Novel cross-linked alginic acid

Abstract

According to the present application, it is possible to provide alginic acid derivatives represented by formula (I) and formula (II), and a novel cross-linked alginic acid obtained by performing a Whistler reaction using the alginic acid derivative of formula (I) and the alginic acid derivative of formula (II). Thus, it is possible to provide a novel alginic acid derivative and a novel cross-linked alginic acid.[Chem. 201]

Description

[0001] This application is a divisional application of the invention patent application with application number 201980038664.8, application date June 13, 2019, entitled "Novel Crosslinked Alginate". Technical Field

[0002] This invention relates to novel alginate derivatives, novel cross-linked alginate, and methods for their manufacture. Background Technology

[0003] Alginic acid is obtained from macroalgae (レッソニア), macrocystis (macrocystis), and kelp (ラミナリア), Ascophyllum nodosum (アスコフィラム), Clusterweed (ダービリア), Fucosides ( Ecklonia cava Alginic acid is a high-molecular-weight acidic polysaccharide extracted from the cell walls of natural brown algae such as kelp, black seaweed, and kombu. It is a linear hybrid polymer composed of two uronic acids: β-D-mannuronic acid (component M) and α-L-glucuronic acid (component G), its C-5 epimer, bonded together in a 1-4 bond. Specifically, its chemical structure is a block copolymer obtained by complexly bonding mannuronic acid homopolymer blocks (MM), glucuronic acid homopolymer blocks (GG), and blocks (MG) obtained by random arrangement of mannuronic acid and glucuronic acid in arbitrary arrangements and proportions. Alginic acid is widely used in the fields of medicine, biotechnology, cosmetics, fiber, papermaking, and food.

[0004] Monovalent salts of alginate (e.g., sodium alginate) are water-soluble, but divalent salts of alginate (e.g., calcium alginate) have the property of gelling (insoluble) through cross-linking with metal ions. Attempts have been made to modify or shape alginate into alkali metal salts suitable for various applications by taking advantage of this property.

[0005] In order to explore the possibility of modifying or shaping polysaccharides (e.g., hyaluronic acid, chondroitin sulfate, alginate, etc.) into various materials and to improve their physical properties (e.g., strength, swelling properties, etc.), various studies have been carried out to date, such as on cross-linked polysaccharides obtained by covalent bonding.

[0006] Specifically, methods for obtaining cross-linked polysaccharides include (1) cross-linking methods using aldehyde cross-linking agents such as formaldehyde (Patent Document 1: International Publication No. 2011 / 028031), (2) self-cross-linking methods utilizing carboxyl and hydroxyl groups in polysaccharides (Patent Document 2: International Publication No. 1989 / 10941), and (3) cross-linking methods using the same bifunctional cross-linking agent (diepoxide, divinyl sulfone, diamine, or dihydrazide, etc.) or different bifunctional cross-linking agents (ephalo alcohol, etc.) (Patent Document 3: International Publication No. 2009 / 073437).

[0007] In addition, it is known that (4) a crosslinking method involving light irradiation after introducing photoreactive groups (cinnamic acid, substituted cinnamic acid, acrylic acid, maleic acid, fumaric acid, furanylacrylic acid, thiopheneacrylic acid, cinnamylacetic acid, sorbic acid, thymine, or coumarin, etc.) (Patent Documents 4, 5: International Publication No. 2005 / 026214, Japanese Patent Application Publication No. 09-87236), and (5) a crosslinking method in which polysaccharides with introduced thiol groups are crosslinked with each other by disulfide bonds, and a crosslinking method in which Michael addition reaction is carried out by using polysaccharides with introduced thiol groups and polysaccharides with introduced maleimide groups (Patent Document 6: International Publication No. 2008 / 071058), etc.

[0008] Furthermore, as a method for crosslinking polysaccharides through covalent bonds, it is known (6) to crosslink by using polysaccharides with alkynyl groups and polysaccharides with azide groups to carry out the Huisgen reaction (1,3-dipolar addition cyclization reaction).

[0009] Cross-linked polysaccharides that cross-link polysaccharides via the Wheatstone reaction are disclosed in (i) International Publication No. 2008 / 031525 (Patent Document 7), (ii) International Publication No. 2012 / 165462 (Patent Document 8), (iii) International Publication No. 2015 / 020206 (Patent Document 9), and (iv) Chinese Patent Application Publication No. 106140040 (Patent Document 10).

[0010] However, (i) Patent Document 7 relates to cross-linked polysaccharides, wherein the first polysaccharide is hyaluronic acid and the second polysaccharide is a polysaccharide selected from chondroitin, sulfated dermatin, alginate or its salts, and the chain alkyne and azide groups introduced by the linking group in each polysaccharide are subjected to a Wheatstone reaction in the presence of a copper catalyst, without disclosing the novel cross-linked alginate described later.

[0011] Furthermore, (ii) Patent Document 8 relates to cross-linked polysaccharides, wherein the first and second polysaccharides are selected from polysaccharides of hyaluronic acid, carboxymethyl dextran, cellulose derivatives, and chitosan (the first and second polysaccharides may be of the same or different types), and a cyclic alkyne group and azide group introduced by a linker group (the polysaccharide and the linker group are bonded by an ester bond) are introduced in each polysaccharide via a Wheatstone reaction, without disclosing the novel cross-linked alginic acid described later.

[0012] In addition, (iii) Patent Document 9 relates to cross-linked polysaccharides, which are obtained by making the first polysaccharide hyaluronic acid and the second polysaccharide chondroitin sulfate, and by subjecting the cyclic alkyne group and azide group introduced by the linking group in each polysaccharide to a Wheatstone reaction, without disclosing the novel cross-linked alginic acid described later.

[0013] Furthermore, (iv) Patent Document 10 relates to cross-linked polysaccharides, wherein the polysaccharide is obtained by making the first polysaccharide chitosan and the second polysaccharide sodium alginate, and by subjecting the cyclic alkyne and azide groups introduced in the middle of each polysaccharide by the linking group (the polysaccharide and the linking group are bonded by an ester bond) to a Wheatstone reaction, without disclosing the novel cross-linked alginic acid described later.

[0014] (v) Patent document 11 relates to a method for derivatizing sugars by bonding them to an 8-membered ring alkyne group, wherein the sugar is a non-natural sugar (capsular sugar derived from Streptococcus agaris), not alginic acid, and the terminus of the bonded 8-membered ring alkyne group does not form an amide bond relative to the carboxyl group of the sugar, thus differing from the novel alginic acid derivatives and their manufacturing methods described later.

[0015] (vi) Non-patent literature 1 describes a branched alginic acid (bAlg-DBCO) with a cyclooctyne side chain introduced into the side chain. However, it is obtained by reacting branched alginic acid (bAlg) synthesized from alginic acid and branched polyethylene glycol (4-arm PEG-NH2) with amino-modified cyclooctyne (DBCO-PEG-amine). It has a different structure from the novel alginic acid derivatives described later, and its intended use is also different.

[0016] Existing technical documents

[0017] Patent documents

[0018] [Patent Document 1] International Publication No. 2011 / 028031

[0019] [Patent Document 2] International Publication No. 1989 / 10941

[0020] [Patent Document 3] International Publication No. 2009 / 073437

[0021] [Patent Document 4] International Publication No. 2005 / 026214

[0022] [Patent Document 5] Japanese Patent Application Publication No. 09-87236

[0023] [Patent Document 6] International Publication No. 2008 / 071058

[0024] [Patent Document 7] International Publication No. 2008 / 031525

[0025] [Patent Document 8] International Publication No. 2012 / 165462

[0026] [Patent Document 9] International Publication No. 2015 / 020206

[0027] [Patent Document 10] Chinese Patent Application Publication No. 106140040

[0028] [Patent Document 11] International Publication No. 2014 / 111344

[0029] Non-patent literature

[0030] [Patent Document 1] Nat Commun. 9(1), p2195-, 2018. Summary of the Invention

[0031] The problem that the invention aims to solve

[0032] In the above context, there is a need to obtain novel alginate derivatives or novel cross-linked alginates. Additionally, methods for their manufacture are also required.

[0033] means for solving problems

[0034] In order to solve the above-mentioned problems, the inventors conducted in-depth research and discovered novel alginate derivatives represented by formula (I) or formula (II). Furthermore, using novel cross-linked alginate obtained by subjecting the novel alginate derivatives of formula (I) and formula (II) to a Wheatstone reaction, beads (pigment-containing beads) were formed as a cross-linked alginate structure. These beads exhibited high stability, and compared to conventional gels, they could be adjusted to have a permeability corresponding to the target, thus completing the present invention.

[0035] The novel alginate derivatives (formulas (I) and (II)) provided herein can be used, for example, for chemical cross-linking formation, i.e., by introducing a reactive group that can be used for chemical cross-linking formation or a complementary reactive group of such reactive group.

[0036] The aforementioned chemical crosslinking can be carried out, for example, by a crosslinking reaction based on the Wheatstone reaction (1,3-dipolar addition cyclization reaction), for example, between alginate derivatives of formula (I) and formula (II), or for example, between alginate derivatives of formula (I) and other molecules having an azide group, or between alginate derivatives of formula (II) and other molecules having an alkynyl group.

[0037] The Wheatstone reaction based on terminal alkyne and terminal azide groups is unsuitable for the chemical modification of biomolecules due to the generally high heating temperatures above 100°C. However, the reaction conditions under which a copper catalyst (e.g., by coexisting with Cu(I)) forms a cyclization adduct (triazole ring) in near 100% yield at room temperature (Angew. Chem. Int. Ed. Engl., 14, pp. 2596-2599, 2002; J. Org. Chem., 9, pp. 3057-3064, 2002) can be utilized for the chemical modification of biomolecules. On the other hand, when using the aforementioned Wheatstone reaction in the presence of a copper catalyst to obtain cross-linked alginate, there is a possibility of trace amounts of copper catalyst remaining in the cross-linked alginate, raising concerns about copper-derived cytotoxicity in the cross-linked alginate or its structure.

[0038] In a preferred embodiment, to avoid copper-derived cytotoxicity in cross-linked alginate, the Wheatstone reaction, which does not require a copper catalyst, is used to obtain cross-linked alginate. Specifically, by using a cyclooctyne derivative (a high-strain cyclic alkynyl group) as the alkynyl group introduced into the alginate derivative, the reaction can be carried out without high-temperature conditions above 100°C and a copper catalyst. Therefore, the novel cross-linked alginate of the preferred embodiment does not contain a copper catalyst, which is excellent from the viewpoint that copper-derived toxicity does not occur even when forming the final product (cross-linked alginate structure).

[0039] Hereinafter, we provide alginate derivatives of formula (I) or (II) in which a cyclic alkyne or azide group is introduced onto any one or more carboxyl groups of alginate via an amide bond and a divalent linker, novel cross-linked alginates obtained by performing a Wheatstone reaction (1,3-dipolar addition cyclization reaction) on alginate derivatives of formula (I) and (II), and methods for manufacturing the aforementioned alginate derivatives and cross-linked alginates. That is, the exemplified methods are as described below in [1] to

[17] .

[0040] [1] The alginate derivative shown in formula (I) below, wherein one or more carboxyl groups of alginate are connected by an amide bond and a divalent linker (-L) 1 -) and introduced a cyclic alkynyl group (Akn),

[0041] [Chemistry 1]

[0042]

[0043] In formula (I), (ALG) and -L 1 - The definition of Akn is the same as the definition in the first method described later.

[0044] [2] [1] The alginic acid derivative represented by formula (I), wherein Akn-L 1 -NH2 group (Akn, and -L) 1 The import rate (same as the definition in Method 1 described below) is 0.1% to 30%.

[0045] [3] [1] The alginic acid derivative shown in formula (I) has a weight-average molecular weight of 100,000 Da to 3,000,000 Da as determined by gel filtration chromatography.

[0046] [4] Alginate derivatives represented by formula (II) below, wherein one or more carboxyl groups of alginate are connected by an amide bond and a divalent linker (-L) 2 -) And an azide group was introduced.

[0047] [Chemistry 2]

[0048]

[0049] In formula (II), (ALG) and -L 2 The definition of - is the same as that in the fourth method described later.

[0050] [5] [4] The alginic acid derivative represented by formula (II), wherein N3-L 2 -NH2 group (-L 2 The import rate (same as the definition in Method 4 described later) is 0.1% to 30%.

[0051] [6] [4] The alginic acid derivatives shown in formula (II) have a weight-average molecular weight of 100,000 Da to 3,000,000 Da as determined by gel filtration chromatography.

[0052] [7] Crosslinked alginate, wherein any carboxyl group of the first alginate and any carboxyl group of the second alginate are bonded by the following formula (III-L):

[0053] [Chemistry 3]

[0054]

[0055] In formula (III-L), the -CONH- and -NHCO- at both ends represent amide bonds derived from any carboxyl group of alginate; -L 1 -、-L 2 - and X are defined in the same way as in the 7th method described later.

[0056] [8] A method for manufacturing cross-linked alginate, comprising performing a Wheatstone reaction by mixing an alginate derivative of formula (I) of any one of [1] to [3] and an alginate derivative of formula (II) of any one of [4] to [6] to obtain the cross-linked alginate described in [7].

[0057] [8-1] Cross-linked alginic acid, which includes chemical cross-linking based on triazole rings formed by the Wheatstone reaction and ionic cross-linking partially formed by calcium ions as cross-linking.

[0058] [9] A cross-linked alginate structure is obtained by adding a mixed solution of an alginate derivative obtained by mixing the alginate derivative of formula (I) of any one of [1] to [3] and the alginate derivative of formula (II) of any one of [4] to [6] to a calcium chloride solution.

[0059]

[10] The cross-linked alginate structure described in [9] above comprises chemical cross-linking based on triazole rings formed by the Wheatstone reaction and ionic cross-linking partially formed by calcium ions as cross-linking.

[0060]

[11] A method for manufacturing a cross-linked alginate structure, comprising adding a mixed solution of an alginate derivative obtained by mixing the alginate derivative of formula (I) of any one of [1] to [3] and the alginate derivative of formula (II) of any one of [4] to [6] to a calcium chloride solution to obtain the cross-linked alginate structure described in [9] or

[10] .

[0061]

[12] The cross-linked alginate structure described in [9] or

[10] above is a bead or near-spherical gel.

[0062]

[13] Medical materials comprising the cross-linked alginate structure described in [9] or

[10] above.

[0063]

[14] The medical material described in

[13] above is a bead or a near-spherical gel.

[0064]

[15] The alginate derivatives described in any one of [1] to [6] above, the cross-linked alginate described in [7] or [8-1] above, and the cross-linked alginate structures described in any one of [9],

[10] and

[12] above, are biocompatible.

[0065]

[16] An amino compound represented by the formula (AM-1), or a pharmaceutically acceptable salt thereof, or a solvate thereof,

[0066] [Chemistry 4]

[0067]

[0068] In formula (AM-1), -L 1 The definitions of - and Akn are the same as those in the 16th method described later.

[0069]

[17] An amino compound represented by the formula (AM-2), or a pharmaceutically acceptable salt thereof, or a solvate thereof,

[0070] [Chemistry 5]

[0071]

[0072] In formula (II), -L 2 The definition of - is the same as that in the 17th method described later.

[0073] Invention Effects

[0074] This invention provides, for example, novel alginate derivatives and novel cross-linked alginate that can be used for chemical cross-linking formation.

[0075] Even if the preferred alginate derivative introduces reactive groups not present in living organisms and leaves unreacted groups, its safety can be expected for living organisms that do not pose a risk of cross-linking with biological components such as cells. Furthermore, the preferred cross-linking reaction is completed at room temperature without the use of a metal catalyst, thus ensuring safe and easy use.

[0076] Some cross-linked alginates are chemically cross-linked via the Wheatstone reaction (1,3-dipolar addition cyclization). Chemical cross-linking and, for example, cross-linking using divalent metal ions (e.g., calcium ions) can be combined, and by adjusting the reaction conditions, their stability can be improved compared to non-cross-linked alginates or non-chemically cross-linked alginates (e.g., cross-linked alginates obtained by calcium ion cross-linking).

[0077] In addition, the gel properties of the crosslinked material can be adjusted, as can the permeability of the material.

[0078] The present invention has at least one of these effects. Attached Figure Description

[0079] [ Figure 1 [Figure showing the evaluation of the stability of the gel with cross-linked alginate structure]

[0080] [ Figure 2 [Graph showing the evaluation of the permeability of the gel with cross-linked alginate structure.]

[0081] [ Figure 3 [Graph showing the evaluation of the permeability of the gel with cross-linked alginate structure.]

[0082] [ Figure 4 [Figure showing the evaluation of the stability of the gel with cross-linked alginate structure]

[0083] [ Figure 5 [Graph showing the evaluation of the permeability of the gel with cross-linked alginate structure.]

[0084] [ Figure 6 [Graph showing the evaluation of the permeability of the gel with cross-linked alginate structure.]

[0085] [ Figure 7 [Graph showing the biocompatibility evaluation of the gel of cross-linked alginate derivative]

[0086] [ Figure 8 [Figure showing the evaluation of the stability of the gel with cross-linked alginate structure]

[0087] [ Figure 9 [Figure showing the evaluation of the stability of the gel under EDTA with cross-linked alginate structure.]

[0088] [ Figure 10 [Figure showing the evaluation of the stability of the gel with cross-linked alginate structure]

[0089] [ Figure 11 [Figure showing the evaluation of the stability of the gel under EDTA with cross-linked alginate structure.]

[0090] [ Figure 12 [Figure showing the evaluation of the stability of the gel with cross-linked alginate structure]

[0091] [ Figure 13 [Figure showing the evaluation of the stability of the gel under EDTA with cross-linked alginate structure.]

[0092] [ Figure 14 [Graph showing the evaluation of the permeability of the gel with cross-linked alginate structure.]

[0093] [ Figure 15 [Graph showing the evaluation of the permeability of the gel with cross-linked alginate structure.]

[0094] [ Figure 16 [Graph showing the evaluation of the permeability of the gel with cross-linked alginate structure.]

[0095] [ Figure 17 [Graph showing the biocompatibility evaluation of the gel of cross-linked alginate derivative] Detailed Implementation

[0096] [Specific methods]

[0097] Including the following methods[1]~

[17] .

[0098] [1] The first method is as follows. The alginate derivative shown in formula (I) below, wherein one or more carboxyl groups of the alginate are connected by an amide bond and a divalent linker (-L). 1 -) and introduced a cyclic alkynyl group (Akn),

[0099] [Chemistry 6]

[0100]

[0101] In formula (I), (ALG) represents alginate; -NHCO- represents an amide bond derived from any carboxyl group of alginate; -L 1 - indicates a binary linker selected from the following partial structural formulas [in each formula, the outer edges of the wavy lines at both ends are not included]:

[0102] [Chemistry 7]

[0103]

[0104] Akn represents a cyclic alkynyl group selected from the following structural formulas [in each formula, the right side of the wavy line is not included]:

[0105] [Chemistry 8]

[0106]

[0107] An asterisk (*) indicates a chiral center.

[0108] [1-1] In the alginate derivative of formula (I) of the aforementioned method [1], -L 1 - Preferably, the dual-valent linker is selected from the following partial structural formulas [in each formula, the outer sides of the wavy lines at both ends are not included]:

[0109] [Chemistry 9]

[0110]

[0111] More preferably, it is a dual-valent linker selected from the following partial structural formulas [in each formula, the outer side of the wavy line at both ends is not included]:

[0112] [Chemistry 10]

[0113]

[0114] A further preferred option is a dual-valent linker selected from the following partial structural formulas [in each formula, the outer sides of the wavy lines at both ends are not included]:

[0115] [Chemistry 11]

[0116] .

[0117] [1-2] In the alginate derivative of the aforementioned formula (I) of the aforementioned method [1],

[0118] Akn is preferably a cyclic alkynyl group selected from the following partial structural formulas [in each formula, the right side of the wavy line is not included]:

[0119] [Chemistry 12]

[0120]

[0121] More preferably, it is a cyclic alkynyl group selected from the following partial structural formulas [in each formula, the right side of the wavy line is not included]:

[0122] [Chemistry 13]

[0123] .

[0124] [1-3] In the alginate derivative of formula (I) of the aforementioned method [1], Akn and -L 1 - The preferred combination is as shown by groups selected from the following partial structural formulas [in each formula, the right side of the wavy line (imino side) is not included]:

[0125] [Chemistry 14]

[0126]

[0127] [Chemistry 15]

[0128]

[0129] More preferably, it is represented by groups selected from the following partial structural formulas [in each formula, the right side of the wavy line (imino side) is not included]:

[0130] [Chemistry 16]

[0131]

[0132] Further preferred are those represented by groups selected from the following partial structural formulas [in each formula, the right side of the wavy line (imino side) is not included]:

[0133] [Chemistry 17]

[0134] .

[0135] [1-1a] In the alginate derivative of the aforementioned formula (I) of the aforementioned method [1], -L 1 - Preferably, the dual-valent linker is selected from the following partial structural formulas [in each formula, the outer sides of the wavy lines at both ends are not included]:

[0136] [Chemistry 18]

[0137]

[0138] More preferably, it is a dual-valent linker selected from the following partial structural formulas [in each formula, the outer side of the wavy line at both ends is not included]:

[0139] [Chemistry 19]

[0140]

[0141] A further preferred option is a dual-valent linker selected from the following partial structural formulas [in each formula, the outer sides of the wavy lines at both ends are not included]:

[0142] [Chemistry 20]

[0143] .

[0144] [1-2a] In the alginate derivative of the aforementioned formula (I) of the aforementioned method [1], Akn is preferably a cyclic alkynyl group selected from the following partial structural formulas [in each formula, the right side of the wavy line is not included]:

[0145] [Chemistry 21]

[0146]

[0147] More preferably, it is a cyclic alkynyl group selected from the following partial structural formulas [in each formula, the right side of the wavy line is not included]:

[0148] [Chemistry 22]

[0149] .

[0150] [1-3a] In the alginate derivative of formula (I) of the aforementioned method [1], Akn and -L 1 - The preferred combination is as shown by groups selected from the following partial structural formulas [in each formula, the right side of the wavy line (imino side) is not included]:

[0151] [Chemistry 23]

[0152]

[0153] [Chemistry 24]

[0154]

[0155] More preferably, it is represented by groups selected from the following partial structural formulas [in each formula, the right side of the wavy line (imino side) is not included]:

[0156] [Chemistry 25]

[0157]

[0158] [Chemistry 26]

[0159]

[0160] Further preferred are those represented by groups selected from the following partial structural formulas [in each formula, the right side of the wavy line (imino side) is not included]:

[0161] [Chemistry 27]

[0162] .

[0163] [1-1b] In the alginate derivative of formula (I) of the aforementioned method [1], -L 1 - Preferably, the dual-valent linker is selected from the following partial structural formulas [in each formula, the outer sides of the wavy lines at both ends are not included]:

[0164] [Chemistry 28]

[0165]

[0166] More preferably, it is a dual-valent linker selected from the following partial structural formulas [in each formula, the outer side of the wavy line at both ends is not included]:

[0167] [Chemistry 29]

[0168]

[0169] A further preferred option is a dual-valent linker selected from the following partial structural formulas [in each formula, the outer sides of the wavy lines at both ends are not included]:

[0170] [Chemistry 30]

[0171] .

[0172] [1-2b] In the alginate derivative of the aforementioned formula (I) of the aforementioned method [1], Akn is preferably a cyclic alkynyl group selected from the following partial structural formulas [in each formula, the right side of the wavy line is not included]:

[0173] [Chemistry 31]

[0174]

[0175] More preferably, it is a cyclic alkynyl group selected from the following partial structural formulas [in each formula, the right side of the wavy line is not included]:

[0176] [Chemistry 32]

[0177] .

[0178] [1-3b] In the alginate derivative of formula (I) of the aforementioned method [1], Akn and -L 1 The preferred combinations are shown in the table below:

[0179] [Table 1]

[0180]

[0181] Any combination of items (in the table -L) 1 - or Akn as described in the foregoing [1], [1-1], [1-1a], [1-2], [1-2a], and [1-1b], or groups selected from the following formulas [in each formula, the right side of the wavy line (imino side) is not included];

[0182] [Chemistry 33]

[0183]

[0184] The preferred options are as shown in the table below:

[0185] [Table 2]

[0186]

[0187] Any combination of items (in the table -L) 1 - or various forms of Akn as described in the foregoing [1], [1-1], [1-1a], [1-2], [1-2a], and [1-1b];

[0188] Further preferred options are shown in the table below:

[0189] [Table 3]

[0190]

[0191] Any combination of items (in the table -L) 1 - or various forms of Akn as described in the foregoing [1], [1-1], [1-1a], [1-2], [1-2a], and [1-1b];

[0192] Particularly preferred are those represented by groups selected from the following partial structural formulas [in each formula, the right side of the wavy line (imino side) is not included]:

[0193] [Chemistry 34]

[0194] .

[0195] The preferred method of the aforementioned method [1] is achieved by further appropriately combining Akn and -L 1- is defined as a preferred mode that can optionally form the alginate derivative shown in the aforementioned formula (I) of the aforementioned manner [1].

[0196] [2] The second method is as follows. The alginate derivative of formula (I) described in the aforementioned method [1], wherein Akn-L 1 -NH2 group (Akn, and -L) 1 The import rate (same as the definition in the aforementioned method [1]) is 0.1%~30%.

[0197] [2-1] In the aforementioned method [2], Akn-L 1 The introduction rate of the -NH2 group is preferably 2% to 20%; more preferably 3% to 10%.

[0198] [2-1a] In the aforementioned method [2], Akn-L 1 The introduction rate of the -NH2 group is preferably 0.3% to 20%; more preferably 0.5% to 10%.

[0199] [3] The third method is as follows. The alginate derivative of formula (I) described in the aforementioned method [1] has a weight-average molecular weight of 100,000 Da to 3,000,000 Da as determined by gel filtration chromatography.

[0200] [3-1] In the aforementioned method [3], the weight-average molecular weight of the alginate derivative determined by gel filtration chromatography is preferably 300,000 Da to 2,500,000 Da, and more preferably 500,000 Da to 2,000,000 Da.

[0201] [3-1a] In the aforementioned method [3], the weight-average molecular weight of the alginate derivative determined by gel filtration chromatography is preferably 300,000 Da to 2,500,000 Da, and more preferably 1,000,000 Da to 2,000,000 Da.

[0202] [4] The fourth method is as follows. The alginate derivative shown in formula (II) below, wherein one or more carboxyl groups of the alginate are connected by an amide bond and a divalent linker (-L). 2 -) And an azide group was introduced.

[0203] [Chemistry 35]

[0204]

[0205] In formula (II), (ALG) represents alginate; -NHCO- represents an amide bond derived from any carboxyl group of alginate; -L 2 - indicates a binary linker selected from the following partial structural formulas [in each formula, the outer edges of the wavy lines at both ends are not included]:

[0206] [Chemistry 36]

[0207] ].

[0208] [4-1] In the alginate derivative of formula (II) of the aforementioned method [4], -L 2 - Preferably, the connecting base is selected from the following partial structural formulas:

[0209] [Chemistry 37]

[0210]

[0211] [In each formula, the outer edges of the wavy lines at both ends are not included];

[0212] More preferably, the connecting base is selected from the following partial structural formulas:

[0213] [Chemistry 38]

[0214] .

[0215] [4-1a] In the alginate derivative of formula (II) of the aforementioned method [4], -L 2 - Preferably, the connecting base is selected from the following partial structural formulas:

[0216] [Chemistry 39]

[0217]

[0218] [In each formula, the outer edges of the wavy lines at both ends are not included];

[0219] More preferably, the connecting base is selected from the following partial structural formulas:

[0220] [Chemistry 40]

[0221] .

[0222] [4-1b] In the alginate derivative of formula (II) of the aforementioned method [4], -L 2 - Preferably, the connecting base is selected from the following partial structural formulas:

[0223] [Chemistry 41]

[0224]

[0225] [In each formula, the outer edges of the wavy lines at both ends are not included];

[0226] More preferably, the connecting base is selected from the following partial structural formulas:

[0227] [Chemistry 42]

[0228]

[0229] [In each formula, the outer edges of the wavy lines at both ends are not included.]

[0230] The preferred method of the aforementioned method [4] is achieved by further appropriate combination of azide groups and -L 2 - is defined as a preferred method that can optionally form the alginate derivative shown in the aforementioned formula (II) of the aforementioned manner [4].

[0231] [5] The fifth method is as follows. The alginate derivative of formula (II) described in the aforementioned method [4], wherein N3-L 2 -NH2 group (-L 2 The import rate (same as the definition in the aforementioned method [4]) is 0.1% to 30%.

[0232] [5-1] In the aforementioned method [5], N3-L 2 The introduction rate of the -NH2 group is preferably 2% to 20%; more preferably 3% to 10%.

[0233] [5-1a] In the aforementioned method [5], N3-L 2 The introduction rate of the -NH2 group is preferably 0.3% to 20%; more preferably 0.5% to 15%.

[0234] [6] The sixth method is as follows. The alginate derivative of formula (II) described in the aforementioned method [4], wherein the weight-average molecular weight of the alginate derivative determined by gel filtration chromatography is 100,000 Da to 3,000,000 Da.

[0235] [6-1] In the aforementioned method [6], the weight-average molecular weight of the alginate derivative of formula (II) determined by gel filtration chromatography is preferably 300,000 Da to 2,500,000 Da, more preferably 500,000 Da to 2,000,000 Da.

[0236] [6-1a] In the aforementioned method [6], the weight-average molecular weight of the alginate derivative of formula (II) determined by gel filtration chromatography is preferably 300,000 Da to 2,500,000 Da, more preferably 1,000,000 Da to 2,000,000 Da.

[0237] [7] The seventh method is as follows. Crosslinked alginate is obtained by bonding any carboxyl group of the first alginate and any carboxyl group of the second alginate using the following formula (III-L):

[0238] [Chemistry 43]

[0239]

[0240] In formula (III-L), -CONH- and -NHCO- at both ends represent amide bonds derived from any carboxyl group of alginate;

[0241] -L 1 - Same as the definition in the aforementioned method [1];

[0242] -L 2 - Same as the definition in the aforementioned method [4];

[0243] X is a cyclic group selected from the following partial structural formulas:

[0244] [Chemistry 44]

[0245]

[0246] (In each formula, the outer side of the wavy line at both ends is not included), and the asterisk indicates the chiral center.

[0247] [7-1] In the aforementioned formula (III-L) of the aforementioned method [7], it is preferred that -L 1 -A binary linker selected from the following partial structural formulas [in each formula, the outer edges of the wavy lines at both ends are not included]:

[0248] [Chemistry 45]

[0249] ;

[0250] -L 2 -A binary linker selected from the following partial structural formulas:

[0251] [Chemistry 46]

[0252]

[0253] [In each formula, the outer edges of the wavy lines at both ends are not included];

[0254] X is a cyclic group selected from the following partial structural formulas:

[0255] [Chemistry 47]

[0256]

[0257] (In each formula, the outer edges of the wavy lines at both ends are not included).

[0258] [7-2] In the aforementioned formula (III-L) of the aforementioned method [7], -L is more preferred. 1 -A binary linker selected from the following partial structural formulas [in each formula, the outer edges of the wavy lines at both ends are not included]:

[0259] [Chemistry 48]

[0260] ;

[0261] -L 2 -A binary linker selected from the following partial structural formulas:

[0262] [Chemistry 49]

[0263]

[0264] [In each formula, the outer edges of the wavy lines at both ends are not included];

[0265] X is a cyclic group selected from the following partial structural formulas:

[0266] [Transformation 50]

[0267]

[0268] (In each formula, the outer edges of the wavy lines at both ends are not included).

[0269] [7-3] In the aforementioned formula (III-L) of the aforementioned method [7], -L is further preferred. 1 -A binary linker selected from the following partial structural formulas [in each formula, the outer edges of the wavy lines at both ends are not included]:

[0270] [Chemistry 51]

[0271] ;

[0272] -L 2 -A binary linker selected from the following partial structural formulas:

[0273] [Chemistry 52]

[0274]

[0275] (In each formula, the outer edges of the wavy lines at both ends are not included);

[0276] X is a cyclic group selected from the following partial structural formulas:

[0277] [Chemistry 53]

[0278]

[0279] (In each formula, the outer edges of the wavy lines at both ends are not included).

[0280] [7-3-1] In the aforementioned formula (III-L) of the aforementioned method [7], -L is particularly preferred. 1 -A dual-valent linker for the following partial structural formula:

[0281] [Chemistry 54]

[0282]

[0283] (In the formula, the outer sides of the wavy lines at both ends are not included);

[0284] -L 2 -A dual-valent linker for the following partial structural formula:

[0285] [Chemistry 55]

[0286]

[0287] (In the formula, the outer sides of the wavy lines at both ends are not included);

[0288] X is any cyclic base in the following partial structural formula:

[0289] [Chemistry 56]

[0290]

[0291] (In each formula, the outer edges of the wavy lines at both ends are not included).

[0292] [7-4] In the aforementioned formula (III-L) of the aforementioned method [7], -L is preferred. 2 -XL 1 - Combinations are shown in partial structures selected from the following partial structures [in each formula, the outer sides of the wavy lines at both ends are not included]:

[0293] [Chemistry 57]

[0294] ;

[0295] More preferably -L 2 -XL 1 - Combinations are shown in any of the following partial structural formulas [where the outer edges of the wavy lines at both ends are not included]:

[0296] [Chem.58]

[0297] .

[0298] [7-1a] In the aforementioned formula (III-L) of the aforementioned method [7], it is preferred that -L 1 -A binary linker selected from the following partial structural formulas [in each formula, the outer edges of the wavy lines at both ends are not included]:

[0299] [Chemistry 59]

[0300] ;

[0301] -L 2-A binary linker selected from the following partial structural formulas:

[0302] [Transformation 60]

[0303]

[0304] [In each formula, the outer edges of the wavy lines at both ends are not included];

[0305] X is a cyclic group selected from the following partial structural formulas:

[0306] [Chemistry 61]

[0307]

[0308] (In each formula, the outer edges of the wavy lines at both ends are not included).

[0309] [7-2a] In the aforementioned formula (III-L) of the aforementioned method [7], -L is more preferred. 1 -A binary linker selected from the following partial structural formulas [in each formula, the outer edges of the wavy lines at both ends are not included]:

[0310] [Chemistry 62]

[0311] ;

[0312] -L 2 -A binary linker selected from the following partial structural formulas:

[0313] [Chemistry 63]

[0314]

[0315] [In each formula, the outer edges of the wavy lines at both ends are not included];

[0316] X is a cyclic group selected from the following partial structural formulas:

[0317] [Chemistry 64]

[0318]

[0319] (In each formula, the outer edges of the wavy lines at both ends are not included).

[0320] [7-3a] In the aforementioned formula (III-L) of the aforementioned method [7], -L is further preferred. 1 -A binary linker selected from the following partial structural formulas [in each formula, the outer edges of the wavy lines at both ends are not included]:

[0321] [Chemistry 65]

[0322] ;

[0323] -L 2 -A binary linker selected from the following partial structural formulas:

[0324] [Chemistry 66]

[0325]

[0326] (In each formula, the outer edges of the wavy lines at both ends are not included);

[0327] X is a cyclic group selected from the following partial structural formulas:

[0328] [Chemistry 67]

[0329]

[0330] (In each formula, the outer edges of the wavy lines at both ends are not included).

[0331] [7-3a-1] In the aforementioned formula (III-L) of the aforementioned method [7], -L is particularly preferred. 1 -A binary linker selected from the following partial structural formulas:

[0332] [Chemistry 68]

[0333]

[0334] (In the formula, the outer sides of the wavy lines at both ends are not included);

[0335] -L 2 -A binary linker selected from the following partial structural formulas:

[0336] [Chemistry 69]

[0337]

[0338] (In the formula, the outer sides of the wavy lines at both ends are not included);

[0339] X is a cyclic group selected from the following partial structural formulas:

[0340] [Chemistry 70]

[0341]

[0342] (In each formula, the outer edges of the wavy lines at both ends are not included).

[0343] [7-4a] In the aforementioned formula (III-L) of the aforementioned method [7], -L is preferred. 2 -XL 1 - Combinations are shown in the partial structures selected from the formulas in the table below:

[0344] [Table 4]

[0345]

[0346]

[0347] (in the table -L) 1 -、-L 2 -or -X- as described in the aforementioned manner [1], [1-1], [1-1a], [1-1b], [4], [4-1], [4-1a], [4-1b], [7], [7-1], [7-2], [7-3], [7-3-1], [7-1a], [7-2a], [7-3a], and [7-3a-1]);

[0348] More preferably -L 2 -XL 1 - Combinations are shown in partial structures selected from the following partial structures [where the outer sides of the wavy lines at both ends are not included]:

[0349] [Chemistry 71]

[0350]

[0351] [7-1b] In the aforementioned formula (III-L) of the aforementioned method [7], it is preferred that -L 1 -A binary linker selected from the following partial structural formulas [in each formula, the outer edges of the wavy lines at both ends are not included]:

[0352] [Chemistry 72]

[0353] ;

[0354] -L 2 -A binary linker selected from the following partial structural formulas:

[0355] [Chemistry 73]

[0356]

[0357] [In each formula, the outer edges of the wavy lines at both ends are not included];

[0358] X is a cyclic group selected from the following partial structural formulas:

[0359] [Chemistry 74]

[0360]

[0361] (In each formula, the outer edges of the wavy lines at both ends are not included).

[0362] [7-2b] In the aforementioned formula (III-L) of the aforementioned method [7], -L is more preferred. 1 -A binary linker selected from the following partial structural formulas [in each formula, the outer edges of the wavy lines at both ends are not included]:

[0363] [Chemistry 75]

[0364] ;

[0365] -L 2 -A binary linker selected from the following partial structural formulas:

[0366] [Chemistry 76]

[0367]

[0368] [In each formula, the outer edges of the wavy lines at both ends are not included];

[0369] X is a cyclic group selected from the following partial structural formulas:

[0370] [Chemistry 77]

[0371]

[0372] (In each formula, the outer edges of the wavy lines at both ends are not included).

[0373] [7-3b] In the aforementioned formula (III-L) of the aforementioned method [7], -L is further preferred. 1 -A binary linker selected from the following partial structural formulas [in each formula, the outer edges of the wavy lines at both ends are not included]:

[0374] [Chemistry 78]

[0375] ;

[0376] -L 2 -A binary linker selected from the following partial structural formulas:

[0377] [Chemistry 79]

[0378]

[0379] (In each formula, the outer edges of the wavy lines at both ends are not included);

[0380] X is a cyclic group selected from the following partial structural formulas:

[0381] [Chemistry 80]

[0382]

[0383] (In each formula, the outer edges of the wavy lines at both ends are not included).

[0384] [7-4b] In the aforementioned formula (III-L) of the aforementioned method [7], -L is preferred. 1 -XL 2 - Combinations are shown in partial structures selected from the following table:

[0385] [Table 5]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392] (in the table -L) 1 -、-L 2 -or -X- as described in the foregoing manner [1], [1-1], [1-1a], [1-1b], [1-1b], [4], [4-1], [4-1a], [4-1b], [7][7-1], [7-2], [7-3], [7-3-1], [7-1a], [7-2a], [7-3a], [7-3a-1], [7-1b], [7-2b], and [7-3b]);

[0393] More preferably -L 2 -XL 1 - The combination is shown in the partial structure of the following partial structure [where the outer side of the wavy lines at both ends is not included]:

[0394] [Chemistry 81]

[0395]

[0396] [Chemistry 82]

[0397] .

[0398] The preferred method of method [7] is further appropriately combined -L 1 -、-L 2 - and X are defined as preferred methods that can be arbitrarily formed in the aforementioned manner [7].

[0399] [8] The eighth method is as follows. A method for manufacturing cross-linked alginate includes performing a Wheatstone reaction by mixing an alginate derivative of formula (I) as described in the aforementioned method [1] and an alginate derivative of formula (II) as described in the aforementioned method [4] to obtain the cross-linked alginate as described in the aforementioned method [7].

[0400] [8-1] Method 8-1 is described below. Crosslinked alginate, comprising chemical crosslinking based on triazole rings formed by the Wheatstone reaction and ionic crosslinking partially formed by calcium ions as crosslinking.

[0401] [9] The ninth method is as follows. A cross-linked alginate structure is obtained by adding a mixed solution of an alginate derivative obtained by mixing the alginate derivative of formula (I) of method [1] and the alginate derivative of formula (II) of method [4] to a calcium chloride solution.

[0402]

[10] The tenth method is as follows. The cross-linked alginate structure described in the aforementioned method [9] comprises chemical cross-linking based on a triazole ring formed by the Wheatstone reaction and ionic cross-linking partially formed by calcium ions as cross-linking.

[0403]

[11] The 11th method is as follows. A method for manufacturing a cross-linked alginate structure, comprising adding a mixed solution of an alginate derivative obtained by mixing the alginate derivative of formula (I) of the aforementioned method [1] and the alginate derivative of formula (II) of the aforementioned method [4] dropwise into a calcium chloride solution to obtain the cross-linked alginate structure of the aforementioned method [9] or

[10] .

[0404]

[12] The 12th method is as follows. The cross-linked alginate structure described in the aforementioned method [9] or

[10] is a bead or near-spherical gel.

[0405]

[13] The 13th method is as follows. A medical material comprising a cross-linked alginate structure as described in any one of the foregoing methods [9],

[10] and

[12] .

[0406]

[14] The 14th method is as follows. The medical material described in the aforementioned method

[13] is a bead or a near-spherical gel.

[0407]

[15] The 15th method is as follows. The alginate derivative of any one of the preceding methods [1] to [6], the cross-linked alginate of any one of the preceding methods [7] or [8-1], and the cross-linked alginate structure of any one of the preceding methods [9],

[10] and

[12] , are biocompatible.

[0408]

[16] The 16th method is as follows. Amino compounds represented by the following formula (AM-1), or pharmaceutically acceptable salts thereof, or solvates thereof:

[0409] [Chemistry 83]

[0410]

[0411] In formula (AM-1), -L 1 - The combination of Akn is any combination of any item in the following table:

[0412] [Table 6-1]

[0413]

[0414]

[0415] [Table 6-2]

[0416]

[0417]

[0418] (The definitions of each form are the same as those in the aforementioned method [1]).

[0419] [16-1] In the aforementioned formula (AM-1) of the aforementioned method

[16] , Akn-L is preferred. 1 - The combination is any combination of items in the table below:

[0420] [Table 7]

[0421]

[0422]

[0423] (The forms are as described in the foregoing methods [1-1], [1-2], [1-1a], [1-2a], [1-1b], and [1-2b]);

[0424] More preferably, it is a combination of any of the items in the table below:

[0425] [Table 8]

[0426]

[0427] (The definitions of each form are the same as those of the aforementioned methods [1-1], [1-2], [1-1a], [1-2a], [1-1b], and [1-2b]);

[0428] Further preferred options are combinations of any of the items in the table below:

[0429] [Table 9]

[0430]

[0431] (The definitions of each form are the same as those of the aforementioned methods [1-1], [1-2], [1-1a], [1-2a], [1-1b], and [1-2b]);

[0432] For example, as shown in any of the following structural formulas:

[0433] [Chemistry 84]

[0434] .

[0435] The preferred method of

[16] is achieved by further appropriate combination of Akn and -L 1 - is defined as a preferred method for forming cross-linked alginate derivatives in the aforementioned manner

[16] .

[0436]

[17] Method 17 is described below. Amino compounds represented by the following formula (AM-2), or pharmaceutically acceptable salts thereof, or solvates thereof:

[0437] [Chemistry 85]

[0438]

[0439] In formula (II), -L 2 - is formula (LK-1) (wherein, the substitution of the benzene ring in the formula is para-substitution, except for n1=1 and n2=3), formula (LK-2), formula (LK-3), formula (LK-4) (wherein, the substitution of the benzene ring in the formula is para-substitution, except for n7=3, and the substitution of the benzene ring in the formula is para-substitution, except for n7=2, 3, 4, 6), formula (LK-5) (wherein, the substitution of the benzene ring in the formula is para-substitution, except for n8=1 and n9=2), formula (LK-6), and formula (LK-7) [each formula is the same as the definition of the aforementioned formula [4]].

[0440] [17-1] In the aforementioned formula (AM-2) of the aforementioned method

[17] , -L is preferred. 2 - These are equations (LK-1-1) (excluding n1=1 and n2=3), (LK-2-1), (LK-3-1), (LK-4-1) (excluding n7=2, 3, 4, 6), (LK-5-1) (excluding n8=1 and n9=2), (LK-6-1), and (LK-7-1) [each equation is defined in the same way as the aforementioned methods [4-1], [4-1a] or [4-1b]];

[0441] More preferably, formulas (LK-1-1-a), (LK-2-1-a), (LK-3-1-a), (LK-5-1-a), (LK-6-1-a), (LK-7-1-a), and (LK-7-1-b) [each formula is defined in the same way as the aforementioned methods [4-1], [4-1a], or [4-1b]].

[0442] The preferred method of

[17] is achieved by further appropriate combination of azide group and -L 2 - is defined as a preferred method that can optionally form a cross-linked alginate derivative in the manner described above

[17] .

[0443] The methods are explained in more detail below.

[0444] 1.Alginic acid

[0445] In this specification, when referred to as alginic acid, it means at least one alginic acid (sometimes called "alginic acid class") selected from alginic acid, alginic acid esters, and their salts (e.g., sodium alginate). The alginic acid used can be derived from natural sources or be synthetic, preferably from natural sources. The preferred alginic acid class is a bioavailable polysaccharide extracted from brown algae such as giant kelp, giant kelp, kelp, bladderwort, fucoidanum, black kelp, and kelp, which is a linear polymer of two uronic acids, D-mannuronic acid (M) and L-glucuronic acid (G). More specifically, it is a block copolymer obtained by arbitrary bonding of the homopolymer component of D-mannuronic acid (MM component), the homopolymer component of L-glucuronic acid (GG component), and the component obtained by the random arrangement of D-mannuronic acid and L-glucuronic acid (M / G component).

[0446] In this specification, alginate is sometimes referred to as (ALG), any one of the carboxyl groups of alginate is referred to as -COOH, and alginate is referred to as (ALG)-COOH.

[0447] In some embodiments, alginate is sodium alginate. Commercially available sodium alginate can be used. In the examples described below, the sodium alginate used is sodium alginate of types A-1, A-2, A-3, B-1, B-2, and B-3 as listed in the table below (issued by Mochida Pharmaceutical Co., Ltd.). The viscosity, weight-average molecular weight, and M / G ratio of a 1 w / w% aqueous solution of each sodium alginate are shown in Table 1.

[0448] [Table 10]

[0449]

[0450]

[0451] The physical properties of sodium alginate A-1, A-2, A-3, B-1, B-2, and B-3 were determined using the methods described below. The determination methods are not limited to these methods; sometimes the physical property values ​​differ from those described above depending on the method used.

[0452] [Viscosity Measurement of Sodium Alginate]

[0453] Viscosity determination was performed using a rotational viscometer (cone-plate rotational viscometer) according to the Japanese Pharmacopoeia (16th edition). Specific measurement conditions are as follows: The sample solution was prepared using MilliQ water. The measuring instrument used was a cone-plate rotational viscometer (Viscosity and Viscoelasticity Measurement Apparatus RS600 (Thermo Haake GmbH) sensor: 35 / 1). The rotational speed was 1 rpm when measuring with 1 w / w% sodium alginate solution. The reading time was set to 2 minutes, averaging the readings from the first 1 minute to 2 minutes. The average of three measurements was taken as the measured value. The measurement temperature was set to 20°C.

[0454] [Determination of weight-average molecular weight of sodium alginate]

[0455] The determination was performed using two methods: (1) gel permeation chromatography (GPC) and (2) GPC-MALS. The determination conditions are as follows.

[0456] [Preprocessing Method]

[0457] After the sample is dissolved by adding a dissolving agent, the solution obtained by filtering through a 0.45 μm membrane filter is used as the test solution.

[0458] (1) Gel permeation chromatography (GPC) determination

[0459] [Determination Conditions (Relative Molecular Weight Distribution)]

[0460] Columns: TSKgel GMPW-XL×2 + G2500PW-XL (7.8mm ID×300mm×3 pieces)

[0461] Eluent: 200mM sodium nitrate aqueous solution

[0462] Flow rate: 1.0 mL / min

[0463] Concentration: 0.05%

[0464] Detector: RI detector

[0465] Column temperature: 40℃

[0466] Injection volume: 200μL

[0467] Molecular weight standards: Standard amylopectin, glucose.

[0468] (2) GPC-MALS determination

[0469] [Determination of Refractive Index Increment (dn / dc) (Measurement Conditions)]

[0470] Differential refractive index meter: Optilab T-rEX

[0471] Measurement wavelength: 658nm

[0472] Measurement temperature: 40℃

[0473] Solvent: 200mM sodium nitrate aqueous solution

[0474] Sample concentration: 0.5~2.5 mg / mL (5 concentration).

[0475] [Determination Conditions (Absolute Molecular Weight Distribution)]

[0476] Column: TSKgel GMPW-XL×2+G2500PW-XL (7.8mm ID×300mm×3 books)

[0477] Eluent: 200mM sodium nitrate aqueous solution

[0478] Flow rate: 1.0 mL / min

[0479] Concentration: 0.05%

[0480] Detectors: RI detector, MALS (Massively Oriented Light scattering detector)

[0481] Column temperature: 40℃

[0482] Injection volume: 200μL

[0483] In this specification, the molecular weights of alginic acid, alginic acid derivatives, cross-linked alginic acid, and cross-linked alginic acid are sometimes denoted as Da (Daltons).

[0484] The ratio of D-mannuronic acid to L-glucuronic acid (M / G ratio) in alginates varies primarily depending on the species of organism from which they originate, such as seaweed. Furthermore, it is influenced by the habitat and season of the organism, ranging from a high G-type (M / G ratio of approximately 0.2) to a high M-type (M / G ratio of approximately 5). The gelling ability of alginates and the properties of the resulting gel are affected by the M / G ratio; it is generally known that a higher G ratio results in higher gel strength. In addition, the M / G ratio also affects the hardness, brittleness, water absorption, and softness of the gel. The M / G ratio of the alginates and / or their salts used is typically 0.2 to 4.0, more preferably 0.4 to 3.0, and even more preferably 0.5 to 3.0.

[0485] In this specification, the numerical range represented by "~" indicates the range of values ​​before and after "~" as their respective minimum and maximum values.

[0486] In this specification, the terms "alginate" and "alginate salt" are not particularly limited. However, in order to react with the crosslinking agent, they need to be functional groups that do not hinder the crosslinking reaction. Propylene glycol alginate is a preferred example of an alginate.

[0487] In this specification, examples of alginates include, for instance, monovalent and divalent salts of alginate. Preferred monovalent salts of alginate include sodium alginate, potassium alginate, and ammonium alginate; more preferably, sodium alginate or potassium alginate; and particularly preferably, sodium alginate. Preferred divalent salts of alginate include calcium alginate, magnesium alginate, barium alginate, and strontium alginate.

[0488] Alginic acid is a high-molecular-weight polysaccharide, and its molecular weight is difficult to determine accurately. Generally, its weight-average molecular weight is in the range of 10 million to 10 million, preferably 10,000 to 8 million, and more preferably 20,000 to 3 million. In the determination of the molecular weight of high-molecular-weight substances derived from natural sources, it is known that different determination methods can produce different values.

[0489] For example, the weight-average molecular weight, as determined by gel permeation chromatography (GPC) or gel filtration chromatography (which are also collectively referred to as size exclusion chromatography), is preferably 100,000 or more, more preferably 500,000 or more, and more preferably 5 million or less, more preferably 3 million or less. Its preferred range is 100,000 to 5 million, more preferably 150,000 to 3 million.

[0490] Furthermore, for example, the absolute weight-average molecular weight can be determined according to the GPC-MALS method. The weight-average molecular weight (absolute molecular weight) measured by the GPC-MALS method is preferably 10,000 or more, more preferably 50,000 or more, further preferably 60,000 or more, and preferably 1,000,000 or less, more preferably 800,000 or less, further preferably 700,000 or less, and particularly preferably 500,000 or less. Its preferred range is 10,000 to 1,000,000, more preferably 50,000 to 800,000, further preferably 60,000 to 700,000, and particularly preferably 60,000 to 500,000.

[0491] When calculating the molecular weight of high-molecular-weight polysaccharides using the above method, a measurement error of 10% to 20% typically occurs. For example, if the molecular weight is 400,000, the value will fluctuate within the range of approximately 320,000 to 480,000; if the molecular weight is 500,000, the value will fluctuate within the range of approximately 400,000 to 600,000; and if the molecular weight is 1,000,000, the value will fluctuate within the range of approximately 800,000 to 1,200,000.

[0492] The molecular weight of alginic acid can be determined using conventional methods.

[0493] Representative conditions for using gel filtration chromatography in molecular weight determination are as described in the examples below. For example, a Superose6 Increase 10 / 300 GL column (GE Healthcare) can be used as the developing solvent. A 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl can be used as the molecular weight standard. Blue dextran, thyroglobulin, ferritin, aldolase, conalbumin, ovalbumin, ribonuclease A, and aprotinin can also be used.

[0494] The viscosity of the alginate used in this specification is not particularly limited. When measuring the viscosity as a 1 w / w% aqueous solution of alginate, it is preferably 10 mPa·s to 1000 mPa·s, more preferably 50 mPa·s to 800 mPa·s.

[0495] The viscosity of aqueous solutions of alginate can be determined using conventional methods. For example, rotational viscometers such as coaxial double-cylinder type, single-cylinder type (Brookfield type), and cone-plate type (cone-plate type) can be used. Viscosity determination according to the Japanese Pharmacopoeia (16th edition) is preferred. A cone-plate type viscometer is even more preferred.

[0496] Alginic acids are extracted from brown algae. Initially, they have a large molecular weight and high viscosity, but through heat-based drying and purification processes, the molecular weight decreases and the viscosity decreases. Alginic acids with different molecular weights can be produced by managing conditions such as temperature during manufacturing, selecting the appropriate brown algae as raw materials, and classifying the molecular weight during manufacturing. Furthermore, by mixing them with other types of alginic acids with different molecular weights or viscosities, alginic acids with the target molecular weight can also be formed.

[0497] The alginic acid used in this specification is, in some ways, untreated alginic acid, or in others, treated alginic acid with low endotoxin levels. Low endotoxin levels are defined as endotoxin levels so low as to not substantially cause inflammation or fever. More preferably, it is desirable to use alginic acid treated with low endotoxin levels.

[0498] Low endotoxin treatment can be carried out by known methods or methods based thereon. For example, it can be carried out by methods such as those of Suga et al. for purifying sodium hyaluronate (e.g., see Japanese Patent Application Publication No. 9-324001), Yoshida et al. for purifying β1,3-glucan (e.g., see Japanese Patent Application Publication No. 8-269102), William et al. for purifying large molecular salts of organisms such as sodium alginate and gellan gum (e.g., see Japanese Patent Application Publication No. 2002-530440), James et al. for purifying polysaccharides (e.g., see International Publication No. 93 / 13136), Lewis et al. (e.g., see U.S. Patent No. 5589591), Herman Frank et al. for purifying alginate (e.g., see Appl Microbiol Biotechnol (1994) 40:638-643), or methods based thereon. Low endotoxin treatment is not limited to these methods and can be carried out by washing, filtration based on filters (endotoxin removal filters, electrostatic filters, etc.), ultrafiltration, purification using columns (endotoxin adsorption affinity columns, gel filtration columns, ion exchange resin-based columns, etc.), adsorption in hydrophobic substances, resins, or activated carbon, organic solvent treatment (organic solvent-based extraction, precipitation / sedimentation caused by the addition of organic solvents, etc.), surfactant treatment (for example, see Japanese Patent Application Laid-Open No. 2005-036036, etc.), or appropriate combinations thereof. Known methods such as centrifugation can be appropriately combined in these treatment steps. It is desirable to select appropriate methods based on the type of alginate.

[0499] Endotoxin levels can be confirmed using known methods, such as by using the horseshoe crab reagent (LAL) or by using the EndoSpecimen ES-24S Set (Biochemical Industrial Co., Ltd.).

[0500] The method for treating endotoxins is not particularly limited. As a result, when performing endotoxin determination using Limulus amebocyte lysate (LAL) reagent, the endotoxin content of alginate is preferably 500 endotoxin units (EU) / g or less, more preferably 100 EU / g or less, particularly preferably 50 EU / g or less, and especially preferably 30 EU / g or less. Low-endotoxin treated sodium alginate can be obtained from, for example, Sea Matrix (registered trademark) (Mochida Pharmaceutical Co., Ltd.), PRONOVA... TM Obtained through commercially available products such as UP LVG (FMCBioPolymer).

[0501] 2. Alginic acid derivatives

[0502] This specification provides novel alginate derivatives. In this specification, the alginate derivative is an alginate derivative obtained by introducing a reactive group in the Wheatstone reaction or a complementary reactive group of such reactive group onto any one or more carboxyl groups of alginate via an amide bond and a divalent linker.

[0503] More specifically, the alginate derivatives shown in formula (I) below:

[0504] [Chemistry 86]

[0505]

[0506] In formula (I), (ALG) and -L 1 - Akn is defined in the same way as in the first method above, and the alginate derivative shown in the following formula (II):

[0507] [Chemistry 87]

[0508]

[0509] In formula (II), (ALG) and -L 2 The definition of - is the same as that in the aforementioned fourth method).

[0510] The aforementioned divalent linker (-L) 1 -or-L 2 - Any straight-chain group may be used, provided it does not impair the reaction of the reactive group and the reactive group complementary to it. Specifically, straight-chain alkylene groups (-(CH2)) can be cited as examples. n - (n=1~30) The -CH2- in this group can be replaced by multiple (e.g., 1~10 or 1~5) groups such as -C(=O)-, -CONH-, -O-, -NH-, -S-, benzene ring, heterocycle (pyridine ring, piperidine ring, piperazine ring, etc., 5~6 membered aromatic heterocycles or 5~6 membered non-aromatic heterocycles), and the hydrogen atom of the -CH2- can be selected from oxo (=O), C 1-6 Substitution of multiple (e.g., 1 to 10 or 1 to 5) groups from groups such as alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, etc.), halogen atoms (e.g., fluorine, chlorine, bromine, iodine, etc.), and hydroxyl (-OH).

[0511] The novel alginate derivatives described in this specification, namely the alginate derivatives shown in formulas (I) and (II), can be manufactured, for example, using the methods described below (see the general manufacturing method described below for details).

[0512] [Chemistry 88]

[0513]

[0514] The weight-average molecular weight of the alginate derivatives represented by formula (I) or formula (II) in this specification is 100,000 Da to 3,000,000 Da, preferably 300,000 Da to 2,500,000 Da, and more preferably 500,000 Da to 2,000,000 Da. The molecular weight of the two alginate derivatives can be determined using the method described later.

[0515] In this specification, Akn-L in formula (I) 1 The -NH- group does not need to be bonded to all the carboxyl groups of the alginate constituent units, and the N3-L of formula (II) 2 The -NH- group does not need to be bonded to all the carboxyl groups of the alginate constituent units.

[0516] In this specification, Akn-L in formula (I) 1 When the -NH- group is called a reactive group, N3-L in formula (II) 2 -NH- groups serve as complementary reactive groups. Furthermore, conversely, the N3-L group of formula (II) is used... 2 When the -NH- group is called a reactive group, Akn-L in formula (I) 1 -NH- group serves as a complementary reactive group.

[0517] In this specification, the introduction rate of reactive groups or complementary reactive groups is 0.1% to 30% or 1% to 30%, preferably 2% to 20%, and more preferably 3% to 10%.

[0518] The introduction rate of the aforementioned reactive groups or complementary reactive groups is a percentage of the number of uronic acid monosaccharide units in which each reactive group has been introduced, which are repeating units of alginate. In this specification, unless otherwise specified, the % used for the introduction rate of reactive groups or complementary reactive groups in alginate derivatives (Formula (I) or Formula (II)) refers to mol%. The introduction rate of each reactive group or complementary reactive group can be determined using the method described in the examples below.

[0519] In this specification, the cyclic alkynyl group (Akn) in formula (I) and the azide group in formula (II) form a triazole ring through the Wheatstone reaction, thereby forming a crosslink.

[0520] 3. Wheatstone reaction

[0521] The Wheatstone reaction (1,3-dipolar addition cyclization) is a condensation reaction between compounds having terminal azido and terminal alkynyl groups as shown in the following formula. The reaction is characterized by the good yield of a disubstituted 1,2,3-triazole ring with no other byproducts. It is believed that the reaction can generate 1,4- or 1,5-disubstituted triazole rings, with positional selectivity for the triazole ring achieved by using a copper catalyst.

[0522] [Chemistry 89]

[0523]

[0524] Furthermore, Wittig and Krebs reported a Wheatstone reaction without the use of a copper catalyst. That is, a reaction in which only cyclooctynylene and phenyl azide are mixed to obtain the cycloadduct (in the following formula, R...). 3 =phenyl). Due to the significant twisting of the triple bond in cyclooctylene, the reaction with phenyl azide eliminates this twisting, which becomes the driving force. The reaction proceeds spontaneously and therefore does not require a catalyst.

[0525] [Chemistry 90]

[0526]

[0527] As described above, the Wheatstone reaction can use azide compounds with substituted primary, secondary, tertiary, and aromatic azide groups, as well as compounds with terminal or cyclic alkynyl groups as complementary reactive groups to the azide group. Furthermore, in the Wheatstone reaction, since essentially only the azide and alkynyl groups react, various functional groups (e.g., ester, carboxyl, alkenyl, hydroxyl, amino, etc.) can be substituted in the reaction matrix.

[0528] In some approaches, in order to avoid generating undesirable byproducts and to avoid cytotoxicity based on copper catalysts, and in order to form crosslinks based on 1,2,3-triazole rings between alginate molecules in a short time, easily and efficiently, the alkynyl group as the Wheatstone reaction is used, for example, the cyclic alkynyl group (cyclooctyl) described in the aforementioned approach [1].

[0529] In the preferred cross-linking method for alginate derivatives, the reaction (Wheatstone reaction) produces virtually no undesirable byproducts. Thus, various bioactive molecules can be introduced into the preparation of novel biocompatible materials using alginate and into the formation of alginate hydrogels. Furthermore, cellular substances can be introduced using alginate hydrogels for reconstructive surgery or gene therapy.

[0530] 4. Cross-linked alginate

[0531] Cross-linked alginate exists in the following forms: (i) cross-linked alginate obtained by bonding with divalent metal ions, (ii) cross-linked alginate obtained by chemical bonding, or (iii) cross-linked alginate obtained by a combination of bonding with divalent metal ions and chemical bonding. All cross-linked alginates exhibit the property of forming a semi-solid morphology from a gel-like state, and sometimes a sponge-like morphology.

[0532] Cross-linked alginate, obtained through bonding with divalent metal ions, undergoes a reversible reaction at ultra-high speed. In contrast, cross-linked alginate, obtained through chemical bonding, reacts slowly under milder conditions and is irreversible. The physical properties of cross-linked alginate can be adjusted, for example, by changing the concentration of the aqueous solution containing divalent metal ions (e.g., calcium chloride solution) or the introduction rate of reactive groups into the alginate.

[0533] Various alginate structures can be fabricated using the aforementioned cross-linking reactions. For example, specific structures can be instantaneously fabricated from an alginate solution using ionic cross-linking reactions. To strengthen the structure of these structures (e.g., to achieve long-term stability), chemically bonded cross-linking reactions can be employed. Furthermore, for example, in cross-linked alginate structures involving both divalent metal ion bonding and chemical bonding, the divalent metal ions obtained through ionic cross-linking can be reversibly released, allowing the fabrication of structures with only chemically bonded cross-linking remaining.

[0534] Cross-linked alginate in a certain manner can be obtained by mixing alginate derivatives of the aforementioned formula (I) and formula (II) and carrying out a Wheatstone reaction.

[0535] Cross-linked alginate, through a certain method, forms a three-dimensional mesh structure via chemical cross-linking (based on the cross-linking of triazole rings formed by alkynyl and azide groups). Preferably, the alginate derivative is a substance whose stability is improved after cross-linking.

[0536] Some cross-linked alginates are formed by the following formula (III-L) between any carboxyl group of the first alginate and any carboxyl group of the second alginate:

[0537] [Chemistry 91]

[0538]

[0539] In formula (III-L), the -CONH- and -NHCO- at both ends represent amide bonds derived from any carboxyl group of alginate; -L 1 -、-L 2 - Cross-linked alginate obtained by amide bonding of X and X as defined in the aforementioned method 7.

[0540] In some methods, the mixing ratio of the alginate derivative of formula (I) to the alginate derivative of formula (II) during the preparation of crosslinked alginate is expressed as the weight ratio of the derivative of formula (I) to the derivative of formula (II), for example, 1 to 1.5:1, preferably 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1.

[0541] In some methods, the mixing ratio of the alginate derivative of formula (II) to the alginate derivative of formula (I) during the preparation of crosslinked alginate is expressed as the weight ratio of the derivative of formula (II) to the derivative of formula (I), for example, 1 to 4.0:1, preferably 1.5 to 4.0:1, or 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1.

[0542] In some methods, the mixing ratio of the alginate derivative of formula (I) to the alginate derivative of formula (II) during the preparation of crosslinked alginate is more preferably based on the ratio of the introduction rate (mol%) of the reactive groups of the alginate derivative of formula (I) to the alginate derivative of formula (II), for example, 1 to 1.5:1, preferably 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1.

[0543] In some methods, the mixing ratio of the alginate derivative of formula (II) to the alginate derivative of formula (I) during the preparation of crosslinked alginate is more preferably based on the ratio of the introduction rate (mol%) of the reactive groups of the alginate derivative of formula (II) to the alginate derivative of formula (I), for example, 1 to 4.0:1, preferably 1.5 to 4.0:1, or 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1.

[0544] It should be noted that in the aforementioned mixing ratio, the alginate derivative of formula (I) can be replaced with the alginate derivative of formula (II), and the alginate derivative of formula (II) can be replaced with the derivative of formula (I).

[0545] Crosslinked alginate does not require all carboxyl groups of the constituent units of alginate to have the crosslinking of the above formula (III-L). The incorporation rate (also called crosslinking rate) of the crosslinking shown in the above formula (III-L) in crosslinked alginate is, for example, in the range of 0.1~80%, 0.3~60%, 0.5~30%, or 1.0~10%.

[0546] The concentration of the alginate derivative of formula (I) or formula (II) used in the Wheatstone reaction to obtain cross-linked alginate is typically 1 to 500 mg / mL, preferably in the range of 5 to 100 mg / mL.

[0547] The reaction temperature of the Wheatstone reaction is typically 4 to 60°C, preferably in the range of 15 to 40°C.

[0548] The stirring time used to form cross-linked alginate (hydrogel) is, for example, several seconds to 24 hours, several seconds to 12 hours, several seconds to 30 minutes, or several seconds to 10 minutes.

[0549] The reaction solvent or reaction solution used in the Wheatstone reaction is not particularly limited. Examples include tap water, pure water (e.g., distilled water, ion-exchange water, RO water, RO-EDI water, etc.), ultrapure water, cell culture medium, phosphate-buffered saline (PBS), and physiological saline, with ultrapure water being preferred.

[0550] Some types of cross-linked alginate include chemical cross-linking based on triazole rings formed by the Wheatstone reaction, and ionic cross-linking partially formed by calcium ions.

[0551] 5. Cross-linked alginate structure

[0552] Cross-linked alginate structures can be obtained by methods including performing a cross-linking reaction on the aforementioned alginate derivatives. For example, they can be prepared using the following methods, but are not limited to them.

[0553] [Hybrid Method]

[0554] By adding a mixed solution of alginate derivatives of formula (I) and formula (II) to a solution containing divalent metal ions, a specific structure, namely a cross-linked alginate structure, can be obtained, which forms chemical cross-links (cross-linking based on triazole rings formed by alkyne and azide groups via the Wheatstone reaction) and ionic cross-links (cross-linking partially formed by divalent metal ions).

[0555] [Coating Method]

[0556] A partially cross-linked specific structure is obtained by dropwise addition of a solution containing an alginate derivative of formula (I) to a solution containing divalent metal ions. A cross-linked alginate structure is obtained by adding a structure such as a gel obtained above to the aforementioned solution containing an alginate derivative of formula (II), and further performing a cross-linking reaction (Wheatstone reaction) on the surface of the aforementioned structure. It should be noted that this method can also be implemented by replacing the alginate derivative of formula (I) with the alginate derivative of formula (II), or by replacing the alginate derivative of formula (II) with the alginate derivative of formula (I), respectively.

[0557] The divalent metal ion used in the aforementioned method is not particularly limited. Examples include calcium ions, magnesium ions, barium ions, strontium ions, zinc ions, etc., with calcium ions being preferred.

[0558] The calcium ion-containing solution used in the aforementioned method is not particularly limited. For example, aqueous solutions such as calcium chloride, calcium carbonate, and calcium gluconate are available, with calcium chloride aqueous solution being preferred.

[0559] The concentration of calcium ions in the solution containing calcium ions used in the aforementioned method is not particularly limited. For example, 1 mM to 1 M can be mentioned, preferably 5 mM to 500 mM, and more preferably 10 mM to 300 mM.

[0560] The solvents or solutions used in the aforementioned methods are not particularly limited. Examples include tap water, pure water (e.g., distilled water, ion-exchange water, RO water, RO-EDI water, etc.), ultrapure water, cell culture medium, phosphate-buffered saline (PBS), and physiological saline, with ultrapure water being preferred.

[0561] Examples of specific cross-linked alginate structures include fibrous structures, fibers, beads, gels, and near-spherical gels. Cross-linked alginate structures are preferred because they offer improved stability. Furthermore, cross-linked alginate structures possess the ability to retain their contents (content retention).

[0562] The physical properties of alginate gel can be adjusted by adjusting values ​​such as hardness, elasticity, repulsive force, breaking force, and stress upon rupture.

[0563] 6. Biocompatibility of Alginic Acid Derivatives and Photocrosslinked Alginic Acid Derivatives

[0564] In this specification, the alginate derivative or photocrosslinked alginate structure is biocompatible. In this specification, biocompatibility refers to the property of not causing interactions between the material used in the organism (here, the alginate derivative with the photoreactive group shown in formula (I) and the photocrosslinked alginate structure manufactured using the alginate derivative) and the organism, local reactions in tissues adjacent to the aforementioned material in the organism, or systemic reactions.

[0565] In this specification, the biocompatibility of alginate derivatives or photocrosslinked alginate structures is confirmed using the examples of biocompatibility described later.

[0566] 7. Stability of cross-linked alginate structures

[0567] The stability of cross-linked alginate structures can be confirmed by measuring gel stability, and permeability can be confirmed by measuring gel permeability.

[0568] [Methods for determining gel stability]

[0569] Phosphate-buffered saline (PBS) was added to the cross-linked alginate structure gel in a container, and the concentration of alginate leaking from the PBS was measured (μg / mL). The disintegration rate was obtained by dividing the measured alginate concentration by the total alginate concentration obtained by decomposing the cross-linked alginate structure gel. Specifically, gel stability can be determined using the method described in the examples below.

[0570] In this specification, the gel disintegration rate of the cross-linked alginate structure is preferably 0% to 90%, more preferably 0% to 70%, and even more preferably 0% to 50%. The stability of the cross-linked alginate structure refers to the lower the concentration of alginate leaked in the aqueous solution, i.e., the lower the gel disintegration rate, the higher the stability.

[0571] [Method for determining gel permeability]

[0572] A cross-linked alginate structure gel containing fluorescein isothiocyanate-dextran was prepared. Physiological saline was added to the gel in a container, and the concentration of dextran leaked from the saline was measured. The gel permeability was calculated as a percentage by dividing the measured dextran concentration by the total dextran concentration obtained by decomposing the cross-linked alginate structure gel containing fluorescein isothiocyanate-dextran. Specifically, the gel permeability can be determined using the method described in the examples below.

[0573] The gel permeability after 24 hours of adding physiological saline containing cross-linked alginate, for example, when encapsulating dextran with a molecular weight of 2 million, is preferably 0% to 90%, more preferably 0% to 70%, and even more preferably 0% to 50%. Furthermore, when encapsulating dextran with a molecular weight of 150,000, for example, if the purpose of using the cross-linked alginate structure gel is the release or production of proteins or antibodies, it is preferably 1% to 100%, more preferably 10% to 100%, and even more preferably 30% to 100%. Furthermore, if the purpose is to create an immune barrier, it is preferably 0% to 90%, more preferably 0% to 70%, and even more preferably 0% to 50%.

[0574] The permeability of cross-linked alginate structures refers to the permeability of contents and substances outside the gel. The lower the permeability, the lower the permeability of contents and substances outside the gel. The higher the permeability, the higher the permeability of contents and substances outside the gel.

[0575] The permeability of the gel can be adjusted according to the molecular weight and concentration of the alginate used, the type and rate of cross-linking groups introduced into the alginate, the type and concentration of divalent metal ions used in gelation, or a combination thereof.

[0576] [Preparation method of cross-linked alginate structure gel containing internal contents]

[0577] For example, a cross-linked alginate structure gel containing fluorescein isothiocyanate-dextran can be prepared using the following method.

[0578] (1) A solution of the alginate derivative shown in formula (I) and a solution of fluorescein isothiocyanate-dextran.

[0579] (2) A solution of the alginate derivative shown in formula (II) mixed in the mixed solution obtained in (1).

[0580] (When equation (I) of (1) is changed to equation (II), equation (II) of (2) is changed to equation (I))

[0581] (3) The mixed solution obtained in (2) is added dropwise to a solution containing calcium ions to form a gel that forms chemical cross-links and ionic cross-links in the solution, thereby obtaining a cross-linked alginate structure gel containing fluorescein isothiocyanate.

[0582] 8. Synthetic methods of alginate derivatives

[0583] In this specification, the alginate derivatives represented by formula (I) or formula (II) can respectively make H2N-L 1 -Akn(where L is the formula) 1 And Akn (as defined in the aforementioned method [1]) represents an amine derivative (AM-1) or H2N-L 2 -N3(where L is the formula) 2 The amine derivative (AM-2) shown in the above method [4] is produced by condensation reaction of any carboxyl group of alginate with a condensing agent.

[0584] [Chemistry 92]

[0585]

[0586] [Preparation method of alginate derivative of formula (I)]

[0587] Using a 0.5%–1% by weight aqueous solution of alginate and an amine of formula (AM-1), the amine was prepared according to methods known in the literature, such as those described in “Experimental Chemistry Lectures 5th Edition 16, Synthesis of Organic Compounds IV, Carboxylic Acids and Derivatives, Esters, p35–70, Acid Amides and Acid Imidacyls, p118–154, Amino Acids and Peptides, p258–283, 2007, Maruzen”, and selected from 1,3-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC·HCl), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent), bis(2-oxo-3-oxazolyl)phosphine A condensation reaction of formula (I) is carried out in the presence of a condensing agent such as chloro(BOP-Cl), 2-chloro-1,3-dimethylimidazoline hexafluorophosphate (CIP), or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), in a mixed solvent of water and a solvent selected from those that do not precipitate alginate, such as tetrahydrofuran, ether solvents such as 1,4-dioxane, alcohol solvents such as methanol, ethanol, 2-propanol, and polar solvents such as N,N-dimethylformamide, in the presence or absence of inorganic bases such as sodium bicarbonate and sodium carbonate, or organic bases such as triethylamine and pyridine, at a temperature between 0°C and 50°C.

[0588] [Preparation method of alginate derivative of formula (II)]

[0589] Using an aqueous solution of alginate of 0.5% to 1% by weight and an amine of formula (AM-2), the alginate derivative of formula (II) can be prepared by reacting according to the aforementioned [Preparation method of alginate derivative of formula (I)].

[0590] In the preparation methods of the alginate derivatives of formula (I) or formula (II) mentioned above, the introduction rate of the amine of formula (AM-1) or formula (AM-2) can be adjusted by appropriately selecting the following reaction conditions (i) to (v) taking into account the properties of the amine: (i) equal increase or decrease of the amount of condensing agent, (ii) increase or decrease of the reaction temperature, (iii) extension or shortening of the reaction time, (iv) adjustment of the concentration of alginate in the reaction matrix, and the addition of an organic solvent mixed with water to improve the solubility of the amine of formula (AM-1) or formula (AM-2) in (v).

[0591] The following describes a more specific method for manufacturing amines represented by formula (AM-1) or formula (AM-2).

[0592] It should be noted that in the following manufacturing methods, R A =Methyl, ethyl, etc. C 1~6 Alkyl; P1 It is a protecting group selected from amino groups such as -C(O)O-tert-Bu, -C(O)O-Bn, -C(O)CH3, and -C(O)CF3; P 2 Protecting groups are amino groups selected from -C(O)O-tert-Bu, -C(O)O-Bn, -C(O)CH3, -C(O)CF3, -SO2Ph, -SO2PhMe, -SO2Ph(NO2) and other amino groups; E = detaching groups such as halogen atoms (fluorine, chlorine, bromine, iodine, etc.), -OTs, -OMs and other halogen atoms.

[0593] Furthermore, in the following manufacturing methods, the protective base P 1 and P 2 Protection and deprotection can be performed according to methods known in the literature, such as the deprotection methods described in books like "Protective Groups in Organic Synthesis 4th Edition, 2007, John Wiley & Sons, Greene, etc."

[0594] [Manufacturing Method A]

[0595] Method for producing amines as shown in formula (AM-OL-1):

[0596] [Chemistry 93]

[0597]

[0598] Using a compound of formula (SM-1) [the compound of formula (SM-1) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature] and a compound of formula (RG-1) [the compound of formula (RG-1) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; m1 = an integer from 2 to 6], according to a method known in the literature, such as the method described in "Carbohydrate Polymers, 169, p332-340, 2017", (i) in the presence of AgO3SCF3, in a solvent such as toluene that does not participate in the reaction, (ii) a debromination reaction is carried out using DBU, thereby forming an alkynyl group, and further (iii) by making the protecting group P 1 Deprotection can then be performed to produce amine compounds of formula (AM-OL-1) or salts of formula (AM-OL-1).

[0599] [Manufacturing Method B]

[0600] Method for manufacturing amines as shown in formula (AM-OL-2):

[0601] [Chemistry 94]

[0602]

[0603] <Step 1>

[0604] Using a compound of formula (SM-2) [the compound of formula (SM-2) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature] and a compound of formula (RG-2) [the compound of formula (RG-2) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature], according to a method known in the literature, such as the method described in "European Journal of Organic Chemistry, 2014(6), p1280-1286; 2014", (i) a photo-extending reaction is carried out in a solvent such as tetrahydrofuran that does not participate in the reaction in the presence of PPh3 and N2(CO2CHMe2)2 reagents, followed by (ii) hydrolysis is carried out in a solvent such as methanol, ethanol, tetrahydrofuran, water, or a mixture thereof that does not participate in the reaction in the presence of a base such as sodium hydroxide, thereby producing a compound shown in formula (IM-1).

[0605] <Step 2>

[0606] Using the compound of formula (IM-1) obtained by [Manufacturing Method B] <Step 1> and the compound of formula (RG-3) [the compound of formula (RG-3) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; m5 = an integer from 2 to 6], (iii) a condensation reaction is carried out in the same manner as described above [Preparation method of alginate derivative of formula (I)], followed by (iv) by making the protecting group P 1 Deprotection can produce amine compounds of formula (AM-OL-2) or salts of formula (AM-OL-2).

[0607] [Manufacturing Method C]

[0608] Method for manufacturing amines as shown in formula (AM-OL-3):

[0609] [Chem. 95]

[0610]

[0611] <Step 1>

[0612] Using the compound of formula (SM-1) and the compound of formula (RG-4) [the compound of formula (RG-4) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; m8 = an integer from 1 to 6], according to the method known in the literature, such as the method described in "Journal of the American Chemical Society, 126(46), pp. 15046-15047, 2004", (i) in the presence of AgClO4 in a solvent that does not participate in the reaction, such as toluene, the compound of formula (RG-4) is substituted, then (ii) debromination is carried out using NaOMe, thereby forming an alkynyl group, and (iii) hydrolysis is carried out in the presence of a base such as lithium hydroxide or sodium hydroxide in a solvent that does not participate in the reaction, such as methanol, ethanol, tetrahydrofuran, water, or a mixture thereof, thereby producing the compound shown in formula (IM-2).

[0613] <Step 2>

[0614] Using the compound of formula (IM-2) obtained by [Manufacturing Method C] <Step 1> and the compound of formula (RG-5) [the compound of formula (RG-5) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; m9 = an integer from 2 to 6], a condensation reaction is carried out in the same manner as described above in [Preparation Method of Alginic Acid Derivative of Formula (I)]. Then, the protecting group P is... 1 Deprotection can then be performed to produce amine compounds of formula (AM-OL-3) or salts of formula (AM-OL-3).

[0615] [Manufacturing Method D]

[0616] Method for manufacturing amines as shown in formula (AM-OL-5):

[0617] [Chemistry 96]

[0618]

[0619] <Step 1>

[0620] Using a compound of formula (SM-3) [the compound of formula (SM-3) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature], according to a method known in the literature, such as the method described in "Faming Zhuanli Shenqing, 104529898, 22 Apr 2015", (i) in the presence of a base such as pyridine, in a solvent that does not participate in the reaction, H2NOH-HCl is reacted to form an oxime group, then (ii) P2O5 is reacted with phosphorus pentoxide in methanesulfonic acid to carry out Beckmann transfer, thereby forming an 8-membered ring lactam, then (iii) in a solvent that does not participate in the reaction, the amide group is reduced using a reducing agent such as BH3 or LiAlH4, thereby producing a compound of formula (SM-3).

[0621] <Step 2>

[0622] Using compounds of formula (IM-3) and formula (RG-6) obtained by [Manufacturing Method D] <Step 1> [the compound of formula (RG-6) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; m3 = an integer from 1 to 6], (iv) a condensation reaction is carried out in the same manner as described above [Preparation method of alginate derivative of formula (I)] to obtain a condensate. Then, (v) after adding bromine, a debromination reaction is carried out using tertiary BuOK to form an alkynyl group. Then, (vi) the protecting group P is... 1 Deprotection can then be performed to produce amine compounds of formula (AM-OL-5) or salts of formula (AM-OL-5).

[0623] [Manufacturing Method E]

[0624] Methods for manufacturing amines as shown in formulas (AM-OL-6) and (AM-OL-7):

[0625] [Chemistry 97]

[0626]

[0627] <Step 1>

[0628] Using the compound of formula (IM-4) and the compound of formula (RG-7) obtained in step (ii) of [Manufacturing Method D] [the compound of formula (RG-7) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; m2' = an integer from 2 to 6], the compound shown in formula (IM-5) can be manufactured by reacting in a solvent that does not participate in the reaction, such as in the presence of a base such as sodium hydroxide and a phase-shifting catalyst such as tetrabutylammonium bromide, in the presence of a base such as sodium hydroxide and a phase-shifting catalyst such as tetrabutylammonium bromide.

[0629] <Step 2>

[0630] The compound of formula (IM-5) obtained in [Manufacturing Method E] <Step 1> is subjected to bromine addition, followed by debromination using a base such as tertiary Buok to form an alkynyl group. Then, the protecting group P is... 2 Deprotection can then be performed to produce amine compounds of formula (AM-OL-6) or salts of formula (AM-OL-6).

[0631] <Step 3>

[0632] Using the compound of formula (IM-5) obtained in [Manufacturing Method E] <Step 1>, the compound of formula (IM-6) can be produced by reacting it according to the reduction method of (iii) in [Manufacturing Method D] <Step 1>.

[0633] <Step 4>

[0634] Using the compound of formula (IM-6) obtained in [Manufacturing Method E] <Step 3>, the same reaction as in [Manufacturing Method E] <Step 2> can be carried out, thereby producing an amine compound of formula (AM-OL-7) or a salt of formula (AM-OL-7).

[0635] [Manufacturing Method F]

[0636] Method for manufacturing amines as shown in formula (AM-OL-8):

[0637] [Chem. 98]

[0638]

[0639] <Step 1>

[0640] Using a compound of formula (SM-4) [a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature], according to a method known in the literature, such as the method described in "Synthesis, (9), pp. 1191-1194; 2002", after the addition of bromine, a debromination reaction is carried out using tertiary Buok to form an alkyne group, thereby producing a compound of formula (IM-7).

[0641] <Step 2>

[0642] Using the compound of formula (IM-7) and the compound of formula (RG-8) obtained in [Manufacturing Method F] <Step 1> [the compound of formula (RG-8) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature (see Manufacturing Method H described below for details); m6 = an integer from 1 to 6; m7 = an integer from 2 to 6], the Wheatstone reaction is carried out according to a method known in the literature, such as the method described in "Journal. American. Chemical. Society., 126, pp. 15046-15047, 2004" or "Chem. Ber., 94, pp. 3260-3275, 1961", etc., and then the protecting group P is... 1 Deprotection can then be performed to produce amine compounds of formula (AM-OL-8) or salts of formula (AM-OL-8).

[0643] [Manufacturing Method G]

[0644] Method for manufacturing amines as shown in formula (AM-OL-9):

[0645] [Chemistry 99]

[0646]

[0647] Using a compound of formula (SM-5) [the compound of formula (SM-5) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature], according to a method known in the literature, such as the method described in "U.S. Patent Application Publication No. 2013-0137861", in a solvent that does not participate in the reaction, in the presence / absence of a base such as pyridine, it is reacted with p-nitrobenzene chloroformic acid to obtain a carbonate body. Next, in the presence of triethylamine, in an N,N-dimethylformamide solvent, it is reacted with a compound of formula (RG-9) [the compound of formula (RG-9) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; m4 = an integer from 1 to 6] to obtain a carbamoyl body. Further, by applying a protecting group P... 1Deprotection can then be performed to produce amine compounds of formula (AM-OL-9) or salts of formula (AM-OL-9).

[0648] [Manufacturing Method H]

[0649] The method for manufacturing the amine represented by formula (AM-LK-1) [in formula (AM-LK-1), para-substituted amines with n1=1 and n2=3 can also be manufactured according to the methods described in International Publication No. 2016 / 152980, etc.]:

[0650] [Chemistry 100]

[0651]

[0652] <Step 1>

[0653] Using a compound of formula (SM-6) [the compound of formula (SM-6) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; n1 = an integer from 1 to 6] and a compound of formula (RG-10) [the compound of formula (RG-10) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; n2 = an integer from 2 to 6], the same condensation reaction as described above [the preparation method of the alginate derivative of formula (I)] can be carried out, thereby producing formula (IM-8).

[0654] <Step 2>

[0655] Using the compound of formula (IM-8) obtained in [Manufacturing Method H] <Step 1>, according to a method known in the literature, such as the method described in "Organometallics, 29(23), p6619-6622; 2010", the compound is reacted with NaN3 in a solvent that does not participate in the reaction, such as dimethyl sulfoxide, to introduce an azide group, thereby protecting the P group. 1 Deprotection can then be performed to produce amine compounds of formula (AM-LK-1) or salts of formula (AM-LK-1).

[0656] [Manufacturing Method J]

[0657] Method for manufacturing amines as shown in formula (AM-LK-2):

[0658] [Chemistry 101]

[0659]

[0660] <Step 1>

[0661] Using a compound of formula (SM-7) [the compound of formula (SM-7) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature] and a compound of formula (RG-11) [the compound of formula (RG-11) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; n4 = an integer from 2 to 6], a photoelectrophoresis reaction based on [Manufacturing Method B] <Step 1> is carried out, and then, in the presence of an alkaline base such as sodium hydroxide, the ester group is hydrolyzed in a solvent that does not participate in the reaction, such as methanol, ethanol, tetrahydrofuran, water, or a mixture thereof, thereby producing a compound of formula (IM-9).

[0662] <Step 2>

[0663] Using the compound of formula (IM-9) obtained in [Manufacturing Method J] <Step 1> and the compound of formula (RG-12) [the compound of formula (RG-12) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; n3 = an integer from 2 to 6], a condensation reaction is carried out in the same manner as described above in [Preparation Method of Alginic Acid Derivative of Formula (I)] to obtain a condensate. Then, the protecting group P is... 1 Deprotection can then be performed to produce amine compounds of formula (AM-LK-2) or salts of formula (AM-LK-2).

[0664] [Manufacturing Method K]

[0665] The method for producing the amine shown in formula (AM-LK-3):

[0666] [Chemistry 102]

[0667]

[0668] <Step 1>

[0669] Using the compound of formula (SM-7) and the compound of formula (RG-13) of [Manufacturing Method J] <Step 1> [the compound of formula (RG-13) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; n6 = an integer from 2 to 6], a photoelectrophoresis reaction based on [Manufacturing Method B] <Step 1> is carried out, and then, in the presence of a base such as sodium hydroxide, the ester group is hydrolyzed in a solvent that does not participate in the reaction, such as methanol, ethanol, tetrahydrofuran, water, or a mixture thereof, thereby producing the compound shown in formula (IM-10).

[0670] <Step 2>

[0671] Using the compound of formula (IM-10) obtained in [Manufacturing Method K] <Step 1> and the compound of formula (RG-14) [the compound of formula (RG-14) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; n5 = an integer from 1 to 6], a condensation reaction is carried out in the same manner as described above in [Preparation Method of Alginic Acid Derivative of Formula (I)] to obtain a condensate. Then, the protecting group P is... 1 Deprotection can then be performed to produce amine compounds of formula (AM-LK-3) or salts of formula (AM-LK-3).

[0672] [Manufacturing Method L]

[0673] Method for manufacturing amines as shown in formula (AM-OL-4):

[0674] [Chemistry 103]

[0675]

[0676] <Step 1>

[0677] Using a compound of formula (SM-8) [a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature], after addition with bromine according to a method known in the literature, such as the method described in “International Publication No. 2009 / 067663”, debromination is carried out using LiN(i-Pr)2, thereby producing a compound of formula (IM-11).

[0678] <Step 2>

[0679] Using the compound of formula (IM-11) obtained in [Manufacturing Method L] <Step 1> and the compound shown in formula (RG-15) [the compound of formula (RG-15) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; m1 = an integer from 2 to 6], the compound with a side chain is reacted in the presence of a base such as sodium hydride in a solvent that does not participate in the reaction, such as tetrahydrofuran, thereby obtaining a compound with a side chain introduced. Next, the protecting group P... 1 Deprotection can then be performed to produce amine compounds of formula (AM-OL-4) or salts of formula (AM-OL-4).

[0680] [Manufacturing Method M]

[0681] The method for manufacturing the amine shown in formula (AM-LK-4):

[0682] [Chemistry 104]

[0683]

[0684] <Step 1>

[0685] Using the compound of formula (SM-M) and the compound of formula (RG-M-1) [the compound of formula (SM-M) and the compound of formula (RG-M-1) are commercially available compounds or compounds that can be manufactured from commercially available compounds using manufacturing methods known in the literature; n7 = an integer from 2 to 6], the same condensation reaction as described above [the preparation method of the alginate derivative of formula (I)] is carried out, thereby producing the compound shown in formula (IM-M-1).

[0686] Furthermore, according to methods known in the literature, such as those described in "Experimental Chemistry Lectures 5th Edition 16, Carboxylic Acids and Derivatives, Acid Halides, Acid Anhydrides, pp. 99-118, 2007, Maruzen", the carboxylic acid represented by formula (SM-M) is converted into an acid halide or anhydride. Using a compound of formula (RG-M-1), in the presence of a base such as triethylamine or pyridine, in a solvent selected from halogen solvents such as dichloromethane and chloroform, ether solvents such as diethyl ether and tetrahydrofuran, aromatic hydrocarbon solvents such as toluene and benzene, and polar solvents such as N,N-dimethylformamide, the reaction is carried out at a temperature from 0°C to solvent reflux. In this way, a compound of formula (IM-M-1) can be prepared in the same way.

[0687] <Step 2>

[0688] Using the compound of formula (IM-M-1) obtained in [Manufacturing Method M] <Step 1>, the reaction can be carried out according to a method known in the literature, such as the method described in "Protective Groups in Organic Synthesis" 4th Edition, 2007, John Wiley & Sons, by selecting an appropriate deprotection method according to the type of protecting group, thereby producing a compound of formula (AM-LK-4) or a salt of formula (AM-LK-4).

[0689] [Manufacturing Method N]

[0690] The method for producing the amine shown in formula (AM-OL-17):

[0691] [Chemistry 105]

[0692]

[0693] <Step 1>

[0694] Using the compound of formula (SM-N) and the compound of formula (RG-N-1) [the compound of formula (SM-N) and the compound of formula (RG-N-1) are commercially available compounds or compounds that can be manufactured from commercially available compounds using manufacturing methods known in the literature; m10 = 1~4, m11 = 1~6, m12 = 1~6], the same condensation reaction as described in [Manufacturing Method M] <Step 1> above is carried out, thereby producing the compound shown in formula (IM-N-1).

[0695] <Step 2>

[0696] Using the compound of formula (IM-N-1) obtained in [Manufacturing Method N] <Step 1>, the compound of formula (AM-OL-17) or a salt of formula (AM-OL-17) can be produced by following a method known in the literature, such as the method described in "Protective Groups in Organic Synthesis" 4th Edition, 2007, John Wiley & Sons, according to the type of protecting group, by selecting an appropriate deprotection method.

[0697] [Manufacturing Method P]

[0698] The method for manufacturing the amine represented by formula (AM-OL-18) [In formula (AM-OL-18), amines with m13=1 and m14=2 can also be manufactured according to the methods described in International Publication No. 2015 / 143092, etc.]:

[0699] [Chemistry 106]

[0700]

[0701] <Step 1>

[0702] Using a compound of formula (SM-P) and a compound of formula (RG-P-1) [the compound of formula (SM-P) and the compound of formula (RG-P-1) are commercially available compounds or compounds that can be manufactured from commercially available compounds using manufacturing methods known in the literature; m13 = 1 to 4, m14 = 2 to 6 integers], the same condensation reaction as described in [Manufacturing Method M] <Step 1> is carried out, thereby producing the compound shown in formula (IM-P-1).

[0703] <Step 2>

[0704] Using the compound of formula (IM-P-1) obtained in [Manufacturing Method P] <Step 1>, the compound of formula (AM-OL-18) or a salt of formula (AM-OL-18) can be produced by following a method known in the literature, such as the method described in "Protective Groups in Organic Synthesis" 4th Edition, 2007, John Wiley & Sons, according to the type of protecting group, by selecting an appropriate deprotection method.

[0705] [Manufacturing Method Q]

[0706] Method for manufacturing amines as shown in formula (AM-OL-19):

[0707] [Chemistry 107]

[0708]

[0709] <Step 1>

[0710] Using the compound of formula (SM-Q) and the compound of formula (RG-Q-1) [the compound of formula (SM-Q) and the compound of formula (RG-Q-1) are commercially available compounds or compounds that can be manufactured from commercially available compounds using manufacturing methods known in the literature; m15 = 1 to 4, m16 = 1 to 6 integers], the same condensation reaction as described in [Manufacturing Method M] <Step 1> is carried out, thereby producing the compound shown in formula (IM-Q-1).

[0711] <Step 2>

[0712] Using the compound of formula (IM-Q-1) obtained in [Manufacturing Method Q] <Step 1>, the compound of formula (AM-OL-19) or a salt of formula (AM-OL-19) can be produced by following a method known in the literature, such as the method described in "Protective Groups in Organic Synthesis" 4th Edition, 2007, John Wiley & Sons, according to the type of protecting group, by selecting an appropriate deprotection method.

[0713] [Manufacturing Method R]

[0714] The method for manufacturing the amine shown in formula (AM-LK-5) [In formula (AM-LK-5), amines with n8=1 and n9=2 can also be manufactured according to the methods described in International Publication No. 2016 / 152980, etc.]:

[0715] [Chemistry 108]

[0716]

[0717] <Step 1>

[0718] Using a compound of formula (SM-R) [the compound of formula (SM-R) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; n8 = an integer from 1 to 4] and a compound of formula (RG-R-1) [the compound of formula (RG-R-1) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; n9 = an integer from 1 to 6], the same condensation reaction as described above in [Manufacturing Method M] <Step 1> is carried out, thereby producing the compound shown in formula (IM-R-1).

[0719] <Step 2>

[0720] Using the compound of formula (IM-R-1) obtained in [Manufacturing Method R] <Step 1>, and reacting it with NaN3 in the same manner as in the aforementioned [Manufacturing Method H] <Step 2> to introduce an azide group, the protecting group P is then... 1 Deprotection can then be performed to produce amine compounds of formula (AM-LK-5) or salts of formula (AM-LK-5).

[0721] [Manufacturing Method S]

[0722] Method for manufacturing amines as shown in formula (AM-LK-6):

[0723] [Chemistry 109]

[0724]

[0725] <Step 1>

[0726] [When E = OTs basis or OMs basis]:

[0727] Using a compound of formula (SM-S) [the compound of formula (SM-S) is a commercially available compound or a compound that can be manufactured from a commercially available compound by a manufacturing method known in the literature; n10 = an integer from 1 to 4] and reagents such as methanesulfonyl chloride, toluenesulfonyl chloride, and toluenesulfonic anhydride, the compound shown in formula (IM-S-1) can be manufactured by a method known in the literature, such as the method described in "Journal of the American Chemical Society, 136(29), pp. 10450-10459, 2014", in the presence of a base such as triethylamine, N,N-diisopropylethylamine, or pyridine, using a halogen solvent such as dichloromethane or chloroform, an ether solvent such as diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, or 1,4-dioxane, an aromatic hydrocarbon solvent such as benzene or toluene, or a mixture thereof, or in the absence of a solvent, at a temperature from -78°C to solvent reflux.

[0728] [E = halogens (chlorine, bromine, iodine)]:

[0729] Using a compound of formula (SM-S), and following methods known in the literature, such as those described in "Experimental Chemistry Lectures 4th Edition 19, Organic Synthesis I, Hydrocarbons and Halogens, pp. 363-482, 1992, Maruzen", various halogenating agents (chlorinating agents, brominating agents, iodizing agents) and solvents that do not participate in the reaction are appropriately selected, and the reaction is carried out at a temperature from 0°C to solvent reflux, thereby producing a halogenated compound of formula (IM-S-1) (E = chlorine, bromine, iodine).

[0730] <E=Chlorine>

[0731] As chlorinating agents, reagents such as hydrogen chloride / zinc chloride (HCl / ZnCl2), hydrogen chloride / hexamethylphosphine triamide (HCl / HMPA), thionyl chloride (SOCl2), carbon tetrachloride / triphenylphosphine (CCl4 / PPh3), triphosgene / triphenylphosphine ((CCl3)2CO / PPh3), and triphosgene / N,N-dimethylformamide (POCl3 / DMF) can be used to produce the desired chloride.

[0732] <X=bromine>

[0733] As brominating agents, reagents such as 48% hydrobromic acid (48% HBr), 48% hydrobromic acid / sulfuric acid (48% HBr / H2SO4), hydrogen bromide / lithium bromide (HBr / LiBr), sodium bromide / sulfuric acid (NaBr / H2SO4), and phosphorus tribromide (PBr3) can be used to produce the desired chloride. Furthermore, in formula (IM-S-1), compounds in which E=OTs or OMs react with sodium bromide (NaBr) to produce the desired bromide.

[0734] <X = Iodine>

[0735] As iodizing agents, reagents such as hydroiodic acid (HI) and iodine / triphenylphosphine (I₂ / PPh₃) can be used to produce desired iodides. Alternatively, compounds in formula (IM-S-1) that cause E=OTs or OMs to react with sodium iodide (NaI) can also produce desired iodides.

[0736] <Step 2>

[0737] Using the compound of formula (IM-S-1) obtained in [Manufacturing Method S] <Step 1>, it is reacted with NaN3 in the same manner as in the aforementioned [Manufacturing Method H] <Step 2>, thereby producing a compound of formula (IM-S-2).

[0738] <Step 3>

[0739] Using the compound of formula (IM-S-2) obtained in [Manufacturing Method S] <Step 2>, hydrolysis is carried out in the same manner as the hydrolysis reaction of the ester group in the aforementioned [Manufacturing Method B] <Step 1>, thereby producing the compound of formula (IM-S-3).

[0740] <Step 4>

[0741] Using the compounds of formula (IM-S-3) and formula (RG-S-1) obtained in [Manufacturing Method S] <Step 3> [the compound of formula (RG-S-1) is a commercially available compound or a compound that can be manufactured from a commercially available compound using a manufacturing method known in the literature; n11 = an integer from 1 to 6], the same condensation reaction as described in [Manufacturing Method M] <Step 1> is carried out, thereby producing the compound shown in formula (IM-S-4).

[0742] <Step 5>

[0743] Protecting group P of the compound of formula (IM-S-4) obtained in [Manufacturing Method S] <Step 4> 1 Deprotection can then be performed to produce amine compounds of formula (AM-LK-6) or salts of formula (AM-LK-6).

[0744] [Manufacturing Method T]

[0745] The method for producing the amine shown in formula (AM-LK-7):

[0746] [Chemical 110]

[0747]

[0748] <Step 1>

[0749] Using the compound of formula (SM-M) and the compound of formula (RG-T-1) [the compound of formula (SM-M) and the compound of formula (RG-T-1) are commercially available compounds or compounds that can be manufactured from commercially available compounds using manufacturing methods known in the literature; n12 = an integer from 1 to 6], the same condensation reaction as described in [manufacturing method M] <step 1> is carried out, thereby producing the compound shown in formula (IM-T-1).

[0750] Furthermore, according to methods known in the literature, such as those described in "Experimental Chemistry Lectures 5th Edition 16, Carboxylic Acids and Derivatives, Acid Halides, Acid Anhydrides, pp. 99-118, 2007, Maruzen", the carboxylic acid represented by formula (SM-M) is converted into an acid halide or anhydride. Using a compound of formula (RG-T-1), in the presence of a base such as triethylamine or pyridine, in a solvent selected from halogen solvents such as dichloromethane and chloroform, ether solvents such as diethyl ether and tetrahydrofuran, aromatic hydrocarbon solvents such as toluene and benzene, and polar solvents such as N,N-dimethylformamide, the compound of formula (IM-T-1) can be similarly prepared by reacting the compound at a temperature from 0°C to solvent reflux in the presence of a base such as triethylamine or pyridine.

[0751] <Step 2>

[0752] Using the compound of formula (IM-T-1) obtained in [Manufacturing Method T] <Step 1>, the compound of formula (AM-LK-7) or a salt of formula (AM-LK-7) can be produced by following a method known in the literature, such as the method described in "Protective Groups in Organic Synthesis" 4th Edition, 2007, John Wiley & Sons, according to the type of protecting group, by selecting an appropriate deprotection method.

[0753] Regarding the alkyne-introduced amine (Akn-L) used for the manufacture of alginic acid derivatives of formula (I) or (II) 1 -NH2) or amines with an azide group (N3-L) 2The reactions described in [Manufacturing Method A] to [Manufacturing Method N] and [Manufacturing Method P] to [Manufacturing Method T] can be appropriately combined with methods known in the literature, such as those described in "Lectures on Experimental Chemistry, 5th Edition, 2007, Maruzen", "Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 3rd Edition (Edited by Richard C. Larock), 2018", and "Strategic Applications of Named Reactions in Organic Synthesis, (Edited by Laszlo Kurti, Barbara Czako), Academic Press, 2005", to produce the desired amine. It should be noted that the amines in the table below can also be produced using the methods described in the prior art literature listed in the table.

[0754] [Table 11]

[0755]

[0756] In this specification, amine compounds represented by formula (AM-1) or formula (AM-2) (including the lower formulas of each formula) sometimes form pharmaceutically acceptable salts (e.g., acid addition salts). There are no particular limitations on such salts, as long as they are pharmaceutically acceptable; examples include salts with inorganic acids, salts with organic acids, and salts with acidic amino acids. Suitable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Suitable examples of salts with organic acids include salts of aliphatic monocarboxylic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, butyric acid, valeric acid, heptanoic acid, decanoic acid, myristic acid, palmitic acid, stearic acid, lactic acid, sorbic acid, and mandelic acid; salts of aliphatic dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, and tartaric acid; salts of aliphatic tricarboxylic acids such as citric acid; salts of aromatic monocarboxylic acids such as benzoic acid and salicylic acid; salts of aromatic dicarboxylic acids such as phthalic acid; salts of organic carboxylic acids such as cinnamic acid, glycolic acid, pyruvic acid, oxyferric acid, salicylic acid, and N-acetylcysteine; salts of organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid; and acid addition salts of acidic amino acids such as aspartic acid and glutamic acid. Suitable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, etc. Among these, pharmaceutically acceptable salts are preferred.

[0757] According to conventional methods, for example, the compounds of the present invention are mixed with a solution containing an appropriate amount of acid or base to form the target salt, and then the mixed solvent is filtered off or distilled off, thereby obtaining the aforementioned salt. As a general overview of salts, a detailed description has been provided in the Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Stahl & Wermuth (Wiley-VCH, 2002).

[0758] In this specification, the amine compound (including the hypoforms of each formula) or its salt represented by formula (AM-1) or formula (AM-2) can form a solvate with solvents such as water, ethanol, and glycerol.

[0759] In this specification, unless otherwise specified, when a cyclic group is substituted with a variable substituent, the variable substituent refers to a group that does not bond to a specific carbon atom of the cyclic group. For example, in Formula A below, the variable substituent Rs refers to a group that can substitute for any one of carbon atoms i, ii, iii, iv, or v in Formula A.

[0760] [Chemistry 111]

[0761]

[0762] 9. Applications of alginate derivatives and cross-linked alginate structures

[0763] Alginic acid derivatives can replace traditional alginic acid in a wide range of fields, including food, medicine, cosmetics, fiber, and papermaking. Specifically, preferred applications of alginic acid derivatives or photocrosslinked alginic acid structures include medical materials such as wound dressings, postoperative anti-adhesion materials, drug release substrates, cell culture substrates, and cell transplantation substrates.

[0764] Examples of shapes for cross-linked alginate structures used as medical materials include tubular, fibrous, fiber, bead, gel, and near-spherical gels. Beads, gels, or near-spherical gels are preferred, and near-spherical gels are even more preferred.

[0765] It should be noted that all documents and publications mentioned in this specification, regardless of their purpose, are incorporated herein by reference in their entirety.

[0766] Furthermore, the objectives, features, advantages, and concepts of the present invention will be readily apparent to those skilled in the art from the description herein, and any person skilled in the art can easily implement the invention based on this description. The preferred embodiments and specific examples illustrating the invention are for illustrative purposes only, and the invention is not limited thereto. It will be apparent to those skilled in the art that various modifications can be made based on the description herein within the intent and scope of the invention disclosed herein. Example

[0767] Next, embodiments and test examples are given to further illustrate the present invention in detail. These examples are merely embodiments and test examples and do not limit the present invention. Moreover, variations may be made within the scope of the present invention.

[0768] Nuclear magnetic resonance (NMR) spectroscopy was performed using a JEOL JNM-ECX400 FT-NMR (Japan Electronics). Liquid chromatography-mass spectrometry (LC-Mass) was performed using the following method: A Waters AQUITY UPLC system and a BEH C18 column (2.1 mm × 50 mm, 1.7 μm) (Waters) were used, with acetonitrile:0.05% trifluoroacetic acid aqueous solution = 5:95 (0 min) ~ 95:5 (1.0 min) ~ 95:5 (1.6 min) ~ 5:95 (2.0 min) as the mobile phase and gradient conditions.

[0769] 1 In H-NMR data, in the NMR signal modes, s represents singlet state, d represents doublet state, t represents triplet state, q represents quartet state, m represents multiplyt state, br represents wide range, J represents coupling constant, Hz represents Hertz, CDCl3 represents deuterated chloroform, DMSO-D6 represents deuterated dimethyl sulfoxide, and D2O represents deuterated water. 1 In the H-NMR data, protons of hydroxyl (OH), amino (NH2), and carboxyl (COOH) groups were not recorded because their signals were broadband and could not be confirmed.

[0770] In LC-Mass data, M refers to molecular weight, RT refers to retention time, and [M+H] represents molecular weight. ＋ [M+Na] ＋ Refers to the molecular ion peak.

[0771] In the examples, “room temperature” typically refers to a temperature of about 0°C to about 35°C.

[0772] The reactive substituent introduction rate (mol%) in the examples represents the number of moles of reactive substituents introduced relative to the total number of moles introduced. 1The molar ratio of the monosaccharide glucuronic acid and mannuronic acid units that make up alginate, calculated by H-NMR (D2O).

[0773] In the embodiments, the sodium alginate before the introduction of reactive groups or complementary reactive groups uses sodium alginate with the physical property values ​​shown in Table 10 above.

[0774] Table 12 shows the physical properties (specifically, reactive group introduction rate (mol%), molecular weight, and weight-average molecular weight (10,000 Da)) of the alginate derivatives with introduced reactive groups obtained in Examples 1 to 15 (Examples 1a, 1b, 1c, 1d, 1e, 1f, 2, 3a, 3b, 3c, 3d, 3e, 3f, 4, 5a, 5b, 6, 7a, 7b, 8, 9a, 9b, 9c, 10, 11, 12, 13, 14 and 15).

[0775] (Example 1)

[0776] Synthesis of alginic acid with dibenzocyclooctynylamine group (Examples 1a, 1b, 1c, 1d, 1e, 1f, and 1g):

[0777] [Chemistry 112]

[0778]

[0779] (Example 1a) Synthesis of alginic acid (EX1-(I)-A-2) with dibenzocyclooctyne-amino group introduced:

[0780] To prepare a 1% (w / w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (111.65 mg) and 1 mol sodium bicarbonate solution (403.5 μL) were added. A commercially available ethanol solution of dibenzocyclooctyne-amine [CAS: 1255942-06-3] (EX1-SM, 83.62 mg) was added dropwise to this solution, and the mixture was stirred at room temperature for 18 hours. After adding sodium chloride (400 mg), ethanol (87.2 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX1-(I)-A-2 (376 mg) as a pale yellow solid.

[0781] The introduction rate of the reactive substituent (dibenzocyclooctyn-amino) was 6.9 mol% (NMR integral ratio).

[0782] (Example 1b) Synthesis of alginic acid (EX1-(I)-A-1) with dibenzocyclooctyne-amino group introduced:

[0783] To prepare a 1% (w / w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-1) (19.32 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (49.47 mg) and 1 mol sodium bicarbonate solution (178.8 μL) were added dropwise. An ethanol solution (4 mL) of commercially available dibenzocyclooctyne-amine [CAS: 1255942-06-3] (EX1-SM, 37.05 mg) was added dropwise, and the mixture was stirred at room temperature for 20 hours. After adding sodium chloride (200 mg), ethanol (38.64 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX1-(I)-A-1 (184 mg) as a pale yellow solid.

[0784] The introduction rate of the reactive substituent (dibenzocyclooctyn-amino) was 6.5 mol% (NMR integral ratio).

[0785] (Example 1c) Synthesis of alginic acid (EX1-(I)-A-3) with dibenzocyclooctyne-amino group:

[0786] To prepare a 1% (w / w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-3) (15.06 mL), 38.57 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) and 139.4 μL of sodium bicarbonate solution (1 mol) were added dropwise. A 2 mL ethanol solution of commercially available dibenzocyclooctyne-amine [CAS: 1255942-06-3] (EX1-SM, 28.88 mg) was added dropwise, and the mixture was stirred at room temperature for 23 hours. After adding 150 mg of sodium chloride, 60.24 mL of ethanol was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX1-(I)-A-3 (164 mg) as a pale yellow solid.

[0787] The introduction rate of the reactive substituent (dibenzocyclooctyn-amino) was 6.6 mol% (NMR integral ratio).

[0788] (Example 1d) Synthesis of alginic acid (EX1-(I)-B-2a) with dibenzocyclooctyne-amino group introduced:

[0789] To prepare a 1% (w / w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: B-2) (53.0 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (111.0 mg), a dibenzocyclooctyne-amine [CAS: 1255942-06-3] (EX1-SM, 36.9 mg) in ethanol (5.3 mL), and 1 mol sodium bicarbonate solution (113.7 μL) were added. The mixture was stirred at 30 °C for 3 hours. After adding sodium chloride (530 mg), ethanol (101 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX-(I)-B-2a (465 mg) as a white solid.

[0790] The introduction rate of the reactive substituent (dibenzocyclooctyn-amino) was 4.9 mol% (NMR integral ratio).

[0791] (Example 1e) Synthesis of alginic acid (EX1-(I)-B-2b) with dibenzocyclooctyne-amino group introduced:

[0792] To prepare a 1% (wt%) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: B-2), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (14.7 mg), dibenzocyclooctyne-amine [CAS: 1255942-06-3] (EX1-SM, 4.9 mg), 1 mol sodium bicarbonate solution (17.7 μL), and ethanol (3.5 mL) were added, and the mixture was stirred at 30 °C for 3.5 h. After adding sodium chloride (350 mg), ethanol (70 mL) was added, and the mixture was stirred at room temperature for 30 min. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX-(I)-B-2 (329 mg) as a white solid.

[0793] The introduction rate of the reactive substituent (dibenzocyclooctyn-amino) was 0.8 mol% (NMR integral ratio).

[0794] (Example 1f) Synthesis of alginic acid (EX1-(I)-B-2c) with dibenzocyclooctyne-amino group introduced:

[0795] To a 1% (wt%) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: B-2) (60.0 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (67.0 mg), dibenzocyclooctyne-amine [CAS: 1255942-06-3] (EX1-SM, 16.7 mg), 1 mol sodium bicarbonate solution (60.5 μL), and ethanol (6.0 mL) were added, and the mixture was stirred at 30 °C for 3 hours. After adding sodium chloride (600 mg), ethanol (120 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX-(I)-B-2c (558 mg) as a white solid.

[0796] The introduction rate of the reactive substituent (dibenzocyclooctyn-amino) was 1.9 mol% (NMR integral ratio).

[0797] (Example 1g) Synthesis of alginic acid (EX1-(I)-A-2b) with dibenzocyclooctyne-amino group introduced:

[0798] Using the same method as in (Example 1a), the title compound (EX1-(I)-A-2b) with an NMR integral ratio of 4.9 mol% for the introduction of reactive substituents was obtained.

[0799] (Example 2) Synthesis of N-(1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl-1,8-diamino-3,6-dioxaoctyl alginate (compound EX2-(I)-A-2):

[0800] [Chemistry 113]

[0801]

[0802] To prepare a 1% (w / w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (10.9 mL), 27.91 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) and 100.9 μL of sodium bicarbonate solution were added at room temperature. A commercially available solution of N-(1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl-1,8-diamino-3,6-dioxaoctane [CAS1263166-93-3] (EX-2-SM, 24.54 mg) in ethanol (2 mL) and water (1 mL) was added dropwise to this solution at room temperature, and the mixture was stirred for 21 hours at the same temperature. After adding 100 mg of sodium chloride, 21.8 mL of ethanol was added, and the mixture was stirred for 30 minutes at room temperature. The precipitate was filtered off, washed with ethanol, and dried under reduced pressure to give the title compound EX2-(I)-A-2 (100 mg) as a pale yellow solid.

[0803] The introduction rate of the reactive substituent (N-(1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl-1,8-diamino-3,6-dioxaoctyl) was 5.8 mol% (NMR integral ratio).

[0804] (Example 3) Synthesis of alginate with 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group (Examples 3a, 3b, 3c, 3d, 3e, 3f, and 3g):

[0805] [Chemistry 114]

[0806]

[0807] <Step 1> Synthesis of methyl 4-(2-((tert-butoxycarbonyl)amino)ethoxy)benzoate (compound EX3-IM-1):

[0808] [Chemistry 115]

[0809]

[0810] A solution of diethyl azodicarboxylate (40% toluene solution, 1.92 mL) was added to a solution of triphenylphosphine (0.96 g) in tetrahydrofuran (2.59 mL) under ice-cooled stirring, and the mixture was stirred at room temperature for 20 minutes. To this solution, a solution of commercially available methyl 4-hydroxybenzoate [CAS: 99-76-3] (compound EX3-SM, 0.37 g) and 2-(tert-butoxycarbonyl)ethanolamine [CAS: 26690-80-2] (0.39 g) in tetrahydrofuran (1.1 mL) was added under ice-cooled stirring, and the mixture was stirred at room temperature for 17 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (5% ethyl acetate / n-heptane ~ 40% ethyl acetate / n-heptane) to give a mixture of compounds 1 and 2. This mixture was dissolved in methyl tert-butyl ether (20 mL), washed twice with 1 equivalent concentration sodium hydroxide aqueous solution (5 mL), and then washed with saturated saline solution (5 mL). After drying the organic layer with anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure to obtain compound EX3-IM-1 (0.45 g) as a pink oily substance.

[0811] NMR data (CDCl3) (δ: ppm): 7.98 (2H, d, J = 8.8 Hz), 6.90 (2H, d, J = 8.8Hz), 4.97 (1H, br s), 4.07 (2H, t, J = 5.2 Hz), 3.88 (3H, s), 3.56 (2H, q, J= 5.2 Hz),1.45 (9H, s)

[0812] <Step 2> Synthesis of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide hydrochloride (compound EX3-IM-3):

[0813] [Chemistry 116]

[0814]

[0815] Lithium hydroxide monohydrate (0.25 g) was added to a methanol (4.4 mL) solution of compound EX3-IM-1 (0.44 g) obtained in (Example 3) <Step 1>, and the mixture was stirred at 60°C for 3 hours and 30 minutes. Hydrochloric acid (5 mL) of 1 equivalent concentration was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (10 mL). The organic layer was washed successively with water (5 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was dissolved in acetonitrile (4.4 mL), and 3-azidopropane-1-amine [CAS: 88192-19-2] (0.15 g) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureonium hexafluorophosphate (0.57 g) were added. Then, N,N-diisopropylethylamine (0.52 mL) was added under ice-cooled stirring, and the mixture was stirred at room temperature for 5 hours. Water (10 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (15 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane ~ 100% ethyl acetate) to obtain a fraction containing compound EX3-IM-2 (0.71 g).

[0816] The fraction containing compound EX3-IM-2 (0.71 g) was mixed with 4.9 mL of 4 equimolar concentration hydrogen chloride / 1,4-dioxane and stirred at room temperature for 20 minutes. After adding diisopropyl ether to the reaction mixture, the precipitate was filtered to give the title compound EX3-IM-3 (0.49 g) as a white solid.

[0817] NMR data (CDCl3) (δ: ppm): 7.60 (2H, d, J = 8.8 Hz), 6.93 (2H, d, J = 8.8 Hz), 4.19 (2H, t, J = 4.8 Hz), 3.31–3.29 (6H, m), 1.77–1.71 (2H, m). LC-MS: M (free amine) = 263, RT = 0.54 (min), [M+H] ＋ =264

[0818] (Example 3a) Synthesis of alginate (compound EX3-(II)-A-2) with introduction of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group:

[0819] [Chemistry 117]

[0820]

[0821] In a 1% (wt%) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (19.6 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (50.19 mg), compound EX3-IM-3 obtained in (Example 3) <Step 2> (54.37 mg), and 1 mol concentration sodium bicarbonate solution (181.4 μL) were added under ice-cooled stirring, and the mixture was stirred at room temperature for 5 hours. After adding sodium chloride (200 mg), ethanol (39.2 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX3-(II)-A-2 (198 mg) as a white solid.

[0822] The introduction rate of the reactive substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide) was 6.1 mol% (NMR integral ratio).

[0823] (Example 3b) Synthesis of alginate (compound EX3-(II)-A-1) with introduction of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group:

[0824] [Chemistry 118]

[0825]

[0826] In a 1% (wt%) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-1) (19.32 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (49.47 mg), compound EX3-IM-3 obtained in (Example 3) <Step 2> (53.39 mg), and 1 mol concentration sodium bicarbonate solution (178.8 μL) were added under ice-cooled stirring, and the mixture was stirred at room temperature for 20 hours. After adding sodium chloride (200 mg), ethanol (38.64 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give compound EX-(II)-A-1 (221 mg) as a white solid.

[0827] The introduction rate of the reactive substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide) was 9.4 mol% (NMR integral ratio).

[0828] (Example 3c) Synthesis of alginate (compound EX3-(II)-A-3) with introduction of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group:

[0829] [Chemistry 119]

[0830]

[0831] In a 1% (wt%) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-3) (15.06 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (38.57 mg), compound EX3-IM-3 obtained in (Example 3) <Step 2> (41.78 mg), and 1 mol concentration sodium bicarbonate solution (139.4 μL) were added under ice-cooled stirring, and the mixture was stirred at room temperature for 5 hours. After adding sodium chloride (150 mg), ethanol (60.24 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give compound EX3-(II)-A-3 (155 mg) as a white solid.

[0832] The introduction rate of the reactive substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide) was 6.9 mol% (NMR integral ratio).

[0833] (Example 3d) Synthesis of alginate (compound EX3-(II)-B-2a) with introduction of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group:

[0834] [Chemistry 120]

[0835]

[0836] To a 1% (w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: B-2) (60.0 mL), 125.6 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), 45.4 mg of compound EX3-IM-3 obtained in step 2 of Example 3, and 211.8 μL of sodium bicarbonate solution were added, and the mixture was stirred at 30°C for 3 hours. After adding 600 mg of sodium chloride, 120 mL of ethanol was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX3-(II)-A-2 (553 mg) as a white solid.

[0837] The introduction rate of the reactive substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide) was 3.7 mol% (NMR integral ratio).

[0838] (Example 3e) Synthesis of alginate (compound EX3-(II)-B-2b) with introduction of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group:

[0839] [Chemistry 121]

[0840]

[0841] To a 1% (w / w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: B-2), 14.7 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), 5.3 mg of compound EX3-IM-3 obtained in step 2 of Example 3, and 26.5 μL of sodium bicarbonate solution were added, and the mixture was stirred at 30°C for 3.5 hours. After adding 350 mg of sodium chloride, 70 mL of ethanol was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX3-(II)-A-2 (304 mg) as a white solid.

[0842] The introduction rate of the reactive substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide) was 0.6 mol% (NMR integral ratio).

[0843] (Example 3f) Synthesis of alginate (compound EX3-(II)-B-2c) with introduction of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group:

[0844] [Chemistry 122]

[0845]

[0846] To a 1% (w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: B-2) (60.0 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (67.0 mg), compound EX3-IM-3 obtained in (Example 3) <Step 2> (18.1 mg), and 1 mol concentration sodium bicarbonate solution (90.8 μL) were added, and the mixture was stirred at 30°C for 3 hours. Sodium chloride (600 mg) was added, followed by ethanol (120 mL), and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX3-(II)-A-2 (568 mg) as a white solid.

[0847] The introduction rate of the reactive substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide) was 1.5 mol% (NMR integral ratio).

[0848] (Example 3g) Synthesis of alginate (EX3-(II)-A-2b) with 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group:

[0849] Using the same method as in (Example 3a), the title compound (EX3-(II)-A-2b) with an NMR integral ratio of 4.3 mol% for the introduction of reactive substituents was obtained.

[0850] (Example 4) Synthesis of alginate (compound EX4-(II)-A-2) with introduction of 4-(3-aminopropoxy)-N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)benzamide group:

[0851] [Chemistry 123]

[0852]

[0853] <Step 1> Synthesis of 4-(3-((tert-butoxycarbonyl)amino)propoxy)benzoic acid (compound EX4-IM-2):

[0854] [Chemistry 124]

[0855]

[0856] To a solution of triphenylphosphine (2.07 g) in tetrahydrofuran (7 mL), diisopropyl azodicarboxylate (40% toluene solution, 4.15 mL) was added, and the mixture was stirred until a precipitate formed. After stirring for another hour, a solution of commercially available tert-butyl (3-hydroxypropyl)carbamate [CAS: 58885-58-8] (1.15 g) and methyl 4-hydroxybenzoate [CAS: 99-76-3] (compound EX4-SM, 1 g) in tetrahydrofuran (3 mL) was added, and the mixture was stirred for 3 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (8% ethyl acetate / n-heptane ~ 66% ethyl acetate / n-heptane). The purified product was dissolved in methyl tert-butyl ether (20 mL), washed twice with 1 equivalent concentration sodium hydroxide aqueous solution (5 mL), and then washed with saturated brine (5 mL). The organic layer was dried with anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure to obtain a component containing compound EX4-IM-1 (2.94 g) as a white solid.

[0857] For a methanol (15.6 mL) solution containing 2.94 g of compound EX4-IM-1, lithium hydroxide monohydrate (1.06 g) was added with stirring at room temperature, and the mixture was stirred at 60 °C for 3 hours. After cooling to room temperature, the solvent was distilled off under reduced pressure. The residue was then extracted twice with methyl tert-butyl ether (20 mL) after adding water (20 mL). The aqueous layer was acidified with 1 equivalent hydrochloric acid (25 mL) and extracted three times with ethyl acetate (20 mL), followed by washing with water (10 mL) and saturated brine (10 mL) sequentially. The organic layer was dried over anhydrous sodium sulfate and then distilled off under reduced pressure. Methyl tert-butyl ether (30 mL) and 1 equivalent sodium hydroxide aqueous solution (20 mL) were added to the residue, and the mixture was extracted twice with methyl tert-butyl ether (20 mL). The aqueous layer was acidified with 1 equivalent hydrochloric acid (20 mL) and extracted twice with ethyl acetate (20 mL). The organic layer was dried with anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure to give compound 4-3 (1.4 g) as a white solid.

[0858] NMR data (CDCl3) (δ: ppm): 8.03 (2H, d, J = 7.6 Hz), 6.92 (2H, d, J = 8.8Hz), 4.73 (1H, br s), 4.09 (2H, t, J = 6.0 Hz), 3.34 (2H, q, J = 6.3 Hz),2.05-1.98 (2H,m), 1.45 (9H, s)

[0859] <Step 2> Synthesis of 4-(3-aminopropoxy)-N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethyl)benzamide hydrochloride (compound EX4-IM-4):

[0860] [Chemistry 125]

[0861]

[0862] For a 20 mL acetonitrile solution of compound EX4-IM-2 (1 g) obtained in Step 1 of Example 4, commercially available 2-(2-(2-azidoethoxy)ethoxy)ethane-1-amine [CAS: 166388-57-4] (0.62 g) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureon hexafluorophosphate (1.35 g), N,N-diisopropylethylamine (1.24 mL) was added dropwise under ice-cooled stirring, and the mixture was stirred at room temperature for 1 hour. Water (20 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (20 mL), washed successively with water (10 mL) and saturated brine (10 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane ~ 100% ethyl acetate) to obtain a fraction containing compound EX4-IM-3 (1.37 g).

[0863] For the fraction containing compound EX4-IM-3 (1.37 g), 1,4-dioxane (9.58 mL) was added. For this solution, 4 equivalence hydrogen chloride / 1,4-dioxane (9.58 mL) was added with water cooling and stirring, and the mixture was stirred at room temperature for 1 hour. Diisopropyl ether (100 mL) was added to the reaction mixture, and the suspension was stirred at room temperature for 1 hour. The solvent was distilled off under reduced pressure, and the residue was ground with ethyl acetate (20 mL) and methyl tert-butyl ether (10 mL). The resulting solid was filtered and dried under reduced pressure, thus giving the title compound EX4-IM-4 (1.23 g) as a white solid.

[0864] NMR data (D₂O) (δ: ppm): 7.66–7.64 (2H, m), 6.98–6.94 (2H, m), 4.12 (2H, t, J = 5.6 Hz), 3.66–3.57 (6H, m), 3.57–3.52 (2H, m), 3.47 (2H, t, J = 5.2 Hz), 3.29 (2H, t, J = 4.8 Hz), 3.12 (2H, t, J = 7.2 Hz), 2.10–2.04 (2H, m); LC-MS: M (free amine) = 351, RT = 0.57 (min), [M+H] ＋ =352

[0865] <Step 3> Synthesis of alginate (compound EX4-(II)-A-2) with 4-(3-aminopropoxy)-N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)benzamide group:

[0866] [Chemistry 126]

[0867]

[0868] In a 1% (w / w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (19.6 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (50.19 mg), compound EX4-IM-4 obtained in (Example 4) <Step 2> (70.35 mg), and 1 mol concentration sodium bicarbonate solution (181.4 μL) were added under ice-cooled stirring, and the mixture was stirred at room temperature for 5 hours. After adding sodium chloride (200 mg), ethanol (39.2 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX4-(II)-A-2 (199 mg) as a white solid.

[0869] The introduction rate of the reactive substituent (4-(3-aminopropoxy)-N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethyl)benzamide) was 4.3 mol% (NMR integral ratio).

[0870] (Example 5) Synthesis of alginic acid with N-(2-aminoethyl)-4-(azidomethyl)benzamide group (Examples 5a, 5b and 5c):

[0871] [Chemistry 127]

[0872]

[0873] <Step 1> Synthesis of tert-butyl (2-(4-(chloromethyl)benzamido)ethyl)carbamate (compound EX5-IM-1):

[0874] [Chemistry 128]

[0875]

[0876] EX5-SM (4-(chloromethyl)benzoyl chloride, [CAS: 876-08-4] (2.0 g)) was dissolved in tetrahydrofuran (10.0 mL). Under ice-water cooling, a solution of (2-aminoethyl)carbamate tert-butyl ester [CAS: 57260-73-8] (1.7 g) and N,N'-diisopropylethylamine (3.7 mL) in tetrahydrofuran (10.0 mL) was added dropwise, and the mixture was stirred at room temperature for 1.5 hours. Ethyl acetate (30 mL) and water (10 mL) were added to the reaction mixture, and the mixture was separated. The organic layer was washed successively with semi-saturated sodium bicarbonate solution (10 mL), water (10 mL), and saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was ground with tert-butyl methyl ether, and the resulting solid was filtered off and washed with tert-butyl methyl ether to give the title compound EX5-IM-1 (2.9 g) as a white solid.

[0877] NMR data (CDCl3) (δ: ppm): 7.81 (2H, d, J = 8 Hz), 7.44 (2H, d, J = 8 Hz), 7.24 (1H, brs), 4.96 (1H, brs), 4.60 (2H, s), 3.56 (2H, q, J = 5 Hz), 3.45-3.38 (2H, m), 1.43 (9H, s)

[0878] <Step 2> Synthesis of tert-butyl carbamate (2-(4-(azidomethyl)benzamido)ethyl)carbamate (EX-IM-2):

[0879] [Chemistry 129]

[0880]

[0881] Sodium azide (100 mg) was dissolved in dimethyl sulfoxide (6.0 mL), and compound EX5-IM-1 (400 mg) obtained in step 1 of Example 5 was added. The mixture was stirred at room temperature for 2.5 hours. Water (12 mL) was added to the reaction solution under ice-water cooling, and the precipitated solid was filtered off and washed with water. The resulting solid was dried under reduced pressure at 50 °C to give the title compound EX5-IM-2 (380 mg) as a white solid.

[0882] NMR data (CDCl3) (δ: ppm): 7.84 (2H, d, J = 8 Hz), 7.37 (2H, d, J = 8 Hz), 7.22 (1H, brs), 4.95 (1H, brs), 4.39 (2H, s), 3.56 (2H, q, J = 5 Hz), 3.45-3.38 (2H, m), 1.43 (9H, s)

[0883] <Step 3> Synthesis of N-(2-aminoethyl)-4-(azidomethyl)benzamide hydrochloride (compound EX5-IM-3):

[0884] [Chemistry 130]

[0885]

[0886] Compound EX5-IM-2 (250 mg) obtained in (Example 5) <Step 2> was added to 4 equimolar concentrations of 1.75 mL of hydrogen chloride / 1,4-dioxane under ice-water cooling, and stirred at room temperature for 1 hour. Diisopropyl ether (5.25 mL) was added to the reaction solution, the resulting precipitate was filtered off, washed with diisopropyl ether, and dried under reduced pressure to give the title compound EX5-IM-3 (192 mg) as a white solid.

[0887] NMR data (DMSO-d6) (δ: ppm): 8.68 (1H, t, J = 6 Hz), 7.91 (2H, d, J = 8 Hz), 7.80 (3H, brs), 7.47 (2H, d, J = 8 Hz), 4.53 (2H, s), 3.51 (2H, q, J = 6 Hz), 2.98 (2H, t, J = 6 Hz); LC-MS: M (free amine) = 219, RT = 0.56 (min), [M+H] ＋ =220

[0888] <Step 4-1> (Example 5a) Synthesis of alginic acid (compound EX5-(II)-A-2) with N-(2-aminoethyl)-4-(azidomethyl)benzamide group:

[0889] [Chemistry 131]

[0890]

[0891] To a 1% (wt%) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (20 mL), 84 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), 52 mg of compound EX5-IM-3 obtained in step 3 of Example 5, and 252 μL of sodium bicarbonate solution were added, and the mixture was stirred at 30°C for 3 hours. After adding 200 mg of sodium chloride, 40 mL of ethanol was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX5-(II)-A-2 (185 mg) as a white solid.

[0892] The introduction rate of the reactive substituent (N-(2-aminoethyl)-4-(azidomethyl)benzamido) was 9.4 mol% (NMR integral ratio).

[0893] <Step 4-2> (Example 5b) Synthesis of alginic acid (compound EX5-(II)-B-2) with N-(2-aminoethyl)-4-(azidomethyl)benzamide group:

[0894] [Chemistry 132]

[0895]

[0896] To a 1% (w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: B-2) (20 mL), 84 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), 26 mg of compound EX5-IM-3 obtained in step 3 of Example 5, and 151 μL of sodium bicarbonate solution were added, and the mixture was stirred at 30°C for 3 hours. After adding 200 mg of sodium chloride, 40 mL of ethanol was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX5-(II)-B-2 (187 mg) as a white solid.

[0897] The introduction rate of the reactive group (N-(2-aminoethyl)-4-(azidomethyl)benzamide) was 11 mol% (NMR integral ratio).

[0898] (Example 5c) Synthesis of alginic acid (EX5-(II)-A-2b) with N-(2-aminoethyl)-4-(azidomethyl)benzamide group:

[0899] Using the same method as in (Example 5a), the title compound (EX5-(II)-A-2b) with an NMR integral ratio of 4.9 mol% for the introduction of reactive substituents was obtained.

[0900] (Example 6) Synthesis of N-(3-aminopentynyl)-5,6-dihydro-11,12-disodehydrodibenzo[b,f]azacyclooctenyl (ADIBO-C3-amine)alginic acid (compound EX6-(I)-B-2):

[0901] [Chemistry 133]

[0902]

[0903] <Step 1> Synthesis of N-trifluoroacetyl-5-aminopentanoic acid (EX6-IM-1):

[0904] [Chemistry 134]

[0905]

[0906] 2.0 g of 5-aminovaleric acid (EX6-SM1, [CAS: 660-88-8]), ethyl trifluoroacetate (3.1 mL), and triethylamine (3.6 mL) were dissolved in methanol (90.0 mL) and stirred at 40 °C for 5 hours. The reaction solution was concentrated under reduced pressure, and the residue was concentrated twice under reduced pressure by adding 10 mL of ethanol. The concentrated residue was dissolved in 200 mL of ethyl acetate, washed three times with 70 mL of 0.1 mol sodium dihydrogen phosphate aqueous solution, and washed with 50 mL of saturated brine. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and dried under reduced pressure to give the title compound EX6-IM-1 (1.8 g) as a white solid.

[0907] NMR data (DMSO-d6) (δ: ppm): 12.04 (1H, brs), 9.43 (1H, brs), 3.17 (2H, q, J = 6Hz), 2.22 (2H, H, tt, J = 7, 2 Hz), 1.51-1.46 (4H, m)

[0908] <Step 2> Synthesis of (Z)-N-(5-(dibenzo[b,f]azacyclooctene-5(6H)-yl)-5-oxopentyl-trifluoroacetamide (compound EX6-IM-2):

[0909] [Chemistry 135]

[0910]

[0911] In Example 6, 440 μL of thionyl chloride and 2 μL of N,N-dimethyl sulfoxide were added to compound EX6-IM-1 (617 mg) obtained in Step 1. The mixture was stirred at 80 °C for 1.5 hours, and the reaction solution was concentrated under reduced pressure. The remaining dichloromethane (1.0 mL) solution was added to a solution of compound EX6-SM2 [CAS:23294-93-6] (500 mg) synthesized from 5-dibenzocycloheptenone [CAS:2222-33-5] and pyridine (585 μL) in dichloromethane (5.0 mL) under ice-water cooling. The mixture was stirred at room temperature for 30 minutes. The reaction solution was diluted with tert-butylmethyl ether (20 mL), washed successively with water (10 mL), 1 equivalent hydrochloric acid (10 mL), water (10 mL), and saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (heptane ~ 60% ethyl acetate / heptane), and the resulting solid was ground with tert-butylmethyl methyl ether / heptane. After filtration, the solid was washed with heptane to give the title compound EX6-IM-2 (840 mg) as a white solid.

[0912] NMR data (CDCl3) (δ: ppm): 7.37-7.27 (4H, m), 7.22-7.14 (4H, m), 7.08 (1H, brs), 6.76 (1H, d, J = 13 Hz), 6.57 (1H, d, J = 13 Hz), 5.43 (1H, d, J = 15 Hz), 4.17 (1H, d, J = 15 Hz), 3.22 (1H, dt, J = 13, 6 Hz), 2.83 (1H, dt, J = 13, 6 Hz), 2.22-2.12 (1H, m), 1.87 (1H, dq, J = 16, 5 Hz), 1.68-1.58 (1H, m), 1.52-1.36 (2H, m), 1.28-1.16 (1H, m), LC-MS: M=402, RT=1.05 (min), [M+H] ＋ =403

[0913] <Step 3> Synthesis of N-(5-(11,12-dibromo-11,12-dihydrodibenzo[b,f]azacyclooctene-5(6H)-yl)-5-oxopentyl-trifluoroacetamide (compound EX6-IM-3):

[0914] [Chemistry 136]

[0915]

[0916] In a dichloromethane (2.8 mL) solution of compound EX6-IM-2 (700 mg) obtained in (Example 6) <Step 2>, pyridinium tribromide (612 mg) was added under ice-water cooling. After stirring at room temperature for 1.5 hours, pyridinium tribromide (111 mg) was added, and the mixture was stirred further at room temperature for 1 hour. The reaction solution was diluted with ethyl acetate (20 mL) and washed successively with 2 equivalence hydrochloric acid (10 mL) and saturated brine (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the title crude compound EX6-IM-3 (1.03 g) in a yellow amorphous form.

[0917] LC-MS: M=562, RT=1.10(min), [M+H] ＋ =563(561:563:565=1:2:1)

[0918] <Step 4> Synthesis of N-(3-aminopentynyl)-5,6-dihydro-11,12-disodehydrodibenzo[b,f]azacyclooctene-trifluoroacetamide (EX6-IM-4):

[0919] [Chemistry 137]

[0920]

[0921] In a tetrahydrofuran (1.5 mL) solution of the crude compound EX6-IM-3 (100 mg) obtained in (Example 6) <Step 3>, potassium tert-butoxy (100 mg) was added in small amounts over 8 hours at room temperature with stirring. The reaction solution was diluted with ethyl acetate (15 mL) and washed successively with water (3 mL) and saturated brine (2 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the title crude compound EX6-IM-4 (58 mg) as a light brown gel.

[0922] NMR data (CDCl3) (δ: ppm): 7.69 (1H, d, J = 7 Hz), 7.44-7.23 (7H, m), 5.17 (1H, d, J = 14 Hz), 3.70 (1H, d, J = 14 Hz), 3.21 (1H, dt, J = 13, 6 Hz), 2.57 (1H, dq, J = 19, 5Hz), 2.36-2.28 (1H, m), 1.82 (1H, dq, J = 16, 5 Hz), 1.46-1.34(2H,m), 1.29-1.24(1H,m), 1.15-1.05(1H,m), LC-MS: M=400, RT=1.08(min), [M+H] ＋ =401, [M + Na]＋ =423

[0923] <Step 5> Synthesis of N-(3-aminopentynyl)-5,6-dihydro-11,12-disdehydrodibenzo[b,f]azacyclooctene (compound EX6-IM-5):

[0924] [Chemistry 138]

[0925]

[0926] In Example 6, a methanol (1.2 mL) solution of the crude compound EX6-IM-4 (58 mg) obtained in step 4 was added to a water solution containing 40 mg of potassium carbonate (0.25 mL), and the mixture was stirred at room temperature for 23 hours. The reaction solution was concentrated by adding ethyl acetate (10 mL), dichloromethane (1 mL), and semi-saturated saline (2 mL), and the mixture was separated. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting gel was purified by silica gel chromatography (ethyl acetate ~ 50% methanol / ethyl acetate) to obtain the title compound EX6-IM-5 (22 mg) as a colorless gel.

[0927] NMR data (CDCl3) (δ: ppm): 7.70 (1H, d, J = 8 Hz), 7.43-7.23 (7H, m), 5.18 (1H, d, J = 14 Hz), 3.65 (1H, d, J = 14 Hz), 2.45 (2H, t, J = 7 Hz), 2.24-2.16 (1H, m), 1.96-1.89 (1H, m), 1.48-1.38 (2H, m), 1.21-1.10 (2H, m), LC-MS: M=304, RT=0.76 (min), [M+H] ＋ =305

[0928] <Step 6> Synthesis of alginate (EX6-(I)-B-2) with N-(3-aminopentynyl)-5,6-dihydro-11,12-disodehydrodibenzo[b,f]azacyclooctene (ADIBO-C3-amino) group:

[0929] [Chemistry 139]

[0930]

[0931] To a 1% (w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: B-2) (28.5 mL), 60 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), 2.9 mL of an ethanol solution of compound EX6-IM-5 (22 mg) obtained in (Example 6) <Step 5>, and 72 μL of 1 mol sodium bicarbonate solution were added, and the mixture was stirred at 30°C for 3 hours. After adding 285 mg of sodium chloride, 57 mL of ethanol was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX5-(II)-B-2 (277 mg) as a white solid.

[0932] The introduction rate of the reactive group (N-(3-aminopentynyl)-5,6-dihydro-11,12-disodehydrodibenzo[b,f]azacyclooctene (ADIBO-C3-amino) group) was 2.7 mol% (NMR integral ratio).

[0933] (Example 7) Synthesis of alginic acid with N-(2-aminoethyl)-4-azidobenzamide group (Examples 7a, 7b, and 7c):

[0934] [Chemistry 140]

[0935]

[0936] <Step 1> Synthesis of tert-butyl (2-(4-azidobenzamido)ethyl)carbamate (compound EX7-IM-1):

[0937] [Chemistry 141]

[0938]

[0939] 700 mg of 4-azidobenzoic acid (EX7-SM, [CAS: 6427-66-3]) was mixed with thionyl chloride (783 μL) and N,N-dimethyl sulfoxide (3 μL), and stirred at 70 °C for 1 hour. The reaction solution was concentrated under reduced pressure, and 1 mL of dichloromethane was added to the residue. A solution of 825 mg of tert-butyl carbamate [CAS: 57260-73-8] and 1.04 mL of pyridine in dichloromethane (7.0 mL) was added under ice-water cooling, and the mixture was stirred at room temperature for 1 hour. The reaction solution was diluted with tert-butyl methyl ether (30 mL) and washed sequentially with water (10 mL), saturated sodium bicarbonate solution (5 mL), 0.5 equivalence citric acid (5 mL, twice), water (5 mL), and saturated saline solution (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was ground with tert-butylmethyl methyl ether / heptane, the solid was filtered, and then washed with tert-butylmethyl methyl ether / heptane to give the title compound EX7-IM-1 (1.1 g) as a white solid.

[0940] NMR data (CDCl3) (δ: ppm): 7.83 (2H, d, J = 8 Hz), 7.26 (1H, brs), 7.05 (2H, d, J = 8 Hz), 4.97 (1H, brs), 3.55 (2H, q, J = 5 Hz), 3.45-3.37 (2H, m), 1.43 (9H, s), LC-MS: M=305, RT=0.90 (min), [M+H] ＋ =306, [M + Na] ＋ =328

[0941] <Step 2> Synthesis of N-(2-aminoethyl)-4-azidobenzamide hydrochloride (compound EX7-IM-2):

[0942] [Chemistry 142]

[0943]

[0944] The compound obtained in (Example 7) <Step 1> (EX7-IM-1, 500 mg) was suspended in 1,6-dioxane (1.5 mL). A 4 equivalence hydrogen chloride / dioxane solution (3.5 mL) was added under ice-water cooling, and the mixture was stirred at room temperature for 1 hour. Diisopropyl ether (10.5 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 50 minutes. The solid was filtered, washed with diisopropyl ether, and dried under reduced pressure to give the title compound EX7-IM-2 (365 mg) as a pale beige solid.

[0945] NMR data (DMSO-d6) (δ: ppm): 8.68 (1H, t, J = 6 Hz), 7.93 (2H, d, J = 9 Hz), 7.82 (1H, brs), 7.22 (2H, d, J = 9 Hz), 3.49 (2H, q, J = 6 Hz), 2.97 (2H, t, J = 6 Hz); LC-MS: M (free amine) = 205, RT = 0.56 (min), [M+H] ＋ =206

[0946] <Step 3-1> (Example 7a) Synthesis of alginate (EX7-(II)-B-2a) with N-(2-aminoethyl)-4-azidobenzamide group:

[0947] [Chemistry 143]

[0948]

[0949] To a 1% (w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: B-2) (30.0 mL), 63 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), 18 mg of compound EX7-IM-2 obtained in step 2 of Example 7, and 114 μL of sodium bicarbonate solution were added, and the mixture was stirred at 30°C for 3 hours. After adding 300 mg of sodium chloride, 60 mL of ethanol was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX7-(II)-B-2a (282 mg) as a white solid.

[0950] The introduction rate of the reactive group (N-(2-aminoethyl)-4-azidobenzamide) was 5.1 mol% (NMR integral ratio).

[0951] <Step 3-2> (Example 7b) Synthesis of alginic acid (EX7-(II)-B-2b) with N-(2-aminoethyl)-4-azidobenzamide group:

[0952] [Chemistry 144]

[0953]

[0954] To a 1% (w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: B-2) (60.0 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (67 mg), compound EX7-IM-2 (15 mg) obtained in step 2 of Example 7, and 1 mol sodium bicarbonate solution (91 μL) were added, and the mixture was stirred at 30°C for 3 hours. After adding sodium chloride (600 mg), ethanol (120 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX7-(II)-B-2b (560 mg) as a white solid.

[0955] The introduction rate of the reactive group (N-(2-aminoethyl)-4-azidobenzamide) was 2.0 mol% (NMR integral ratio).

[0956] (Example 7c) Synthesis of alginate (EX7-(II)-A-2) with N-(2-aminoethyl)-4-azidobenzamide group introduced:

[0957] By changing alginate to A-2, the title compound (EX7-(II)-A-2) with a reactive substituent incorporation rate (NMR integral ratio) of 5.0 mol% was obtained using the same method as in (Example 7a).

[0958] (Example 8) Synthesis of alginic acid (EX8-(I)-B-2) with N-(4-(aminoethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group:

[0959] [Chemistry 145]

[0960]

[0961] <Step 1> Synthesis of tert-butyl carbamate (4-(4((2,2,2-trifluoroacetamide)methyl)benzyl)carbamate (compound EX8-IM-1):

[0962] [Chemistry 146]

[0963]

[0964] For the method known in the reference (Bioorganic & Medicinal Chemistry (2003) 11: 4189-4206), a mixture of tert-butyl carbamate (4-(aminoethyl)benzenemethyl)carbamate [CAS: 108468-80-4] (EX8-SM1, 0.67 g), triethylamine (0.39 mL), and methanol (6.67 mL) was synthesized from 1,4-bis(aminomethyl)benzene [CAS: 539-48-0], and ethyl trifluoroacetate (0.44 mL) was added dropwise with stirring under ice-cooled conditions. The reaction mixture was heated to room temperature and stirred at the same temperature for 5 hours. The reaction was stopped with water (10 mL) and extracted three times with ethyl acetate (10 mL). The recovered organic layer was washed with saturated brine (5 mL) and dried with anhydrous sodium sulfate. The dried organic layer was filtered and concentrated to give the title crude compound EX8-IM-1 (0.671 g) as a pale yellow amorphous form.

[0965] NMR data (CDCl3) (δ: ppm): δ: 7.29 (2H, d, J = 8.4 Hz), 7.25 (2H, d, J =7.6 Hz), 6.51 (1H, br s), 4.86 (1H, br s), 4.51 (2H, d, J = 5.2 Hz), 4.31 (2H, d, J =6.0 Hz), 1.46 (9H, s). LC-MS: M=332, RT=0.97 (min), [M+Na]+=355.

[0966] <Step 2> Synthesis of N-(4-(aminoethyl)benzyl)-2,2,2-trifluoroacetamide hydrochloride (compound EX8-IM-2):

[0967] [Chemistry 147]

[0968]

[0969] A 1,4-dioxane solution (3.5 mL) of compound EX8-IM-1 (0.5 g) obtained in step 1 of Example 8 was mixed with 4 equivalence hydrogen chloride / 1,4-dioxane (3.5 mL) under water cooling and stirring, and stirred at room temperature for 3 hours. After adding diisopropyl ether (40 mL) to the reaction mixture, the precipitate was filtered off, yielding the title compound EX8-IM-2 (0.36 g) as a white solid.

[0970] NMR data (D2O) (δ: ppm): δ: 7.29 (2H, d, J = 8.0 Hz), 7.25 (2H, d, J = 8.4 Hz), 4.38 (2H, s), 4.02 (2H, s). LC-MS: M (free amine) = 232, RT = 0.53 (min), [M+H]+ = 233.

[0971] <Step 3> Synthesis of N-(4-((2-(cyclooct-2-yn-1-yloxy)acetamide)methyl)benzyl)-2,2,2-trifluoroacetamide (compound EX8-IM-3):

[0972] [Chemistry 148]

[0973]

[0974] For a carboxylic acid [CAS: 917756-42-4] (EX8-SM2, 0.17 g) synthesized from cycloheptene [CAS: 628-92-2] according to a known method (Org. Process Res. Dev. (2018) 22: 108-110) and an acetonitrile (1.7 mL) solution of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureon hexafluorophosphate (0.26 g), EX8-IM-2 (0.26 g) and N,N-diisopropylethylamine (0.51 mL) obtained in step 2 of Example 8 were added dropwise under ice-cooled stirring, and the mixture was stirred at room temperature for 1 hour and 30 minutes. After the reaction was stopped by adding water (5 mL), the mixture was extracted three times with ethyl acetate (5 mL). The organic layer was washed with saturated brine (3 mL) and dried over anhydrous sodium sulfate. After filtering the dried organic layer, the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (12% ethyl acetate / n-heptane ~ 100% ethyl acetate) to give the title compound EX8-IM-3 (0.189 g) as a white amorphous form.

[0975] NMR data (CDCl3) (δ: ppm): δ: 7.31 (2H, d, J = 8.4 Hz), 7.26 (2H, d, J = 8.0 Hz, overlapping with solvent peak), 6.84 (1H, br s), 6.52 (1H, br s), 4.52 (2H, d, J = 6.0 Hz), 4.49 (2H, d, J = 6.4 Hz), 4.26–4.23 (1H, m), 4.11 (1H, d, J = 15.2 Hz), 3.94 (1H, d, J = 15.2 Hz), 2.26–2.09 (3H, m), 2.00–1.58 (6H, m), 1.48–1.44 (1H, m).LC-MS: M=396, RT=0.99(min), [M+H]+=397.

[0976] <Step 4> Synthesis of N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (compound EX8-IM-4):

[0977] [Chemistry 149]

[0978]

[0979] A mixture of compound EX8-IM-3 (0.18 g) obtained in step 3 of Example 8 and methanol (1.8 mL) was added dropwise with an aqueous solution of potassium carbonate (0.126 g) (0.9 mL) under ice-cooled stirring, and stirred at room temperature for 17 hours and 30 minutes. The methanol was distilled off under reduced pressure, and the mixture was extracted three times with ethyl acetate (5 mL). The organic layer was washed with saturated brine (5 mL) and dried over anhydrous sodium sulfate. After filtering the organic layer, the solvent was distilled off under reduced pressure to give the crude compound EX8-IM-4 (0.13 g) as a pale yellow oil.

[0980] NMR data (CDCl3) (δ: ppm): δ: 7.28–7.28 (4H, m), 6.80 (1H, br s), 4.48 (2H, d, J = 6.0 Hz), 4.26–4.21 (1H, m), 4.11 (1H, d, J = 15.2 Hz), 3.93 (1H, d, J = 15.2 Hz), 3.86 (2H, s), 2.28–2.07 (3H, m), 1.99–1.40 (7H, m, overlapping with solvent peak). LC-MS: M = 300, RT = 0.68 (min), [M+H]+ = 301.

[0981] <Step 5> Synthesis of alginic acid (EX8-(I)-B-2) with N-(4-(aminoethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group:

[0982] [Chemistry 150]

[0983]

[0984] 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (0.118 g) was added to a 1% (w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd., B-2) with stirring at room temperature. Next, a solution of 0.035 g of compound EX8-IM-4 (0.035 g) obtained in step 4 of Example 8 was added dropwise at the same temperature, and the mixture was stirred at 40°C for 4 hours. After cooling to room temperature, sodium chloride (500 mg) was added, followed by ethanol (101.72 mL), and the mixture was stirred for 30 minutes. The resulting precipitate was filtered, washed three times with ethanol (2 mL), and dried under reduced pressure to give the title compound EX8-(I)-B-2 (521 mg) as a white solid.

[0985] The introduction rate of the reactive substituent (N-(4-(aminoethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamido) was 4.46 mol% (NMR integral ratio).

[0986] (Example 8b) Synthesis of alginic acid (EX8-(I)-A-2) with N-(4-(aminoethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group:

[0987] By replacing alginate with A-2, the title compound (EX8-(I)-A-2) with a reactive substituent incorporation rate (NMR integral ratio) of 4.4 mol% was obtained using the same method as in (Example 8).

[0988] (Example 9) Synthesis of alginate (9a, 9b, 9c) with N-(2-aminoethyl)-2-(cyclooct-2-yn-1-oxy)acetamide group:

[0989] [Chemistry 151]

[0990]

[0991] (Example 9a) Synthesis of alginic acid (EX9-(I)-A-2) with N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group:

[0992] <Step 1> Synthesis of tert-butyl carbamate (2-(2,2,2-trifluoroacetamide)carbamate (EX9-IM-1):

[0993] [Chemistry 152]

[0994]

[0995] Ethyl trifluoroacetate (2.24 mL) was added dropwise to a commercially available solution of tert-butyl carbamate (EX9-SM1, 3.00 g, [CAS: 57260-73-8]) in tetrahydrofuran (12.0 mL). The reaction mixture was stirred at room temperature for 14.5 hours. The reaction mixture was concentrated under reduced pressure, and tert-butyl methyl ether (5 mL) and heptane (25 mL) were added to the residue. The mixture was then ground. The solid was filtered and washed with heptane to give the title compound EX9-IM-1 (4.36 g) as a white solid.

[0996] NMR data (CDCl3) (δ: ppm): 7.80 (1H, brs), 4.93 (1H, brs), 3.45 (2H, q, J = 5 Hz), 3.41–3.34 (2H, m), 1.44 (9H, s)

[0997] <Step 2> Synthesis of N-(2-aminoethyl)-2,2,2-trifluoroacetamide hydrochloride (EX9-IM-2):

[0998] [Chemistry 153]

[0999]

[1000] Formic acid (3.1 mL) was added to compound EX9-IM-1 (0.50 g) obtained in (Example 9a) <Step 1>, and the mixture was stirred at room temperature for 22.5 hours. The formic acid was distilled off, and the mixture was azeotropically treated with toluene. Hydrogen chloride / methanol was added to the resulting oil, and the mixture was concentrated under reduced pressure. After azeotropic treatment with ethyl acetate and tert-butyl methyl ether, the mixture was dried under reduced pressure to obtain the title crude compound EX9-IM-2 (0.35 g) as a colorless oil.

[1001] NMR data (DMSO-d6) (δ: ppm): 3.42 (2H, d, J = 6 Hz), 2.92 (2H, d, J = 6 Hz)

[1002] <Step 3> Synthesis of N-(2-(2-(cyclooct-2-yn-1-yloxy)acetamide)ethyl)-2,2,2-trifluoroacetamide (EX9-IM-3):

[1003] [Chemistry 154]

[1004]

[1005] Carboxylic acid (EX8-SM2, 100 mg) synthesized according to a known method (Org. Process Res. Dev. (2018) 22: 108-110) was mixed with ethanol (1.0 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (304 mg), compound EX9-IM-2 (159 mg) obtained in step 2 of Example 9a), and triethylamine (153 μL), and stirred at room temperature for 3.5 hours. Water (4 mL) was added, and the mixture was extracted with ethyl acetate (15 mL, 5 mL). The organic layer was washed successively with 0.5 equivalence citric acid (5 mL), water (5 mL × 2), and saturated brine (3 mL), dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (10% ethyl acetate / n-heptane ~ 60% ethyl acetate / n-heptane) to give the title compound EX9-IM-3 (103 mg) as a white solid.

[1006] NMR data (DMSO-d6) (δ: ppm): 9.42 (1H, brs), 7.83 (1H, brs), 4.29-4.24 (1H, m), 3.87 (2H, d, J = 15 Hz), 3.73 (1H, d, J = 15 Hz), 3.28-3.20 (4H, m), 2.27-2.04(3H, m), 1.96-1.70(4H, m), 1.67-1.50(2H, m), 1.43-1.34(1H, m)

[1007] <Step 4> Synthesis of N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (EX9-IM-4):

[1008] [Chemistry 155]

[1009]

[1010] A methanol (1.55 mL) solution of compound EX9-IM-3 (103 mg) obtained in (Example 9a) <Step 3> was added to a water solution of potassium carbonate (89 mg) (515 μL), and the mixture was stirred at room temperature for 6 hours. The methanol was distilled off under reduced pressure, and water (2 mL) was added to saturate the solution with sodium chloride. The solution was extracted with ethyl acetate (15 mL, 10 mL × 5), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to give the title compound EX9-IM-4 (75 mg) as a colorless oil.

[1011] NMR data (CDCl3) (δ: ppm): 6.83 (1H, brs), 4.28-4.22 (1H, m), 4.06 (1H, d, J = 15Hz), 3.90 (1H, d, J = 15 Hz), 3.42-3.30 (2H, m), 2.86 (2H, t, J = 6 Hz), 2.31-2.12(3H,m), 2.04-1.78(4H,m), 1.75-1.57(2H,m), 1.51-1.43(1H,m)

[1012] <Step 5> Synthesis of alginic acid (EX9-(I)-A-2) with N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group:

[1013] [Chemistry 156]

[1014]

[1015] In a 1% (wt%) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd., A-2) (30 mL), an ethanol solution (3 mL) of compound EX9-IM-4 (17 mg) obtained in step 4 of Example 9a) was added sequentially with stirring at room temperature to 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (84 mg), and 1 mol% sodium bicarbonate solution (76 μL). The mixture was stirred at 30°C for 3 hours. Sodium chloride (0.3 g) was added to the reaction solution, followed by ethanol (60 mL), and the mixture was stirred for 1.5 hours. The resulting precipitate was filtered off, washed with ethanol (10 mL × 5), and dried under reduced pressure to obtain the title compound EX9-(I)-A-2 (290 mg) as a white solid.

[1016] The introduction rate of the reactive substituent (N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide) was 4.3 mol% (NMR integral ratio).

[1017] (Example 9b) Synthesis of alginic acid (EX9-(I)-B-2a) with N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group:

[1018] <Step 1> Synthesis of N-(2-aminoethyl)-2,2,2-trifluoroacetamide hydrochloride (EX9-IM-2):

[1019] [Chemistry 157]

[1020]

[1021] The compound EX9-IM-1 (0.50 g) obtained in (Example 9a) <Step 1> was suspended in 1,4-dioxane (3.0 mL). Under ice-water cooling, 4 equivalence hydrogen chloride / 1,4-dioxane (7.0 mL) was added, and the mixture was stirred at room temperature for 3 hours. Diisopropyl ether (30.0 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 50 minutes. The solid was filtered off, washed with diisopropyl ether, and dried under reduced pressure to give the title compound EX9-IM-2 (0.70 g) as a white solid.

[1022] NMR data (DMSO-d6) (δ: ppm): 9.56 (1H, brs), 8.00 (3H, brs), 3.45 (2H, d, J = 6 Hz), 2.95 (2H, d, J = 6 Hz)

[1023] <Step 2> Synthesis of N-(2-(2-(cyclooct-2-yn-1-yloxy)acetamide)ethyl)-2,2,2-trifluoroacetamide (EX9-IM-3):

[1024] [Chemistry 158]

[1025]

[1026] Using carboxylic acid (EX8-SM2, 300 mg) synthesized according to a known method (Org. Process Res. Dev. (2018) 22: 108-110) and compound EX9-IM-2 (380 mg) obtained in (Example 9b) <Step 1>, the same operation as in (Example 9a) <Step 3> was performed to obtain the title compound EX9-IM-3 (322 mg) as a white solid.

[1027] NMR data (CDCl3) (δ: ppm): 7.95 (1H, brs), 6.95 (1H, brs), 4.28-4.23 (1H, m), 4.08 (2H, d, J = 15 Hz), 3.91 (1H, d, J = 15 Hz), 3.56-3.50 (4H, m), 2.31-2.12(3H, m), 2.03-1.78(4H, m), 1.75-1.61(2H, m), 1.52-1.42(1H, m)

[1028] <Step 3> Synthesis of N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (EX9-IM-4):

[1029] [Chemistry 159]

[1030]

[1031] The compound EX9-IM-3 (322 mg) obtained in (Example 9b) <Step 2> was subjected to the same operation as in (Example 9b) <Step 4> to obtain the title compound EX9-IM-4 (238 mg) as a colorless oil.

[1032] NMR data (CDCl3) (δ: ppm): 6.82 (1H, brs), 4.28-4.22 (1H, m), 4.06 (1H, d, J = 15Hz), 3.90 (1H, d, J = 15 Hz), 3.40-3.31 (2H, m), 2.86 (2H, t, J = 6 Hz), 2.31-2.12(3H,m), 2.02-1.78(4H,m), 1.75-1.57(2H,m), 1.52-1.41(1H,m)

[1033] <Step 4> Synthesis of alginic acid (EX9-(I)-B-2a) with N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group:

[1034] [Chemistry 160]

[1035]

[1036] In a 1% (wt%) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd., B-2) (120 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (335 mg), an ethanol solution (12 mL) of compound EX9-IM-4 (68 mg) obtained in (Example 9b) <Step 3>, and 1 mol% sodium bicarbonate solution (303 μL) were added sequentially under stirring at room temperature. The mixture was stirred at 30°C for 3 hours. Sodium chloride (1.2 g) was added to the reaction solution, followed by ethanol (240 mL), and the mixture was stirred for 1.5 hours. The resulting precipitate was filtered off, washed with ethanol (20 mL × 5), and dried under reduced pressure to obtain the title compound EX9-(I)-B-2a (1.16 g) as a white solid.

[1037] The introduction rate of the reactive substituent (N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide) was 4.2 mol% (NMR integral ratio).

[1038] (Example 9c) Synthesis of alginic acid (EX9-(I)-B-2b) with N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group:

[1039] [Chemistry 161]

[1040]

[1041] In a 1% (wt%) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd., B-2) (120 mL), a solution of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (167 mg), a 12 mL ethanol solution of compound EX9-IM-4 (34 mg) obtained in step 3 of Example 9b), and 151 μL of 1 mol% sodium bicarbonate solution were added sequentially with stirring at room temperature. The mixture was stirred at 30°C for 3 hours. Sodium chloride (1.2 g) was added to the reaction solution, followed by ethanol (240 mL), and the mixture was stirred for 1.5 hours. The resulting precipitate was filtered, washed with ethanol (20 mL × 5), and dried under reduced pressure to obtain the title compound EX9-(I)-B-2b (1.12 g) as a white solid.

[1042] The introduction rate of the reactive substituent (N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide) was 2.1 mol% (NMR integral ratio).

[1043] (Example 10) Synthesis of alginic acid (EX10-(II)-A-2) with N-(2-(2-aminoethoxy)ethyl)-4-(azidomethyl)benzamide group:

[1044] [Chemistry 162]

[1045]

[1046] <Step 1> Synthesis of tert-butyl carbamate (2-(2-(4-(chloromethyl)benzamido)ethoxy)ethyl)carbamate (EX10-IM-1):

[1047] [Chemistry 163]

[1048]

[1049] EX5-SM (4-(chloromethyl)benzoyl chloride, 0.50 g) was dissolved in tetrahydrofuran (5.0 mL), and a solution of (2-(2-aminoethoxy)ethyl)carbamate tert-butyl ester (0.54 g, [CAS: 127828-22-2]) and diisopropylethylamine (0.92 mL) in tetrahydrofuran (5.0 mL) was added. The mixture was stirred at room temperature for 3 hours. Ethyl acetate (25 mL) and water (10 mL) were added to the reaction mixture, and the mixture was separated. The organic layer was washed successively with water (5 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was ground with a mixed solvent of tert-butyl methyl ether / n-heptane, and the resulting solid was filtered off and washed with n-heptane to give the title compound EX10-IM-1 (0.79 g) as a white solid.

[1050] NMR data (CDCl3) (δ: ppm): 7.79 (2H, d, J = 8 Hz), 7.46 (2H, d, J = 8 Hz), 6.62 (1H, brs), 4.83 (1H, brs), 4.61 (2H, s), 3.68-3.62 (4H, m), 3.55 (2H, t, J = 5 Hz), 3.33 (2H, t, J = 5 Hz), 1.42 (9H, s)

[1051] <Step 2> Synthesis of tert-butyl carbamate (2-(2-(4-(azidomethyl)benzamido)ethoxy)ethyl)carbamate (EX10-IM-2):

[1052] [Chemistry 164]

[1053]

[1054] Sodium azide (109 mg) was dissolved in dimethyl sulfoxide (7.5 mL), and compound EX10-IM-1 (500 mg) obtained in step 1 of Example 10 was added. The mixture was stirred at room temperature for 3 hours. Water (15 mL) was added to the reaction solution under ice-water cooling, and the precipitated solid was filtered off and washed with water. The resulting solid was dried to give the title compound EX10-IM-2 (478 mg) as a white solid.

[1055] NMR data (CDCl3) (δ: ppm): 7.82 (2H, d, J = 8 Hz), 7.39 (2H, d, J = 8 Hz), 6.63 (1H, brs), 4.83 (1H, brs), 4.40 (2H, s), 3.68-3.62 (4H, m), 3.55 (2H, t, J = 5 Hz), 3.33 (2H, q, J = 5 Hz), 1.42 (9H, s)

[1056] <Step 3> Synthesis of N-(2-(2-aminoethoxy)ethyl)-4-(azidomethyl)benzamide hydrochloride (EX10-IM-3):

[1057] [Chemistry 165]

[1058]

[1059] In (Example 10) <Step 2>, compound EX10-IM-2 (400 mg) was added to 4 equimolar concentrations of hydrogen chloride / 1,4-dioxane (2.8 mL) under ice-water cooling, and the mixture was stirred at room temperature for 1.75 hours. Diisopropyl ether (8.4 mL) was added to the reaction mixture to obtain a gel. The supernatant was removed by decantation, and the mixture was washed with diisopropyl ether by decantation and dried under reduced pressure to give the title compound EX10-IM-3 (298 mg) as a beige solid.

[1060] NMR data (DMSO-d6) (δ: ppm): 8.60 (1H, t, J = 6 Hz), 7.89 (2H, d, J = 8 Hz), 7.90 (3H, brs), 7.45 (2H, d, J = 8 Hz), 4.52 (2H, s), 3.62 (2H, t, J = 5 Hz), 3.58 (2H, t, J = 6 Hz), 3.47 (2H, q, J = 6 Hz), 2.98 (2H, t, J = 5 Hz); LC-MS (free amine): RT = 0.58 (min), [M+H] ＋ =264

[1061] <Step 4> Synthesis of N-(2-(2-aminoethoxy)ethyl)-4-(azidomethyl)benzamide-based alginate (EX10-(II)-A-2):

[1062] [Chemistry 166]

[1063]

[1064] To a 1% (w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (40 mL), 112 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), 30 mg of compound EX10-IM-3 obtained in step 3 of Example 10, and 151 μL of sodium bicarbonate solution were added, and the mixture was stirred at 30°C for 3 hours. After adding 400 mg of sodium chloride, 80 mL of ethanol was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX10-(II)-A-2 (408 mg) as a white solid.

[1065] The introduction rate of the reactive substituent (N-(2-(2-aminoethoxy)ethyl)-4-(azidomethyl)benzamido) was 4.7 mol% (NMR integral ratio).

[1066] (Example 11) Synthesis of alginic acid (EX11-(II)-A-2) with N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(azidomethyl)benzamide group:

[1067] [Chemistry 167]

[1068]

[1069] <Step 1> Synthesis of tert-butyl carbamate (2-(2-(2-(4-(chloromethyl)benzamido)ethoxy)ethoxy)ethyl)carbamate (EX11-IM-1):

[1070] [Chemistry 168]

[1071]

[1072] EX5-SM (4-(chloromethyl)benzoyl chloride, 0.50 g) was dissolved in tetrahydrofuran (5.0 mL), and a solution of (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate tert-butyl ester (0.66 g) and diisopropylethylamine (0.92 mL) in tetrahydrofuran (5.0 mL) was added dropwise. The mixture was stirred at room temperature for 4.7 hours. Ethyl acetate (25 mL) and water (10 mL) were added to the reaction mixture, and the layers were separated. The organic layer was washed successively with semi-saturated sodium bicarbonate solution (10 mL), water (10 mL), and saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Tert-butyl methyl ether was added to the residue, and the solid was removed by filtration. The filtrate obtained by concentration under reduced pressure was purified by silica gel column chromatography (20% ethyl acetate / n-heptane~ethyl acetate) to give the title compound EX11-IM-1 (0.82 g) as a colorless oil.

[1073] NMR data (CDCl3) (δ: ppm): 7.79 (2H, d, J = 8 Hz), 7.45 (2H, d, J = 8 Hz), 6.71 (1H, brs), 4.97 (1H, brs), 4.60 (2H, s), 3.70-3.60 (8H, m), 3.55 (2H, t, J = 5 Hz), 3.31 (2H, q, J = 6 Hz), 1.43 (9H, s)

[1074] <Step 2> Synthesis of tert-butyl carbamate (EX11-IM-2):

[1075] [Chemistry 169]

[1076]

[1077] Sodium azide (152 mg) was added to a dimethyl sulfoxide solution of compound EX11-IM-1 (0.82 g) obtained in step 1 of Example 11, and the mixture was stirred at room temperature for 3.5 hours. Water (23 mL) was added to the reaction mixture under ice-water cooling, and the mixture was stirred at the same temperature for 30 minutes. The mixture was extracted with ethyl acetate (30 mL, 10 mL), and the organic layer was washed successively with water (10 mL × 3) and saturated brine (5 mL). The organic layer was dried over anhydrous sodium sulfate, and the precipitated solid was filtered off and dried under reduced pressure to obtain the title compound EX11-IM-2 (0.80 g) as a colorless oil.

[1078] NMR data (CDCl3) (δ: ppm): 7.81 (2H, d, J = 8 Hz), 7.39 (2H, d, J = 8 Hz), 6.73 (1H, brs), 4.97 (1H, brs), 4.40 (2H, s), 3.72-3.60 (8H, m), 3.55 (2H, t, J = 5 Hz), 3.31 (2H, q, J = 5 Hz), 1.43 (9H, s)

[1079] <Step 3> Synthesis of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(azidomethyl)benzamide hydrochloride (EX11-IM-3):

[1080] [Chemistry 170]

[1081]

[1082] Compound EX11-IM-2 (0.80 g) obtained in (Example 11) <Step 2> was added to 4 equimolar concentrations of 1,4-dioxane (5.3 mL) under ice-water cooling, and stirred at room temperature for 1.75 hours. Diisopropyl ether (16.0 mL) was added to the reaction mixture, and the mixture was stirred for 30 minutes. The solvent was removed by decantation, and the residue was washed with diisopropyl ether. The resulting residue was dried under reduced pressure to obtain the title compound EX11-IM-3 (0.73 g) as a colorless gel.

[1083] NMR data (DMSO-d6) (δ: ppm): 8.62 (1H, t, J = 6 Hz), 7.95 (3H, brs), 7.88 (2H, d, J = 8 Hz), 7.45 (2H, d, J = 8 Hz), 4.52 (2H, s), 3.62–3.52 (8H, m), 3.43 (2H, q, J = 6 Hz), 2.98–2.89 (2H, m); LC-MS (free amine): RT = 0.59 (min), [M+H] ＋ =308

[1084] <Step 4> Synthesis of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(azidomethyl)benzamide-based alginate (EX11-(II)-A-2):

[1085] [Chemistry 171]

[1086]

[1087] To a 1% (w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (40 mL), a solution of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (112 mg), a solution of compound EX11-IM-3 (38 mg) obtained in step 3 of Example 11 (4.0 mL) in ethanol, and 1 mol sodium bicarbonate solution (151 μL) were added. The mixture was stirred at 30°C for 3 hours. After adding sodium chloride (0.4 g), ethanol (80 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX11-(II)-A-2 (416 mg) as a white solid.

[1088] The introduction rate of the reactive substituent (N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(azidomethyl)benzamido) was 4.2 mol% (NMR integral ratio).

[1089] (Example 12) Synthesis of alginic acid (EX12-(II)-A-2) with N-(2-(2-aminoethoxy)ethyl)-6-(azidomethyl)nicotinamide group:

[1090] [Chemistry 172]

[1091]

[1092] <Step 1> Synthesis of methyl 6-(azidomethyl)nicotinic acid (EX12-IM-1):

[1093] [Chemistry 173]

[1094]

[1095] Following a known method (Angew. Chem. Int. Ed. (2012) 51: 5852-5856), a mixture of commercially available methyl 6-(hydroxymethyl)nicotinic acid [CAS: 56026-36-9] (EX12-SM1, 0.5 g) and tetrahydrofuran (5 mL) was added with p-toluenesulfonyl chloride (0.68 g) and triethylamine (0.63 mL) under ice-cooled stirring. The reaction mixture was stirred at room temperature for 20 hours and 30 minutes, followed by the addition of sodium azide (0.29 g) at the same temperature and stirring for 4 hours at room temperature. After the reaction was complete, ethyl acetate (10 mL) and water (10 mL) were added to dilute the reaction mixture, and the aqueous layer was extracted three times with ethyl acetate (10 mL). The combined organic layers were washed successively with water (5 mL) and saturated brine (5 mL) and dried over anhydrous sodium sulfate. The organic layers were filtered and concentrated under reduced pressure to obtain the crude product. The crude product was prepared by silica gel column chromatography (n-heptane / ethyl acetate), yielding the title compound EX12-IM-1 (0.34 g) in a pale yellow amorphous form.

[1096] NMR data (CDCl3) (δ: ppm): 9.18 (1H, d, J = 2.0 Hz), 8.33 (1H, dd, J = 8.0, 2.0 Hz), 7.45 (1H, d, J = 8.0 Hz), 4.57 (2H, s), 3.96 (3H, s). LC-MS: M = 192, RT = 0.78 (min), [M+H]+ = 193.

[1097] <Step 2> Synthesis of 6-(azidomethyl)nicotinic acid (EX12-IM-2):

[1098] [Chemistry 174]

[1099]

[1100] A mixture of compound EX12-IM-1 (0.342 g) obtained in step 1 of Example 12 and methanol (6.84 mL) was added with 1 mol of lithium hydroxide monohydrate (5.34 mL) at room temperature, and the mixture was stirred for 30 minutes at the same temperature. After the reaction was complete, acetic acid (0.41 mL) was added, and the reaction solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-heptane / ethyl acetate ~ ethyl acetate / methanol) to give the title compound EX12-IM-2 (0.28 g) as a pale yellow amorphous form.

[1101] NMR data (CD3OD) (δ: ppm): 9.09 (1H, d, J = 2.4 Hz), 8.38 (1H, dd, J = 8.0, 2.4 Hz), 7.56 (1H, d, J = 8.0 Hz), 4.57 (2H, s). LC-MS: M=178, RT=0.60 (min), [M+H]+=179.

[1102] <Step 3> Synthesis of tert-butyl carbamate (2-(2-(6-(azidomethyl)nicotinamide)ethoxy)ethyl)carbamate (EX12-IM-3):

[1103] [Chemistry 175]

[1104]

[1105] For the mixture of compound 12-IM-2 (100 mg) obtained in (Example 12) <Step 2>, commercially available N-(tert-butoxycarbonyl)-2-(2-aminoethoxy)ethylamine [CAS: 127828-22-2] (108.07 μL) and acetonitrile (2000 μL), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureon hexafluorophosphate (213.43 mg) and triethylamine (156.48 μL) were added under ice-cooled stirring, and the mixture was stirred at room temperature for 1 hour and 45 minutes. Then, N-(tert-butoxycarbonyl)-2-(2-aminoethoxy)ethylamine (54 μL) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureon hexafluorophosphate (106.7 mg) were added with stirring at room temperature, and the mixture was stirred for 17 hours at the same temperature. The reaction was stopped by adding water (5 mL), followed by adding ethyl acetate (10 mL). The aqueous layer was extracted three times with ethyl acetate (10 mL) and dried over anhydrous sodium sulfate. The organic layer was filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (n-heptane / ethyl acetate) to give the title compound EX12-IM-3 (187 mg) as a colorless oil.

[1106] NMR data (CDCl3) (δ: ppm): 9.00 (1H, d, J = 2.0 Hz), 8.18 (1H, dd, J = 8.0, 2.0Hz), 7.43 (1H, d, J = 8.0 Hz), 6.83 (1H, br s), 4.82 (1H, br s) s), 4.55(2H, s), 3.69-3.65(4H, m), 3.56(2H, t, J = 5.2 Hz), 3.34(1H, d, J = 5.6 Hz), 3.32(1H, d, J = 5.6 Hz), 1.41 (9H, s). LC-MS: M=364, RT=0.78 (min), [M+H]+=365.

[1107] <Step 4> Synthesis of N-(2-(2-aminoethoxy)ethyl)6-(azidomethyl)nicotinamide 2-hydrochloride (EX12-IM-4):

[1108] [Chemistry 176]

[1109]

[1110] A mixture of compound EX12-IM-3 (0.187 g) obtained in step 3 of Example 12 and a solution of 1,4-dioxane (1.31 mL) was added with 4 equivalence hydrogen chloride / 1,4-dioxane (1.31 mL) under water cooling and stirring, and stirred at room temperature for 3 hours. After adding diisopropyl ether (20 mL) to the reaction solution, the precipitate was filtered to give the title compound EX12-IM-4 (0.16 g) as a grayish-white solid.

[1111] NMR data (DMSO-d6) (δ: ppm): 9.02-9.02 (1H, m), 8.80 (1H, br s), 8.27-8.25 (1H, m), 7.89 (3H, br s), 7.54 (1H, d, J = 8.4 Hz), 4.59 (2H, s), 3.64-3.57 (4H, m), 3.51-3.47 (2H, m), 3.01-2.97 (2H, m). LC-MS (free amine): M = 264, RT = 0.49 (min), [M+H]+ = 265.

[1112] <Step 5> Introduction of the synthesis of N-(2-(2-aminoethoxy)ethyl)-6-(azidomethyl)nicotinamide alginate (EX12-(II)-A-2):

[1113] [Chemistry 177]

[1114]

[1115] To a 1% (w / w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (39.55 mL), 91.52 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) and 1 mol of sodium bicarbonate solution (183 μL) were added with stirring at room temperature. Next, a mixture of compound EX12-IM-4 (30 mg), water (1 mL), and ethanol (1 mL) obtained in step 4 of Example 12 was added at the same temperature, and the mixture was stirred at 40°C for 4 hours. After adding sodium chloride (400 mg), 79.1 mL of ethanol was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX12-(II)-A-2 (378 mg) as a white solid.

[1116] The introduction rate of the reactive group N-(2-(2-aminoethoxy)ethyl)-6-(azidomethyl)nicotinamide was 4.8 mol% (NMR integral ratio).

[1117] (Example 13) Synthesis of alginic acid (EX13-(II)-A-2) with N-(2-(2-aminoethoxy)ethyl)-4-azidobenzamide group:

[1118] [Chemistry 178]

[1119]

[1120] <Step 1> Synthesis of tert-butyl carbamate (2-(2-(4-azidobenzamido)ethoxy)ethyl)carbamate (EX13-IM-1):

[1121] [Chemistry 179]

[1122]

[1123] 4-Azide-benzoic acid (EX7-SM, 300 mg) and (2-(2-aminoethoxy)ethyl)carbamate tert-butyl ester [CAS: 127828-22-2] (376 mg) were dissolved in acetonitrile (6.0 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureon hexafluorophosphate (0.77 g) and diisopropylethylamine (707 μL) were added, and the mixture was stirred at room temperature for 16 hours. Ethyl acetate (20 mL) and water (10 mL) were added to the reaction mixture, and the mixture was separated. The organic layer was washed sequentially with water (10 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (20% ethyl acetate / n-heptane ~ 70% ethyl acetate / n-heptane) to give the title compound EX13-IM-1 (673 mg) as a pale yellow gel.

[1124] NMR data (CDCl3) (δ: ppm): 7.83 (2H, d, J = 9 Hz), 7.08 (2H, d, J = 9 Hz), 6.61 (1H, brs), 4.84 (1H, brs), 3.68-3.64 (4H, m), 3.56 (2H, t, J = 5 Hz), 3.34 (2H, q, J = 5Hz), 1.44 (9H, s)

[1125] <Step 2> Synthesis of N-(2-(2-aminoethoxy)ethyl)-4-azidobenzamide hydrochloride (EX13-IM-2):

[1126] [Chemistry 180]

[1127]

[1128] In the reaction mixture obtained in (Example 13) <Step 1>, 670 mg of compound EX13-IM-1 was added under ice-water cooling with 4.7 mL of 4-equivalent hydrogen chloride / 1,4-dioxane, and stirred at room temperature for 2 hours. Diisopropyl ether (14.0 mL) was then added to the reaction mixture, and the mixture was stirred for 30 minutes. The resulting solid was filtered, washed with diisopropyl ether, and dried under reduced pressure to give the title compound EX13-IM-2 (604 mg) as a pale beige solid.

[1129] NMR data (DMSO-d6) (δ: ppm): 8.61 (1H, t, J = 6 Hz), 7.95 (3H, brs), 7.93 (2H, d, J = 9 Hz), 7.20 (2H, d, J = 9 Hz), 3.62 (2H, t, J = 5 Hz), 3.57 (2H, t, J = 6 Hz), 3.46 (2H, q, J = 6 Hz), 3.02–2.93 (2H, m); LC-MS (free amine): RT = 0.57 (min), [M+H] ＋ =250

[1130] <Step 3> Synthesis of N-(2-(2-aminoethoxy)ethyl)-4-azidobenzamide-based alginate (EX13-(II)-A-2):

[1131] [Chemistry 181]

[1132]

[1133] To a 1% (w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (112 mg), compound EX13-IM-2 obtained in step 2 of Example 13 (31 mg), and 1 mol sodium bicarbonate solution (151 μL) were added, and the mixture was stirred at 30°C for 3 hours. Sodium chloride (0.4 g) was added, followed by ethanol (80 mL), and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to obtain the title compound EX13-(II)-A-2 (400 mg) as a white solid.

[1134] The introduction rate of the reactive group (N-(2-(2-aminoethoxy)ethyl)-4-azidobenzamide) was 3.9 mol% (NMR integral ratio).

[1135] (Example 14) Synthesis of alginic acid (EX14-(II)-A-2) with N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-azidobenzamide group:

[1136] [Chemistry 182]

[1137]

[1138] <Step 1> Synthesis of tert-butyl carbamate (2-(2-(2-(4-azidobenzamido)ethoxy)ethoxy)ethyl)carbamate (EX14-IM-1):

[1139] [Chemistry 183]

[1140]

[1141] 4-Azide-benzoic acid (EX7-SM, 300 mg) and (2-(2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate tert-butyl ester (457 mg) were dissolved in acetonitrile (6.0 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureonium hexafluorophosphate (0.77 g) and diisopropylethylamine (707 μL) were added, and the mixture was stirred at room temperature for 16 hours. Ethyl acetate (20 mL) and water (10 mL) were added to the reaction mixture, and the mixture was separated. The organic layer was washed successively with water (10 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (40% ethyl acetate / n-heptane ~ 90% ethyl acetate / n-heptane) to give the title compound EX14-IM-1 (603 mg) as a pale yellow oil.

[1142] NMR data (DMSO-d6) (δ: ppm): 8.53 (1H, t, J = 6 Hz), 7.89 (2H, d, J = 9 Hz), 7.19 (2H, d, J = 9 Hz), 6.76 (1H, t, J = 5 Hz), 3.55-3.47(6H, m), 3.42-3.33(4H, m), 3.04(2H, q, J = 6 Hz), 1.36(9H, s)

[1143] <Step 2> Synthesis of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-azidobenzamide hydrochloride (EX14-IM-2):

[1144] [Chemistry 184]

[1145]

[1146] In Example 14, <Step 1>, the compound (EX14-IM-1, 600 mg) was added to a 4-equivalent hydrogen chloride / 1,4-dioxane solution (4.2 mL) under ice-water cooling, and the mixture was stirred at room temperature for 2 hours. Diisopropyl ether (12.0 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. The solvent was removed by decantation, and the residue was washed with diisopropyl ether. The resulting residue was dried under reduced pressure to obtain the title compound EX14-IM-2 (596 mg) as a beige gel.

[1147] NMR data (DMSO-d6) (δ: ppm): 8.62 (1H, t, J = 6 Hz), 7.97 (3H, brs), 7.91 (2H, d, J = 9 Hz), 7.20 (2H, d, J = 9 Hz), 3.62–3.51 (8H, m), 3.42 (2H, q, J = 6 Hz), 2.97–2.89 (2H, m); LC-MS (free amine): RT = 0.58 (min), [M+H] ＋ =294

[1148] <Step 3> Synthesis of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-azidobenzamide-based alginate (EX14-(II)-A-2):

[1149] [Chemistry 185]

[1150]

[1151] To a 1% (w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (40 mL), a solution of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (112 mg), a solution of compound EX14-IM-2 (45 mg) obtained in step 2 of Example 14 (4) in ethanol (4.0 mL), and 1 mol sodium bicarbonate solution (151 μL) were added, and the mixture was stirred at 30°C for 3 hours. After adding 0.4 g, ethanol (80 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX14-(II)-A-2 (408 mg) as a white solid.

[1152] The introduction rate of the reactive group (N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-azidobenzamide) was 4.2 mol% (NMR integral ratio).

[1153] (Example 15) Synthesis of alginic acid (EX15-(I)-A-2) with N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2-yn-1-oxy)acetamide group:

[1154] [Chemistry 186]

[1155]

[1156] <Step 1> Synthesis of tert-butyl (2-(2-(2,2,2-trifluoroacetamide)ethoxy)ethylcarbamate (EX15-IM-1):

[1157] [Chemistry 187]

[1158]

[1159] Ethyl trifluoroacetate (0.6 mL) was added dropwise to a tetrahydrofuran (4.0 mL) solution of tert-butyl (2-aminoethyl)carbamate (EX9-SM1, 1.0 g, [CAS: 57260-73-8]). The reaction mixture was stirred at room temperature for 3.5 hours. The reaction solution was concentrated under reduced pressure to give the title crude compound EX15-IM-1 (1.5 g) as a colorless oil.

[1160] NMR data (CDCl3) (δ: ppm): 7.01 (1H, brs), 4.84 (1H, brs), 3.62–3.51 (6H, m), 3.31 (2H, q, J = 5 Hz), 1.45 (9H, s)

[1161] <Step 2> Synthesis of N-(2-(2-aminoethoxy)ethyl)-2,2,2-trifluoroacetamide hydrochloride (EX15-IM-2):

[1162] [Chemistry 188]

[1163]

[1164] In (Example 15) <Step 1>, compound EX15-IM-1 (1.5 g) was added to a 4 equivalence hydrogen chloride / 1,4-dioxane solution (10.3 mL) under ice-water cooling, and the mixture was stirred at room temperature for 1 hour. Diisopropyl ether (30 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. The solvent was removed by distillation under reduced pressure, followed by azeotropic treatment with diisopropyl ether, and then drying under reduced pressure to obtain the title compound EX15-IM-2 (1.3 g) as a colorless oil.

[1165] NMR data (DMSO-d6) (δ: ppm): 9.55 (1H, brs), 8.05 (3H, brs), 3.61 (2H, d, J = 5Hz), 3.54 (2H, t, J = 6 Hz), 3.39 (2H, q, J = 6Hz), 3.00-2.91 (2H, m)

[1166] <Step 3> Synthesis of N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamide)ethoxy)ethyl)-2,2,2-trifluoroacetamide (EX15-IM-3):

[1167] [Chemistry 189]

[1168]

[1169] The carboxylic acid (EX8-SM2, 300 mg) synthesized according to a known method (Org. Process Res. Dev. (2018) 22: 108-110) and the compound obtained in step 2 of Example 14 (443 mg) were dissolved in acetonitrile (6.0 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureon hexafluorophosphate (0.75 g) and diisopropylethylamine (920 μL) were added, and the mixture was stirred at room temperature for 2.5 hours. Ethyl acetate (20 mL) and water (10 mL) were added to the reaction mixture, and the mixture was separated. The organic layer was washed successively with water (10 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50% ethyl acetate / n-heptane ~ 70% ethyl acetate / n-heptane) to give the title compound EX15-IM-3 (469 mg) as a colorless gel.

[1170] NMR data (DMSO-d6) (δ: ppm): 9.45 (1H, brs), 7.61 (1H, t, J = 6 Hz), 4.29-4.25 (1H, m), 3.87 (2H, d, J = 15 Hz), 3.75 (1H, d, J = 15 Hz), 3.50 (2H, t, J = 6 Hz), 3.43 (2H, t, J = 6 Hz), 3.37-3.31 (2H, m), 3.24 (2H, q, J = 6 Hz), 2.27-2.03 (3H, m), 1.96-1.69 (4H, m), 1.67-1.50 (2H, m), 1.43-1.35 (1H, m)

[1171] <Step 4> Synthesis of N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (EX15-IM-4):

[1172] [Chemistry 190]

[1173]

[1174] In a methanol (3.0 mL) solution of compound EX15-IM-3 (220 mg) obtained in (Example 15) <Step 3>, a water solution of potassium carbonate (103 mg) (0.99 mL) was added, and the mixture was stirred at room temperature for 4.5 hours. The methanol was distilled off under reduced pressure, and water (2 mL) was added, followed by saturation with sodium chloride. The mixture was extracted with ethyl acetate (15 mL, 10 mL × 4), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was dissolved in ethyl acetate (10 mL), filtered to remove insoluble matter, and concentrated under reduced pressure to obtain the crude compound EX15-IM-4 (140 mg) as a pale yellow gel.

[1175] NMR data (CDCl3) (δ: ppm): 6.89 (1H, brs), 4.27-4.22 (1H, m), 4.07 (1H, d, J = 15Hz), 3.88 (1H, d, J = 15 Hz), 3.58-3.47 (6H, m), 2.87 (2H, t, J = 5 Hz), 2.31-2.10 (3H, m), 2.03-1.77 (4H, m), 1.73-1.59 (2H, m), 1.51-1.43 (1H, m), LC-MS: RT=0.60 (min), [M+H] ＋ =269

[1176] <Step 5> Synthesis of alginic acid (EX15-(I)-A-2) with N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group:

[1177] [Chemistry 191]

[1178]

[1179] In a 1% (w) aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd., A-2) (40 mL), a 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (112 mg), an ethanol solution (4.0 mL) of compound EX15-IM-4 (30 mg) obtained in step 4 of Example 15), and 1 mol% sodium bicarbonate solution (101 μL) were added sequentially under stirring at room temperature. The mixture was stirred at 30°C for 3 hours. Sodium chloride (0.4 g) was added to the reaction solution, followed by ethanol (80 mL), and the mixture was stirred for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure to give the title compound EX15-(I)-A-2 (410 mg) as a white solid.

[1180] The introduction rate of the reactive substituent (N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2-yn-1-yloxy)acetamido) was 3.2 mol% (NMR integral ratio).

[1181] [Table 12]

[1182] Example Measurement wavelength (nm) Molecular weight (Da) Weight-average molecular weight (Da) Importation rate (mol%) 1a 280 12,000 to 2,650,000 1.53 million 6.9(*) 1b 280 5,000 to 2.62 million 1.15 million 6.5(*) 1c 280 27,000~2.66 million Da 1.71 million 6.6(*) 1d 288 37.3 million to 2.85 million 1.41 million 4.9(*) 1e 287 13,700 to 2,520,000 1.4 million 0.8(*) 1f 287 22.3 million to 2.57 million 1.42 million 1.9(*) 2 Differential refractometer 6,000~2.35 million Da 920,000 Da 5.8(*) 3a 255 15,000 to 2,530,000 1.51 million 6.1(*) 3b 255 5,000 to 2,590,000 1.14 million 9.4(*) 3c 255 18,000 to 2,690,000 1.65 million 6.9(*) 3d 249 76.3 million to 2.59 million 1.42 million 3.7(*) 3e 249 22.9 million to 2.56 million 1.41 million 0.6(*) 3f 249 11,800 to 2,540,000 1.42 million 1.5(*) 4 255 10,000 to 2,850,000 1.46 million 4.3(*) 5a 255 11 million to 2.66 million 1.53 million 9.4(*) 5b 232 51.9 million to 2.66 million 1.39 million 11(*) 6 287 20.8 million to 2.57 million 1.4 million 2.7(*) 7a 267 64.3 million to 2.59 million 1.41 million 5.1(*) 7b 267 18.2 million to 2.56 million 1.41 million 2.0(*) 8 215 18.5 million to 2.83 million 1.38 million 4.46(*) 9a Differential refractometer 13,000 to 2,820,000 1.42 million 4.3(*) 9b Differential refractometer 13,000 to 2,590,000 1.41 million 4.2(*) 9c Differential refractometer 13,000 to 2,670,000 1.41 million 2.1(*) 10 230 77.4 million to 2.66 million 1.43 million 4.7(*) 11 230 71.2 million to 2.71 million 1.45 million 4.2(*) 12 270 76.8 million to 2.59 million 1.41 million 4.8(*) 13 270 51.3 million to 2.69 million 1.41 million 3.9(*) 14 270 50.2 million to 2.67 million 1.43 million 4.2(*) 15 Differential refractometer 13,000 to 3,640,000 1.4 million 3.2(*)

[1183] (*) NMR integration ratio.

[1184] [Determination of the introduction rate of reactive groups or complementary reactive groups]

[1185] The reactive group or complementary reactive group introduction rate refers to the percentage of reactive groups or complementary reactive groups introduced into each uronic acid monosaccharide unit, which is a repeating unit of alginate.

[1186] In this embodiment, the introduction rate (mol%) of reactive groups or complementary reactive groups is determined by... 1 The integral ratio of H-NMR was calculated. Furthermore, the amount of alginate necessary for the inoculation rate was determined using the carbazole-sulfuric acid method with a calibration curve, and the amount of reactive or complementary reactive groups was also determined using absorbance measurement with a calibration curve.

[1187] [Determination of molecular weight]

[1188] The solid alginate derivatives with incorporated reactive or complementary reactive groups obtained in the examples were dissolved in 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl to prepare 0.1% or 0.2% solutions. Insoluble matter was removed by passing the solutions through a 0.22 μm polyethersulfone filter (Minisart HighFlow Filter, Sartorius) and the resulting samples were used for gel filtration. The spectra of each sample were measured using a DU-800 spectrophotometer (Beckman-Coulter) to determine the detection wavelength for each compound during gel filtration. For compounds without specific absorption wavelengths, a differential refractometer was used.

[1189] 200 μL of gel filtration sample was fed onto a Superose6 Increase 10 / 300 GL column (GE Healthcare). Gel filtration was performed using an AKTA Explorer 10S as the chromatographic apparatus, with 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl as the developing solvent, at a flow rate of 0.8 mL / min at room temperature. Isosorbite uptake at determined wavelengths for each compound was monitored, and elution profiles were constructed. The resulting chromatograms were analyzed using Unicorn 5.31 software (GE Healthcare) to determine peak ranges.

[1190] The molecular weights of alginate with incorporated reactive or complementary reactive groups were determined using blue dextran (2 million Da, SIGMA), thyroglobulin (669,000 Da, GE Healthcare), ferritin (440,000 Da, GE Healthcare), aldolase (158,000 Da, GE Healthcare), conalbumin (75,000 Da, GE Healthcare), ovalbumin (44,000 Da, GE Healthcare), ribonuclease A (13,700 Da, GE Healthcare), and aprotinin (6,500 Da, GE Healthcare) as standards. Gel filtration was performed under the same conditions as for alginate with incorporated reactive or complementary reactive groups, and the eluent volume for each component was determined using Unicorn software. The eluent volume for each component was plotted on the x-axis, and the logarithm of the molecular weight was plotted on the y-axis. Linear regression was then performed to create a calibration curve. Two calibration curves were created: one from blue dextran to ferritin, and the other from ferritin to aprotinin.

[1191] Using this calibration curve, first calculate the molecular weight (Mi) at elution time i in the obtained chromatography. Next, read the absorbance at elution time i and use it as Hi. Based on these data, calculate the weight-average molecular weight (Mw) as follows.

[1192] [Number 1]

[1193] .

[1194] [Determination of gel stability]

[1195] (Determination of gel stability (1))

[1196] The alginate derivatives (EX1-(I)-A-2) obtained in Example 1a and (EX3-(II)-A-2) obtained in Example 3a> were dissolved in water at a concentration of 1% by weight to obtain (alginate aqueous solution 1-1) and (alginate aqueous solution 2-1), respectively. Equal volumes of (alginate aqueous solution 1-1) and (alginate aqueous solution 2-1) were mixed, and the resulting aqueous solution was added to a syringe equipped with an 18-gauge syringe. The syringe was placed on a syringe pump set to a flow rate of 1 mL / min, and the solution was added dropwise to a 30 mmol / L calcium chloride solution for 30 seconds. After stirring for 5 minutes, an alginate gel was obtained. The gel was washed once with 10 mL of phosphate-buffered saline (PBS), and then allowed to stand in PBS at 37°C for 10 minutes for chemical cross-linking, resulting in an alginate gel that underwent both chemical and ionic cross-linking. [The text then abruptly shifts to a different topic:] ...add to this gel... Add 20 mL of PBS, shake at 37 °C, and recover the aqueous solution over time. Replenish with the same amount of PBS as the recovered solution. After the experiment, add 5 μL of alginate lyase (Nipponjin, 319-08261) to the test solution, shake at 37 °C for 2 hours to completely disintegrate the gel, and recover the aqueous solution. The concentration of alginate in the recovered aqueous solution was determined by the carbazole-sulfuric acid method. The value obtained by correcting the concentration of alginate in the aqueous solution at each time point with the recovered alginate concentration, divided by the total alginate concentration calculated from the alginate concentration at all time points and the alginate concentration at the end of the experiment, was expressed as a percentage and used as the disintegration rate, serving as an indicator of gel stability. Similarly, as a control, cross-linked alginate gels (beads) were prepared using alginate (A-2), (EX1-(I)-A-2), (A-2), and (EX3-(II)-A-2), and the disintegration rates were measured.

[1197] The results are shown in Figure 1 As a control, the cross-linked alginate gels prepared using (A-2), (EX1-(I)-A-2), (A-2), and (EX3-(II)-A-2) essentially dissolved after 4 hours. In contrast, the cross-linked alginate gels prepared using the alginate derivatives of (EX1-(I)-A-2) and (EX3-(II)-A-2) did not disintegrate even after 144 hours, further improving the stability of the gel. This indicates that the structure formed by cross-linking using the Wheatstone reaction can maintain its structure even after long-term use in solutions without calcium ions (below physiological concentrations depending on the organism).

[1198] It should be explained that Figure 1The disintegration rate on the vertical axis refers to the relative disintegration rate (%). The maximum value of the measured disintegration rate (only for the alginate gel prepared in (A-2): measured value after 8 hours) was corrected to 100%, and the disintegration rate at each point was set as a relative value to this maximum value.

[1199] (Determination of gel stability (2))

[1200] The alginate derivatives (EX1-(I)-B-2a) obtained in Example 1d, (EX3-(II)-B-2a) obtained in Example 3d, (EX7-(II)-B-2a) obtained in Example 7a, and (EX8-(I)-B-2) obtained in Example 8 were dissolved in water at a concentration of 1.0 w / w% to obtain alginate aqueous solutions (1d-1), (3d-1), (7a-1), and (8a-1). Next, equal volumes of mixed solutions of alginate aqueous solution (1d-1) and alginate aqueous solution (3d-1), equal volumes of mixed solutions of alginate aqueous solution (1d-1) and alginate aqueous solution (7a-1), equal volumes of mixed solutions of alginate aqueous solution (8a-1) and alginate aqueous solution (3d-1), and equal volumes of mixed solutions of alginate aqueous solution (8a-1) and alginate aqueous solution (7a-1) were used to obtain chemically cross-linked and ionicly cross-linked alginate gels (beads) (A1, B1, C1, D1) according to the following alginate gel (bead) preparation method (see Table 13).

[1201] [Preparation method of alginate gel (beads)]

[1202] The prepared mixed solution was added to a syringe equipped with an 18-gauge needle. The syringe was placed on a syringe pump set to a flow rate of 1 mL / min, and the solution was dropped into a 30 mmol / L calcium chloride solution for 30 seconds. After stirring for 5 minutes, alginate gel (beads) was obtained. The gel was washed once with 10 mL of phosphate-buffered saline (PBS) and then allowed to stand in pure water at 37°C for 10 minutes for chemical cross-linking, resulting in alginate gel (beads) that underwent both chemical and ionic cross-linking.

[1203] [Table 13]

[1204]

[1205] [Stability determination of alginate gels (A1~D1)]

[1206] Each alginate gel (bead) (A1~D1) obtained above underwent chemical and ionic crosslinking. 19.5 mL of PBS was added, and the mixture was shaken at 37°C. The aqueous solution was recovered after 1, 2, 4, 8, 24, 48, 72, and 144 hours, and the same amount of PBS was replenished. After the experiment, 20 μL of alginate lyase (Creative Enzymes, NATE-1563) was added to the test solution, and the mixture was shaken overnight at 37°C to completely disintegrate the gel. The aqueous solution was then recovered. The concentration of alginate in the recovered aqueous solution was determined by the carbazole-sulfuric acid method. The alginate concentration at each time point was divided by the total alginate concentration calculated from the alginate concentrations at all time points and the concentration at the end of the experiment. The percentage obtained was used as the disintegration rate, which served as an indicator of gel stability. As a control, alginate without the introduced reactive groups (B-2) was used. Alginate gel (beads) (REF) were prepared according to the aforementioned method, and the disintegration rate was measured.

[1207] The results are shown in Figure 4 As a control, the alginate gel (gel REF) prepared using alginate (B-2) without the introduction of reactive groups showed more than 90% disintegration after 144 hours. In contrast, the aforementioned cross-linked alginate gels (beads) (A1~D1) obtained using various alginate derivatives (EX1-(I)-B-2a, EX3-(II)-B-2a, EX7-(II)-B-2a, and EX8-(I)-B-2) did not disintegrate even after 144 hours, further improving the stability of the gel. That is, it indicates that the structure formed by cross-linking using the Wheatstone reaction can maintain its structure even after a long period of time in solutions without calcium ions (below the physiological concentration depending on the organism).

[1208] (Determination of gel stability (3): Determination of stability in the presence of gel EDTA)

[1209] 19.5 mL of 5 mM EDTA-2K / PBS solution was added to each of the chemically and ionicly crosslinked alginate gels (beads) (A1-D1) obtained using the aforementioned gel stability determination method (2). The mixture was shaken at 37°C, and the aqueous solution was recovered after 24 hours to prepare EDTA-treated crosslinked alginate gels (beads) (A2-D2). After the experiment, 10 μL of alginate lyase (CreativeEnzymes, NATE-1563) was added to the test solution, and the mixture was shaken overnight at 37°C to completely disintegrate the gel. The aqueous solution was then recovered. The concentration of alginate in the recovered aqueous solution was determined using the carbazole-sulfuric acid method. The alginate concentration in the aqueous solution after 24 hours was divided by the total alginate concentration calculated from the alginate concentration after 24 hours and the alginate concentration after the experiment. The result was expressed as a percentage and used as the disintegration rate, which was used as an indicator of gel stability. Similarly, as a control, alginate gels (beads) (REF2) were prepared using alginate (B-2) without the introduction of reactive groups, and the disintegration rate was determined.

[1210] [Table 14]

[1211]

[1212] The results are shown in Table 14. As a control, the cross-linked alginate gel (beads) (REF2) prepared using alginate gel with unreactive groups (B-2) and treated with EDTA showed 100% disintegration after 24 hours. In contrast, the cross-linked alginate gel (beads) (A1~D1) obtained using the aforementioned cross-linked alginate gel (beads) (A2~D2) prepared using various alginate derivatives (EX1-(I)-B-2a, EX3-(II)-B-2a, EX7-(II)-B-2a, and EX8-(I)-B-2) and treated with EDTA did not disintegrate even after 24 hours, further improving gel stability. This indicates that by forming chemical cross-links based on the Wheatstone reaction, the prepared (bead) structure can maintain its structure over a long period even without calcium ion-based ionic cross-links.

[1213] (Determination of gel stability (4))

[1214] Alginate with a reactive substituent obtained in Example 1g (EX1-(I)-A-2b), Alginate with a reactive substituent obtained in Example 3g (EX3-(II)-A-2b), Alginate with a reactive substituent obtained in Example 5c (EX5-(II)-A-2b), Alginate with a reactive substituent obtained in Example 7c (EX7-(II)-A-2), and Alginate with a reactive substituent obtained in Example 9a (EX9-(I)-A-2) were dissolved in water to prepare 1.0% aqueous solutions of alginate (1g-1), (3g-1), (5c-1), (7c-1), and (9a-1). The aforementioned alginate aqueous solution was mixed in equal volumes using combinations of (9a-1) and (5c-1), (9a-1) and (7c-1), (9a-1) and (3g-1), and (1g-1) and (3g-1). Each mixture was added to a syringe equipped with an 18-gauge needle. The syringe was placed on a syringe pump set to a flow rate of 1 mL / min, and the solution was dropped into a 30 mmol / L calcium chloride solution for 30 seconds, followed by stirring for 5 minutes to obtain alginate gels. Each gel was washed once with 10 mL of PBS and then allowed to stand in PBS at 37°C for 10 minutes for chemical cross-linking to obtain chemically cross-linked alginate gels. 19.5 mL of PBS was added to each gel, and the mixture was shaken at 37°C. The aqueous solution was recovered over time, and the same amount of PBS was replenished to compensate for the recovered solution. After the experiment, 10 μL of alginate lyase (Nipponjin, 319-08261) was added to the test solution, and the gel was shaken overnight at 37°C to completely disintegrate. The aqueous solution was then recovered. The concentration of alginate in the recovered aqueous solution was determined using the carbazole-sulfuric acid method. The value obtained by correcting the concentration of alginate in the aqueous solution at each time point with the recovered alginate concentration was divided by the total alginate concentration calculated from the alginate concentration at all time points and the alginate concentration at the end of the experiment. The result was expressed as a percentage and used as the disintegration rate, which served as an indicator of gel stability.

[1215] The results are shown in Figure 8 The stability of the aforementioned cross-linked alginate gels (beads) after 96 hours ranged from 0.3% to 4.8%, indicating that the structure could be maintained even after long-term use (the gels prepared in (Example 1g) and (Example 3g) served as controls, with a stability of 0.3% after 96 hours).

[1216] Determination of gel stability (5): Determination of stability in the presence of gel EDTA

[1217] Alginate with a reactive substituent obtained in Example 1g (EX1-(I)-A-2b), Alginate with a reactive substituent obtained in Example 3g (EX3-(II)-A-2b), Alginate with a reactive substituent obtained in Example 5c (EX5-(II)-A-2b), Alginate with a reactive substituent obtained in Example 7c (EX7-(II)-A-2), and Alginate with a reactive substituent obtained in Example 9a (EX9-(I)-A-2) were dissolved in water to prepare 1.0% aqueous solutions of alginate (1g-1), (3g-1), (5c-1), (7c-1), and (9a-1). The aforementioned alginate aqueous solution was mixed in equal volumes using combinations of (9a-1) and (5c-1), (9a-1) and (7c-1), (9a-1) and (3g-1), and (1g-1) and (3g-1). Each mixture was then added to a syringe equipped with an 18-gauge needle. The syringe was placed on a syringe pump set to a flow rate of 1 mL / min, and the solution was dropped into a 30 mmol / L calcium chloride solution over 30 seconds, followed by stirring for 5 minutes to obtain alginate gels. Each gel was washed once with 10 mL of PBS and then allowed to stand in PBS at 37°C for 10 minutes for chemical cross-linking to obtain chemically cross-linked alginate gels.

[1218] Add 19.5 mL of 5 mM EDTA-2K dihydrate / physiological saline to each of the aforementioned gels, shake at 37°C, and recover the aqueous solution over time. Replenish with the same amount of 5 mM EDTA-2K / physiological saline as the recovered solution. After the experiment (24 hours later), add 30 μL of alginate lyase (Nipponjin, 319-08261) to the test solution, shake overnight at 37°C to completely disintegrate the gel, and recover the aqueous solution. Determine the alginate concentration in the recovered aqueous solution using the carbazole-sulfuric acid method. Divide the value obtained by correcting the alginate concentration in the aqueous solution at each time point using the recovered alginate concentration by the total alginate concentration calculated from the alginate concentration at all time points and the alginate concentration at the end of the experiment. Express the result as a percentage, and use this value as the disintegration rate, as an indicator of gel stability.

[1219] get Figure 9 The results showed that the aforementioned cross-linked alginate gel (beads) did not disintegrate even after 24 hours, confirming the stability of the gel. This indicates that the (bead) structure produced by forming chemical cross-links using the Wheatstone reaction can maintain its structure over a long period.

[1220] (Determination of gel stability (6))

[1221] A 1.0% aqueous solutions of alginate (1 g-1), (3 g-1), (5 c-1), (7 c-1), and (8 b-1) were prepared by dissolving the alginate with a reactive substituent obtained in (Example 1 g) (EX1-(I)-A-2b), (Example 3 g) (EX3-(II)-A-2b), (Example 5 c) (EX5-(II)-A-2b), (Example 7 c) (EX7-(II)-A-2), and (Example 8 b) (EX8-(I)-A-2) in water. The aforementioned alginate aqueous solution was mixed in equal volumes with combinations of (1 g⁻¹) and (5 c⁻¹), (1 g⁻¹) and (7 c⁻¹), (8 b⁻¹) and (5 c⁻¹), (8 b⁻¹) and (7 c⁻¹), (8 b⁻¹) and (3 g⁻¹), and (1 g⁻¹) and (3 g⁻¹). Each mixture was then added dropwise to a 30 mmol / L calcium chloride solution over 30 seconds, followed by stirring for 5 minutes to obtain alginate gels. Each gel was washed once with 10 mL of PBS and then allowed to stand in PBS at 37°C for 10 minutes for chemical cross-linking to obtain chemically cross-linked alginate gels. Add 19.5 mL of PBS to the gel, shake at 37°C, and recover the aqueous solution over time. Replenish with the same amount of PBS as the recovered amount. After the experiment, add 10 μL of alginate lyase (Nipponjin, 319-08261) to the test solution, shake overnight at 37°C to completely disintegrate the gel, and recover the aqueous solution. The concentration of alginate in the recovered aqueous solution is determined using the carbazole-sulfuric acid method. The percentage obtained by dividing the amount of dissolved alginate up to each time point by the total alginate concentration calculated from the alginate concentrations at all time points and the alginate concentration at the end of the experiment is used as the disintegration rate, which is an indicator of gel stability.

[1222] The results are shown in Figure 10 The stability of each of the aforementioned cross-linked alginate gels (beads) was approximately 20% after 96 hours, indicating that the structure could be maintained even after a long period of time (the stability of the gels prepared in (Example 1g) and (Example 3g) as controls was 20.4% after 96 hours).

[1223] (Determination of gel stability (7): Determination of stability in the presence of gel EDTA)

[1224] A 1.0% aqueous solutions of alginate (1 g-1), (3 g-1), (5 c-1), (7 c-1), and (8 b-1) were prepared by dissolving the alginate with a reactive substituent obtained in (Example 1 g) (EX1-(I)-A-2b), (Example 3 g) (EX3-(II)-A-2b), (Example 5 c) (EX5-(II)-A-2b), (Example 7 c) (EX7-(II)-A-2), and (Example 8 b) (EX8-(I)-A-2) in water. The aforementioned alginate aqueous solution was mixed in equal volumes with combinations of (1 g⁻¹) and (5 c⁻¹), (1 g⁻¹) and (7 c⁻¹), (8 b⁻¹) and (5 c⁻¹), (8 b⁻¹) and (7 c⁻¹), (8 b⁻¹) and (3 g⁻¹), and (1 g⁻¹) and (3 g⁻¹). Each mixture was added to a syringe equipped with an 18-gauge needle. The syringe was placed on a syringe pump set to a flow rate of 1 mL / min, and the solution was added dropwise to a 30 mmol / L calcium chloride solution for 30 seconds. The mixture was stirred for 5 minutes to obtain alginate gels. 19.5 mL of 5 mM EDTA-2K dihydrate / physiological saline was added to each gel, and the mixture was shaken at 37°C. The aqueous solution was recovered over time, and the same amount of 5 mM EDTA-2K / physiological saline was replenished. After the experiment (24 hours later), 30 μL of alginate lyase (Nipponjin, 319-08261) was added to the test solution, and the gel was shaken overnight at 37°C to completely disintegrate. The aqueous solution was then recovered. The concentration of alginate in the recovered aqueous solution was determined using the carbazole-sulfuric acid method. The percentage obtained by dividing the amount of dissolved alginate up to each time point by the total alginate concentration calculated from the alginate concentration at all time points and the alginate concentration at the end of the experiment was used as the disintegration rate, which served as an indicator of gel stability.

[1225] get Figure 11 The results showed that the aforementioned cross-linked alginate gel (beads) did not disintegrate even after 24 hours, confirming the stability of the gel. This indicates that the (bead) structure produced by forming chemical cross-links using the Wheatstone reaction can maintain its structure over a long period.

[1226] (Determination of gel stability (8))

[1227] The reactive substituent-containing alginic acid (EX1-(I)-A-2b) obtained in Example 1g, the reactive substituent-containing alginic acid (EX3-(II)-A-2b) obtained in Example 3g, the reactive substituent-containing alginic acid (EX10-(II)-A-2) obtained in Example 10, the reactive substituent-containing alginic acid (EX11-(II)-A-2) obtained in Example 11, and the reactive substituent-containing alginic acid (EX12-(II)-A-2) obtained in Example 12 were compared. Alginate with reactive substituents obtained in Example 13 (EX13-(II)-A-2), Alginate with reactive substituents obtained in Example 14 (EX14-(II)-A-2), and Alginate with reactive substituents obtained in Example 15 (EX15-(I)-A-2) were dissolved in water to prepare 1.0% aqueous solutions of alginate (1 g-1), (3 g-1), (10 g-1), (11 g-1), (12 g-1), (13 g-1), (14 g-1), and (15 g-1). The aforementioned alginate aqueous solution was mixed in equal volumes using combinations of (3 g⁻¹) and (15 g⁻¹), (1 g⁻¹) and (10 g⁻¹), (1 g⁻¹) and (11 g⁻¹), (1 g⁻¹) and (12 g⁻¹), (1 g⁻¹) and (13 g⁻¹), (1 g⁻¹) and (14 g⁻¹), and (1 g⁻¹) and (3 g⁻¹). Each mixture was then added to a syringe equipped with an 18-gauge needle. The syringe was placed on a syringe pump set to a flow rate of 1 mL / min, and the solution was dropped into a 30 mmol / L calcium chloride solution for 30 seconds, followed by stirring for 5 minutes to obtain alginate gels. Each gel was washed once with 10 mL of PBS and then allowed to stand in PBS at 37°C for 10 minutes for chemical cross-linking to obtain chemically cross-linked alginate gels. Add 19.5 mL of PBS to the gel, shake at 37°C, and recover the aqueous solution over time. Replenish with the same amount of PBS as the recovered amount. After the experiment, add 10 μL of alginate lyase (Nipponjin, 319-08261) to the test solution, shake overnight at 37°C to completely disintegrate the gel, and recover the aqueous solution. The concentration of alginate in the recovered aqueous solution is determined using the carbazole-sulfuric acid method. The percentage obtained by dividing the amount of dissolved alginate up to each time point by the total alginate concentration calculated from the alginate concentrations at all time points and the alginate concentration at the end of the experiment is used as the disintegration rate, which is an indicator of gel stability.

[1228] The results are shown in Figure 12The stability of each of the aforementioned cross-linked alginate gels (beads) was less than 0.5% after 96 hours, indicating that the structure could be maintained even after a long period of time (the stability of the gels prepared in (Example 1g) and (Example 3g) as controls was 0.3% after 96 hours).

[1229] (Determination of gel stability (9): Determination of stability in the presence of EDTA gel)

[1230] The reactive substituent-containing alginic acid (EX1-(I)-A-2b) obtained in Example 1g, the reactive substituent-containing alginic acid (EX3-(II)-A-2b) obtained in Example 3g, the reactive substituent-containing alginic acid (EX10-(II)-A-2) obtained in Example 10, the reactive substituent-containing alginic acid (EX11-(II)-A-2) obtained in Example 11, and the reactive substituent-containing alginic acid (EX12-(II)-A-2) obtained in Example 12 were compared. Alginate with reactive substituents obtained in Example 13 (EX13-(II)-A-2), Alginate with reactive substituents obtained in Example 14 (EX14-(II)-A-2), and Alginate with reactive substituents obtained in Example 15 (EX15-(I)-A-2) were dissolved in water to prepare 1.0% aqueous solutions of alginate (1 g-1), (3 g-1), (10 g-1), (11 g-1), (12 g-1), (13 g-1), (14 g-1), and (15 g-1). The aforementioned alginate aqueous solution was mixed in equal amounts with combinations of (3g-1) and (15-1), (1g-1) and (10-1), (1g-1) and (11-1), (1g-1) and (12-1), (1g-1) and (13-1), (1g-1) and (14-1), and (1g-1) and (3g-1). Each mixed solution was added to another syringe equipped with an 18-gauge needle. The syringe was set on an injection pump with a flow rate of 1 mL / min, and the solution was dropped into a 30 mmol / L calcium chloride solution for 30 seconds. The mixture was stirred for 5 minutes to obtain each alginate gel.

[1231] Add 19.5 mL of 5 mM EDTA-2K dihydrate / physiological saline to each gel, shake at 37°C, and recover the aqueous solution over time. Replenish with the same amount of 5 mM EDTA-2K / physiological saline as the recovered solution. After the experiment (24 hours later), add 30 μL of alginate lyase (Nipponjin, 319-08261) to the test solution, shake overnight at 37°C to completely disintegrate the gel, and recover the aqueous solution. Determine the alginate concentration in the recovered aqueous solution using the carbazole-sulfuric acid method. The percentage obtained by dividing the amount of dissolved alginate up to each time point by the total alginate concentration calculated from the alginate concentrations at all time points and the alginate concentration at the end of the experiment is used as the disintegration rate, which is an indicator of gel stability.

[1232] get Figure 13 The results showed that the aforementioned cross-linked alginate gel (beads) did not disintegrate even after 24 hours, confirming the stability of the gel. This indicates that the (bead) structure produced by forming chemical cross-links using the Wheatstone reaction can maintain its structure over a long period.

[1233] [Gel permeability determination]

[1234] (Gel permeability determination (1))

[1235] The alginate derivatives (EX1-(I)-A-2) obtained in (Example 1a) and (EX3-(II)-A-2) obtained in (Example 3a) were dissolved in water at a concentration of 2% to obtain (alginate aqueous solution 1-2) and (alginate aqueous solution 2-2). Further, an equal amount of fluorescein isothiocyanate-glucan (FD2000S) with a molecular weight of 2 million or fluorescein isothiocyanate-glucan (FD150S) with a molecular weight of 1 mg / mL was added to (alginate aqueous solution 1-2) to obtain (alginate aqueous solution 3) or (alginate aqueous solution 4). In addition, an equal amount of physiological saline was added to (alginate aqueous solution 2-2) to obtain (alginate aqueous solution 5).

[1236] Equal volumes of (alginic acid aqueous solution 3) and (alginic acid aqueous solution 5) were mixed. This mixture was then added to a syringe fitted with an 18-gauge needle. The syringe was placed on a syringe pump set to a flow rate of 1 mL / min, and the solution was dropped into a 30 mmol / L calcium chloride solution over 30 seconds. The mixture was stirred for approximately 20 minutes to obtain an alginate gel. The gel was washed once with 10 mL of physiological saline to obtain chemically cross-linked alginate gel (beads) encapsulating fluorescein isothiocyanate (molecular weight 2 million)-dextran. Furthermore, equal volumes of (alginic acid aqueous solution 4) and (alginic acid aqueous solution 5) were mixed, and chemically cross-linked alginate gel (beads) encapsulating fluorescein isothiocyanate (molecular weight 150,000)-dextran were obtained using the same method as described above.

[1237] Add 20 mL of physiological saline to the obtained gel (beads), shake at 37°C, and recover the aqueous solution after a period of time. After the experiment, add 10 μL of alginate lyase (Nipponjin, 319-08261) to the test solution, shake at 37°C for more than 2 hours to completely disintegrate the gel, and recover the aqueous solution. Measure the dextran concentration in the recovered aqueous solution using quantitative fluorescence method (excitation light: 485 nm, fluorescence: 535 nm). Divide the dextran concentration at each time point by the dextran concentration at the end of the experiment, and express the result as a percentage. This value is used as the transmittance.

[1238] The results of gels (EX1-(I)-A-2 / EX3-(II)-A-2 / glucan (molecular weight: 2 million)) obtained by adding equal amounts of (alginic acid aqueous solution 3) and (alginic acid aqueous solution 5) are shown in the figure. Figure 2 The pass rate was confirmed to be 6.4% over 24 hours.

[1239] The results of gels (EX1-(I)-A-2 / EX3-(II)-A-2 / glucan (molecular weight: 150,000)) obtained by adding equal amounts of (alginic acid aqueous solution 4) and (alginic acid aqueous solution 5) are shown in the figure. Figure 3 The leakage rate was confirmed to be 23.6% after 3 hours and 31.9% after 24 hours.

[1240] (Gel permeability determination (2))

[1241] The alginate derivatives (EX1-(I)-B-2a) obtained in Example 1d, (EX3-(II)-B-2a) obtained in Example 3d, (EX7-(II)-B-2a) obtained in Example 7a, and (EX8-(I)-B-2) obtained in Example 8 were dissolved in water at a concentration of 1.5 w / w% to obtain alginate aqueous solutions (1d-2), (3d-2), (7a-2), and (8a-2). Further, fluorescein isothiocyanate-glucan (FD2000S) with a molecular weight of 2 million or fluorescein isothiocyanate-glucan (FD150S) with a molecular weight of 1 mg / mL was added to (alginic acid aqueous solution 3d-2) to obtain (alginic acid aqueous solution 3d-2-A) or (alginic acid aqueous solution 3d-2-B). Similarly, by adding fluorescein isothiocyanate-glucan (FD2000S) with a molecular weight of 2 million or fluorescein isothiocyanate-glucan (FD150S) with a molecular weight of 1 mg / mL to (alginic acid aqueous solution 7a-2), we obtain (alginic acid aqueous solution 7a-2-A) or (alginic acid aqueous solution 7a-2-B).

[1242] These aqueous solutions were mixed in the combinations shown in Table 15 to achieve a final concentration of alginic acid of 1.0 w / w% and a final concentration of fluorescein isothiocyanate-dextran of 100 μg / mL. The resulting aqueous solution was added to a syringe fitted with an 18-gauge needle. The syringe was placed on a syringe pump set to a flow rate of 1 mL / min and added dropwise to a 30 mmol / L calcium chloride solution for 30 seconds, followed by stirring for 5 minutes to obtain an alginic acid gel. The gel was washed once with 10 mL of phosphate-buffered saline (PBS) and then allowed to stand in pure water at 37°C for 10 minutes for chemical cross-linking, resulting in chemically cross-linked alginic acid gels (beads) containing fluorescein isothiocyanate (molecular weight 2 million)-dextran and chemically cross-linked alginic acid gels (beads) containing fluorescein isothiocyanate (molecular weight 150,000)-dextran (gels a~h).

[1243] [Table 15]

[1244] (1d-2) (8a-2) (3d-2-A) Gel a Gel b (7a-2-A) Gel C Gel d (3d-2-B) Gel e Gel f (7a-2-B) Gel g Gel h

[1245] Add 19.5 mL of physiological saline to each gel from a to h, shake at 37°C, and recover the aqueous solution after 3 and 24 hours. Replenish with the same amount of physiological saline as the recovered solution. After the experiment, add 20 μL of alginate lyase (CreativeEnzymes, NATE-1563) to the test solution, shake overnight at 37°C to completely disintegrate the gel, and recover the aqueous solution. Measure the dextran concentration in the recovered aqueous solution using quantitative fluorescence method (excitation light: 485 nm, fluorescence: 535 nm). Divide the dextran concentration at each time point by the total dextran concentration calculated from the total dextran concentration at all time points and the dextran concentration at the end of the experiment, and express the result as a percentage. This value is used as the transmittance.

[1246] The results of mixing (alginic acid aqueous solution 3d-2-A) and (alginic acid aqueous solution 7a-2-A) with (alginic acid aqueous solution 1d-2) and (alginic acid aqueous solution 8a-2) respectively to obtain gels (gel a, gel b, gel c, and gel d in Table 15) are shown in Table 15. Figure 5 The permeability was 0% at both 3 hours and 24 hours. The results of mixing (alginic acid aqueous solution 3d-2-B) and (alginic acid aqueous solution 7a-2-B) with (alginic acid aqueous solution 1d-2) and (alginic acid aqueous solution 8a-2), respectively, to obtain gels (gels e, f, g, and h in Table 15) are shown below. Figure 6 The transmittance is 2.5%~4.6% after 3 hours and 6.8%~8.6% after 24 hours.

[1247] (Gel permeability determination (3))

[1248] Alginate with a reactive substituent obtained in Example 3g (EX3-(II)-A-2b), alginate with a reactive substituent obtained in Example 5c (EX5-(II)-A-2b), and alginate with a reactive substituent obtained in Example 7c (EX7-(II)-A-2) were dissolved in water to a concentration of 2.0% to prepare an aqueous solution of alginate. Two-fifths of the volume of fluorescein isothiocyanate-glucan (FD150S) with a molecular weight of 150,000 prepared at 1 mg / mL and three-fifths of the volume of water were added to the aqueous solution of alginate to prepare 1.0% aqueous solutions of alginate containing 0.2 mg / mL fluorescein isothiocyanate-glucan (3g-2), (5c-2), and (7c-2). Further, the alginic acid with the introduced reactive substituent (EX1-(I)-A-2b) obtained in (Example 1g) and the alginic acid with the introduced reactive substituent (EX9-(I)-A-2) obtained in (Example 9a) were dissolved in water to a concentration of 1.0% to prepare alginic acid aqueous solutions (1g-1) and (9a-1), respectively. Equal volumes of the aforementioned alginic acid aqueous solutions were mixed in combinations of (9a-1) and (5c-2), (9a-1) and (7c-2), (9a-1) and (3g-2), and (1g-1) and (3g-2), and 40 mL of a 30 mmol / L calcium chloride solution was added. The mixture was stirred for 5 minutes to obtain an alginic acid gel. The gel was washed once with 10 mL of physiological saline and then allowed to stand at 37°C for 10 minutes for chemical cross-linking to obtain chemically cross-linked alginic acid gels containing fluorescein isothiocyanate-dextran. Add 19.5 mL of physiological saline to each gel, shake at 37°C, and recover the aqueous solution over time. Replenish with the same amount of physiological saline as the recovered solution. After the experiment (24 hours later), add 10 μL of alginate lyase (Nipponjin, 319-08261) to the test solution, shake at 37°C for at least 3 hours to completely disintegrate the gel, and recover the aqueous solution. Measure the dextran concentration in the recovered aqueous solution using quantitative fluorescence method (excitation light: 485 nm, fluorescence: 535 nm). Divide the dextran concentration at each time point by the dextran concentration at the end of the experiment, and express the result as a percentage. This value is used as the transmittance.

[1249] get Figure 14 The results showed that the transmittance was approximately 30% after 3 hours. Furthermore, the transmittance was approximately 40% after 24 hours.

[1250] (Gel permeability determination (4))

[1251] Alginate with a reactive substituent obtained in Example 3g (EX3-(II)-A-2b), alginate with a reactive substituent obtained in Example 5c (EX5-(II)-A-2b), and alginate with a reactive substituent obtained in Example 7c (EX7-(II)-A-2) were dissolved in water to a concentration of 2.0% to prepare an aqueous solution of alginate. Two-fifths volume of fluorescein isothiocyanate-glucan (FD150S) with a molecular weight of 1 mg / mL and three-fifths volume of water were added to the aqueous solution of alginate to obtain 1.0% aqueous solutions of alginate containing 0.2 mg / mL fluorescein isothiocyanate-glucan (3g-2), (5c-2), and (7c-2). Further, the alginic acid with the introduced reactive substituent (EX1-(I)-A-2b) obtained in (Example 1g) and the alginic acid with the introduced reactive substituent (EX8-(I)-A-2) obtained in (Example 8b) were dissolved in water to a concentration of 1.0% to prepare alginic acid aqueous solutions (1g-1) and (5c-2), (1g-1) and (7c-2), (8b-1) and (5c-2), (8b-1) and (7c-2), (8b-1) and (3g-2), and (1g-1) and (3g-2), respectively. Equal volumes of these combinations were mixed, and 40 mL of a 30 mmol / L calcium chloride solution was added. The mixture was stirred for 5 minutes to obtain an alginic acid gel. The gel was washed once with 10 mL of physiological saline. It was then allowed to stand at 37°C for 10 minutes to undergo chemical cross-linking, resulting in chemically cross-linked alginate gels containing fluorescein isothiocyanate-dextran. 19.5 mL of physiological saline was added to each gel, and the mixture was shaken at 37°C. The aqueous solution was recovered over time, and the same amount of physiological saline was replenished. After the experiment (24 hours later), 10 μL of alginate lyase (Nipponjin, 319-08261) was added to the test solution, and the mixture was shaken at 37°C for at least 3 hours to completely disintegrate the gel. The aqueous solution was recovered. The concentration of dextran in the recovered aqueous solution was measured using quantitative fluorescence spectroscopy (excitation light: 485 nm, fluorescence: 535 nm). The percentage obtained by dividing the dextran concentration at each time point by the dextran concentration at the end of the experiment was used as the transmittance.

[1252] get Figure 15 The results showed that the transmittance was approximately 35% after 3 hours. Furthermore, the transmittance was approximately 45% after 24 hours.

[1253] (Gel permeability determination (5))

[1254] Alginic acid with a reactive substituent obtained in (Example 3g) (EX3-(II)-A-2b), alginic acid with a reactive substituent obtained in (Example 10) (EX10-(II)-A-2), alginic acid with a reactive substituent obtained in (Example 11) (EX11-(II)-A-2), alginic acid with a reactive substituent obtained in (Example 12) (EX12-(II)-A-2), alginic acid with a reactive substituent obtained in (Example 13) (EX13-(II)-A-2), and alginic acid with a reactive substituent obtained in (Example 14) (EX14-(II)-A-2) were dissolved in water to a concentration of 2.0% to prepare an aqueous solution of alginic acid. 2 / 5 of the volume of this aqueous solution was added to prepare a solution of... To prepare 1.0% alginate aqueous solutions containing 0.2 mg / mL fluorescein isothiocyanate-dextran (FD150S) with a molecular weight of 150,000, using fluorescein isothiocyanate-dextran (FD150S) at a concentration of 1 mg / mL and 3 / 5 of the volume of water, we prepared alginate aqueous solutions (3 g-2), (10 g-2), (11 g-2), (12 g-2), (13 g-2), and (14 g-2) respectively. Further, alginate aqueous solutions (1 g-1) and (15 g-1) were prepared by dissolving the reactively substituent-modified alginate (EX1-(I)-A-2b) obtained in (Example 1 g) and the reactively substituent-modified alginate (EX15-(I)-A-2) obtained in (Example 15) in water at a concentration of 1.0%. They were mixed in equal amounts with combinations of (15-1) and (3g-2), (1g-1) and (10-2), (1g-1) and (11-2), (1g-1) and (12-2), (1g-1) and (13-2), (1g-1) and (14-2), and (1g-1) and (3g-2), respectively. 40 mL of a 30 mmol / L calcium chloride solution was added, and the mixture was stirred for 5 minutes to obtain alginate gels. Each gel was washed once with 10 mL of physiological saline and then allowed to stand at 37°C for 10 minutes for chemical cross-linking, yielding chemically cross-linked alginate gels containing fluorescein isothiocyanate-dextran. 19.5 mL of physiological saline was added to each gel, and the mixture was shaken at 37°C. The aqueous solution was recovered over time, and the same amount of physiological saline was replenished as recovered. After the experiment (24 hours later), 10 μL of alginate lyase (Nipponjin, 319-08261) was added to the test solution, and the solution was shaken at 37°C for at least 3 hours to completely disintegrate the gel. The aqueous solution was then recovered. The concentration of dextran in the recovered aqueous solution was measured using quantitative fluorescence method (excitation light: 485 nm, fluorescence: 535 nm). The percentage obtained by dividing the dextran concentration at each time point by the dextran concentration after the experiment was used as the transmittance.

[1255] get Figure 16 The results showed that the transmittance was approximately 17% to 28% after 3 hours. Furthermore, the transmittance was approximately 25% to 35% after 24 hours.

[1256] [Biocompatibility evaluation of cross-linked alginate derivatives (gels)]

[1257] Alginate derivatives (EX1-(I)-B-2-L) with an NMR integral ratio of 0.9 mol% prepared using the same method as in (Example 1d), alginate derivatives (EX3-(II)-B-2-L) with an NMR integral ratio of 0.6 mol% prepared using the same method as in (Example 3d), alginate derivatives (EX7-(II)-B-2-L) with an NMR integral ratio of 0.9 mol% prepared using the same method as in (Example 7a), and alginate derivatives (EX8-(I)-B-2-L) with an NMR integral ratio of 0.3 mol% prepared using the same method as in (Example 8) were dissolved in physiological saline to obtain alginate aqueous solutions (1d-L), (3d-L), (7a-L), and (8a-L), respectively.

[1258] Further, 1.5 × 10⁻⁶ alginate aqueous solution was added to (3 d-L) and (7 a-L) respectively. 7 CHO cells were suspended in PBS at a concentration of cells / mL to obtain (3d-LC of alginate aqueous solution) or (7a-LC of alginate aqueous solution).

[1259] These aqueous solutions were mixed in the combinations shown in Table 16 to achieve a final alginate concentration of 1.0 w / w% and a final CHO cell concentration of 5.0 × 10⁻⁶. 6 Cells / mL. The resulting aqueous solution was added to a syringe fitted with an 18-gauge needle. The syringe was set to a syringe pump with a flow rate of 1 mL / min. The solution was added dropwise to a 50 mmol / L calcium chloride solution for 30 seconds. After stirring for 5 minutes, the solution was washed once with 10 mL of phosphate-buffered saline (PBS) to obtain alginate gels (beads) containing CHO cells (gels CHO-1 to CHO-4).

[1260] [Table 16]

[1261] 1d-L 8a-L 3D-LC Gel CHO-1 Gel CHO-2 7a-LC Gel CHO-3 Gel CHO-4

[1262] Gels (CHO-1) to (CHO-4) were seeded in 6-well plates (FALCON, Cat #351146). The medium composition listed in Table 17 below was added at 5 mL / well to impregnate the gels. The plates were then incubated at 37°C with shaking at 125 rpm for 2 days. After the experiment, the gels were recovered and impregnated again in 5 mL of fresh medium. 50 μL of alginate lyase (CreativeEnzymes, NATE-1563) was added, and the plates were shaken at 37°C for 1 hour to completely disintegrate the gels. The medium was then recovered. The number of viable and dead cells in the recovered medium was counted using trypan blue staining. The percentage obtained by dividing the number of viable cells by the sum of the number of viable and dead cells was used as the cell viability, serving as an indicator of the gel's biocompatibility. Similarly, as a control, CHO cells were prepared with alginate gels (beads) (REF-CHO) containing unreactive groups (B-2) to measure cell viability.

[1263] [Table 17]

[1264]

[1265] The results are shown in Figure 7 The cell viability of the alginate gels ((gel CHO-1) to (gel CHO-4)) obtained from the combinations of alginate derivatives described in Table 16 above was 87.2% to 89.0%. Furthermore, as a control, the cell viability of the alginate gel (REF-CHO) prepared using alginate without the introduced reactive group (B-2) was 90.5%. Based on these results, it is indicated that alginate derivatives with introduced reactive groups and chemically cross-linked alginate structures (beads) formed using the Wheatstone reaction possess the same high degree of biocompatibility as alginate without the introduced reactive groups.

[1266] [Biocompatibility evaluation of cross-linked alginate derivatives (gels) (2)]

[1267] The alginic acid with a reactive substituent obtained in (Example 1g) (EX1-(I)-A-2b), the alginic acid with a reactive substituent obtained in (Example 3g) (EX3-(II)-A-2b), the alginic acid with a reactive substituent obtained in (Example 5c) (EX5-(II)-A-2b), the alginic acid with a reactive substituent obtained in (Example 7c) (EX7-(II)-A-2), the alginic acid with a reactive substituent obtained in (Example 8b) (EX8-(I)-A-2), the alginic acid with a reactive substituent obtained in (Example 9a) (EX9-(I)-A-2), and the alginic acid with a reactive substituent obtained in (Example 10) Alginic acid with introduced reactive substituents (EX10-(II)-A-2), alginic acid with introduced reactive substituents obtained in (Example 11) (EX11-(II)-A-2), alginic acid with introduced reactive substituents obtained in (Example 12) (EX12-(II)-A-2), alginic acid with introduced reactive substituents obtained in (Example 13) (EX13-(II)-A-2), alginic acid with introduced reactive substituents obtained in (Example 14) (EX14-(II)-A-2), and alginic acid with introduced reactive substituents obtained in (Example 15) (EX15-(I)-A-2) were dissolved in water to prepare alginic acid solutions with introduced cross-linking groups. After sterilization by filtration using a Minisarthighflow (Zaltrick, 16532GUK), an aqueous solution of alginic acid / physiological saline with introduced cross-linking groups of 1.0% was prepared. HeLa cells, obtained by seeding them in 96-well plates at a cell concentration of 5 × 10³ cells / well and culturing for 1 day, were then treated with alginate / physiological saline solution containing 1.0% cross-linking group, in combination with (Example 1g), (Example 8b), (Example 9a), or (Example 15) and (Example 3g), or in combination with (Example 1g) and (Example 5c), (Example 7c), (Example 10), (Example 11), (Example 12), (Example 13), or (Example 14) to a final concentration of 0.1%. After culturing for 1 day, ATP activity was evaluated as an indicator of cytotoxicity using a CellTiter-Glo luminescent cell viability assay (Promega, G7571).

[1268] get Figure 17 The results showed that ATP activity was confirmed in all the aforementioned cross-linked alginate gels, indicating that there was no cytotoxicity in the cross-linked alginate gels and that the chemically cross-linked alginate structures (beads) formed by the Wheatstone reaction are biocompatible.

Claims

1. Alginic acid derivatives, represented by formulas selected from the following: [Chemistry 3] In each formula, (ALG) represents alginic acid; the -NHCO- bonded to (ALG) represents an amide bond derived from any carboxyl group of alginic acid.

2. The alginate derivative according to claim 1, wherein, The introduction rate of each of the following groups is 0.1%~30%. 。 3. The alginate derivative according to claim 1, wherein, The weight-average molecular weights of alginic acid derivatives, determined by gel filtration chromatography, ranged from 100,000 Da to 3,000,000 Da.

4. The alginate derivative according to any one of claims 1 to 3, which is biocompatible.

5. Alginic acid derivatives, represented by formulas selected from the following: [Chemistry 7] In each formula, (ALG) represents alginic acid; the -NHCO- bonded to (ALG) represents an amide bond derived from any carboxyl group of alginic acid.

6. The alginate derivative according to claim 5, wherein, The introduction rate of each of the following groups is 0.1%~30%. 。 7. The alginate derivative according to claim 5, wherein, The weight-average molecular weights of alginic acid derivatives, determined by gel filtration chromatography, ranged from 100,000 Da to 3,000,000 Da.

8. The alginate derivative according to any one of claims 5 to 7, which is biocompatible.

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