Degradable polyethylene glycol bond

Abstract

The present application provides a bio-related substance bonded with a high molecular weight polyethylene glycol derivative that does not cause cellular cavitation. The bio-related substance is bonded with the following formula (A): (wherein, m is 1 to 7, n1 and n2 are independently 45 to 682, p is 1 to 4, R is an alkyl group with 1 to 4 carbon atoms, Z is an oligopeptide with 2 to 8 residues composed of neutral amino acids excluding cysteine, Q is a residue of a compound with 2 to 5 active hydrogens, D is a bio-related substance, L 1 , L 2 , L 3 , L 4 and L 5 Each of them is independently a single bond or a divalent spacer group, and y is 1 to 40. A bio-related substance of a degradable polyethylene glycol derivative represented by ).

Description

Technical Field

[0001] The present invention relates to a bio-related substance to which a degradable polyethylene glycol derivative that is degraded within cells is bonded. Background Art

[0002] Medicines using biological related substances such as hormones, cytokines, antibodies, enzymes, etc., are usually rapidly discharged from the body due to the glomerular filtration in the kidney, the uptake of macrophages in the liver or spleen, etc. Therefore, there are many cases where the blood half-life is short and it is difficult to obtain sufficient pharmacological effects. To solve this problem, attempts to chemically modify biological related substances by hydrophilic polymers such as sugar chains, polyethylene glycols, or albumin, etc. have been carried out. As a result, the blood half-life of biological related substances can be extended by the increase of molecular weight, the formation of hydration layers, etc. In addition, by modifying with polyethylene glycol, the effects such as the reduction of toxicity or antigenicity of biological related substances, the improvement of the solubility of poorly soluble water-soluble agents, etc. are also widely known.

[0003] Bio-related substances modified by polyethylene glycol are covered by the hydration layer formed by the ether bond of polyethylene glycol and the hydrogen bond of water molecules, and the molecular size increases, which can avoid glomerular filtration in the kidney. It is further known that the interaction between it and the opsonin and the cell surface constituting each tissue is reduced, and the migration in each tissue is reduced. Known polyethylene glycol is an excellent material that can prolong the blood half-life of bio-related substances, and its performance is that the larger the molecular weight, the higher the effect. So far, more research on bio-related substances modified with high molecular weight polyethylene glycol of molecular weight more than 40,000 has been carried out, and the result that the blood half-life can be significantly prolonged has been obtained.

[0004] Polyethylene glycol is used as the best standard in the modifier for improving the performance of biological related substances. Now there are multiple polyethylene glycol modified preparations entering the market and used in medical sites. On the other hand, the European Medicines Agency (EMA) reported in 2012 that when animals were given a biological related substance modified with a high molecular weight polyethylene glycol of more than 40,000 molecular weight for a long time with a prescribed dosage, a phenomenon of cavitation would be generated in the cells of a part of the tissue (non-patent literature 1). At present, considering that there is no report that the generation of cavitation itself has adverse effects on the human body, in addition, the dosage used in the above-mentioned EMA report is an extremely high dosage compared to the dosage usually applied in the medical site, etc., and it can be said that the safety of the therapeutic preparation modified with polyethylene glycol of more than 40,000 molecular weight currently manufactured and sold is not a problem. However, it can also be imagined that in the treatment of very special diseases (such as dwarfism, etc.), it is necessary to adopt a high dosage and a long-term treatment plan of administering polyethylene glycol modified preparations to patients. Therefore, it is expected that there is a potential demand for the development of polyethylene glycol modified preparations that can also be applied in the above-mentioned special conditions and do not produce cavitation in cells.

[0005] In Non-Patent Document 2, when a large excess of polyethylene glycol compared to the dosage of a conventional polyethylene glycol-modified preparation was administered alone to animals for a long period of time, no cavitation was observed at a molecular weight of 20,000, but cavitation was confirmed at a molecular weight of 40,000. As a means of inhibiting cavitation, it is conceivable to reduce the molecular weight of polyethylene glycol, but there is a problem that the blood half-life of the bio-related substance cannot be sufficiently improved if the molecular weight is reduced.

[0006] There are reported examples of technologies that promote the degradation of high molecular weight polyethylene glycol into low molecular weight polyethylene glycol in the body and excrete it through the kidneys.

[0007] Patent Document 1 describes a polyethylene glycol derivative having a thioether bond or peptide bond site that is cut in vivo. It describes that the polyethylene glycol derivative is decomposed in vivo to a molecular weight suitable for excretion from the kidney. However, there is no data showing specific decomposition at all, nor is there any data showing that excretion from the kidney is promoted. Furthermore, there is no description of vacuoles in cells.

[0008] Patent Document 2 describes a polyethylene glycol derivative having an acetal site that can be hydrolyzed under a low pH environment in vivo. It describes that the polyethylene glycol derivative is decomposed in vivo to a molecular weight suitable for excretion from the kidney. However, there is no data specifically promoting excretion from the kidney, and further, there is no record of vacuoles in the relevant cells. In addition, it is known that these acetal sites that can be hydrolyzed can also be slowly decomposed in the blood, and it is estimated that the blood half-life of the modified biological related substance cannot be fully improved.

[0009] On the other hand, there are reported examples of polyethylene glycol derivatives introduced with degradable oligopeptides or hydrogels that degrade in vivo in order to release drugs efficiently.

[0010] Non-patent document 3 describes a polyethylene glycol derivative having an oligopeptide site that is decomposed by an enzyme. It is reported that the oligopeptide is introduced as a linker between the anticancer agent and the polyethylene glycol, and the oligopeptide is decomposed by an enzyme specifically expressed around the tumor, and the anticancer agent can be released efficiently. The purpose is to release the anticancer agent, not to give decomposability to the polyethylene glycol for the purpose of inhibiting cell vacuoles.

[0011] Non-patent document 4 describes a hydrogel using a cross-linked molecule having an oligopeptide site that is decomposed by an enzyme and a multi-branched polyethylene glycol derivative. Among them, the oligopeptide is used as a cross-linked molecule that links the multi-branched polyethylene glycol derivative, and further, the hydrogel can be given degradability by an enzyme. Its purpose is to prepare a degradable hydrogel, not to give degradability to polyethylene glycol for the purpose of inhibiting cell vacuoles.

[0012] Patent document 3 describes a branched polyethylene glycol derivative with an oligopeptide as a skeleton. The oligopeptide is used as the basic skeleton of the polyethylene glycol derivative, rather than imparting decomposability by enzymes. In addition, the oligopeptide is characterized in that it contains amino acids such as lysine and aspartic acid having amino and carboxyl groups in the side chains, and the purpose is to synthesize a branched polyethylene glycol derivative that utilizes these amino acids for reactions. It is not a polyethylene glycol derivative intended to inhibit cellular vacuoles.

[0013] As described above, there is a need for a high molecular weight polyethylene glycol derivative that is stable in the blood, can sufficiently improve the blood half-life of the modified bio-related substance, can be specifically degraded in the cell when taken up into the cell, and can inhibit the generation of cellular vacuoles.

[0014] Prior art literature

[0015] Patent Literature

[0016] Patent Document 1: Japanese Patent Application No. 2009-527581

[0017] Patent Document 2: WO2005 / 108463

[0018] Patent Document 3: WO2006 / 088248

[0019] Non-patent literature

[0020] Non-patent literature 1: EMA / CHMP / SWP / 647258 / 2012

[0021] Non-patent document 2: Daniel G. Rudmann, et al., Toxicol. Pathol., 41, 970-983 (2013)

[0022] Non-patent document 3: Francesco M Veronese, et al., Bioconjugate Chem., 16, 775-784 (2005)

[0023] Non-patent document 4: Jiyuan Yang, et al., Marcomol. Biosci., 10(4), 445-454 (2010) Summary of the invention

[0024] Problems to be solved by the invention

[0025] The present invention aims to provide a bio-related substance that does not cause cellular cavitation and is bonded with a high molecular weight polyethylene glycol derivative. More specifically, the present invention aims to provide a bio-related substance that is stable in the blood in a living body and is modified with a degradable polyethylene glycol derivative that is degraded in cells to sufficiently improve the blood half-life.

[0026] Means for solving problems

[0027] The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have invented a bio-related substance in which a degradable polyethylene glycol derivative having an oligopeptide that is degraded within cells is bonded.

[0028] That is, the present invention is as follows.

[0029] [1] A bio-related substance, comprising a degradable polyethylene glycol derivative represented by the following formula (A) bonded thereto:

[0030]

Chemistry 1

[0031]

[0032] (wherein, m is 1 to 7, n1 and n2 are independently 45 to 682, p is 1 to 4, R is an alkyl group having 1 to 4 carbon atoms, Z is an oligopeptide having 2 to 8 residues composed of neutral amino acids excluding cysteine, Q is a residue of a compound having 2 to 5 active hydrogen atoms, D is a bio-related substance, and L 1 , L 2 , L 3 , L 4 and L 5 Each of them is independently a single bond or a divalent spacer group (spacer), and y is 1 to 40. ).

[0033] [2] The bio-related substance according to [1], which is a bio-related substance to which a degradable polyethylene glycol derivative represented by the following formula (1) is bonded:

[0034]

Chemistry 2

[0035]

[0036] (wherein, m is 1 to 7, n1 and n2 are independently 45 to 682, p is 1 to 4, R is an alkyl group having 1 to 4 carbon atoms, Z is an oligopeptide having 2 to 8 residues composed of neutral amino acids excluding cysteine, Q is a residue of a compound having 2 to 5 active hydrogen atoms, D is a bio-related substance, and L 1 , L 2 , L 3 , L 4 and L 5 are each independently a single bond or a divalent spacer group. ).

[0037] [3] The bio-related substance according to [1] or [2], wherein Q is a residue of ethylene glycol, lysine, aspartic acid, glutamic acid, glycerol, pentaerythritol, diglycerol or xylitol.

[0038] [4] The bio-related substance according to [1] or [2], wherein Q is a residue of an oligopeptide, which is a 4-8 residue oligopeptide containing any one of lysine, aspartic acid and glutamic acid, and the amino acids other than these are neutral amino acids excluding cysteine.

[0039] [5] The bio-related substance according to [4], wherein the oligopeptide Q is an oligopeptide having glycine as the amino acid at the C-terminus.

[0040] [6] The bio-related substance according to [4] or [5], wherein the oligopeptide Q is an oligopeptide having at least one hydrophobic neutral amino acid with a hydrophilic index of 2.5 or more.

[0041] [7] The bio-related substance according to any one of [1] to [6], wherein the oligopeptide Z is an oligopeptide having glycine as the amino acid at the C-terminus.

[0042] [8] The bio-related substance according to any one of [1] to [7], wherein the oligopeptide Z is an oligopeptide having at least one hydrophobic neutral amino acid with a hydrophilic index of 2.5 or more.

[0043] [9] The bio-related substance according to any one of [1] to [8], wherein the molecular weight of one molecule of the degradable polyethylene glycol derivative bonded to D is 30,000 or more.

[0044]

[10] The bio-related substance according to any one of [1] to [9], wherein L1 , L 2 , L 3 , L 4 and L 5 Each independently represents a single bond, a phenylene group, an amide bond, an ether bond, a thioether bond, a carbamate bond, a secondary amino group, a carbonyl group, a urea bond, a triazine group, a bond between maleimide and thiol, an oxime bond, or an alkylene group which may contain these bonds and groups.

[0045]

[11] The bio-related substance according to any one of [1] to

[10] , wherein the bio-related substance D is a hormone, a cytokine, an antibody, an aptamer or an enzyme.

[0046]

[12] The bio-related substance according to any one of [1] to

[11] , wherein the bio-related substance D is insulin, calcitonin, human growth hormone, interferon, granulocyte colony stimulating factor, erythropoietin or an antibody fragment.

[0047]

[13] The bio-related substance according to [2], which is a compound having Z in formula (1) bonded thereto and having Z 1 A bio-related substance of a degradable polyethylene glycol derivative represented by the following formula (2):

[0048]

Chemistry 3

[0049]

[0050] (wherein, m is 1 to 7, n1 and n2 are independently 45 to 682, p is 1 to 4, R is an alkyl group having 1 to 4 carbon atoms, and Z 1 is an oligopeptide of 2 to 6 residues composed of neutral amino acids excluding cysteine, A is a neutral amino acid excluding cysteine, a and b are independently 0 or 1, and (a+b)≥1, Q is a residue of a compound having 2 to 5 active hydrogen atoms, D is a bio-related substance, L 1 , L 2 , L 3 , L 4 and L 5 are each independently a single bond or a divalent spacer group. ).

[0051]

[14] The bio-related substance according to

[13] , wherein Q is a residue of ethylene glycol, lysine, aspartic acid, glutamic acid, glycerol, pentaerythritol, diglycerol or xylitol.

[0052]

[15] The bio-related substance according to

[13] , wherein Q is a residue of an oligopeptide, which is a 4-8 residue oligopeptide containing any one of lysine, aspartic acid and glutamic acid, and the amino acids other than these are neutral amino acids excluding cysteine.

[0053]

[16] The bio-related substance according to

[15] , wherein the oligopeptide Q is an oligopeptide having glycine as the amino acid at the C-terminus.

[0054]

[17] The bio-related substance according to

[15] or

[16] , wherein the oligopeptide Q is an oligopeptide having at least one hydrophobic neutral amino acid with a hydrophilic index of 2.5 or greater.

[0055]

[18] The bio-related substance according to any one of

[13] to

[17] , wherein Z 1 The oligopeptide is an oligopeptide having at least one hydrophobic neutral amino acid with a hydropathic index of 2.5 or more.

[0056]

[19] The bio-related substance according to any one of

[13] to

[18] , wherein the neutral amino acid in A is a hydrophobic neutral amino acid having a hydrophilic index of 2.5 or more.

[0057]

[20] The bio-related substance according to any one of

[13] to

[19] , wherein the molecular weight of one molecule of the degradable polyethylene glycol derivative bonded to D is 30,000 or more.

[0058]

[21] The bio-related substance according to any one of

[13] to

[20] , wherein L 1 , L 2 , L 3 , L 4 and L 5 Each independently represents a single bond, a phenylene group, an amide bond, an ether bond, a thioether bond, a carbamate bond, a secondary amino group, a carbonyl group, a urea bond, a triazine group, a bond between maleimide and thiol, an oxime bond, or an alkylene group which may contain these bonds and groups.

[0059]

[22] The bio-related substance according to any one of

[13] to

[21] , wherein the bio-related substance D is a hormone, a cytokine, an antibody, an aptamer or an enzyme.

[0060]

[23] The bio-related substance according to any one of

[13] to

[22] , wherein the bio-related substance D is insulin, calcitonin, human growth hormone, interferon, granulocyte colony stimulating factor, erythropoietin or an antibody fragment.

[0061]

[24] The bio-related substance according to

[13] , which is a bio-related substance to which a degradable polyethylene glycol derivative represented by the following formula (3) is bonded, wherein m in formula (2) is 1:

[0062]

Chemistry 4

[0063]

[0064] (wherein n3 and n4 are independently 45 to 682, p is 1 to 4, R is an alkyl group having 1 to 4 carbon atoms, and Z 1 is an oligopeptide of 2 to 6 residues composed of neutral amino acids excluding cysteine, A is a neutral amino acid excluding cysteine, a and b are independently 0 or 1, and (a+b)≥1, Q is a residue of a compound having 2 to 5 active hydrogen atoms, D is a bio-related substance, L 1 , L 2 , L 3 , L 4 and L 5 are each independently a single bond or a divalent spacer group. ).

[0065]

[25] The bio-related substance according to

[24] , wherein Q is a residue of ethylene glycol, lysine, aspartic acid, glutamic acid, glycerol, pentaerythritol, diglycerol or xylitol.

[0066]

[26] The bio-related substance according to

[24] , wherein Q is a residue of an oligopeptide, which is a 4-8 residue oligopeptide containing any one of lysine, aspartic acid and glutamic acid, and the amino acids other than these are neutral amino acids excluding cysteine.

[0067]

[27] The bio-related substance according to

[26] , wherein the oligopeptide Q is an oligopeptide having glycine as the amino acid at the C-terminus.

[0068]

[28] The bio-related substance according to

[26] or

[27] , wherein the oligopeptide Q is an oligopeptide having at least one hydrophobic neutral amino acid with a hydrophilic index of 2.5 or more.

[0069]

[29] The bio-related substance according to any one of

[24] to

[28] , wherein Z 1 The oligopeptide is an oligopeptide having at least one hydrophobic neutral amino acid having a hydropathic index of 2.5 or more.

[0070]

[30] The bio-related substance according to any one of

[24] to

[29] , wherein the neutral amino acid in A is a hydrophobic neutral amino acid having a hydrophilic index of 2.5 or more.

[0071]

[31] The bio-related substance according to any one of

[24] to

[30] , wherein the molecular weight of one molecule of the degradable polyethylene glycol derivative bonded to D is 30,000 or more.

[0072]

[32] The bio-related substance according to any one of

[24] to

[31] , wherein L 1 , L 2 , L 3 , L 4 and L 5Each independently represents a single bond, a phenylene group, an amide bond, an ether bond, a thioether bond, a carbamate bond, a secondary amino group, a carbonyl group, a urea bond, a triazine group, a bond between maleimide and thiol, an oxime bond, or an alkylene group which may contain these bonds and groups.

[0073]

[33] The bio-related substance according to any one of

[24] to

[32] , wherein the bio-related substance D is a hormone, a cytokine, an antibody, an aptamer or an enzyme.

[0074]

[34] The bio-related substance according to any one of

[24] to

[33] , wherein the bio-related substance D is insulin, calcitonin, human growth hormone, interferon, granulocyte colony stimulating factor, erythropoietin or an antibody fragment.

[0075]

[35] The bio-related substance according to [2], which is a residue of ethylene glycol in the formula (1), 1 A bio-related substance of a degradable polyethylene glycol derivative represented by the following formula (4), wherein CH2CH2O and p is 1:

[0076]

Chemistry 5

[0077]

[0078] (wherein, m is 1 to 7, n5 and n6 are independently 113 to 682, R is an alkyl group having 1 to 4 carbon atoms, Z is an oligopeptide of 2 to 8 residues consisting of neutral amino acids excluding cysteine, D is a bio-related substance, L 2 , L 3 , L 4 and L 5 are each independently a single bond or a divalent spacer group. ).

[0079]

[36] The bio-related substance according to

[35] , wherein the oligopeptide Z is an oligopeptide having glycine as the amino acid at the C-terminus.

[0080]

[37] The bio-related substance according to

[35] or

[36] , wherein the oligopeptide Z is an oligopeptide having at least one hydrophobic neutral amino acid with a hydropathic index of 2.5 or greater.

[0081]

[38] The bio-related substance according to any one of

[35] to

[37] , wherein the molecular weight of one molecule of the degradable polyethylene glycol derivative bonded to D is 30,000 or more.

[0082]

[39] The bio-related substance according to any one of

[35] to

[38] , wherein L 2 , L 3 , L 4 and L 5Each independently represents a single bond, a phenylene group, an amide bond, an ether bond, a thioether bond, a carbamate bond, a secondary amino group, a carbonyl group, a urea bond, a triazine group, a bond between maleimide and thiol, an oxime bond, or an alkylene group which may contain these bonds and groups.

[0083]

[40] The bio-related substance according to any one of

[35] to

[39] , wherein the bio-related substance D is a hormone, a cytokine, an antibody, an aptamer or an enzyme.

[0084]

[41] The bio-related substance according to any one of

[35] to

[40] , wherein the bio-related substance D is insulin, calcitonin, human growth hormone, interferon, granulocyte colony stimulating factor, erythropoietin or an antibody fragment.

[0085]

[42] The bio-related substance according to

[13] , which is a residue of ethylene glycol in the formula (2), 1 A bio-related substance of a degradable polyethylene glycol derivative represented by the following formula (5), wherein CH2CH2O and p is 1:

[0086]

Chemistry 6

[0087]

[0088] (wherein, m is 1 to 7, n5 and n6 are independently 113 to 682, R is an alkyl group having 1 to 4 carbon atoms, and Z 1 is an oligopeptide of 2 to 6 residues composed of neutral amino acids excluding cysteine, A is a neutral amino acid excluding cysteine, a and b are independently 0 or 1, and (a+b)≥1, D is a biologically related substance, L 2 , L 3 , L 4 and L 5 are each independently a single bond or a divalent spacer group. ).

[0089]

[43] The bio-related substance according to

[42] , wherein Z 1 The oligopeptide is an oligopeptide having at least one hydrophobic neutral amino acid having a hydropathic index of 2.5 or more.

[0090]

[44] The bio-related substance according to

[42] or

[43] , wherein the neutral amino acid in A is a hydrophobic neutral amino acid having a hydrophilic index of 2.5 or more.

[0091]

[45] The bio-related substance according to any one of

[42] to

[44] , wherein the molecular weight of one molecule of the degradable polyethylene glycol derivative bonded to D is 30,000 or more.

[0092]

[46] The bio-related substance according to any one of

[42] to

[45] , wherein L 2 , L 3 , L 4 and L 5 Each independently represents a single bond, a phenylene group, an amide bond, an ether bond, a thioether bond, a carbamate bond, a secondary amino group, a carbonyl group, a urea bond, a triazine group, a bond between maleimide and thiol, an oxime bond, or an alkylene group which may contain these bonds and groups.

[0093]

[47] The bio-related substance according to any one of

[42] to

[46] , wherein the bio-related substance D is a hormone, a cytokine, an antibody, an aptamer or an enzyme.

[0094]

[48] ​​The bio-related substance according to any one of

[42] to

[47] , wherein the bio-related substance D is insulin, calcitonin, human growth hormone, interferon, granulocyte colony stimulating factor, erythropoietin or an antibody fragment.

[0095]

[49] The bio-related substance according to

[24] , wherein Q in formula (3) is a residue of ethylene glycol, L 1 A bio-related substance of a degradable polyethylene glycol derivative represented by the following formula (6), wherein CH2CH2O and p is 1:

[0096]

Chemistry 7

[0097]

[0098] (wherein n7 and n8 are independently 226 to 682, R is an alkyl group having 1 to 4 carbon atoms, and Z 1 is an oligopeptide of 2 to 6 residues composed of neutral amino acids excluding cysteine, A is a neutral amino acid excluding cysteine, a and b are independently 0 or 1, and (a+b)≥1, D is a biologically related substance, L 2 , L 3 , L 4 and L 5 are each independently a single bond or a divalent spacer group. ).

[0099]

[50] The bio-related substance according to

[49] , wherein Z 1 The oligopeptide is an oligopeptide having at least one hydrophobic neutral amino acid having a hydropathic index of 2.5 or more.

[0100]

[51] The bio-related substance according to

[49] or

[50] , wherein the neutral amino acid in A is a hydrophobic neutral amino acid having a hydrophilic index of 2.5 or more.

[0101]

[52] The bio-related substance according to any one of

[49] to

[51] , wherein the molecular weight of one molecule of the degradable polyethylene glycol derivative bonded to D is 30,000 or more.

[0102]

[53] The bio-related substance according to any one of

[49] to

[51] , wherein L 2 , L 3 , L 4 and L 5 Each independently represents a single bond, a phenylene group, an amide bond, an ether bond, a thioether bond, a carbamate bond, a secondary amino group, a carbonyl group, a urea bond, a triazine group, a bond between maleimide and thiol, an oxime bond, or an alkylene group which may contain these bonds and groups.

[0103]

[54] The bio-related substance according to any one of

[49] to

[52] , wherein the bio-related substance D is a hormone, a cytokine, an antibody, an aptamer or an enzyme.

[0104]

[55] The bio-related substance according to any one of

[49] to

[54] , wherein the bio-related substance D is insulin, calcitonin, human growth hormone, interferon, granulocyte colony stimulating factor, erythropoietin or an antibody fragment.

[0105] Effects of the Invention

[0106] The bio-related substance to which the degradable polyethylene glycol derivative is bonded of the present invention is stable in the blood in a living body, and has oligopeptides between polyethylene glycol chains that are degraded by intracellular enzymes, so it is stable in the blood, and can give the bio-related substance a blood half-life equivalent to that of the existing non-degradable polyethylene glycol derivatives. Therefore, when it is taken up into cells, the oligopeptide sites between the polyethylene glycol chains are rapidly degraded, thereby suppressing the generation of cell vacuoles, which is an unsolved problem. BRIEF DESCRIPTION OF THE DRAWINGS

[0107]

Figure 1

[0108]

Figure 2

[0109]

Figure 3

[0110]

Figure 4

[0111]

Figure 5

[0112]

Figure 6

[0113]

Figure 7

[0114]

Figure 8

[0115]

Fig. 9

[0116]

Fig.10

[0117]

Fig.11

[0118]

Fig.12

[0119]

Fig.13

[0120]

Fig.14

[0121]

Fig.15

[0122]

Fig.16

[0123]

Fig.17

[0124]

Fig.18

[0125] The present invention is described in detail below.

[0126] The bio-related substance to which the degradable polyethylene glycol derivative is bonded according to the present invention is represented by the following formula (A).

[0127]

Chemistry 8

[0128]

[0129] (wherein, m is 1 to 7, n1 and n2 are independently 45 to 682, p is 1 to 4, R is an alkyl group having 1 to 4 carbon atoms, Z is an oligopeptide having 2 to 8 residues composed of neutral amino acids excluding cysteine, Q is a residue of a compound having 2 to 5 active hydrogen atoms, D is a bio-related substance, and L 1 , L 2 , L 3 , L 4 and L 5 Each is independently a single bond or a divalent spacer group, and y is 1 to 40. )

[0130] In the present invention, the number of molecules of the degradable polyethylene glycol derivative bonded to the bio-related substance is represented by y in formula (A), and is usually 1-40, preferably 1-20, more preferably 1-10, and particularly preferably 1.

[0131] As the effects obtained by increasing the number of molecules of the degradable polyethylene glycol derivative bonded to the bio-related substance, there are the extension of the blood half-life, the reduction of antigenicity, etc., but there is a possibility of reduced activity depending on the bio-related substance. It is known that the activity of bio-related substances such as enzymes will not be reduced even if they are bonded to multiple polyethylene glycol derivatives.

[0132] Hereinafter, the case where the number of molecules of the degradable polyethylene glycol derivative bonded to the bio-related substance is 1, that is, the case where y in formula (A) is 1, will be described.

[0133] The bio-related substance to which the degradable polyethylene glycol derivative is bonded according to the present invention is represented by the following formula (1).

[0134]

Chemistry 9

[0135]

[0136] (wherein, m is 1 to 7, n1 and n2 are independently 45 to 682, p is 1 to 4, R is an alkyl group having 1 to 4 carbon atoms, Z is an oligopeptide having 2 to 8 residues composed of neutral amino acids excluding cysteine, Q is a residue of a compound having 2 to 5 active hydrogen atoms, D is a bio-related substance, and L 1 , L 2 , L 3 , L 4 and L 5 are each independently a bond or a divalent spacer group. )

[0137] The molecular weight of one molecule of the polyethylene glycol derivative bonded to the bio-related substance D of formula (1) of the present invention is usually 4,000 to 160,000, preferably 10,000 to 120,000, and more preferably 30,000 to 80,000. In a preferred embodiment of the present invention, the molecular weight of one molecule of the polyethylene glycol derivative in formula (1) of the present invention is 30,000 or more. The molecular weight here refers to the number average molecular weight (Mn).

[0138] In formula (1), n1 and n2 are the numbers of repeating units of polyethylene glycol, and are usually independently 45 to 682, preferably independently 113 to 568, and more preferably independently 180 to 525. n1 and n2 may be different or the same.

[0139] p in formula (1) is 1 to 4. When p is 1, the polyethylene glycol derivative part in formula (1) is a straight chain type, and when p is 2 to 4, the polyethylene glycol derivative part in formula (1) is a branched type.

[0140] R in formula (1) is an alkyl group having 1 to 4 carbon atoms, and specific examples thereof include methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, etc., preferably an alkyl group having 1 to 3 carbon atoms, more preferably a methyl group or an ethyl group, and still more preferably a methyl group.

[0141] There are no particular restrictions on Z in formula (1) if it is an oligopeptide that is stable in the blood of a living body and decomposed by intracellular enzymes, but it is preferably an oligopeptide of 2 to 8 residues consisting of neutral amino acids excluding cysteine, more preferably an oligopeptide of 2 to 6 residues consisting of neutral amino acids excluding cysteine, and still more preferably an oligopeptide of 2 to 4 residues consisting of neutral amino acids excluding cysteine.

[0142] In addition, Z in formula (1) is preferably an oligopeptide composed of neutral amino acids that do not contain amino acids having amino or carboxyl groups on the side chains, specifically, lysine, aspartic acid, and glutamic acid. In the synthesis of the polyethylene glycol derivative part in formula (1) of the present invention, when the oligopeptide is introduced into the polyethylene glycol derivative, the reaction between the amino group at the N-terminus or the carboxyl group at the C-terminus of the oligopeptide and the polyethylene glycol derivative is utilized. However, if the oligopeptide contains an amino acid having an amino or carboxyl group on the side chain, the polyethylene glycol derivative is introduced not only into the amino group at the N-terminus or the carboxyl group at the C-terminus as the target, but also into the amino group or carboxyl group of the side chain. Since the impurities are difficult to remove by conventional purification steps such as extraction and crystallization, it is ideal to use an oligopeptide composed of amino acids that do not have amino or carboxyl groups on the side chains in order to obtain a target product with good purity. The amino acids used here are α-amino acids, and are basically L-type.

[0143] Furthermore, since cysteine, which is a neutral amino acid, has a thiol group and forms a disulfide bond with another thiol group, Z in formula (1) is preferably an oligopeptide composed of neutral amino acids that do not contain cysteine.

[0144] Furthermore, Z in formula (1) is preferably an oligopeptide having glycine as the amino acid at the C-terminus. When the carboxyl group at the C-terminus is reacted with a polyethylene glycol derivative, it is basically necessary to activate the carboxyl group at the C-terminus with a condensing agent or the like. It is known that in the activation process, amino acids other than glycine are prone to undergo epimerization to generate stereoisomers as byproducts. By making the amino acid at the C-terminus of the oligopeptide achiral glycine, a high-purity target product can be obtained without generating stereoisomers as byproducts.

[0145] Furthermore, Z in formula (1) 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, and isoleucine, and more preferably an oligopeptide having phenylalanine. The hydropathy index (hydropathy index) completed by Kyte and Doolittle quantitatively represents the hydrophobicity of amino acids, and the larger the value, the more hydrophobic the amino acid is (Kyte J & Doolittle RF, 1982, J Mol Biol, 157: 105-132.).

[0146] In formula (1), Z is not particularly limited as long as it is an oligopeptide of 2 to 8 residues composed of neutral amino acids excluding cysteine, which is stable in the blood of a living body and has the ability to be decomposed by an intracellular enzyme. Specific examples include glycine-phenylalanine-leucine-glycine, glycine-glycine-phenylalanine-glycine, glycine-phenylalanine-glycine, glycine-leucine-glycine, valine-citrulline-glycine, valine-alanine-glycine, glycine-glycine-glycine, phenylalanine-glycine, and the like, preferably glycine-phenylalanine- The amino acids are preferably selected from the group consisting of valine-citrulline-glycine, valine-alanine-glycine, phenylalanine-glycine, glycine-glycine-phenylalanine-glycine, glycine-glycine-leucine-glycine, glycine-phenylalanine-glycine, glycine-glycine-glycine, valine-citrulline-glycine, phenylalanine-glycine, more preferably glycine-phenylalanine-leucine-glycine, glycine-phenylalanine-glycine, valine-citrulline-glycine, phenylalanine-glycine, and still more preferably glycine-phenylalanine-leucine-glycine, valine-citrulline-glycine, phenylalanine-glycine.

[0147] Q in formula (1) is a residue of a compound having 2 to 5 active hydrogen atoms. Examples of active hydrogen atoms include hydrogen atoms such as hydroxyl, carboxyl, and amino groups (however, in the present invention, in the case of a primary amino group (-NH2), the active hydrogen atoms are counted as one). Examples of "residues of compounds having 2 active hydrogen atoms" include residues of ethylene glycol, etc., examples of "residues of compounds having 3 active hydrogen atoms" include residues of oligopeptides, lysine, aspartic acid, glutamic acid, glycerol, etc., examples of "residues of compounds having 4 active hydrogen atoms" include residues of pentaerythritol, diglycerol, etc., examples of "residues of compounds having 5 active hydrogen atoms" include residues of xylitol, etc., preferably residues of ethylene glycol, lysine, aspartic acid, glutamic acid, glycerol, pentaerythritol, diglycerol, or xylitol, or residues of oligopeptides, more preferably residues of ethylene glycol, lysine, glutamic acid, glycerol, or oligopeptides, and particularly preferably residues of ethylene glycol or lysine.

[0148] In addition, the relationship between v, which is the number of active hydrogen atoms of Q, and p, which represents the number of polyethylene glycol chain roots of the polyethylene glycol derivative portion in formula (1), is preferably v=p+1, where v=2 when p=1 and Q is a residue of ethylene glycol or the like; v=3 when p=2 and Q is a residue of glycerol, lysine, aspartic acid, glutamic acid or the like; v=4 when p=3 and Q is a residue of pentaerythritol, diglycerol or the like; and v=5 when p=4 and Q is a residue of xylitol or the like.

[0149] When Q is an oligopeptide residue, the oligopeptide 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 contains any one of lysine, aspartic acid and glutamic acid, and the amino acids other than these are composed of neutral amino acids excluding cysteine. Specific examples include glutamic acid-glycine-phenylalanine-leucine-glycine, glycine-glutamic acid-phenylalanine-leucine-glycine, glutamic acid-glycine-glycine-phenylalanine-glycine, glutamic acid-phenylalanine-glycine, glutamic acid-phenylalanine-glycine, Glutamic acid-glycine-phenylalanine-glycine, glutamate-glycine-leucine-glycine, glutamate-valine-citrulline-glycine, glutamate-valine-alanine-glycine, glutamate-phenylalanine-glycine, etc., preferably glutamate-glycine-phenylalanine-leucine-glycine, glutamate-glycine-phenylalanine-glycine, glutamate-valine-citrulline-glycine, glutamate-valine-alanine-glycine, more preferably glutamate-glycine-phenylalanine-leucine-glycine, glutamate-valine-citrulline-glycine. In addition, the oligopeptide is preferably an oligopeptide having glycine as an amino acid at the C-terminus. The oligopeptide is further preferably an oligopeptide having at least one hydrophobic neutral amino acid with a hydrophilic index of more than 2.5. In addition, for a detailed description of the oligopeptide, please refer to the description of the oligopeptide of Z above.

[0150] L in formula (1) 1 , L 2 , L 3 , L 4 and L 5 Each of them is independently a single bond or a divalent spacer group. These spacers are not particularly limited if they are groups that can form a covalent bond, but are preferably a single bond, a phenylene group, an amide bond, an ether bond, a thioether bond, a carbamate bond, a secondary amino group, a carbonyl group, a urea bond, a triazine group, a bond between maleimide and thiol, an oxime bond, or an alkylene group that can contain these bonds and groups. More preferably, they are a single bond, an amide bond, an ether bond, a thioether bond, a carbamate bond, a secondary amino group, a carbonyl group, a triazine group, a bond between maleimide and thiol, an oxime bond, or a group formed by bonding these bonds and groups with 1 to 3 alkylene groups. A particularly preferred embodiment is a substance shown in the following group (I). Ester bonds and carbonate bonds are not suitable because they are slowly decomposed in the blood of a living body.

[0151] Group (I):

[0152]

Chemistry 10

[0153]

[0154] In the formulae (z1) to (z13), s represents an integer of 0 to 10, preferably an integer of 0 to 6, and more preferably an integer of 0 to 3. In the formulae (z2) to (z13), 2 to 3 s may be the same or different.

[0155] L in formula (1) 1 , L 2 , L 3 and L 4 Preferably, they are (z1), (z2), (z3), (z4), (z5), (z6), (z7), (z8), and (z11) of the above group (I), and more preferably (z1), (z2), (z3), (z4), (z5), and (z6).

[0156] L in formula (1) 5 Preferred are (z1), (z2), (z3), (z4), (z5), (z6), (z7), (z8), (z9), (z10), (z12), and (z13) of the above group (I), more preferred are (z2), (z3), (z4), (z5), (z6), (z7), (z10), (z12), and (z13), and particularly preferred are (z3), (z5), and (z12).

[0157] In a preferred embodiment of the present invention, L 1 , L 2 , L3 , L 4 and L 5 Each independently represents a single bond, a phenylene group, a carbamate bond, an amide bond, an ether bond, a thioether bond, a secondary amino group, a carbonyl group, a urea bond, a triazine group, a bond between maleimide and thiol, an oxime bond, or an alkylene group which may contain these bonds and groups.

[0158] In other preferred embodiments of the present invention, L 1 , L 2 , L 3 , L 4 and L 5 Each is independently a single bond, an amide bond, an ether bond, a secondary amino group, a carbonyl group, or an alkylene group that may contain these bonds and groups (e.g., a single bond, -O-, -(CH2)2-, -(CH2)3-, -(CH2)2-O-, -(CH2)3-O-, -(CH2)2-CO-NH-, -(CH2)2-CO-NH-(CH2)3-, -CO-, -(CH2)5-CO-, -NH-), and more preferably, L 1 is a single bond, an ether bond, or an alkylene group which may contain an ether bond (e.g., a single bond, -O-, -(CH2)2-O-), L 2 is a carbonyl group or an alkylene group which may contain a carbonyl group (e.g., -CO-, -(CH2)5-CO-), L 3 is a secondary amino group (-NH-), L 4 is an alkylene group which may contain an ether bond (e.g., -(CH2)3-O-), L 5 It is an alkylene group which may contain a single bond or an amide bond (e.g., a single bond, -(CH2)2-, -(CH2)3-, -(CH2)2-CO-NH-, -(CH2)2-CO-NH-(CH2)3-).

[0159] In formula (1), m is the number of repeating units when the bonded product of the polyethylene glycol chain and the oligopeptide is one structural unit, and is preferably 1-7, more preferably 1-5, and even more preferably 1-3.

[0160] D in formula (1) is a biologically related substance, which is not particularly limited and is a substance related to the diagnosis, cure, alleviation, treatment or prevention of diseases 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.

[0161] More specifically, as cytokines, interferon type I, II, and III, interleukins, tumor necrosis factors, and their receptor antagonists can be cited as immunomodulatory interferons. As growth factors, erythropoietin as a hematopoietic growth factor, granulocyte colony stimulating factor (GCSF) as a stimulating factor can be cited, and as coagulation factors, factor V, factor VII, factor VIII, factor IX, factor X, and factor XII can be cited. As hormones, calcitonin, insulin, its analogs, agonist peptides, GLP-1, and somatostatin, human growth hormone, etc. can be cited. As antibodies, antibody fragments as fragments thereof can include Fab, svFV, etc., as aptamers, DNA aptamers, RNA aptamers, etc. can be cited, and as enzymes, superoxide dismutase, uricase, etc. can be cited. It is ideal that these proteins have low stability in the blood and are modified with polyethylene glycol to extend the blood half-life. Suitable proteins include interferon, interleukin, erythropoietin, GCSF, factor VIII, factor IX, calcitonin, insulin, agonist peptides, human growth hormone, and antibody fragments. More preferred proteins include insulin, calcitonin, human growth hormone, interferon, GCSF, erythropoietin, and antibody fragments (especially Fab). Further preferred proteins include calcitonin (especially salmon calcitonin), insulin, human growth hormone, and GCSF. Especially preferred proteins include calcitonin (especially salmon calcitonin), human growth hormone, and GCSF.

[0162] The following formula (2) represents a bio-related substance to which a polyethylene glycol derivative is bonded in a suitable manner. The bio-related substance to which a polyethylene glycol derivative is bonded in a suitable manner is a bio-related substance to which a polyethylene glycol derivative of formula (1) is bonded, wherein the oligopeptide Z is an oligopeptide having glycine as a C-terminal amino acid, i.e., Z in formula (1) is Z 1 , A and glycine residues.

[0163]

Chemistry 11

[0164]

[0165] (Where Z 1 is an oligopeptide of 2 to 6 residues consisting of neutral amino acids excluding cysteine, A is a neutral amino acid excluding cysteine, a and b are independently 0 or 1, and (a+b) ≥ 1, m, n1 and n2, p, R, Q, D, L 1 , L 2 , L 3 , L 4 and L 5 Same meaning as above.)

[0166] The molecular weight of one molecule of the polyethylene glycol derivative bonded to the bio-related substance D of formula (2) of the present invention is usually 4,000 to 160,000, preferably 10,000 to 120,000, and more preferably 30,000 to 80,000. In a preferred embodiment of the present invention, the molecular weight of one molecule of the polyethylene glycol derivative in formula (2) of the present invention is 30,000 or more. The molecular weight here refers to the number average molecular weight (Mn).

[0167] Z in formula (2) 1 It is an oligopeptide of 2 to 6 residues consisting of neutral amino acids excluding cysteine, preferably an oligopeptide of 2 to 4 residues consisting of neutral amino acids excluding cysteine, and more preferably an oligopeptide of 2 to 3 residues consisting of neutral amino acids excluding cysteine. In addition, it 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, and isoleucine, and more preferably an oligopeptide having phenylalanine.

[0168] Z in formula (2) 1 It is an oligopeptide that is stable in the blood of a living body and is decomposed by an enzyme in the cell. Specific examples include glycine-phenylalanine-leucine, glycine-glycine-phenylalanine, glycine-phenylalanine, valine-citrulline, valine-alanine, and glycine-glycine. Preferably, glycine-phenylalanine-leucine, glycine-glycine-phenylalanine, glycine-phenylalanine, glycine-glycine, valine-citrulline, and valine-alanine are present. More preferably, glycine-phenylalanine-leucine, glycine-phenylalanine, valine-citrulline, and valine-alanine are present. Still more preferably, glycine-phenylalanine-leucine and valine-citrulline are present.

[0169] A in formula (2) is a neutral amino acid excluding cysteine, preferably a hydrophobic neutral amino acid having a hydrophilic index of 2.5 or more, specifically phenylalanine, leucine, valine, isoleucine, more preferably phenylalanine and leucine.

[0170] In addition, m, n1 and n2, R, Q, D, L 1 , L 2 , L 3 , L 4 and L 5 Preferred embodiments are as described in the description of the above formula (1).

[0171] In addition, the following formula (3) represents a bio-related substance to which a polyethylene glycol derivative is bonded in an appropriate manner, that is, in the bio-related substance to which a polyethylene glycol derivative of formula (2) is bonded, m=1.

[0172]

Chemistry 12

[0173]

[0174] (where n3 and n4 are independently 45 to 682, p, R, Z 1 , A, a, b, Q, D, L 1 , L 2 , L 3 , L 4 and L 5 Same meaning as above.)

[0175] The molecular weight of one molecule of the polyethylene glycol derivative bonded to the bio-related substance D of formula (3) of the present invention is usually 4,000 to 160,000, preferably 10,000 to 120,000, and more preferably 30,000 to 80,000. In a preferred embodiment of the present invention, the molecular weight of one molecule of the polyethylene glycol derivative in formula (3) of the present invention is 30,000 or more. The molecular weight here refers to the number average molecular weight (Mn).

[0176] In formula (3), n3 and n4 are the numbers of repeating units of polyethylene glycol, and are usually independently 45 to 682, and preferably independently 113 to 568.

[0177] In addition, R, Z 1 , A, Q, D, L 1 , L 2 , L 3 , L 4 and L 5 Preferred embodiments are as described in the description of the above formulae (1) and (2).

[0178] [Biologically relevant substance to which the linear polyethylene glycol derivative of formula (1) is bonded]

[0179] The following formula (4) represents a bio-related substance to which a linear polyethylene glycol derivative is bonded in an appropriate manner. The bio-related substance to which a linear polyethylene glycol derivative is bonded in an appropriate manner is a bio-related substance to which a polyethylene glycol derivative of formula (1) is bonded, wherein Q is a residue of ethylene glycol, and L is 1 is CH2CH2O, and p is 1.

[0180]

Chemistry 13

[0181]

[0182] (where n5 and n6 are independently 113 to 682, m, R, Z, D, L 2 , L 3 , L 4 and L5 Same meaning as above.)

[0183] The molecular weight of one molecule of the polyethylene glycol derivative bonded to the bio-related substance D of formula (4) of the present invention is usually 20,000 to 120,000, preferably 25,000 to 80,000, and more preferably 30,000 to 60,000. In a preferred embodiment of the present invention, the molecular weight of one molecule of the polyethylene glycol derivative in formula (1) of the present invention is 30,000 or more. The molecular weight here refers to the number average molecular weight (Mn).

[0184] In formula (4), n5 and n6 are each independently the number of repeating units of polyethylene glycol, and are usually independently 113 to 682, and preferably independently 180 to 525. n5 and n6 may be different or the same.

[0185] In addition, m, R, Z, D, L 2 , L 3 , L 4 and L 5 Preferred embodiments are as described for the above formula (1).

[0186] In addition, the following formula (5) represents a bio-related substance to which a linear polyethylene glycol derivative is bonded in an appropriate manner. The bio-related substance to which a linear polyethylene glycol derivative is bonded in an appropriate manner is: In the bio-related substance to which a polyethylene glycol derivative of formula (2) is bonded, Q is a residue of ethylene glycol, and L is 1 is CH2CH2O, and p is 1.

[0187]

Chemistry 14

[0188]

[0189] (where n5 and n6 are independently 113 to 682, m, R, Z 1 , A, a, b, D, L 2 , L 3 , L 4 and L 5 Same meaning as above.)

[0190] In formula (5), n5 and n6 are each independently the number of repeating units of polyethylene glycol, and are usually independently 113 to 682, and preferably independently 180 to 525. n5 and n6 may be different or the same.

[0191] The molecular weight of one molecule of the polyethylene glycol derivative bonded to the bio-related substance D of formula (5) of the present invention is usually 20,000 to 60,000, preferably 25,000 to 55,000, and more preferably 30,000 to 50,000. In a preferred embodiment of the present invention, the molecular weight of one molecule of the polyethylene glycol derivative in formula (5) of the present invention is 30,000 or more. The molecular weight here refers to the number average molecular weight (Mn).

[0192] In addition, m, R, Z 1 , A, D, L 2 , L 3 , L 4 and L 5 Preferred embodiments are as described for the above formulae (1) and (2).

[0193] In addition, the following formula (6) represents a bio-related substance to which a linear polyethylene glycol derivative is bonded in an appropriate manner. The bio-related substance to which a linear polyethylene glycol derivative is bonded in an appropriate manner is a bio-related substance to which a polyethylene glycol derivative of formula (3) is bonded, wherein Q is a residue of ethylene glycol, and L is 1 is CH2CH2O, and p is 1.

[0194]

Chemistry 15

[0195]

[0196] (where n7 and n8 are independently 226 to 682, R, Z 1 , A, a, b, D, L 2 , L 3 , L 4 and L 5 Same meaning as above.)

[0197] The molecular weight of one molecule of the polyethylene glycol derivative bonded to the bio-related substance D of formula (6) of the present invention is usually 20,000 to 60,000, preferably 25,000 to 55,000, and more preferably 30,000 to 50,000. In a preferred embodiment of the present invention, the molecular weight of one molecule of the polyethylene glycol derivative in formula (3) of the present invention is 30,000 or more. The molecular weight here refers to the number average molecular weight (Mn).

[0198] In formula (6), n7 and n8 are each independently the number of repeating units of polyethylene glycol, and are usually independently 226 to 682, and preferably independently 340 to 568. n7 and n8 may be different or the same.

[0199] In addition, R, Z 1 , A, D, L2 , L 3 , L 4 and L 5 Preferred embodiments are as described for the above formulae (1) and (2).

[0200] [Bio-related substance to which the branched polyethylene glycol derivative of formula (1) is bonded]

[0201] Among the bio-related substances bonded with the polyethylene glycol derivative of formula (1), p is 2 to 4 and Q is a residue of a compound having 3 to 5 active hydrogen atoms. Such bio-related substances bonded with the polyethylene glycol derivative are bio-related substances bonded with a branched polyethylene glycol derivative in an appropriate form.

[0202] The residue of the compound having 3 to 5 active hydrogen atoms of Q is preferably a residue of lysine, aspartic acid, glutamic acid, glycerol, pentaerythritol, diglycerol or xylitol, or a residue of an oligopeptide, and is particularly preferably a residue of lysine or glutamic acid. Here, a preferred embodiment of the oligopeptide is as described for the above formula (1).

[0203] In addition, m, R, Z, D, L 1 , L 2 , L 3 , L 4 and L 5 Preferred embodiments are as described for the above formula (1).

[0204] In addition, among the bio-related substances bonded with the polyethylene glycol derivative of formula (2), p is 2 to 4 and Q is a residue of a compound having 3 to 5 active hydrogen atoms. Such bio-related substances bonded with the polyethylene glycol derivative are bio-related substances bonded with a branched polyethylene glycol derivative in an appropriate manner.

[0205] The residue of the compound having 3 to 5 active hydrogen atoms of Q is preferably a residue of lysine, aspartic acid, glutamic acid, glycerol, pentaerythritol, diglycerol or xylitol, or a residue of an oligopeptide, and is particularly preferably a residue of lysine or glutamic acid. Preferred embodiments of the oligopeptide are as described for the above formula (1).

[0206] In addition, m, R, Z 1 , A, D, L 1 , L 2 , L 3 , L 4 and L 5 Preferred embodiments are as described for the above formulae (1) and (2).

[0207] Furthermore, in the bio-related substance bonded with the polyethylene glycol derivative of formula (3), n3 and n4 are independently 113 to 682, p is 2 to 4, and Q is a residue of a compound having 3 to 5 active hydrogen atoms. Such a bio-related substance bonded with the polyethylene glycol derivative is a bio-related substance bonded with a branched polyethylene glycol derivative in a suitable manner.

[0208] n3 and n4 are each independently the number of repeating units of polyethylene glycol, and are preferably each independently 226 to 455. n3 and n4 may be different or the same.

[0209] The residue of the compound having 3 to 5 active hydrogen atoms of Q is preferably a residue of lysine, aspartic acid, glutamic acid, glycerol, pentaerythritol, diglycerol or xylitol, or a residue of an oligopeptide, and is particularly preferably a residue of lysine or glutamic acid. Here, a preferred embodiment of the oligopeptide is as described for the above formula (1).

[0210] In addition, R, Z 1 , A, D, L 1 , L 2 , L 3 , L 4 and L 5 Preferred embodiments are as described for the above formulae (1) and (2).

[0211] Preferred examples of the bio-related substance to which the polyethylene glycol derivative of formula (1) is bonded according to the present invention include the following polyethylene glycol derivatives.

[0212] [Biologically related substances bonded with polyethylene glycol derivatives (1-1)]

[0213] A bio-related substance to which a polyethylene glycol derivative of formula (1) is bonded,

[0214] m is 1 to 3;

[0215] n1 and n2 are independently 113 to 568;

[0216] p is 1 or 2;

[0217] R is an alkyl group having 1 to 3 carbon atoms (e.g., methyl);

[0218] Z is an oligopeptide of 2 to 4 residues consisting of neutral amino acids excluding cysteine ​​(e.g., glycine-phenylalanine-leucine-glycine, glycine-glycine-phenylalanine-glycine, glycine-phenylalanine-glycine, glycine-glycine-glycine, valine-citrulline-glycine, valine-alanine-glycine, phenylalanine-glycine);

[0219] Q is an ethylene glycol residue or a lysine residue;

[0220] D is calcitonin, human growth hormone, or granulocyte colony-stimulating factor;

[0221] L 1 , L 2 , L 3 , L 4 and L 5 Each independently represents a single bond, an amide bond, an ether bond, a secondary amino group, a carbonyl group, or an alkylene group which may contain these bonds and groups [preferably, each independently represents a group selected from

[0222]

Chemistry 16

[0223]

[0224] (wherein s is an integer from 0 to 6, and the two s's in (z2), (z3), (z5) and (z6) may be the same or different.) spacer groups] (for example, single bond, -O-, -(CH2)2-, -(CH2)3-, -(CH2)2-O-, -(CH2)3-O-, -(CH2)2-CO-NH-, -(CH2)2-CO-NH-(CH2)3-, -CO-, -(CH2)5-CO-, -NH-).

[0225] [Biologically related substances bonded with polyethylene glycol derivatives (1-2)]

[0226] A bio-related substance to which a polyethylene glycol derivative of formula (1) is bonded,

[0227] m is 1 to 3;

[0228] n1 and n2 are independently 113 to 568;

[0229] p is 1 or 2;

[0230] R is an alkyl group having 1 to 3 carbon atoms (e.g., methyl);

[0231] Z is an oligopeptide of 2 to 4 residues consisting of neutral amino acids excluding cysteine ​​(e.g., glycine-phenylalanine-leucine-glycine, glycine-glycine-phenylalanine-glycine, glycine-phenylalanine-glycine, glycine-glycine-glycine, valine-citrulline-glycine, valine-alanine-glycine, phenylalanine-glycine);

[0232] Q is an ethylene glycol residue or a lysine residue;

[0233] D is calcitonin, human growth hormone, or granulocyte colony-stimulating factor;

[0234] L 1is a single bond, an ether bond or an alkylene group which may contain an ether bond [preferably

[0235]

Chemistry 17

[0236]

[0237] (wherein s is an integer from 0 to 6, and the two s in (z2) may be the same or different.)] (e.g., a single bond, -O-, -(CH2)2-O-);

[0238] L 2 is a carbonyl group or an alkylene group which may contain a carbonyl group [preferably

[0239]

Chemistry 18

[0240]

[0241] (wherein, the two s may be the same or different and are integers from 0 to 6.)] (e.g., -CO-, -(CH2)5-CO-);

[0242] L 3 is a secondary amino group (-NH-);

[0243] L 4 is an alkylene group which may contain an ether bond [preferably

[0244]

Chemistry 19

[0245]

[0246] (wherein, the two s may be the same or different and are integers from 0 to 6.)] (e.g. -(CH2)3-O-);

[0247] L 5 is an alkylene group which may contain a single bond or an amide bond [preferably

[0248]

Chemistry 20

[0249]

[0250] (wherein s is an integer from 0 to 6, and the two s in (z3) may be the same or different.)] (e.g., single bond, -(CH2)2-, -(CH2)3-, -(CH2)2-CO-NH-, -(CH2)2-CO-NH-(CH2)3-).

[0251] Preferred examples of the bio-related substance to which the polyethylene glycol derivative of formula (2) is bonded according to the present invention include the following bio-related substances to which the polyethylene glycol derivative is bonded.

[0252] [Biologically related substances bonded with polyethylene glycol derivatives (2-1)]

[0253] A bio-related substance to which a polyethylene glycol derivative of formula (2) is bonded,

[0254] m is 1 to 3;

[0255] p is 1 or 2;

[0256] n1 and n2 are independently 113 to 568;

[0257] R is an alkyl group having 1 to 3 carbon atoms (e.g., methyl);

[0258] Z 1 An oligopeptide of 2 to 3 residues consisting of neutral amino acids excluding cysteine ​​(e.g., glycine-phenylalanine-leucine, glycine-glycine-phenylalanine, glycine-phenylalanine, glycine-glycine, valine-citrulline, valine-alanine);

[0259] A is phenylalanine or leucine;

[0260] a and b are independently 0 or 1, and (a+b)≥1;

[0261] Q is an ethylene glycol residue or a lysine residue;

[0262] D is calcitonin, human growth hormone, or granulocyte colony-stimulating factor;

[0263] L 1 , L 2 , L 3 , L 4 and L 5 Each independently represents a single bond, an amide bond, an ether bond, a secondary amino group, a carbonyl group, or an alkylene group which may contain these bonds and groups [preferably each independently represents a group selected from

[0264]

Chemistry 21

[0265]

[0266] (wherein s is an integer from 0 to 6, and the two s's in (z2), (z3), (z5) and (z6) may be the same or different.) spacer groups] (for example, single bond, -O-, -(CH2)2-, -(CH2)3-, -(CH2)2-O-, -(CH2)3-O-, -(CH2)2-CO-NH-, -(CH2)2-CO-NH-(CH2)3-, -CO-, -(CH2)5-CO-, -NH-).

[0267] [Biologically related substances bonded with polyethylene glycol derivatives (2-2)]

[0268] A biologically relevant substance bonded with a polyethylene glycol derivative of formula (2),

[0269] m is 1 to 3;

[0270] p is 1 or 2;

[0271] n1 and n2 are each independently 113 to 568;

[0272] R is an alkyl group having 1 to 3 carbon atoms (e.g., methyl);

[0273] Z 1 is an oligopeptide composed of 2 to 3 residues of neutral amino acids excluding cysteine (e.g., glycine - phenylalanine - leucine, glycine - glycine - phenylalanine, glycine - phenylalanine, glycine - glycine, valine - citrulline, valine - alanine);

[0274] A is phenylalanine or leucine;

[0275] a and b are each independently 0 or 1, and (a + b) ≥ 1;

[0276] Q is a residue of ethylene glycol or a residue of lysine;

[0277] D is calcitonin, human growth hormone or granulocyte colony - stimulating factor;

[0278] L 1 is a single bond, an ether bond or an alkylene group which may contain an ether bond [preferably

[0279]

Chemical formula 22

[0280]

[0281] (wherein, s is an integer from 0 to 6, and the two s in (z2) may be the same or different.) (e.g., a single bond, -O-, -(CH2)2 - O-);

[0282] L 2 is a carbonyl group or an alkylene group which may contain a carbonyl group [preferably

[0283]

Chemical formula 23

[0284]

[0285] (wherein, the two s may be the same or different and are integers from 0 to 6.) (e.g., -CO-, -(CH2)5 - CO-);

[0286] L 3 is a secondary amino group (-NH-);

[0287] L 4 is an alkylene group which may contain an ether bond [preferably

[0288]

Chemistry 24

[0289]

[0290] (wherein, the two s may be the same or different and are integers from 0 to 6.)] (e.g. -(CH2)3-O-);

[0291] L 5 is an alkylene group which may contain a single bond or an amide bond [preferably

[0292]

Chemistry 25

[0293]

[0294] (wherein s is an integer from 0 to 6, and the two s in (z3) may be the same or different.)] (e.g., single bond, -(CH2)2-, -(CH2)3-, -(CH2)2-CO-NH-, -(CH2)2-CO-NH-(CH2)3-).

[0295] Preferred examples of the bio-related substance to which the polyethylene glycol derivative of formula (3) is bonded according to the present invention include the following bio-related substances to which the polyethylene glycol derivative is bonded.

[0296] [Biologically related substances bonded with polyethylene glycol derivatives (3-1)]

[0297] A bio-related substance to which a polyethylene glycol derivative of formula (3) is bonded,

[0298] p is 1 or 2;

[0299] n3 and n4 are independently 340 to 568;

[0300] R is an alkyl group having 1 to 3 carbon atoms (e.g., methyl);

[0301] Z 1 An oligopeptide of 2 to 3 residues consisting of neutral amino acids excluding cysteine ​​(e.g., glycine-phenylalanine-leucine, glycine-glycine-phenylalanine, glycine-phenylalanine, glycine-glycine, valine-citrulline, valine-alanine);

[0302] A is phenylalanine or leucine;

[0303] a and b are independently 0 or 1, and (a+b)≥1;

[0304] Q is an ethylene glycol residue or a lysine residue;

[0305] D is calcitonin, human growth hormone, or granulocyte colony-stimulating factor;

[0306] L 1 , L 2 , L 3 , L 4 and L 5 Each independently represents a single bond, an amide bond, an ether bond, a secondary amino group, a carbonyl group, or an alkylene group which may contain these bonds and groups [preferably each independently represents a group selected from

[0307]

Chemistry 26

[0308]

[0309] (wherein s is an integer from 0 to 6, and the two s's in (z2), (z3), (z5) and (z6) may be the same or different.) spacer groups] (for example, single bond, -O-, -(CH2)2-, -(CH2)3-, -(CH2)2-O-, -(CH2)3-O-, -(CH2)2-CO-NH-, -(CH2)2-CO-NH-(CH2)3-, -CO-, -(CH2)5-CO-, -NH-).

[0310] [Biologically related substances bonded with polyethylene glycol derivatives (3-2)]

[0311] A bio-related substance to which a polyethylene glycol derivative of formula (3) is bonded,

[0312] p is 1 or 2;

[0313] n3 and n4 are independently 340 to 568;

[0314] R is an alkyl group having 1 to 3 carbon atoms (e.g., methyl);

[0315] Z 1 An oligopeptide of 2 to 3 residues consisting of neutral amino acids excluding cysteine ​​(e.g., glycine-phenylalanine-leucine, glycine-glycine-phenylalanine, glycine-phenylalanine, glycine-glycine, valine-citrulline, valine-alanine);

[0316] A is phenylalanine or leucine;

[0317] a and b are independently 0 or 1, and (a+b)≥1;

[0318] Q is an ethylene glycol residue or a lysine residue;

[0319] D is calcitonin, human growth hormone, or granulocyte colony-stimulating factor;

[0320] L 1 is a single bond, an ether bond or an alkylene group which may contain an ether bond [preferably

[0321] [Chemical Formula 27]

[0322]

[0323] (In the formula, s is an integer from 0 to 6, and the two s's in (z2) may be the same or different.) (Examples include a single bond, -O-, -(CH2)2-O-);

[0324] L 2 is a carbonyl group or an alkylene group that may contain a carbonyl group [preferably

[0325] [Chemical Formula 28]

[0326]

[0327] (In the formula, the two s's may be the same or different and are integers from 0 to 6.) (Examples include -CO-, -(CH2)5-CO-);

[0328] L 3 is a secondary amino group (-NH-);

[0329] L 4 is an alkylene group that may contain an ether bond [preferably

[0330] [Chemical Formula 29]

[0331]

[0332] (In the formula, the two s's may be the same or different and are integers from 0 to 6.) (Examples include -(CH2)3-O-);

[0333] L 5 is an alkylene group that may contain a single bond or an amide bond [preferably

[0334] [Chemical Formula 30]

[0335]

[0336] (In the formula, s is an integer from 0 to 6, and the two s's in (z3) may be the same or different.) (Examples include a single bond, -(CH2)2-, -(CH2)3-, -(CH2)2-CO-NH-, -(CH2)2-CO-NH-(CH2)3-).

[0337] As suitable examples of the biologically relevant substance bonded with the polyethylene glycol derivative of formula (4) of the present invention, the following biologically relevant substances bonded with a polyethylene glycol derivative can be cited.

[0338] [Biologically relevant substance (4-1) bonded with a polyethylene glycol derivative]

[0339] A bio-related substance to which a polyethylene glycol derivative of formula (4) is bonded,

[0340] m is 1 to 3;

[0341] n5 and n6 are independently 180 to 525;

[0342] R is an alkyl group having 1 to 3 carbon atoms (e.g., methyl);

[0343] Z is an oligopeptide of 2 to 4 residues consisting of neutral amino acids excluding cysteine ​​(e.g., glycine-phenylalanine-leucine-glycine, glycine-glycine-phenylalanine-glycine, glycine-phenylalanine-glycine, glycine-glycine-glycine, valine-citrulline-glycine, valine-alanine-glycine, phenylalanine-glycine);

[0344] D is calcitonin, human growth hormone, or granulocyte colony-stimulating factor;

[0345] L 2 , L 3 , L 4 and L 5 Each independently represents a single bond, an amide bond, an ether bond, a secondary amino group, a carbonyl group, or an alkylene group which may contain these bonds and groups [preferably each independently represents a group selected from

[0346]

Chemistry 31

[0347]

[0348] (wherein s is an integer from 0 to 6, and the two s's in (z2), (z3), (z5) and (z6) may be the same or different.) spacer groups] (for example, single bond, -O-, -(CH2)2-, -(CH2)3-, -(CH2)2-O-, -(CH2)3-O-, -(CH2)2-CO-NH-, -(CH2)2-CO-NH-(CH2)3-, -CO-, -(CH2)5-CO-, -NH-).

[0349] [Biologically related substances bonded with polyethylene glycol derivatives (4-2)]

[0350] A bio-related substance to which a polyethylene glycol derivative of formula (4) is bonded,

[0351] m is 1 to 3;

[0352] n5 and n6 are independently 180 to 525;

[0353] R is an alkyl group having 1 to 3 carbon atoms (e.g., methyl);

[0354] Z is an oligopeptide composed of 2 to 4 residues of neutral amino acids excluding cysteine (e.g., glycine - phenylalanine - leucine - glycine, glycine - glycine - phenylalanine - glycine, glycine - phenylalanine - glycine, glycine - glycine - glycine, valine - citrulline - glycine, valine - alanine - glycine, phenylalanine - glycine);

[0355] D is calcitonin, human growth hormone or granulocyte colony - stimulating factor;

[0356] L 2 is a carbonyl group or an alkylene group that may contain a carbonyl group [preferably

[0357]

Chemical Formula 32

[0358]

[0359] (wherein the two s may be the same or different and are integers from 0 to 6.) (e.g., -CO-, -(CH2)5 - CO-);

[0360] L 3 is a secondary amino group (-NH-);

[0361] L 4 is an alkylene group that may contain an ether bond [preferably

[0362]

Chemical Formula 33

[0363]

[0364] (wherein the two s may be the same or different and are integers from 0 to 6.) (e.g., -(CH2)3 - O-);

[0365] L 5 is an alkylene group that may contain a single bond or an amide bond [preferably

[0366]

Chemical Formula 34

[0367]

[0368] (wherein s is an integer from 0 to 6, and the two s in (z3) may be the same or different.) (e.g., single bond, -(CH2)2 -, -(CH2)3 -, -(CH2)2 - CO - NH -, -(CH2)2 - CO - NH -(CH2)3 -).

[0369] As suitable examples of the biologically - relevant substance bonded with the polyethylene glycol derivative of formula (5) of the present invention, the following biologically - relevant substances bonded with polyethylene glycol derivatives can be cited.

[0370] [Biologically - relevant substance (5 - 1) bonded with polyethylene glycol derivative]

[0371] A bio-related substance to which a polyethylene glycol derivative of formula (5) is bonded,

[0372] m is 1 to 3;

[0373] n5 and n6 are independently 180 to 525;

[0374] R is an alkyl group having 1 to 3 carbon atoms (e.g., methyl);

[0375] Z 1 An oligopeptide of 2 to 3 residues consisting of neutral amino acids excluding cysteine ​​(e.g., glycine-phenylalanine-leucine, glycine-glycine-phenylalanine, glycine-phenylalanine, glycine-glycine, valine-citrulline, valine-alanine);

[0376] A is phenylalanine or leucine;

[0377] a and b are independently 0 or 1, and (a+b)≥1;

[0378] D is calcitonin, human growth hormone, or granulocyte colony-stimulating factor;

[0379] L 2 , L 3 , L 4 and L 5 Each independently represents a single bond, an amide bond, an ether bond, a secondary amino group, a carbonyl group, or an alkylene group which may contain these bonds and groups [preferably each independently represents a group selected from

[0380]

Chemistry 35

[0381]

[0382] (wherein s is an integer from 0 to 6, and the two s's in (z2), (z3), (z5) and (z6) may be the same or different.) spacer groups] (for example, single bond, -O-, -(CH2)2-, -(CH2)3-, -(CH2)2-O-, -(CH2)3-O-, -(CH2)2-CO-NH-, -(CH2)2-CO-NH-(CH2)3-, -CO-, -(CH2)5-CO-, -NH-).

[0383] [Biologically related substances bonded with polyethylene glycol derivatives (5-2)]

[0384] A bio-related substance to which a polyethylene glycol derivative of formula (5) is bonded,

[0385] m is 1 to 3;

[0386] n5 and n6 are independently 180 to 525;

[0387] R is an alkyl group having 1 to 3 carbon atoms (such as methyl);

[0388] Z 1 is an oligopeptide composed of 2 to 3 residues of neutral amino acids excluding cysteine (such as glycine - phenylalanine - leucine, glycine - glycine - phenylalanine, glycine - phenylalanine, glycine - glycine, valine - citrulline, valine - alanine);

[0389] A is phenylalanine or leucine;

[0390] a and b are each independently 0 or 1, and (a + b) ≥ 1;

[0391] D is calcitonin, human growth hormone or granulocyte colony - stimulating factor;

[0392] L 2 is a carbonyl group or an alkylene group which may contain a carbonyl group [preferably

[0393]

Chemical formula 36

[0394]

[0395] (In the formula, the two s may be the same or different and are integers from 0 to 6.) (such as -CO-, -(CH2)5 - CO-);

[0396] L 3 is a secondary amino group (-NH-);

[0397] L 4 is an alkylene group which may contain an ether bond [preferably

[0398]

Chemical formula 37

[0399]

[0400] (In the formula, the two s may be the same or different and are integers from 0 to 6.) (such as -(CH2)3 - O-);

[0401] L 5 is an alkylene group which may contain a single bond or an amide bond [preferably

[0402]

Chemical formula 38

[0403]

[0404] (In the formula, s is an integer from 0 to 6, and the two s in (z3) may be the same or different.) (such as a single bond, -(CH2)2 -, -(CH2)3 -, -(CH2)2 - CO - NH -, -(CH2)2 - CO - NH -(CH2)3 -).

[0405] As suitable examples of the biologically relevant substance bonded with the polyethylene glycol derivative of formula (6), the following biologically relevant substances bonded with the polyethylene glycol derivative can be mentioned.

[0406] [Biologically relevant substance (6-1) bonded with polyethylene glycol derivative]

[0407] A biologically relevant substance bonded with the polyethylene glycol derivative of formula (6),

[0408] n7 and n8 are each independently 340 to 568;

[0409] R is an alkyl group having 1 to 3 carbon atoms (for example, methyl);

[0410] Z 1 is an oligopeptide composed of 2 to 3 residues of neutral amino acids excluding cysteine (for example, glycine-phenylalanine-leucine, glycine-glycine-phenylalanine, glycine-phenylalanine, glycine-glycine, valine-citrulline, valine-alanine);

[0411] A is phenylalanine or leucine;

[0412] a and b are each independently 0 or 1, and (a + b) ≥ 1;

[0413] D is calcitonin, human growth hormone or granulocyte colony-stimulating factor;

[0414] L 2 、L 3 、L 4 和L 5 are each independently a single bond, an amide bond, an ether bond, a secondary amino group, a carbonyl group, or an alkylene group that may contain these bonds and groups [preferably each independently a spacer group selected from

[0415]

Chemical formula 39

[0416]

[0417] (wherein s is an integer from 0 to 6, and two of (z2), (z3), (z5) and (z6) may be the same or different.) (for example, a single bond, -O-, -(CH2)2-, -(CH2)3-, -(CH2)2-O-, -(CH2)3-O-, -(CH2)2-CO-NH-, -(CH2)2-CO-NH-(CH2)3-, -CO-, -(CH2)5-CO-, -NH-).

[0418] [Biologically relevant substance (6-2) bonded with polyethylene glycol derivative]

[0419] A bio-related substance to which a polyethylene glycol derivative of formula (6) is bonded,

[0420] n7 and n8 are independently 340 to 568;

[0421] R is an alkyl group having 1 to 3 carbon atoms (e.g., methyl);

[0422] Z 1 An oligopeptide of 2 to 3 residues consisting of neutral amino acids excluding cysteine ​​(e.g., glycine-phenylalanine-leucine, glycine-glycine-phenylalanine, glycine-phenylalanine, glycine-glycine, valine-citrulline, valine-alanine);

[0423] A is phenylalanine or leucine;

[0424] a and b are independently 0 or 1, and (a+b)≥1;

[0425] D is calcitonin, human growth hormone, or granulocyte colony-stimulating factor;

[0426] L 2 is a carbonyl group or an alkylene group which may contain a carbonyl group [preferably

[0427]

Chemistry 40

[0428]

[0429] (wherein, the two s may be the same or different and are integers from 0 to 6.)] (e.g., -CO-, -(CH2)5-CO-);

[0430] L 3 is a secondary amino group (-NH-);

[0431] L 4 is an alkylene group which may contain an ether bond [preferably

[0432]

Chemistry 41

[0433]

[0434] (wherein, the two s may be the same or different and are integers from 0 to 6.)] (e.g. -(CH2)3-O-);

[0435] L 5 is an alkylene group which may contain a single bond or an amide bond [preferably

[0436]

Chemistry 42

[0437]

[0438] (wherein s is an integer from 0 to 6, and the two s in (z3) may be the same or different.)] (e.g., single bond, -(CH2)2-, -(CH2)3-, -(CH2)2-CO-NH-, -(CH2)2-CO-NH-(CH2)3-).

[0439] The bio-related substance of the present invention to which the polyethylene glycol derivative of formula (1) is bonded can be obtained by reacting a degradable polyethylene glycol derivative represented by the following formula (7) with a bio-related substance.

[0440]

Chemistry 43

[0441]

[0442] (where X is a functional group that can react with biologically related substances, m, n1 and n2, p, R, Z, Q, L 1 , L 2 , L 3 , L 4 and L 5 Same meaning as above.)

[0443] X in formula (7) is not particularly limited as long as it is a functional group that reacts with a functional group present in a biologically related substance such as a physiologically active protein, peptide, antibody, nucleic acid, etc. to be chemically modified to form a covalent bond. For example, functional groups described in “Harris, JM Poly (Ethylene Glycol) Chemistry; Plenum Press: New York, 1992”, “Hermanson, G.T. 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” can be cited.

[0444] The "functional group capable of reacting with a bio-related substance" represented by X in formula (7) is not particularly limited as long as it is a functional group that can chemically bond with a functional group such as an amino group, a thiol group, an aldehyde group, a carboxyl group, an unsaturated bond or an azido group possessed by the bio-related substance. Specifically, active ester groups, active carbonate groups, aldehyde groups, isocyanate groups, isothiocyanate groups, epoxide groups, carboxyl groups, thiol groups, maleimide groups, substituted maleimide groups, hydrazide groups, dithiopyridyl groups, substituted sulfonate groups, vinyl sulfone groups, amino groups, oxyamino groups, iodoacetamide groups, alkylcarbonyl groups, alkenyl groups, alkynyl groups, azido groups, acrylic groups, sulfonyloxy groups, α-haloacetyl groups, allyl groups, vinyl groups, etc. are preferred, and active ester groups, active carbonate groups, aldehyde groups, isocyanate groups, isothiocyanate groups, epoxide groups (epoxide The invention relates to a group comprising a maleimide group, a vinyl sulfone group, an acrylic 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 azido group, more preferably an active ester group, an active carbonate group, an aldehyde group, a maleimide group and an amino group, and particularly preferably an aldehyde group, a maleimide group and an amino group.

[0445] In a suitable embodiment, such functional group X can be classified into the following group (II), group (III), group (IV), group (V), group (VI) and group (VII).

[0446] Group (II): Functional groups that can react with amino groups in bio-related substances

[0447] The following (a), (b), (c), (d), (e), (f), (g), (j), (k)

[0448] Group (III): Functional groups that can react with thiol groups of biologically relevant substances

[0449] The following (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l)

[0450] Group (IV): Functional groups that can react with aldehyde groups in biologically relevant substances

[0451] The following (h), (m), (n), (p)

[0452] Group (V): Functional groups that can react with carboxyl groups of biologically relevant substances

[0453] The following (h), (m), (n), (p)

[0454] Group (VI): Functional groups that can react with unsaturated bonds in bio-related substances

[0455] The following (h), (m), (o)

[0456] Group (VII): Functional groups that can react with azide groups in biologically relevant substances

[0457] The following (l)

[0458]

Chemistry 44

[0459]

[0460] In the functional group (j), W in the formula represents a halogen atom such as a chlorine atom (Cl), an iodine atom (Br) or an iodine atom (I), and is preferably Br or I, and more preferably I.

[0461] In addition, in the functional group (e) and the functional group (l), Y in the formula 1 , Y 3 Each 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 methyl, ethyl, propyl, isopropyl, butyl, and tert-butyl, preferably methyl and ethyl.

[0462] 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 fluorine atoms, and specifically includes methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, hexyl, nonyl, vinyl, phenyl, benzyl, 4-methylphenyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-(trifluoromethoxy)phenyl, etc., preferably methyl, vinyl, 4-methylphenyl, and 2,2,2-trifluoroethyl.

[0463] The active ester group refers to 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.

[0464] Active carbonate group refers to a carbonate group having an alkoxy group with high leaving ability. As an alkoxy group with high leaving ability, an alkoxy group derived from nitrophenol, N-hydroxysuccinimide, pentafluorophenol, etc. can be enumerated. Active carbonate group is preferably a carbonate group having an alkoxy group derived from nitrophenol or N-hydroxysuccinimide.

[0465] The substituted maleimide group refers to a maleimide group in which a hydrocarbon group is bonded to one of the carbon atoms of the double bonds of the maleimide group. Specific examples of the hydrocarbon group include methyl, ethyl, propyl, isopropyl, butyl, and tert-butyl groups, and methyl and ethyl groups are preferred.

[0466] The substituted sulfonate group refers to a sulfonate group in which a hydrocarbon group which may contain a fluorine atom is bonded to the sulfur atom of the sulfonate group. Specific examples of the hydrocarbon group which may contain a fluorine atom include methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, hexyl, nonyl, vinyl, phenyl, benzyl, 4-methylphenyl, trifluoromethyl, 2,2,2-trifluoroethyl, and 4-(trifluoromethoxy)phenyl, and preferably methyl, vinyl, 4-methylphenyl, and 2,2,2-trifluoroethyl.

[0467] The degradable polyethylene glycol derivative of the bio-related substance bonded with the polyethylene glycol derivative used in the present invention can be produced, for example, by the route shown in the following process diagram (process diagram (I)) when it is a linear type.

[0468] Process diagram (I)

[0469]

Chemistry 45

[0470]

[0471] (In the process, PEG1 and PEG2 are polyethylene glycol derivatives, Peptide is an oligopeptide, X1, X2, and X3 are functional groups that can react with the oligopeptide of the polyethylene glycol derivative, Pro is a protecting group, and R has the same meaning as above.)

[0472] PEG1 and PEG2 in process diagram (I) are polyethylene glycol derivatives, and their molecular weights are defined by the numbers n1 and n2 of repeating units of the polyethylene glycol, that is, since n is 113 to 682, their molecular weights range from 5,000 to 30,000.

[0473] Peptide in process diagram (I) is an oligopeptide having the same meaning as the above Z. In process diagram (I), an oligopeptide in which the amino group at the N-terminus is protected by a protecting group or an oligopeptide in which the carboxyl group at the C-terminus is protected by a protecting group is used.

[0474] In process diagram (I), X1, X2, and X3 are functional groups of polyethylene glycol derivatives that can react with the carboxyl group or amino group of the peptide.

[0475] Pro in process diagram (I) is a protecting group, where the protecting group refers to a component that prevents or blocks the reaction of a functional group in a molecule that can undergo a specific chemical reaction under certain reaction conditions. The protecting group varies depending on the type of the protected functional group that can undergo a chemical reaction, 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 common books, such as "Wuts, PGM; Greene, TW Protective Groups in Organic Synthesis, 4th ed.; Wiley-Interscience: New York, 2007 (PGM Wus; TW Greene, Protective Groups in Organic Synthesis, Fourth Edition; Wiley-Interscience: New York, 2007)". In addition, the functional group protected by the protecting group can be deprotected, i.e., chemically reacted, by using reaction conditions suitable for each protecting group, and the original functional group can be regenerated. Typical deprotection conditions for protecting groups are recorded in the above-mentioned literature.

[0476] The reaction between the polyethylene glycol derivative and the oligopeptide in process diagram (I) is not particularly limited, and the polyethylene glycol derivative and the oligopeptide are bonded to each other by covalent bonds through chemical reactions. The bonding between the oligopeptide and the polyethylene glycol depends on the combination of functional groups used in the reaction, but is basically carried out via the L 1 , L 2 , L 3 , L 4 , L 5 The divalent spacer group is bonded to an alkylene group including a urethane bond or an amide bond.

[0477] Reaction A-1 in process diagram (I) is the reaction of an oligopeptide having a single terminal protected by a protecting group and a polyethylene glycol derivative having a single terminal R. By subsequent deprotection B-1, a polyethylene glycol derivative having a single terminal R and an oligopeptide having a single terminal can be obtained.

[0478] Reaction C-1 in process diagram (I) is a reaction of a polyethylene glycol derivative having a single terminal protected by a protecting group and a polyethylene glycol derivative having an oligopeptide at the terminal obtained by deprotection B-1. By subsequent deprotection D-1, a polyethylene glycol derivative having two polyethylene glycol chains and one oligopeptide linked can be obtained.

[0479] Reaction A-2 in process diagram (I) is a reaction of an oligopeptide protected by a single terminal protecting group with a polyethylene glycol derivative in which two polyethylene glycol chains obtained by deprotection D-1 and one oligopeptide are linked. By subsequent deprotection B-2, a polyethylene glycol derivative having an oligopeptide at the terminal can be obtained.

[0480] Reaction C-2 in process diagram (I) is a reaction of a polyethylene glycol derivative having a single terminal protected by a protecting group and a polyethylene glycol derivative having an oligopeptide at the terminal obtained by deprotection B-2. By subsequent deprotection D-2, a polyethylene glycol derivative having three polyethylene glycol chains and two oligopeptides connected can be obtained.

[0481] By repeating the cycle of reaction A→deprotection B→reaction C→deprotection D in process diagram (I), the degradable polyethylene glycol derivative of the present invention can be obtained.

[0482] The terminal functional group of the degradable polyethylene glycol derivative can be chemically converted as needed. A conventionally known method can be used for the reaction for converting the functional group, but it is necessary to appropriately select conditions that do not decompose the oligopeptide or the divalent spacer group.

[0483] As a typical example of the synthesis of the degradable polyethylene glycol derivative, the following process can be cited. Here, as a representative example of process diagram (I), the synthesis method of an oligopeptide using an amino group protected by a protecting group at the N-terminus is described. 6 , L 7 , L 8 , L 9 , L 10 , L 11 , L 12 With the L 1 , L 2 , L 3 , L 4 , L 5 The divalent spacer groups shown have the same meanings.

[0484]

Chemistry 46

[0485]

[0486] Reaction A-1 is a step of obtaining a polyethylene glycol derivative (5) by bonding the carboxyl group of an oligopeptide whose N-terminal amino group is protected by a protecting group to the amino group of a polyethylene glycol derivative (4) having a single terminal R by a condensation reaction.

[0487] The protecting group of the amino group at the N-terminus of the oligopeptide is not particularly limited, and examples thereof include acyl protecting groups and carbamate protecting groups, and specific examples thereof include trifluoroacetyl, 9-fluorenylmethoxycarbonyl, and tert-butoxycarbonyl.

[0488] There is no particular restriction on the condensation reaction, and it is desirable to use a reaction using a condensing agent. As a condensing agent, a condensing agent of a carbodiimide system such as dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) can be used alone, or it can be used in combination with reagents such as N-hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt). In addition, condensing agents such as HATU, HBTU, TATU, TBTU, COMU, DMT-MM with higher reactivity can also be used. In addition, in order to promote the reaction, a base such as triethylamine and dimethylaminopyridine can also be used.

[0489] Impurities generated by side reactions during the reaction or oligopeptides and condensing agents that are not consumed during the reaction and remain are preferably purified and removed. The purification is not particularly limited and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, etc.

[0490]

Chemistry 47

[0491]

[0492] Deprotection B-1 is a step of deprotecting the protecting group of the polyethylene glycol derivative (5) obtained in Reaction A-1 to obtain the polyethylene glycol derivative (6). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use a method that does not cause oligopeptide, L 6 The divalent spacer group does not decompose.

[0493] Impurities generated by side reactions during the deprotection reaction are preferably removed by purification. The purification is not particularly limited, and purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, and the like.

[0494]

Chemistry 48

[0495]

[0496] Reaction C-1 is a step of bonding the amino group of the polyethylene glycol derivative (6) obtained in Deprotection B-1 with the active ester group or active carbonate group of the polyethylene glycol derivative (7) to obtain a polyethylene glycol derivative (8) having a structure in which two polyethylene glycol chains are linked by an oligopeptide.

[0497] The polyethylene glycol derivative (7) has an amino group protected by a protecting group at one end and an active ester group or an active carbonate group at the other end. As the leaving group of the active ester group and the active carbonate group, succinimidyloxy, phthalimidyloxy, 4-nitrophenoxy, 1-imidazole, pentafluorophenoxy, benzotriazole-1-yloxy and 7-azabenzotriazole-1-yloxy can be mentioned. In addition, the polyethylene glycol derivative (7) does not necessarily have to have an activated functional group. When it has a carboxyl group at the end, a reaction using a condensing agent can be used in the same way as reaction A-1. In order to promote the reaction, a base such as triethylamine and dimethylaminopyridine can also be used. The protecting group of the polyethylene glycol derivative (7) is not particularly limited, and for example, acyl protecting groups and carbamate protecting groups can be mentioned, and specifically, trifluoroacetyl, 9-fluorenylmethoxycarbonyl and tert-butoxycarbonyl can be mentioned.

[0498] Impurities generated by side reactions during the reaction or polyethylene glycol derivatives that are not consumed during the reaction and remain are preferably purified and removed. The purification is not particularly limited and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, and the like.

[0499] As a method for removing polyethylene glycol impurities having different molecular weights and functional groups from the polyethylene glycol derivative (8), the purification techniques described in Japanese Patent Application Laid-Open Nos. 2014-208786 and 2011-79934 can be used.

[0500]

Chemistry 49

[0501]

[0502] Deprotection D-1 is a step of deprotecting the protecting group of the polyethylene glycol derivative (8) obtained in reaction C-1 to obtain a polyethylene glycol derivative (9). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use a method that does not allow the oligopeptide and L to react. 6 , L 7 , L 8 The conditions for decomposition of the divalent spacer group.

[0503] Impurities generated by side reactions during the deprotection reaction are preferably removed by purification. The purification is not particularly limited, and purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, and the like.

[0504]

Chemistry 50

[0505]

[0506] Reaction A-2 is a step of obtaining polyethylene glycol derivative (10) by bonding the carboxyl group of an oligopeptide whose N-terminal amino group is protected by a protecting group to the amino group of the polyethylene glycol derivative (9) obtained in deprotection B-1 through a condensation reaction. The reaction and purification can be carried out under the same conditions as those of reaction A-1.

[0507]

Chemistry 51

[0508]

[0509] Deprotection B-2 is a step of deprotecting the protecting group of the polyethylene glycol derivative (10) obtained in Reaction A-2 to obtain a polyethylene glycol derivative (11). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use a method that does not allow the oligopeptide and L to react. 6 , L 7 , L 8 The reaction and purification can be carried out under the same conditions as those of the deprotection B-1.

[0510]

Chemistry 52

[0511]

[0512] Reaction C-2 is a step of reacting the amino group of the polyethylene glycol derivative (11) obtained in the deprotection step B-2 to bond with the active ester group or active carbonate group of the polyethylene glycol derivative (7) to obtain a polyethylene glycol derivative (12) having a structure in which three polyethylene glycol chains are linked by two oligopeptides. The reaction and purification can be carried out under the same conditions as those of the reaction C-1.

[0513]

Chemistry 53

[0514]

[0515] Deprotection D-2 is a step of deprotecting the protecting group of the polyethylene glycol derivative (12) obtained in reaction C-2 to obtain a polyethylene glycol derivative (13). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use a method that does not allow the oligopeptide and L to react. 6 , L 7 , L 8 The reaction and purification can be carried out under the same conditions as those for the deprotection of D-1.

[0516] The above reactions are summarized as the following process diagram (II): By repeating the cycle of reaction A→deprotection B→reaction C→deprotection D, degradable polyethylene glycol derivatives (9), (13), (14), and (15) can be obtained.

[0517] Process Diagram (II)

[0518]

Chemical 54

[0519]

[0520] The degradable polyethylene glycol derivatives of (9), (13), (14), and (15) obtained in Process Diagram (II) are specifically as follows.

[0521] Polyethylene glycol derivative (9): A polyethylene glycol derivative having a structure in which two polyethylene glycol chains are connected by one oligopeptide

[0522] Polyethylene glycol derivative (13): A polyethylene glycol derivative having a structure in which three polyethylene glycol chains are connected by two oligopeptides

[0523] Polyethylene glycol derivative (14): A polyethylene glycol derivative having a structure in which four polyethylene glycol chains are connected by three oligopeptides

[0524] Polyethylene glycol derivative (15): A polyethylene glycol derivative having a structure in which five polyethylene glycol chains are connected by four oligopeptides

[0525] The obtained degradable polyethylene glycol derivatives of (9), (13), (14), and (15) have an amino group at the end, and various functional groups can be converted using this. This reaction will be described later.

[0526] In addition, by changing the protecting group of the amino group of the polyethylene glycol derivative (7) used in Reaction C-1, Reaction C-2, Reaction C-3, and Reaction C-4 in Process Diagram (II) to, for example, an acetal group (specifically, 3,3-diethoxypropyl, etc.) as a protecting group for an aldehyde group, an alkyl ester-based protecting group (specifically, methyl ester, tert-butyl ester, benzyl ester, etc.) as a protecting group for a carboxyl group, etc., degradable polyethylene glycol derivatives having different functional groups can be obtained. At this time, in Deprotection D-1, Deprotection D-2, Deprotection D-3, and Deprotection D-4, by using known methods suitable for their respective protecting groups for deprotection, the target product can be obtained.

[0527] Regarding the polyethylene glycol derivative in place of the polyethylene glycol derivative (7), for example, substances having the following structures can be cited.

[0528]

Chemical 55

[0529]

[0530]

Chemical 56

[0531]

[0532] As a different manufacturing method of the degradable polyethylene glycol derivative of the present invention, for example, it can also be manufactured through the route shown in the following Process Diagram (Process Diagram (III)).

[0533] Process Diagram (III)

[0534]

Chemistry 57

[0535]

[0536] (In the process, PEG1, PEG2, PEG3 are polyethylene glycol derivatives, Peptide is an oligopeptide, X1, X2, X3, X4, X5 are functional groups of polyethylene glycol derivatives, Pro, Pro2, Pro3 are protecting groups, and R has the same meaning as above.)

[0537] PEG1, PEG2, and PEG3 in process diagram (III) are polyethylene glycol derivatives, and their respective molecular weights are defined as n1 and n2, which are the repeating unit numbers of the polyethylene glycol. That is, since n is 113 to 682, their molecular weights range from 5,000 to 30,000.

[0538] The peptide in the process diagram (III) is an oligopeptide having the same definition as that of Z. In the process diagram (III), an oligopeptide in which the amino group at the N-terminus is protected by a protecting group or an oligopeptide in which the carboxyl group at the C-terminus is protected by a protecting group is used.

[0539] In step (III), X1, X2, X3, X4, and X5 are functional groups of polyethylene glycol derivatives that can react with the carboxyl group or amino group of the peptide.

[0540] Pro, Pro2, and Pro3 in process diagram (III) are protecting groups, and Pro in process diagram (III) is a protecting group that is stable under the deprotection conditions of Pro2, and Pro2 is a protecting group that is stable under the deprotection conditions of Pro3. The combination of these protecting groups can be selected from the protecting groups described in "Wuts, PGM; Greene, TW Protective Groups in Organic Synthesis, 4th ed.; Wiley-Interscience: New York, 2007 (PGM Wus; TW Greene, Protective Groups in Organic Synthesis, 4th ed.; Wiley-Interscience: New York, 2007)".

[0541] The reaction between the polyethylene glycol derivative and the oligopeptide in process diagram (III) is not particularly limited, and the polyethylene glycol derivative and the oligopeptide can be covalently bonded by chemical reaction. The bonding between the oligopeptide and the polyethylene glycol depends on the combination of functional groups used in the reaction, but is basically carried out via the L 1 , L 2 , L 3 , L 4, L 5 The divalent spacer group is bonded to an alkylene group including a urethane bond or an amide bond.

[0542] Reaction E in process diagram (III) is a reaction of an oligopeptide protected by a protecting group Pro3 at a single terminal and a polyethylene glycol derivative protected by a protecting group Pro2 at a single terminal. By subsequent deprotection F, only the protecting group Pro3 on the peptide side is deprotected, and a polyethylene glycol derivative having an oligopeptide at a single terminal and a protecting group Pro2 at a single terminal can be obtained.

[0543] Reaction G-1 in process diagram (III) is a reaction of a polyethylene glycol derivative having a single terminal protected by a protecting group Pro and a polyethylene glycol derivative having a single terminal oligopeptide and a single terminal protecting group Pro2 obtained by deprotection F. By the subsequent deprotection H-1, only the protecting group Pro2 on one side of the polyethylene glycol derivative is deprotected, and a polyethylene glycol derivative in which two polyethylene glycol chains and one oligopeptide are linked can be obtained.

[0544] Reaction G-2 in process diagram (III) is a reaction of a polyethylene glycol derivative having a single terminal protected by a protecting group Pro obtained by deprotection of H-1 and a polyethylene glycol derivative having a single terminal oligopeptide and a single terminal protecting group Pro2 obtained by deprotection of F. By the subsequent deprotection of H-2, only the protecting group Pro2 on one side of the polyethylene glycol derivative is deprotected, and a polyethylene glycol derivative having three polyethylene glycol chains and two oligopeptides linked thereto can be obtained.

[0545] By using, in the steps of reactions G-1, G-2, and G-3, a polyethylene glycol derivative having an oligopeptide at a single terminal and a protecting group Pro2 at a single terminal obtained by deprotection F after reaction E in step (III) as a raw material, and repeatedly performing reactions G and deprotection H, the precursor of the degradable polyethylene glycol derivative of the present invention can be efficiently obtained.

[0546] Reaction A-1 and deprotection B-1 in process diagram (III) can obtain a polyethylene glycol derivative having R at one terminal and an oligopeptide at one terminal in the same manner as in process diagram (I).

[0547] Reaction J in process diagram (III) is a reaction of a polyethylene glycol derivative (precursor) having a single terminal protected by a protecting group Pro obtained by repeating reactions G and deprotection H, and a polyethylene glycol derivative having R at a single terminal and an oligopeptide at a single terminal obtained by deprotection B-1. The degradable polyethylene glycol derivative of the present invention can be obtained by subsequent deprotection K.

[0548] The terminal functional group of the degradable polyethylene glycol derivative can be chemically converted as needed. A conventionally known method can be used for the reaction for converting the functional group, but it is necessary to appropriately select conditions that do not decompose the oligopeptide or the divalent spacer group.

[0549] As a typical example of the synthesis of the degradable polyethylene glycol derivative, the following process can be cited: Here, as a representative example of process diagram (III), a method for synthesizing an oligopeptide in which the amino group at the N-terminus is protected by a protecting group is described.

[0550]

Chemistry 58

[0551]

[0552] Reaction E is a step of obtaining a polyethylene glycol derivative (17) by bonding the carboxyl group of an oligopeptide whose N-terminal amino group is protected by a protecting group Pro3 and the amino group of a polyethylene glycol derivative (16) whose single terminal carboxyl group is protected by a protecting group Pro2 through a condensation reaction.

[0553] The protecting group Pro3 of the amino group at the N-terminus of the oligopeptide is not particularly limited, and examples thereof include acyl protecting groups and carbamate protecting groups, and specifically include trifluoroacetyl, 9-fluorenylmethoxycarbonyl, etc. Next, the protecting group Pro2 of the carboxyl group of the polyethylene glycol derivative (16) is not particularly limited, and examples thereof include tert-butyl, etc.

[0554] The reaction and purification can be carried out under the same conditions as those of the reaction A-1.

[0555]

Chemistry 59

[0556]

[0557] Deprotection F is a step of deprotecting the amino group Pro3 of the polyethylene glycol derivative (17) obtained in reaction E to obtain a polyethylene glycol derivative (18). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use a method that does not allow the protective group Pro2, oligopeptide, L 9 , L 10 For example, when Pro3 is 9-fluorenylmethoxycarbonyl and Pro2 is tert-butyl, Pro3 can be selectively deprotected by using an appropriate base compound such as piperidine. The reaction and purification can be carried out under the same conditions as those of the deprotection B-1.

[0558]

Chemistry 60

[0559]

[0560] Reaction G-1 is a step of reacting the amino group of the polyethylene glycol derivative (18) obtained in deprotection F with the active ester group or active carbonate group of the polyethylene glycol derivative (7) to bond to obtain a polyethylene glycol derivative (19) having a structure in which two polyethylene glycol chains are linked by one oligopeptide. The polyethylene glycol derivative (7) is as described above. The reaction and purification can be carried out under the same conditions as in the above-mentioned reaction C-1.

[0561]

Chemistry 61

[0562]

[0563] Deprotection H-1 is a step of deprotecting the carboxyl protecting group Pro2 of the polyethylene glycol derivative (19) obtained in reaction G-1 to obtain the polyethylene glycol derivative (20). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use a method that does not protect the protecting group Pro, oligopeptide, L 7 , L 8 , L 9 , L 10 For example, when Pro is a trifluoroacetyl group and Pro2 is a tert-butyl group, Pro2 can be selectively deprotected under the conditions of using a suitable acidic compound.

[0564] Impurities generated by side reactions during the deprotection reaction are preferably removed by purification. The purification is not particularly limited, and purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, and the like.

[0565]

Chemistry 62

[0566]

[0567] Reaction G-2 is a step of condensing the amino group of the polyethylene glycol derivative (18) obtained in deprotection F with the carboxyl group of the polyethylene glycol derivative (20) obtained in deprotection H-1 to obtain a polyethylene glycol derivative (21) having a structure in which three polyethylene glycol chains are linked by two oligopeptides. The reaction and purification can be carried out under the same conditions as those of the above reaction C-1.

[0568] As with the above reaction A-1, it is preferable to use a condensing agent for the reaction. In order to promote the reaction, a base such as triethylamine and dimethylaminopyridine may be used.

[0569] Impurities generated by side reactions during the reaction or polyethylene glycol derivatives that are not consumed during the reaction and remain are preferably purified and removed. The purification is not particularly limited and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, and the like.

[0570] As a method for removing polyethylene glycol impurities having different molecular weights or functional groups from the polyethylene glycol derivative (21), purification techniques described in Japanese Patent Application Laid-Open Nos. 2014-208786 and 2011-79934 can be used.

[0571]

Chemistry 63

[0572]

[0573] Deprotection H-2 is a step of deprotecting the carboxyl protecting group Pro2 of the polyethylene glycol derivative (21) obtained in reaction G-2 to obtain the polyethylene glycol derivative (22). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use a method that does not protect the protecting group Pro, oligopeptide, L 7 , L 8 , L 9 , L 10 The conditions for decomposition of the divalent spacer group.

[0574] The above reactions are summarized as the following process diagram (IV). Using the polyethylene glycol derivative (18) obtained in reaction E and deprotection F as a raw material, by repeating reaction G → deprotection H, for example, a polyethylene glycol derivative (23) having a structure in which four polyethylene glycol chains are linked by three oligopeptides, a polyethylene glycol derivative (24) having a structure in which five polyethylene glycol chains are linked by four oligopeptides, and the like can be obtained as an intermediate of the degradable polyethylene glycol derivative of the present invention.

[0575] Process Diagram (IV)

[0576]

Chemistry 64

[0577]

[0578] The polyethylene glycol derivatives (20), (22), (23) and (24) obtained in step (IV) specifically correspond to the following.

[0579] Polyethylene glycol derivative (20): A polyethylene glycol derivative having a structure in which two polyethylene glycol chains are connected by one oligopeptide

[0580] Polyethylene glycol derivative (22): A polyethylene glycol derivative having a structure in which three polyethylene glycol chains are connected by two oligopeptides

[0581] Polyethylene glycol derivative (23): A polyethylene glycol derivative having a structure in which four polyethylene glycol chains are connected by three oligopeptides

[0582] Polyethylene glycol derivative (24): A polyethylene glycol derivative having a structure in which five polyethylene glycol chains are connected by four oligopeptides

[0583] Next, by using the polyethylene glycol derivatives (20), (22), (23) and (24) obtained in step (IV) as starting materials, and carrying out the following reaction J and deprotection K, a degradable polyethylene glycol derivative can be obtained. The following steps show the case of using the polyethylene glycol derivative (24).

[0584]

Chemistry 65

[0585]

[0586] Reaction J is a step of condensing the amino group of the polyethylene glycol derivative (6) obtained in the deprotection B-1 with the carboxyl group of the polyethylene glycol derivative (24) obtained in the deprotection H-4 to obtain a polyethylene glycol derivative (25) having a structure in which six polyethylene glycol chains are linked by five oligopeptides. The reaction and purification can be carried out under the same conditions as in the reaction C-1.

[0587] As with the above reaction A-1, it is preferable to use a condensing agent for the reaction. In order to promote the reaction, a base such as triethylamine and dimethylaminopyridine may be used.

[0588] Impurities generated by side reactions during the reaction or polyethylene glycol derivatives that are not consumed during the reaction and remain are preferably purified and removed. The purification is not particularly limited and can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, and the like.

[0589] As a method for removing polyethylene glycol impurities having different molecular weights or functional groups from the polyethylene glycol derivative (25), purification techniques described in Japanese Patent Application Laid-Open Nos. 2014-208786 and 2011-79934 can be used.

[0590]

Chemistry 66

[0591]

[0592] Deprotection K is a step of deprotecting the protecting group of the polyethylene glycol derivative (25) obtained in reaction J to obtain a polyethylene glycol derivative (26). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use a method that does not cause oligopeptide or L 6 , L 7 , L 8 , L 9 , L 10 The reaction and purification can be carried out under the same conditions as those for the deprotection of D-1.

[0593] As a different method for producing a degradable polyethylene glycol derivative as a bio-related substance bonded with a polyethylene glycol derivative used in the present invention, for example, in the case of a branched type, it can be produced by the route shown in the following process diagram (process diagram (V)). Here, the route is to obtain a branched degradable polyethylene glycol by reacting the degradable polyethylene glycol obtained in step (I) or step (III) with Q which is a residue of a compound having 3 to 5 active hydrogen atoms.

[0594] Process diagram (V)

[0595]

Chemistry 67

[0596]

[0597] (In the process, PEG1 and PEG2 are polyethylene glycol derivatives, Peptide is an oligopeptide, Q is a residue of a compound having 3 to 5 active hydrogen atoms, X6 and X7 are functional groups, Pro is a protecting group, p is 2 to 4, and R has the same meaning as above.)

[0598] PEG1 and PEG2 in process diagram (V) are polyethylene glycol derivatives, and their respective molecular weights are defined as n1 and n2 as the repeating unit numbers of polyethylene glycol mentioned above, that is, since n is 45 to 682, their molecular weights range from 2000 to 30000.

[0599] The peptide in process diagram (V) is an oligopeptide having the same definition as that of Z described above.

[0600] Q in the process diagram (V) is a residue of a compound having 3 to 5 active hydrogen atoms and having the same definition as Q above.

[0601] In process diagram (V), X6 is a functional group of a polyethylene glycol derivative that can react with a functional group having active hydrogen of Q, such as a hydroxyl group, a carboxyl group or an amino group, and X7 is a functional group that can react with a bio-related substance bonded to Q.

[0602] The reaction of Q and the degradable polyethylene glycol derivative in process diagram (V) is not particularly limited, and Q and the degradable polyethylene glycol derivative are covalently bonded by a chemical reaction. The bonding of Q and the degradable polyethylene glycol derivative depends on the combination of functional groups used in the reaction, but is basically carried out via the L 1 , L 2 , L 3 , L 4 , L 5 The divalent spacer group is bonded to an alkylene group including a urethane bond or an amide bond, etc.

[0603] Reaction L in process diagram (V) is a reaction of Q, which is a residue of a compound having 3 to 5 active hydrogen atoms, in which one functional group having active hydrogen atoms is protected by a protecting group Pro, with a degradable polyethylene glycol derivative having a single terminal R obtained in the above process (I) or process (III). By subsequent deprotection M, the protecting group Pro of Q can be deprotected to obtain a branched degradable polyethylene glycol derivative. The terminal functional group of the degradable polyethylene glycol derivative can be chemically converted as needed. The reaction for the functional group conversion can use a conventionally known method, but it is necessary to appropriately select conditions that do not decompose the oligopeptide and the divalent spacer group.

[0604] As a typical example of the synthesis of the degradable polyethylene glycol derivative, the following process can be cited: Here, as a representative example of process diagram (V), a synthesis method in the case of using a glutamic acid residue of a compound having three active hydrogen atoms as Q is described.

[0605]

Chemistry 68

[0606]

[0607] Reaction L is, for example, a step of bonding the amino group of the polyethylene glycol derivative (9) obtained in deprotection D-1 with two carboxyl groups of a glutamic acid derivative protected by an amino group protecting group by a condensation reaction to obtain a branched polyethylene glycol derivative (27) having a structure in which two degradable polyethylene glycol chains are connected by glutamic acid residues. The reaction and purification can be carried out under the same conditions as those of reaction G-2.

[0608]

Chemistry 69

[0609]

[0610] Deprotection M is a step of deprotecting the protecting group of the polyethylene glycol derivative (27) obtained in reaction L to obtain a polyethylene glycol derivative (28). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use a method that does not cause the oligopeptide or L to be protected. 6 , L 7 , L 8 The reaction and purification can be carried out under the same conditions as those for the deprotection of D-1.

[0611] As another method for producing the branched degradable polyethylene glycol derivative of the present invention, for example, it can be produced by the route shown in the following process diagram (process diagram (VI)). This is a route for obtaining a branched degradable polyethylene glycol by reacting the polyethylene glycol derivative having a single peptide at one end obtained in steps (I) and (III) with a branched polyethylene glycol derivative in which polyethylene glycol is bonded to Q, which is a residue of a compound having 3 to 5 active hydrogen atoms.

[0612] Process Diagram (VI)

[0613]

Chemistry 70

[0614]

[0615] (In the steps, PEG1, PEG2, and PEG3 are polyethylene glycol derivatives, Peptide is an oligopeptide, Q is a residue of a compound having 3 to 5 active hydrogen atoms, X8 and X9 are functional groups, Pro is a protecting group, p is 2 to 4, and R has the same meaning as above.)

[0616] PEG1, PEG2, and PEG3 in process diagram (VI) are polyethylene glycol derivatives, and their molecular weights are defined by the numbers n1 and n2 of repeating units of the polyethylene glycol, that is, since n is 45 to 682, their molecular weights range from 2000 to 30000.

[0617] The peptide in process diagram (VI) is an oligopeptide having the same definition as that of Z described above.

[0618] In process diagram (VI), X8 is a functional group of a polyethylene glycol derivative that can react with a carboxyl group or an amino group of a peptide, and X9 is a functional group that can react with a bio-related substance bonded to Q.

[0619] There are no particular restrictions on the reaction of the polyethylene glycol derivative having a peptide at a single terminal in process diagram (VI) with the branched polyethylene glycol derivative in which polyethylene glycol is bonded to Q, which is a residue of a compound having 3 to 5 active hydrogen atoms. These polyethylene glycol derivatives are bonded by covalent bonds through chemical reactions. The bonding of the branched polyethylene glycol derivative and the degradable polyethylene glycol derivative depends on the combination of functional groups used in the reaction, but is basically carried out via the L 1 , L 2 , L 3 , L 4 , L 5 The divalent spacer group is bonded to an alkylene group including a urethane bond or an amide bond, etc.

[0620] The reaction of a branched polyethylene glycol derivative in which one functional group having an active hydrogen of Q, which is a residue of a compound having 3 to 5 active hydrogens, is protected by a protecting group Pro with the polyethylene glycol derivative having a peptide at one end and R at the other end obtained in the above step (I) or step (III). By subsequent deprotection of P, the protecting group Pro of Q can be deprotected to obtain a branched degradable polyethylene glycol derivative. The terminal functional groups of the degradable polyethylene glycol derivative can be chemically transformed as needed. Reactions for such functional group transformation can use publicly known methods, but conditions that do not decompose the oligopeptide and the divalent spacer group must be appropriately selected.

[0621] As a typical example of synthesizing the degradable polyethylene glycol derivative, the following steps can be cited. Here, as a representative example of step diagram (VI), a synthesis method using a branched polyethylene glycol derivative with Q being a glycerol residue as a raw material will be described.

[0622]

Chemical formula 71

[0623]

[0624] Reaction N is a step of bonding the two carboxyl groups of a branched polyethylene glycol derivative (29) obtained, for example, according to the examples of Patent Document JP-A-2012-25932, to the amino group of the polyethylene glycol derivative (6) obtained in deprotection B-1 by a condensation reaction to obtain a branched polyethylene glycol derivative (30). The reaction and purification can be carried out under the same conditions as in the above reaction G-2.

[0625]

Chemical formula 72

[0626]

[0627] Deprotection P is a step of deprotecting the protecting group of the polyethylene glycol derivative (30) obtained in reaction N to obtain a polyethylene glycol derivative (31). As the deprotection reaction, publicly known methods can be used, but conditions that do not decompose the oligopeptide or the divalent spacer group of L 6 、L 11 、L 12 need to be used. The reaction and purification can be carried out under the same conditions as in the above deprotection D-1.

[0628] Next, the polyethylene glycol derivatives obtained in deprotection D, deprotection K, deprotection M, and deprotection P have an amino group at the terminal, and various functional groups can be converted using this.

[0629] There are no particular restrictions on the process of converting the amino group at the end of the polyethylene glycol derivative into other functional groups. Basically, by using a compound having an active ester group capable of reacting with the amino group, or common reaction reagents such as acid anhydrides and acyl chlorides, it can be easily converted into various functional groups.

[0630] For example, when it is desired to convert the amino group at the end of the polyethylene glycol derivative into a maleimide group, the target product can be obtained by reacting it with the following reagent.

[0631]

Chemical Formula 73

[0632]

[0633] For example, when it is desired to convert the amino group at the end of the polyethylene glycol derivative into a carboxyl group, the target product can be obtained by reacting it with succinic anhydride or glutaric anhydride.

[0634] These reaction reagents are low-molecular-weight reagents. Due to the large difference in solubility with the polyethylene glycol derivative, they can be easily removed by common purification methods such as extraction and crystallization.

[0635] It is required that the degradable polyethylene glycol obtained through the above-described process has the property of being stable in the blood and decomposing only inside cells. To appropriately evaluate its performance, for example, the following tests can be carried out to evaluate the stability of the degradable polyethylene glycol in the blood and its degradability inside cells.

[0636] There are no particular restrictions on the test method for evaluating the stability of the degradable polyethylene glycol derivative in the blood. For example, tests using sera of mice, rats, humans, etc. can be cited. Specifically, by dissolving the polyethylene glycol derivative in serum to a concentration of 1 to 10 mg / mL, incubating at 37 °C for 96 hours, recovering the polyethylene glycol derivative contained in the serum, and measuring GPC, the decomposition rate can be evaluated. The decomposition rate is calculated from the peak area percentage of the GPC main fraction of the polyethylene glycol derivative before the stability test and the peak area percentage of the GPC main fraction of the polyethylene glycol derivative after the stability test. Specifically, the following formula is used.

[0637] Decomposition rate = (peak area percentage before the test - peak area percentage after the test) ÷ peak area percentage before the test × 100

[0638] For example, when the peak area percentage of the GPC main fraction of the degradable polyethylene glycol derivative before the stability test is 95% and the peak area percentage of the GPC main fraction after the test is 90%, the decomposition rate is calculated as follows.

[0639] Decomposition rate = (95 - 90) ÷ 95 × 100 = 5.26 (%)

[0640] If the degradable polyethylene glycol derivative is degraded in the blood, the target blood half-life cannot be achieved. Therefore, in a stability test, the decomposition rate after 96 hours is preferably 10% or less, more preferably 5% or less.

[0641] There are no particular restrictions on the test method for evaluating the degradability of degradable polyethylene glycol derivatives in cells, and examples thereof include tests using a culture medium containing degradable polyethylene glycol derivatives to culture cells. There are no particular restrictions on the cells and culture medium used here, but specifically, the polyethylene glycol derivative can be dissolved in the culture medium RPMI-1640 to a concentration of 1 to 20 mg / mL, and macrophages RAW264.7 can be cultured at 37°C for 96 hours using the culture medium, and then the polyethylene glycol derivative in the cells can be recovered and GPC can be measured to evaluate the decomposition rate. As with the stability test, the decomposition rate can be calculated using the peak area % of the GPC main fraction of the polyethylene glycol derivative before and after the test.

[0642] For example, assuming that the peak area % of the GPC main fraction of the degradable polyethylene glycol derivative before the degradability test using cells is 95% and the peak area % of the GPC main fraction after the test is 5%, the decomposition rate is calculated as follows.

[0643] Decomposition rate = (95-5) ÷ 95 × 100 = 94.7 (%)

[0644] If the degradable polyethylene glycol derivative is not efficiently degraded in cells, vacuolation of target cells cannot be suppressed. Therefore, the degradability rate after 96 hours in the degradability test is preferably 90% or more, more preferably 95% or more.

[0645] There are no particular limitations on the method for bonding the obtained degradable polyethylene glycol derivative to a bio-related substance, and for example, the method described in "Hermanson, GT Bioconjugate Techniques, 3rd ed.; Academic Press: San Diego, CA, 2013" or "Mark, Sonny S. Bioconjugate protocols, strategies and methods; 2011" can be used. Among these, for example, when the amino group of a lysine residue of a protein, peptide, etc., which is a bio-related substance, is used as a target, a polyethylene glycol derivative having an activated ester group or an activated carbonate group is used. In addition, when the thiol group of a cysteine ​​residue of a protein, peptide, etc., which is a bio-related substance, is used as a target, a polyethylene glycol derivative having a maleimide group or an iodoacetamide group is used. Since the number of free cysteine ​​residues contained in natural bio-related substances is extremely small, this method can further selectively bond polyethylene glycol to the bio-related substances. Furthermore, as a method for generating or introducing thiol groups, there are methods such as cutting disulfide bonds of bio-related substances and introducing cysteine ​​residues by genetically modifying bio-related substances. It is known that by combining these techniques, a target number of polyethylene glycol derivatives can be bonded to a target site of a bio-related substance.

[0646] Next, when the amino group at the N-terminus of a bio-related substance such as a protein or peptide is used as a target, a polyethylene glycol derivative having an aldehyde group is used. Specifically, by using a polyethylene glycol derivative having an aldehyde group and a suitable reducing agent in a low pH buffer solution, the polyethylene glycol derivative can be selectively bonded to the amino group at the N-terminus of a protein or peptide.

[0647] The bio-related substances bonded with these polyethylene glycol derivatives can be purified by dialysis, gel permeation chromatography (GPC), ion exchange chromatography (IEC) and the like, which are known as conventional methods. In addition, the bio-related substances obtained can usually be evaluated by analytical methods such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), polyacrylamide gel electrophoresis (SDS-PAGE), and reverse phase chromatography (RPLC).

[0648] There is no particular limitation on the method for evaluating the physiological activity of the bio-related substance to which the degradable polyethylene glycol derivative is bonded. For example, if the bio-related substance is insulin, the blood sugar concentration is measured, and if it is calcitonin, the blood calcium concentration is measured. The evaluation can be performed by periodically sampling blood from animals after administration and measuring the substance in the blood with a suitable analytical instrument. Specifically, if it is insulin, a glucose assay kit can be used to monitor the decrease in glucose concentration after administration, and if it is calcitonin, a calcium assay kit can be used to monitor the decrease in calcium concentration after administration for evaluation.

[0649] The test method for evaluating the blood half-life and in vivo distribution of the bio-related substance to which the degradable polyethylene glycol derivative is bonded is not particularly limited, and examples thereof include tests in which the substance is labeled with a radioisotope or a fluorescent substance, administered to mice or rats, and monitored.

[0650] It is believed that the degradable peptide introduced into the polyethylene glycol derivative gives polyethylene glycol degradability in cells, but the peptide structure may change the in vivo kinetics of the bio-related substance bonded with polyethylene glycol. Therefore, in order to confirm the effect of the introduced peptide structure on the in vivo kinetics, it is necessary to compare the blood half-life and its in vivo distribution with the bio-related substance modified with the polyethylene glycol derivative of the same molecular weight that does not have degradability. Specifically, the bio-related substance labeled with a radioactive isotope can be bonded with a non-degradable polyethylene glycol derivative and a degradable polyethylene glycol derivative, and the two bio-related substances obtained can be administered to mice, and the radiation dose of blood and each organ can be measured at multiple time points for quantitative measurement.

[0651] There are no particular limitations on the test method for evaluating the cellular vacuolation inhibition of the degradable polyethylene glycol derivative, and examples thereof include a test in which a high dose, high frequency, and long-term administration is performed to mice or rats to confirm slice images of organs or organs that are thought to be prone to cavitation, as described in Non-Patent Document 2.

[0652] Specifically, a polyethylene glycol derivative can be dissolved in physiological saline to a concentration of 10 to 250 mg / m, and 20 to 100 μL of the drug can be administered three times a week for more than four weeks via the tail vein of mice. Paraffin sections of organs believed to be prone to cavitation, such as the cerebral choroid plexus or spleen, are prepared and stained, and the section images are confirmed by a pathological method to evaluate cavitation inhibition.

[0653] In addition, in this evaluation, it is necessary to administer a much larger amount of polyethylene glycol than the usual dosage of polyethylene glycol in the technical field.

[0654] Non-patent document 2 records that cavitation of cells caused by high molecular weight polyethylene glycol is related to the accumulation of polyethylene glycol in tissues. There is no particular limitation on the test method for evaluating the accumulation of degradable polyethylene glycol derivatives in cells, but it can be evaluated by slice images made in the same way as the above-mentioned cavitation evaluation. The accumulation of polyethylene glycol can be evaluated by confirming the stained slice images of organs such as the choroid plexus or spleen, which are considered to be prone to cavitation, by pathological methods.

[0655] In this evaluation, it is necessary to administer a much larger amount of polyethylene glycol than the usual dosage of polyethylene glycol in the technical field.

[0656] [Example]

[0657] The following examples obtained 1 H-NMR was obtained from JNM-ECP400 or JNM-ECA600 manufactured by JEOL DATUM Co., Ltd. The sample tube used D2O or CDCl3 and d6-DMSO containing tetramethylsilane (TMS) as the internal standard as the deuterated solvent. The molecular weight and amine purity of the obtained polyethylene glycol derivative were calculated using liquid chromatography (GPC and HPLC). Regarding the liquid chromatography system, GPC used "HLC-8320GPC EcoSEC" manufactured by Tosoh Corporation, and HPLC used "ALLIANCE" manufactured by WATERS. The analysis conditions of GPC and HPLC are shown below.

[0658] GPC analysis (molecular weight determination)

[0659] Detector: Differential refractometer

[0660] Chromatographic columns: Ultrahydrogel 500 and Ultrahydrogel 250 (WATERS)

[0661] Mobile phase: 100 mM acetate buffer + 0.02% NaN3 (pH 5.2)

[0662] Flow rate: 0.5mL / min

[0663] Sample volume: 5 mg / mL, 20 μL

[0664] Column temperature: 30°C

[0665] HPLC analysis (amine purity determination)

[0666] Detector: Differential refractometer

[0667] Chromatographic column: TSKgel SP-5PW (Tosoh)

[0668] Mobile phase: 1 mM sodium phosphate buffer (pH 6.5)

[0669] Flow rate: 0.5 mL / min

[0670] Injection volume: 5 mg / mL, 20 μL

[0671] Column temperature: 40 °C

[0672] [Example 1]

[0673] Synthesis of compound (p1)( ME-200GLFG(L)-200PA )

[0674] [Chemical Formula 74]

[0675]

[0676] [Example 1-1]

[0677] [Chemical Formula 75]

[0678]

[0679] Glycyl-L-phenylalanyl-L-leucyl-glycine with the N-terminus protected by tert-butoxycarbonyl (Boc group) (Boc-Gly-Phe-Leu-Gly) (0.197 g, 4.0×10 -4 moles, manufactured by GenScript Biotech Corp.) and N-hydroxysuccinimide (57.5 mg, 5.0×10 -4 moles, manufactured by Midori Kagaku Co., Ltd.) were dissolved in anhydrous N,N'-dimethylformamide (1.0 g), and then N,N'-dicyclohexylcarbodiimide (0.103 g, 5.0×10 -4 moles, manufactured by Tama Chemical Industry Co., Ltd.) was added, and the reaction was carried out for 1 hour in a nitrogen atmosphere at room temperature. After dilution with anhydrous N,N'-dimethylformamide (3.0 g), methoxy-PEG with a propylamino group at the end (2.0 g, 9.5×10 -5Mole, average molecular weight = about 21,000, "SUNBRIGHT MEPA-20T" manufactured by NOF Corporation), and react it in a nitrogen atmosphere at room temperature for 1 hour. Then, ethyl acetate (20 g) was added to dilute the reaction solution, and suction was filtered using a Kiriyama funnel covered with LS100 filter paper. Ethyl acetate (30 g) was added to the filtrate, and after stirring until it became uniform, hexane (25 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. Filter with 5A filter paper, recover the precipitate, and then dissolve it in ethyl acetate (50 g) again, add hexane (25 g), and stir at room temperature for 15 minutes to precipitate the product. Filter with 5A filter paper, recover the precipitate, wash it with hexane (25 g), filter with 5A filter paper, and vacuum dry to obtain the above-mentioned compound (p2) ( ME-200GLFG(L)-Boc ). The yield was 1.8 g.

[0680] NMR (CDCl3): 0.89ppm (d, 3H, -NH-CO-CH-CH2-CH( CH3 )2), 0.92ppm(d, 3H, -NH-CO-CH-CH2-CH( CH3 )2), 1.36ppm(s, 9H, -NH-CO-OC (CH3)3 ), 1.48ppm (m, 1H, -NH-CO-CH- CH2 -CH(CH3)2), 1.55ppm(m, 1H, -NH-CO-CH- CH2 -CH(CH3)2), 1.80ppm(m, 3H), 3.13ppm(dd, 1H, -NH-CO-CH- CH2 -C6H5), 3.21ppm (dd, 1H, -NH-CO-CH- CH2 -C6H5), 3.33ppm(m, 2H, -CO-NH- CH2 -CH2-CH2-O-(CH2-CH2-O)n-CH3), 3.38ppm(s, 3H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.65ppm(m、about 2,000H、-CO-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3), 3.91ppm(t, 1H, -NH-CO- CH -CH2-CH(CH3)2), 4.43ppm (broad, 1H), 4.55ppm (q, 1H, -NH-CO- CH-CH2-C6H5), 5.77 ppm (broad, 1H), 6.76 ppm (broad, 1H), 6.86 ppm (broad, 1H), 6.90 ppm (broad, 1H), 7.14 ppm (broad, 1H), 7.20 ppm (d, 2H, -NH-CO-CH-CH2- C6H5 ), 7.32 ppm (m, 3H, -NH-CO-CH-CH2- C6H5 )

[0681] [Example 1-2]

[0682]

Chemical Formula 76

[0683]

[0684] The ME-200GLFG(L)-Boc (1.8 g, 8.6×10 -5 moles) obtained in Example 1-1 was dissolved in dichloromethane (9.0 g), and methanesulfonic acid (584 μL, 9.0×10 -3 moles, manufactured by Kanto Chemical Co., Inc.) was added, and the reaction was carried out for 2 hours in a nitrogen atmosphere at room temperature. Then, the reaction solution was diluted with toluene (18 g), ion-exchanged water (18 g) was added, and the mixture was stirred at room temperature for 15 minutes, and the product was extracted into the aqueous layer. An appropriate amount of 1 mol / L aqueous sodium hydroxide solution was added to the obtained aqueous layer to adjust the pH to 12, and then sodium chloride (4.5 g) was dissolved. Chloroform (18 g) was added thereto, and the mixture was stirred at room temperature for 15 minutes, and the product was extracted into the organic layer. After separating the aqueous layer and the organic layer, chloroform (18 g) was added again to the aqueous layer, and the mixture was stirred at room temperature for 15 minutes, and the product was extracted into the organic layer. After concentrating the organic layers obtained in the first and second extractions at 40 °C, ethyl acetate (36 g) was added to the obtained concentrate. To the obtained ethyl acetate solution, sodium sulfate (0.90 g) was added, and the mixture was stirred at 30 °C for 15 minutes, and then suction filtration was carried out using a Kiriyama funnel with Opliite on 5A filter paper. Hexane (18 g) was added to the obtained filtrate, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using 5A filter paper, the precipitate was washed with hexane (18 g), suction filtration was carried out using 5A filter paper, and vacuum drying was carried out to obtain the above compound (p3)( ME-200GLFG(L)-NH2 ). The yield was 1.4 g.

[0685] NMR (CDCl3): 0.89 ppm (d, 3H, -NH-CO-CH-CH2-CH( CH3 ), 0.91 ppm (d, 3H, -NH-CO-CH-CH2-CH( CH3 ), 1.53 ppm (m, 2H, -NH-CO-CH- CH2-CH(CH3)2), 1.70 ppm (m, 1H, -NH-CO-CH-CH2- CH (CH3)2), 1.80 ppm (m, 2H, -CO-NH-CH2- CH2 -CH2-O-(CH2-CH2-O)n-CH3), 3.10 ppm (dd, 1H, -NH-CO-CH- CH2 -C6H5), 3.18 ppm (dd, 1H, -NH-CO-CH- CH2 -C6H5), 3.33 ppm (m, 7H), 3.74 ppm (m, ca. 1,900H, -CO-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3), 4.31 ppm (broad, 1H), 4.55 ppm (t, 1H, -NH-CO- CH -CH2-C6H5), 6.91 ppm (broad, 1H), 7.00 ppm (broad, 1H), 7.28 ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )、7.98 ppm (broad, 1H)

[0686] [Example 1 - 3]

[0687] [Chemical Formula 77]

[0688]

[0689] The ME - 200GLFG(L)-NH2 (1.2 g, 5.7×10 -5 moles) obtained in Examples 1 - 2 was dissolved in chloroform (14.4 g), and then triethylamine (10 μL, 7.1×10 -5 moles, Kanto Chemical Co., Inc.) and a heterobifunctional PEG having a propylamino protected with tert - butoxycarbonyl at one end and a succinimidyl carbonate group at the other end (1.3 g, 6.2×10 -5 moles, average molecular weight = ca. 19,000, "SUNBRIGHT BO - 200TS" manufactured by NOF Corporation) were added, and the mixture was reacted for 2 hours in a nitrogen atmosphere at room temperature. After the reaction, it was concentrated at 40 °C. Ethyl acetate (48 g) was added to the obtained concentrate to dissolve it, and then hexane (24 g) was added and stirred at room temperature for 15 minutes to precipitate the product, which was then suction - filtered using a 5A filter paper. The obtained precipitate was dissolved again in ethyl acetate (48 g), hexane (24 g) was added, and it was stirred at room temperature for 15 minutes to precipitate the product, which was then suction - filtered using a 5A filter paper. After recovering the precipitate, it was washed with hexane (24 g) and suction - filtered using a 5A filter paper, and then dried in vacuo to obtain the above - mentioned compound (p4)( ME-200GLFG(L)-200Boc)。The yield is 2.0 g.

[0690] NMR(CDCl3): 0.90 ppm (t, 6H, -NH-CO-CH-CH2-CH( CH3 )2), 1.44 ppm (s, 9H, -CH2-CH2-CH2-NH-CO-O-C (CH3)3 ), 1.64 ppm (m, 1H), 1.76 ppm (m, 5H), 3.20 ppm (m, 4H), 3.33 ppm (m, 2H, -CO-NH- CH2 -CH2-CH2-O-(CH2-CH2-O)n-CH3), 3.38 ppm (s, 3H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 ), 3.64 ppm (m, approximately 3,800H, -NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3, -NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.10 ppm (m, 2H, -NH-CO-O- CH2 -CH2-O-(CH2-CH2-O)n-CH2-CH2-), 4.32 ppm (m, 1H), 4.50 ppm (q, 1H), 5.02 ppm (broad, 1H), 6.45 ppm (broad, 1H), 6.93 ppm (broad, 1H), 7.06 ppm (broad, 1H), 7.13 ppm (broad, 1H), 7.27 ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )

[0691] [Example 1-4]

[0692]

Chemical Formula 78

[0693]

[0694] Dissolve the ME-200GLFG(L)-200Boc (1.8 g, 4.3×10 -5 moles) obtained in Examples 1-3 in dichloromethane (9.0 g), and add methanesulfonic acid (292 μL, 4.5×10 -3Mol, Kanto Chemical Co., Ltd.), and reacted it in a nitrogen atmosphere at room temperature for 2 hours. Then, the reaction solution was diluted with toluene (18g), ion exchange water (18g) was added, and stirred at room temperature for 15 minutes, and the product was extracted into the water layer. According to the conditions described in Japanese Patent Laid-Open No. 2014-208786, the pH of the water layer was adjusted to 2.0 using 1mol / L hydrochloric acid, and the water layer was washed with a mixed solution of toluene and chloroform to remove polyethylene glycol impurities without amino groups. Then, an appropriate amount of 1mol / L sodium hydroxide aqueous solution was added to the water layer, and after adjusting the pH to 12, sodium chloride (4.5g) was dissolved. Chloroform (18g) was added thereto, stirred at room temperature for 15 minutes, and the product was extracted into the organic layer. After separating the water layer and the organic layer, chloroform (18g) was added to the water layer again, stirred at room temperature for 15 minutes, and the product was extracted into the organic layer. After concentrating the organic layers obtained in the first and second extractions at 40°C, ethyl acetate (27 g) was added to the obtained concentrate. Sodium sulfate (0.90 g) was added to the obtained ethyl acetate solution, and after stirring at 30°C for 30 minutes, suction filtration was performed using a Kiriyama funnel with Oplite on 5A filter paper. Hexane (18 g) was added to the obtained filtrate, and stirred at room temperature for 15 minutes to precipitate the product, and after suction filtration using 5A filter paper, the precipitate was washed with hexane (18 g), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p1) ( ME-200GLFG(L)-200PA ). The yield is 1.5 g. The molecular weight is shown in Table 1. HPLC: amine purity 91%.

[0695] NMR(CDCl3): 0.90ppm(t, 6H, -NH-CO-CH-CH2-CH( CH3 )2), 1.48ppm (width, 1H), 1.62ppm (t, 1H), 1.71ppm (m, 1H), 1.82ppm (m, 2H), 3.12ppm (m, 2H), 3.19ppm (d, 2H), 3.34ppm (m, 2H), 3.38ppm (s, 3H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.64ppm(m、about 3,800H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.10ppm (m, 2H), 4.34ppm (m, 1H), 4.50ppm (q, 1H), 6.46ppm (broad, 1H), 6.94ppm (broad, 1H), 7.08ppm (broad, 1H), 7.27ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )

[0696] [Example 2]

[0697] Synthesis of compound (p5)( ME-200GLFG(L)-200AL )

[0698] [Chemical Formula 79]

[0699]

[0700] [Example 2-1]

[0701] [Chemical Formula 80]

[0702]

[0703] The hetero-bifunctional PEG having 3,3-diethoxypropyl at one end and hydroxyl at the other end (15.0 g, 7.5×10 -4 mol, average molecular weight = about 20,000) synthesized by the method described in Patent No. 3508207 etc. was dissolved in anhydrous dichloromethane (75 g), and then N,N'-disuccinimidyl carbonate (1.2 g, 4.7×10 -3 mol, manufactured by Tokyo Chemical Industry Co., Ltd.) and triethylamine (836 μL, 6.0×10 -3 mol, Kanto Chemical Co., Inc.) were added, and the mixture was reacted at room temperature under a nitrogen atmosphere for 5 hours. After suction filtration using a 5A filter paper, the filtrate was concentrated at 40 °C, ethyl acetate (150 g) and 2,6-di-tert-butyl-p-cresol (30 mg) were added to the obtained concentrate, and the mixture was stirred until homogeneous. Hexane (75 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using a 5A filter paper, the precipitate was recovered, and ethyl acetate (150 g) and 2,6-di-tert-butyl-p-cresol (30 mg) were added again to dissolve it. Hexane (75 g) was added, and the mixture was stirred at room temperature for 15 minutes to precipitate the product, and then suction filtration was performed using a 5A filter paper. After performing the same operation 4 times, the obtained precipitate was washed with hexane (90 g), suction filtered using a 5A filter paper, and then dried under vacuum to obtain the above compound (p6). The yield was 11.8 g.

[0704] NMR(CDCl3): 1.20 ppm (t, 6H, -CH2-CH2-CH(OCH2 CH3 )2), 1.90 ppm (q, 2H, -CH2- CH2 -CH(OCH2CH3)2), 2.84 ppm (s, 4H, -O-CO-O- NC4H4O2 ), 3.65 ppm (m, about 1,900H, -O-CO-O-CH2-CH2-O-( CH2CH2O)n-CH2-), 4.46ppm(t, 2H, -O-CO-O- CH2 -CH2-O-(CH2CH2O)n-CH2-), 4.64ppm(t, 1H, -CH2-CH2- CH (OCH2CH3)2)

[0705] [Example 2-2]

[0706]

Chemistry 81

[0707]

[0708] The ME-200GLFG(L)-NH2 (1.9 g, 9.0 × 10 -5 mol) was dissolved in chloroform (18 g), and triethylamine (13 μL, 9.3×10 -5 mol, Kanto Chemical Co., Ltd.) and succinimidyl-PEG-3,3-diethoxypropyl obtained in Example 2-1 (1.5 g, 7.5 × 10 -5 mole, average molecular weight = about 21,000), and reacted it in a nitrogen atmosphere at room temperature for 2 hours. After the reaction, it was concentrated at 40°C, and the obtained concentrate was dissolved in a mixed solution of toluene and chloroform according to the conditions described in Japanese Patent Laid-Open No. 2011-79934, and the organic layer was washed with 5% saline to remove polyethylene glycol impurities with a molecular weight of about 21,000. The organic layer was concentrated at 40°C, and ethyl acetate (60 g) was added to the obtained concentrate to dissolve it, and then hexane (30 g) was added and stirred at room temperature for 15 minutes to precipitate the product, and 5A filter paper was used for suction filtration. The obtained precipitate was dissolved again in ethyl acetate (60 g), and hexane (30 g) was added and stirred at room temperature for 15 minutes to precipitate the product, and 5A filter paper was used for suction filtration. After recovering the precipitate, it was washed with hexane (30 g), filtered with 5A filter paper, and vacuum dried to obtain the above-mentioned compound (p7) ( ME-200GLFG(L)-200DE ). The yield is 3.1 g.

[0709] NMR(CDCl3): 0.90ppm(t, 6H, -NH-CO-CH-CH2-CH( CH3 )2), 1.20ppm(t, 6H, -CH2-CH2-CH(OCH2 CH3 )2), 1.47ppm (m, 1H, -NH-CO-CH- CH2 -CH(CH3)2), 1.61ppm(m, 1H, -NH-CO-CH- CH2-CH(CH3)2), 1.72ppm(m, 1H), 1.82ppm(m, 2H), 1.90ppm(q, 2H, -CH2- CH2 -CH(OCH2CH3)2), 2.58ppm(m, 1H), 3.19ppm(d, 2H, -NH-CO-CH- CH2 -C6H5), 3.33ppm(m, 2H, -CO-NH- CH2 -CH2-CH2-O-(CH2-CH2-O)n-CH3), 3.38ppm(-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 ), 4.29ppm (m, 1H), 4.50ppm (q, 1H), 4.64ppm (t, 1H, -CH2-CH2- CH (OCH2CH3)2), 6.38ppm (broad, 1H), 6.89ppm (broad, 1H), 6.99ppm (broad, 1H), 7.10ppm (broad, 1H), 7.29ppm (m, 5H)

[0710] [Example 2-3]

[0711]

Chemistry 82

[0712]

[0713] ME-200GLFG(L)-200DE (1.0 g, 2.4 × 10 -5After 200 g of 1,000 mol) was dissolved in distilled water for injection (20 g), the pH was adjusted to 1.50 with 85% phosphoric acid (0.46 g), and the mixture was reacted at 20-25° C. for 2 hours. After the reaction, 0.69 g of a 400 g / L sodium hydroxide aqueous solution was added, the pH was adjusted to 6.70, and sodium chloride (4.0 g) was added to dissolve it. A 1 M sodium hydroxide aqueous solution (1.76 g) was added dropwise to the obtained solution, and after the pH was adjusted to 7.05, chloroform (15 g) in which 2,6-di-tert-butyl-p-cresol (1.5 mg) was previously dissolved was added, and the mixture was 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 after the organic layer was recovered, chloroform (15 g) in which 2,6-di-tert-butyl-p-cresol (1.5 mg) was previously dissolved was added to the aqueous layer again, and the mixture was 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 concentrated at 50°C, ethyl acetate (10 g) was added to the obtained concentrate, and after stirring until uniform, magnesium sulfate (0.25 g) was added, and stirred at 30°C for 15 minutes, and suction filtered using a Kiriyama funnel with Oplite on 5A filter paper, and washed with ethyl acetate (10 g). Hexane (15 g) was added to the obtained liquid, and stirred at room temperature for 15 minutes to precipitate the product, and after suction filtering using 5A filter paper, the precipitate was washed with hexane (10 g) in which 2,6-di-tert-butyl-p-cresol (1.0 mg) was previously dissolved, and suction filtered using 5A filter paper, and vacuum dried to obtain the above-mentioned compound (p5) ( ME-200GLFG(L)-200AL The yield was 0.65 g. The molecular weight is shown in Table 1. The aldehyde purity was 86% ( 1 H-NMR).

[0714] NMR(CDCl3): 0.90ppm(t, 6H, -NH-CO-CH-CH2-CH( CH3 )2), 1.47ppm (m, 1H, -NH-CO-CH- CH2 -CH(CH3)2), 1.61ppm(m, 1H, -NH-CO-CH- CH2 -CH(CH3)2), 1.72ppm(m, 1H), 1.84ppm(m, 5H), 2.68ppm(m, 2H), 3.19ppm(d, 2H, -NH-CO-CH- CH2 -C6H5), 3.33ppm(m, 2H, -CO-NH- CH2 -CH2-CH2-O-(CH2-CH2-O)n-CH3), 3.38ppm(-CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 ), 4.06ppm (m, 1H, -NH-CO-O- CH2-CH2-O-(CH2CH2O)n-CH2CH2-), 4.12ppm(m, 1H, -NH-CO-O- CH2 -CH2-O-(CH2CH2O)n-CH2CH2-), 4.32ppm (m, 1H), 4.51ppm (q, 1H), 6.36ppm (broad, 1H), 6.87ppm (broad, 1H), 6.99ppm (broad, 2H), 7.14ppm (broad, 1H), 7.29ppm (m, 5H), 9.80ppm (s, 1H, -CH2CH2- CHO )

[0715] [Example 3]

[0716] Synthesis of compound (p8) (ME-100GLFG(L)-100GLFG(L)-100GLFG(L)-100PA)

[0717]

Chemistry 83

[0718]

[0719] [Example 3-1]

[0720]

Chemistry 84

[0721]

[0722] The above-mentioned compound (p9) was obtained by the same production method as in Example 1 using glycyl-L-phenylalanyl-L-leucyl-glycine (Boc-Gly-Phe-Leu-Gly) protected at the N-terminus with a tert-butoxycarbonyl group (Boc group), methoxy PEG having a propylamino group at the terminal (average molecular weight = about 10,000, "SUNBRIGHT MEPA-10T" manufactured by NOF Corporation), and heterobifunctional PEG having a propylamino group protected at one terminal with a tert-butoxycarbonyl group and a succinimidyl carbonate group at the other terminal (average molecular weight = about 10,000, "SUNBRIGHT BO-100TS" manufactured by NOF Corporation) as raw materials ( ME- 100GLFG(L)-100PA ). The yield was 1.0 g.

[0723] NMR(CDCl3): 0.90ppm(t, 6H, -NH-CO-CH-CH2-CH( CH3)2), 1.48ppm (width, 1H), 1.62ppm (t, 1H), 1.71ppm (m, 1H), 1.82ppm (m, 2H), 3.12ppm (m, 2H), 3.19ppm (d, 2H), 3.34ppm (m, 2H), 3.38ppm (s, 3H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.64ppm(m、about 1,800H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.10ppm (m, 2H), 4.34ppm (m, 1H), 4.50ppm (q, 1H), 6.46ppm (broad, 1H), 6.94ppm (broad, 1H), 7.08ppm (broad, 1H), 7.27ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )

[0724] [Example 3-2]

[0725]

Chemistry 85

[0726]

[0727] ME-100GLFG(L)-100PA (1.0 g, 5.0 × 10 -5 mol) and Boc-Gly-Phe-Leu-Gly (0.99 g, 2.0 × 10 -4 Anhydrous N,N'-dimethylformamide (10 g) was added to the mixture and dissolved by heating at 30°C. Then, diisopropylethylamine (65 μL, 3.8×10 -4 mole, manufactured by Kanto Chemical Co., Ltd.) and (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinium hexafluorophosphate (COMU) (0.106 g, 2.5 × 10 -4mol, Sigma-Aldrich Company Ltd.), and reacted it in a nitrogen atmosphere at room temperature for 1 hour. After the reaction was completed, it was diluted with chloroform (100g), saturated sodium bicarbonate aqueous solution (50g) was added, and it was stirred at room temperature for 15 minutes for washing. After separation of the aqueous layer and the organic layer, saturated sodium bicarbonate aqueous solution (50g) was added to the organic layer again, stirred at room temperature for 15 minutes for washing, and the organic layer was recovered. Magnesium sulfate (1.0g) was added to the obtained organic layer, and after stirring for 30 minutes for dehydration, it was filtered using a Kiriyama funnel with Oplite on 5A filter paper. The obtained liquid was concentrated at 40°C, ethyl acetate (50g) was added to the concentrate and stirred until uniform, and then hexane (25g) was added, and the product was precipitated by stirring at room temperature for 15 minutes. Filter with 5A filter paper, recover the precipitate, and then dissolve it in ethyl acetate (50g) again, add hexane (25g) at room temperature, and stir at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (25 g), suction filtrated using 5A filter paper, and vacuum dried to obtain the above compound (p10) ( ME-100GLFG(L)-100GLFG(L)-Boc ). The yield was 0.9 g.

[0728] NMR(CDCl3): 0.90ppm(t, 12H, -NH-CO-CH-CH2-CH( CH3 )2), 1.44ppm(s, 9H, -CH2-CH2-CH2-NH-CO-OC (CH3)3 ), 1.64ppm (m, 2H), 1.76ppm (m, 5H), 3.20ppm (m, 4H), 3.33ppm (m, 4H, -CO-NH- CH2 -CH2-CH2-O-(CH2-CH2-O)n-), 3.38ppm(s, 3H, -O-(CH2-CH2-O)n- CH3 )、3.64ppm(m、about 1,800H、-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.10ppm(m, 2H, -NH-CO-O- CH2 -CH2-O-(CH2-CH2-O)n-CH2-CH2-), 4.32ppm (m, 2H), 4.50ppm (q, 2H), 5.02ppm (broad, 2H), 6.45ppm (broad, 2H), 6.93ppm (broad, 2H), 7.06ppm (broad, 2H), 7.13ppm (broad, 2H), 7.27ppm (m, 10H, -NH-CO-CH-CH2- C6H5 )

[0729] [Example 3-3]

[0730]

Chemistry 86

[0731]

[0732] The ME-100GLFG(L)-100GLFG(L)-Boc (0.9 g, 4.5 × 10 -5 mol) was prepared by the same method as in Example 1-2, and the Boc group was deprotected to obtain the above compound (p11) ( ME-100GLFG (L)-100GLFG(L)-NH2 ). The yield is 0.8 g.

[0733] NMR (CDCl3): 0.89ppm (d, 6H, -NH-CO-CH-CH2-CH( CH3 )2), 0.91ppm(d, 6H, -NH-CO-CH-CH2-CH( CH3 )2), 1.53ppm (m, 4H, -NH-CO-CH- CH2 -CH(CH3)2), 1,70ppm(m, 2H, -NH-CO-CH-CH2- CH (CH3)2), 1.80ppm(m, 4H, -CO-NH-CH2- CH2 -CH2-O-(CH2-CH2-O)n-), 3.10ppm(dd, 2H, -NH-CO-CH- CH2 -C6H5), 3.18ppm (dd, 2H, -NH-CO-CH- CH2 -C6H5), 3.33ppm (m, 14H), 3.74ppm (m, about 1,800H, -CO-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-), 4.31ppm (broad, 2H), 4.55ppm (t, 2H, -NH-CO- CH -CH2-C6H5), 6.91ppm (broad, 2H), 7.00ppm (broad, 2H), 7.28ppm (m, 10H, -NH-CO-CH-CH2- C6H5 )、7.98ppm(wide, 2H)

[0734] [Examples 3-4]

[0735]

Chemistry 87

[0736]

[0737] The ME-100GLFG(L)-100GLFG(L)-NH2 (0.8 g, 4.0×10 -5 mol) was reacted with a heterobifunctional PEG (average molecular weight = about 10,000, "SUNBRIGHT BO-100TS" manufactured by NOF Corporation) having a propylamino group protected by a tert-butoxycarbonyl group at one end and a carbonate succinimidyl ester group at the other end by the same production method as in Example 1-3 to obtain the above-mentioned compound (p12) ( ME-100GLFG(L)-100GLFG(L)- 100Boc ). The yield was 1.0 g.

[0738] NMR(CDCl3): 0.90ppm(t, 12H, -NH-CO-CH-CH2-CH( CH3 )2), 1.44ppm(s, 9H, -CH2-CH2-CH2-NH-CO-OC (CH3)3 ), 1.64ppm (m, 2H), 1.76ppm (m, 10H), 3.20ppm (m, 8H), 3.33ppm (m, 6H, -CO-NH- CH2 -CH2-CH2-O-(CH2-CH2-O)n-), 3.38ppm(s, 3H, -(CH2-CH2-O)n- CH3 )、3.64ppm(m、about 2,700H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.10ppm(m, 4H, -NH-CO-O- CH2 -CH2-O-(CH2-CH2-O)n-CH2-CH2-), 4.32ppm (m, 2H), 4.50ppm (q, 2H), 5.02ppm (broad, 2H), 6.45ppm (broad, 2H), 6.93ppm (broad, 2H), 7.06ppm (broad, 2H), 7.13ppm (broad, 2H), 7.27ppm (m, 10H, -NH-CO-CH-CH2- C6H5 )

[0739] [Examples 3-5]

[0740]

Chemistry 88

[0741]

[0742] The ME-100GLFG(L)-100GLFG(L)-100Boc (1.0 g, 3.3 × 10-5 The crude product was purified by ion exchange chromatography filled with a cation exchange resin SP Sepharose FF (manufactured by GE Healthcare) to obtain the above-mentioned compound (p13) ( ME-100GLFG(L)-100GLFG(L)-100PA ). The yield is 0.8 g.

[0743] NMR(CDCl3): 0.90ppm(t, 12H, -NH-CO-CH-CH2-CH( CH3 )2), 1.48ppm (width, 2H), 1.62ppm (t, 2H), 1.71ppm (m, 2H), 1.82ppm (m, 4H), 3.12p pm(m, 4H), 3.19ppm(d, 4H), 3.34ppm(m, 4H), 3.38ppm(s, 3H, -O-(CH2-CH2-O)n- CH3 )、3.64ppm(m、about 2,700H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.10ppm (m, 4H), 4.34ppm (m, 2H), 4.50ppm (q, 2H), 6.46ppm (broad, 2H), 6.94ppm (broad, 2H), 7.08ppm (broad, 2H), 7.27ppm (m, 10H, -NH-CO-CH-CH2- C6H5 )

[0744] [Examples 3-6]

[0745]

Chemistry 89

[0746]

[0747] ME-100GLFG(L)-100GLFG(L)-100PA (0.8 g, 2.7 × 10 -5 mol) as a raw material, and the same production method was repeated in the order of Example 3-2, Example 3-3, Example 3-4, and Example 3-5 to obtain the above compound (p8) ( ME-100GLFG(L)-100GLFG(L)-100GLFG(L)- 100PA ). The yield was 0.4 g. The molecular weight is shown in Table 1. HPLC: amine purity 90%. NMR (CDCl3): 0.90 ppm (t, 18H, -NH-CO-CH-CH2-CH ( CH3)2), 1.48ppm (width, 3H), 1.62ppm (t, 3H), 1.71ppm (m, 3H), 1.82ppm (m, 6H), 3.12pp m(m, 6H),, 3.19ppm(d, 6H), 3.34ppm(m, 6H), 3.38ppm(s, 3H, -O-(CH2-CH2-O)n- CH3 )、3.64ppm(m、about 3,600H、-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.10ppm (m, 8H), 4.34ppm (m, 3H), 4.50ppm (q, 3H), 6.46ppm (broad, 3H), 6.94ppm (broad, 3H), 7.08ppm (broad, 3H), 7.27ppm (m, 15H, -NH-CO-CH-CH2- C6H5 )

[0748] [Example 4]

[0749] Compound (p14) ME-200G(Cit)V-200PA )

[0750]

Chemistry 90

[0751]

[0752] [Example 4-1]

[0753]

Chemistry 91

[0754]

[0755] L-Valyl-L-citrullyl-glycine (Fmoc-Val-(Cit)-Gly) (0.299 g, 5.4 × 10 -4 moles, manufactured by GenScript Biotech Corp.) and methoxy PEG having a propylamino group at the end (2.7 g, 1.3 × 10 -4 Anhydrous N,N'-dimethylformamide (27 g) was added to the mixture and dissolved by heating at 30°C. Then, diisopropylethylamine (172 μL, 1.0×10 -3 mol, manufactured by Kanto Chemical Co., Ltd.) and (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinium hexafluorophosphate (COMU) (0.289 g, 6.810 -4mol, Sigma-Aldrich Company Ltd.), and reacted it in a nitrogen atmosphere at room temperature for 1 hour. After the reaction was completed, it was diluted with chloroform (270g), saturated sodium bicarbonate aqueous solution (108g) was added, and the mixture was stirred at room temperature for 15 minutes for washing. After separation of the aqueous layer and the organic layer, saturated sodium bicarbonate aqueous solution (108g) was added to the organic layer again, stirred at room temperature for 15 minutes for washing, and the organic layer was recovered. Magnesium sulfate (2.7g) was added to the obtained organic layer, and after stirring for 30 minutes for dehydration, a Kiriyama funnel with Oplite on 5A filter paper was used for suction filtration. The obtained liquid was concentrated at 40°C, ethyl acetate (108g) was added to the concentrate and stirred until uniform, and then hexane (54g) was added, and the product was precipitated by stirring at room temperature for 15 minutes. Filter with 5A filter paper, recover the precipitate, and then dissolve it in ethyl acetate (108g) again, add hexane (54g) at room temperature, and stir at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (54 g), and suction filtered using 5A filter paper, and vacuum dried to obtain the above compound (p15) ( ME-200G(Cit)V-Fmoc ). The yield is 2.3 g.

[0756] NMR (d6-DMSO): 0.84ppm (d, 3H, -NH-CO-CH-CH( CH3 )2), 0.85ppm(d, 3H, -NH-CO-CH-CH( CH3 )2), 1.37ppm (m, 2H), 1.52ppm (m, 1H), 1.63ppm (m, 3H), 1.98ppm (m, 1H, -O-CO-NH-CH-C H (CH3)2), 2.93ppm (m, 4H),, 3.09ppm (m, 2H, -NH-CH-CH2-CH2- CH2 -NH-CO-NH2), 3.24ppm(s, 3H, -NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.48ppm(m、about 1,900H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3), 3.89ppm(m, 2H), 4.25ppm(m, 3H, -NH-CO-O- CH2-CH <)、5.35ppm(broad, 2H、-NH-CH-CH2-CH2-CH2-NH-CO- NH2), 5.91ppm (width, 1H), 7.33ppm (t, 2H, Ar), 7.41ppm (m, 3H, Ar), 7.73ppm (m, 3H, Ar), 7.89ppm (d, 2H, Ar), 8.10ppm (d, 1H), 8.20ppm (width, 1H)

[0757] [Example 4-2]

[0758]

Chemistry 92

[0759]

[0760] ME-200G(Cit)V-Fmoc (2.1 g, 1.0×10 -4 mol) was added with N,N'-dimethylformamide (12.6 g) and dissolved by heating at 30°C. Piperidine (0.66 g, 7.8 × 10 -3 mol, Wako Pure Chemical Industries, Ltd.), and reacted it in a nitrogen atmosphere at room temperature for 2 hours. After the reaction was completed, ethyl acetate (150 g) was added and stirred until uniform, hexane (75 g) was added, and the product was precipitated by stirring at room temperature for 15 minutes. Filtered with 5A filter paper, the precipitate was recovered, and then dissolved in ethyl acetate (150 g) again, hexane (75 g) was added, and the product was precipitated by stirring at room temperature for 15 minutes. Filtered with 5A filter paper, the precipitate was recovered, and then washed with hexane (75 g), filtered with 5A filter paper, and vacuum dried to obtain the above-mentioned compound (p16) ( ME-200G(Cit)V-NH2 ). The yield was 1.8 g.

[0761] NMR(d6-DMSO): 0.77ppm(d, 3H, -NH-CO-CH-CH( CH3 )2), 0.87ppm(d, 3H, -NH-CO-CH-CH( CH3 )2), 1.35ppm (m, 2H), 1.51ppm (m, 1H), 1.64ppm (m, 4H), 1.93ppm (m, 1H, -O-CO-NH-CH-C H (CH3)2), 2.96ppm (m, 4H),, 3.10ppm (m, 2H, -NH-CH-CH2-CH2- CH2 -NH-CO-NH2), 3.24ppm(s, 3H, -NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 、)、3.48ppm(m、about 1,900H、-NH-CH2-CH2-CH2-O-( CH2-CH2-O)n-CH3), 4.21ppm (broad, 1H), 5.34ppm (broad, 2H, -NH-CH-CH2-CH2-CH2-NH-CO- NH2 )、5.91ppm(wide, 1H)、7.73ppm(t, 1H)、8.08ppm(wide, 1H)、8.23ppm(t, 1H)

[0762] [Example 4-3]

[0763]

Chemistry 93

[0764]

[0765] The ME-200G(Cit)V-NH2 (1.2 g, 5.7×10 -5 mol) was dissolved in chloroform (7.2 g), and triethylamine (9.2 μL, 6.7×10 -5 mol, manufactured by Kanto Chemical Co., Ltd.) and a heterobifunctional PEG having a propylamino group protected by a tert-butoxycarbonyl group at one end and a succinimidyl carbonate group at the other end (1.2 g, 5.2 × 10 -5 Mole, average molecular weight = about 23,000, "SUNBRIGHT BO-200TS" manufactured by NOF Corporation) was reacted in a nitrogen atmosphere at room temperature for 2 hours. After the reaction, it was concentrated at 40°C, ethyl acetate (120g) was added to the obtained concentrate to dissolve it, and then hexane (60g) was added and stirred at room temperature for 15 minutes to precipitate the product, and 5A filter paper was used for suction filtration. The obtained precipitate was washed with hexane (12g), filtered with 5A filter paper, and vacuum dried to obtain the above-mentioned compound (p17) ( ME-200G(Cit)V-200Boc ). The yield is 2.1 g.

[0766] NMR (CDCl3): 0.96ppm (d, 3H, -NH-CO-CH-CH( CH3 )2),,1.01ppm(d,3H,-NH-CO-CH-CH( CH3 )2), 1.44ppm(s, 9H, -CH2-CH2-CH2-NH-CO-OC (CH3)3 ), 1.59ppm (m, 2H), 1.76ppm (m, 3H), 1.82ppm (q, 2H), 1.92ppm (m, 1H), 3.09ppm (m, 1H), 3.23ppm (m, 2H, -NH-CH-CH2-CH2- CH2 -NH-CO-NH2), 3.38ppm(s, 3H, -NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.64ppm(m、about 3,800H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.07ppm (dd, 1H), 4.15ppm (m, 2H), 4.31ppm (m, 1H), 4.38ppm (m, 1H), 5.02ppm (broad, 1H), 5.24ppm (broad, 2H, -NH-CH-CH2-CH2-CH2-NH-CO- NH2 )、5.74ppm(broad, 1H)、5.81ppm(d, 1H)、7.05ppm(broad, 1H)、7.46ppm(broad, 1H)、8.30ppm(broad, 1H)

[0767] [Example 4-4]

[0768]

Chemistry 94

[0769]

[0770] The ME-200G (Cit) V-200Boc (2.0 g, 4.5 × 10 -5 mol) was dissolved in dichloromethane (10 g), and methanesulfonic acid (291 μL, 4.5×10 -3mol, Kanto Chemical Co., Ltd.), and reacted it in a nitrogen atmosphere at room temperature for 2 hours. Then, the reaction solution was diluted with toluene (20g), ion exchange water (20g) was added, and stirred at room temperature for 15 minutes, and the product was extracted into the water layer. According to the conditions described in Japanese Patent Laid-Open No. 2014-208786, the pH of the water layer was adjusted to 2.0 using 1mol / L hydrochloric acid, and the water layer was washed with a mixed solution of toluene and chloroform to remove polyethylene glycol impurities without amino groups. Then, an appropriate amount of 1mol / L sodium hydroxide aqueous solution was added to the water layer, and after adjusting the pH to 12, sodium chloride (5.0g) was dissolved. Chloroform (10g) was added thereto, stirred at room temperature for 15 minutes, and the product was extracted into the organic layer. After separating the water layer and the organic layer, chloroform (10g) was added to the water layer again, stirred at room temperature for 15 minutes, and the product was extracted into the organic layer. After concentrating the organic layers obtained in the first and second extractions at 40°C, ethyl acetate (100 g) was added to the obtained concentrate. Sodium sulfate (2.0 g) was added to the obtained ethyl acetate solution, and after stirring at 30°C for 15 minutes, suction filtration was performed using a Kiriyama funnel with Oplite on 5A filter paper. Hexane (50 g) was added to the obtained liquid, and stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using 5A filter paper, the precipitate was washed with hexane (20 g), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p14) ( ME-200G(Cit)V-200PA ). The yield is 1.7 g. The molecular weight is shown in Table 1. HPLC: amine purity 92%.

[0771] NMR (CDCl3): 0.96ppm (d, 3H, -NH-CO-CH-CH( CH3 )2),,1.01ppm(d,3H,-NH-CO-CH-CH( CH3 )2), 1.60ppm (m, 2H), 1.75ppm (m, 3H), 1.82ppm (m, 2H), 1.93ppm (m, 1H), 2.23ppm (m, 1H), 2.82ppm (t, 2H, -CH2-CH2-CH2- NH2 ), 3.10ppm (m, 1H), 3.30ppm (m, 2H), 3..38ppm (m, 4H), 3.64ppm (m, about 3,800H, -NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2- CH2-O)n-CH2-CH2-), 3.95ppm (m, 1H), 4.07ppm (dd, 1H), 4.15ppm (m, 1H), 4.31ppm (m, 1H), 4.38ppm (m, 1H), 5.24ppm (wide, 2H, -NH-CH-CH2-CH2-CH2-NH-CO- NH2 )、5.79ppm(wide, 1H)、5.82ppm(wide, 1H)、7.06ppm(wide, 1H)、7.48ppm(wide, 1H)、8.31ppm(wide, 1H)

[0772] [Example 5]

[0773] Compound (p18) ME-200G(Cit)V-200MA )

[0774]

Chemistry 95

[0775]

[0776] ME-200G(Cit)V-200PA (0.2 g, 4.5×10 -6 Toluene (1.1 g) and acetonitrile (0.16 g) were added to the solution and dissolved by heating at 40°C. N-methylmorpholine (2.5 μL, 2.2×10 -5 mol, manufactured by Kanto Chemical Co., Ltd.) and 3-maleimidopropionic acid-N-succinimidyl ester (1.8 mg, 6.8 × 10 -6 The mixture was stirred for 1 hour at 40°C under a nitrogen atmosphere. Ethyl acetate (6.0 g) was added to the reaction solution and stirred until uniform. Hexane (8.0 g) was added at 17°C and stirred at 17°C for 15 minutes to precipitate the product. After suction filtration using 5A filter paper, the precipitate was washed with hexane (8.0 g), suction filtration using 5A filter paper, and vacuum drying to obtain the above-mentioned compound (p18) ( ME-200G(Cit)V-200MA The yield is 0.12 g. The molecular weight is shown in Table 1. The purity of maleimide is 88% ( 1 H-NMR).

[0777] NMR (CDCl3): 0.96ppm (d, 3H, -NH-CO-CH-CH( CH3 )2),,1.01ppm(d,3H,-NH-CO-CH-CH( CH3 )2), 1.59ppm (width, 2H), 1.76ppm (m, 5H), 2.46ppm (t, 2H, -CH2-CH2- CH2-NH-CO-CH2-CH2-C4NO2H2), 3.12ppm (m, 1H), 3.34ppm (m, 5H), 3.64ppm (m, about 3,800H, -NH-CH2-CH2-CH2-O-( CH2- CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.02ppm (m, 1H), 4.13ppm (m, 1H), 4.28ppm (m, 1H), 4.37ppm (m, 1H) ), 5.86ppm (width, 1H), 6.42ppm (width, 1H), 6.68ppm (s, 2H, -CH2-CH2-CH2-NH-CO-CH2-CH2- C4NO2H2 )、7.04ppm(wide, 1H)、7.46ppm(wide, 1H)、8.20ppm(wide, 1H)

[0778] [Example 6]

[0779] Compound (p19) ME-200GGG-200PA )

[0780]

Chemistry 96

[0781]

[0782] [Example 6-1]

[0783]

Chemistry 97

[0784]

[0785] To glycyl-glycyl-glycine (2.0 g, 1.1 × 10 -2 After adding ion exchange water (60 g) and sodium bicarbonate (4.5 g) to dissolve, tetrahydrofuran (60 g) and di-tert-butyl dicarbonate (Boc2O) (4.6 g, 2.1×10 -2Mol, Tokyo Chemical Industry Co., Ltd.), and reacted it at 40°C for 23 hours. Water (120g) was added to the reaction solution, and after cooling to 5°C, phosphoric acid was added in an appropriate amount to adjust the pH to 7, and hexane (120g) was added and stirred at room temperature for 15 minutes. After separating the organic layer and the aqueous layer, hexane (120g) was added to the aqueous layer again and stirred at room temperature for 15 minutes. After recovering the aqueous layer, ethanol (100g) was added and concentrated at 50°C, and ethanol (100g) was added again and concentrated at 50°C. Ethanol (100g) was added to the obtained concentrate, and after heating and dissolving at 50°C, it was filtered using a Kiriyama funnel with Oplite on 5A filter paper. After concentrating the obtained filtrate at 50°C, vacuum drying was obtained to obtain Boc-glycyl-glycyl-glycine. The yield was 1.5g.

[0786] NMR(D2O): 1.45ppm(s, 9H, -NH-CO-OC (CH3)3 )、3.78ppm(s、2H、- CH2 -NH-CO-OC(CH3)3), 3.84ppm(s, 2H, -CO-NH- CH2 -COOH), 4.00ppm(s, 2H, -CO-NH- CH2 -CO-NH-)

[0787] [Example 6-2]

[0788]

Chemistry 98

[0789]

[0790] To Boc-glycyl-glycyl-glycine (0.174 g, 6.0 × 10 -4 moles) and methoxy PEG with propylamino at the end (3.0 g, 1.4 × 10 -4 To a mixture of 27 g of anhydrous N,N'-dimethylformamide (27 g) and heated at 30°C was added diisopropylethylamine (191 μL, 1.1 × 10 -3 mole, manufactured by Kanto Chemical Co., Ltd.) and (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinium hexafluorophosphate (COMU) (0.321 g, 7.5 × 10 -4mol, Sigma-Aldrich Company Ltd.), and reacted it in a nitrogen atmosphere at room temperature for 1 hour. After the reaction was completed, it was diluted with chloroform (300g), saturated sodium bicarbonate aqueous solution (120g) was added, and the mixture was stirred at room temperature for 15 minutes for washing. After separation of the aqueous layer and the organic layer, saturated sodium bicarbonate aqueous solution (120g) was added to the organic layer again, and the organic layer was recovered after stirring at room temperature for 15 minutes for washing. Magnesium sulfate (3.0g) was added to the obtained chloroform solution, and after stirring for 30 minutes for dehydration, a Kiriyama funnel with Oplite on 5A filter paper was used for suction filtration. The obtained liquid was concentrated at 40°C, ethyl acetate (120g) was added to the concentrate and stirred until uniform, and then hexane (60g) was added, and the product was precipitated by stirring at room temperature for 15 minutes. Filter with 5A filter paper, recover the precipitate, dissolve it in ethyl acetate (120g) again, add hexane (60g), and stir at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (60 g), suction filtrated using 5A filter paper, and vacuum dried to obtain the above compound (p21) ( ME-200GGG-Boc ). The yield is 2.6 g.

[0791] NMR (CDCl3): 1.45ppm (s, 9H, -NH-CO-OC (CH3)3 ), 1.80ppm(m, 2H, -CO-NH-CH2- CH2 -CH2-O-(CH2-CH2-O)n-CH3), 3.38ppm (m, 5H), 3.64ppm (m, about 1,900H, -CO-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3), 3.86ppm (d, 2H, - CH2 -NH-CO-OC(CH3)3), 3.91ppm(d, 2H), 3.99ppm(d, 2H), 5.69ppm(broad, 1H), 7.10ppm(broad, 1H), 7.44ppm(broad, 2H)

[0792] [Example 6-3]

[0793]

Chemistry 99

[0794]

[0795] The ME-200GGG-Boc (2.4 g, 1.1 × 10 -4 mol) was dissolved in dichloromethane (12 g), and methanesulfonic acid (723 μL, 1.1×10 -2mol, Kanto Chemical Co., Ltd.), and reacted it in a nitrogen atmosphere at room temperature for 2 hours. Then, the reaction solution was diluted with toluene (24g), ion exchange water (24g) was added, stirred at room temperature for 15 minutes, and the product was extracted into the water layer. An appropriate amount of 1mol / L sodium hydroxide aqueous solution was added to the obtained water layer, and after adjusting the pH to 12, sodium chloride (6.0g) was dissolved. Chloroform (12g) was added thereto, stirred at room temperature for 15 minutes, and the product was extracted into the organic layer. After separating the water layer and the organic layer, chloroform (12g) was added to the water layer again, stirred at room temperature for 15 minutes, and the product was extracted into the organic layer. After concentrating the organic layers obtained in the first and second extractions at 40°C, ethyl acetate (120g) was added to the obtained concentrate. Sodium sulfate (2.4g) was added to the obtained ethyl acetate solution, and after stirring at 30°C for 15 minutes, suction filtration was performed using a Kiriyama funnel with Oplite on 5A filter paper. Hexane (60 g) was added to the obtained liquid, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using 5A filter paper, the precipitate was washed with hexane (24 g), suction filtration using 5A filter paper, and vacuum drying to obtain the above-mentioned compound (p22) ( ME-200GGG-NH2 ). The yield is 2.2 g.

[0796] NMR (CDCl3): 1.53 ppm (broad, 1H), 1.79 ppm (m, 2H, -CO-NH-CH2- CH2 -CH2-O-(CH2-CH2-O)n-CH3), 3.38ppm (m, 5H), 3.64ppm (m, about 1,900H, -CO-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3), 3.92ppm (d, 2H), 4.01ppm (broad, 1H), 7.06ppm (broad, 1H), 7.24ppm (broad, 1H), 7.86ppm (broad, 1H)

[0797] [Example 6-4]

[0798]

Chemistry 100

[0799]

[0800] The ME-200GGG-NH2 (1.5 g, 7.1 × 10 -5 mol) was dissolved in chloroform (7.5 g), and triethylamine (11.6 μL, 8.4 × 10 -5 Mol, Kanto Chemical Co., Ltd.) and a heterobifunctional PEG having a propylamino group protected by a tert-butoxycarbonyl group at one end and a succinimidyl carbonate group at the other end (1.8 g, 7.8 × 10-5 mol, average molecular weight = about 23,000, "SUNBRIGHT BO-200TS" manufactured by NOF Corporation) was reacted in a nitrogen atmosphere at room temperature for 2 hours. After the reaction, it was concentrated at 40°C, ethyl acetate (150g) was added to the obtained concentrate to dissolve it, and then hexane (75g) was added and stirred at room temperature for 15 minutes to precipitate the product, and 5A filter paper was used for suction filtration. The obtained precipitate was washed with hexane (75g), filtered with 5A filter paper, and vacuum dried to obtain the above compound (p23) ( ME- 200GGG-200Boc ). The yield is 3.0 g.

[0801] NMR (CDCl3): 1.44ppm (s, 9H, -CH2-CH2-CH2-NH-CO-OC (CH3)3 ), 1.78ppm(m, 4H, -CO-NH-CH2- CH2 -CH2-O-(CH2-CH2-O)n-CH3, -CH2- CH2 -CH2-NH-CO-OC(CH3)3), 3.23ppm(m, 2H, -CH2-CH2- CH2 -NH-CO-OC(CH3)3), 3.35ppm(m, 2H), 3.38ppm(s, 3H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.65ppm(m、about 3,800H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 3.97ppm (d, 2H), 4.23ppm (wide, 2H, -NH-CO-O- CH2 -CH2-O-(CH2-CH2-O)n-CH2-CH2-), 5.01ppm (broad, 1H), 6.21ppm (broad, 1H), 7.00ppm (broad, 1H), 7.47ppm (broad, 1H), 7.61ppm (broad, 1H)

[0802] [Example 6-5]

[0803]

Chemistry 101

[0804]

[0805] The ME-200GGG-200Boc (2.5 g, 5.8 × 10 -5mol) was dissolved in dichloromethane (12.5 g), and methanesulfonic acid (365 μL, 5..6×10 -3 Mol, Kanto Chemical Co., Ltd.), and reacted it in a nitrogen atmosphere at room temperature for 2 hours. Then, the reaction solution was diluted with toluene (25g), ion exchange water (25g) was added, stirred at room temperature for 15 minutes, and the product was extracted into the water layer. According to the conditions described in Japanese Patent Laid-Open No. 2014-208786, the pH of the water layer was adjusted to 2.0 using 1mol / L hydrochloric acid, and the water layer was washed with a mixed solution of toluene and chloroform to remove polyethylene glycol impurities without amino groups. Then, an appropriate amount of 1mol / L sodium hydroxide aqueous solution was added to the water layer, and after adjusting the pH to 12, sodium chloride (6.3g) was dissolved. Chloroform (12.5g) was added thereto, stirred at room temperature for 15 minutes, and the product was extracted into the organic layer. After separating the water layer and the organic layer, chloroform (12.5g) was added to the water layer again, stirred at room temperature for 15 minutes, and the product was extracted into the organic layer. After concentrating the organic layers obtained in the first and second extractions at 40°C, ethyl acetate (125 g) was added to the obtained concentrate. Sodium sulfate (2.5 g) was added to the obtained ethyl acetate solution, and after stirring at 30°C for 15 minutes, suction filtration was performed using a Kiriyama funnel with Oplite on 5A filter paper. Hexane (62.5 g) was added to the obtained liquid, and stirred at room temperature for 15 minutes to precipitate the product, and after suction filtration using 5A filter paper, the precipitate was washed with hexane (25 g), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p19) ( ME-200GGG-200PA ). The yield is 2.4 g. The molecular weight is shown in Table 1. HPLC: amine purity 91%.

[0806] NMR (CDCl3): 1.79ppm (m, 4H, -CO-NH-CH2- CH 2-CH2-O-(CH2-CH2-O)n-CH3, -CH2- CH2 -CH2-NH2), 2.91ppm (broad, 2H), 3.37ppm (m, 5H), 3.65ppm (m, about 3,800H, -NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 3.90ppm (t, 3H), 3.97ppm (d, 2H), 4.23ppm (wide, 2H, -NH-CO-O- CH2-CH2-O-(CH2-CH2-O)n-CH2-CH2-), 6.19ppm (broad, 1H), 7.00ppm (broad, 1H), 7.45ppm (broad, 1H), 7.61ppm (broad, 1H)

[0807] [Example 7]

[0808] Compound (p24) ME-200GF-200PA )

[0809]

Chemistry 102

[0810]

[0811] [Example 7-1]

[0812]

Chemistry 103

[0813]

[0814] L-phenylalanyl-glycine (Fmoc-Phe-Gly) (0.533 g, 1.2 × 10 -3 mole, manufactured by Watanabe Chemical Industry Co., Ltd.) and methoxy PEG having a propylamino group at the end (6.0 g, 2.9 × 10 -4 To the mixture was added anhydrous N,N'-dimethylformamide (60 g) and heated at 30°C to dissolve the mixture. Then, diisopropylethylamine (383 μL, 2.3×10 -3 mole, manufactured by Kanto Chemical Co., Ltd.) and (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinium hexafluorophosphate (COMU) (0.642 g, 1.5 × 10 -3mol, Sigma-Aldrich Company Ltd. system), and reacted it in a nitrogen atmosphere at room temperature for 1 hour. After the reaction was completed, it was diluted with chloroform (600g), saturated sodium bicarbonate aqueous solution (240g) was added, and it was stirred at room temperature for 15 minutes for washing. After separation of the aqueous layer and the organic layer, a saturated sodium bicarbonate aqueous solution (240g) was added to the organic layer again, and it was stirred at room temperature for 15 minutes for washing, and the organic layer was recovered. Magnesium sulfate (2.4g) was added to the obtained chloroform solution, and after stirring for 30 minutes for dehydration, a Kiriyama funnel with Oplite on 5A filter paper was used for suction filtration. The obtained liquid was concentrated at 40°C, and ethyl acetate (240g) was added to the concentrate and stirred until uniform, and then hexane (120g) was added, and the product was precipitated by stirring at room temperature for 15 minutes. Filter with 5A filter paper, recover the precipitate, dissolve it in ethyl acetate (240g) again, add hexane (120g), and stir at room temperature for 15 minutes to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (120 g), suction filtrated using 5A filter paper, and vacuum dried to obtain the above compound (p25) ( ME-200GF-Fmoc ). The yield was 5.1 g.

[0815] NMR (d6-DMSO): 1.62ppm (m, 2H, -CO-NH-CH2- CH2 -CH2-O-(CH2-CH2-O)n-CH3), 2.80ppm(m, 1H, -NH-CO-CH- CH2 -C6H5), 3.04ppm(m, 1H, -NH-CO-CH- CH2 -C6H5), 3.10ppm (m, 2H, -CO-NH- CH2 -CH2-CH2-O-(CH2-CH2-O)n-CH3),, 3.24ppm(s, 3H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.48ppm(m、about 1,900H、-CO-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3), 4.20ppm (m, 4H), 7.33ppm (m, 9H), 7.66ppm (m, 4H, Ar), 7.88ppm (d, 2H, Ar), 8.27ppm (t, 1H)

[0816] [Example 7-2]

[0817]

Chemistry 104

[0818]

[0819] ME-200GF-Fmoc (4.9 g, 2.3 × 10 -4 mol) was added with N,N'-dimethylformamide (29.4 g) and dissolved by heating at 30°C. Piperidine (1.55 g, 1.8 × 10 -2 mol, Wako Pure Chemical Industries, Ltd.), and reacted it in a nitrogen atmosphere at room temperature for 2 hours. After the reaction was completed, ethyl acetate (300 g) was added and stirred until uniform, hexane (150 g) was added, and the product was stirred at room temperature for 15 minutes to precipitate. Filtered with 5A filter paper, the precipitate was recovered, and then dissolved in ethyl acetate (300 g) again, hexane (150 g) was added, and the product was stirred at room temperature for 15 minutes to precipitate. Filtered with 5A filter paper, the precipitate was recovered, and then washed with hexane (150 g), filtered with 5A filter paper, and vacuum dried to obtain the above-mentioned compound (p26) ( ME-200GF-NH2 ). The yield is 3.9 g.

[0820] NMR (d6-DMSO): 1.62ppm (m, 2H, -CO-NH-CH2- CH2 -CH2-O-(CH2-CH2-O)n-CH3), 1.64ppm (broad, 1H), 2.59ppm (dd, 1H, -NH-CO-CH- CH2 -C6H5), 2.98ppm (dd, 1H, -NH-CO-CH- CH2 -C6H5), 3.10ppm(q, 2H, -CO-NH- CH2 -CH2-CH2-O-(CH2-CH2-O)n-CH3), 3.24ppm(s, 3H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.48ppm(m、about 1,900H、-CO-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3), 7.24ppm(m, 6H, -NH-CO-CH-CH2- C6H5 , -NH-), 7.73ppm(t, 1H), 8.12ppm(wide, 1H)

[0821] [Example 7-3]

[0822]

Chemistry 105

[0823]

[0824] The ME-200GF-NH2 (0.98 g, 4.7 × 10 -5mol) was dissolved in chloroform (4.9 g), and triethylamine (7.5 μL, 5.4×10 -5 Mol, Kanto Chemical Co., Ltd.) and a heterobifunctional PEG having a propylamino group protected by a tert-butoxycarbonyl group at one end and a succinimidyl carbonate group at the other end (1.3 g, 6.2 × 10 -5 mol, average molecular weight = about 23,000, "SUNBRIGHT BO-200TS" manufactured by NOF Corporation) was reacted in a nitrogen atmosphere at room temperature for 2 hours. After the reaction, it was concentrated at 40°C, ethyl acetate (98g) was added to the obtained concentrate to dissolve it, and then hexane (49g) was added and stirred at room temperature for 15 minutes to precipitate the product, which was filtered using 5A filter paper. The obtained precipitate was washed with hexane (49g), filtered using 5A filter paper, and vacuum dried to obtain the above compound (p27) ( ME- 200GF-200Boc ). The yield is 2.0 g.

[0825] NMR (CDCl3): 1.44ppm (s, 9H, -CH2-CH2-CH2-NH-CO-OC (CH3)3 ), 1.77ppm(m, 4H, -CO-NH-CH2- CH2 -CH2-O-(CH2-CH2-O)n-CH3, -CH2- CH2 -CH2-NH-CO-OC(CH3)3), 3.02ppm(m, 1H), 3.21ppm(m, 3H), 3.36ppm(m, 4H), 3.65ppm(m, about 3,800H, -NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.18ppm(m, 2H, -NH-CO-O- CH2 -CH2-O-(CH2-CH2-O)n-CH2-CH2-), 4.39ppm(q, 1H, -NH-CO- CH -CH2-C6H5), 5.03ppm (broad, 1H), 5.55ppm (d, 1H), 6.86ppm (broad, 1H), 7.02ppm (broad, 1H), 7.25ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )

[0826] [Example 7-4]

[0827]

Chemistry 106

[0828]

[0829] The ME-200GF-200Boc (1.8 g, 4.1 × 10 -5 mol) was dissolved in dichloromethane (9.0 g), and methanesulfonic acid (262 μL, 4.3×10 -3 Mol, Kanto Chemical Co., Ltd.), and reacted it in a nitrogen atmosphere at room temperature for 2 hours. Then, the reaction solution was diluted with toluene (18g), ion exchange water (18g) was added, and stirred at room temperature for 15 minutes, and the product was extracted into the water layer. According to the conditions described in Japanese Patent Laid-Open No. 2014-208786, the pH of the water layer was adjusted to 2.0 using 1mol / L hydrochloric acid, and the water layer was washed with a mixed solution of toluene and chloroform to remove polyethylene glycol impurities without amino groups. Then, an appropriate amount of 1mol / L sodium hydroxide aqueous solution was added to the water layer, and after adjusting the pH to 12, sodium chloride (4.5g) was dissolved. Chloroform (18g) was added thereto, stirred at room temperature for 15 minutes, and the product was extracted into the organic layer. After separating the water layer and the organic layer, chloroform (18g) was added to the water layer again, stirred at room temperature for 15 minutes, and the product was extracted into the organic layer. After concentrating the organic layers obtained in the first and second extractions at 40°C, ethyl acetate (90 g) was added to the obtained concentrate. Sodium sulfate (1.8 g) was added to the obtained ethyl acetate solution, and after stirring at 30°C for 15 minutes, suction filtration was performed using a Kiriyama funnel with Oplite on 5A filter paper. Hexane (45 g) was added to the obtained liquid, and stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using 5A filter paper, the precipitate was washed with hexane (18 g), suction filtration using 5A filter paper, and vacuum drying to obtain the above-mentioned compound (p24) ( ME-200GF-200PA ). The yield is 1.7 g. The molecular weight is shown in Table 1. HPLC: amine purity 87%.

[0830] NMR (CDCl3): 1.77ppm (m, 4H, -CO-NH-CH2- CH 2-CH2-O-(CH2-CH2-O)n-CH3, -CH2- CH2 -CH2-NH2), 2.87ppm (m, 2H), 3.07ppm (m, 1H), 3.30ppm (m, 3H), 3.38ppm (s, 3H, -NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.64ppm(m、about 3,800H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2-O)n-CH2-CH2-), 4.18ppm(m, 2H, -NH-CO-O- CH2 -CH2-O-(CH2-CH2-O)n-CH2-CH2-), 4.32ppm(q, 1H, -NH-CO- CH -CH2-C6H5), 5.56ppm (broad, 1H), 6.83ppm (broad, 1H), 6.96ppm (broad, 1H), 7.25ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )

[0831] [Example 8]

[0832] Compound (p28) ME-200GAV-200PA )

[0833]

Chemistry 107

[0834]

[0835] The same preparation method as in Example 4 was used as a raw material, using L-valine-L-alanine-glycine (Fmoc-Val-Ala-Gly) whose N-terminus was protected by a 9-fluorenylmethoxycarbonyl group (Fmoc group) to obtain the above compound (p28) ( ME- 200GAV-200PA ). The yield is 1.0 g. The molecular weight is shown in Table 1. HPLC: amine purity 90%.

[0836] NMR(d6-DMSO): 0.82ppm(dd, 6H, -NH-CO-CH-CH2-CH( CH3 )2), 1.21ppm (d, 3H, -NH-CO-CH(CH3)-), 1.62ppm (m, 4H, -CO-NH-CH2- CH2 -CH2-O-(CH2-CH2-O)n-CH3, -CH2- CH2 -CH2-NH2), 1.95ppm (m, 1H), 3.10ppm (m, 2H), 3.23ppm (s, 3H, -O-(CH2-CH2-O)n- CH3 )、3.60ppm(m、about 3,800H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.05ppm (m, 2H), 4.22ppm (m, 1H), 7.24ppm (broad, 1H), 7.69ppm (broad, 1H), 8.10ppm (broad, 2H)

[0837] [Example 9]

[0838] Compound (p29) ME-200GFGG-200PA )

[0839]

Chemistry 108

[0840]

[0841] The same preparation method as in Example 4 was used as a raw material, using glycine-glycine-L-phenylalanine-glycine (Fmoc-Gly-Gly-Phe-Gly) whose N-terminus was protected by a 9-fluorenylmethoxycarbonyl group (Fmoc group) to obtain the above compound (p29) ( ME-200GFGG-200PA ). The yield is 1.2 g. The molecular weight is shown in Table 1. HPLC: amine purity 91%.

[0842] NMR (d6-DMSO): 1.62ppm (m, 4H, -CO-NH-CH2- CH2 -CH2-O-(CH2-CH2-O)n-CH3, -CH2- CH2 -CH2-NH2), 2.80ppm (m, 1H), 3.10ppm (m, 2H), 3.23ppm (s, 3H, -O-(CH2-CH2-O)n- CH3 )、3.60ppm(m、about 3,800H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2- CH2 -O)n-CH2-CH2-), 4.05ppm (m, 2H), 4.47ppm (m, 1H), 7.20ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )、7.43ppm(wide, 1H)、7.62ppm(wide, 1H)、8.00ppm(wide, 1H)、8.12ppm(wide, 1H)、8.24ppm(wide, 1H)

[0843] [Example 10]

[0844] Compound (p30) ME-200GFG-200PA )

[0845]

Chemistry 109

[0846]

[0847] The same preparation method as in Example 4 was used as a raw material, using glycine-L-phenylalanine-glycine (Fmoc-Gly-Phe-Gly) whose N-terminus was protected by a 9-fluorenylmethoxycarbonyl group (Fmoc group) to obtain the above compound (p30) ( ME- 200GFG-200PA ). The yield is 1.1 g. The molecular weight is shown in Table 1. HPLC: amine purity 89%.

[0848] NMR (d6-DMSO): 1.62ppm (m, 4H, -CO-NH-CH2- CH2 -CH2-O-(CH2-CH2-O)n-CH3, -CH2- CH2 -CH2-NH2), 2.80ppm (m, 1H), 3.10ppm (m, 2H), 3.23ppm (s, 3H, -O-(CH2-CH2-O)n- CH3 )、3.60ppm(m、about 3,800H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2- CH2 -O)n-CH2-CH2-), 4.05ppm (m, 2H), 4.47ppm (m, 1H), 7.20ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )、7.34ppm(wide, 1H)、7.64ppm(wide, 1H)、8.10ppm(wide, 1H)、8.30ppm(wide, 1H)

[0849] [Example 11]

[0850] Compound (p31) ME-200GF-200PA(amide) )

[0851]

Chemistry 110

[0852]

[0853] The same manufacturing method as in Example 7 was used as raw materials, using L-phenylalanyl-glycine (Fmoc-Phe-Gly) whose N-terminus was protected by 9-fluorenylmethoxycarbonyl (Fmoc group) and a heterobifunctional PEG having a propylamino group protected by tert-butoxycarbonyl at one end and hexanoic acid-N-succinimidyl active ester at the other end (average molecular weight = about 21,000, "SUNBRIGHT BO-200HS" manufactured by NOF Corporation) to obtain the above-mentioned compound (p31) (ME-200GF-200PA (amide)) in which the heterobifunctional PEG was introduced by an amide bond instead of the carbamate bond of Example 7. The yield was 1.0 g. The molecular weight is shown in Table 1. HPLC: amine purity 91%.

[0854] NMR (d6-DMSO): 1.11ppm (m, 2H, -NH-CO-CH2-CH2- CH2 -CH2-CH2-O-(CH2-CH2-O)n-), 1.38ppm(m, 2H, -NH-CO-CH2- CH2 -CH2-CH2-CH2-O-(CH2-CH2-O)n-), 1.62ppm(m, 6H, -NH-CO-CH2-CH2-CH2- CH2 -CH2-O-(CH2-CH2-O)n-, -CO-NH-CH2- CH2 -CH2-O-(CH2-CH2-O)n-CH3, -CH2- CH2 -CH2-NH2), 2.02ppm(m, 2H, -NH-CO- CH2 -CH2-CH2-CH2-CH2-O-(CH2-CH2-O)n-), 2.80ppm(m, 1H), 3.10ppm(m, 2H), 3.23ppm(s, 3H, -O-(CH2-CH2-O)n- CH3 )、3.60ppm(m、about 3,800H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.47ppm (m, 1H), 7.20ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )、7.26ppm(wide, 1H)、7.64ppm(wide, 1H)、8.10ppm(wide, 1H)、8.30ppm(wide, 1H)

[0855] [Example 12]

[0856] Compound (p32)ME-200GLFG(D)-200PA )

[0857]

Chemistry 111

[0858]

[0859] The same manufacturing method as in Example 1 was used as a raw material, using glycine-D-phenylalanine-D-leucine-glycine with the N-terminus protected by a tert-butyloxycarbonyl (Boc) group, to obtain the above compound (p32) (ME-200GLFG(D)-200PA) having an amino acid as an optical isomer of D type. The yield was 1.1 g. The molecular weight is shown in Table 1. HPLC: Amine purity 93%.

[0860] NMR(CDCl3): 0.90ppm(t, 6H, -NH-CO-CH-CH2-CH( CH3 )2), 1.48ppm (width, 1H), 1.62ppm (t, 1H), 1.71ppm (m, 1H), 1.82ppm (m, 2H), 3.12ppm (m, 2H), 3.19ppm (d, 2H), 3.34ppm (m, 2H), 3.38ppm (s, 3H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.64ppm(m、about 3,800H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.10ppm (m, 2H), 4.34ppm (m, 1H), 4.50ppm (q, 1H), 6.46ppm (broad, 1H), 6.94ppm (broad, 1H), 7.08ppm (broad, 1H), 7.27ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )

[0861] [Example 13]

[0862] Compound (p34) LY-(ME-100GLFG(L)-100)2-PA )

[0863]

Chemistry 112

[0864]

[0865] [Example 13-1]

[0866]

Chemistry 113

[0867]

[0868] Glycyl-L-phenylalanyl-L-leucyl-glycine (Boc-Gly-Phe-Leu-Gly) (0.438 g, 8.8 × 10 -4 mol, manufactured by GenScript Biotech Corp.) and N-hydroxysuccinimide (0.128 g, 1.1 × 10 -3 mol, manufactured by Midori Kagaku Co., Ltd.) was dissolved in anhydrous N,N'-dimethylformamide (1.0 g), and N,N'-dicyclohexylcarbodiimide (0.229 g, 1.1 × 10 -3 Mole, Tama Chemical Industry Co., Ltd.) was reacted in a nitrogen atmosphere at room temperature for 1 hour. After adding anhydrous N,N'-dimethylformamide (3.0 g) for dilution, methoxy PEG having a propylamino group at the end (2.0 g, 2.2 × 10 -4 Mole, average molecular weight = about 9,000, "SUNBRIGHT MEPA-10T" manufactured by NOF Corporation), and reacted it in a nitrogen atmosphere at room temperature for 1 hour. Then, ethyl acetate (20 g) was added to dilute the reaction solution, and suction was filtered using a Kiriyama funnel covered with LS100 filter paper. Ethyl acetate (30 g) was added to the filtrate, and after stirring until uniform, hexane (25 g) was added, and the product was stirred at room temperature for 15 minutes to precipitate. Filter with 5A filter paper, recover the precipitate, and then dissolve it in ethyl acetate (50 g) again, add hexane (25 g), and stir at room temperature for 15 minutes to precipitate the product. Filter with 5A filter paper, recover the precipitate, wash it with hexane (25 g), filter with 5A filter paper, and vacuum dry to obtain the above-mentioned compound (p35) ( ME-100GLFG(L)-Boc ). The yield was 1.8 g.

[0869] NMR (CDCl3): 0.89ppm (d, 3H, -NH-CO-CH-CH2-CH( CH3 )2), 0.92ppm(d, 3H, -NH-CO-CH-CH2-CH( CH3 )2), 1.36ppm(s, 9H, -NH-CO-OC (CH3)3 )1.48ppm(m, 1H, -NH-CO-CH- CH2 -CH(CH3)2), 1.55ppm(m, 1H, -NH-CO-CH- CH2 -CH(CH3)2), 1.80ppm(m, 3H), 3.13ppm(dd, 1H, -NH-CO-CH- CH2 -C6H5), 3.21ppm (dd, 1H, -NH-CO-CH-CH2 -C6H5), 3.33ppm(m, 2H, -CO-NH- CH2 -CH2-CH2-O-(CH2-CH2-O)n-CH3), 3.38ppm(s, 3H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.65ppm(m、about 820H、-CO-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3), 3.91ppm(t, 1H, -NH-CO- CH -CH2-CH(CH3)2), 4.43ppm (broad, 1H), 4.55ppm (q, 1H, -NH-CO- CH -CH2-C6H5), 5.77ppm (broad, 1H), 6.76ppm (broad, 1H), 6.86ppm (broad, 1H), 6.90ppm (broad, 1H), 7.14ppm (broad, 1H), 7.20ppm (d, 2H, -NH-CO-CH-CH2- C6H5 ), 7.32ppm (m, 3H, -NH-CO-CH-CH2- C6H5 )

[0870] [Example 13-2]

[0871]

Chemistry 114

[0872]

[0873] The ME-100GLFG(L)-Boc (1.8 g, 2.0×10 -4 mol) was dissolved in dichloromethane (9.0 g), and methanesulfonic acid (1.3 mL, 2.0 × 10 -2mol, Kanto Chemical Co., Ltd.), and reacted it in a nitrogen atmosphere at room temperature for 2 hours. Then, the reaction solution was diluted with toluene (18g), ion exchange water (18g) was added, and the mixture was stirred at room temperature for 15 minutes, and the product was extracted into the water layer. An appropriate amount of 1 mol / L sodium hydroxide aqueous solution was added to the obtained water layer, and after adjusting the pH to 12, sodium chloride (4.5g) was dissolved. Chloroform (18g) was added thereto, stirred at room temperature for 15 minutes, and the product was extracted into the organic layer. After separating the water layer and the organic layer, chloroform (18g) was added to the water layer again, stirred at room temperature for 15 minutes, and the product was extracted into the organic layer. After concentrating the organic layers obtained in the first and second extractions at 40°C, ethyl acetate (36g) was added to the obtained concentrate. Sodium sulfate (0.90g) was added to the obtained ethyl acetate solution, and after stirring at 30°C for 15 minutes, suction filtration was performed using a Kiriyama funnel with Opliite on 5A filter paper. Hexane (18 g) was added to the obtained filtrate, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using 5A filter paper, the precipitate was washed with hexane (18 g), suction filtration using 5A filter paper, and vacuum dried to obtain the above-mentioned compound (p36) ( ME-100GLFG(L)-NH2 ). The yield is 1.4 g.

[0874] NMR (CDCl3): 0.89ppm (d, 3H, -NH-CO-CH-CH2-CH( CH3 )2), 0.91ppm(d, 3H, -NH-CO-CH-CH2-CH( CH3 )2), 1.53ppm (m, 2H, -NH-CO-CH- CH2 -CH(CH3)2), 1,70ppm(m, 1H, -NH-CO-CH-CH2- CH (CH3)2), 1.80ppm(m, 2H, -CO-NH-CH2- CH2 -CH2-O-(CH2-CH2-O)n-CH3), 3.10ppm(dd, 1H, -NH-CO-CH- CH2 -C6H5), 3.18ppm (dd, 1H, -NH-CO-CH- CH2 -C6H5), 3.33ppm(m, 7H), 3.74ppm(m, about 820H, -CO-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3), 4.31ppm (broad, 1H), 4.55ppm (t, 1H, -NH-CO- CH -CH2-C6H5), 6.91ppm (broad, 1H), 7.00ppm (broad, 1H), 7.28ppm (m, 5H, -NH-CO-CH-CH2-C6H5 )、7.98ppm(wide, 1H)

[0875] [Example 13-3]

[0876]

Chemistry 115

[0877]

[0878] A heterobifunctional PEG (5 g, 6.0 × 10 -5 mol) was dissolved in dichloromethane (12 g), and di(N-succinimidyl) carbonate (0.513 g, 2.0 × 10 -3 mol, Tokyo Chemical Industry Co., Ltd.) and pyridine (243 μL, 3.0 × 10 -3 Mol, Kanto Chemical Co., Ltd.), reacted for 7 hours under a nitrogen atmosphere at 27°C. Then, the reaction solution was diluted with dichloromethane (20g), filtered using 5A filter paper, and 2,6-di-tert-butyl-p-cresol (4.3mg) was added to the obtained liquid and stirred for 5 minutes. Then, a 5wt% sodium chloride aqueous solution (6g, pH5) was added, and the mixture was stirred at room temperature for 15 minutes for washing. After separating the aqueous layer and the organic layer, magnesium sulfate (1.0g) was added to the organic layer, and after stirring at room temperature for 30 minutes, it was filtered using a Kiriyama funnel with Oplite on 5A filter paper. After concentrating the obtained filtrate at 35°C, ethyl acetate (50g) was added to the obtained concentrate for dissolution. After dissolution, hexane (25g) was added, stirred at room temperature for 30 minutes to precipitate the product, and filtered using 5A filter paper. Hexane (25 g) was added to the obtained precipitate, and the mixture was stirred for 30 minutes, filtered using 5A filter paper, and vacuum dried to obtain the above-mentioned compound (p37) ( THP-10TS ). The yield is 4.3 g.

[0879] NMR (CDCl3): 1.52ppm (m, 2H, -tetrahydropyranyl )、1.59ppm(m、2H、 -tetrahydropyranyl )、1.73ppm(m、1H、 -tetrahydropyranyl )、1.85ppm(m、1H、 -tetrahydropyranyl )、2.85ppm(t、4H、 -succinimide )、3.64ppm(m、about 820H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2-O)n-CH2-CH2-), 3.80ppm (m, 2H), 3.88ppm (m, 2H, -tetrahydropyranyl ), 4.47ppm (m, 2H), 4.64ppm (t, 1H, -tetrahydropyranyl )

[0880] [Example 13-4]

[0881]

Chemistry 116

[0882]

[0883] The ME-100GLFG(L)-NH2 (3.64 g, 3.4×10 -4 mol) was dissolved in chloroform (17 g), and triethylamine (56.5 μL, 4.1×10 -4 mole, Kanto Chemical Co., Ltd.), THP-10TS obtained in Example 13-3 (2.80 g, 3.1 × 10 -4 mol), and reacted it in a nitrogen atmosphere at room temperature for 2 hours. After the reaction, it was concentrated at 40°C, and a 0.1 mol / L sodium citrate aqueous solution (120g, pH2.5) was added to the obtained concentrate to dissolve it, and then reacted it in a nitrogen atmosphere at room temperature for 4 hours. After the reaction, chloroform (50g) and 2,6-di-tert-butyl-p-cresol (5.0mg) were added and stirred for 10 minutes. After separating the aqueous layer and the organic layer, magnesium sulfate (3.2g) was added to the organic layer, and after stirring at 40°C for 30 minutes, it was filtered using a Kiriyama funnel with Oplite on 5A filter paper. After concentrating the obtained filtrate, ethyl acetate (50g) was added to the obtained concentrate to dissolve it. After dissolution, hexane (25g) was added, stirred at room temperature for 15 minutes to precipitate the product, and filtered using 5A filter paper. Hexane (25 g) was added to the obtained precipitate, stirred for 15 minutes, filtered using 5A filter paper, and vacuum dried to obtain the above compound (p38) (ME-100GLFG(L)-100HO). The yield was 5.30 g.

[0884] NMR(CDCl3): 0.90ppm(t, 6H, -NH-CO-CH-CH2-CH( CH3 )2), 1.48ppm (width, 1H), 1.62ppm (t, 1H), 1.71ppm (m, 1H), 1.82ppm (m, 2H), 3.19ppm (d, 2H), 3.34ppm (m, 2H), 3.38ppm (s, 3H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3)、3.64ppm(m、about 820H、-NH-CH2-CH2-CH2-O-( CH2- CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.10ppm (m, 2H), 4.34ppm (m, 1H), 4.50ppm (q, 1H), 6.46ppm (broad, 1H), 6.94ppm (broad, 1H), 7.08ppm (broad, 1H), 7.27ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )

[0885] [Example 13-5]

[0886]

Chemistry 117

[0887]

[0888] ME-100GLFG(L)-100HO (5.0 g, 2.8 × 10 -4 mol) was dissolved in dichloromethane (12 g), and di(N-succinimidyl) carbonate (0.285 g, 1..1×10 -3 mol, Tokyo Chemical Industry Co., Ltd.) and pyridine (135 μL, 1.7 × 10 -3 mol, Kanto Chemical Co., Ltd.), and reacted it under a nitrogen atmosphere at 27°C for 7 hours. Then, the reaction solution was diluted with dichloromethane (20g), filtered using 5A filter paper, and 2,6-di-tert-butyl-p-cresol (4.3mg) was added to the obtained liquid and stirred for 5 minutes. Then, a 5wt% sodium chloride aqueous solution (6g, pH5) was added, and the mixture was stirred at room temperature for 15 minutes for washing. After separating the aqueous layer and the organic layer, magnesium sulfate (1.0g) was added to the organic layer, and after stirring at room temperature for 30 minutes, it was filtered using a Kiriyama funnel with Oplite on 5A filter paper. After concentrating the obtained filtrate at 35°C, ethyl acetate (50g) was added to the obtained concentrate for dissolution. After dissolution, hexane (25g) was added, stirred at room temperature for 30 minutes to precipitate the product, and filtered using 5A filter paper. Hexane (25 g) was added to the obtained precipitate, and the mixture was stirred for 30 minutes, and suction was performed using 5A filter paper, and vacuum dried to obtain the above-mentioned compound (p39) ( ME-100GLFG(L)- 100TS ). The yield is 4..2g.

[0889] NMR(CDCl3): 0.90ppm(t, 6H, -NH-CO-CH-CH2-CH( CH3)2), 1.44ppm(s, 9H, -CH2-CH2-CH2-NH-CO-OC (CH3)3 ), 1.64ppm (m, 1H), 1.76ppm (m, 5H), 2.85ppm (t, 4H, -succinimide ), 3.20ppm (m, 4H), 3.33ppm (m, 2H, -CO-NH- CH2 -CH2-CH2-O-(CH2-CH2-O)n-CH3), 3.38ppm(s, 3H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.64ppm(m、about 820H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.10ppm(m, 2H, -NH-CO-O- CH2 -CH2-O-(CH2-CH2-O)n-CH2-CH2-), 4.32ppm (m, 1H), 4.50ppm (q, 1H), 5.02ppm (broad, 1H), 6.45ppm (broad, 1H), 6.93ppm (broad, 1H), 7.06ppm (broad, 1H), 7.13ppm (broad, 1H), , 7.27ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )

[0890] [Example 13-6]

[0891]

Chemistry 118

[0892]

[0893] The ME-100GLFG(L)-100TS (3.6 g, 2.0×10 --4 mol) was dissolved in N,N'-dimethylformamide (7.0 g), and L-lysine ethyl ester dihydrochloride (26 mg, 1.1 × 10 -4 mol, manufactured by Sigma-Aldrich Company Ltd.), triethylamine (73 mg, 7.2 × 10 -4mol), and reacted at 40°C for 5 hours under a nitrogen atmosphere. The reaction solution was diluted with ethyl acetate (36 g), and then filtered using a Kiriyama funnel covered with 5A filter paper. Hexane (18 g) was added to the obtained filtrate, and the product was precipitated by stirring at room temperature for 15 minutes. After filtering with 5A filter paper, the precipitate was washed with hexane (18 g), filtered with 5A filter paper, and vacuum dried to obtain the above-mentioned compound (p40) (LY- (ME- 100GLFG(L)-100)2-CE ). The yield is 3.1 g.

[0894] NMR(CDCl3): 0.90ppm(t, 6H, -NH-CO-CH-CH2-CH( CH3 )2), 1.28ppm(t, 3H, -CO-O-CH2- CH3 ), 1.36ppm (m, 2H), 1.48ppm (width, 1H), 1.52ppm (m, 2H), 1.62ppm (t, 1H), 1.70ppm (m, 2H), 1.82ppm (m, 3H), 3 .15ppm (q, 2H), 3.19ppm (d, 2H), 3.34ppm (m, 2H), 3.38ppm (s, 6H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.64ppm(m、about 1,600H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.10ppm (m, 2H), 4.21ppm (m, 6H), 4.34ppm (m, 2H), 4.50ppm (q, 1H), 4.90ppm (broad, 1H), 5.37ppm (d, 1H), 6.46ppm (broad, 1H), 6.94ppm (broad, 1H), 7.08ppm (broad, 1H), 7.27ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )

[0895] [Example 13-7]

[0896]

Chemistry 119

[0897]

[0898] LY-(ME-100GLFG(L)-100)2-CE (1.8 g, 5.0×10 -5mol) was dissolved in a 0.13 mol / L sodium hydroxide aqueous solution (18 g), and it was reacted at room temperature for 3 hours under a nitrogen atmosphere. After dissolving sodium chloride (4.5 g) in the reaction solution, the pH of the aqueous layer was adjusted to 8.5 using 85% phosphoric acid, and dichloromethane (11 g) was added thereto, stirred at room temperature for 15 minutes, and the product was extracted into an organic layer. After separating the aqueous layer and the organic layer, dichloromethane (11 g) was added to the aqueous layer again, stirred at room temperature for 15 minutes, and the product was extracted into an organic layer. After concentrating the organic layers obtained in the first and second extractions at 40°C, ethyl acetate (36 g) was added to the obtained concentrate. To the ethyl acetate solution obtained, magnesium sulfate (0.90 g) was added, and after stirring at 30°C for 30 minutes, suction filtration was performed using a Kiriyama funnel with Oplite on 5A filter paper. Hexane (18 g) was added to the obtained filtrate, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After suction filtration using 5A filter paper, the precipitate was washed with hexane (18 g), suction filtration using 5A filter paper, and vacuum dried to obtain the above-mentioned compound (p41) ( LY-(ME-100GLFG(L)-100)2-C2 ). The yield is 1.4 g.

[0899] NMR(CDCl3): 0.90ppm(t, 6H, -NH-CO-CH-CH2-CH( CH3 )2),, 1.36ppm (m, 2H), 1.48ppm (width, 1H), 1.52ppm (m, 2H), 1.62ppm (t, 1H), 1.70ppm (m, 2H), 1.82ppm (m, 3H), 3.15ppm (m, 2H), 3.19ppm (d, 2H), 3.34ppm (m, 2H), 3.38ppm (s, 6H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.64ppm(m、about 1,600H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.10ppm (m, 2H), 4.21ppm (m, 4H), 4.34ppm (m, 2H), 4.50ppm (q, 1H), 4.90ppm (broad, 1H), 5.37ppm (d, 1H), 6.46ppm (broad, 1H), 6.94ppm (broad, 1H), 7.08ppm (broad, 1H), 7.27ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )

[0900] [Example 13-8]

[0901]

Chemistry 120

[0902]

[0903] LY-(ME-100GLFG(L)-100)2-C2 (1.5 g, 4.0 × 10 -5 mol) and 2,6-di-tert-butyl-p-cresol (1.2 mg) were dissolved in toluene (6.0 g), and N-hydroxysuccinimide (16 mg, 1.4 × 10 -4 mol), 1,3-dicyclohexylcarbodiimide (20 mg, 1.0×10 -4 mol), and reacted at 40°C for 4 hours under a nitrogen atmosphere. Next, N-(tert-butoxycarbonyl)-1,3-diaminopropane was added, and reacted at 40°C for 4 hours under a nitrogen atmosphere. The reaction solution was diluted with toluene (12 g), and filtered using a Kiriyama funnel with Oplite on 5A filter paper. The obtained filtrate was diluted with ethyl acetate (18 g), and hexane (18 g) was added. The product was stirred at room temperature for 15 minutes to precipitate, and filtered using 5A filter paper. The precipitate was washed with hexane (18 g), filtered using 5A filter paper, and vacuum dried to obtain the above-mentioned compound (p42) ( LY-(ME-100GLFG(L)-100)2-Boc ). The yield is 1.1 g.

[0904] NMR(CDCl3): 0.90ppm(t, 6H, -NH-CO-CH-CH2-CH( CH3 )2),, 1.36ppm (m, 2H), 1.44ppm (s, 9H), 1.48ppm (width, 1H), 1.52ppm (m, 2H), 1.62ppm (m, 3H), 1.70ppm (m, 2H), 1.82ppm (m, 3H), 3.15ppm (m, 4H), 3.19ppm (d, 2H), 3.29ppm (m, 2H), 3.34ppm (m, 2H), 3.38ppm (s, 6H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.64ppm(m、about 1,600H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2-O)n-CH2-CH2-), 4.10ppm (m, 3H), 4.21ppm (m, 4H), 4.34ppm (m, 1H), 4.50ppm (q, 1H), 5.08ppm (broad, 1H), 5.56ppm (d, 1H), 6.46ppm (broad, 1H), 6.94ppm (broad, 1H), 7.08ppm (broad, 1H), 7.27ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )

[0905] [Example 13-9]

[0906]

Chemistry 121

[0907]

[0908] LY-(ME-100GLFG(L)-100)2-Boc (0.90 g, 2.5×10 -5 mol) was dissolved in dichloromethane (4.5 g), and methanesulfonic acid (162 μL, 2.5×10 -3 mol), and reacted it at room temperature for 2 hours under a nitrogen atmosphere. After diluting the reaction solution with toluene (9.0g), add ion exchange water (23g), stir at room temperature for 15 minutes, and extract the product into the water layer. Then, wash the water layer with a mixed solution of toluene and chloroform to remove polyethylene glycol impurities that do not have amino groups. Then, add an appropriate amount of 1mol / L sodium hydroxide aqueous solution to the water layer, adjust the pH to 12, and dissolve sodium chloride (2.3g). Add chloroform (9.0g) thereto, stir at room temperature for 15 minutes, and extract the product into the organic layer. After separating the water layer and the organic layer, add chloroform (9.0g) to the water layer again, stir at room temperature for 15 minutes, and extract the product into the organic layer. After concentrating the organic layers obtained in the first and second extractions at 40°C, add ethyl acetate (36g) to the obtained concentrate. Sodium sulfate (0.90 g) was added to the obtained ethyl acetate solution, and the mixture was stirred at 30°C for 30 minutes, and then filtered using a Kiriyama funnel with Oplite on 5A filter paper. Hexane (18 g) was added to the obtained filtrate, and the mixture was stirred at room temperature for 15 minutes to precipitate the product. After filtering using 5A filter paper, the precipitate was washed with hexane (18 g), filtered using 5A filter paper, and vacuum dried to obtain the above-mentioned compound (p34) ( LY-(ME-100GLFG(L)-100)2-PA ). The yield is 0.8 g. The molecular weight is shown in Table 1. HPLC: amine purity 92%.

[0909] NMR(CDCl3): 0.90ppm(t, 6H, -NH-CO-CH-CH2-CH( CH3)2),, 1.37ppm (m, 2H), 1.48ppm (width, 1H), 1.52ppm (m, 2H), 1.62ppm (m, 3H), 1.70ppm (m, 2H), 1.82ppm (m, 3H), 2.84ppm (m, 2H), 3.15ppm (m, 2H), 3.19ppm (d, 2H), 3.34ppm (m, 2H), 3.38ppm (s, 6H, -CO-NH-CH2-CH2-CH2-O-(CH2-CH2-O)n- CH3 )、3.64ppm(m、about 1,600H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2-CH2 -O)n-CH2-CH2-), 4.10ppm (m, 3H), 4.21ppm (m, 4H), 4.34ppm (m, 1H), 4.50ppm (q, 1H), 5.58ppm (broad, 1H), 6.46ppm (broad, 1H), 6.94ppm (broad, 1H), 7.08ppm (broad, 1H), 7.27ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )

[0910] [Comparative Example 1]

[0911] Compound (p33) ME-200F-200PA )

[0912]

Chemistry 122

[0913]

[0914] The above compound (p33) (ME-200F-200PA) was obtained by the same production method as in Example 4, using L-phenylalanine (Fmoc-Phe) protected at the N-terminus by 9-fluorenylmethoxycarbonyl (Fmoc group) as a raw material. The yield was 1.8 g. The molecular weight is shown in Table 1. HPLC: amine purity 92%.

[0915] NMR (d6-DMSO): 1.62ppm (m, 4H, -CO-NH-CH2- CH2 -CH2-O-(CH2-CH2-O)n-CH3, -CH2- CH2 -CH2-NH2), 2.80ppm (m, 1H), 3.10ppm (m, 2H), 3.23ppm (s, 3H, -O-(CH2-CH2-O)n- CH3 )、3.60ppm(m、about 3,800H、-NH-CH2-CH2-CH2-O-( CH2-CH2 -O)n-CH3、-NH-CO-O-CH2-CH2-O-( CH2- CH2 -O)n-CH2-CH2-), 3.95ppm (m, 2H), 4.13ppm (m, 1H), 7.20ppm (m, 5H, -NH-CO-CH-CH2- C6H5 )、7.38ppm(wide, 1H)、、7.91ppm(wide, 1H)

[0916] The average molecular weights of the polyethylene glycol derivatives obtained in Examples 1 to 13 and Comparative Example 1 are shown below.

[0917]

Table 1

[0918]

[0919] [Example 14]

[0920] Stability test in serum

[0921] Add 1 mL of mouse or human serum to a 1.5 mL microcentrifuge tube, and add various polyethylene glycol derivatives to a concentration of 5.0 mg / mL. After incubation at 37 ° C for 96 hours, sample 200 μL, add acetonitrile thereto, stir with a vortexer for 1 minute to precipitate the protein in the serum, and recover the supernatant after centrifugation. Then, in order to remove hydrophobic substances such as fatty acids, hexane is added to the recovered liquid, stirred with a vortexer for 1 minute, and the lower layer is recovered after centrifugation. The solution is concentrated under vacuum conditions to recover polyethylene glycol derivatives from the serum. Then, GPC analysis is performed to calculate the decomposition rate of the degradable polyethylene glycol derivatives.

[0922] The decomposition rate is calculated as follows.

[0923] Decomposition rate = (40 kDa peak area % before the test - 40 kDa peak area % after the test) ÷ (40 kDa peak area % before the test) × 100

[0924] The results are shown in Table 2 below.

[0925]

Table 2

[0926]

[0927] This test revealed that the decomposition rate of any degradable polyethylene glycol derivative after 96 hours was less than 20%, and in particular, GLFG(L), G(cit)V, etc. had a low decomposition rate and were stable in the blood.

[0928] [Example 15]

[0929] Dissociation assay using cells

[0930] 10 mL of RPMI-1640 (10% FBS Pn / St) was used to inoculate 10 × 10 6 After culturing RAW264.7 of cell at 37°C for 24 hours, the medium was replaced with a medium in which various polyethylene glycol derivatives were dissolved to a concentration of 10 mg / mL, and cultured at 37°C for 96 hours. After culturing, the cells were dissolved in a 1% SDS solution, diluted with PBS, acetonitrile was added thereto, and stirred with a vortex for 1 minute to precipitate the protein in the cell lysate. After centrifugation, the supernatant was recovered. Then, in order to remove hydrophobic substances such as fatty acids, hexane was added to the recovered solution, stirred with a vortex for 1 minute, and the lower layer was recovered after centrifugation. The solution was concentrated under vacuum conditions to recover the polyethylene glycol derivatives from the cells.

[0931] Furthermore, in order to confirm the decomposition in the medium used for cell culture, the cells were cultured only in a medium in which various polyethylene glycol derivatives were dissolved to a concentration of 10 mg / mL at 37°C for 96 hours, and the polyethylene glycol derivatives were recovered in the same manner as above.

[0932] Then, the various polyethylene glycol derivatives recovered were subjected to GPC analysis, and the decomposition rate of the degradable polyethylene glycol derivative was calculated using the same calculation formula as in Example 14.

[0933] The results are shown in Table 3. In addition, the GPC spectra before and after the cell experiment are shown in Table 3. Figure 1 and Figure 2 shown.

[0934]

Table 3

[0935]

[0936] In the polyethylene glycol derivative having an oligopeptide as a degradable linker, it was confirmed that it was effectively degraded in cells from a molecular weight of 40,000 to 20,000. In addition, ME-200GLFG(D)-200PA using D-amino acids, which are less naturally present, was also confirmed to be degraded, although the degradation rate was low. On the other hand, the polyethylene glycol derivative using phenylalanine as a linker of Comparative Example 1 and the non-degradable methoxy PEG amine 40 kDa were not confirmed to be degraded in cells.

[0937] [Example 16]

[0938] PEGylation of salmon calcitonin (sCT)

[0939] The amino acid sequence:

[0940] The salmon calcitonin (sCT) (5 mg, 1.5 × 10 -6 mol, PH Japan Co., Ltd.) was dissolved in 100 mM sodium acetate buffer (pH 5.0), and ME-200GL FG (L) -200AL obtained in Example 2 or methoxy PEG aldehyde 40 kDa (180 mg, 4.5 × 10 -6 mole) and the reducing agent 2-picolyl borane (2.0×10 -5 The reaction mixture was dialyzed using a 10 mM sodium acetate buffer (pH 5.0) and purified by ion exchange chromatography using HiTrap SPHP (5 mL, manufactured by GE Healthcare) to obtain ME-200GLFG(L)-200-sCT or methoxy PEG40kDa-sCT. The molar yield was 44%.

[0941] RPLC analysis

[0942] Device: "ALLIANCE" manufactured by Waters Corporation

[0943] Detector: UV (280nm)

[0944] Chromatographic column: Inertsil WP300 C18 (GL Sciences)

[0945] Mobile phase A: 0.05% TFA-H2O

[0946] Mobile phase B: 0.05% TFA-ACN

[0947] Gradient: B30% (0min) → B40% (5min) → B50% (15min) → B100% (16min) → B100% (20min)

[0948] Flow rate: 1.0mL / min

[0949] Column temperature: 40°C

[0950] The purity of PEGylated sCT was calculated according to the above RPLC analysis conditions. Figure 3 shown.

[0951] RPLC purity of ME-200GLFG(L)-200-sCT: 99%

[0952] RPLC purity of methoxy PEG 40kDa-sCT 99%

[0953] MALDI-TOF-MS analysis

[0954] Device: "autoflex3" manufactured by Bruker Corporation

[0955] Sample: 0.5 mg / mL, PBS solution

[0956] Matrix: α-cyano-4-hydroxycinnamic acid (CHCA) saturated solution (0.01% TFA-H2O: ACN = 2:1)

[0957] Mix the sample (1 μL) and matrix (19 μL) and take 1 μL as the target spot.

[0958] The molecular weights of raw PEG and PEGylated sCT were determined under the above-mentioned MALDI-TOF-MS analysis conditions.

[0959] Figure 4 In FIG. 1 , the MALDI-TOF-MS results of ME-200GLFG(L)-200AL and ME-200GLFG(L)-200-sCT, which are raw materials, are shown together.

[0960] The molecular weight of ME-200GLFG(L)-200-sCT is 46,289

[0961] The molecular weight of ME-200GLFG(L)-200AL is 43,210

[0962] Figure 5 In FIG. 1 , the MALDI-TOF-MS results of methoxy PEG aldehyde 40 kDa and methoxy PEG 40 kDa-sCT as raw materials are shown together.

[0963] The molecular weight of methoxy PEG 40kDa-sCT is 46,427

[0964] Methoxy PEG aldehyde 40kDa molecular weight 43,303

[0965] It was confirmed that the molecular weight of PEGylated sCT increased compared to the molecular weight of the PEG derivative as a raw material by almost only the molecular weight of sCT.

[0966] SDS-PAGE analysis

[0967] Kit: NuPAGE (registered trademark) Bis-Tris Precast Gel (gel concentration 4-12%) manufactured by Thermo Fisher Scientific Inc.

[0968] Staining solution: Coomassie Brilliant Blue solution (CBB solution) or iodine staining solution (BaCl2+I2 solution)

[0969] The evaluation of PEGylated sCT was performed according to the recommended assay conditions of the above-mentioned SDS-PAGE kit. Figure 6 As shown. In PEGylated sCT, bands were observed by CBB staining, which selectively stains proteins and peptides, and further, bands were also observed by iodine staining, which stains polyethylene glycol. The observation of bands by both stainings confirmed that the polyethylene glycol derivative was bonded to sCT.

[0970] [Example 17]

[0971] PEGylation of human growth hormone (hGH)

[0972] The amino acid sequence:

[0973] MFPTIPLSRL FDNAMLRAHR LHQLAFDTYQ EFEEAYIPKE QKYSFLQNPQ TSLCFSESIPTPSNREETQQ KSNLELLRIS LLLIQSWLEP VQFLRSVFAN SLVYGASDSN VYDLLKDLEE GIQTLMGRLEDGSPRTGQIF KQTYSKFDTN SHNDDALLKN YGLLYCFRKD MDKVETFLRI VQCRSVEGSC GF (SEQ ID NO: 2) Human Growth Hormone (hGH) (4 mg, 1.8 × 10 -7 mol, Shenandoah Biotechnology) was dissolved in 100 mM sodium acetate buffer (pH 5.5), and ME-200GL FG (L) -200AL obtained in Example 2 or methoxy PEG aldehyde 40 kDa (36 mg, 9.0 × 10 -7 mole) and sodium cyanoborohydride as a reducing agent (9.0×10 -6The hGH concentration was adjusted to 1.0 mg / mL and the mixture was reacted at 25°C for 24 hours. The reaction solution was then dialyzed using a 10 mM sodium acetate buffer (pH 4.7) and purified by ion exchange chromatography using HiTrap SP HP (5 mL, manufactured by GE Healthcare) to obtain ME-200GLFG(L)-200-hGH or methoxy PEG40kDa-hGH. The molar yield was 30%.

[0974] RPLC analysis

[0975] Installation: “ALLIANCE” by WATERS CORPORATION

[0976] Detector: UV (280nm)

[0977] Chromatographic column: Inertsil WP300 C18 (CL SCIENCES)

[0978] Mobile phase A: 0.05% TFA-H2O

[0979] Mobile phase B: 0.05% TFA-ACN

[0980] Gradient: B40% (0 min) → B80% (25 min) → B90% (27 min) → B40% (27.1 min)

[0981] Flow rate: 1.0mL / min

[0982] Column temperature: 40°C

[0983] Under the above RPLC analysis conditions, the purity of PEGylated hGH was calculated.

[0984] RPLC purity of ME-200GLFG(L)-200-hGH: 90%

[0985] RPLC purity of methoxy PEG40kDa-hGH is 97%

[0986] MALDI-TOF-MS analysis

[0987] Device: "autoflex3" manufactured by Bruker Corporation

[0988] Sample: 0.5 mg / mL, PBS solution

[0989] Matrix: cinnamic acid (SA) saturated solution (0.01% TFA-H2O: ACN = 2:1)

[0990] Mix the sample (1 μL) and matrix (19 μL) and take 1 μL as the target spot.

[0991] Under the above MALDI-TOF-MS analysis conditions, the molecular weight of PEGylated hGH was determined. Figure 7 and Figure 8 shown.

[0992] The molecular weight of ME-200GLFG(L)-200-hGH is 65,153

[0993] The molecular weight of methoxy PEG40kDa-hGH is 65,263

[0994] It was confirmed that the molecular weight of PEGylated hGH was higher than that of the PEG derivative used as the raw material (reference Figure 4 and Figure 5 )'s molecular weight, roughly only the molecular weight of hGH has increased.

[0995] SDS-PAGE analysis

[0996] Kit: NuPAGE (registered trademark) Bis-Tris Precast Gel (gel concentration 4-12%) manufactured by Thermo Fisher Scientific Inc.

[0997] Staining solution: Coomassie Brilliant Blue solution (CBB solution) or iodine staining solution (BaCl2+I2 solution)

[0998] The evaluation of PEGylated hGH was performed according to the recommended assay conditions of the above-mentioned SDS-PAGE kit. Fig. 9 As shown. In PEGylated hGH, a band was observed under CBB staining that selectively stains proteins and peptides, and further, a band was also observed under iodine staining that stains polyethylene glycol. The bands were observed under both stainings, confirming that the polyethylene glycol derivative was bonded to hGH.

[0999] [Example 18]

[1000] PEGylation of GCSF

[1001] The amino acid sequence:

[1002] Granulocyte colony stimulating factor (GCSF) (100 μg, 5.3×10 -9 mol, PeproTech, Inc.) was dissolved in 10 mM sodium acetate buffer (pH 4.6, containing 5% sorbitol), and ME-200GL FG (L) -200AL obtained in Example 2 and sodium cyanoborohydride (5.3 × 10 -7 The reaction mixture was diluted with 10 mM sodium acetate buffer (pH 4.6) and purified by ion exchange chromatography using HiTrap SP HP (5 mL, manufactured by GE Healthcare) to obtain ME-200GLFG(L)-200-GCSF. The molar yield was 64%.

[1003] RPLC analysis

[1004] Installation: “ALLIANCE” by WATERS CORPORATION

[1005] Detector: UV (280nm)

[1006] Chromatographic column: Inertsil WP300 C18 (CL SCIENCES)

[1007] Mobile phase A: 0.1% TFA-H2O

[1008] Mobile phase B: 0.1% TFA-ACN

[1009] Gradient: B40% (0min) → B70% (25min) → B90% (27min) → B40% (29min)

[1010] Flow rate: 1.0mL / min

[1011] Column temperature: 40°C

[1012] Under the above RPLC analysis conditions, the purity of PEGylated GCSF was calculated.

[1013] RPLC purity of ME-200GLFG(L)-200-GCSF: 97%

[1014] MALDI-TOF-MS analysis

[1015] Device: "autoflex3" manufactured by Bruker Corporation

[1016] Sample: 0.5 mg / mL, PBS solution

[1017] Matrix: cinnamic acid (SA) saturated solution (0.01% TFA-H2O: ACN = 2:1)

[1018] Mix the sample (1 μL) and matrix (19 μL) and take 1 μL as the target spot.

[1019] The molecular weight of PEGylated GCSF was measured under the above-mentioned MALDI-TOF-MS analysis conditions. The molecular weight of ME-200GLFG(L)-200-GCSF was 62,266. It was confirmed that the molecular weight of PEGylated GCSF increased by almost only the molecular weight of GCSF compared to the molecular weight of the PEG derivative as the raw material.

[1020] SDS-PAGE analysis

[1021] Kit: NuPAGE (registered trademark) Bis-Tris Precast Gel (gel concentration 4-12%) manufactured by Thermo Fisher Scientific Inc.

[1022] Staining solution: Coomassie Brilliant Blue solution (CBB solution) or iodine staining solution (BaCl2+I2 solution)

[1023] The evaluation of PEGylated GCSF was performed according to the recommended assay conditions of the above-mentioned SDS-PAGE kit. Fig. 9 As shown. In PEGylated GCSF, bands were observed under CBB staining that selectively stains proteins and peptides, and further, bands were also observed under iodine staining that stains polyethylene glycol. The observation of bands under both stainings confirmed that the polyethylene glycol derivative was bonded to GCSF.

[1024] [Example 19]

[1025] Evaluation of the physiological activity of PEGylated salmon calcitonin (sCT)

[1026] The physiological activities of four groups of ME-200GLFG(L)-200-sCT obtained in Example 16, which is sCT bonded with a degradable polyethylene glycol derivative with a molecular weight of 40,000, methoxy PEG40kDa-sCT bonded with non-degradable methoxy PEG40kDa, unmodified sCT, and normal saline, were compared and evaluated using animal experiments. The mice were Balb / c (8 weeks old, male), and the sCT solution and the PEGylated sCT solution were each prepared with normal saline to a sCT concentration of 0.5 mg / mL, and the solution was administered until the sCT dosage reached 40 μg / kg. A small amount of blood was collected from the mice at 1, 3, 6, and 24 hours, and the calcium concentration was measured using Calcium E-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The results are as follows. Fig.10 shown.

[1027] All sCTs significantly reduced calcium concentrations compared to the saline (PBS) group. Six hours after administration, an increase in calcium concentration was observed in unmodified sCT, but low calcium concentrations were maintained in ME-200GLFG(L)-200-sCT and methoxyPEG40kDa-sCT. This suggests that PEGylation prolongs the blood half-life of sCT and extends its physiological activity.

[1028] [Example 20]

[1029] In vivo kinetic studies using animal models (radioactive isotopes)

[1030] The sCT obtained in Example 16, ME-200GLFG(L)-200-sCT, which is a degradable polyethylene glycol derivative with a molecular weight of 40,000, and methoxy PEG40kDa-sCT, which is a non-degradable methoxy PEG40kDa, were dissolved in a 50mM sodium bicarbonate aqueous solution to a concentration of 0.1mg / mL, and Bolton-Hunter reagent (0.4625MBq) was added thereto. After stirring with a vortexer, the mixture was allowed to react overnight at room temperature. The reaction solution was separated using a PD-10 column, and the fractions were confirmed to contain polyethylene glycol colorimetric reagents (ammonium thiocyanate and cobalt nitrate) and a gamma counter. 125 I fraction, is recovered.

[1031] The in vivo dynamics were evaluated by animal experiments using the two radioactive isotope-labeled PEGylated sCT obtained and sCT labeled in the same manner. The mouse species was Balb / c (8-week-old, male), and the PEGylated sCT solution was prepared with physiological saline to a concentration of 50 mg / mL for unlabeled PEGylated sCT, and a trace amount of radioactive isotope-labeled PEGylated sCT was added, and 20 μL was administered via the tail vein of the mouse. Then, blood and various organs (liver, kidney, spleen, lung, brain, heart, stomach, pancreas, intestine, testis, thyroid, etc.) were taken from the mouse at 1, 3, 6, and 48 hours, and the retention amount of the labeled PEGylated sCT was measured using a gamma counter.

[1032] As a result of in vivo kinetic studies of radioactive isotope-labeled sCT bound to a degradable polyethylene glycol derivative, ME-200GLFG(L)-200-sCT, and methoxy PEG40kDa-sCT bound to a non-degradable methoxy PEG40kDa, Fig.11 The blood concentration is shown in Figures 12 to 15 The retention amounts in representative organs, namely, the liver, kidney, spleen, and lung, are shown in FIG.

[1033] This result demonstrates that ME-200GLFG(L)-200-sCT has a similar blood half-life to that of methoxy PEG40 kDa-sCT, which is generally not degradable, and also has a similar distribution tendency in the body.

[1034] [Example 21]

[1035] Cavitation evaluation test in animal experiments

[1036] Using the degradable polyethylene glycol derivative ME-200GLFG(L)-200PA with a molecular weight of 40,000 and an amino group at the end obtained in Example 1 and the non-degradable methoxy PEG amine 40kDa, an evaluation of cavitation was performed via animal experiments. The mouse species was Balb / c (8 weeks old, male), and the polyethylene glycol solution was prepared with physiological saline to a concentration of 100 mg / mL of the polyethylene glycol derivative, and 20 μL was administered via the tail vein of the mouse. The administration was continued 3 times a week for 4 weeks. After the administration, the mice were perfused and fixed with a 4% paraformaldehyde aqueous solution, and paraffin sections were prepared. HE staining and immunostaining with anti-PEG antibodies were performed to evaluate cavitation in the choroid plexus epithelial cells of the brain. As immunostaining, an immunostaining kit (BOND Refine Polymer Detection Kit, manufactured by Leica) and an anti-PEG antibody (B-47 antibody, manufactured by Abcam Inc.) were used for implementation. The image of the choroid plexus section of the brain after immunostaining with anti-PEG antibodies is shown as follows Fig.16 and Fig.17 shown.

[1037] The results showed that the degradable polyethylene glycol ME-200GLFG(L)-200PA significantly inhibited the formation of vacuoles compared to methoxy PEG amine 40 kDa.

[1038] Furthermore, the amount of polyethylene glycol administered in this example was optimized for evaluating cavitation, and was a very large amount compared to the usual dosage of polyethylene glycol in the technical field.

[1039] [Example 22]

[1040] Animal experiment on the accumulation of polyethylene glycol

[1041] The accumulation of polyethylene glycol was evaluated through animal experiments using a degradable polyethylene glycol derivative ME-200GLFG(L)-200PA with a molecular weight of 40,000 and an amino group at the end, non-degradable methoxy PEG amine 20kDa, methoxy PEG amine 40kDa, and PBS as a control. The mouse species was Balb / c (8 weeks old, male), and the polyethylene glycol solution was prepared using physiological saline to a concentration of 62.5 mg / mL of the polyethylene glycol derivative, and 100 μL was administered through the tail vein of the mouse. The administration was continued 3 times a week for 4 weeks. After the administration, the mice were perfused and fixed with a 4% paraformaldehyde aqueous solution, and paraffin sections were prepared. Immunostaining with anti-PEG antibodies was performed to evaluate the accumulation in the choroid plexus epithelial cells of the brain. The images of the choroid plexus sections of each brain after immunostaining are shown below. Fig.18 shown.

[1042] In the choroid plexus sections of mice administered with PBS without polyethylene glycol, no staining occurred. In contrast, the result of non-degradable methoxy PEG amine 40kDa was that the sections were stained brown over a large area. This stained area indicates that PEG has accumulated. On the other hand, in the sections of degradable polyethylene glycol ME-200GLFG(L)-200PA, the brown staining area was small, showing the same accumulation as methoxy PEG amine 20kDa, which has half the molecular weight. As a result, degradable polyethylene glycol significantly inhibited the accumulation of polyethylene glycol in tissues compared with non-degradable methoxy PEG amine 40kDa of the same molecular weight due to its degradability.

[1043] In order to quantify these accumulations, image analysis was performed using the following analysis software, and the scores were calculated.

[1044] Device: All in one microscopy BZ-X710

[1045] Analysis software: BZ-X Analyzer

[1046] Specifically, the area of ​​the choroid plexus tissue and the area of ​​the stained portion showing accumulation of brown in the image were extracted using analysis software, and the scores were performed using the following calculation formula. The calculated scores are shown in Table 4.

[1047] Accumulation rate = area of ​​brown staining / area of ​​choroid plexus tissue

[1048] Score = accumulation rate / accumulation rate of PBS

[1049] The results showed that degradable polyethylene glycol can significantly inhibit the accumulation of polyethylene glycol in tissues.

[1050] Furthermore, the amount of polyethylene glycol administered in this example was optimized for evaluating the accumulation, and was a very large amount compared to the usual dosage of polyethylene glycol in the technical field.

[1051]

Table 4

[1052] Fraction PBS 1.0 Methoxy PEG Amine 40kDa 35.9 Methoxy PEG Amine 20kDa 2.3 ME-200GLFG(L)-200PA 4.2

[1053]

Industrial Applicability

[1054] The bio-related substance to which the degradable polyethylene glycol derivative is bonded of the present invention is stable in the blood in a living body, and has an oligopeptide between the polyethylene glycol chains that is degraded by an enzyme in the cell, so it is stable in the blood, and can give the bio-related substance the same blood half-life as that of the existing non-degradable polyethylene glycol derivative. Therefore, when taken up into cells, the oligopeptide site between the polyethylene glycol chains is rapidly degraded, and the generation of cellular vacuoles, which has been a problem so far, can be suppressed.

[1055] This application is based on Japanese Patent Application No. 2018-064306 filed in Japan, and all the contents are included in this specification. Sequence Listing <110> NOF CORPORATION Tokyo Institute of Technology <120> Degradable polyethylene glycol bond <130> 092887 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 32 <212> PRT <213> Atlantic salmon (Salmo salar) <400> 1 Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Gln Glu Leu 1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro 20 25 30 <210> 2 <211> 192 <212> PRT <213> Homo sapiens <400> 2 Met Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu 1 5 10 15 Arg Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe 20 25 30 Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn 35 40 45 Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn 50 55 60 Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser 65 70 75 80 Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser 85 90 95 Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr 100 105 110 Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg 115 120 125 Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr 130 135 140 Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn 145 150 155 160 Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr 165 170 175 Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 180 185 190 <210> 3 <211> 174 <212> PRT <213> Homo sapiens <400> 3 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 35 40 45 Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys 50 55 60 Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser 65 70 75 80 Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser 85 90 95 Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp 100 105 110 Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro 115 120 125 Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 130 135 140 Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe 145 150 155 160 Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 165 170

Claims

1. A bio-related substance, which is bonded with a degradable polyethylene glycol derivative represented by the following formula (A): In formula (A), m is 1 to 7, n1 and n2 are independently 180 to 682, p is 1 to 4, R is an alkyl group having 1 to 4 carbon atoms, Z is glycine-phenylalanine-leucine-glycine, valine-citrulline-glycine, phenylalanine-glycine, valine-alanine-glycine, glycine-glycine-phenylalanine-glycine or glycine-phenylalanine-glycine, Q is a residue of ethylene glycol, glycerol, pentaerythritol, diglycerol or xylitol, D is a bio-related substance, L 1 , L 2 , L 3 , L 4 and L 5 Each of them is independently a single bond or a divalent spacer group, and y is 1-40.

2. The bio-related substance according to claim 1, which is a bio-related substance to which a degradable polyethylene glycol derivative represented by the following formula (1) is bonded: In formula (1), m is 1 to 7, n1 and n2 are independently 180 to 682, p is 1 to 4, R is an alkyl group having 1 to 4 carbon atoms, Z is glycine-phenylalanine-leucine-glycine, valine-citrulline-glycine, phenylalanine-glycine, valine-alanine-glycine, glycine-glycine-phenylalanine-glycine or glycine-phenylalanine-glycine, Q is a residue of ethylene glycol, glycerol, pentaerythritol, diglycerol or xylitol, D is a bio-related substance, L 1 , L 2 , L 3 , L 4 and L 5 Each independently represents a single bond or a divalent spacer group. 3 . The bio-related substance according to claim 1 , wherein the molecular weight of one molecule of the degradable polyethylene glycol derivative bonded to D is 30,000 or more.

4. The bio-related substance according to claim 1 or 2, L 1 , L 2 , L 3 , L 4 and L 5 Each independently represents a single bond, a phenylene group, a carbamate bond, an amide bond, an ether bond, a thioether bond, a secondary amino group, a carbonyl group, a urea bond, a triazine group, a bond between maleimide and thiol, an oxime bond, or an alkylene group which may contain these bonds and groups.

5. The bio-related substance according to claim 1 or 2, wherein the bio-related substance D is a hormone, a cytokine, an antibody, an aptamer or an enzyme.

6. The bio-related substance according to claim 2, which is a compound having Z in formula (1) bonded thereto and consisting of Z 1 A bio-related substance of a degradable polyethylene glycol derivative represented by the following formula (2): In formula (2), m is 1 to 7, n1 and n2 are each independently 180 to 682, p is 1 to 4, R is an alkyl group having 1 to 4 carbon atoms, and Z 1 a -A b is glycine-phenylalanine-leucine, valine-citrulline, phenylalanine, valine-alanine, glycine-glycine-phenylalanine or glycine-phenylalanine, Q is a residue of ethylene glycol, glycerol, pentaerythritol, diglycerol or xylitol, D is a biologically relevant substance, L 1 , L 2 , L 3 , L 4 and L 5 Each independently represents a single bond or a divalent spacer group.

7. The bio-related substance according to claim 6, which is a bio-related substance to which a degradable polyethylene glycol derivative represented by the following formula (3) is bonded, wherein m in formula (2) is 1: In formula (3), n3 and n4 are independently 180 to 682, p is 1 to 4, R is an alkyl group having 1 to 4 carbon atoms, and Z 1 a -A b is glycine-phenylalanine-leucine, valine-citrulline, phenylalanine, valine-alanine, glycine-glycine-phenylalanine or glycine-phenylalanine, Q is a residue of ethylene glycol, glycerol, pentaerythritol, diglycerol or xylitol, D is a biologically relevant substance, L 1 , L 2 , L 3 , L 4 and L 5 Each independently represents a single bond or a divalent spacer group.

8. The bio-related substance according to claim 2, which is a residue in which Q in the formula (1) is an ethylene glycol residue, L 1 A bio-related substance of a degradable polyethylene glycol derivative represented by the following formula (4), wherein CH2CH2O and p is 1: In formula (4), m is 1 to 7, n5 is 182 to 684, n6 is 180 to 682, R is an alkyl group having 1 to 4 carbon atoms, Z is glycine-phenylalanine-leucine-glycine, valine-citrulline-glycine, phenylalanine-glycine, valine-alanine-glycine, glycine-glycine-phenylalanine-glycine or glycine-phenylalanine-glycine, D is a bio-related substance, L 2 , L 3 , L 4 and L 5 Each independently represents a single bond or a divalent spacer group.

9. The bio-related substance according to claim 6, which is a residue in which Q in formula (2) is an ethylene glycol residue, L 1 A bio-related substance of a degradable polyethylene glycol derivative represented by the following formula (5), wherein CH2CH2O and p is 1: In formula (5), m is 1 to 7, n5 is 182 to 684, n6 is 180 to 682, R is an alkyl group having 1 to 4 carbon atoms, and Z is 1 a -A b is glycine-phenylalanine-leucine, valine-citrulline, phenylalanine, valine-alanine, glycine-glycine-phenylalanine or glycine-phenylalanine, D is a biologically relevant substance, L 2 , L 3 , L 4 and L 5 Each independently represents a single bond or a divalent spacer group.

10. The bio-related substance according to claim 7, which is a residue in which Q in formula (3) is an ethylene glycol residue, L 1 A bio-related substance of a degradable polyethylene glycol derivative represented by the following formula (6), wherein CH2CH2O and p is 1: In formula (6), n7 is 228 to 684, n8 is 226 to 682, R is an alkyl group having 1 to 4 carbon atoms, and Z 1 a -A b is glycine-phenylalanine-leucine, valine-citrulline, phenylalanine, valine-alanine, glycine-glycine-phenylalanine or glycine-phenylalanine, D is a biologically relevant substance, L 2 , L 3 , L 4 and L 5 Each independently represents a single bond or a divalent spacer group.

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