suction filtration using 5A filter paper, and vacuum dried to obtain the above compound (p15). Yield: 4.6 g. HPLC: Amine purity was 92%. 1 H-NMR(d6-DMSO): 1.54ppm(m, 2H, -NH-CO-CH(NH2)- CH 2-CH2-), 1.62ppm(m, 4H, -CO-NH-CH2- CH 2-CH2-), 1.97ppm(m, 2H, -NH-CO-CH(NH2)-CH2- CH 2-), 2.74ppm(dd, 2H, -CO-NH-CH- CH 2-C6H5), 2.81ppm(dd, 2H, -CO-NH-CH- CH 2-C6H5), 3.11ppm(m, 11H), 3.64ppm(m, approx. 1,900H, -CO-NH-CH2-CH2-CH2-(O- CH 2 -CH 2)n-OH), 4.49ppm(m, 4H, -CO-NH-CH- CH 2-C6H5), 4.57ppm(m, 2H, -CO-NH- CH -CH2-C6H5), 7.25ppm(m, 10H, -CO-NH-CH-CH2- C 6 H5), 7.74ppm(m, 2H), 8.44ppm(m, 2H), 8.61ppm(m, 2H)

[0267] [Example 4-3] Synthesis of compound (p16)

[0268] [ka]

[0269] The compound (p15) (1.0 g) obtained in Example 4-2 and glutaric acid (3.3 mg) were dissolved in acetonitrile (8.0 g), and then diisopropylethylamine (8.4 mg) and DMT-MM (22.6 mg) were added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 3 hours. After the reaction was completed, the reaction solution was diluted with toluene (100 g), and then hexane (60 g) was added and stirred at room temperature for 15 minutes to precipitate the product. The mixture was filtered by suction using 5A filter paper, the precipitate was collected, and then dissolved again in toluene (100 g), hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The mixture was filtered by suction using 5A filter paper, and the precipitate was collected, and then washed with hexane (50 g) containing BHT (10 mg), filtered by suction using 5A filter paper, and dried in vacuum to obtain the above compound (p16). Yield: 683 mg. 1 H-NMR(d6-DMSO): 1.54ppm(m, 4H, -NH-CO-CH(NH)- CH 2-CH2-), 1.62ppm(m, 8H), 1.97ppm(m, 4H, -NH-CO-CH(NH)-CH2- CH 2-), 2.05ppm(m, 4H, -NH-CO- CH 2 -CH 2-CH2-CO-NH-), 2.30ppm(t, 2H, -NH-CO-CH2-CH2- CH 2-CO-NH-), 2.74ppm(dd, 2H, -CO-NH-CH- CH 2-C6H5), 2.81ppm(dd, 2H, -CO-NH-CH- CH2-C6H5), 3.11ppm(m, 22H), 3.64ppm(m, approx. 3,800H, -CO-NH-CH2-CH2-CH2-(O- CH 2 -CH 2)n-OH), 4.49ppm(m, 8H, -CO-NH-CH- CH 2-C6H5), 4.57ppm(m, 4H, -CO-NH- CH -CH2-C6H5), 7.25ppm(m, 20H, -CO-NH-CH-CH2- C 6 H 5), 7.74ppm(m, 4H), 8.44ppm(m, 4H), 8.61ppm(m, 4H)

[0270] [Example 4-4] Synthesis of compound (p17)

[0271] [ka]

[0272] The compound (p16) (500 mg) obtained in Example 4-3 was dissolved in toluene (10 g) at 30° C. and dehydrated azeotropically under reduced pressure. The concentrate was then dissolved in chloroform (3.0 g), and N-hydroxyphthalimide (7.2 mg), triphenylphosphine (36 mg), and diisopropyl azodicarboxylate (24 mg) were added and reacted at room temperature under a nitrogen atmosphere for 4 hours. After the reaction was completed, methanol (10 mg) was added to the reaction solution and stirred at 25° C. for 30 minutes, and then ethylenediamine monohydrate (20 mg) was added and reacted at 40° C. under a nitrogen atmosphere for 1 hour. After the reaction was completed, the reaction solution was diluted with toluene (50 g), hexane (30 g) was added, and the mixture was stirred at room temperature for 30 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (20 g) containing BHT (4 mg), suction filtration using 5A filter paper, and vacuum dried to obtain the above compound (p17). Yield 263 mg. HPLC: Purity of oxyamine was 90%. 1 H-NMR(d6-DMSO): 1.54ppm(m, 4H, -NH-CO-CH(NH)- CH2-CH2-), 1.62ppm (m, 8H), 1.97ppm (m, 4H, -NH-CO-CH(NH)-CH2- CH 2-), 2.05ppm(m, 4H, -NH-CO- CH 2 -CH 2-CH2-CO-NH-), 2.30ppm(t, 2H, -NH-CO-CH2-CH2- CH 2-CO-NH-), 2.74ppm(dd, 2H, -CO-NH-CH- CH 2-C6H5), 2.81ppm (dd, 2H, -CO-NH-CH- CH 2-C6H5), 3.11ppm(m, 22H), 3.64ppm(m, about 3,800H, -CO-NH-CH2-CH2-CH2-(O- CH 2 -CH 2)n-ONH2), 4.49ppm (m, 8H, -CO-NH-CH- CH 2-C6H5), 4.57ppm (m, 4H, -CO-NH- CH -CH2-C6H5), 7.25ppm(m, 20H, -CO-NH-CH-CH2- C 6 H 5), 7.74ppm (m, 4H), 8.44ppm (m, 4H), 8.61ppm (m, 4H)

[0273] [Example 5] Synthesis of compound (p23)

[0274]

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[0275] [Example 5-1] Synthesis of compound (p18)

[0276]

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[0277] Acetonitrile (16 g) was added to L-glutamic acid (Fmoc-Glu-OH) (18 mg) whose N-terminus was protected with a 9-fluorenylmethyloxycarbonyl group (Fmoc group) and the compound (p5) (4.0 g) obtained in Example 2-2, and the mixture was dissolved by heating at 30°C. Then, diisopropylethylamine (17 mg) and DMT-MM (36 mg) were added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 1 hour. Then, piperidine (212 mg) was added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 2 hours. After the reaction was completed, the reaction solution was diluted with toluene (150 g), and hexane (90 g) was added, and the mixture was stirred at room temperature for 30 minutes to precipitate the product. The mixture was filtered by suction using 5A filter paper, and the precipitate was collected, and then dissolved in toluene (200 g) containing BHT (40 mg), and hexane (100 g) was added, and the mixture was stirred at room temperature for 30 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (150 g) containing BHT (30 mg), suction filtration using 5A filter paper, and vacuum dried to obtain the above compound (p18). Yield: 2.7 g. HPLC: Amine purity was 91%. 1 H-NMR(d6-DMSO):0.89ppm(d,6H, -NH-CO-CH-CH2-CH( CH 3)2), 0.91ppm(d, 6H, -NH-CO-CH-CH2-CH( CH 3)2), 1.44ppm(s, 18H, -CH2-CH2-CH2-NH-CO-OC (CH 3 ) 3), 1.48 ppm (m, 2H, -NH-CO- CH -CH2-CH(CH3)2), 1.73ppm(m, 12H), 2.05ppm(m, 4H), 3.18ppm(t, 4H, -CH2-CH2- CH 2-NH-CO-OC(CH3)3), 3.64ppm(m, approx. 3,800H, -NH-CH2-CH2-CH2-O-( CH 2 -CH 2-O)n-), 4.09ppm(s, 8H), 4.44ppm(m, 2H), 4.92ppm(m, 2H), 6.76ppm(broad, 2H), 7.25ppm(m, 20H, -CO-NH-CH-CH2- C 6 H5), 8.01ppm (broad, 2H), 8.32ppm (broad, 4H), 8.70ppm (broad, 2H), 9.04ppm (broad, 4H)

[0278] [Example 5-2] Synthesis of compound (p19)

[0279] [ka]

[0280] The above compound (p19) was obtained by the same method as in Example 4-2, using L-glutamic acid (Fmoc-Glu-OH) (18 mg) whose N-terminus was protected with a 9-fluorenylmethyloxycarbonyl group (Fmoc group) and the compound (p1) (4.0 g) obtained in Example 1-1 as raw materials. Yield: 3.2 g. HPLC: Amine purity was 90%. 1 H-NMR(d6-DMSO): 1.54ppm(m, 2H, -NH-CO-CH(NH2)- CH 2-CH2-), 1.62ppm(m, 4H, -CO-NH-CH2- CH 2-CH2-), 1.97ppm(m, 2H, -NH-CO-CH(NH2)-CH2- CH 2-), 2.74ppm(dd, 2H, -CO-NH-CH- CH 2-C6H5), 2.81ppm(dd, 2H, -CO-NH-CH- CH 2-C6H5), 3.11ppm(m, 11H), 3.64ppm(m, approx. 1,900H, -CO-NH-CH2-CH2-CH2-(O- CH 2 -CH 2)n-OH), 4.49ppm(m, 4H, -CO-NH-CH- CH 2-C6H5), 4.57ppm(m, 2H, -CO-NH- CH -CH2-C6H5), 7.25ppm(m, 10H, -CO-NH-CH-CH2- C 6 H 5), 7.74ppm(m, 2H), 8.44ppm(m, 2H), 8.61ppm(m, 2H)

[0281] [Example 5-3] Synthesis of compound (p20)

[0282] [ka]

[0283] The compound (p19) (3.0 g) obtained in Example 5-2, sodium acetate (60 mg), and glutaric anhydride (86 mg) were dissolved in toluene (10 g) and reacted at 40° C. for 6 hours under a nitrogen atmosphere. After the reaction was completed, the mixture was diluted with toluene (150 g) and suction filtered using 5A filter paper. Hexane (100 g) was added to the obtained filtrate and stirred at room temperature for 30 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper. The obtained precipitate was dissolved in toluene (200 g), hexane (100 g) was added, and stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (150 g) containing BHT (30 mg), suction filtered using 5A filter paper, and vacuum dried to obtain the above compound (p20). Yield: 2.3 g. 1 H-NMR(d6-DMSO): 1.62ppm(m, 4H, -CO-NH-CH2- CH 2-CH2-(O-CH2-CH2)n-OH), 1.97ppm(m, 2H, -NH-CO-CH(NH2)-CH2- CH 2-), 2.02ppm(m, 8H), 2.30ppm(t, 2H, -CH2-CH2- CH 2-COOH), 2.74ppm(dd, 2H, -CO-NH-CH- CH 2-C6H5), 2.81ppm(dd, 2H, -CO-NH-CH- CH 2-C6H5), 3.64ppm(m, approx. 3,800H, -CO-NH-CH2-CH2-CH2-(O- CH 2 -CH 2)n-OH), 4.57ppm(m, 2H, -CO-NH- CH -CH2-C6H5), 7.25ppm(m, 10H, -CO-NH-CH-CH2- C 6 H5), 7.74ppm(m, 2H), 8.44ppm(m, 3H), 8.61ppm(m, 2H)

[0284] [Example 5-4] Synthesis of compound (p21)

[0285] [ka]

[0286] The compound (p18) (1.8 g) obtained in Example 5-1 and the compound (p20) (1.8 g) obtained in Example 5-3 were dissolved in acetonitrile, and then diisopropylethylamine (14 mg) and DMT-MM (373 mg) were added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 3 hours. After the reaction was completed, the reaction solution was diluted with toluene (200 g), and then hexane (100 g) was added and stirred at room temperature for 30 minutes to precipitate the product. The mixture was suction filtered using 5A filter paper, the precipitate was collected, and then dissolved in toluene (200 g) containing BHT (40 mg), hexane (100 g) was added, and the mixture was stirred at room temperature for 30 minutes to precipitate the product. The mixture was suction filtered using 5A filter paper, the precipitate was collected, and then washed with hexane (100 g) containing BHT (20 mg), suction filtered using 5A filter paper, and vacuum dried to obtain the above compound (p21). Yield 2.4 g. 1 H-NMR(d6-DMSO):0.89ppm(d,6H, -NH-CO-CH-CH2-CH( CH 3)2), 0.91ppm(d, 6H, -NH-CO-CH-CH2-CH( CH 3)2), 1.44ppm(s, 18H, -CH2-CH2-CH2-NH-CO-OC (CH 3 ) 3), 1.48 ppm (m, 2H, -NH-CO- CH -CH2-CH(CH3)2), 1.73ppm(m, 16H), 2.05ppm(m, 14H), 3.64ppm(m, approx. 7,600H, -CO-NH-CH2-CH2-CH2-(O- CH 2 -CH2)n-), 4.09ppm(s, 12H), 4.44ppm(m, 4H), 4.57ppm(m, 4H, -CO-NH- CH -CH2-C6H5), 6.76ppm(broad, 2H), 7.25ppm(m, 20H, -CO-NH-CH-CH2- C 6 H 5), 8.01ppm (broad, 4H), 8.32ppm (broad, 8H), 9.04ppm (broad, 6H)

[0287] [Example 5-5] Synthesis of compound (p22)

[0288] [ka]

[0289] The compound (p20) (2.0 g) obtained in Example 5-4 was dissolved in acetonitrile (20 g), p-nitrophenyl chloroformate (20 mg) and N-phenylmorpholine (20 mg) were added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 3 hours. Then, ion-exchanged water (7 mg) and N-phenylmorpholine (41 mg) were added, and the mixture was stirred, and then 3-aminopropanoic acid aqueous solution (36 mg) and 10N sodium hydroxide aqueous solution (2.5 mL) were added and reacted at room temperature for 3 hours. After the reaction was completed, toluene (100 g) was added, and the mixture was concentrated and azeotropically dehydrated. Then, the concentrated liquid was diluted with toluene (30 g), suction filtered using 5A filter paper, and hexane (30 g) was added to the filtrate and stirred at room temperature for 30 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, redissolved in toluene (50 g) containing BHT (10 mg), and hexane (30 g) was added and stirred at room temperature for 30 minutes. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (30 g) containing BHT (6 mg), suction filtration using 5A filter paper, and vacuum dried to obtain the above compound (p22). Yield: 1.3 g. 1 H-NMR(d6-DMSO):0.89ppm(d,6H, -NH-CO-CH-CH2-CH( CH3)2), 0.91 ppm (d, 6H, -NH-CO-CH-CH2-CH( CH 3)2), 1.44 ppm (s, 18H, -CH2-CH2-CH2-NH-CO-O-C (CH 3 ) 3), 1.48 ppm (m, 2H, -NH-CO- CH -CH2-CH(CH3)2), 1.73 ppm (m, 16H), 2.05 ppm (m, 14H), 2.49 ppm (t, 4H, -CO-NH-CH2- CH 2-COOH) 3.64 ppm (m, about 7,600H, -CO-NH-CH2-CH2-CH2-O-( CH 2 -CH 2-O)n-), 4.09 ppm (s, 12H), 4.44 ppm (m, 4H), 4.57 ppm (m, 4H, -CO-NH- CH -CH2-C6H5), 6.76 ppm (broad, 2H), 7.25 ppm (m, 20H, -CO-NH-CH-CH2- C 6 H 5), 8.01 ppm (broad, 4H), 8.32 ppm (broad, 8H), 9.04 ppm (broad, 6H)

[0290] [Example 5-6] Synthesis of compound (p23)

[0291] [Chemical formula]

[0292] The compound (p22) (1.0 g) obtained in Example 5-5 was dissolved in ion-exchanged water (4.5 g), 6N hydrochloric acid (0.46 g) was added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 3 hours. After the reaction was completed, 1N aqueous sodium hydroxide solution was added, the pH was adjusted to 6.5, and then sodium chloride (1.0 g) was added and dissolved. 1N aqueous sodium hydroxide solution was added to the obtained solution, the pH was adjusted to 7.10, and then chloroform (30 g) containing BHT (6 mg) was added and stirred at room temperature for 20 minutes to extract the product into the organic layer. The organic layer and the aqueous layer were separated, the organic layer was recovered, and then chloroform (30 g) containing BHT (6 mg) was added again to the aqueous layer and stirred at room temperature for 20 minutes to extract the product into the organic layer. The organic layers obtained in the first and second extractions were combined and concentrated at 40° C., and the obtained concentrate was dissolved in toluene (100 g), hexane (50 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (50 g) containing BHT (10 mg), suction filtration using 5A filter paper, and vacuum dried to obtain the above compound (p23). Yield: 532 mg. HPLC: Amine purity was 84%. 1 H-NMR(d6-DMSO): 0.89ppm(d, 6H, -NH-CO-CH-CH2-CH( CH 3)2), 0.91ppm(d, 6H, -NH-CO-CH-CH2-CH( CH 3)2), 1.48ppm(m, 2H, -NH-CO- CH -CH2-CH(CH3)2), 1.73ppm(m, 16H), 2.05ppm(m, 14H), 2.49ppm(t, 4H, -CO-NH-CH2- CH 2-COOH), 2.68ppm(t, 4H, -CH2-CH2- CH 2-NH2), 3.64ppm(m, approx. 7,600H, -CO-NH-CH2-CH2-CH2-O-( CH 2 -CH 2-O)n-), 4.09ppm(s, 12H), 4.44ppm(m, 4H), 4.57ppm(m, 4H, -CO-NH- CH -CH2-C6H5), 6.76ppm(broad, 2H), 7.25ppm(m, 20H, -CO-NH-CH-CH2-C 6 H 5), 8.01ppm (broad, 4H), 8.32ppm (broad, 8H), 9.04ppm (broad, 6H)

[0293] [Table 1]

[0294] [Example 6] Stability test in serum 1 mL of mouse or human serum was added to a 1.5 mL Eppendorf tube, and various polyethylene glycol derivatives were added to a concentration of 5.0 mg / mL. After 96 hours of incubation at 37°C, 200 μL was sampled, acetonitrile was added, and the serum was stirred for 1 minute with a vortex to precipitate proteins, and the supernatant was collected after centrifugation. Next, in order to remove hydrophobic substances such as fatty acids, hexane was added to the collected solution, which was stirred for 1 minute with a vortex, and the lower layer was collected after centrifugation. This solution was concentrated under vacuum conditions to collect the polyethylene glycol derivatives from the serum. Then, GPC analysis was performed to calculate the decomposition rate of the degradable polyethylene glycol derivatives. The decomposition rate was calculated using the following formula. Decomposition rate = (peak area% of main fraction before test - peak area% of main fraction after test) ÷ (peak area% of main fraction before test) × 100 The results are shown in Table 2 below.

[0295] [Table 2]

[0296] According to Table 2, the degradable polyethylene glycol derivatives (p2) and (p16) were not degraded in serum, similar to the non-degradable polyethylene glycol derivative methoxy PEG 40 kDa, indicating that the degradable polyethylene glycol derivatives are stable in blood.

[0297] [Example 7] Degradation test using cells 10 x 106 cells of RAW264.7 were seeded on a 100 mm dish using 10 mL of RPMI-1640 (10% FBS Pn / St) medium, and after 24 hours of culture at 37 ° C, the medium was replaced with one in which various polyethylene glycol derivatives were dissolved to a concentration of 10 mg / mL, and cultured at 37 ° C for 96 hours. After 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 for 1 minute to precipitate proteins in the cell lysate. After centrifugation, the supernatant was collected. Next, in order to remove hydrophobic substances such as fatty acids, hexane was added to the collected solution, which was stirred with a vortex for 1 minute, and the lower layer was collected after centrifugation. This solution was concentrated under vacuum conditions, and the polyethylene glycol derivatives were collected from the cells. In addition, to confirm decomposition in the medium used for cell culture, various polyethylene glycol derivatives were dissolved in the medium to a concentration of 10 mg / mL, and the cells were cultured at 37°C for 96 hours, and the polyethylene glycol derivatives were recovered using the same procedure as above. Thereafter, the various polyethylene glycol derivatives thus recovered were subjected to GPC analysis, and the decomposition rates of the degradable polyethylene glycol derivatives were calculated using the same calculation formula as in Example 7. The results are shown in Table 3 below.

[0298] [Table 3]

[0299] According to Table 3, it was confirmed that compounds (p2) and (p16), which are degradable polyethylene glycol derivatives, were effectively degraded within cells (degradation rate 99%), with (p2) degrading from a molecular weight of 40,000 to 20,000, and (p16) degrading from a molecular weight of 40,000 to 10,000. These degradable polyethylene glycol derivatives were not degraded in the medium used for cell culture, confirming that they were specifically degraded within cells. On the other hand, no degradation was observed within cells for either methoxy PEG 40 kDa, a non-degradable polyethylene glycol derivative.

[0300] [Example 8] PEGylation of salmon calcitonin (sCT) Amino acid sequence: CSNLSTCVLG KLSQELHKLQ TYPRTNTGSG TP (SEQ ID NO: 1) Salmon calcitonin (sCT) (0.5 mg, 1.5 × 10 -7 100 mM sodium borate buffer (pH 9.0) was added to the sCT solution to adjust the sCT concentration to 2.0 mg / mL, and the compound (p3) obtained in Example 1 (0.6 mg, 1.5 × 10 -8 mol) was added and reacted at 4°C for 24 hours. The reaction solution was then dialyzed against 10 mM sodium acetate buffer (pH 5.0) and purified by ion exchange chromatography using HiTrap SP HP (5 mL, GE Healthcare) to obtain PEGylated sCT. The molar yield was 39%.

[0301] RP-HPLC analysis Equipment: WATERS "ALLIANCE" Detector: UV (280 nm) Column: Inertsil WP300 C18 (GL Science) Mobile phase A: 0.05%TFA-H2O Mobile phase B: 0.05%TFA-ACN Gradient: B30% (0 min), B40% (5 min), B50% (15 min), B100% (16 min), B100% (20 min) in that order. Flow rate: 1.0mL / min Column temperature: 40℃ The purity of the PEGylated sCT was calculated under the above RP-HPLC analysis conditions. RPLC purity of PEGylated sCT: 98%

[0302] MALDI-TOF-MS analysis Equipment: Bruker "autoflex3" Sample: 0.5mg / mL, PBS solution Matrix: α-cyano-4-hydroxycinnamic acid (CHCA) saturated solution (0.01% TFA-H2O:ACN=2:1) Mix sample (1 μL) and matrix (19 μL) and spot 1 μL onto the target. The molecular weight of the PEGylated sCT was measured under the above MALDI-TOF-MS analysis conditions. Molecular weight of PEGylated sCT: 48,585 Compound (p3), a multi-arm polyethylene glycol derivative, has two active carbonate groups and can bind two molecules of sCT. It was confirmed that the molecular weight of the PEGylated sCT was increased by approximately the molecular weight of two molecules of sCT compared to the molecular weight of the raw material compound (p3).

[0303] SDS-PAGE analysis Kit: Thermo Fisher Scientific NuPAGE® Bis-Tris Precast Gel (gel concentration 4-12%) Staining solution: Coomassie Brilliant Blue solution (CBB solution) or iodine staining solution (BaCl2+I2 solution) PEGylated sCT was evaluated according to the measurement conditions recommended for the SDS-PAGE kit. PEGylated sCT showed bands in CBB staining, which selectively stains proteins and peptides, and also in iodine staining, which stains polyethylene glycol. As bands were observed in both stainings, it was confirmed that compound (p3), a polyethylene glycol derivative, was bound to sCT. [Industrial Applicability]

[0304] The degradable polyethylene glycol derivative of the present invention is a high molecular weight polyethylene glycol derivative that does not cause cellular vacuoles, can be effectively used for modifying biologically relevant substances, is stable in the blood of the living body, and is degraded within the cells.

Claims

1. Below formula (1): 【Chemistry 1】 (In the formula, n1 and n2 are each independently 45 to 950; W １ and W ２ are each independently an oligopeptide having 2 to 47 residues, the oligopeptides W 1 and W 2 are oligopeptides having glycine as the C-terminal amino acid, a1 and a2 are each independently an oligopeptide having 1 to 8, Q is a hydrocarbon chain having 2 to 12 carbon atoms which may contain an oxygen atom and / or a nitrogen atom, and X １ and X ２ are each independently a functional group capable of reacting with a biologically relevant substance, and L １ and L ２ and L ３ and L ４ and L ５ and L ６ Each of the groups independently represents a divalent spacer.

2. W １ and W ２ 2. The degradable polyethylene glycol derivative according to claim 1, wherein the oligopeptide is an oligopeptide having at least one hydrophobic neutral amino acid having a hydropathic index of 2.5 or more.

3. Below formula (2): 【Chemistry 2】 (In the formula, n3 and n4 are each independently 110 to 950; W ３ and W ４ are each independently an oligopeptide of 2 to 5 residues, the oligopeptides W 3 and W 4 are oligopeptides composed only of neutral amino acids and having glycine as the C-terminal amino acid, Q is a hydrocarbon chain having 2 to 12 carbon atoms which may contain an oxygen atom and / or a nitrogen atom, and X １ and X ２ are each independently a functional group capable of reacting with a biologically relevant substance, and L １ and L ２ and L ３ and L ４ and L ５ and L ６ Each of the groups independently represents a divalent spacer.

4. W ３ and W ４ 4. The degradable polyethylene glycol derivative according to claim 3, wherein the oligopeptide is an oligopeptide having at least one hydrophobic neutral amino acid having a hydropathic index of 2.5 or more.

5. Below formula (3): 【Chemistry 3】 (In the formula, n1 and n2 are each independently 45 to 950; W ５ and W ６ are each independently an oligopeptide of 5 to 47 residues having a symmetric structure with glutamic acid at its center, and the oligopeptides of W 5 and W 6 having a symmetric structure with glutamic acid at its center have the following structure of v1, v2, or v3: 【Chemistry 4】 【Chemistry 5】 【Chemistry 6】 (wherein Glu is a residue of glutamic acid, and Z is a degradable oligopeptide of 2 to 5 residues consisting of neutral amino acids excluding cysteine), b1 and b2 each independently are 2 to 8, Q is a hydrocarbon chain having 2 to 12 carbon atoms which may contain oxygen and / or nitrogen atoms, and X １ and X ２ are each independently a functional group capable of reacting with a biologically relevant substance, and L １ and L ２ and L ３ and L ４ and L ５ and L ６ Each of the groups independently represents a divalent spacer.

6. 6. The degradable polyethylene glycol derivative according to claim 5, wherein the degradable oligopeptide Z is an oligopeptide having glycine as the C-terminal amino acid.

7. 7. The degradable polyethylene glycol derivative according to claim 5 or 6, wherein the degradable oligopeptide Z is an oligopeptide having at least one hydrophobic neutral amino acid having a hydropathic index of 2.5 or more.

8. The degradable polyethylene glycol derivative according to any one of claims 1 to 7, which has a total molecular weight of 20,000 or more.

9. X １ and X ２ are each independently 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 vinylsulfonyl group, an acryl group, a sulfonyloxy group, a carboxyl group, a thiol group, a dithiopyridyl group, an α-haloacetyl group, an alkynyl group, an allyl group, a vinyl group, an amino group, an oxyamino group, a hydrazide group, and an azide group. The degradable polyethylene glycol derivative according to any one of claims 1 to 8.

10. Below formula (8): 【Chemistry 7】 (wherein n3 and n4 are each independently 110 to 950; W3 and W4 are each independently an oligopeptide of 2 to 5 residues; the oligopeptides of W3 and W4 are composed only of neutral amino acids and have glycine as the C-terminal amino acid; Q is a hydrocarbon chain having 2 to 12 carbon atoms which may contain an oxygen atom and a nitrogen atom; D １ and D. ２ are each independently a bio-related substance, L ３ and L ４ and L ５ and L ６ and L １１ and L １２ each independently represents a divalent spacer.) 【Chemistry 8】 (wherein n1 and n2 are each independently 45 to 950, W 5 and W 6 are each independently an oligopeptide of 5 to 47 residues having a symmetric structure with glutamic acid at its center, and the symmetric oligopeptide of W 5 and W 6 having a glutamic acid at its center has the following structure of v1, v2, or v3: 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 (wherein Glu is a residue of glutamic acid, and Z is a degradable oligopeptide of 2 to 5 residues consisting of a neutral amino acid excluding cysteine), b1 and b2 each independently are 2 to 8, Q is a hydrocarbon chain having 2 to 12 carbon atoms which may contain an oxygen atom and / or a nitrogen atom, X 1 and X 2 each independently are a functional group capable of reacting with a bio-relevant substance, and L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 each independently are a divalent spacer.

11. L １１ and L １２ The bio-related substance according to claim 10, wherein each of the groups independently represents a urethane bond, an amide bond, an ether bond, a thioether bond, a secondary amino group, a carbonyl group, a urea bond, a triazolyl group, a maleimide and mercapto bond, an oxime bond, or an alkylene group which may contain these bonds and groups.

12. The bio-related substance according to claim 10 or 11, wherein D is a hormone, a cytokine, an antibody, an aptamer or an enzyme.

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