Novel crosslinked alginic acid

Abstract

To provide novel alginic acid derivatives and a novel crosslinked alginic acid.SOLUTION: The alginic acid derivatives are represented by formula (I) and formula (II). The novel crosslinked alginic acid is obtained by Huisgen reaction using the alginic acid derivative of the formula (I) and the alginic acid derivative of the formula (II).SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to novel alginic acid derivatives, novel crosslinked alginic acids, novel crosslinked alginic acid structures, and methods for producing the same.

Background Art

[0002] Alginic acid is a high molecular weight acidic polysaccharide molecule extracted from the cell walls of natural brown algae such as Lessonia, Macrocystis, Laminaria, Ascophyllum, Darbilla, Hijikia, Arame, and Kombu. It is a linear heteropolymer in which two types of uronic acids, β-D-mannuronic acid (M component) and its C-5 epimer, α-L-guluronic acid (G component), are linked by 1-4 bonds. Specifically, its chemical structure is a block copolymer in which homopolymer blocks of mannuronic acid (MM), homopolymer blocks of guluronic acid (GG), and blocks (MG) in which mannuronic acid and guluronic acid are randomly arranged are complexly bonded in any order and ratio. Alginic acid is widely used in fields such as medicine, biotechnology, cosmetics, fibers, paper making, and food.

[0003] Alkaline metal salts of alginic acid monovalent salts (for example, sodium alginate, etc.) are water-soluble, but alkaline earth metal salts of alginic acid divalent salts (for example, calcium alginate, etc.) have the property of being crosslinked by metal ions and gelling (insolubilizing), and attempts have been made to modify or mold them into suitable materials for various applications using this property.

[0004] In order to explore the possibility of modifying or molding various materials with polysaccharides (for example, hyaluronic acid, chondroitin sulfate, alginic acid, etc.) and improving their physical properties (for example, strength, swelling property, etc.), various studies have been conducted so far on crosslinked polysaccharides crosslinked by covalent bonds.

[0005] As methods for obtaining crosslinked polysaccharides, specifically, (1) a crosslinking method using an aldehyde crosslinking agent such as formaldehyde (Patent Document 1: WO 2011 / 028031 pamphlet), (2) a self-crosslinking method using carboxy groups and hydroxyl groups in polysaccharides (Patent Document 2: WO 89 / 10941 pamphlet), and (3) a crosslinking method using a homo-bifunctional crosslinking agent (diepoxide, divinyl sulfone, diamine, or dihydrazide, etc.) or a hetero-bifunctional crosslinking agent (epihalohydrin, etc.) (Patent Document 3: WO 2009 / 073437 pamphlet) are known.

[0006] Also, (4) a crosslinking method by irradiating light after introducing a photoreactive group (cinnamic acid, substituted cinnamic acid, acrylic acid, maleic acid, fumaric acid, furylacrylic acid, thiopheneacrylic acid, cinnamylideneacetic acid, sorbic acid, thymine, or coumarin, etc.) (Patent Documents 4 and 5: WO 2005 / 026214 pamphlet, JP-A-9-87236), and (5) a crosslinking method of crosslinking polysaccharides having thiol groups with disulfide bonds and a crosslinking method by Michael addition reaction using polysaccharides having thiol groups and polysaccharides having maleimide groups (Patent Document 6: WO 2008 / 071058 pamphlet), etc. are known.

[0007] Furthermore, as a method for crosslinking polysaccharides by covalent bonds, a crosslinking method by subjecting a polysaccharide having an alkyne group and a polysaccharide having an azide group to a Huisgen reaction (1,3-dipolar cycloaddition reaction) is known.

[0008] Crosslinked polysaccharides obtained by crosslinking polysaccharides by the Huisgen reaction are disclosed in (i) WO 2008 / 031525 pamphlet (Patent Document 7), (ii) WO 2012 / 165462 pamphlet (Patent Document 8), (iii) WO 2015 / 020206 pamphlet (Patent Document 9), (iv) Chinese Patent Application Publication No. 106140040 specification (Patent Document 10), and (v) WO 2019 / 240219 pamphlet (Patent Document 13), etc.

[0009] However, (i) Patent Document 7 relates to a crosslinked polysaccharide obtained by subjecting a chain alkyne group and an azide group introduced into each polysaccharide via a linker to a Huisgen reaction using a copper catalyst, with the first polysaccharide being hyaluronic acid and the second polysaccharide being a polysaccharide selected from chondroitin, dermatan sulfate, alginic acid or its salts, etc., and does not disclose the novel crosslinked alginic acid described below.

[0010] Also, (ii) Patent Document 8 relates to a crosslinked polysaccharide obtained by subjecting a cyclic alkyne group and an azide group introduced into each polysaccharide via a linker (the polysaccharide and the linker are in an ester bond) to a Huisgen reaction, with the first polysaccharide and the second polysaccharide being polysaccharides selected from hyaluronic acid, carboxymethyl dextran, cellulose derivatives, and chitosan (the first polysaccharide and the second polysaccharide may be the same or different), and does not disclose the novel crosslinked alginic acid described below.

[0011] Also, (iii) Patent Document 9 relates to a crosslinked polysaccharide obtained by subjecting a cyclic alkyne group and an azide group introduced into each polysaccharide via a linker to a Huisgen reaction, with the first polysaccharide being hyaluronic acid and the second polysaccharide being chondroitin sulfate, and does not disclose the novel crosslinked alginic acid described below.

[0012] Also, (iv) Patent Document 10 relates to a crosslinked polysaccharide obtained by subjecting a cyclic alkyne group and an azide group introduced into each polysaccharide via a linker (the polysaccharide and the linker are in an ester bond) to a Huisgen reaction, with the first polysaccharide being chitosan and the second polysaccharide being sodium alginate, and does not disclose the novel crosslinked alginic acid described below.

[0013] Also, International Publication No. 2016 / 019391 pamphlet (Patent Document 11) and International Publication No. 2017 / 165389 pamphlet (Patent Document 12) describe alginic acid with an azide group introduced into the side chain, but do not disclose a crosslinked alginic acid structure formed from alginic acid with an alkyne group introduced into the side chain, and the purpose of its use is also different from that of the present invention.

[0014] Furthermore, Non-Patent Document 1 describes branched alginic acid (bAlg-DBCO) with a cyclooctyne side chain introduced into the side chain. However, it is obtained by reacting branched alginic acid (Branched alginic acid: bAlg) synthesized from alginic acid and branched polyethylene glycol (4-arm PEG-NH2) with amino cyclooctyne (DBCO-PEG-amine). Its structure is different from the novel alginic acid derivative described later, and its purpose of use is also different.

Prior Art Documents

Patent Documents

[0015]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Patent Document 10

Patent Document 11

Patent Document 12

Patent Document 13

Non-Patent Document

[0016]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0017] Under the above circumstances, a novel alginic acid derivative, a novel crosslinked alginic acid formed from the novel alginic acid derivative, a crosslinked alginic acid structure, and a method for producing them have been demanded.

Means for Solving the Problems

[0018] As a result of intensive studies to solve the above problems, the present inventors have each found a novel alginic acid derivative represented by formula (I) or formula (II). Furthermore, by using a novel crosslinked alginic acid obtained by subjecting the novel alginic acid derivatives of formula (I) and formula (II) to the Huisgen reaction, beads (dye-containing beads), which are one of the crosslinked alginic acid structures, were molded. As a result, it was found that the beads have high stability and can be adjusted to a gel having a transmittance according to the purpose as compared with conventional gels, and the present invention has been completed.

[0019] The novel alginic acid derivatives (formulas (I) and (II)) provided here can be used, for example, for forming chemical crosslinks, that is, those into which a reactive group that can be used for forming chemical crosslinks or a complementary reactive group of the reactive group has been introduced.

[0020] The chemical crosslinking formation is carried out, for example, by a crosslinking reaction using the Huisgen reaction (1,3-dipolar cycloaddition reaction), and may be carried out, for example, between the alginic acid derivatives of formula (I) and formula (II), or may be carried out, for example, between the alginic acid derivative of formula (I) and another molecule having an azide group, or may be carried out between the alginic acid derivative of formula (II) and another molecule having an alkyne group.

[0021] Since the Huisgen reaction using terminal alkyne groups and terminal azide groups generally requires heating at 100 °C or higher, it has not been suitable to use this reaction for the chemical modification of biomolecules. However, by coexisting a copper catalyst (for example, Cu(I)) in the reaction, reaction conditions have been found in which a cycloadduct (triazole ring) is formed at room temperature with a yield of almost 100% (Angew. Chem. Int. Ed. Engl., 41, p2596 - 2599, 2002; J. Org. Chem., 67, p3057 - 3064, 2002), and it has become possible to use it for the chemical modification of biomolecules. On the other hand, when attempting to obtain crosslinked alginic acid by the Huisgen reaction in the presence of the copper catalyst, there is a possibility that a trace amount of the copper catalyst may remain in the crosslinked alginic acid, and there is a concern that copper-derived cytotoxicity may be expressed in the crosslinked alginic acid or the crosslinked alginic acid structure.

[0022] In a preferred embodiment, in order to avoid the expression of copper-derived cytotoxicity in the crosslinked alginic acid, a copper-catalyst-free Huisgen reaction is utilized to obtain crosslinked alginic acid. Specifically, by using a cyclooctyne derivative (highly strained cyclic alkyne group) for the alkyne group introduced into the alginic acid derivative, it was possible to carry out the reaction without the need for high-temperature conditions of 100 °C or higher and a copper catalyst. Therefore, the novel crosslinked alginic acid of the preferred embodiment is also excellent from the viewpoint that since it does not contain a copper catalyst, copper-derived toxicity does not occur even when formed into a final shaped article (crosslinked alginic acid structure).

[0023] Here, provided are alginic acid derivatives of formula (I) or formula (II) in which a cyclic alkyne group or an azide group is introduced into any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker, a novel cross-linked alginic acid, a cross-linked alginic acid structure obtained by performing a Huisgen reaction (1,3-dipolar cycloaddition reaction) using the alginic acid derivatives of formula (I) and formula (II), and methods for producing each of the above alginic acid derivatives, cross-linked alginic acid, and cross-linked alginic acid structure. That is, exemplary embodiments can be as follows in [1] to

[23] below.

[0024] [1] An alginic acid derivative represented by the following formula (I) in which a cyclic alkyne group (Akn) is introduced into any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker (-L 1 -): [Chemical formula] [In formula (I), Akn, -L 1 -, -NHCO-, and (ALG) are the same as defined in the first embodiment described below], and an alginic acid derivative represented by the following formula (II) in which an azide group is introduced into any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker (-L 2 -): [Chemical formula] [In formula (II), -L 2 -, -NHCO-, and (ALG) are the same as defined in the first embodiment described below]. A cross-linked alginic acid obtained by subjecting the alginic acid derivative to a cross-linking reaction.

[0025] [1-Ia] The alginic acid derivative represented by the above formula (I) according to [1], wherein the introduction rate of the Akn-L 1 -NH2 group (Akn and -L 1 - are the same as defined in the first embodiment described below) is 0.1% to 30%.

[0026] The alginic acid derivative represented by the formula (I) according to the above [1], wherein the weight average molecular weight measured by gel filtration chromatography of the [1-Ib] alginic acid derivative is 100,000 Da to 3,000,000 Da.

[0027] 〔1-IIa〕N3-L 2 -NH2 group (-L 2 - is the same as the definition in the first aspect described later), and the introduction rate of the alginic acid derivative represented by the formula (II) according to the above [1] is 0.1% to 30%.

[0028] The alginic acid derivative represented by the formula (II) according to the above [1], wherein the weight average molecular weight measured by gel filtration chromatography of the [1-IIb] alginic acid derivative is 100,000 Da to 3,000,000 Da.

[0029] 〔2〕The following formula (I) in which a cyclic alkyne group (Akn) is introduced into any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker (-L 1 -):

Chemical formula

[0030] 〔3〕The introduction rate of the Akn-L 1 -NH2 group (Akn and -L 1 - are the same as the definitions in the third aspect described later) is 0.1% to 30%, and the alginic acid derivative represented by the formula (I) according to the above [2].

[0031] The alginic acid derivative represented by the formula (I) according to the above [2], wherein the weight average molecular weight measured by gel filtration chromatography of the alginic acid derivative is 100,000 Da to 3,000,000 Da.

[0032] [5] An amide bond and a divalent linker (-L 2 -) are introduced into any one or more carboxyl groups of alginic acid, and the azide group is introduced into the following formula (II):

Chemical formula

[0033] [6] The introduction rate of the N3-L 2 -NH2 group (-L 2 - is the same as the definition in the sixth aspect described later) is 0.1% to 30%, and the alginic acid derivative represented by the formula (II) described in [5] above.

[0034] [7] The weight average molecular weight measured by the gel filtration chromatography method of the alginic acid derivative is 100,000 Da to 3,000,000 Da, and the alginic acid derivative represented by the formula (II) described in [5] above.

[0035] [8] Any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (III-L):

Chemical formula

[0036] [8a] Any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (III-L):

Chemical formula

[0037] [8-1] The crosslinked alginic acid according to [1] or [8a] above, including chemical crosslinking by a triazole ring formed by the Huisgen reaction as the crosslinking and ionic crosslinking partially formed by a divalent metal ion. [8-2] The crosslinked alginic acid according to [1] or [8a] above, including chemical crosslinking by a triazole ring formed by the Huisgen reaction as the crosslinking.

[0038] [8-3-1] In the above [8-1], the divalent metal ion is an ion selected from the group consisting of calcium ion, magnesium ion, barium ion, strontium ion, and zinc ion.

[0039] [8-3-2] In the above [8-1], the source of the divalent metal ion is an aqueous solution selected from the group consisting of an aqueous calcium chloride solution, an aqueous calcium carbonate solution, an aqueous calcium gluconate solution, an aqueous barium chloride solution, etc.

[0040] [9] A method for producing crosslinked alginic acid, which includes obtaining the crosslinked alginic acid according to [1] or [8a] above by mixing the alginic acid derivative represented by formula (I) described in [1] and the alginic acid derivative represented by formula (II) and performing a crosslinking reaction (Huisgen reaction).

[0041] [9-1] A method for producing crosslinked alginic acid, which includes obtaining the crosslinked alginic acid according to [1] or [8a] above by adding a solution of the alginic acid derivative represented by formula (I) described in [1] to a solution of the alginic acid derivative represented by formula (II) described in [1] and performing a crosslinking reaction (Huisgen reaction).

[0042] 〔9-2〕A method for producing crosslinked alginic acid, which comprises adding a solution of the alginic acid derivative represented by the formula (II) described in [1] to a solution of the alginic acid derivative represented by the formula (I) described in [1] and performing a crosslinking reaction (Huisgen reaction) to obtain the crosslinked alginic acid described in [1] or [8a].

[0043] 〔10〕The chemical crosslinking formed by performing the Huisgen reaction (crosslinking reaction) using the alginic acid derivative represented by the formula (I) and the alginic acid derivative represented by the formula (II) described in [1] is represented by the following formula (III-L):

Chemical formula

[0044] 〔11〕A crosslinked alginic acid structure obtained by dropping a mixed solution of an alginic acid derivative obtained by mixing the alginic acid derivative represented by the formula (I) and the alginic acid derivative represented by the formula (II) described in [1] into a solution containing a divalent metal ion.

[0045] 〔11-1〕A crosslinked alginic acid structure obtained by adding a gel obtained by dropping a solution of the alginic acid derivative represented by the formula (I) described in [1] into a solution containing a divalent metal ion to a solution of the alginic acid derivative represented by the formula (II) described in [1] and performing a crosslinking reaction.

[0046] 〔11-2〕A crosslinked alginic acid structure obtained by adding a gel obtained by dropping a solution of the alginic acid derivative represented by the formula (II) described in [1] into a solution containing a divalent metal ion to a solution of the alginic acid derivative represented by the formula (I) described in [1] and performing a crosslinking reaction.

[0047] The crosslinked alginate structure according to any one of

[11] to [11-2], comprising a chemical crosslink by a triazole ring formed by a Huisgen reaction as the crosslink and an ionic crosslink partially formed by a divalent metal ion.

[0048] The crosslinked alginate structure according to any one of

[11] to [11-2], comprising a chemical crosslink by a triazole ring formed by a Huisgen reaction as the crosslink.

[0049] 〔12-3-1〕In the above [12-1], the divalent metal ion is an ion selected from the group consisting of calcium ion, magnesium ion, barium ion, strontium ion, and zinc ion.

[0050] 〔12-3-2〕In the above [12-1], the source of the divalent metal ion is an aqueous solution selected from the group consisting of an aqueous calcium chloride solution, an aqueous calcium carbonate solution, an aqueous calcium gluconate solution, an aqueous barium chloride solution, and the like.

[0051] A crosslinked alginate structure having a retention property of the content, obtained by subjecting the alginate derivative represented by the formula (I) and the alginate derivative represented by the formula (II) according to [1] above to ionic crosslinking with a divalent metal ion and chemical crosslinking by a Huisgen reaction.

[0052] A crosslinked alginate structure having a retention property of the content, obtained by subjecting the alginate derivative represented by the formula (I) and the alginate derivative represented by the formula (II) according to [1] above to chemical crosslinking by a Huisgen reaction.

[0053] 〔13-3-1〕In the above [13-1], the divalent metal ion is an ion selected from the group consisting of calcium ion, magnesium ion, barium ion, strontium ion, and zinc ion.

[0054] [13-3-2] In the above [13-1], the source of divalent metal ions is an aqueous solution selected from the group consisting of an aqueous calcium chloride solution, an aqueous calcium carbonate solution, an aqueous calcium gluconate solution, an aqueous barium chloride solution, and the like.

[0055]

[14] By performing the Huisgen reaction using the alginic acid derivative represented by the formula (I) described in the above [1], and the alginic acid derivative represented by the formula (II), the chemical crosslinking formed is represented by the following formula (III-L): [Chemical formula] [In the formula (III-L), the -CONH- and -NHCO- at both ends, -L 1 -, and -L 2 - are the same as the definitions in the 14th aspect described later], and the crosslinked alginic acid structure described in the above

[11] to [13-2] has such a structure.

[0056]

[15] A method for producing a crosslinked alginic acid structure, which is obtained by dropping a mixed solution of alginic acid derivatives obtained by mixing the alginic acid derivative represented by the formula (I) described in the above [1] and the alginic acid derivative represented by the formula (II) into a solution containing divalent metal ions and performing a crosslinking reaction.

[0057] [15-1] A method for producing a crosslinked alginic acid structure, which is obtained by adding a gel obtained by dropping a solution of the alginic acid derivative represented by the formula (I) described in the above [1] into a solution containing divalent metal ions to a solution of the alginic acid derivative represented by the formula (II) described in the above [1] and performing a crosslinking reaction.

[0058] [15-2] A method for producing a crosslinked alginic acid structure, which is obtained by adding a gel obtained by dropping a solution of the alginic acid derivative represented by the formula (II) described in the above [1] into a solution containing divalent metal ions to a solution of the alginic acid derivative represented by the formula (I) described in the above [1] and performing a crosslinking reaction.

[0059] A method for producing the crosslinked alginic acid structure according to any one of the above items

[15] to [15-2], including chemical crosslinking by a triazole ring formed by the Huisgen reaction as the crosslinking and ionic crosslinking partially formed by a divalent metal ion.

[0060] A method for producing the crosslinked alginic acid structure according to any one of the above items

[15] to [15-2], including chemical crosslinking by a triazole ring formed by the Huisgen reaction as the crosslinking.

[0061] 〔16-3-1〕In the above [16-1], the divalent metal ion is an ion selected from the group consisting of calcium ion, magnesium ion, barium ion, strontium ion, and zinc ion.

[0062] 〔16-3-2〕In the above [16-1], as the source of the divalent metal ion, an aqueous solution selected from the group consisting of an aqueous solution of calcium chloride, an aqueous solution of calcium carbonate, an aqueous solution of calcium gluconate, an aqueous solution of barium chloride, etc. is used.

[0063] 〔17〕When the Huisgen reaction is carried out using the alginic acid derivative represented by the formula (I) and the alginic acid derivative represented by the formula (II) according to any one of the above items [1], the chemical crosslinking formed is the following formula (III-L):

Chemical formula

[15] to [16-2].

[0064] 〔18〕The crosslinked alginic acid structure according to any one of the above items

[11] to

[14] , which is beads or a substantially spherical gel.

[0065] 〔19〕The medical material containing the crosslinked alginic acid structure according to any one of 〔11〕~〔14〕 above.

[0066] 〔20〕The medical material according to 〔19〕 above, which is beads or a substantially spherical gel.

[0067] 〔21〕The crosslinked alginic acid according to 〔1〕 or 〔8a〕 above, the alginic acid derivative according to 〔2〕 or 〔5〕 above, or the crosslinked alginic acid structure according to any one of 〔11〕~〔14〕 above, which has biocompatibility.

[0068] 〔22〕The amino compound represented by the following formula (AM-1):

Chemical formula

[0069] 〔23〕The amino compound represented by the following formula (AM-2):

Chemical formula

Advantages of the Invention

[0070] The present invention provides, for example, a novel alginic acid derivative, a novel crosslinked alginic acid, a novel crosslinked alginic acid structure, etc., which can be used for forming a chemical crosslink. Preferably, the alginic acid derivative has a reactive group that does not exist in a living body. Even if unreacted groups remain, it is expected to be safe for living organisms such as cells because there is no risk of a cross-linking reaction with living body components. Further, preferably, the cross-linking reaction is completed at room temperature without using a metal catalyst, so that it can be used safely and easily. The cross-linked alginic acid in some embodiments is chemically cross-linked by the Huisgen reaction (1,3-dipolar cycloaddition reaction). The cross-linking can be used in combination with chemical cross-linking and ionic cross-linking using a divalent metal ion (for example, calcium ion). By adjusting the reaction conditions, preferably its stability is improved as compared with non-cross-linked alginic acid or non-chemically cross-linked alginic acid (for example, cross-linked alginic acid cross-linked with calcium ion). Further, preferably, the gel physical properties of the cross-linked product can be adjusted, and the substance permeability can also be adjusted. The present invention has at least one or more of these effects.

Brief Description of Drawings

[0071]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Mode for Carrying Out the Invention

[0072] [Specific Embodiments] The following embodiments [1] to

[23] may be included. [1] The first embodiment is as follows. An alginic acid derivative represented by the following formula (I) in which a cyclic alkyne group (Akn) is introduced via an amide bond and a divalent linker (-L 1 -), and an alginic acid derivative represented by the following formula (II) in which an azide group is introduced via an amide bond and a divalent linker (-L 2 -) are used to perform a crosslinking reaction to obtain crosslinked alginic acid.

[0073] [Alginic Acid Derivative Represented by Formula (I)] The following formula (I):

Chemical Formula

Table 1-1

Table 1-2

Table 2

[0074] [Alginic acid derivative represented by formula (II)] The following formula (II):

Chemical formula

Table 3-1

Table 3-2

[0075] [1-1-1] In the formula (I) of the above aspect [1], -L 1 - is preferably the following table:

Table 4-1

Table 4-2

[0076] [1-1-2]In formula (I) of the above aspect [1], -L 1 -is more preferably a linker selected from the group consisting of the partial structural formulas described in the following table:

Table 5-1

Table 5-2

[0077] [1-1-3]In formula (I) of the above aspect [1], -L 1 -is even more preferably a linker selected from the group consisting of the partial structural formulas described in the following table:

Table 6-1

Table 6-2

[0078] [1-2-1] In the formula (I) of the above aspect [1], Akn is preferably the following table:

Table 7

[0079] [1-2-2] In the formula (I) of the above aspect [1], Akn is more preferably the following table:

Table 8

[0080] [1-2-3] In the formula (I) of the above aspect [1], Akn is even more preferably the following table:

Table 9

[0081] [1-3-1] In the formula (II) of the above aspect [1], -L 2 - is preferably a linker selected from the group consisting of the partial structural formulas described in the following table:

Table 10

[0082] [1-3-2] In the formula (II) of the above aspect [1], -L 2 - is more preferably the following table:

Table 11

[0083] [1-3-3] In the formula (II) of the above aspect [1], -L 2 - is even more preferably the following table:

Table 12

[0084] [1-4-1] In the formula (I) of the above aspect [1], Akn and -L 1 - combinations preferably have the formulae in the table below: [Table 13] As shown by the partial structure selected from the group of Akn, -L 1 The formulas of -L are as described in the above embodiment [1]. 2 When an alginic acid derivative represented by formula (II) having a linker selected from the group consisting of (L2-10), (L2-10-p1), (L2-10-p2) and (L2-10-X) is used, -L in the above table is 1 Of these, the linkers (L1-1), (L1-2a), (L1-2b), (L1-11) and (L1-12) are excluded from the preferred embodiments.

[0085] [1-4-2] In the formula (I) of the above aspect [1], Akn and -L 1 More preferably, the combination of formulae in the following table: [Table 14] As shown by the partial structure selected from the group of Akn, -L 1 The formulas of -L are as described in the above embodiment [1-1] (provided that, when the crosslinking reaction is carried out, -L 2- When using an alginate derivative represented by formula (II) having a linker selected from any one of the group consisting of (L2-10), (L2-10-p1), (L2-10-p2), or (L2-10-X), -L in the table 1 - Among them, the linkers of (L1-1-1), (L1-2a-1), (L1-2b-1), (L1-11-1), and (L1-12-p1) are excluded from more preferred embodiments).

[0086] [1-4-3] In formula (I) of the above aspect [1], the combination of Akn and -L 1 - is more preferably the formula in the following table:

Table 15

[0087] [1-4-4] In formula (I) of the above aspect [1], the combination of Akn and -L 2 - is particularly preferably the following partial structural formula:

Chemical formula

[0088] [1-Ia] The first-Ia embodiment is as follows. Akn-L 1 -NH2 group (Akn, and -L 1 - is the same as defined in the above embodiment [1]) The introduction rate is about 0.1% to about 30%, and the alginic acid derivative represented by the formula (I) described in the above embodiment [1].

[0089] [1-Ia-1] In the above embodiment [1-Ia], Akn-L 1 The introduction rate of the -NH2 group is preferably about 1.0% to about 20%; more preferably, it is about 2.0 to 10%.

[0090] [1-Ib] The first-Ib embodiment is as follows. The weight average molecular weight measured by the gel filtration chromatography method of the alginic acid derivative is about 100,000 Da to about 3,000,000 Da, and the alginic acid derivative represented by the formula (I) described in the above embodiment [1].

[0091] [1-Ib-1] In the above embodiment [1-Ib], the weight average molecular weight measured by the gel filtration chromatography method of the alginic acid derivative is preferably about 300,000 Da to about 2,500,000 Da, and more preferably about 500,000 Da to about 1,000,000 Da.

[0092] [1-Ic] The first-Ic embodiment is as follows. N3-L 2 -NH2 group (-L 2 - is the same as defined in the above embodiment [1]) The introduction rate is about 0.1% to about 30%, and the alginic acid derivative represented by the formula (II) described in the above embodiment [1].

[0093] [1-Ic-1] In the above embodiment [1-Ic], N3-L 2 The introduction rate of the -NH2 group is preferably about 1.0% to about 20%; more preferably, it is about 2.0 to 10%.

[0094] [1-Id]Aspect [1-Id] is as follows. An alginic acid derivative represented by formula (II) described in the above aspect [1], wherein the weight-average molecular weight measured by gel filtration chromatography of the alginic acid derivative is from about 100,000 Da to about 3,000,000 Da.

[0095] [1-Id-1]In the above aspect [1-Id], the weight-average molecular weight of the alginic acid derivative of formula (II) measured by gel filtration chromatography is preferably from about 300,000 Da to about 2,500,000 Da, more preferably from about 500,000 Da to about 1,000,000 Da.

[0096] The preferred aspect of the above aspect [1], further Akn, -L 1 - and -L 2 By appropriately combining the definitions of -, the preferred aspect of the crosslinked alginic acid of the above aspect [1] can be arbitrarily formed.

[0097] [2]The second aspect is as follows. A cyclic alkyne group (Akn) is introduced into any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker (-L 1 -), the following formula (I):

Chemical formula

Table 16-1

Table 16-2

Table 17

[0098] [2-1] In the formula (I) of the above aspect [2], -L 1 - is preferably the following table:

Table 18-1

Table 18-2

Table 19-1

Table 19-2

Table 20-1

Table 20-2

[0099] [2-2] In the formula (I) of the above aspect [2], Akn is preferably the following table:

Table 21

Table 22

Table 23

[0100] [2-3] In the formula (I) of the above aspect [2], Akn and -L 1 - the combination of is preferably the following table:

Table 24

Table 25

Table 26

Chemical formula

[0101] In a preferred embodiment of the above aspect [2], more preferably Akn, and -L 1 By appropriately combining the definitions of -, a preferred embodiment of the alginic acid derivative represented by the formula (I) in the above aspect [2] can be arbitrarily formed.

[0102] [3] The third aspect is as follows. Akn-L 1 -NH2 group (Akn, and -L 1 - is the same as the definition described in the above aspect [2]), and the introduction rate of the alginic acid derivative of the formula (I) described in the above aspect [2] is about 0.1% to about 30%.

[0103] [3-1] In the above aspect [3], for Akn-L 1 The introduction rate of the -NH2 group is preferably about 1.0% to about 20%; more preferably about 2.0 to 10%.

[0104] [4] The fourth aspect is as follows. The alginic acid derivative of the formula (I) described in the above aspect [2], wherein the weight average molecular weight measured by the gel filtration chromatography method of the alginic acid derivative is about 100,000 Da to about 3,000,000 Da.

[0105] [4-1] In the above aspect [4], the weight average molecular weight measured by the gel filtration chromatography method of the alginic acid derivative is preferably about 300,000 Da to about 2,500,000 Da, and more preferably about 500,000 Da to about 1,000,000 Da.

[0106] [5] The fifth aspect is as follows. The following formula (II) in which an azide group is introduced via an amide bond and a divalent linker (-L 2 -) to any one or more carboxyl groups of alginic acid: [Chemical formula] [In formula (II), (ALG) represents alginic acid; -NHCO- represents an amide bond through any carboxyl group of alginic acid; -L 2 - represents the following table:

Table 27-1

Table 27-2

[0107] [5-1] In the alginic acid derivative of formula (II) of the above aspect [5], -L 2 - is preferably the following table:

Table 28

Table 29

Table 30

[0108] [5a] The 5a aspect is as follows. An azide group is introduced through an amide bond and a divalent linker (-L 2 -) to any one or more carboxyl groups of alginic acid, and the following formula (II):

Chemical formula

[0109] [5a-1] In the alginic acid derivative of formula (II) of the above aspect [5a], -L 2 - is preferably a linker selected from the group consisting of the partial structural formulas described in the following table: [Table 32] - (excluding the outside of the dashed lines at both ends in each formula); More preferably, it is a linker selected from the group consisting of the partial structural formulas described in the following table: [Table 33] - (excluding the outside of the dashed lines at both ends in each formula); Even more preferably, it is a linker selected from the group consisting of the partial structural formulas described in the following table: [Table 34] - (excluding the outside of the dashed lines at both ends in each formula).

[0110] [6] The sixth aspect is as follows. N3-L 2 -NH2 group (-L 2 - is the same as defined in the above aspect [5]) of the alginic acid derivative of formula (II) described in the above aspect [5] or aspect [5a], with an introduction rate of about 0.1% to about 30%.

[0111] [6-1] In the above aspect [6], N3-L 2 The introduction rate of the -NH2 group is preferably about 1.0% to about 20%; more preferably, about 2.0 to 10%.

[0112] [7] The seventh aspect is as follows. The alginic acid derivative of formula (II) described in the above aspect [5] or aspect [5a], wherein the weight average molecular weight measured by gel filtration chromatography of the alginic acid derivative is about 100,000 Da to about 3,000,000 Da.

[0113] [7-1] In the above aspect [7], the weight average molecular weight measured by gel filtration chromatography of the alginic acid derivative of formula (II) is preferably about 300,000 Da to about 2,500,000 Da, more preferably about 500,000 Da to about 1,000,000 Da.

[0114] [8] The eighth aspect is as follows. Any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are bonded via the following formula (III-L): [Chemical formula] [In formula (III-L), -CONH- and -NHCO- at both ends represent amide bonds via any carboxyl group of alginic acid; -L 1 - is the same as the definition in the above aspect [1]; -L 2 - is the same as the definition in the above aspect [1]; X is a cyclic group selected from the group of partial structural formulas described in the following table: [Table 35-1] [Table 35-2] and is bonded via (the outer side of the broken lines at both ends is not included) and is the cross-linked alginic acid described in the above aspect [1]. (However, in formula (III-L), -L 1- When it is any one linker selected from the group of (L1-1), (L1-2a), (L1-2b), (L1-11) or (L1-12), the corresponding -L 2 - in which the linker of (L2-10) is excluded).

[0115] [8a] The 8a-th aspect is as follows. Any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (III-L): [Chemical formula] [In formula (III-L), the -CONH- and -NHCO- at both ends represent amide bonds through any carboxyl group of alginic acid; -L 1 - is the same as the definition in the above aspect [1]; -L 2 - is the same as the definition in the above aspect [1]; X is the same as the definition in the above aspect [8]] Crosslinked alginic acid bonded through. (However, in formula (III-L), when -L 1 - is any one linker selected from the group of (L1-1), (L1-2a), (L1-2b), (L1-11) or (L1-12), the corresponding -L 2 - in which the linker of (L2-10) is excluded).

[0116] [8-1-1] In formula (III-L) of the above aspect [8] or aspect [8a], preferably, -L 1 - is the same as the linker selected from the group of formulas representing -L 1 - described in the above aspect [1-1-1].

[0117] [8-1-2] In formula (III-L) of the above aspect [8] or aspect [8a], more preferably, -L 1 - is the same as the linker selected from the group of formulas representing -L 1 - described in the above aspect [1-1-2].

[0118] [8-1-3] In formula (III-L) of the above aspect [8] or [8a], more preferred -L 1 - is the -L described in the above aspect [1-1-3] 1 - is the same as the linker selected from the group consisting of formulas representing -L

[0119] [8-2-1] In formula (III-L) of the above aspect [8] or aspect [8a], preferred -L 2 - is the -L described in the above aspect [1-2-1] 2 - is the same as the linker selected from the group consisting of formulas representing -L

[0120] [8-2-2] In formula (III-L) of the above aspect [8] or aspect [8a], more preferred -L 2 - is the -L described in the above aspect [1-2-2] 2 - is the same as the linker selected from the group consisting of formulas representing -L

[0121] [8-2-3] In formula (III-L) of the above aspect [8] or aspect [8a], further preferred -L 2 - is the -L described in the above aspect [1-2-3] 2 - is the same as the linker selected from the group consisting of formulas representing -L

[0122] [8-3-1] In formula (III-L) of the above aspect [8] or aspect [8a], X is preferably a cyclic group selected from the group consisting of the partial structural formulas (TZ-1), (TZ-2), (TZ-3), (TZ-4), (TZ-5), (TZ-6), (TZ-10), (TZ-1-r), (TZ-2-r), (TZ-3-r), (TZ-4-r), (TZ-5-r), (TZ-6-r), and (TZ-10-r) described in the above aspect [8].

[0123] [8-3-2] In the formula (III-L) of the said aspect [8] or aspect [8a], X is more preferably a cyclic group selected from the group consisting of partial structural formula (TZ-2), formula (TZ-3), formula (TZ-6), formula (TZ-10), formula (TZ-2-r), formula (TZ-3-r), formula (TZ-6-r), and formula (TZ-10-r).

[0124] [8-3-3] In the formula (III-L) of the said aspect [8] or aspect [8a], X is even more preferably a cyclic group selected from the group consisting of partial structural formula (TZ-2), formula (TZ-6), formula (TZ-2-r), and formula (TZ-6-r).

[0125] [8-4-1] In the formula (III-L) of the said aspect [8] or aspect [8a], preferably, -L 2 -X-L 1 - combination is as shown by the partial structures selected from the group of the following formulas:

Table 36-1

Table 36-2

[0126] [8-4-2] In the formula (III-L) of the said aspect [8] or aspect [8a], more preferably, -L 2 -X-L 1 - combination is as shown by the partial structures selected from the group of the following formulas:

Table 37-1

Table 37-2

[0127] [8-4-3] In the formula (III-L) of the above aspect [8] or aspect [8a], more preferably, the combination of -L 2 -X-L 1 - is the formula in the following table:

Table 38

[0128] [8-4-4] In the formula (III-L) of the above aspect [8] or aspect [8a], particularly preferably, the combination of -L 2 -X-L 1 - is the following partial structural formula [wherein, the outside of the broken lines at both ends is not included]:

Chemical formula

[0129] [8-5-1] In the crosslinked alginic acid described in the above aspect [1] or aspect [8a], the crosslinking is a chemical crosslinking by a triazole ring formed by a Huisgen reaction and an ionic crosslinking partially formed by a divalent metal ion.

[0130] [8-5-2] In the crosslinked alginic acid described in the above aspect [1] or aspect [8a], the crosslinking is a chemical crosslinking by a triazole ring formed by a Huisgen reaction.

[0131] [8-5-3] In the above aspect [8-5-1], the divalent metal ion is preferably a divalent metal ion selected from the group consisting of calcium ion, magnesium ion, barium ion, strontium ion or zinc ion; more preferably, calcium ion or barium ion; still more preferably, calcium ion.

[0132] [8-5-4] In the above aspect [8-5-1], the divalent metal ion used for forming the ionic crosslinking can preferably be supplied from an aqueous solution selected from the group consisting of an aqueous calcium chloride solution, an aqueous calcium carbonate solution, an aqueous calcium gluconate solution, and an aqueous barium chloride solution; more preferably, an aqueous calcium chloride solution or an aqueous barium chloride solution; still more preferably, an aqueous calcium chloride solution.

[0133] Preferred embodiment of aspect [8], further -L 1 -, -L 2 -, and by appropriately combining the definitions of X, a preferred embodiment of the crosslinked alginic acid of the above aspect [8] can be arbitrarily formed. Preferred embodiment of aspect [8a], further -L 1 -, -L 2 -, and by appropriately combining the definitions of X, a preferred embodiment of the crosslinked alginic acid of the above aspect [8a] can be arbitrarily formed.

[0134] [9] The ninth aspect is as follows. A method for producing crosslinked alginic acid, comprising obtaining the crosslinked alginic acid according to the aspect [1] or aspect [8a] by mixing the alginic acid derivative represented by the formula (I) described in the aspect [1] and the alginic acid derivative represented by the formula (II) and performing a crosslinking reaction (Huisgen reaction).

[0135] [9-1] The ninth - first aspect is as follows. A method for producing crosslinked alginic acid, comprising obtaining the crosslinked alginic acid according to the aspect [1] or aspect [8a] by adding a solution of the alginic acid derivative represented by the formula (I) described in the aspect [1] to a solution of the alginic acid derivative represented by the formula (II) described in the aspect [1] and performing a crosslinking reaction (Huisgen reaction).

[0136] [9-2] The ninth - second aspect is as follows. A method for producing crosslinked alginic acid, comprising obtaining the crosslinked alginic acid according to the aspect [1] or aspect [8a] by adding a solution of the alginic acid derivative represented by the formula (II) described in the aspect [1] to a solution of the alginic acid derivative represented by the formula (I) described in the aspect [1] and performing a crosslinking reaction (Huisgen reaction).

[0137]

[10] The chemical crosslinking formed by performing the Huisgen reaction (crosslinking reaction) using the alginic acid derivative represented by the formula (I) and the alginic acid derivative represented by the formula (II) described in the aspect [1] is of the following formula (III-L):

Chemical formula

[0138]

[11] The 11th aspect is as follows. A crosslinked alginate structure obtained by dropping a mixed solution of an alginate derivative represented by the formula (I) described in [1] and an alginate derivative represented by the formula (II) into a solution containing a divalent metal ion.

[0139] [11-1] The 11-1st aspect is as follows. A crosslinked alginate structure obtained by adding a gel obtained by dropping a solution of an alginate derivative represented by the formula (I) described in any one of [1] into a solution containing a divalent metal ion to a solution of an alginate derivative represented by the formula (II) described in [1] and performing a crosslinking reaction.

[0140] [11-2] The 11-2nd aspect is as follows. A crosslinked alginate structure obtained by adding a gel obtained by dropping a solution of an alginate derivative represented by the formula (II) described in [1] into a solution containing a divalent metal ion to a solution of an alginate derivative represented by the formula (I) described in [1] and performing a crosslinking reaction.

[0141] [12-1] The 12-1st aspect is as follows. A crosslinked alginate structure according to any one of the aspects

[11] to [11-2], including chemical crosslinking by a triazole ring formed by a Huisgen reaction as crosslinking and ionic crosslinking partially formed by a divalent metal ion.

[0142] [12-2] The 12-2nd aspect is as follows. A crosslinked alginate structure according to any one of the aspects

[11] to [11-2], including chemical crosslinking by a triazole ring formed by a Huisgen reaction as crosslinking.

[0143] [12-3-1] In the aspect [12-1], the divalent metal ion is preferably a divalent metal ion selected from the group consisting of calcium ion, magnesium ion, barium ion, strontium ion or zinc ion; more preferably, calcium ion or barium ion; still more preferably, calcium ion.

[0144] [12-3-2] In the aspect [12-1], the divalent metal ion used for forming an ionic crosslink is preferably an aqueous solution selected from the group consisting of an aqueous calcium chloride solution, an aqueous calcium carbonate solution, an aqueous calcium gluconate solution, and an aqueous barium chloride solution; more preferably, an aqueous calcium chloride solution or an aqueous barium chloride solution; still more preferably, an aqueous calcium chloride solution.

[0145]

[13] The 13th aspect is as follows. A crosslinked alginate structure having a retention property of the content, obtained by chemically crosslinking the alginate derivative represented by the formula (I) and the alginate derivative represented by the formula (II) described in the aspect [1] by ionic crosslinking with a divalent metal ion and / or Huisgen reaction.

[0146] [13-1-1] In the aspect

[13] , the divalent metal ion is preferably a divalent metal ion selected from the group consisting of calcium ion, magnesium ion, barium ion, strontium ion, or zinc ion; more preferably, calcium ion or barium ion; still more preferably, calcium ion.

[0147] [13-1-2] In the aspect

[13] , the divalent metal ion used for forming an ionic crosslink is preferably an aqueous solution selected from the group consisting of an aqueous calcium chloride solution, an aqueous calcium carbonate solution, an aqueous calcium gluconate solution, and an aqueous barium chloride solution; more preferably, an aqueous calcium chloride solution or an aqueous barium chloride solution; still more preferably, an aqueous calcium chloride solution.

[0148]

[14] The 14th aspect is as follows. The chemical crosslink formed by performing the Huisgen reaction using the alginate derivative represented by the formula (I) and the alginate derivative represented by the formula (II) described in the aspect [1] is the following formula (III-L): [Chemical formula] [In formula (III-L), the -CONH- and -NHCO- at both ends, X, -L 1 -, and -L 2 - are the same as the definitions in the above aspect [8], and the crosslinked alginate structure according to any one of the above aspects

[11] to

[13] having the structure of].

[0149]

[15] The 15th aspect is as follows. A method for producing a crosslinked alginate structure, which is obtained by dropping a mixed solution of an alginate derivative represented by formula (I) and an alginate derivative represented by formula (II) described in the above aspect [1] into a solution containing a divalent metal ion and performing a crosslinking reaction.

[0150] [15-1] The 15-1st aspect is as follows. A method for producing a crosslinked alginate structure, which is obtained by adding a gel obtained by dropping a solution of an alginate derivative represented by formula (I) described in the above aspect [1] into a solution containing a divalent metal ion to a solution of an alginate derivative represented by formula (II) described in the above aspect [1] and performing a crosslinking reaction.

[0151] [15-2] The 15-2nd aspect is as follows. A method for producing a crosslinked alginate structure, which is obtained by adding a gel obtained by dropping a solution of an alginate derivative represented by formula (II) described in the above aspect [1] into a solution containing a divalent metal ion to a solution of an alginate derivative represented by formula (I) described in the above aspect [1] and performing a crosslinking reaction.

[0152] [16-1] The 16-1st aspect is as follows. A method for producing a crosslinked alginate structure according to any one of the above aspects

[15] to [15-2], which includes chemical crosslinking by a triazole ring formed by a Huisgen reaction as crosslinking and ionic crosslinking partially formed by a divalent metal ion.

[0153] [16-2]Aspect 16-2 is as follows. A method for producing the crosslinked alginic acid structure according to any one of aspects

[15] to [15-2], which includes chemical crosslinking by a triazole ring formed by a Huisgen reaction as the crosslinking.

[0154] [16-3-1]In the aspect [16-1], the divalent metal ion is preferably a divalent metal ion selected from the group consisting of calcium ion, magnesium ion, barium ion, strontium ion, or zinc ion; more preferably, calcium ion or barium ion; still more preferably, calcium ion.

[0155] [16-3-2]In the aspect [16-1], the divalent metal ion used for forming the ionic crosslinking can preferably use an aqueous solution selected from the group consisting of an aqueous calcium chloride solution, an aqueous calcium carbonate solution, an aqueous calcium gluconate solution, and an aqueous barium chloride solution as a supply source; more preferably, an aqueous calcium chloride solution or an aqueous barium chloride solution; still more preferably, an aqueous calcium chloride solution.

[0156]

[17] Aspect 17 is as follows. By using the alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to aspect [1], the chemical crosslinking formed by performing a Huisgen reaction is the following formula (III-L): [Chemical formula] [In formula (III-L), the -CONH- and -NHCO- at both ends, X, -L 1 -, and -L 2 - are the same as the definitions in aspect [8]] having the structure of, a method for producing the crosslinked alginic acid structure according to any one of aspects

[15] to [16-2].

[0157]

[18] Aspect 18 is as follows. The crosslinked alginic acid structure according to any one of aspects

[11] to

[14] , which is beads or a substantially spherical gel.

[0158]

[19] The 19th aspect is as follows. A medical material containing the crosslinked alginic acid structure according to any one of aspects

[11] to

[14] .

[0159]

[20] The 20th aspect is as follows. The medical material according to aspect

[19] , which is beads or a substantially spherical gel.

[0160]

[21] The 21st aspect is as follows. The crosslinked alginic acid according to aspect [1] or aspect [8a], the alginic acid derivative according to aspect [2] or aspect [5], or the crosslinked alginic acid structure according to any one of aspects

[11] to

[14] , which has biocompatibility.

[0161]

[22] The 22nd aspect is as follows. The following formula (AM-1): [Chemical formula] [In formula (AM-1), -L 1 -, and the definition of Akn are the same as those described in aspect [2]] An amino compound represented by, or a pharmaceutically acceptable salt thereof, or a solvate thereof. However, the following table: [Table 39] Excluding the amino compound or a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0162] [22-1] The compound represented by formula (AM-1) of aspect

[22] is preferably an amino compound in which -L 1 - is the same as the preferred -L 1 - defined in aspect [2-1], and Akn is the same as the preferred definition of Akn described in aspect [2-2], or a pharmaceutically acceptable salt thereof, or a solvate thereof. However, the following table: [Table 40] Excluding the amino compound or a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0163] [22-2] The compound represented by formula (AM-1) of the above aspect

[22] is more preferably -L 1 - is the more preferred -L described in the above aspect [2-1] 1 - has the same definition as the more preferred -L described in the above aspect [2-2], and Akn has the same definition as the more preferred Akn described in the above aspect [2-2], and is an amino compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof. However, the following table:

Table 41

[0164] [22-3] The compound represented by formula (AM-1) of the above aspect

[22] is even more preferably -L 1 - is the even more preferred -L described in the above aspect [2-1] 1 - has the same definition as the even more preferred -L described in the above aspect [2-2], and Akn has the same definition as the even more preferred Akn described in the above aspect [2-2], and is an amino compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof. However, the following table:

Table 42

[0165] [22-4] The compound represented by the above formula (AM-1) of the above aspect

[22] is particularly preferably the following formula:

Chemical formula

[0166]

[23] The 23rd aspect is as follows. The following formula (AM-2):

Chemical formula

Table 43

[0167] [23-1] The compound represented by formula (AM-2) of the above aspect

[23] is preferably -L 2 - is the same as the definitions of (L2-2a), (L2-2b-A), (L2-3), (L2-4-p1), (L2-5a-p1), and (L2-5b-p1) described in the above aspect [5-1], an amino compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof. However, the following table:

Table 44

[0168] [23-2] The compound represented by formula (AM-2) of the above aspect

[23] is more preferably -L 2 - is the same as the definitions of (L2-2a-1), (L2-2b-B), (L2-3-1), (L2-4-p2), (L2-5a-p2), and (L2-5b-p2) described in the above aspect [5-1], an amino compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof. However, the following table:

Table 45

[0169] [23-3] The compound represented by the formula (AM-2) of the above aspect

[23] is more preferably the following formula:

Chemical formula

[0170] Hereinafter, each aspect will be described in more detail.

[0171] 1. Alginate In this specification, when referring to alginate, it means at least one alginate (sometimes referred to as "alginate-like substances") selected from the group consisting of alginate, alginate esters, and salts thereof (e.g., sodium alginate). The alginate used may be of natural origin or synthetic, but is preferably of natural origin. The preferably used alginate-like substances are biocompatible polysaccharides extracted from brown algae such as Lessonia, Macrocystis, Laminaria, Ascophyllum, Darbilla, Kadjika, Arame, and Kombu, and are polymers in which two types of uronic acids, D-mannuronic acid (M) and L-guluronic acid (G), are polymerized linearly. More specifically, it is a block copolymer in which a homopolymer fraction of D-mannuronic acid (MM fraction), a homopolymer fraction of L-guluronic acid (GG fraction), and a fraction in which D-mannuronic acid and L-guluronic acid are randomly arranged (M / G fraction) are arbitrarily combined.

[0172] Alginic acid is a kind of natural polysaccharide extracted and purified from brown algae. It is a polymer formed by the polymerization of D-mannuronic acid (M) and L-guluronic acid (G). The composition ratio of D-mannuronic acid and L-guluronic acid in alginic acid (M / G ratio), that is, the gel strength, varies mainly depending on the type of organism such as seaweed from which it is derived, and is also affected by the growth location and season of the organism. The M / G ratio ranges widely from a high G type with an M / G ratio of about 0.2 to a high M type with an M / G ratio of about 5. The physicochemical properties of alginic acid vary depending on the M / G ratio, the way of arranging M and G, etc., and there may be different preferred uses. The gelation ability of alginic acids and the properties of the generated gels are affected by the M / G ratio. Generally, it is known that when the G ratio is high, the gel strength increases. The M / G ratio also affects other properties such as the hardness, brittleness, water absorbency, and flexibility of the gel. Therefore, the alginic acid used in the present invention is preferably one with an appropriate M / G ratio and appropriate viscosity according to its final use.

[0173] Industrial production methods of alginic acid include the acid method and the calcium method, etc. In the present invention, those produced by any production method can be used. By purification, those with a quantitative value by HPLC method in the range of 80 to 120% by mass are preferred, those in the range of 90 to 110% by mass are more preferred, and those in the range of 95 to 105% by mass are even more preferred. In the present invention, those with a quantitative value by HPLC method within the above range are referred to as high-purity alginic acid. The alginic acid or its salt used in the present invention is preferably high-purity alginic acid. As commercially available products, for example, as the Kimica Algin series, those sold by Kimica Corporation, preferably those of high-purity food and pharmaceutical grades can be purchased and used. It is also possible to further purify the commercially available products as appropriate and then use them. For example, it is preferable to perform low endotoxin treatment. As the purification method and the low endotoxin treatment method, for example, the methods described in JP-A-2007-75425 can be adopted.

[0174] As the salt of "alginic acid" used in the present invention, it is a "monovalent metal salt of alginic acid", which is a salt formed by ion-exchanging the hydrogen ions of the carboxylic acid of D-mannuronic acid or L-guluronic acid of alginic acid with monovalent metal ions such as Na+ and K+. Specific examples of the monovalent metal salt of alginic acid include sodium alginate and potassium alginate, and among them, sodium alginate is particularly preferred.

[0175] In this specification, alginic acid may be represented as (ALG), and when one of any carboxyl groups of alginic acid is represented as -COOH, it may be denoted as (ALG)-COOH.

[0176] For the alginic acid used in the present invention, it is advisable to use one with an appropriate weight-average molecular weight according to its final intended use. For example, it is preferable to use one with a weight-average molecular weight of 10,000 to 10 million, more preferably 100,000 or more and 5 million or less, and even more preferably 150,000 or more and 3 million or less.

[0177] In some embodiments, the alginic acid is sodium alginate. As the sodium alginate, commercially available sodium alginate can be used. Here, in the examples described later, the sodium alginate used is sodium alginate of A-1, A-2, A-3, B-1, B-2, and B-3 (manufactured by Motoda Pharmaceutical Co., Ltd.) described in the following table. The viscosities, weight-average molecular weights, and M / G ratios of 1 w / w% aqueous solutions of each sodium alginate are shown in the following table.

[0178]

Table 46

[0179] The physical property values of the above-mentioned sodium alginate A-1, A-2, A-3, B-1, B-2, and B-3 were measured by the following various methods. The measurement methods are not limited to these methods, but the physical property values may differ from the above depending on the measurement methods.

[0180] [Measurement of the Viscosity of Sodium Alginate] The measurement was carried out using a rotational viscometer method (cone and plate type rotational viscometer) in accordance with the viscosity measurement method of the Japanese Pharmacopoeia (16th Edition). The specific measurement conditions are as follows. The sample solution was prepared using MilliQ water. The measuring instrument used was a cone and plate type rotational viscometer (viscosity and viscoelasticity measuring device Rheostress RS600 (Thermo Haake GmbH) sensor: 35 / 1). The rotation speed was set to 1 rpm when measuring the 1 w / w% sodium alginate solution. The reading time was measured for 2 minutes, and the average value from 1 minute to 2 minutes after the start was taken. The average value of three measurements was used as the measured value. The measurement temperature was set to 20 °C.

[0181] [Measurement of the Weight-Average Molecular Weight of Sodium Alginate] (1) Gel permeation chromatography (GPC) and (2) two measurement methods of GPC-MALS were used for the measurement. The measurement conditions are as follows.

[0182] [Pretreatment Method] After adding the eluent to the sample and dissolving it, the solution filtered through a 0.45 μm membrane filter was used as the measurement solution.

[0183] (1) Gel Permeation Chromatography (GPC) Measurement [Measurement Conditions (Relative Molecular Weight Distribution Measurement)] Column: TSKgel GMPW-XL × 2 + G2500PW-XL (7.8 mm I.D. × 300 mm × 3 columns) Eluent: 200 mM aqueous sodium nitrate solution Flow rate: 1.0 mL / min Concentration: 0.05% Detector: RI detector Column temperature: 40 °C Injection volume: 200 μL Molecular weight standard: Standard pullulan, glucose

[0184] (2) GPC-MALS Measurement [Measurement of Refractive Index Increment (dn / dc) (Measurement Conditions)] Differential refractometer: Optilab T-rEX Measurement wavelength: 658 nm Measurement temperature: 40 °C Solvent: 200 mM aqueous sodium nitrate solution Sample concentration: 0.5 - 2.5 mg / mL (5 concentrations)

[0185] [Measurement conditions (absolute molecular weight distribution measurement)] Column: TSKgel GMPW-XL × 2 + G2500PW-XL (7.8 mm I.D. × 300 mm × 3 columns) Eluent: 200 mM aqueous sodium nitrate solution Flow rate: 1.0 mL / min Concentration: 0.05% Detector: RI detector, light scattering detector (MALS) Column temperature: 40 °C Injection volume: 200 μL

[0186] In this specification, when indicating the molecular weight of alginic acid, alginic acid derivatives, crosslinked alginic acid, and crosslinked alginic acid, Da (dalton) may be appended as the unit.

[0187] The composition ratio (M / G ratio) of D-mannuronic acid and L-guluronic acid in alginic acids varies mainly depending on the type of organism from which they are derived, such as seaweed, and is also affected by the growth location and season of that organism. The M / G ratio ranges widely from a high G type with an M / G ratio of about 0.2 to a high M type with an M / G ratio of about 5. The gelation ability of alginic acids and the properties of the gels formed are affected by the M / G ratio. Generally, it is known that when the G ratio is high, the gel strength is high. The M / G ratio also affects other properties such as the hardness, brittleness, water absorbency, and flexibility of the gel. The M / G ratio of the alginic acids and / or their salts used is usually 0.1 - 4.0, in certain embodiments 0.1 - 3.0, in certain embodiments 0.1 - 2.0, in certain embodiments 0.5 - 1.8, in certain embodiments 0.8 - 1.2. In another embodiment, it is 0.1 - 0.5.

[0188] Also, the alginic acid used in the present invention preferably has an appropriate viscosity and an appropriate M / G ratio according to its final intended use.

[0189] In this specification, the numerical range indicated by "~" indicates a range that includes the numerical values described before and after "~" as the minimum value and the maximum value, respectively.

[0190] In this specification, the "alginate ester" and "alginate" used, although not particularly limited, need to have no functional group that inhibits the cross-linking reaction in order to react with the cross-linking agent. Preferred examples of the alginate ester include propylene glycol alginate, etc.

[0191] In this specification, examples of the alginate include monovalent salts of alginic acid and divalent salts of alginic acid. Preferred examples of the monovalent salt of alginic acid include sodium alginate, potassium alginate, ammonium alginate, etc., more preferably sodium alginate or potassium alginate, and particularly preferably sodium alginate. Preferred examples of the divalent salt of alginic acid include calcium alginate, magnesium alginate, barium alginate, strontium alginate, etc.

[0192] Alginic acid is a high molecular polysaccharide, and although it is difficult to accurately determine the molecular weight, generally the weight average molecular weight is in the range of 1000 to 10 million, preferably 10,000 to 8 million, and more preferably 20,000 to 3 million. In the measurement of the molecular weight of natural product-derived high molecular substances, it is known that the values may vary depending on the measurement method.

[0193] When specifying the molecular weight of the alginate derivative, alginic acid or its salt of the present invention in this specification, unless otherwise specified, it is the weight average molecular weight calculated by size exclusion chromatography (SEC). Also for the alginic acid or its salt used in the present invention, it is desirable to use one with an appropriate molecular weight distribution according to its final use.

[0194] For example, under the measurement conditions of gel permeation chromatography (GPC) or gel filtration chromatography (collectively also referred to as size exclusion chromatography (SEC)) described in the following examples, it is preferably from 100,000 to 5,000,000, more preferably from 150,000 to 3,000,000. In another aspect, it is in the range of 500,000 to 3,000,000, more preferably from 1,000,000 to 2,500,000, and even more preferably in the range of 1,000,000 to 2,000,000.

[0195] Also, for example, according to the GPC-MALS (SEC-MALS) method, the absolute weight average molecular weight can be measured. 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, even more preferably 60,000 or more, and also preferably 1,000,000 or less, more preferably 800,000 or less, even more preferably 700,000 or less, and particularly preferably 500,000 or less. The preferred range is from 10,000 to 1,000,000, more preferably from 50,000 to 800,000, and even more preferably from 60,000 to 500,000.

[0196] Generally, when calculating the molecular weight of a high molecular polysaccharide by a method using SEC or SEC-MALS as described above, a measurement error of about 10% to about 30% may occur. For example, if it is 500,000, the value may vary in the range of about 350,000 to 650,000, and if it is 1,000,000, the value may vary in the range of about 700,000 to 1,300,000. In this specification, when "about" is described in the description of molecular weight measurement, values up to ±10% of the numerical value, and in some aspects, values up to ±20% of the numerical value may also be included.

[0197] Here, generally, a high molecular substance derived from a natural product is not a substance having a single molecular weight, but an aggregate of molecules having various molecular weights, and thus is measured as a molecular weight distribution having a certain width. A typical measurement method is gel filtration chromatography. Representative information on the molecular weight distribution obtained by gel filtration chromatography includes the weight average molecular weight (Mw), the number average molecular weight (Mn), and the dispersion ratio (Mw / Mn).

[0198] The weight-average molecular weight emphasizes the contribution to the average molecular weight of high-molecular-weight polymers and is represented by the following formula.

[0199] Mw = Σ(WiMi) / W = Σ(HiMi) / Σ(Hi) The number-average molecular weight is calculated by dividing the total weight of the polymer by the total number of polymers.

[0200] Mn = W / ΣNi = Σ(MiNi) / ΣNi = Σ(Hi) / Σ(Hi / Mi) Here, W is the total weight of the polymer, Wi is the weight of the i-th polymer, Mi is the molecular weight at the elution time of the i-th polymer, Ni is the number of molecules with molecular weight Mi, and Hi is the height at the elution time of the i-th polymer.

[0201] In the measurement of the molecular weight of natural product-derived polymer substances, it is known that the values may vary depending on the measurement method (example of hyaluronic acid: Chikako YOMOTA et.al. Bull.Natl.Health Sci., Vol.117, pp135-139(1999), Chikako YOMOTA et.al. Bull.Natl.Inst. Health Sci., Vol.121, pp30-33(2003)). Regarding the measurement of the molecular weight of alginic acid, there are documents describing methods calculated from intrinsic viscosity and methods calculated by SEC-MALLS (Size Exclusion Chromatography with Multiple Angle Laser Light Scattering Detection) (ASTM F2064-00(2006), published by ASTM International). In the present invention, the weight-average molecular weight can be a value measured by a conventional method as shown in the above documents, for example, by measuring the molecular weight by size exclusion chromatography (SEC) and calculating it using a calibration curve with pullulan as a standard substance. Also, in the present invention, the weight-average molecular weight can be the absolute molecular weight measured by a conventional method as shown in the above documents, for example, by SEC-MALS.

[0202] The molecular weight of alginic acids can be measured according to a conventional method.

[0203] When specifying the molecular weight of alginic acid or its salt in this specification, unless otherwise specified, it is the weight-average molecular weight calculated by gel filtration chromatography. Representative conditions when using gel filtration chromatography for molecular weight measurement can, for example, adopt the conditions of the present examples described below. As the column, for example, a Superose6 Increase 10 / 300 GL column (GE Healthcare Sciences) can be used. As the developing solvent, for example, a 10 mmol / L phosphate buffer solution (pH 7.4) containing 0.15 mol / L NaCl can be used. As the molecular weight standards, blue dextran, thyroglobulin, ferritin, aldolase, conalbumin, ovalbumin, ribonuclease A, and aprotinin can be used.

[0204] The viscosity of alginic acid used in this specification is not particularly limited. However, when measuring the viscosity as an aqueous solution of 1 w / w% alginic acids, it is preferably 10 mPa·s to 1000 mPa·s, more preferably 50 mPa·s to 800 mPa·s.

[0205] The viscosity of an aqueous solution of alginic acid can be measured according to a conventional method. For example, it can be measured using a coaxial double cylindrical rotational viscometer, a single cylindrical rotational viscometer (Brookfield type viscometer), a conical - plate rotational viscometer (cone - plate type viscometer), etc. of the rotational viscometer method. Preferably, it is desirable to follow the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). More preferably, a cone - plate type viscometer is used.

[0206] When initially extracted from brown algae, alginates have a large molecular weight and a high viscosity. However, during processes such as drying and purification by heat, the molecular weight decreases and the viscosity becomes lower. Alginates with different molecular weights can be produced by methods such as controlling the conditions such as temperature in the manufacturing process, selecting the brown algae used as the raw material, and fractionating the molecular weight in the manufacturing process. Furthermore, it is also possible to obtain alginates with the desired molecular weight by mixing alginates from different lots having different molecular weights or viscosities.

[0207] As used herein, alginic acid is, in some embodiments, alginic acid that has not been treated with low endotoxin, or in some other embodiments, alginic acid that has been treated with low endotoxin. Low endotoxin means that the endotoxin level is low to the extent that it does not substantially cause inflammation or fever. More preferably, it is desirable to use alginates that have been treated with low endotoxin.

[0208] The endotoxin reduction treatment can be carried out by a known method or a method similar thereto. For example, the purification of sodium hyaluronate can be carried out by the method of Suga et al. (see, for example, Japanese Patent Application Laid-Open No. 9-324001, etc.), the purification of β1,3-glucan by Yoshida et al. (see, for example, Japanese Patent Application Laid-Open No. 8-269102, etc.), the purification of biopolymer salts such as alginate and gellan gum by Williams et al. (see, for example, Japanese Translation of PCT International Publication No. 2002-530440, etc.), the purification of polysaccharides by James et al. (see, for example, the pamphlet of International Publication No. 93 / 13136, etc.), the method of Lewis et al. (see, for example, the specification of U.S. Patent No. 5,589,591, etc.), the purification of alginate by Herman Frank et al. (see, for example, Appl Microbiol Biotechnol (1994) 40:638-643, etc.), etc., or methods similar thereto. The low endotoxin treatment is not limited to these, but may be performed by known methods such as washing, filtration with a filter (endotoxin removal filter, charged filter, etc.), ultrafiltration, purification using a column (endotoxin adsorption affinity column, gel filtration column, column with ion exchange resin, etc.), adsorption to hydrophobic substances, resins or activated carbon, organic solvent treatment (extraction with organic solvent, precipitation / sedimentation by adding organic solvent, etc.), surfactant treatment (see, for example, JP 2005-036036 A, etc.), or by appropriately combining these. These treatment steps may be appropriately combined with known methods such as centrifugation. It is desirable to select an appropriate method according to the type of alginic acid.

[0209] The endotoxin level can be confirmed by known methods, and can be measured, for example, by a method using Limulus reagent (LAL) or a method using Endospecy (registered trademark) ES-24S set (Seikagaku Corporation).

[0210] The method for treating the endotoxin used is not particularly limited, but as a result, the endotoxin content of the alginic acids is preferably 500 endotoxin units (EU) / g or less, more preferably 100 EU / g or less, particularly preferably 50 EU / g or less, and most preferably 30 EU / g or less when endotoxin measurement is performed using the Limulus reagent (LAL). In the present invention, "substantially endotoxin-free" means that the endotoxin value measured by the Japanese Pharmacopoeia endotoxin test is within the above numerical range. Sodium alginate treated with low endotoxin can be obtained, for example, from commercially available products such as Sea Matrix (registered trademark) (Mochida Pharmaceutical Co., Ltd.), PRONOVA TM UP LVG (FMCBioPolymer).

[0211] 2. Alginate derivatives In the present specification, novel alginate derivatives are provided. In the present specification, as the alginate derivative, a reactive group in the Huisgen reaction or a complementary reactive group of the reactive group is introduced into any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker. More specifically, the following formula (I):

Chemical formula

Chemical formula

[0212] The above divalent linker (-L 1 - or -L 2-) can use any linear group as long as it does not inhibit the reaction between the reactive group and the reactive group complementary to the reactive group. Specifically, for example, a linear alkylene group (-(CH2) n -, n = 1 to 30) (in the group, -CH2- can be replaced by a plurality of (for example, 1 to 10, or 1 to 5) groups such as -C(=O)-, -CONH-, -O-, -NH-, -S-, benzene ring, heterocyclic ring (5- to 6-membered aromatic heterocyclic ring or 5- to 6-membered non-aromatic heterocyclic ring such as pyridine ring, piperidine ring, piperazine ring, etc.), and the hydrogen atom of -CH2- can be an oxo group (=O), C 1-6 alkyl group (for example, groups such as methyl group, ethyl group, n-propyl group, iso-propyl group, etc.), halogen atom (for example, fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), hydroxyl group (-OH), etc., and can be substituted by a plurality of (for example, 1 to 10, or 1 to 5) groups selected from such groups), but is not limited thereto.

[0213] It is possible to substitute the hydrogen atom of the imino group (-NH-) in the -NH-CO- group of the alginic acid derivative represented by the formula (I) or formula (II) with a methyl group to form a -N(Me)-CO- group. In the alginic acid derivative represented by the formula (I) or formula (II), the bonding mode of the linker (-L 1 -, -L 2 -) and alginic acid is a -NH-CO- bond or -N(Me)-CO-; preferably, it is a -NH-CO- bond.

[0214] The alginic acid derivatives represented by the formula (I) and formula (II), which are novel alginic acid derivatives in this specification, can be produced, for example, by the method of the following formula (for details, refer to the general production method described later).

[0215]

Chemical formula

[0216] The weight-average molecular weight of the alginic acid derivative represented by formula (I) or formula (II) in this specification is from 100,000 Da to 3,000,000 Da, preferably from 300,000 Da to 2,500,000 Da, more preferably from 500,000 Da to 2,000,000 Da. The molecular weight of both of these alginic acid derivatives can be determined by the method described below.

[0217] In this specification, Akn-L in formula (I) 1 The -NH- group does not necessarily bind to all of the carboxyl groups of the alginic acid constituent unit, and N3-L in formula (II) 2 The -NH- group does not necessarily bind to all of the carboxyl groups of the alginic acid constituent unit.

[0218] In this specification, when the -NH- group in Akn-L of formula (I) 1 is referred to as a reactive group, the -NH- group in N3-L of formula (II) 2 becomes a complementary reactive group. Conversely, when the -NH- group in N3-L of formula (II) 2 is referred to as a reactive group, the -NH- group in Akn-L of formula (I) 1 becomes a complementary reactive group.

[0219] In this specification, the introduction rate of the reactive group or the complementary reactive group is 0.1% to 30% or 1% to 30% respectively, preferably 2% to 20%, more preferably 3% to 10%.

[0220] The introduction rate of the reactive group or the complementary reactive group is a value expressed as a percentage of the number of uronic acid monosaccharide units into which each reactive group has been introduced among the uronic acid monosaccharide units that are the repeating units of alginic acids. In this specification, unless otherwise specified, the % used for the introduction rate of the reactive group or the complementary reactive group in the alginic acid derivative (formula (I) or formula (II)) means mol%. The introduction rate of each reactive group or the complementary reactive group can be determined by the method described in the examples below.

[0221] In this specification, the cyclic alkyne group (Akn) in formula (I) and the azide group in formula (II) form a triazole ring by the Huisgen reaction, thereby forming a crosslink.

[0222] 3. Huisgen Reaction The Huisgen reaction (1,3-dipolar cycloaddition reaction) is a condensation reaction between compounds having a terminal azide group and a terminal alkyne group as shown in the following formula. As a result of the reaction, a disubstituted 1,2,3-triazole ring is obtained in good yield, and it has the characteristic that no extra by-products are formed. Although it is considered that a 1,4- or 1,5-disubstituted triazole ring can be formed in this reaction, it is possible to obtain a triazole ring regioselectively by using a copper catalyst.

[0223]

Chemical formula

[0224] In addition, a Huisgen reaction without using a copper catalyst has been reported by Wittig and Krebs. That is, it is a reaction in which a cycloaddition adduct can be obtained simply by mixing cyclooctyne and phenyl azide (in the following formula, R 3 = phenyl). In this reaction, since the triple bond of cyclooctyne is highly strained, the driving force is the relief of strain by the reaction with phenyl azide, and the reaction proceeds spontaneously, making the catalyst unnecessary.

[0225]

Chemical formula

[0226] As described above, for the Huisgen reaction, azide compounds having substituted primary azides, secondary azides, tertiary azides, aromatic azides, etc., and compounds having a terminal or cyclic alkyne group which is a complementary reactive group to the azide group can be used. Also, in the Huisgen reaction, since almost only the azide group and the alkyne group react, it is possible to substitute various functional groups (for example, ester group, carboxyl group, alkenyl group, hydroxyl group, amino group, etc.) in the reaction substrate.

[0227] In some embodiments, in order to form crosslinks between alginate molecules through a 1,2,3-triazole ring without generating undesirable by-products and avoiding cytotoxicity caused by copper catalysts, and to achieve this easily and efficiently in a short time without using a copper catalyst, as the alkyne group in the Huisgen reaction, for example, the cyclic alkyne group (cyclooctyl group) described in the above aspect [1] is used.

[0228] In the method for crosslinking an alginate derivative in a preferred embodiment, almost no undesirable by-products are formed in the reaction (Huisgen reaction). In this case, in the preparation of a novel form of biocompatible material using alginate and the formation of an alginate hydrogel, it is possible to incorporate various bioactive molecules, and in the alginate hydrogel for reconstructive surgery or gene therapy, it is possible to incorporate cell substances.

[0229] 4. Crosslinked Alginate Crosslinked alginate includes those via (i) divalent metal ion bonds, (ii) chemical bonds, or (iii) both divalent metal ion bonds and chemical bonds. Any of the crosslinked alginates has the property of forming a gel-like to semi-solid, and in some cases, a sponge-like form.

[0230] The crosslinked alginate via divalent metal ion bonds reacts at an ultra-high speed and is reversible, while the crosslinked alginate via chemical bonds reacts slowly under relatively mild conditions and is irreversible. The physical properties of the crosslinked alginate can be adjusted, for example, by changing the concentration of an aqueous solution containing the divalent metal ion used (e.g., calcium chloride aqueous solution), or the introduction rate of the reactive group introduced into the alginate.

[0231] By utilizing the above crosslinking reaction, it becomes possible to create various alginate structures. For example, through an ionic crosslinking reaction, a specific structure can be instantaneously formed from an alginate solution, and for the purpose of strengthening the structure of the said structure (for example, obtaining long-term stability, etc.), it is possible to utilize a crosslinking reaction by chemical bonding. Also, for example, in a crosslinked alginate structure via both divalent metal ion bonding and chemical bonding, the divalent metal ions incorporated by ionic crosslinking can be reversibly released to create a structure in which only crosslinking by chemical bonding remains.

[0232] Crosslinked alginate in one embodiment can be obtained by mixing the alginate derivatives of the above formula (I) and the above formula (II) and performing the Huisgen reaction.

[0233] Crosslinked alginate in one embodiment forms a three-dimensional network structure through chemical crosslinking (crosslinking by a triazole ring formed from an alkyne group and an azide group). Preferred alginate derivatives are those with improved stability of the crosslinked alginate after crosslinking.

[0234] In some embodiments of crosslinked alginate, an amide bond is formed between any carboxyl group of the first alginate and any carboxyl group of the second alginate via the following formula (III-L):

Chemical formula

[0235] In some embodiments, when preparing crosslinked alginate, the mixing ratio of the alginate derivative of formula (I) and the alginate derivative of formula (II) is, in terms of 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.

[0236] In some embodiments, when preparing crosslinked alginic acid, the mixing ratio of the alginic acid derivative of formula (II) to the alginic acid derivative of formula (I) is, in terms of 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.

[0237] In some embodiments, when preparing crosslinked alginic acid, the mixing ratio of the alginic acid derivative of formula (I) to the alginic acid derivative of formula (II) is more preferably in terms of the ratio of the introduction rate (mol%) of the reactive groups of the alginic acid derivative of formula (I) to the alginic acid 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.

[0238] In some embodiments, when preparing crosslinked alginic acid, the mixing ratio of the alginic acid derivative of formula (II) to the alginic acid derivative of formula (I) is more preferably in terms of the ratio of the introduction rate (mol%) of the reactive groups of the alginic acid derivative of formula (II) to the alginic acid 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.

[0239] It should be noted that in the above mixing ratio, it is also possible to replace the alginic acid derivative of formula (I) with the alginic acid derivative of formula (II), and the alginic acid derivative of formula (II) with the derivative of formula (I), respectively.

[0240] For crosslinked alginic acid, it is not necessary that all carboxyl groups of the structural units of alginic acid have the crosslinking of the above formula (III-L). The introduction rate (also referred to as the crosslinking rate) of the crosslinking represented by the above formula (III-L) in crosslinked alginic acid is, for example, in the range of about 0.1 to about 80%, about 0.3 to about 60%, about 0.5 to about 30%, or about 1.0 to about 10%.

[0241] The concentration of the alginic acid derivative of formula (I) or formula (II) in the Huisgen reaction for obtaining crosslinked alginic acid is usually about 1 to about 500 mg / mL, preferably in the range of about 5 to about 100 mg / mL.

[0242] The reaction temperature of the Huisgen reaction is usually an external temperature of about 4 to about 60 °C, preferably in the range of an external temperature of about 15 to about 40 °C.

[0243] The stirring time for forming crosslinked alginic acid (hydrogel) is, for example, several seconds to about 24 hours, several seconds to about 12 hours, several seconds to about 30 minutes, or several seconds to about 10 minutes.

[0244] The reaction solvent or reaction solution used in the Huisgen reaction is not particularly limited, and examples thereof include tap water, pure water (e.g., distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water, cell culture medium, phosphate buffered saline (PBS), and physiological saline, etc., and preferably ultrapure water.

[0245] Crosslinked alginic acid in some embodiments is crosslinked alginic acid comprising chemical crosslinking by a triazole ring formed by the Huisgen reaction as crosslinking and ionic crosslinking partially formed by divalent metal ions.

[0246] 5. Crosslinked alginic acid structure The crosslinked alginic acid structure can be obtained by a method including subjecting the alginic acid derivative of formula (I) and the alginic acid derivative of formula (II) to a crosslinking reaction.

[0247] In this specification, "performing a crosslinking reaction" or "conducting a crosslinking reaction" means that by performing a Huisgen reaction using the alginic acid derivative of the formula (I) and the alginic acid derivative of the formula (II), a chemical crosslink (chemical bond) is formed between the alginic acid derivative of the formula (I) and the alginic acid derivative of the formula (II), or by coexisting a divalent metal ion with the alginic acid derivative of the formula (I) and the alginic acid derivative of the formula (II), an ionic crosslink (ionic bond) is formed between the respective derivatives of the alginic acid derivative of the formula (I) and / or the alginic acid derivative of the formula (II), or both a chemical crosslink by the Huisgen reaction and an ionic crosslink by a divalent metal ion are formed.

[0248] The crosslinked alginic acid structure can be prepared, for example, by the following methods, but is not limited thereto.

[0249] [Mixing method] By dropping a mixed solution of the alginic acid derivative obtained by mixing the alginic acid derivative of the formula (I) and the alginic acid derivative of the formula (II) into a solution containing a divalent metal ion, a crosslinked alginic acid structure, which is a specific structure in which a chemical crosslink (crosslinking by a triazole ring formed from an alkyne group and an azide group by the Huisgen reaction) and an ionic crosslink (crosslinking partially formed by a divalent metal ion) are formed, can be obtained.

[0250] [Coating method] A specific structure that is partially crosslinked can be obtained by, for example, dropping a solution containing the alginic acid derivative of the formula (I) into a solution containing a divalent metal ion. By adding the structure obtained above, such as a gel, to a solution containing the alginic acid derivative of the formula (II) and performing a further crosslinking reaction (Huisgen reaction) on the surface of the structure or the like, a crosslinked alginic acid structure can be obtained. Incidentally, this method can also be carried out by replacing the alginic acid derivative of the formula (I) with the alginic acid derivative of the formula (II) and the alginic acid derivative of the formula (II) with the alginic acid derivative of the formula (I), respectively.

[0251] The divalent metal ions used in the above method are not particularly limited, and examples thereof include divalent metal ions selected from the group consisting of calcium ions, magnesium ions, barium ions, strontium ions, zinc ions, etc.; preferably, calcium ions or barium ions; more preferably, calcium ions.

[0252] The solution containing divalent metal ions used in the above method is not particularly limited, and examples thereof include aqueous solutions selected from the group consisting of aqueous calcium chloride solution, aqueous calcium carbonate solution, aqueous calcium gluconate solution, or aqueous barium chloride solution, etc.; preferably, aqueous calcium chloride solution or aqueous barium chloride solution; more preferably, aqueous calcium chloride solution.

[0253] The divalent metal ion concentration of the solution containing divalent metal ions used in the above method is not particularly limited, and examples thereof include about 1 mM to about 1 M; preferably, about 5 mM to about 500 mM; more preferably, about 10 mM to about 300 mM.

[0254] The solvent or solution used in the above method is also not particularly limited, and examples thereof include tap water, pure water (e.g., distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water, cell culture medium, phosphate buffered saline (PBS), and physiological saline, etc.; preferably, ultrapure water.

[0255] Examples of specific crosslinked alginate structures include fibrous structures, fibers, beads, gels, substantially spherical gels, etc. Preferred crosslinked alginate structures have improved stability. Also, the crosslinked alginate structure may have the ability to retain contents inside (content retention).

[0256] The physical properties of the alginate gel can be adjusted by physical property values such as hardness, elasticity, resilience, breaking force, stress at break, etc.

[0257] 6. Biocompatibility of Alginate Derivatives and Crosslinked Alginate Structures In the present specification, an alginic acid derivative, crosslinked alginic acid or crosslinked alginic acid structure has biocompatibility. In the present specification, biocompatibility refers to the property of not causing reactions such as the interaction between a biomaterial (here, an alginic acid derivative represented by formula (I) or formula (II), and a crosslinked alginic acid or crosslinked alginic acid structure produced using both alginic acid derivatives) and a living body, the local reaction of tissues adjacent to the biomaterial, or a systemic reaction, and is said to have biocompatibility.

[0258] In the present specification, regarding the biocompatibility of an alginic acid derivative, crosslinked alginic acid or crosslinked alginic acid structure, it is confirmed in the examples regarding biocompatibility described later.

[0259] 7. Stability of Crosslinked Alginic Acid Structure The stability of the crosslinked alginic acid structure can be confirmed, for example, by measuring the gel stability and measuring the gel permeability.

[0260] [Method for Measuring Gel Stability] Phosphate buffered saline (PBS) is added to the crosslinked alginic acid structure gel placed in a container, and the concentration (μg / mL) of alginic acid eluted in PBS is measured. The amount of eluted alginic acid is calculated from the measured alginic acid concentration, and the value obtained by dividing by the total amount of alginic acid calculated from the total alginic acid concentration obtained by decomposing the crosslinked alginic acid structure gel and expressed as a percentage is defined as the disintegration rate. Specifically, the gel stability can be determined by the method described in the examples below.

[0261] In the present specification, the gel disintegration rate of the crosslinked alginic acid structure is preferably 0% to about 90%, more preferably 0% to about 70%, and still more preferably 0% to about 50%. The stability of the crosslinked alginic acid structure means that the lower the concentration of alginic acid leaking into the aqueous solution, that is, the lower the gel disintegration rate, the higher the stability.

[0262] [Method for Measuring Gel Permeability] A crosslinked alginate structure gel encapsulating fluorescein isothiocyanate-dextran is prepared, physiological saline is added to the gel placed in a container, and the concentration of dextran leaked into the physiological saline is measured. The percentage value obtained by dividing the amount of dextran calculated from the measured concentration of dextran by the total amount of dextran calculated from the total dextran concentration obtained by decomposing the fluorescein isothiocyanate-dextran encapsulated crosslinked alginate structure gel is the gel permeability. Specifically, the gel permeability can be determined by the method described in the examples below.

[0263] The gel permeability of crosslinked alginate 24 hours after adding physiological saline is, for example, preferably 0% to about 90%, more preferably 0% to about 70%, and even more preferably 0% to about 50% when encapsulating dextran with a molecular weight of about 2 million. Also, when encapsulating dextran with a molecular weight of about 150,000, for example, if the purpose of using the crosslinked alginate structure gel is the release / production of proteins or antibodies, it is preferably about 1% to about 100%, more preferably about 10% to about 100%, and even more preferably about 30% to about 100%. Also, if the purpose of use is an immunological barrier, it is preferably 0% to about 90%, more preferably 0% to about 70%, and even more preferably 0% to about 50%.

[0264] The permeability of the crosslinked alginate structure means that the lower the permeability, the lower the permeability of the content and gel external substances, and the higher the permeability, the higher the permeability of the content and gel external substances.

[0265] The permeability of the gel can be adjusted by the molecular weight, concentration of the alginate used, the type and introduction rate of the reactive groups introduced into the alginate, the type and concentration of the divalent metal ions used for gelation, or a combination thereof.

[0266] [Method for preparing a crosslinked alginate structure gel encapsulating a content] For example, a crosslinked alginate structure gel encapsulating fluorescein isothiocyanate-dextran as a content can be prepared by the following method.

[0267] (1) Mix the solution of the alginic acid derivative represented by formula (I) with the fluorescein isothiocyanate-dextran solution. (2) Mix the solution of the alginic acid derivative represented by formula (II) with the mixed solution obtained in (1). (When formula (I) in (1) is changed to formula (II), formula (II) in (2) will be changed to formula (I)) (3) Drop the mixed solution obtained in (2) into a solution containing calcium ions, and the gel obtained will form chemical cross-links and ionic cross-links in the solution, thereby obtaining a cross-linked alginic acid structure gel encapsulating fluorescein isothiocyanate-dextran.

[0268] In the description herein, when "about" is described, unless otherwise specified, values within ±20% of the numerical value, preferably within ±10% of the numerical value, may also be included.

[0269] 8. Method for synthesizing alginic acid derivative In this specification, the alginic acid derivative represented by formula (I) or formula (II) is each an amine derivative (AM-1) represented by H2N-L 1 -Akn (wherein L 1 and Akn are the same as defined in the above aspect [1]), or an amine derivative (AM-2) represented by H2N-L 2 -N3 (wherein L 2 is the same as defined in the above aspect [4]), and can be produced by a condensation reaction using a condensing agent between any carboxyl group of the alginic acids.

[0270] [Production method of the alginic acid derivative of formula (I)]

[0271] [Production method of the alginic acid derivative of formula (I)] Using an aqueous solution of alginic acid at 0.5% to 1% by weight and an amine represented by the formula (AM-1), according to a method known in the literature, for example, the method described in "Experimental Chemistry Course, 5th Edition, 16, Synthesis of Organic Compounds IV, Carboxylic Acids and Derivatives, Esters, p35 - 70, Acid Amides and Acid Imides, p118 - 154, Amino Acids and Peptides, p258 - 283, 2007, Maruzen", etc., in the presence of a condensing agent 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 - oxazolidinyl)phosphinic chloride (BOP - Cl), 2 - chloro - 1,3 - dimethylimidazolinium hexafluorophosphate (CIP), or 4 - (4,6 - dimethoxy - 1,3,5 - triazin - 2 - yl) - 4 - methylmorpholinium chloride (DMT - MM), etc., in a mixed solvent of a solvent selected from ether solvents such as tetrahydrofuran and 1,4 - dioxane, alcohol solvents such as methanol, ethanol, 2 - propanol, etc., and polar solvents such as N,N - dimethylformamide and water, to such an extent that alginic acid does not precipitate, in the presence or absence of an inorganic base such as sodium hydrogen carbonate and sodium carbonate, or an organic base such as triethylamine and pyridine, a condensation reaction is carried out at a temperature between 0°C and 50°C to produce an alginic acid derivative of the formula (I).

[0272] [Process for Producing the Alginic Acid Derivative of the Formula (II)] Using an aqueous solution of alginic acid at 0.5% to 1% by weight and an amine represented by the formula (AM - 2), by carrying out the reaction according to the above - mentioned [Process for Producing the Alginic Acid Derivative of the Formula (I)], an alginic acid derivative of the formula (II) can be produced.

[0273] In the method for producing the alginic acid derivative of the formula (I) or the alginic acid derivative of the formula (II) described above, the introduction rate of the amine of the formula (AM-1) or the formula (AM-2) can be adjusted by appropriately selecting and combining the reaction conditions such as the following (i) to (v) in consideration of the properties of the amine and the like. (i) Increase or decrease the equivalent amount of the condensing agent, (ii) Raise or lower the reaction temperature, (iii) Lengthen or shorten the reaction time, (iv) Adjust the concentration of the alginic acid as the reaction substrate, (v) Add an organic solvent miscible with water to increase the solubility of the amine of the formula (AM-1) or the formula (AM-2), etc.

[0274] The following shows a more specific method for producing the amine represented by the formula (AM-1) or the formula (AM-2).

[0275] In addition, in each of the following production methods, the definitions of m1, n1, m2a, n2a, p2a, m2b, n2b, p2b, m3, n3, p3, m4a, n4a, m4b, n4b, m5a, n5a, p5a, q5a, m5b, n5b, p5b, q5b, m6a, n6a, p6a, m6b, n6b, p6b, m7, n7, m8a, n8a, m8b, n8b, m9a, n9a, p9a, m9b, n9b, p9b, m10, n10, p10, m11, m12, n12, p12, x1, x2, x2a, y2a, x2b, y2b, x3, y3, x4, y4, z4, x5a, y5a, x5b, y5b, x6a, y6a, z6a, v6a, x6b, y6b, z6b, v6b, x7a, y7a, z7a, x7b, y7b, z7b, x8a, y8a, z8a, x8b, y8b, z8b, x9a, y9a, z9a, x9b, y9b, z9b, x10 and y10 are the same as the definitions described in the above aspect [1]; P 1 is an amino protecting group selected from -C(O)O-tertBu group, -C(O)O-Bn group, -C(O)CH3 group, -C(O)CF3 group, -SO2Ph, -SO2PhMe group, -SO2Ph(NO2) group, etc.; E is a leaving group such as a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), -OTs group, -OMs group, etc.

[0276] Also, in each of the following production methods, the protecting group P 1The protection and deprotection can be carried out according to the methods known from the literature, for example, the deprotection methods described in the book "Protective Groups in Organic Synthesis 4th Edition, 2007, John Wiley & Sons, Greene et al.".

[0277] [Production Method AM-A] Method for producing an amine represented by formula (AM-1-B1): [Chemical formula] Using the compound of formula (SM-B1) and the compound of formula (RG-B1) [the compound of formula (SM-B1) and the compound of formula (RG-B1) are commercially available compounds or compounds that can be produced from commercially available compounds by methods known from the literature], a condensation reaction similar to the above [production method of the alginic acid derivative of formula (I)] is carried out, and then the protecting group P 1 is deprotected to produce an amine compound represented by formula (AM-1-B1) or a salt thereof.

[0278] [Production Method AM-B] Method for producing amines represented by formula (AM-1-B2a) and formula (AM-1-B2b): [Chemical formula]

[0279] Using the compound of formula (SM-B2a) and the compound of formula (RG-B2a) [the compound of formula (SM-B2a) and the compound of formula (RG-B2b) are commercially available compounds or compounds that can be prepared from commercially available compounds by production methods known from the literature], in the same manner as [Production Method AM-A], by reacting, an amine compound represented by formula (AM-1-B2a) or a salt thereof can be produced. Similarly, by reacting in the same manner using the compound of formula (SM-B2b) and the compound of formula (RG-B2b) [the compound of formula (SM-B2b) and the compound of formula (RG-B2b) are commercially available compounds or compounds that can be prepared from commercially available compounds by production methods known from the literature], an amine compound represented by formula (AM-1-B2b) or a salt thereof can be produced.

[0280] [Production Method AM-C] Method for producing an amine represented by formula (AM-1-B3):

Chemical Formula

[0281] Using the compound of formula (SM-B3) [the compound of formula (SM-B3) is a commercially available compound or a compound that can be prepared from a commercially available compound by a production method known from the literature], by carrying out the reaction according to the above synthetic scheme (the reaction in each step is in accordance with the reaction described in [Production Method AM-A]), an amine compound represented by formula (AM-1-B3) or a salt thereof can be produced. In the above scheme, the compounds of formula (RG-B3), formula (RG-B3-1), and formula (RG-B3-2) are commercially available compounds or compounds that can be prepared from commercially available compounds by production methods known from the literature.

[0282] [Production Method AM-D] Method for producing amines represented by formula (AM-1-B5a) and formula (AM-1-B5b):

Chemical Formula

[0283] By carrying out the reaction according to the above synthetic scheme (the reactions in each step are in accordance with the reactions described in [Production Method AM-A]), an amine compound represented by formula (AM-1-B5a) and formula (AM-1-B5b), or a salt thereof can be produced. In the above scheme, the compounds of formula (SM-B5a), formula (RG-B5a), formula (RG-B5a-1), formula (RG-B5a-2), formula (SM-B5b), formula (RG-B5b), formula (RG-B5b-1), and formula (RG-B5b-2) are commercially available compounds or compounds that can be produced from commercially available compounds by production methods known from the literature.

[0284] [Production Method AM-E] Production method of the amine represented by formula (AM-1-B6a) and formula (AM-1-B6b):

Chemical formula

[0285] By carrying out the reaction according to the above synthetic scheme (the reactions in each step are in accordance with the reactions described in [Production Method AM-A]), an amine compound represented by formula (AM-1-B6a) and formula (AM-1-B6b), or a salt thereof can be produced. In the above scheme, the compounds of formula (SM-B6a), formula (RG-B6a), formula (SM-B6b), and formula (RG-B6b) are commercially available compounds or compounds that can be produced from commercially available compounds by production methods known from the literature.

[0286] [Production Method AM-F] Production method of the amine represented by formula (AM-1-B10):

Chemical formula

[0287] By carrying out the reaction according to the above synthetic scheme (the reactions in each step are in accordance with the reactions described in [Production Method AM-A]), an amine compound represented by formula (AM-1-B10), or a salt thereof can be produced. In the above scheme, the compounds of formula (SM-B10), formula (RG-B10), formula (RG-B10-1), and formula (RG-B10-2) are commercially available compounds or compounds that can be produced from commercially available compounds by production methods known from the literature.

[0288] [Production method AM-G] Method for producing amines represented by formula (AM-1-B4a) and formula (AM-1-B4b):

Chemical formula

[0289] Using the compound of formula (SM-B4) and the compound of formula (RG-B4a) [the compound of formula (SM-B4) and the compound of formula (RG-B4a) are commercially available compounds or compounds that can be produced from commercially available compounds by methods known in the literature], according to a method known in the literature, for example, the method described in 'Journal of the American Chemical Society, 126(46), p15046-15047, 2004', etc., <Step 1a> In the presence of reagents such as silver trifluoromethanesulfonate and AgClO4, react in a solvent that does not participate in the reaction, such as toluene and dichloromethane, to obtain a compound of formula (IM-B4a-1), <Step 2a> Subsequently, perform a dehalogenation reaction using a base such as sodium hydride and NaOMe to obtain a compound of formula (IM-B4a-2), <Step 3a> Further, by removing the protecting group P 1 An amine compound represented by formula (AM-1-B4a) or a salt thereof can be produced. Similarly, by using formula (RG-B4b) instead of formula (RG-B4a) and performing the reaction according to the above scheme, an amine compound represented by formula (AM-1-B4b) or a salt thereof can be produced.

[0290] [Production method AM-H] Method for producing amines represented by formula (AM-1-B8a) and formula (AM-1-B8b):

Chemical formula

[0291] By carrying out the reaction according to the above synthesis scheme (the reactions in each step are in accordance with the reactions described in [Production Method AM-A]), an amine compound represented by formula (AM-1-B8a) or formula (AM-1-B8b), or a salt thereof can be produced. In the above scheme, formula (SM-B8a) or formula (SM-B8b) is a commercially available compound or a compound that can be produced in accordance with the reactions described in [Production Method AM-G], and the compound of formula (RG-B8a) or formula (RG-B8b) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known in the literature.

[0292] [Production Method AM-J] Method for producing an amine represented by formula (AM-1-B7):

Chemical formula

[0293] Using the compound of formula (SM-B7) and the compound of formula (RG-B7) [the compound of formula (SM-B7) and the compound of formula (RG-B7) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known in the literature], a condensation reaction similar to the above [Production method of the alginic acid derivative of formula (I)] is carried out, and then the protecting group P 1 is deprotected to produce an amine compound represented by formula (AM-1-B7), or a salt thereof.

[0294] [Production Method AM-J-2] Method for producing an amine represented by formula (AM-1-B7):

Chemical formula

[0295] Using the compound of formula (SM-B7) and the compound of formula (RG-B7-2) [the compound of formula (SM-B7) and the compound of formula (RG-B7-2) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known in the literature], a condensation reaction similar to the above [Production method of the alginic acid derivative of formula (I)] is carried out (<Step 1> and <Step 2>), and then the protecting group P 1Deprotect, and then, using a compound of formula (RG-B7-3) (which is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known from the literature), perform a condensation reaction similar to the above <Step 1>, and then deprotect the protecting group P 1 to produce an amine compound represented by formula (AM-1-B7), or a salt thereof.

[0296] [Production Method AM-K] Method for producing amines represented by formula (AM-1-B9a) and formula (AM-1-B9b):

Chemical Formula

[0297] By carrying out the reaction according to the above synthetic scheme (the reactions in each step are in accordance with the reactions described in [Production Method AM-J]), an amine compound represented by formula (AM-1-B9a) and formula (AM-1-B9b), or a salt thereof can be produced. In the above scheme, the compound of formula (SM-B7), formula (RG-B9a) or formula (RG-B9b) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known from the literature.

[0298] [Production Method AM-L] Method for producing an amine represented by formula (AM-2-Z1):

Chemical Formula

[0299] Using a compound of formula (SM-Z1) [the compound of formula (SM-Z1) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known from the literature], according to a method known from the literature, for example, the method described in 'Organometallics, 29(23), p6619 - 6622; 2010', etc., react NaN3 in a solvent that does not participate in the reaction, such as dimethyl sulfoxide, to introduce an azide group, and then deprotect the protecting group P 1 to produce an amine compound represented by formula (AM-2-Z1), or a salt thereof. Some amine compounds represented by formula (AM-2-Z1) or their salts are also available as commercially available compounds.

[0300] [Production method AM-M] Production method of amines represented by formula (AM-2-Z2a) and formula (AM-2-Z2b):

Chemical formula

[0301] Using a compound of formula (SM-Z2a) or a compound of formula (SM-Z2b) [the compound of formula (SM-Z2a) and the compound of formula (SM-Z2b) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known from the literature], in the same manner as [Production method AM-L], after reacting with NaN3 to introduce an azide group, the protecting group P 1 is deprotected to produce an amine compound represented by formula (AM-2-Z2a) or formula (AM-2-Z2b), or a salt thereof. Some amine compounds represented by formula (AM-2-Z2a) or formula (AM-2-Z2b), or their salts are also available as commercially available compounds.

[0302] [Production method AM-N] Production method of amines represented by formula (AM-2-Z3):

Chemical formula

[0303] Using a compound of formula (SM-Z3) and a compound of formula (RG-Z3) [the compound of formula (SM-Z3) and the compound of formula (RG-Z3) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known from the literature], a condensation reaction similar to [Production method of the alginic acid derivative of formula (I)] is carried out, and then the protecting group P 1 is deprotected to produce an amine compound represented by formula (AM-2-Z3), or a salt thereof.

[0304] [Production method AM-O] Production method of amines represented by formula (AM-2-Z4): [Chemical formula]

[0305] Using the compound of formula (SM-Z4) and the compound of formula (RG-Z4) [the compound of formula (SM-Z4) and the compound of formula (RG-Z4) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known from the literature], a condensation reaction similar to the above [production method of the alginic acid derivative of formula (I)] is carried out, and then the protecting group P 1 is deprotected to produce an amine compound represented by formula (AM-2-Z4), or a salt thereof.

[0306] [Production method AM-P] Production method of amines represented by formula (AM-2-Z5a) and formula (AM-2-Z5b): [Chemical formula]

[0307] Using the compound of formula (SM-Z5a) and the compound of formula (RG-Z5a) [the compound of formula (SM-Z5a) and the compound of formula (RG-Z5a) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known from the literature], in the presence of a base such as sodium hydride or potassium carbonate, in a solvent that does not participate in the reaction such as tetrahydrofuran, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, etc., a reaction is carried out to obtain a compound with a side chain introduced. Subsequently, the protecting group P 1 is deprotected to produce an amine compound represented by formula (AM-2-Z5a), or a salt thereof. Similarly, by using the compound of formula (SM-Z5b) and the compound of formula (RG-Z5b) [the compound of formula (SM-Z5b) and the compound of formula (RG-Z5b) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known from the literature] and carrying out the reaction in the same manner, an amine compound represented by formula (AM-2-Z5b), or a salt thereof, can be produced.

[0308] The amino compounds represented by formula (AM-2-Z6a), formula (AM-2-Z6b), formula (AM-2-Z7a), formula (AM-2-Z7b), formula (AM-2-Z8a), formula (AM-2-Z8b), formula (AM-2-Z9a), and formula (AM-2-Z9b), or salts thereof, can be produced by the production methods shown in the following scheme according to the above [Production Method AM-A] to [Production Method AM-P].

[0309]

Chemical formula

[0310]

Chemical formula

[0311]

Chemical formula

[0312]

Chemical formula

[0313] [Production Method AM-Q] Production method of the amine represented by formula (AM-2-Z10):

Chemical formula

[0314] <Process 1> Compounds of formula (SM-Q) [The compounds of formula (SM-Q) are commercially available compounds or compounds that can be prepared from commercially available compounds by methods known in the literature] and compounds of formula (RG-Q1) [The compounds of formula (RG-Q1) are commercially available compounds or compounds that can be prepared from commercially available compounds by methods known in the literature] are used. According to a method known in the literature, for example, the method described in "European Journal of Organic Chemistry, 2014(6), p1280-1286; 2014", etc., (i) in the presence of reagents such as PPh3 and N2(CO2CHMe2)2, in a solvent not involved in the reaction such as tetrahydrofuran, the Mitsunobu reaction is carried out. Subsequently, in the presence of a base such as sodium hydroxide, in a solvent not involved in the reaction such as methanol, ethanol, tetrahydrofuran, water or a mixed solvent thereof, hydrolysis of the ester group is carried out to produce a compound represented by formula (IM-Q1).

[0315] <Process 2> [Production method AM-Q] Using the compound of formula (IM-Q1) obtained in <Process 1> and the compound of formula (RG-Q2) [The compound of formula (RG-Q2) is a commercially available compound or a compound that can be prepared from a commercially available compound by a method known in the literature], a condensation reaction similar to the above [production method of the alginic acid derivative of formula (I)] is carried out to obtain a condensate. Subsequently, by removing the protecting group P 1 An amine compound represented by formula (AM-2-Z10), or a salt thereof, can be produced.

[0316] [Production method AM-R] Method for producing an amine represented by formula (AM-1-B10):

Chemical formula

[0317] <Project 1> Using a compound of formula (SM-R) [the compound of formula (SM-R) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known from the literature], according to a method known from the literature, for example, the method described in 'Faming Zhuanli Shenqing, 104529898, 22 Apr 2015', etc., (i) in the presence of a base such as pyridine, in a solvent that does not participate in the reaction such as ethanol, react H2NOH-HCl to form an oxime, and then (ii) react diphosphorus pentoxide in P2O5, methanesulfonic acid to perform a Beckmann rearrangement to form an 8-membered lactam, and then (iii) in a solvent that does not participate in the reaction such as diethyl ether, use a reducing agent such as BH3, LiAlH4 to reduce the amide group to produce a compound represented by formula (IM-R1).

[0318] <Project 2> [Production method AM-R] Using the compound of formula (IM-R1) and the compound of formula (RG-R1) obtained in <Project 1> [the compound of formula (RG-R1) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known from the literature], perform a condensation reaction similar to the above [production method of the alginic acid derivative of formula (I)] to obtain a condensate, then add bromine, and then perform a debromination reaction using tert-BuOK to form an alkyne group, and then 1 By deprotecting the protecting group P, an amine compound represented by formula (AM-1-B10), or a salt thereof, can be produced.

[0319] [Production method AM-S] Method for producing an amine represented by formula (AM-2-Z11):

Chemical formula

[0320] Using the compound of formula (SM-S) and the compound of formula (RG-S1) [the compound of formula (SM-S) and the compound of formula (RG-S1) are commercially available compounds or compounds that can be produced from commercially available compounds by production methods known from the literature], perform a condensation reaction similar to the above [production method of the alginic acid derivative of formula (I)], and then the protecting group P 1By deprotecting, a compound represented by formula (AM-2-Z11) or a salt thereof can be produced.

[0321] [Production method AM-T] Production method of amines represented by formula (AM-1-T1) and formula (AM-1-T2): [Chemical formula]

[0322] <Step 1> Using a compound of formula (SM-T) and a compound of formula (RG-T-1) [the compound of formula (SM-T) and the compound of formula (RG-T-1) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known from the literature], a Grignard reaction is carried out according to a method known from the literature, for example, the method described in WO2004 / 035017 etc., and subsequently an oxidation reaction is carried out to obtain a compound of formula (IM-T-1).

[0323] <Step 2> After protecting the carbonyl group of the compound of formula (IM-T-1) (for example, an acetal group etc.), bromine is added to the cyclooctene ring, and then a de-bromination reaction is carried out using a base such as tert-BuOK, and subsequently the protecting group of the carbonyl group and the protecting group P 1 are deprotected to produce an amine represented by formula (AM-1-T1) or a salt thereof.

[0324] <Step 3> Using an amine of formula (AM-1-T1) or a salt thereof and a compound of formula (RG-T-2) [the compound of formula (RG-T-2) is a commercially available compound or a compound that can be produced from commercially available compounds by a production method known from the literature], a condensation reaction is carried out, and the protecting group P 1 is deprotected to produce an amine represented by formula (AM-1-T2) or a salt thereof.

[0325] [Production method AM-U] Production method of amines represented by formula (AM-1-U1) and formula (AM-1-U2): [Chemical formula]

[0326] In the [Manufacturing Method AM-T], by replacing the compound of formula (SM-T) with the compound of formula (SM-U) [the compound of formula (SM-U) is a commercially available compound or a compound that can be produced from a commercially available compound by a manufacturing method known from the literature], and carrying out the reaction according to the method described in [Manufacturing Method AM-T], an amine represented by formula (AM-1-U1) and formula (AM-1-U2), or a salt thereof is produced.

[0327] By replacing the aldehyde used in <Step 1> of the [Manufacturing Method AM-T] and [Manufacturing Method AM-U] with the aldehyde of the following formula (RG-T-3) or formula (RG-T-4) [the compound of formula (RG-T-3) and the compound of formula (RG-T-4) are commercially available compounds or compounds that can be produced from commercially available compounds by a manufacturing method known from the literature], an amine having a corresponding linker, or a salt thereof is produced.

Chemical formula

Chemical formula

[0328] <Step 1> Using a compound of formula (SM-V) [the compound of formula (SM-V) is a commercially available compound or can be produced by a method known from the literature, for example, the method described in Bioorganic & Medicinal Chemistry, 23(22), p7150 - 7157, 2015, etc.], converting it to an acid chloride according to a conventional method, then carrying out a Grignard reaction using a compound of formula (RG-V-1) [the compound of formula (RG-V-1) is a commercially available compound or a compound that can be produced from a commercially available compound by a manufacturing method known from the literature], and subsequently deprotecting the protecting group P 1 to produce an amine represented by formula (AM-1-V1), or a salt thereof.

[0329] <Process 2> A condensation reaction is carried out using an amine of formula (AM-1-V1) or a salt thereof and a compound of formula (RG-T-1), and the protecting group P 1 is deprotected to produce an amine represented by formula (AM-1-V2), or a salt thereof.

[0330] By replacing the compound used in <Process 1> of the above [Production Method AM-V] with a compound of the following formula [each compound is a commercially available compound or can be produced from a commercially available compound by a production method known from the literature], an amine having a corresponding linker, or a salt thereof, is produced by carrying out the reaction.

Chemical formula

[0331] [Production Method AM-W] Production method of amines represented by formula (AM-1-W1) and formula (AM-1-W2):

Chemical formula

[0332] In the above [Production Method AM-V], the compound of formula (SM-V) is replaced with a compound of formula (SM-W) [the compound of formula (SM-W) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known from the literature], and the reaction is carried out according to the method described in [Production Method AM-V] to produce an amine represented by formula (AM-1-W1) and formula (AM-1-W2), or a salt thereof.

[0333] By replacing the compound used in <Process 1> of the above [Production Method AM-W] with a compound of the following formula [each compound is a commercially available compound or can be produced from a commercially available compound by a production method known from the literature], an amine having a corresponding linker, or a salt thereof, is produced by carrying out the reaction.

Chemical formula

[0334] An alkyne group-introduced amine (Akn-L 1 -NH2) or an azide group-introduced amine (N3-L 2 -NH2) used for producing an alginic acid derivative represented by the formula (I) or the formula (II) can produce a desired amine by appropriately combining each reaction described in the above [Production Method AM-A] to [Production Method AM-P] and the methods known from the literature, for example, the methods described in "Experimental Chemistry Course, 5th Edition, each volume, 2007, Maruzen", "Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 3rd Edition (Edited by Richard C. Larock), 2018", "Strategic Applications of Named Reactions in Organic Synthesis, (Edited by Laszlo Kurti, Barbara Czako), Academic Press, 2005", etc.

[0335] In this specification, the amine compounds represented by formula (AM-1) or formula (AM-2) (including the sub-formulas of each formula) may form pharmaceutically acceptable salts (for example, acid addition salts). Such salts are not particularly limited as long as they are pharmaceutically acceptable salts, and examples include salts with inorganic acids, salts with organic acids, salts with acidic amino acids, and the like. Preferable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Preferable examples of salts with organic acids include salts with aliphatic monocarboxylic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, butyric acid, valeric acid, enanthic acid, capric acid, myristic acid, palmitic acid, stearic acid, lactic acid, sorbic acid, mandelic acid, and the like; salts with aliphatic dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, tartaric acid, and the like; salts with aliphatic tricarboxylic acids such as citric acid; salts with aromatic monocarboxylic acids such as benzoic acid, salicylic acid, and the like; salts with aromatic dicarboxylic acids such as phthalic acid; salts with organic carboxylic acids such as cinnamic acid, glycolic acid, pyruvic acid, oxalic acid, salicylic acid, N-acetylcysteine, and the like; salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; and acid addition salts with acidic amino acids such as aspartic acid, glutamic acid, and the like. Preferable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, and the like. Among these, pharmaceutically acceptable salts are preferred.

[0336] The salt can be obtained by following a conventional method. For example, after forming the target salt by mixing a solution containing an appropriate amount of acid or base with the compound of the present invention, it can be obtained by fractional filtration or by distilling off the mixed solvent. As a general review of salts, Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Stahl & Wermuth (Wiley-VCH, 2002) has been published, and detailed descriptions are provided in this book.

[0337] In this specification, the amine compound represented by the formula (AM-1) or the formula (AM-2) (including the sub-formulas of each formula) or a salt thereof can form a solvate with a solvent such as water, ethanol, glycerol, etc.

[0338] In this specification, unless otherwise specified, when a variable substituent is substituted on a cyclic group, it means that the variable substituent is not bonded to a specific carbon atom of the cyclic group. For example, the variable substituent Rs in the following formula A can be substituted on any of the carbon atoms i, ii, iii, iv, or v in the formula A.

Chemical formula

[0339] In this specification, in the linker (-L 1 - or -L 2 -) in the chemically modified alginic acid derivative represented by the formula (I) or the formula (II), when an asymmetric carbon exists, it means that each optical isomer thereof is also included.

[0340] For example, when -L 1 - in the formula (I) is the formula (L1-8a), m8a = 2, n8a = 1, and R 1 = Me, the following formula (L1-8a-M) (where the outside of both dashed lines is not included):

Chemical formula

Chemical formula

[0341] The linker (-L 1 - or -L 2When an asymmetric carbon is present (when it is an optically active substance) in (-), in the step of synthesizing the amine derivative (AM-1) corresponding to formula (I) or formula (II), it can be separated from its racemate into each optically active substance by ordinary optical resolution means (separation methods). Also, in the step of synthesizing formula (AM-1) or formula (AM-2) which is an amine derivative corresponding to formula (I), one of the optical isomers can be selectively synthesized by using asymmetric synthesis, and each optically active substance can be synthesized.

[0342] 9. Use of alginate derivatives and crosslinked alginate structures Alginate derivatives can be used in place of conventional alginic acid in a wide range of fields such as food, medicine, cosmetics, fibers, and paper. Specific preferred uses of alginate derivatives or photo-crosslinked alginate structures include medical materials such as wound dressings, postoperative adhesion prevention materials, drug sustained release substrates, cell culture substrates, and cell transplantation substrates.

[0343] Examples of the shape of the crosslinked alginate structure when used as a medical material include tube shape, fiber shape, fiber, beads, gel, and substantially spherical gel. It is preferably beads, gel, or substantially spherical gel, and more preferably substantially spherical gel.

[0344] All documents and publications described in this specification are hereby incorporated by reference in their entirety regardless of their purpose.

[0345] Also, the objects, features, advantages, and ideas of the present invention are clear to those skilled in the art from the description of this specification, and those skilled in the art can easily implement the present invention from the description of this specification. The best mode for carrying out the invention and specific examples, etc. show preferred embodiments of the present invention and are shown for illustration or explanation, and the present invention is not limited thereto. It is clear to those skilled in the art that various modifications can be made within the intention and scope of the present invention disclosed in this specification based on the description of this specification.

Examples

[0346] Next, examples and test examples are given to explain the present invention in more detail. However, these examples are merely examples and test examples, and do not limit the present invention, and may be changed without departing from the scope of the present invention.

[0347] For the measurement of nuclear magnetic resonance spectrum (NMR), JEOL JNM-ECX400 FT-NMR (JEOL Ltd.) was used. Liquid chromatography-mass spectrometry (LC-Mass) was measured by the following method. [UPLC] Waters AQUITY UPLC system and BEH C18 column (2.1 mm × 50 mm, 1.7 μm) (Waters) were used, and the mobile phase and gradient conditions of 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) were used.

[0348] 1 In the 1H-NMR data, in the pattern of NMR signals, s means singlet, d means doublet, t means triplet, q means quartet, m means multiplet, br means broad, J means coupling constant, Hz means Hertz, CDCl3 means deuterochloroform, DMSO-d6 means deuterodimethyl sulfoxide, and D2O means heavy water. 1 In the 1H-NMR data, for signals that cannot be confirmed because they are broad bands, such as protons of hydroxyl group (OH), amino group (NH2), carboxyl group (COOH), etc., they are not described in the data.

[0349] In the LC-Mass data, M means molecular weight, RT means retention time, [M+H] + ,[M+Na] + means the molecular ion peak.

[0350] "Room temperature" in the examples shall generally indicate a temperature of about 0°C to about 35°C. The introduction rate (mol%) of the reactive substituent in the examples is 1It shall indicate the ratio of the number of moles of the introduced reactive substituent to the number of moles of the monosaccharide (glucuronic acid and mannuronic acid) units constituting alginic acid calculated from 1H-NMR (D2O).

[0351] In the examples, sodium alginate before the introduction of the reactive group or the complementary reactive group was the sodium alginate showing the physical property values described in Table 46 above.

[0352] Table 48 shows the physical property values (specifically, the reactive group introduction rate (mol%), molecular weight, and weight average molecular weight (×10,000 Da)) of the alginic acid derivatives having the reactive groups obtained in Examples (1) to (20). Tables 49-1 to 49-5 show the 1H-NMR of the intermediates in Examples (1) to (20), and Table 50 shows the LCM-Mass of the intermediates in Examples (1) to (20).

[0353] (Example 1) Synthesis of 3-azidopropylamino group-introduced alginic acid (EX1-A2): [Chemical formula]

[0354] To an aqueous solution (20 mL) of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) prepared to 1 wt%, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (56 mg), an ethanol (2 mL) solution of commercially available 3-azidopropylamine [CAS REGISTRY NO.: 88192-19-2] (1-1, 5.1 mg), and 1 molar concentration-sodium bicarbonate water (50 μL) were added. After stirring at 30°C for 3 hours, sodium chloride (0.2 g) and ethanol (40 mL) were sequentially added, and the mixture was stirred at room temperature for 30 minutes. The obtained precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained solid was dissolved in water and freeze-dried to obtain the title compound EX1-A2 (187 mg) as a white solid.

[0355] (Example 2) Synthesis of alginic acid (EX2 - A2) with 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine group introduced: [Chemical formula]

[0356] To an aqueous solution of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: A - 2) (10.9 mL) prepared to 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (55.83 mg) and 1 molar concentration of sodium bicarbonate solution (252.17 μL) were added under ice-cooling and stirring. Subsequently, a solution of commercially available 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine [CAS REGISTRY NO.: 166388-57-4] (2-1, 26.36 mg) in ethanol (1 mL) and water (1 mL) was added, and after stirring at room temperature for 15 hours, sodium chloride (100 mg) and ethanol (21.8 mL) were sequentially added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound EX2 - A2 (99 mg) as a white solid.

[0357] (Example 3) Synthesis of alginic acid (EX3 - A2) with 2-amino-N-(3-azidopropyl)acetamide group introduced: [Chemical formula]

[0358] <Step 1> Synthesis of tert-butyl (2-(3-azidopropyl)amino)-2-oxoethyl)carbamate (3-2): [Chemical formula] To a solution of commercially available 3-azidopropylamine [CAS REGISTRY NO.: 88192-19-2] (1-1, 41 μL) and N-(tert-butoxycarbonyl)glycine [CAS REGISTRY NO.: 4530-20-5] (3-1, 100 mg) in ethanol (2 mL) was added 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (197 mg), and the mixture was stirred at room temperature for 18 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting oil was dissolved in methyl-tert-butyl ether (10 mL) and washed successively with saturated aqueous sodium bicarbonate, water, and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the title compound 3-2 (95 mg) as a colorless oil.

[0359] <Step 2> Synthesis of 2-amino-N-(3-azidopropyl)acetamide hydrochloride (3-3):

Chemical formula

[0360] (Example 3) To the compound (3-2, 95 mg) obtained in <Step 1> was added 4 N-hydrogen chloride / 1,4-dioxane (665 μL) under ice-cooling, and the mixture was stirred at room temperature for 1 hour. Diisopropyl ether (2.0 mL) was added to the reaction mixture, and the mixture was concentrated under reduced pressure. The resulting oil was decanted and washed with methyl-tert-butyl ether and then concentrated under reduced pressure to obtain the title compound 3-3 (62 mg) as a colorless gum.

[0361] <Step 3> Synthesis of alginic acid with 2-amino-N-(3-azidopropyl)acetamide group introduced (EX3-A2):

Chemical formula

[0362] To an aqueous solution (20 mL) of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: A-2) prepared at 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (56 mg), a solution of the compound (3-3, 10.6 mg) obtained in <Step 2> of (Example 3) in ethanol (2 mL), and 1 molar concentration - sodium bicarbonate solution (76 μL) were added. After stirring at 30 °C for 3 hours, sodium chloride (0.2 g) and ethanol (40 mL) were sequentially added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained solid was dissolved in water and freeze-dried to obtain the title compound EX3-A2 (207 mg) as a white solid.

[0363] (Example 4) Synthesis of 3-amino-N-(3-azidopropyl)propanamide group-introduced alginic acid (EX4-A2):

Chemical formula

[0364] <Step 1> Synthesis of tert-butyl (3-((3-azidopropyl)amino)-3-oxopropyl)carbamate (4-2):

Chemical formula

[0365] To a solution of commercially available 3-azidopropylamine [CAS REGISTRY NO.: 88192-19-2] (1-1, 38 μL) and N-(tert-butoxycarbonyl)-β-alanine [CAS REGISTRY NO.: 3303-84-2] (4-1, 100 mg) in ethanol (2 mL) was added 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (146 mg), and the mixture was stirred at room temperature for 18 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was successively washed with water and saturated brine. After drying the organic layer over anhydrous sodium sulfate, it was concentrated under reduced pressure to obtain the title compound 4-2 (124 mg) as a white wax-like substance.

[0366] <Step 2> Synthesis of 3-amino-N-(3-azidopropyl)propanamide hydrochloride (4-3):

Chemical formula

[0367] (Example 4) To the compound (4-2, 124 mg) obtained in <Step 1> was added 4N-hydrogen chloride / 1,4-dioxane (868 μL) under ice-cooling, and the mixture was stirred at room temperature for 1 hour. Diisopropyl ether (2.6 mL) was added to the reaction mixture, and it was concentrated under reduced pressure. The obtained oily substance was decanted and washed with methyl tert-butyl ether, and then concentrated under reduced pressure to obtain the title compound 4-3 (93 mg) as a colorless gum-like substance.

[0368] <Step 3> Synthesis of 3-amino-N-(3-azidopropyl)propanamide group-introduced alginic acid (EX4-A2):

Chemical formula

[0369] To an aqueous solution (20 mL) of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: A-2) prepared at 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (56 mg), a solution of the compound (4-3, 12.6 mg) obtained in <Step 2> of (Example 4) in ethanol (2 mL), and 1 molar concentration-sodium bicarbonate solution (76 μL) were added. After stirring at 30 °C for 3 hours, sodium chloride (0.2 g) and ethanol (40 mL) were sequentially added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained solid was dissolved in water and freeze-dried to obtain the title compound EX4-A2 (211 mg) as a white solid.

[0370] (Example 5) Synthesis of N-(4-(aminomethyl)benzyl)-2-azidoacetamide group-introduced alginic acid (EX5-A2): [Chemical formula]

[0371] <Step 1> Synthesis of tert-butyl (4-((2-azidoacetamido)methyl)benzyl)carbamate (5-2): [Chemical formula]

[0372] From commercially available 2-azidoacetic acid [CAS REGISTRY NO.: 18523-48-3] (1-1, 41 μL), a solution of azidoacetyl chloride in methylene chloride (1.0 mL) prepared in the same manner as described in Organic Letters (2017), 19(23), 6400-6403 was added to a solution of commercially available 1-(N-tert-butoxycarbonyl-aminomethyl)-4-(aminomethyl)benzene [CAS REGISTRY NO.: 108468-00-4] (5-1, 100 mg) and triethylamine (118 μL) in methylene chloride (1.0 mL) under ice-cooling, and the mixture was stirred at room temperature for 2.5 hours. Ethyl acetate (20 mL) and water (5 mL) were added to the reaction mixture. After liquid separation, the organic layer was washed successively with water, saturated aqueous sodium hydrogen carbonate, water, and saturated brine. The insoluble matter was filtered off, the filtrate was dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was triturated with methyl tert-butyl ether / n-heptane. The obtained solid was collected by filtration to give the title compound 5-2 (91 mg) as a pale beige solid.

[0373] <Step 2> Synthesis of N-(4-(aminomethyl)benzyl)-2-azidoacetamide hydrochloride (5-3):

Chemical formula

[0374] (Example 5) To the compound (5-2, 91 mg) obtained in <Step 1>, 4N-hydrogen chloride / 1,4-dioxane (637 μL) was added under ice-cooling, then 1,4-dioxane (627 μL) was added, and the mixture was stirred at room temperature for 3.5 hours. Diisopropyl ether (3.8 mL) was added to the reaction mixture and stirred for 10 minutes. The obtained solid was filtered to give the title compound 5-3 (62 mg) as a beige solid.

[0375] <Step 3> Synthesis of N-(4-(aminomethyl)benzyl)-2-azidoacetamide group-introduced alginic acid (EX5-A2):

Chemical formula

[0376] To an aqueous solution (25 mL) of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: A-2) prepared to 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (70 mg), the compound obtained in <Step 2> of (Example 5) (5-3, 16.1 mg), and 1 molar concentration - sodium bicarbonate solution (95 μL) were added. After stirring at 30 °C for 3 hours, sodium chloride (0.25 g) and ethanol (50 mL) were sequentially added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained solid was dissolved in water and freeze-dried to obtain the title compound EX5-A2 (239 mg) as a white solid.

[0377] (Example 6) Synthesis of 2-(4-azidophenoxy)ethane-1-amino group-introduced alginic acid (EX6-A2):

Chemical formula

[0378] <Step 1> Synthesis of tert-butyl (2-(4-azidophenoxy)ethyl)carbamate (6-3):

Chemical formula

[0379] A mixture of commercially available 4-azidophenol [CAS REGISTRY NO.: 24541-43-3] (6-1, 0.3 g), commercially available tert-butyl (2-bromoethyl)carbamate [CAS REGISTRY NO.: 39684-80-5] (6-2, 0.6 g) and N-methylpyrrolidone (3 mL) was added with potassium carbonate (0.61 g) at room temperature. The reaction mixture was stirred at 80 °C for 6 hours and 30 minutes, cooled to room temperature, and then water (10 mL) and methyl tert-butyl ether (20 mL) were added. The resulting suspension was filtered through celite, and the residue was washed twice with methyl tert-butyl ether (5 mL). The filtrate was separated, and the organic layer was concentrated under reduced pressure to obtain a crude product. This crude product was dissolved in methyl tert-butyl ether (20 mL), washed successively twice with 1 N aqueous sodium hydroxide solution (5 mL), twice with water (5 mL), and once with saturated brine (5 mL), and dried over anhydrous sodium sulfate. After filtering the organic layer, the title compound 6-3 (0.411 g) was obtained as a purple oil by concentrating under reduced pressure.

[0380] <Step 2> Synthesis of 2-(4-azidophenoxy)ethane-1-amine hydrochloride (6-4): [Chemical formula]

[0381] (Example 6) To a mixture of the compound obtained in <Step 1> (6-3, 0.41 g) and 1,4-dioxane (2.87 mL), 4 N hydrogen chloride / 1,4-dioxane (2.87 mL) was added under stirring with water cooling, and then the mixture was stirred at room temperature for 18 hours. Diisopropyl ether (40 mL) was added to the reaction solution, and the suspension was stirred at room temperature for 30 minutes. The precipitate was filtered, and the recovered solid was dried under reduced pressure to obtain the title compound 6-4 (0.2834 g) as a pale purple solid.

[0382] <Step 3> Synthesis of 2-(4-azidophenoxy)ethane-1-aminogroup-introduced alginic acid (EX6-A2): [Chemistry] To an aqueous solution (29.66 mL) of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) prepared to 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (91.52 mg) and 1 M sodium bicarbonate aqueous solution (68.63 μL) were added at room temperature. Subsequently, a solution of the compound (6-4, 14.73 mg) obtained in <Step 2> of (Example 6) in water (1 mL) and ethanol (1 mL) was added at room temperature, and the mixture was stirred at the same temperature for 42 hours. Then, sodium chloride (300 mg) and ethanol (59.3 mL) were sequentially added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained solid was dissolved in water and freeze-dried to obtain the title compound EX6-A2 (269 mg) as a pink solid.

[0383] (Example 7) Synthesis of N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX7-B2): [Chemistry]

[0384] <Step 1> Synthesis of tert-butyl (2-(2,2,2-trifluoroacetamido)carbamate (7-2): [Chemistry]

[0385] To a solution of commercially available tert-butyl (2-aminoethyl) carbamate (7-1, 3.00 g, [CAS REGISTRY NO.: 57260-73-8]) in tetrahydrofuran (12.0 mL) was added dropwise ethyl trifluoroacetate (2.24 mL). The reaction mixture was stirred at room temperature for 14.5 h. The reaction solution was concentrated under reduced pressure, and tert-butyl methyl ether (5 mL) and heptane (25 mL) were added to the residue, followed by trituration. The solid was collected by filtration and washed with heptane to obtain the title compound 7-2 (4.36 g) as a white solid.

[0386] <Step 2> Synthesis of N-(2-aminoethyl)-2,2,2-trifluoroacetamide hydrochloride (7-3):

Chemical formula

[0387] (Example 7) The compound 7-2 (0.50 g) obtained in <Step 1> was suspended in 1,4-dioxane (3.0 mL). Under ice-cooling, 4 N-hydrogen chloride / 1,4-dioxane (7.0 mL) was added, and the mixture was stirred at room temperature for 3 h. Diisopropyl ether (30.0 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 50 min. The solid was collected by filtration, washed with diisopropyl ether, and dried under reduced pressure to obtain the title compound 7-3 (0.70 g) as a white solid.

[0388] <Step 3> Synthesis of N-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)-2,2,2-trifluoroacetamide (7-5):

Chemical formula

[0389] To a solution of carboxylic acid (7-4, 300 mg) synthesized according to a method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110) in ethanol (2 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (1.09 g), the compound 7-3 (380 mg) obtained in <Example 7> <Step 2>, and triethylamine (321 μL) were added. After stirring at 30 °C for 3 hours, triethylamine (229 μL) was added, and the mixture was stirred at the same temperature for 1 hour. After further stirring at room temperature for 15.5 hours, water (10 mL) and ethyl acetate (50 mL) were added, and the layers were separated. The aqueous layer was extracted with ethyl acetate (10 mL). The organic layer was washed successively with 0.5 N-citric acid, water, and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. tert-Butyl methyl ether was added to the residue, the insoluble matter was filtered off, the filtrate was concentrated, and then purified by silica gel column chromatography (10% - ethyl acetate / n-heptane to 40% ethyl acetate / n-heptane) to obtain the title compound 7-5 (322 mg) as a white solid.

[0390] <Step 4> Synthesis of N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (7-6): [Chemical formula]

[0391] (Example 7) To a solution of the compound 7-5 (322 mg) obtained in <Step 3> in methanol (4.8 mL), a solution of potassium carbonate (278 mg) in water (1.6 mL) was added, and the mixture was stirred at room temperature for 7.5 hours. The reaction solution was concentrated under reduced pressure, water (3 mL) was added, and then saturated with sodium chloride. The aqueous layer was extracted with ethyl acetate (30 mL, 10 mL × 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the title compound 7-6 (238 mg) as a colorless oil.

[0392] <Step 5> Synthesis of N-(2-aminoethyl)-2-(cyclooct-2-en-1-yloxy)acetamide group-introduced alginic acid (EX7-B2):

Chemical formula

[0393] To an aqueous solution of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: B-2) (120 mL) prepared to 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (335 mg), a solution of compound 7-6 (68 mg) obtained in <Step 4> of (Example 7) in ethanol (12 mL), and 1 M sodium bicarbonate solution (303 μL) were sequentially added under stirring at room temperature, and the mixture was stirred at 30 °C for 3 hours. After adding sodium chloride (1.2 g) to the reaction solution, ethanol (240 mL) was added, and the mixture was stirred for 1.5 hours. The obtained precipitate was collected by filtration, washed with ethanol (20 mL × 5), and dried under reduced pressure. The obtained solid was dissolved in water and freeze-dried to obtain the title compound EX7-B2 (1.16 g) as a white solid.

[0394] (Example 8) Synthesis of N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2-en-1-yloxy)acetamide group-introduced alginic acid (EX8-A2):

Chemical formula

[0395] <Step 1> Synthesis of tert-butyl (2-(2-(2,2,2-trifluoroacetamido)ethoxy)ethylcarbamate (8-2):

Chemical formula

[0396] To a solution of tert-butyl (2-aminoethyl)carbamate (8-1, 1.0 g, [CAS REGISTRY NO.:57260-73-8]) in tetrahydrofuran (4.0 mL) was added dropwise ethyl trifluoroacetate (0.6 mL). The reaction mixture was stirred at room temperature for 3.5 hours and concentrated under reduced pressure to obtain the title crude compound 8-2 (1.5 g) as a colorless oil.

[0397] <Step 2> Synthesis of N-(2-(2-aminoethoxy)ethyl)-2,2,2-trifluoroacetamide hydrochloride (8-3): [Chemical formula]

[0398] (Example 8) To the compound 8-2 (1.5 g) obtained in <Step 1> was added 4 N-hydrogen chloride / 1,4-dioxane solution (10.3 mL) under ice-cooling, and the mixture was stirred at room temperature for 1 hour. Diisopropyl ether (30 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes. The solvent was distilled off under reduced pressure, azeotroped with diisopropyl ether, and then dried under reduced pressure to obtain the title compound 8-3 (1.3 g) as a colorless oil.

[0399] <Step 3> Synthesis of N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)ethyl)-2,2,2-trifluoroacetamide (8-4): [Chemical formula]

[0400] The carboxylic acid (7-4, 300 mg) synthesized according to the method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), and the compound 8-3 (443 mg) obtained in <Step 2> of (Example 8) were dissolved in acetonitrile (6.0 mL). O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (0.75 g) and N,N-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 solution, and liquid separation was performed. The organic layer was successively washed 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 to 70% ethyl acetate / n-heptane) to obtain the title compound 8-4 (469 mg) as a colorless gum.

[0401] <Step 4> Synthesis of N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2-in-1-yloxy)acetamide (8-5): [Chemical formula]

[0402] (Example 8) To a solution of the compound 8-4 (220 mg) obtained in <Step 3> in methanol (3.0 mL), a solution of potassium carbonate (103 mg) in water (0.99 mL) was added, and the mixture was stirred at room temperature for 4.5 hours. Methanol was distilled off under reduced pressure, water (2 mL) was added, and the solution was saturated with sodium chloride. It 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), the insoluble matter was removed by filtration, and then concentrated under reduced pressure to obtain the title crude compound 8-5 (140 mg) as a pale yellow gum.

[0403] <Step 5> Synthesis of N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2-en-1-yloxy)acetamide group-introduced alginic acid (EX8-A2):

Chemical formula

[0404] To an aqueous solution of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: A-2) (40 mL) prepared to 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (112 mg), a solution of compound 8-5 (30 mg) obtained in <Step 4> of (Example 8) in ethanol (4.0 mL), and 1 molar concentration-sodium bicarbonate water (101 μL) were sequentially added under stirring at room temperature, and the mixture was stirred at 30 °C for 3 hours. After adding sodium chloride (0.4 g) to the reaction solution, ethanol (80 mL) was added, and the mixture was stirred for 30 minutes. The obtained precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained solid was dissolved in water and freeze-dried to obtain the title compound EX8-A2 (410 mg) as a white solid.

[0405] (Examples 9a, 9b) Synthesis of N-(2-aminoethyl)-2-(2-(cyclooct-2-en-1-yloxy)acetamido)acetamide group-introduced alginic acid (EX9a-A2, EX9b-B2):

Chemical formula

[0406] <Step 1> Synthesis of tert-butyl (2-oxo-2-((2-(2,2,2-trifluoroacetamido)ethyl)amino)ethyl)carbamate (9-1):

Chemical formula

[0407] N-(tert-Butoxycarbonyl)glycine (91 mg, [CAS REGISTRY NO.: 4530-20-5]), the compound (7-3, 100 mg) obtained in <Step 2> of (Example 7) was dissolved in acetonitrile (3.0 mL). O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (217 mg) and N,N-diisopropylethylamine (281 μL) were added, and the mixture was stirred at room temperature for 3.5 hours. Ethyl acetate (15 mL) and water (5 mL) were added to the reaction solution. After liquid separation, the organic layer was washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (elution solvent: 40% ethyl acetate / n-heptane → ethyl acetate) to obtain the title compound 9-1 (180 mg) as a pale beige amorphous solid.

[0408] <Step 2> Synthesis of N-(2-(2-aminoacetamido)ethyl)-2,2,2-trifluoroacetamide hydrochloride (9-2):

Chemical formula

[0409] (Example 9) To the compound (9-1, 180 mg) obtained in <Step 1>, 4N-hydrogen chloride / 1,4-dioxane (1.2 mL) was added under ice-cooling, and then the mixture was stirred at room temperature for 0.8 hour. Diisopropyl ether (3.6 mL) was added to the reaction solution, and the mixture was stirred for 30 minutes. The obtained solid was filtered to obtain the title compound 9-2 (114 mg) as a white solid.

[0410] <Step 3> Synthesis of N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)acetamido)ethyl)-2,2,2-trifluoroacetamide (9-3):

Chemical formula

[0411] The carboxylic acid (7-4, 80 mg) synthesized according to the method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), the compound (9-2, 110 mg) obtained in <Step 2> of (Example 9) were added with ethanol (1.6 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (219 mg), and triethylamine (67 μL), and stirred at room temperature for 3 hours. To the reaction solution, water (3.2 mL) was added, and after stirring at room temperature for 30 minutes, the solid was filtered and washed with water. To the obtained solid, ethyl acetate / ethanol (1 / 1, 10 mL) was added, and the insoluble matter was filtered off. The filtrate was concentrated under reduced pressure to obtain the title compound 9-3 (101 mg) as a white solid.

[0412] <Step 4> Synthesis of N-(2-(aminoethyl)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)acetamide (9-4): [Chemical formula]

[0413] (Example 9) To a methanol (1.8 mL) solution of the compound (9-3, 60 mg) obtained in <Step 3>, a water (0.3 mL) solution of potassium carbonate (59 mg) was added, and the mixture was stirred at room temperature for 4 hours. After concentrating the reaction solution under reduced pressure, water (2 mL) was added and saturated with sodium chloride. Extraction was performed with ethyl acetate (15 mL, 10 mL × 4), and the extraction layer was concentrated under reduced pressure. To the residue, ethyl acetate (10 mL) and ethanol (1 mL) were added, and the insoluble matter was filtered off. The obtained filtrate was concentrated under reduced pressure to obtain the title compound 9-4 (49 mg) as a colorless gum.

[0414] <Step 5-1> Synthesis of alginic acid (EX9a-A2) introduced with N-(2-(aminoethyl)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)acetamide group: [Chemical formula]

[0415] To an aqueous solution (38 mL) of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) prepared at 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (106 mg), a solution of the compound (9-4, 30.3 mg) obtained in <Step 4> of (Example 9) in ethanol (3.8 mL), and 1 M sodium bicarbonate solution (96 μL) were added. After stirring at 30 °C for 3.2 hours, sodium chloride (0.38 g) and ethanol (76 mL) were sequentially added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained solid was dissolved in water and freeze-dried to obtain the title compound EX9a-A2 (381 mg) as a white solid.

[0416] <Step 5-2> Synthesis of alginic acid with N-(2-(aminoethyl)-2-(2-(cyclooct-2-en-1-yloxy)acetamido)acetamido group introduced (EX9b-B2):

Chemical formula

[0417] To an aqueous solution (38 mL) of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: B-2) prepared at 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (64 mg), the compound (9-4, 18.2 mg) obtained in <Step 4> of (Example 9), and 1 M sodium bicarbonate solution (58 μL) were added. After stirring at 30 °C for 3.2 hours, sodium chloride (0.38 g) and ethanol (76 mL) were sequentially added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained solid was dissolved in water and freeze-dried to obtain the title compound EX9b-B2 (366 mg) as a white solid.

[0418] (Example 10) Synthesis of N-(2-aminoethyl)-3-(2-(cyclooct-2-en-1-yloxy)acetamido)propanamide group-introduced alginic acid (EX10-A2):

Chemical formula

[0419] <Step 1> Synthesis of tert-butyl (3-oxo-3-((2-(2,2,2-trifluoroacetamido)ethyl)amino)propyl)carbamate (10-1):

Chemical formula

[0420] Commercially available N-(tert-butoxycarbonyl)-β-alanine (113 mg, [CAS REGISTRY NO.: 3303-84-2]) and the compound (7-3, 110 mg) obtained in <Step 2> of Example 7 were dissolved in acetonitrile (3.3 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (261 mg) and N,N-diisopropylethylamine (319 μL) were added, and the mixture was stirred at room temperature for 3 hours. Ethyl acetate (15 mL) and water (5 mL) were added to the reaction solution. After liquid separation, the organic layer was washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and then triturated with tert-butyl methyl ether (20 mL). The solid was collected by filtration and dissolved in ethyl acetate (20 mL). The organic layer was washed successively with 1 N-citric acid, water, and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was triturated with tert-butyl methyl ether (10 mL), and then the solid was collected by filtration to obtain the title compound 10-1 (80 mg) as a white solid.

[0421] <Step 2> Synthesis of 3-amino-N-(2-(2,2,2-trifluoroacetamido)ethyl)propanamide hydrochloride (10-2):

Chemical formula

[0422] (Example 10) To the compound (10-1, 80 mg) obtained in <Step 1>, 4N-hydrogen chloride / 1,4-dioxane (1.1 mL) was added under ice-water cooling, and the mixture was stirred at room temperature for 2 hours. Diisopropyl ether (3.4 mL) was added to the reaction solution, and the mixture was stirred for 1.5 hours. The obtained solid was filtered to obtain the title compound 10-2 (61 mg) as a white solid.

[0423] <Step 3> Synthesis of 3-(2-(cyclooct-2-yn-1-yloxy)acetamido)-N-(2-(2,2,2-trifluoroacetamido)ethyl)propanamide (10-3): [Chemical formula]

[0424] The carboxylic acid (7-4, 44 mg) synthesized according to a method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110) and the compound (10-2, 61 mg) obtained in <Step 2> of (Example 10) were added with ethanol (1.2 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (115 mg), and triethylamine (39 μL), and the mixture was stirred at room temperature for 2 hours. Water (3.7 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (15 mL, 5 mL). The organic layer was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. tert-Butyl methyl ether (10 mL) was added to the obtained solid, triturated, and filtered. The obtained solid was purified by column chromatography (80% ethyl acetate / n-heptane → ethyl acetate → 20% methanol / ethyl acetate) to obtain the title compound 10-3 (60 mg) as a pale yellow solid.

[0425] <Step 4> Synthesis of N-(2-(aminoethyl)-3-(2-(cyclooct-2-en-1-yloxy)acetamido)propanamide (10-4):

Chem.

[0426] (Example 10) To a solution of the compound (10-3, 60 mg) obtained in <Step 3> in methanol (3.0 mL) was added a solution of potassium carbonate (42 mg) in water (0.3 mL). After stirring at room temperature for 3 hours, another solution of potassium carbonate (42 mg) in water (0.3 mL) was added, and the mixture was stirred at room temperature for 16.5 hours. The reaction solution was concentrated under reduced pressure, saturated brine (2 mL) was added, and it was further saturated with sodium chloride. It was extracted with ethyl acetate (15 mL, 10 mL × 4), the extract layer was dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. Ethyl acetate (5 mL) and a few drops of methanol were added to the residue, and the insoluble matter was filtered off. The obtained filtrate was concentrated under reduced pressure to obtain the title compound 10-4 (31 mg) as a colorless oil.

[0427] <Step 5> Synthesis of alginic acid with N-(2-aminoethyl)-3-(2-(cyclooct-2-en-1-yloxy)acetamido)propanamide group introduced (EX10-A2):

Chem.

[0428] To an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (41 mL) prepared at 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (114 mg), a solution of the compound obtained in <Step 4> of (Example 10) (10-4, 30.5 mg) in ethanol (4.1 mL), and 1 M sodium bicarbonate solution (103 μL) were added. After stirring at 30 °C for 3 hours, sodium chloride (0.41 g) and ethanol (82 mL) were sequentially added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained solid was dissolved in water and freeze-dried to obtain the title compound EX10-A2 (406 mg) as a white solid.

[0429] (Examples 11a, 11b) Synthesis of 2-(cyclooct-2-yn-1-yloxy)ethane-1-aminogroup-introduced alginic acid (EX11a-A2, EX11b-B2): [Chemical formula]

[0430] <Step 1> Synthesis of (E)-N-(2-((2-bromocyclooct-2-en-1-yl)oxy)ethyl)-2,2,2-trifluoroacetamide (11-3): [Chemical formula]

[0431] The dibromo compound (11-1, 1 g) synthesized according to a method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110) and the alcohol compound (11-2, 5.28 g) synthesized according to a method known in the literature (WO 2015 / 140807 pamphlet) were added to dichloromethane (2 mL) at room temperature. While maintaining the internal temperature at room temperature, the reaction vessel was wrapped with aluminum foil to protect it from light. Subsequently, silver trifluoromethanesulfonate (1.92 g) was added at once at room temperature, and the mixture was stirred at the same temperature for 1 hour. After stirring, saturated brine (5 mL) was added under ice-cooling, and the precipitated silver salt was removed by filtration through Celite, and the residue was washed with methyl tert-butyl ether (10 mL). The filtrate was separated, and the organic layer was washed twice with water (5 mL). Then, it was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. This crude product was purified by silica gel column chromatography (n-heptane / ethyl acetate), and a fraction containing compound 11-3 (0.46 g) was obtained.

[0432] <Step 2> Synthesis of 2-(cyclooct-2-yn-1-yloxy)ethan-1-amine (11-4): [Chemical formula]

[0433] (Example 11) To a mixture of the fraction containing the compound (11-3, 0.46 g) obtained in <Step 1> and dimethyl sulfoxide (1.38 mL), 28% sodium methoxide methanol solution (1.82 mL) was added under water-cooled stirring, and the mixture was stirred at room temperature for 16 hours. Water (10 mL) was added to stop the reaction, and methanol was concentrated under reduced pressure. The resulting solution was extracted three times with methyl tert-butyl ether (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product of the title compound 11-4 (0.196 g) as a brown oil.

[0434] <Step 3-1> Synthesis of 2-(cyclooct-2-en-1-yloxy)ethane-1-aminogroup-introduced alginic acid (EX11a-A2): [Chemical formula]

[0435] To an aqueous solution (69.2 mL) of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: A-2) prepared to 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (213.55 mg) was added at room temperature. Subsequently, a solution of the compound (11-4, 26.78 mg) obtained in <Step 2> of (Example 11) in water (1 mL) and ethanol (1 mL) was added at room temperature, and after stirring at the same temperature for 24 hours, sodium chloride (700 mg) and ethanol (138.4 mL) were sequentially added, and the mixture was stirred at room temperature for 30 minutes. The obtained precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained solid was dissolved in water and then freeze-dried to obtain the title compound EX11a-A2 (661 mg) as a white solid.

[0436] <Step 3-2>[ Synthesis of 2-(cyclooct-2-en-1-yloxy)ethane-1-aminogroup-introduced alginic acid (EX11b-B2): [Chemical formula]

[0437] Using an aqueous solution (70.1 mL) of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: B-2) prepared to 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (216.4 mg), and the compound (11-4, 27.14 mg) obtained in <Step 2> of (Example 11), the same operation as in <Step 3-1> of (Example 11) was performed to obtain the title compound EX11b-B2 (648 mg) as a white solid.

[0438] (Example 12) Synthesis of 2-(2-(Cyclooct-2-en-1-yloxy)ethoxy)ethane-1-aminogroup-introduced alginic acid (EX12-A2):

Chem.

[0439] <Step 1> Synthesis of 2,2,2-trifluoro-N-(2-(2-hydroxyethoxy)ethyl)acetamide (12-2):

Chem.

[0440] Ethyl 2,2,2-trifluoroacetate (2.5 mL) was added dropwise to a solution of commercially available 2-(2-aminoethoxy)ethanol [CAS REGISTRY NO.: 929-06-6] (12-1, 2.0 mL) in tetrahydrofuran (8.0 mL) over 5 minutes, and the mixture was stirred at room temperature for 20 hours. After concentrating the reaction solution under reduced pressure, ethyl acetate (30 mL) and water (10 mL) were added, and the layers were separated. The aqueous layer was extracted with ethyl acetate (10 mL), and the combined organic layers were washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the title compound 12-2 (3.7 g) as a colorless oil.

[0441] <Step 2> Synthesis of (E)-N-(2-(2-((2-bromocyclooct-2-en-1-yl)oxy)ethoxy)ethyl)-2,2,2-trifluoroacetamide (12-3):

Chem.

[0442] The dibromo compound (11-1, 0.30 g) synthesized according to a method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110) was dissolved in methylene chloride (0.54 mL), and in the dark under aluminum foil shading, the compound obtained in <Step 1> of Example 12 (12-2, 1.86 g) and silver trifluoromethanesulfonate (0.52 g) were added. After stirring at room temperature for 1.5 hours in the dark, saturated sodium bicarbonate solution (2.0 mL) and saturated brine (3.0 mL) were sequentially added to the reaction solution under ice-cooling. The solid was filtered off through Celite and washed with tert-butyl methyl ether (10 mL × 3). The filtrate was separated, and the organic layer was washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the title compound 12-3 (424 mg) as a pale brown oily substance.

[0443] <Step 3> Synthesis of N-(2-(2-(cyclooct-2-en-1-yloxy)ethoxy)ethyl)-2,2,2-trifluoroacetamide (12-4): [Chemical formula]

[0444] (Example 12) The compound obtained in <Step 2> (12-3, 100 mg) was dissolved in tetrahydrofuran (0.7 mL) and N,N-dimethylformamide (0.7 mL). 60% sodium hydride (21 mg) was added under ice-cooling, and the mixture was stirred at the same temperature for 3 hours. 60% sodium hydride (10 mg) was added, and the mixture was stirred at room temperature for 1 hour. Further, 60% sodium hydride (10 mg) was added, and the mixture was stirred at room temperature for 20 hours. Water (3 mL) was added, and the mixture was extracted with ethyl acetate (15 mL, 10 mL). The organic layer was washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (n-heptane → 50% ethyl acetate / n-heptane) to obtain the title compound 12-4 (37 mg) as a colorless oily substance.

[0445] <Step 4> Synthesis of 2-(2-(cyclooct-2-en-1-yloxy)ethoxy)ethan-1-amine (12-5):

Chemical formula

[0446] (Example 12) To a solution of the compound (12-4, 37 mg) obtained in <Step 3> in methanol (555 μL) was added a solution of potassium carbonate (50 mg) in water (185 μL), and the mixture was stirred at room temperature for 17 hours. After concentrating the reaction solution under reduced pressure, water (1 mL) was added and saturated with sodium chloride. The mixture was extracted with ethyl acetate (10 mL × 4), and the extract layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. Ethyl acetate (10 mL) and a few drops of methanol were added to the residue, and the insoluble matter was filtered off. The obtained filtrate was concentrated under reduced pressure to obtain the title compound 12-5 (30 mg) as a colorless oil.

[0447] <Step 5> Synthesis of alginic acid (EX12-A2) with 2-(2-(cyclooct-2-en-1-yloxy)ethoxy)ethan-1-amino group introduced:

Chemical formula

[0448] To an aqueous solution (52 mL) of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: A-2) prepared to 1 wt% were added 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (145 mg), a solution of the compound (12-5, 29 mg) obtained in <Step 4> of (Example 12) in ethanol (5.2 mL), and 1 M sodium bicarbonate solution (131 μL). After stirring at 30 °C for 3.2 hours, sodium chloride (0.52 g) and ethanol (104 mL) were sequentially added, and the mixture was stirred at room temperature for 30 minutes. The obtained precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The obtained solid was dissolved in water and freeze-dried to obtain the title compound EX12-A2 (522 mg) as a white solid.

[0449] (Example 13) Synthesis of alginic acid with 3-amino-N-(2-(2-(2-(cyclooct-2-en-1-yloxy)acetamido)ethoxy)ethyl)propanamide group introduced (EX13-A2):

Chemical formula

[0450] <Step 1> Synthesis of 3-(2,2,2-trifluoroacetamido)propanoic acid (13-2):

Chemical formula

[0451] Commercially available β-alanine [CAS REGISTRY NO.: 107-95-9] (13-1, 2.0 g) was dissolved in methanol (40.0 mL), and triethylamine (3.3 mL) was added. Under water cooling, 2,2,2-trifluoroacetic acid (3.4 mL) was added dropwise over 5 minutes, and the mixture was stirred at room temperature for 20.5 hours. The reaction solution was concentrated under reduced pressure, water (20 mL) was added, and the pH was adjusted to 4 with 1 N hydrochloric acid. The solution was extracted with ethyl acetate (100 mL × 2, 50 mL), and the organic layer was washed with saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the title compound 13-2 (2.9 g) as a white solid.

[0452] <Step 2> Synthesis of tert-butyl (2-(2-(3-(2,2,2-trifluoroacetamido)propanamido)ethoxy)ethyl)carbamate (13-3):

Chemical formula

[0453] (Example 13) To a solution of the compound (13-2, 400 mg) obtained in <Step 1> and tert-butyl (2-aminoethyl) carbamate (8-1, 441 mg, [CAS REGISTRY NO.: 57260-73-8]) in ethanol (4.0 mL) was added 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (897 mg), and the mixture was stirred for 3.5 hours. To the reaction solution was added water (5 mL), and the mixture was extracted with ethyl acetate (20 mL, 10 mL). The organic layer was successively washed with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (30% ethyl acetate / n-heptane → ethyl acetate) to obtain the title compound 13-3 (451 mg) as a colorless oil.

[0454] <Step 3> Synthesis of N-(2-(2-aminoethoxy)ethyl)-3-(2,2,2-trifluoroacetamido)propanamide hydrochloride (13-4):

Chemical formula

[0455] (Example 13) To the compound (13-3, 451 mg) obtained in <Step 2> was added 4N-hydrogen chloride / 1,4-dioxane (3.16 mL) under ice-cooling, and the mixture was stirred at room temperature for 3 hours. To the reaction solution was added diisopropyl ether (6.4 mL), and the mixture was concentrated under reduced pressure to obtain the title compound 13-4 (433 mg) as a colorless gum.

[0456] <Step 4> Synthesis of N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)ethyl-3-(2,2,2-trifluoroacetamido)propanamide (13-5):

Chemical formula

[0457] The carboxylic acid (7-4, 111 mg) synthesized according to a method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), and the compound (13-4, 215 mg) obtained in <Step 3> (Example 13) were added with ethanol (1.7 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (253 mg), and triethylamine (102 μL), and stirred at room temperature for 21 hours. To the reaction solution was added water (5 mL), and the mixture was extracted with ethyl acetate (15 mL). The organic layer was successively washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (30% ethyl acetate / n-heptane → ethyl acetate → 15% methanol / ethyl acetate) to obtain the title compound 13-5 (35 mg) as a colorless oil.

[0458] <Step 5> Synthesis of 3-amino-N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)ethyl)propanamide (13-6): [Chemical formula]

[0459] (Example 13) To a methanol (700 μL) solution of the compound (13-5, 35 mg) obtained in <Step 4> was added a water (175 μL) solution of potassium carbonate (33 mg), and the mixture was stirred at room temperature for 16.5 hours. After the reaction solution was concentrated under reduced pressure, water (2 mL) was added, and the solution was saturated with sodium chloride. The mixture was extracted with ethyl acetate (10 mL × 5), and the extracted layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Ethyl acetate (10 mL) and a few drops of methanol were added to the residue, and the insoluble matter was filtered off. The obtained filtrate was concentrated under reduced pressure to obtain the title compound 13-6 (24 mg) as a colorless gum.

[0460] <Step 6> Synthesis of 3 - amino - N-(2-(2-(2-(cyclooct - 2 - en - 1 - yloxy)acetamido)ethoxy)ethyl)propanamide - introduced alginic acid (EX13 - A2):

Chemical formula

[0461] To an aqueous solution of sodium alginate (manufactured by Mita Pharmaceutical Co., Ltd.: A - 2) (28 mL) prepared to 1 wt%, 4-(4,6 - dimethoxy - 1,3,5 - triazin - 2 - yl)-4 - methylmorpholinium chloride (DMT - MM) (78 mg), a solution of the compound (13 - 6, 24 mg) obtained in <Step 5> of (Example 13) in ethanol (2.8 mL), and 1 - molar - concentration sodium bicarbonate solution (71 μL) were added. After stirring at 30 °C for 3.5 hours, sodium chloride (0.28 g) and ethanol (56 mL) were sequentially added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained solid was dissolved in water and then freeze - dried to obtain the title compound EX13 - A2 (272 mg) as a white solid.

[0462] (Example 14) Synthesis of N-(4-(2 - aminoethoxy)benzyl)-2-(cyclooct - 2 - en - 1 - yloxy)acetamide - introduced alginic acid (EX14a - A2, EX14b - B2):

Chemical formula

[0463] <Step 1> Synthesis of N-(2 - bromoethyl)-2,2,2 - trifluoroacetamide (14 - 2):

Chemical formula

[0464] To a solution of commercially available 2-bromoethylamine hydrobromide [CAS REGISTRY NO.: 2576-47-8] (14-1, 1.3 g) in methanol (30 mL), triethylamine (4.29 mL) was added under ice-cooling and stirring. To this mixture, ethyl trifluoroacetate (1.92 mL) was gradually added at the same temperature, and the mixture was stirred at room temperature for 42 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and water (10 mL) was added. The mixture was extracted three times with ethyl acetate (10 mL), and the organic layer was successively washed with water (5 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the title compound 14-2 (2.457 g) as a pale brown solid.

[0465] <Step 2> Synthesis of tert-butyl (4-(2-(2,2,2-trifluoroacetamido)ethoxy)benzyl)carbamate (14-4): [Chemical formula]

[0466] To a mixture of commercially available tert-butyl (4-hydroxybenzyl)carbamate [CAS REGISTRY NO.: 149505-94-2] (14-3, 0.36 g), the compound obtained in <Step 1> of Example 14 (14-2, 0.46 g), potassium iodide (0.35 g), and N-methylpyrrolidone (3.6 mL), potassium carbonate (0.45 g) was added at room temperature, and the mixture was stirred at 140°C for 5 hours. After completion of the reaction, the reaction mixture was cooled to room temperature and diluted with water (10 mL). The mixture was extracted three times with methyl tert-butyl ether (10 mL), and the organic layer was successively washed twice with 1N aqueous sodium hydroxide solution (5 mL), water (5 mL), and saturated brine (5 mL), and dried over anhydrous sodium sulfate. After filtering the organic layer, the organic layer was concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by silica gel column chromatography (n-heptane / ethyl acetate) to obtain the title compound 14-4 (0.202 g) as a white amorphous solid.

[0467] <Step 3> Synthesis of N-(2-(4-(aminomethyl)phenoxy)ethyl)-2,2,2-trifluoroacetamide hydrochloride (14-5): [Chemical formula]

[0468] (Example 14) Using the compound (14-4, 0.2 g) obtained in <Step 2>, the same operation as in <Step 2> of (Example 6) was carried out to obtain the title compound 14-5 (0.147 g) as a white solid.

[0469] <Step 4> Synthesis of N-(2-(4-((2-(cyclooct-2-yn-1-yloxy)acetamido)methyl)phenoxy)ethyl)-2,2,2-trifluoroacetamide (14-6): [Chemical formula]

[0470] To a mixture of the carboxylic acid (7-4, 50 mg) synthesized according to a method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), the compound (14-5, 81.96 mg) synthesized in <Step 3> of (Example 14), and ethanol, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (137.22 mg) and triethylamine (38.25 μL) were added under ice-cooling and stirring, and the mixture was stirred at room temperature for 1 hour and 30 minutes. After completion of the reaction, water (2 mL) was added, the suspension was stirred, and methyl tert-butyl ether (0.5 mL) was added. The separated aqueous layer was extracted twice with methyl tert-butyl ether (5 mL), washed successively with water (5 mL) and saturated brine (5 mL), and dried over anhydrous sodium sulfate. The dried organic layer was filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (n-heptane / ethyl acetate) to obtain the title compound 14-6 (99 mg) as a white amorphous solid.

[0471] <Step 5> Synthesis of N-(4-(2-aminoethoxy)benzyl)-2-(cyclooct-2-en-1-yloxy)acetamide (14-7):

Chemical formula

[0472] (Example 14) To a mixture of the compound obtained in <Step 4> (14-6, 99 mg) and methanol (1485 μL), potassium carbonate (64.17 mg) and water (495 μL) were added under water-cooled stirring, and the mixture was stirred at room temperature for 15 hours. After completion of the reaction, methanol was concentrated under reduced pressure, and the resulting aqueous layer was extracted 3 times with ethyl acetate (5 mL). The organic layer was washed successively with water (5 mL) and saturated brine (5 mL), and dried over anhydrous sodium sulfate. After filtering the dried organic layer, it was concentrated under reduced pressure to obtain a crude product of the title compound 14-7 (68 mg) as a yellow oil.

[0473] <Step 6-1> Synthesis of alginic acid with N-(4-(2-aminoethoxy)benzyl)-2-(cyclooct-2-en-1-yloxy)acetamide group introduced (EX14a-A2):

Chemical formula

[0474] Using an aqueous solution of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: A-2) prepared to 1 wt% (49.44 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (152.54 mg), and the compound obtained in <Step 5> of (Example 14) (14-7, 37.79 mg), the same operation as in <Step 3-1> of (Example 11) was performed to obtain the title compound EX14a-A2 (479 mg) as a white solid.

[0475] <Step 6-2> Synthesis of N-(4-(2-aminoethoxy)benzyl)-2-(cyclooct-2-en-1-yloxy)acetamide group-introduced alginic acid (EX14b-B2): [Chemical formula]

[0476] Using an aqueous solution of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: B-2) prepared to 1% by weight (40.08 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (123.66 mg), and the compound obtained in (Example 14) <Step 5> (14-7, 30.64 mg), the same operation as in (Example 11) <Step 3-1> was carried out to obtain the title compound EX14b-B2 (356 mg) as a white solid.

[0477] (Example 15) Synthesis of 2-amino-N-[3-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-3-oxopropyl]acetamide group-introduced alginic acid (EX15-A2): [Chemical formula]

[0478] <Step 1> Synthesis of (9H-fluoren-9-yl)methyl-N-[3-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-3-oxopropyl]acetamide-2-carbamate group (15-2): [Chemical formula]

[0479] Commercially available 3-amino-1-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-1-propanone [CAS REGISTRY NO.: 1255942-06-3] (15-1, 50 mg) and N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine [CAS REGISTRY NO.: 29022-11-5] (54 mg) were dissolved in acetonitrile (1.5 mL). O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (76 mg) and N,N-diisopropylethylamine (70 μL) were added, and the mixture was stirred at room temperature for 4.5 hours. Ethyl acetate (15 mL) and water (5 mL) were added to the reaction solution. After liquid separation, the organic layer was successively washed with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and then purified by column chromatography to obtain the title compound 15-2 (63 mg) as a pale beige amorphous solid.

[0480] <Step 2> Synthesis of 2-amino-N-[3-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-3-oxopropyl]acetamide (15-3): [Chemical formula]

[0481] (Example 15) To the compound (15-2, 63 mg) obtained in <Step 1> was added a solution of piperidine (56 μL) in N,N-dimethylformamide (315 μL), and the mixture was stirred at room temperature for 30 minutes. Ethyl acetate (15 mL) and water (5 mL) were added to the reaction solution. After liquid separation, the organic layer was successively washed with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. tert-Butyl methyl ether (5 mL) was added to the obtained solid, and after trituration, the solid was collected by filtration to obtain the title compound 15-3 (10 mg) as a pale beige solid. The filtrate was recovered to additionally obtain the title compound 15-3 (11 mg) as a pale yellow gum.

[0482] <Step 3> Synthesis of 2-amino-N-[3-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-3-oxopropyl] acetamide group-introduced alginic acid (EX15-A2):

Chem.

[0483] To an aqueous solution (19 mL) of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: A-2) prepared to 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (106 mg), a solution of the compound (15-3, 21 mg) obtained in <Step 2> of (Example 15) in ethanol (1.9 mL), and 1 molar concentration-sodium bicarbonate water (48 μL) were added. After stirring at 30 °C for 3 hours, sodium chloride (0.19 g) and ethanol (38 mL) were sequentially added, and the mixture was stirred at room temperature for 30 minutes. The obtained precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained solid was dissolved in water and freeze-dried to obtain the title compound EX15-A2 (188 mg) as a white solid.

[0484] (Example 16) Synthesis of 2-amino-N-(2-(cyclooct-2-yn-1-yloxy)ethyl) acetamide group-introduced alginic acid (EX16-A2):

Chem.

[0485] <Step 1> Synthesis of (2,2,2-trifluoroacetyl) glycine (16-2):

Chem.

[0486] Glycine (16-1, 2 g) was suspended in methanol (10 mL) and cooled to 4°C. At the same temperature, ethyl trifluoroacetate (3.5 mL) and triethylamine (3.71 mL) were added, and the mixture was stirred at room temperature for 23 hours. After completion of the reaction, 1 N-hydrochloric acid (20 mL) was gradually added until the pH reached 2, and the mixture was extracted three times with ethyl acetate (10 mL) and washed successively with water (5 mL) and saturated brine (5 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a pale yellow oily substance. The obtained oily substance was dissolved in ethyl acetate (20 mL), and n-heptane (10 mL) was added. The solution was concentrated under reduced pressure to obtain the title compound 16-2 (3.22 g) as a white amorphous solid.

[0487] <Step 2> Synthesis of N-(2-((2-(cyclooct-2-en-1-yloxy)ethyl)amino)-2-oxoethyl)-2,2,2-trifluoroacetamide (16-3):

Chemical formula

[0488] To a mixture of compound 11-4 (80 mg) and the compound obtained in <Step 1> (16-2, 81.83 mg) of Example 16, ethanol (1600 μL) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (239.21 mg) were added under ice-cooling with stirring, and the mixture was stirred at room temperature for 3 hours. Water (2 mL) was added to stop the reaction, and the mixture was extracted three times with methyl tert-butyl ether (5 mL). The organic layer was washed successively with water (5 mL) and saturated brine (5 mL), and dried over anhydrous sodium sulfate. After filtering the organic layer, the crude product was obtained by concentrating under reduced pressure. The crude product was triturated with n-heptane (10 mL), filtered, and dried under reduced pressure to obtain the title compound 16-3 (95.1 mg) as a white solid.

[0489] <Step 3> Synthesis of 2-amino-N-(2-(cyclooct-2-en-1-yloxy)ethyl)acetamide (16-4):

Chemical formula

[0490] (Example 16) Using the compound (16-3, 60 mg) obtained in <Step 2>, methanol (900 μL), potassium carbonate (51.78 mg), and water (300 μL), the same operation as in <Step 5> of (Example 14) was carried out to obtain the title compound 16-4 (15 mg) as a pale yellow oil.

[0491] <Step 4> Synthesis of alginic acid with 2-amino-N-(2-(cyclooct-2-en-1-yloxy)ethyl)acetamide group introduced (EX16-A2):

Chemical formula

[0492] Using an aqueous solution of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: A-2) prepared to 1 wt% (29.66 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (91.52 mg), and the compound (16-4, 15 mg) obtained in <Step 3> of (Example 16), the same operation as in <Step 3-1> of (Example 11) was carried out to obtain the title compound EX16-A2 (279 mg) as a white solid.

[0493] (Example 17) Synthesis of alginic acid with (2S)-2-amino-N-(2-(cyclooct-2-en-1-yloxy)ethyl)-3-phenylpropanamide group introduced (EX17-A2):

Chemical formula

[0494] <Step 1> Synthesis of (2,2,2-trifluoroacetyl)-L-phenylalanine (17-2): [Chemical Formula]

[0495] L-Phenylalanine [CAS REGISTRY NO.: 63-91-2] (17-1, 2 g) was dissolved in methanol (10 mL) and cooled to 4°C. Subsequently, ethyl trifluoroacetate (1.59 mL) and triethylamine (1.69 mL) were added at the same temperature, and the mixture was stirred at room temperature for 16 hours. After completion of the reaction, 1 N-hydrochloric acid (10 mL) was gradually added until the pH reached 1, and the suspension was stirred for 30 minutes. The suspension was filtered, and the recovered solid was dried under reduced pressure to obtain the title compound 17-2 (2.53 g) as a white solid.

[0496] <Step 2>[ Synthesis of (2S)-N-(2-(cyclooct-2-yn-1-yloxy)ethyl)-3-phenyl-2-(2,2,2-trifluoroacetamido)propanamide (17-3): [Chemical Formula]

[0497] To a mixture of compound 11-4 (60 mg) and the compound obtained in <Step 1> (Example 17) (17-2, 93.7 mg), ethanol (1200 μL) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (179.41 mg) were added under ice-cooling, and the mixture was stirred at room temperature for 3 hours. Water (2 mL) was added to stop the reaction, and the mixture was extracted three times with methyl tert-butyl ether (5 mL). The organic layer was washed successively with water (5 mL) and saturated brine (5 mL), and dried over anhydrous sodium sulfate. After filtering the organic layer, it was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (n-heptane / ethyl acetate) to obtain the title compound 17-3 (57 mg) as a white amorphous solid.

[0498] <Project 3> Synthesis of (2S)-2-amino-N-(2-(cyclooct-2-en-1-yloxy)ethyl)-3-phenylpropanamide (17-4):

Chemical formula

[0499] (Example 17) Using the compound (17-3, 57 mg) obtained in <Project 2>, methanol (855 μL), potassium carbonate (38.39 mg), and water (285 μL), the same operation as in <Project 5> of (Example 14) was carried out to obtain the title compound 17-4 (35 mg) as a pale yellow oily substance.

[0500] <Project 4> Synthesis of alginic acid with (2S)-2-amino-N-(2-(cyclooct-2-en-1-yloxy)ethyl)-3-phenylpropanamide group introduced (EX17-A2):

Chemical formula

[0501] An aqueous solution of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: A-2) prepared to 1% by weight (47.46 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (146.44 mg), and the compound (17-4, 34.53 mg) obtained in <Project 3> of (Example 17) were used, and the same operation as in <Project 3-1> of (Example 11) was carried out to obtain the title compound EX17-A2 (383 mg) as a white solid.

[0502] (Example 18) Synthesis of alginic acid with 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group introduced (EX18-A2):

Chemical formula

[0503] <Project 1> Synthesis of methyl 4-(2-((tert-butoxycarbonyl)amino)ethoxy)benzoate (18-2):

Chem.

[0504] To a solution of triphenylphosphine (0.96 g) in tetrahydrofuran (2.59 mL), a solution of diethyl azodicarboxylate (40% toluene solution, 1.92 mL) was added under ice-cooling and stirring, and the mixture was stirred at room temperature for 20 minutes. To this solution, a solution of commercially available 4-hydroxybenzoic acid (Compound 18-1, 0.37 g) and 2-(tert-butoxycarbonyl)ethanolamine (0.39 g) in tetrahydrofuran (1.1 mL) was added under ice-cooling and 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 to 40% ethyl acetate / n-heptane) to obtain a mixture of Compound 18-1 and Compound 18-2. This mixture was dissolved in methyl tert-butyl ether (20 mL) and washed successively with 1 N aqueous sodium hydroxide solution (5 mL) twice and saturated brine (5 mL). After drying the organic layer over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure to obtain the title compound 18-2 (0.45 g) as a pink oily substance.

[0505] <Project 2> Synthesis of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide hydrochloride (Compound 18-4):

Chem.

[0506] (Example 18) To a solution of compound 18-2 (0.44 g) obtained in <Step 1> in methanol (4.4 mL) was added lithium hydroxide monohydrate (0.25 g), and the mixture was stirred at 60 °C for 3 hours and 30 minutes. 1 N-Hydrochloric acid (5 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (10 mL). The organic layer was successively washed with water (5 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was dissolved in acetonitrile (4.4 mL), and 3-azidopropan-1-amine (0.15 g) and O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (0.57 g) were added. Subsequently, under ice-cooling and stirring, N,N-diisopropylethylamine (0.52 mL) was added, 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 distilled off under reduced pressure. The residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate), and a fraction containing compound 18-3 (0.71 g) was obtained.

[0507] To the fraction containing compound 18-3 (0.71 g) was added 4 N-hydrogen chloride / 1,4-dioxane (4.9 mL), and the mixture was stirred at room temperature for 20 minutes. Diisopropyl ether was added to the reaction solution, and the precipitate was filtered to obtain the title compound 18-4 (0.49 g) as a white solid.

[0508] <Step 3> Synthesis of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group-introduced alginic acid (Compound EX18-A2): [Chemical formula]

[0509] To an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) prepared at 1% by weight (19.6 mL), under ice-cooling and stirring, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (50.19 mg), the compound 18-4 (54.37 mg) obtained in <Process 2> of Example 18, and 1 molar concentration-sodium bicarbonate aqueous solution (181.4 μL) were used, and the same operation as in <Process 3-1> of Example 11 was carried out to obtain the title compound EX18-A2 (198 mg) as a white solid.

[0510] (Example 19) Synthesis of alginic acid (EX19-A2) into which a 3-amino-1-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-1-propanone group is introduced: [Chemical formula]

[0511] To an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) prepared at 1% by weight (43.6 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (111.7 mg), 1 molar concentration-sodium bicarbonate aqueous solution (403.5 μL), and commercially available 3-amino-1-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-1-propanone [CAS REGISTRY NO.: 1255942-06-3] (19-1, 83.6 mg) were used, and the same operation as in <Process 3-1> of Example 11 was carried out to obtain the title compound EX19-A2 (376 mg) as a pale yellow solid.

[0512] (Example 20) Synthesis of alginic acid (EX20-B2) into which an N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group is introduced: [Chemical formula]

[0513] <Project 1> Synthesis of tert-butyl (4-((2,2,2-trifluoroacetamido)methyl)benzyl)carbamate (Compound 20-2):

Chemical Structure

[0514] To a mixture of tert-butyl (4-(aminoethyl)benzyl)carbamate (20-1, 0.67 g), triethylamine (0.39 mL), and methanol (6.67 mL) synthesized with reference to a method known in the literature (Bioorganic & Medicinal Chemistry (2003) 11:4189-4206), ethyl trifluoroacetate (0.44 mL) was added dropwise under ice-cooling and stirring. The reaction mixture was warmed to room temperature and stirred at the same temperature for 5 hours. The reaction was stopped with water (10 mL), and the mixture was extracted 3 times with ethyl acetate (10 mL). The recovered organic layer was washed with saturated brine (5 mL) and dried over anhydrous sodium sulfate. After filtering the dried organic layer, it was concentrated to obtain the title crude compound 20-2 (0.67 g) as a pale yellow amorphous solid.

[0515] <Project 2> Synthesis of N-(4-(aminoethyl)benzyl)-2,2,2-trifluoroacetamide hydrochloride (Compound 20-3):

Chemical Structure

[0516] (Example 20) To a 1,4-dioxane solution (3.5 mL) of Compound 20-2 (0.5 g) obtained in <Project 1>, 4 N-hydrogen chloride / 1,4-dioxane (3.5 mL) was added under water-cooling and stirring, and the mixture was stirred at room temperature for 3 hours. Diisopropyl ether (40 mL) was added to the reaction solution, and the precipitate was filtered to obtain the title compound 20-3 (0.4 g) as a white solid.

[0517] <Project 3> Synthesis of N-(4-((2-(Cyclooct-2-en-1-yloxy)acetamido)methyl)benzyl)-2,2,2-trifluoroacetamide (Compound 20-4): [Chemical formula]

[0518] To a solution of carboxylic acid (7-4, 0.17 g) and O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (0.26 g) in acetonitrile (1.7 mL) synthesized according to a method known in the literature (Org. Process Res. Dev. (2018) 22:108-110), under ice-cooling and stirring, Compound 20-3 (0.26 g) and N,N-diisopropylethylamine (0.51 mL) obtained in <Step 2> of Example 20 were added dropwise, and the mixture was stirred at room temperature for 1 hour and 30 minutes. After adding water (5 mL) to stop the reaction, the mixture was extracted three times with ethyl acetate (5 mL). The organic layer was washed with saturated brine (3 mL) and then dried over anhydrous sodium sulfate. After filtering the dried organic layer, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (12% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain the title compound 20-4 (0.19 g) as a white amorphous solid.

[0519] <Step 4> Synthesis of N-(4-(Aminomethyl)benzyl)-2-(cyclooct-2-en-1-yloxy)acetamide (Compound 20-5): [Chemical formula]

[0520] (Example 20) To a mixture of Compound 20-4 (0.18 g) obtained in <Step 3> and methanol (1.8 mL), an aqueous solution (0.9 mL) of potassium carbonate (0.13 g) was added dropwise under ice-cooling with stirring, and the mixture was stirred at room temperature for 17 hours and 30 minutes. Methanol was distilled off under reduced pressure, and the residue 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 obtain the title crude compound 20-5 (0.13 g) as a pale yellow oil.

[0521] <Step 5> Synthesis of N-(4-(aminoethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamidogroup-introduced alginic acid (EX20-B2): [Chemical formula]

[0522] Using an aqueous solution (50.9 mL) of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd.: B-2) prepared to 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (0.12 g), and a solution of Compound 20-5 (35 mg) obtained in <Step 4> of (Example 20) in ethanol (3 mL), the same operation as in <Step 3-1> of (Example 11) was performed to obtain the title compound EX20-B2 (521 mg) as a white solid.

[0523] (Examples P1 to P7) The alginic acid derivatives of (Example P1) to (Example P7) shown in the following table are produced using the corresponding amino compounds (which may be pharmaceutically acceptable salts thereof, or solvates thereof) and alginic acid according to the method shown in the above examples. [Table 47]

[0524] Physical property data of alginic acid derivatives [Table 48]

[0525] NMR data of the intermediate compound

Table 49-1

Table 49-2

Table 49-3

Table 49-4

Table 49-5

[0526] LC-Mass data of the intermediate compound

Table 50

[0527] [Measurement of the introduction rate of reactive groups or complementary reactive groups] The introduction rate of reactive groups or complementary reactive groups means the value expressed as a percentage of the number of reactive groups or complementary reactive groups introduced per uronic acid monosaccharide unit, which is the repeating unit of alginic acid. In this example, the introduction rate (mol%) of reactive groups or complementary reactive groups is 1 Calculated by the integration ratio of 1H-NMR. Also, the amount of alginic acid required for calculating the introduction rate is measured by the carbazole-sulfuric acid method using a calibration curve, and the amount of reactive groups or complementary reactive groups can also be measured by the absorbance measurement method using a calibration curve.

[0528] [Measurement of molecular weight] The alginate solid introduced with a reactive group or a complementary reactive group obtained in the examples was dissolved in a 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl to prepare a 0.1% or 0.2% solution. After removing insolubles through a polyethersulfone filtration filter with a pore size of 0.22 μm (Minisart High Flow Filter, Sartorius), it was used as a sample for gel filtration. The spectra of each sample were measured with a spectrophotometer DU-800 (Beckman-Coulter), and the measurement wavelengths in the gel filtration of each compound were determined. For compounds having no specific absorption wavelength, a differential refractometer was used.

[0529] 200 μL of the sample for gel filtration was applied to a Superose6 Increase 10 / 300 GL column (GE Healthcare Sciences). Gel filtration was carried out using an AKTA Explorer 10S as a chromatograph device and a 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl as an elution solvent at a flow rate of 0.8 mL / min at room temperature. The elution profile of the sample was prepared by monitoring the absorption at the wavelength determined for each compound. The obtained chromatogram was analyzed with Unicorn 5.31 software (GE Healthcare Sciences) to determine the peak range.

[0530] The molecular weight of alginic acid into which a reactive group or a complementary reactive group has been introduced was determined by gel filtration under the same conditions as those for the alginic acid into which the reactive group or the complementary reactive group had been introduced, using as standards blue dextran (molecular weight 2,000,000 Da, SIGMA), thyroglobulin (molecular weight 669,000 Da, GE Healthcare Sciences), ferritin (molecular weight 440,000 Da, GE Healthcare Sciences), aldolase (molecular weight 158,000 Da, GE Healthcare Sciences), conalbumin (molecular weight 75,000 Da, GE Healthcare Sciences), ovalbumin (molecular weight 44,000 Da, GE Healthcare Sciences), ribonuclease A (molecular weight 13,700 Da, GE Healthcare Sciences), and aprotinin (molecular weight 6500 Da, GE Healthcare Sciences). The elution volume of each component was determined using Unicorn software. The elution volume of each component was plotted on the horizontal axis and the logarithm of the molecular weight on the vertical axis, and linear regression was performed to create a calibration curve. Two types of calibration curves were created, one from blue dextran to ferritin and the other from ferritin to aprotinin.

[0531] Using this calibration curve, the molecular weight (Mi) at the elution time i of the previously obtained chromatogram was calculated. Next, the absorbance at the elution time i was read as Hi. The weight average molecular weight (Mw) was determined from these data using the following equation.

[0532] [Number]

[0533] [Measurement of gel stability] (Measurement of gel stability (1)): Stability in PBS The alginate derivatives (Ex1-A2), (Ex4-A2), (Ex5-A2), and (Ex19-A2) obtained in Examples 1, 4, 5, and 19 were dissolved in water to a concentration of 1.0% to obtain alginate aqueous solutions (1-1), (4-1), (5-1), and (19-1), respectively. These were mixed in equal amounts in combinations of (1-1) and (19-1), (4-1) and (19-1), and (5-1) and (19-1), placed in a syringe equipped with an 18-gauge injection needle, and this syringe was installed on a syringe pump set to a flow rate of 1 mL / min. They were dropped into a calcium chloride solution with a concentration of 30 mmol / L for 30 seconds and stirred for 5 minutes to obtain alginate gels. The gels were washed once with 10 mL of PBS and left standing in PBS at 37°C for 10 minutes for chemical cross-linking to obtain chemically cross-linked alginate gels. The cross-linked alginate gels (beads) prepared with (Ex18-A2) / (Ex19-A2) were also prepared in the same manner. 19.5 mL of PBS was added to this gel, and it was shaken at 37°C. The aqueous solution was collected over time, and the same amount of PBS as the collected amount was replenished. After the test, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and it was shaken at 37°C for 3 hours or more to completely disintegrate the gel, and the aqueous solution was collected. The alginate concentration in the collected aqueous solution was measured by the carbazole-sulfuric acid method, and the amount of eluted alginate up to each time point was expressed as a percentage by dividing by the total amount of alginate calculated from the alginate concentration at all time points and the alginate concentration after the end of the test. This value was defined as the disintegration rate and used as an index of gel stability.

[0534] The results of Figure 1 were obtained. The cross-linked alginate gel (beads) did not disintegrate even after 96 hours, and the stability of the gel was confirmed. That is, it was suggested that the structure of the prepared (bead) structure was maintained over a long period due to the formation of chemical cross-links by the Huisgen reaction. The cross-linked alginate gel (beads) prepared with (Ex18-A2) / (Ex19-A2) is the control of this test.

[0535] (Measurement of Gel Stability (2)): Stability under EDTA (Measurement of gel stability (1)) The aqueous alginic acid solutions obtained in (1) were mixed in equal amounts in the combinations of (1-1) and (19-1), (4-1) and (19-1), and (5-1) and (19-1), respectively, placed in a syringe equipped with an 18-gauge injection needle, and this syringe was installed on a syringe pump set at a flow rate of 1 mL / min. They were dropped into a calcium chloride solution with a concentration of 30 mmol / L for 30 seconds and stirred for 5 minutes to obtain alginic acid gels. The crosslinked alginic acid gels (beads) prepared with (Ex18-A2) / (Ex19-A2) were also prepared in the same manner as these. This gel was washed once with 10 mL of physiological saline and allowed to stand in physiological saline at 37°C for 10 minutes for chemical crosslinking to obtain a chemically crosslinked alginic acid gel. 19.5 mL of 5 mM dipotassium ethylenediaminetetraacetate dihydrate (EDTA·2K) / physiological saline was added to this gel, and it was shaken at 37°C. The aqueous solution was collected over time, and the same amount of 5 mM EDTA·2K / physiological saline as the collected amount was replenished. After the test ended, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and it was shaken at 37°C for 3 hours or more to completely disintegrate the gel, and the aqueous solution was collected. The alginic acid concentration in the collected aqueous solution was measured by the carbazole-sulfuric acid method, and the amount of eluted alginic acid up to each time point was expressed as a percentage by dividing it by the total amount of alginic acid calculated from the alginic acid concentration at all time points and the alginic acid concentration after the test ended. This value was defined as the disintegration rate and used as an index of gel stability.

[0536] The results are shown in Figure 2. The crosslinked alginic acid gel (beads) had a disintegration rate of 2% or less after 24 hours. That is, it was suggested that the structure prepared by forming crosslinks by the Huisgen reaction maintained its structure even under conditions of a solution without calcium ions (below the physiological concentration for the living body). The crosslinked alginic acid gel (beads) prepared with (Ex18-A2) / (Ex19-A2) was the control of this test.

[0537] (Measurement of gel stability (3)): Stability in PBS The alginate derivatives (Ex3-A2), (Ex5-A2), (Ex6-A2), (Ex9a-A2), (Ex10-A2), (Ex12-A2), and (Ex18-A2) obtained in Examples 3, 5, 6, 9, 10, 12, and 18 were dissolved in water to a concentration of 1.0% to obtain alginic acid aqueous solutions (3-1), (5-1), (6-1), (9-1), (10-1), (12-1), and (18-1), respectively. These were mixed in equal amounts in combinations of (6-1) and (9-1), (3-1) and (10-1), (5-1) and (10-1), and (18-1) and (12-1), placed in a syringe equipped with an 18-gauge injection needle, and this syringe was installed on a syringe pump set to a flow rate of 1 mL / min. They were dropped into a calcium chloride solution with a concentration of 30 mmol / L for 30 seconds and stirred for 5 minutes to obtain alginic acid gels. This gel was washed once with 10 mL of PBS and allowed to stand in PBS at 37°C for 10 minutes for chemical crosslinking to obtain a chemically crosslinked alginic acid gel. The crosslinked alginic acid gel (beads) prepared with (Ex18-A2) / (Ex19-A2) was also prepared in the same manner as these. 19.5 mL of PBS was added to this gel, and it was shaken at 37°C. The aqueous solution was collected over time, and the same amount of PBS as the collected amount was replenished. After the test, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and it was shaken at 37°C for 3 hours or more to completely disintegrate the gel, and the aqueous solution was collected. The alginic acid concentration in the collected aqueous solution was measured by the carbazole-sulfuric acid method, and the amount of eluted alginic acid up to each time point was expressed as a percentage of the total alginic acid amount calculated from the alginic acid concentration at all time points and the alginic acid concentration after the end of the test. This value was defined as the disintegration rate and used as an index of gel stability.

[0538] The results of Figure 3 were obtained. The crosslinked alginic acid gel (beads) did not disintegrate even after 96 hours, and the stability of the gel was confirmed. That is, it was suggested that the structure of the prepared (bead) structure was maintained over a long period due to the formation of chemical crosslinks by the Huisgen reaction. Note that the crosslinked alginic acid gel (beads) prepared with (Ex18-A2) / (Ex19-A2) is the control of this test.

[0539] (Measurement of Gel Stability (4)): Stability under EDTA (Measurement of Gel Stability (3)) The aqueous alginate solutions obtained in (Measurement of Gel Stability (3)) were mixed in equal amounts in the combinations of (6-1) and (9-1), (3-1) and (10-1), (5-1) and (10-1), (18-1) and (12-1), respectively, put into a syringe equipped with an 18-gauge injection needle, placed on a syringe pump set at a flow rate of 1 mL / min, dropped into a calcium chloride solution with a concentration of 30 mmol / L for 30 seconds, and stirred for 5 minutes to obtain an alginate gel. This gel was washed once with 10 mL of physiological saline and allowed to stand in physiological saline at 37°C for 10 minutes for chemical crosslinking to obtain a chemically crosslinked alginate gel. The crosslinked alginate gel (beads) prepared with (Ex18-A2) / (Ex19-A2) was also prepared in the same manner as these. 19.5 mL of 5 mM potassium ethylenediaminetetraacetate dihydrate (EDTA·2K) / physiological saline was added to this gel, shaken at 37°C, and the aqueous solution was collected over time, and the same amount of 5 mM EDTA·2K / physiological saline as the collected amount was replenished. After the test was completed, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and shaken at 37°C for 3 hours or more to completely disintegrate the gel, and the aqueous solution was collected. The alginate concentration in the collected aqueous solution was measured by the carbazole sulfuric acid method, and the amount of eluted alginate up to each time point was divided by the total amount of alginate calculated from the alginate concentration at all time points and the alginate concentration after the test was completed, and the value expressed as a percentage was defined as the disintegration rate and used as an index of gel stability.

[0540] The results are shown in Fig. 4. The crosslinked alginate gel (beads) had a disintegration rate of 4% or less after 24 hours. That is, it was suggested that the structure prepared by crosslinking formation by the Huisgen reaction maintained its structure even under conditions of a solution without calcium ions (below the physiological concentration for the living body). The crosslinked alginate gel (beads) prepared with Ex18-A2 / Ex19-A2 was the control of this test.

[0541] (Measurement of Gel Stability (5)): Stability in PBS The alginate derivatives of (Ex4-A2), (EX9a-A2), (Ex16-A2), (Ex18-A2) and (Ex20-B2) obtained in Examples 4, 9, 16, 18 and 20 were dissolved in water to a concentration of 1.0% to obtain alginate aqueous solutions (4-1), (9-1), (16-1), (18-1), (20-1), respectively. These were mixed in equal amounts in the combinations of (4-1) and (20-1), (18-1) and (9-1), (18-1) and (16-1), respectively, placed in a syringe equipped with an 18-gauge injection needle, and this syringe was placed on a syringe pump set to a flow rate of 1 mL / min, and dropped into a calcium chloride solution with a concentration of 30 mmol / L for 30 seconds and stirred for 5 minutes to obtain an alginate gel. This gel was washed once with 10 mL of PBS and allowed to stand in PBS at 37°C for 10 minutes for chemical crosslinking to obtain a chemically crosslinked alginate gel. The crosslinked alginate gel (beads) prepared with (Ex18-A2) / (Ex19-A2) was also prepared in the same manner as these. 19.5 mL of PBS was added to this gel, shaken at 37°C, and the aqueous solution was collected over time, and the same amount of PBS as the collected amount was replenished. After the test, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and shaken at 37°C for 3 hours or more to completely disintegrate the gel, and the aqueous solution was collected. The alginate concentration in the collected aqueous solution was measured by the carbazole sulfuric acid method, and the amount of eluted alginate up to each time point was divided by the total amount of alginate calculated from the alginate concentration at all time points and the alginate concentration after the test end, and the value expressed as a percentage was defined as the disintegration rate and used as an index of gel stability.

[0542] The results are shown in Figure 5. The crosslinked alginate prepared by the above method had a disintegration rate of about 12% or less even after 96 hours. That is, it was suggested that the structure of the prepared (bead) structure was maintained due to the formation of chemical crosslinking by the Huisgen reaction. The crosslinked alginate gel (beads) prepared with Ex18-A2 / Ex19-A2 is the control of this test.

[0543] (Measurement of gel stability (6)): Stability under EDTA The aqueous alginate solutions obtained in (Measurement of gel stability (5)) were respectively mixed in equal amounts in the combinations of (4-1) and (20-1), (18-1) and (9-1), and (18-1) and (16-1), put into a syringe equipped with an 18-gauge injection needle, and this syringe was placed on a syringe pump set at a flow rate of 1 mL / min. They were dropped into a calcium chloride solution with a concentration of 30 mmol / L for 30 seconds and stirred for 5 minutes to obtain alginate gels. These gels were washed once with 10 mL of physiological saline and left standing in physiological saline at 37°C for 10 minutes for chemical cross-linking to obtain chemically cross-linked alginate gels. The cross-linked alginate gels (beads) prepared with (Ex18-A2) / (Ex19-A2) were also prepared in the same manner as these. 19.5 mL of 5 mM dipotassium ethylenediaminetetraacetate dihydrate (EDTA·2K) / physiological saline was added to this gel, shaken at 37°C, and the aqueous solution was collected over time. The same amount of 5 mM EDTA·2K / physiological saline as the collected amount was replenished. After the test ended, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and it was shaken at 37°C for 3 hours or more to completely disintegrate the gel, and the aqueous solution was collected. The alginate concentration in the collected aqueous solution was measured by the carbazole-sulfuric acid method, and the amount of eluted alginate up to each time point was divided by the total amount of alginate calculated from the alginate concentration at all time points and the alginate concentration after the test ended, and the value expressed as a percentage was taken as the disintegration rate, which was used as an index of gel stability.

[0544] The results of Fig. 6 were obtained. The cross-linked alginate gel (bead) had a disintegration rate of 18% or less after 24 hours. That is, it was suggested that due to the formation of cross-links by the Huisgen reaction, the prepared structure could maintain its structure even under conditions of a solution without calcium ions (below the physiological concentration for the living body). The cross-linked alginate gel (bead) prepared with Ex18-A2 / Ex19-A2 was the control of this test.

[0545] [Measurement of gel permeability] (Measurement of gel permeability (1)) The alginic acid derivatives (Ex1-A2), (Ex3-A2), (Ex4-A2), (Ex5-A2), and (Ex18-A2) obtained in Examples 1, 3, 4, 5, and 18 were dissolved in water to a concentration of 2.0% to prepare an aqueous alginic acid solution. To this aqueous alginic acid solution, 2 / 5 volume of fluorescein isothiocyanate-dextran with a molecular weight of 150,000 prepared at 1 mg / mL (Sigma-Aldrich, FD150S), and 3 / 5 volume of water were added to obtain 1.0% aqueous alginic acid solutions (1-2), (3-2), (4-2), (5-2), and (18-2) containing 0.2 mg / mL fluorescein isothiocyanate-dextran.

[0546] Furthermore, the alginic acid derivatives (Ex10-A2), (Ex12-A2), and (Ex19-A2) obtained in Examples 10, 12, and 19 were dissolved in water to a concentration of 1.0% to obtain aqueous alginic acid solutions (10-1), (12-1), and (19-1), respectively.

[0547] These were respectively mixed in equal amounts in the combinations of (1-2) and (19-1), (4-2) and (19-1), (5-2) and (19-1), (3-2) and (10-1), (18-2) and (12-1). 40 mL of a calcium chloride solution with a concentration of 30 mmol / L was added, and the mixture was stirred for 5 minutes to obtain an alginate gel. This gel was washed once with 10 mL of physiological saline and left standing in physiological saline at 37°C for 10 minutes for chemical cross-linking to obtain a fluorescein isothiocyanate-dextran encapsulated chemically cross-linked alginate gel. The fluorescein isothiocyanate-dextran encapsulated chemically cross-linked alginate gel prepared with (Ex18-A2) / (Ex19-A2) was also prepared in the same manner. 19.5 mL of physiological saline was added to this gel, and it was shaken at 37°C. The aqueous solution was collected over time, and the same amount of physiological saline as the collected amount was replenished. After the test ended, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and it was shaken at 37°C for 3 hours or more to completely disintegrate the gel, and the aqueous solution was collected. The dextran concentration in the collected aqueous solution was measured by fluorescence quantification (excitation light: 485 nm, fluorescence: 535 nm), and the value obtained by dividing the amount of dextran up to each time point by the total amount of dextran after the test ended and expressing it as a percentage was defined as the transmittance.

[0548] The results shown in Fig. 7 were obtained. The transmittance after 24 hours was in the range of 25% to 40%. Incidentally, the fluorescein isothiocyanate-dextran encapsulated chemically cross-linked alginate gel prepared with (Ex18-A2) / (Ex19-A2) was the control of this test.

[0549] (Measurement of Gel Permeability (2)) The alginate derivatives (Ex4-A2), (Ex5-A2), (Ex6-A2), and (Ex18-A2) obtained in Examples 4, 5, 6, and 18 were dissolved in water to a concentration of 2.0% to prepare an aqueous alginate solution. To this aqueous alginate solution, 2 / 5 volume of fluorescein isothiocyanate-dextran with a molecular weight of 150,000 prepared at 1 mg / mL (Sigma-Aldrich, FD150S), and 3 / 5 volume of water were added to obtain 1.0% aqueous alginate solutions (4-2), (5-2), (6-2), and (18-2) containing 0.2 mg / mL fluorescein isothiocyanate-dextran.

[0550] Furthermore, the alginate derivatives (EX9a-A2), (Ex10-A2), (Ex16-A2), and (Ex20-B2) obtained in Examples 9, 10, 16, and 20 were dissolved in water to a concentration of 1.0% to obtain aqueous alginate solutions (9-1), (10-1), (16-1), and (20-1), respectively.

[0551] These were each mixed in equal amounts in the combinations of (4-2) and (20-1), (5-2) and (10-1), (6-2) and (9-1), (18-2) and (9-1), (18-2) and (16-1), and 40 mL of a calcium chloride solution with a concentration of 30 mmol / L was added, followed by stirring for 5 minutes to obtain an alginate gel. This gel was washed once with 10 mL of physiological saline and allowed to stand in physiological saline at 37 °C for 10 minutes for chemical crosslinking to obtain a fluorescein isothiocyanate-dextran encapsulated chemically crosslinked alginate gel. The fluorescein isothiocyanate-dextran encapsulated chemically crosslinked alginate gel prepared with (Ex18-A2) / (Ex19-A2) was also prepared in the same manner as these. 19.5 mL of physiological saline was added to this gel, and it was shaken at 37 °C. The aqueous solution was collected over time, and the same amount of physiological saline as the collected amount was replenished. After the test was completed, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and it was shaken at 37 °C for 3 hours or more to completely disintegrate the gel, and the aqueous solution was collected. The dextran concentration in the collected aqueous solution was measured by fluorescence quantification (excitation light: 485 nm, fluorescence: 535 nm), and the value obtained by dividing the amount of dextran up to each time point by the total amount of dextran after the test was completed and expressing it as a percentage was defined as the transmittance.

[0552] The results of Fig. 8 were obtained. The transmittance after 24 hours was in the range of 25% to 30%. The fluorescein isothiocyanate-dextran encapsulated chemically crosslinked alginate gel prepared with Ex18-A2 / Ex19-A2 was the control of this test.

[0553] [Evaluation of Biocompatibility of Crosslinked Alginate Derivative (Gel)] Each of the alginate derivatives (EX4-A2), (EX5-A2), (EX12-A2), (EX16-A2), (EX18-A2), (EX19-A2), and (EX20-B2) obtained in Examples 4, 5, 12, 16, 18, 19, and 20 was dissolved in water to obtain a reactive group-introduced alginate solution. After filtering and sterilizing this with a Mini Sart High Flow (Sartorius, 16532GUK), a 1.0% reactive group-introduced alginate / physiological saline aqueous solution was prepared. Cell concentration 5×10 3HeLa cells seeded in a 96-well plate at a density of cells / well and cultured for 1 day were added with an aqueous solution of 1.0% reactive group-introduced alginic acid / sodium chloride at a final concentration of 0.1% in combinations of (EX18-A2) and (EX19-A2), (Ex5-A2) and (EX19-A2), (Ex4-A2) and (Ex20-B2), (EX18-A2) and (Ex12-A2), or (Ex16-A2). After culturing for 1 day, ATP activity was evaluated as an index of cytotoxicity using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571).

[0554] The results shown in Fig. 9 were obtained. Since ATP activity was confirmed in all crosslinked alginic acid gels evaluated by the above method, it was suggested that the crosslinked alginic acid gels had no cytotoxicity, and it was suggested that the alginic acid structure (beads) in which chemical crosslinking was formed by the Huisgen reaction had biocompatibility.

Claims

**Claim 1**: The following formula (I) in which a cyclic alkyne group (Akn) is introduced into any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker (-L1-): 【Chemical Formula 1】 [In formula (I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L1- represents a linker selected from the group consisting of the following table: 【Table 1-1】 【Table 1-2】 The partial structural formulas described [in each formula, the outside of the dashed lines at both ends is not included]; Akn represents a cyclic alkyne group selected from the group consisting of the following table: 【Table 2】 The partial structural formulas described [in each formula, the right side of the dashed line is not included]], which is represented by an alginic acid derivative. **Claim 2**: The alginic acid derivative according to claim 1, wherein the divalent linker (-L1-) is a linker selected from the group consisting of the following table: 【Table 3】 The partial structural formulas described [in each formula, the outside of the dashed lines at both ends is not included]. **Claim 3**: The alginic acid derivative according to claim 1, wherein Akn is a cyclic alkyne group selected from the group consisting of the following table: 【Table 4】 The partial structural formulas described [in each formula, the right side of the dashed line is not included]. **Claim 4**: The alginic acid derivative according to claim 1, wherein the combination of Akn-L1- is a partial structure selected from the group of the following partial structural formulas: [Chemical Formula 2] The partial structures described [in each formula, the right side of the dashed line is not included]. **Claim 5**: The alginic acid derivative according to claim 1, wherein the introduction rate of the Akn-L1-NH2 group (Akn and -L1- are the same as defined in claim 1) is 0.1% to 30%. **Claim 6**: The alginic acid derivative according to claim 1, wherein the weight average molecular weight measured by gel filtration chromatography of the alginic acid derivative is 100,000 Da to 3,000,000 Da. **Claim 7**: The alginic acid derivative according to claim 1, which has biocompatibility.

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