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 a novel alginic acid derivative, a novel crosslinked alginic acid, a novel crosslinked alginic acid structure, and a method for producing the same. [Background technology]

[0002] Alginic acid is a polymeric acidic polysaccharide molecule extracted from the cell walls of natural brown algae such as Lessonia, Macrocystis, Laminaria, Ascophyllum, Durvillea, Ecklonia cava, Eisenia bifidum, and Laminaria kelp. It is a linear heteropolymer consisting of 1-4 bonds between two types of uronic acid, β-D-mannuronic acid (M component) and its C-5 epimer, α-L-guluronic acid (G component). Specifically, its chemical structure is a block copolymer consisting of a homopolymer block of mannuronic acid (MM), a homopolymer block of guluronic acid (GG), and a block of randomly arranged mannuronic acid and guluronic acid (MG) in a complex combination in any order and ratio. Alginic acid is widely used in the fields of medicine, biotechnology, cosmetics, textiles, papermaking, food, etc.

[0003] Monovalent alginic acid salts, such as alkali metal alginates (e.g., sodium alginate, etc.), are water-soluble, but divalent alginic acid salts, such as alkaline earth metal alginates (e.g., calcium alginate, etc.), have the property of being cross-linked by metal ions and gelling (becoming insoluble), and attempts are being made to take advantage of this property to modify or mold them into products suitable for various uses.

[0004] In order to explore the possibility of modifying or molding polysaccharides (e.g., hyaluronic acid, chondroitin sulfate, alginic acid, etc.) into various materials and improving their physical properties (e.g., strength, swelling, etc.), various studies have been conducted, for example, on cross-linked polysaccharides cross-linked by covalent bonds.

[0005] Specific methods for obtaining crosslinked polysaccharides include (1) a crosslinking method using an aldehyde crosslinking agent such as formaldehyde (Patent Document 1: WO 2011 / 028031), (2) a self-crosslinking method using carboxyl groups and hydroxyl groups in polysaccharides (Patent Document 2: WO 89 / 10941), and (3) a crosslinking method using a homobifunctional crosslinking agent (diepoxide, divinyl sulfone, diamine, dihydrazide, etc.) or a heterobifunctional crosslinking agent (epihalohydrin, etc.) (Patent Document 3: WO 2009 / 073437).

[0006] Also known are (4) a crosslinking method in which a photoreactive group (cinnamic acid, substituted cinnamic acid, acrylic acid, maleic acid, fumaric acid, furylacrylic acid, thiopheneacrylic acid, cinnamylideneacetic acid, sorbic acid, thymine, coumarin, or the like) is introduced followed by light irradiation (Patent Documents 4 and 5: International Publication No. WO 2005 / 026214 and JP 09-87236 A), and (5) a crosslinking method in which polysaccharides having thiol groups introduced therein are crosslinked with each other via disulfide bonds, and a crosslinking method in which a polysaccharide having a thiol group introduced therein and a polysaccharide having a maleimide group introduced therein are subjected to a Michael addition reaction (Patent Document 6: International Publication No. WO 2008 / 071058).

[0007] Furthermore, as a method for covalently crosslinking polysaccharides, (6) a crosslinking method using a polysaccharide having an alkyne group introduced therein and a polysaccharide having an azide group introduced therein in a Huisgen reaction (1,3-dipolar cycloaddition reaction) is known.

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

[0009] However, (i) Patent Document 7 relates to a crosslinked polysaccharide obtained by subjecting a chain-like alkyne group and an azide group introduced via a linker to a first polysaccharide selected from hyaluronic acid, a second polysaccharide selected from chondroitin, sulfated dermatan, alginic acid or a salt thereof, and the like, in the presence of a copper catalyst, and does not disclose a novel crosslinked alginic acid, which will be described later.

[0010] Furthermore, (ii) Patent Document 8 relates to a crosslinked polysaccharide obtained by subjecting the first and second polysaccharides, which are selected from hyaluronic acid, carboxymethyldextran, cellulose derivatives, and chitosan (the first and second polysaccharides may be the same or different), to a Huisgen reaction of a cyclic alkyne group and an azide group introduced into each polysaccharide via a linker (an ester bond is formed between the polysaccharide and the linker); however, the document does not disclose a novel crosslinked alginic acid, which will be described later.

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

[0012] Furthermore, (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 (an ester bond between the polysaccharide and the linker) to a Huisgen reaction, in which the first polysaccharide is chitosan and the second polysaccharide is sodium alginate. However, the document does not disclose a novel crosslinked alginic acid, which will be described later.

[0013] In addition, WO 2016 / 019391 (Patent Document 11) and WO 2017 / 165389 (Patent Document 12) describe alginic acid having an azide group introduced into the side chain, but do not disclose a cross-linked alginic acid structure formed from alginic acid having an alkyne group introduced into the side chain, and the purpose of use thereof is also different from that of the present invention.

[0014] Furthermore, Non-Patent Document 1 describes a branched alginic acid (bAlg-DBCO) having a cyclooctyne side chain introduced into the side chain. However, this was obtained by reacting branched alginic acid (bAlg), which was synthesized from alginic acid and branched polyethylene glycol (4-arm PEG-NH2), with aminated cyclooctyne (DBCO-PEG-amine). This has a different structure from the novel alginic acid derivatives described below, and its intended use is also different. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] International Publication No. 2011 / 028031 Brochure [Patent Document 2] International Publication No. 89 / 10941 Brochure [Patent Document 3] International Publication No. 2009 / 073437 Brochure [Patent Document 4] International Publication No. 2005 / 026214 Brochure [Patent Document 5] Japanese Patent Application Publication No. 9-87236 [Patent Document 6] International Publication No. 2008 / 071058 Brochure [Patent Document 7] International Publication No. 2008 / 031525 Brochure [Patent Document 8] International Publication No. 2012 / 165462 Brochure [Patent Document 9] International Publication No. 2015 / 020206 Brochure [Patent Document 10] China Patent Application Publication No. 106140040 [Patent Document 11] International Publication No. 2016 / 019391 Brochure [Patent Document 12] International Publication No. 2017 / 165389 Brochure [Patent Document 13] International Publication No. 2019 / 240219 Brochure [Non-patent literature]

[0016] [Non-Patent Document 1] Nat Commun.9(1), p2195-, 2018. Summary of the Invention [Problem to be solved by the invention]

[0017] In the above circumstances, there has been a demand for 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 the same. [Means for solving the problem]

[0018] As a result of intensive research to solve the above problems, the present inventors have found novel alginic acid derivatives represented by formula (I) or formula (II). Furthermore, they have found that beads (dye-containing beads), which are one type of crosslinked alginic acid structure, are formed using novel crosslinked alginic acids obtained by subjecting the novel alginic acid derivatives of formula (I) and formula (II) to a Huisgen reaction, and have found that the beads have high stability and can be adjusted to have a transmittance according to the purpose compared to conventional gels, and have completed the present invention.

[0019] The novel alginic acid derivatives (formula (I) and formula (II)) provided herein can be used, for example, for chemical crosslinking, i.e., they have a reactive group that can be used for chemical crosslinking or a complementary reactive group to the reactive group introduced therein.

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

[0021] The Huisgen reaction using terminal alkyne groups and terminal azide groups generally requires heating at 100°C or higher, and therefore this reaction was not suitable for chemical modification of biomolecules. However, by allowing a copper catalyst (e.g., Cu(I)) to coexist in the reaction, reaction conditions were found under which a cyclized adduct (triazole ring) was formed at room temperature in almost 100% yield (Angew.Chem.Int.Ed.Engl.,41,p2596-2599,2002; J.Org.Chem.,67,p3057-3064,2002), making it possible to use the reaction for chemical modification of biomolecules. On the other hand, when crosslinked alginic acid is obtained by the Huisgen reaction in the presence of the copper catalyst, there is a possibility that a trace amount of copper catalyst remains in the crosslinked alginic acid, which raises concerns that copper-derived cytotoxicity may be expressed in the crosslinked alginic acid or crosslinked alginic acid structure.

[0022] In a preferred embodiment, in order to avoid the occurrence of copper-derived cytotoxicity in crosslinked alginic acid, crosslinked alginic acid is obtained by utilizing the Huisgen reaction that does not require a copper catalyst. Specifically, by using a cyclooctyne derivative (a highly distorted cyclic alkyne group) as the alkyne group introduced into the alginic acid derivative, the reaction can be carried out without the need for high temperature conditions of 100°C or higher and a copper catalyst. Therefore, since the novel crosslinked alginic acid of a preferred embodiment does not contain a copper catalyst, it is also excellent in terms of not exhibiting copper-derived toxicity even when molded into a final product (crosslinked alginic acid structure).

[0023] Here, there are provided alginic acid derivatives of formula (I) or (II) in which a cyclic alkyne group or azide group has been introduced via an amide bond and a divalent linker to any one or more carboxyl groups of alginic acid shown in the following embodiments, novel crosslinked alginic acid and crosslinked alginic acid structures 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 alginic acid derivatives, crosslinked alginic acid, and crosslinked alginic acid structures. That is, exemplary embodiments may be as follows: [1] to

[23] .

[0024] [1] An amide bond and a divalent linker (-L 1 A cyclic alkyne group (Akn) is introduced via a ring structure represented by the following formula (I): [ka] [In formula (I), Akn, -L 1 -, -NHCO- and (ALG) are defined as in the first embodiment described below], and an alginic acid derivative having an amide bond and a divalent linker (-L 2 A compound of the following formula (II) in which an azide group is introduced via an azide group (-): [ka] [In formula (II), -L 2 -, -NHCO- and (ALG) are the same as those in the first embodiment described below. The alginic acid derivative represented by the formula (I) is used to crosslink the cellulose acetate copolymer. Cross-linked alginate.

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

[0026] [1-Ib] An alginic acid derivative represented by formula (I) described in [1] above, which has a weight-average molecular weight of 100,000 Da to 3,000,000 Da as measured by gel filtration chromatography.

[0027] [1-IIa]N3-L 2 -NH2 group(-L 2 The alginic acid derivative represented by formula (II) described in [1] above, in which the introduction rate of (- is the same as that in the first embodiment described later) is 0.1% to 30%.

[0028] [1-IIb] An alginic acid derivative represented by formula (II) according to [1] above, which has a weight-average molecular weight of 100,000 Da to 3,000,000 Da as measured by gel filtration chromatography.

[0029] [2] An amide bond and a divalent linker (-L 1 A cyclic alkyne group (Akn) is introduced via a cyclic alkyne group (Akn) represented by the following formula (I): [ka] [In formula (I), (ALG), -L 1 -, the definition of Akn is the same as that in the second embodiment described below.

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

[0031] [4] An alginic acid derivative represented by formula (I) according to [2] above, which has a weight-average molecular weight of 100,000 Da to 3,000,000 Da as measured by gel filtration chromatography.

[0032] [5] An amide bond and a divalent linker (-L 2 -), an azide group is introduced via the following formula (II): [ka] [In formula (II), (ALG), -L 2 The definition of - is the same as that in the fifth embodiment described below.

[0033] [6] N3-L 2 -NH2 group(-L 2 An alginic acid derivative represented by formula (II) according to [5] above, in which the introduction rate of the substituent (- is the same as defined in the sixth aspect described below) is 0.1% to 30%.

[0034] [7] An alginic acid derivative represented by formula (II) according to [5] above, which has a weight-average molecular weight of 100,000 Da to 3,000,000 Da as measured by gel filtration chromatography.

[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): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends represent amide bonds via any carboxyl group of alginic acid; 1 -, -L 2 -, and X are as defined in the eighth embodiment described below.

[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): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends represent amide bonds via any carboxyl group of alginic acid; 1 -, -L 2 -, and X is as defined in embodiment 8a below.

[0037] [8-1] The crosslinked alginic acid according to [1] or [8a], which includes chemical crosslinks via triazole rings formed by the Huisgen reaction as crosslinks, and ionic crosslinks partially formed by divalent metal ions. [8-2] The crosslinked alginic acid according to [1] or [8a], which includes a chemical crosslink via a triazole ring formed by the Huisgen reaction as the crosslink.

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

[0040] [9] A method for producing a crosslinked alginic acid, comprising mixing an alginic acid derivative represented by formula (I) described in [1] above with an alginic acid derivative represented by formula (II) and carrying out a crosslinking reaction (Huisgen reaction) to obtain the crosslinked alginic acid described in [1] or [8a] above.

[0041] [9-1] A method for producing crosslinked alginic acid, comprising adding a solution of an alginic acid derivative represented by formula (I) described in [1] above to a solution of an alginic acid derivative represented by formula (II) described in [1] above to perform a crosslinking reaction (Huisgen reaction) to obtain the crosslinked alginic acid described in [1] or [8a] above.

[0042] [9-2] A method for producing crosslinked alginic acid, comprising adding a solution of an alginic acid derivative represented by formula (II) described in [1] above to a solution of an alginic acid derivative represented by formula (I) described in [1] above to perform a crosslinking reaction (Huisgen reaction) to obtain the crosslinked alginic acid described in [1] or [8a] above.

[0043]

[10] The chemical crosslink formed by carrying out a Huisgen reaction (crosslinking reaction) using the alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) described in [1] above is represented by the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends, -L 1 - and -L 2 A method for producing crosslinked alginic acid, comprising obtaining the crosslinked alginic acid described in [1] or [8a] above, wherein - is the same as defined in the tenth aspect described below.

[0044]

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

[0045] [11-1] A crosslinked alginate structure obtained by dropping a solution of an alginate derivative represented by formula (I) described in [1] above into a solution containing a divalent metal ion to obtain a gel, and then adding the gel to a solution of an alginate derivative represented by formula (II) described in [1] above to cause a crosslinking reaction.

[0046] [11-2] A crosslinked alginate structure obtained by dropping a solution of an alginate derivative represented by formula (II) described in [1] above into a solution containing a divalent metal ion to obtain a gel, and then adding the gel to a solution of an alginate derivative represented by formula (I) described in [1] above to cause a crosslinking reaction.

[0047] [12-1] The crosslinked alginate structure according to any one of

[11] to [11-2], wherein the crosslinks are chemical crosslinks via triazole rings formed by the Huisgen reaction, and ionic crosslinks partially formed by divalent metal ions.

[0048] [12-2] The crosslinked alginate structure according to any one of the above

[11] to [11-2], wherein the crosslinks comprise chemical crosslinks via triazole rings formed by a Huisgen reaction.

[0049] [12-3-1] In the above [12-1], the divalent metal ion is an ion selected from the group consisting of a calcium ion, a magnesium ion, a barium ion, a strontium ion, and a 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] [13-1] A crosslinked alginate structure having the ability to retain contents, obtained by subjecting an alginate derivative represented by formula (I) described in [1] above and an alginate derivative represented by formula (II) to ionic crosslinking with a divalent metal ion and chemical crosslinking by the Huisgen reaction.

[0052] [13-2] A crosslinked alginate structure having content retention properties, obtained by chemically crosslinking an alginate derivative represented by formula (I) and an alginate derivative represented by formula (II) described in [1] above through the Huisgen reaction.

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

[0054] [13-3-2] In the above [13-1], 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.

[0055]

[14] The chemical crosslink formed by the Huisgen reaction using the alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) described in [1] above is represented by the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends, -L 1 - and -L 2 The crosslinked alginate structure according to any one of

[11] to [13-2] above, wherein - is as defined in the fourteenth aspect described below.

[0056]

[15] A method for producing a crosslinked alginate structure, comprising dropping a mixed solution of an alginate derivative represented by formula (I) described in [1] above and an alginate derivative represented by formula (II) into a solution containing a divalent metal ion to carry out a crosslinking reaction.

[0057] [15-1] A method for producing a crosslinked alginic acid structure, comprising dropping a solution of an alginic acid derivative represented by formula (I) described in [1] above into a solution containing a divalent metal ion to obtain a gel, and then adding the gel to a solution of an alginic acid derivative represented by formula (II) described in [1] above to cause a crosslinking reaction.

[0058] [15-2] A method for producing a crosslinked alginic acid structure, comprising dropping a solution of an alginic acid derivative represented by formula (II) described in [1] above into a solution containing a divalent metal ion to obtain a gel, and then adding the gel to a solution of an alginic acid derivative represented by formula (I) described in [1] above to cause a crosslinking reaction.

[0059] [16-1] A method for producing a crosslinked alginate structure described in any one of

[15] to [15-2] above, wherein the crosslinks include chemical crosslinks via triazole rings formed by the Huisgen reaction and ionic crosslinks partially formed by divalent metal ions.

[0060] [16-2] A method for producing a crosslinked alginate structure according to any one of the above

[15] to [15-2], wherein the crosslinking is a chemical crosslinking via a triazole ring formed by a Huisgen reaction.

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

[0062] [16-3-2] In the above [16-1], 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.

[0063]

[17] The chemical crosslink formed by the Huisgen reaction using the alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to any one of the above [1] is represented by the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends, -L 1 - and -L 2 The method for producing a crosslinked alginic acid structure according to any one of the above

[15] to [16-2], wherein the structure is:

[0064]

[18] The crosslinked alginate structure according to any one of the above

[11] to

[14] , which is in the form of beads or a roughly spherical gel.

[0065]

[19] A medical material comprising the crosslinked alginate structure according to any one of

[11] to

[14] above.

[0066]

[20] The medical material according to

[19] above, which is a bead or an approximately spherical gel.

[0067]

[21] A biocompatible crosslinked alginic acid according to [1] or [8a], an alginic acid derivative according to [2] or [5], or a crosslinked alginic acid structure according to any one of

[11] to

[14] .

[0068]

[22] The following formula (AM-1): [ka] [In formula (AM-1), -L 1 -, and Akn are defined as in the twenty-second embodiment described below], or a pharma- ceutically acceptable salt thereof, or a solvate thereof.

[0069]

[23] The following formula (AM-2): [ka] [In formula (II), -L 2 The definition of - is as described in the 23rd aspect below. 2 - An amino compound represented by the following definition: wherein R is an integer from 1 to 5; or a pharma- ceutically acceptable salt thereof, or a solvate thereof. Effect 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, and the like that can be used for chemical crosslinking. Preferably, the alginic acid derivative is one in which a reactive group not present in living organisms has been introduced, and even if unreacted groups remain, there is no risk of crosslinking with biological components such as cells, and so it is expected to be safe for living organisms. In addition, preferably, the crosslinking reaction is completed at room temperature without using a metal catalyst, so that it is safe and easy to use. In some embodiments, the crosslinked alginic acid is chemically crosslinked by the Huisgen reaction (1,3-dipolar cycloaddition reaction). The crosslinking can be performed by combining chemical crosslinking with ionic crosslinking using divalent metal gold ions (e.g., calcium ions), and the stability is preferably improved compared to non-crosslinked alginic acid or non-chemically crosslinked alginic acid (e.g., calcium ion-crosslinked crosslinked alginic acid) by adjusting the reaction conditions. In addition, it is preferable that the gel properties of the crosslinked body 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 the drawings]

[0071] [Figure 1] FIG. 1 shows the evaluation of gel stability of cross-linked alginate structures. [Diagram 2] FIG. 13. Evaluation of gel stability under EDTA of cross-linked alginate constructs. [Diagram 3] FIG. 1 shows the evaluation of gel stability of cross-linked alginate structures. [Figure 4] FIG. 13. Evaluation of gel stability under EDTA of cross-linked alginate constructs. [Diagram 5] FIG. 1 shows the evaluation of gel stability of cross-linked alginate structures. [Figure 6] FIG. 13. Evaluation of gel stability under EDTA of cross-linked alginate constructs. [Figure 7] FIG. 1 shows the evaluation of the gel transmittance of a cross-linked alginate structure. [Figure 8] FIG. 1 shows the evaluation of the gel transmittance of a cross-linked alginate structure. [Figure 9]FIG. 1 shows an evaluation of the biocompatibility of a gel of a cross-linked alginic acid derivative. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] [Specific aspects] The following aspects [1] to

[23] may be included. [1] The first aspect is as follows: An amide bond and a divalent linker (-L) are bonded to any one or more carboxyl groups of alginic acid. 1 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 2 A crosslinked alginic acid obtained by carrying out a crosslinking reaction using an alginic acid derivative represented by the following formula (II) having an azide group introduced therein via an alkyl group (R-1).

[0073] [Alginic acid derivative represented by formula (I)] The following formula (I): [ka] [In formula (I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 1 -See the table below: [Table 1-1] [Table 1-2] [In each formula, the areas outside the dashed lines at both ends are not included]; Akn is shown in the table below: [Table 2] An alginic acid derivative represented by the partial structural formula (I) shown below (each formula does not include the right side of the dashed line), which represents a cyclic alkyne group selected from the group consisting of:

[0074] [Alginic acid derivative represented by formula (II)] The following formula (II): [ka] In formula (II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 2 -See the table below: [Table 3-1] [Table 3-2] [In each formula, the outside of the broken lines at both ends is not included]] (provided that in the alginic acid derivative represented by formula (I), -L 1 - is any one linker selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11) and (L1-12); and an alginic acid derivative represented by formula (II), 2 (Excluding cross-linked alginic acid obtained by carrying out a cross-linking reaction using a derivative in which - is the linker of (L2-10).

[0075] [1-1-1] In the formula (I) of the above aspect [1], -L 1 is preferably as shown in the following table: [Table 4-1] [Table 4-2] (In each formula, the areas outside the dashed lines at both ends are not included.) (However, when carrying out the crosslinking reaction, -L 2When 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 Among these, the linkers (L1-1), (L1-2a), (L1-2b), (L1-11) and (L1-12-p1) are excluded from the preferred embodiments.

[0076] [1-1-2] In the formula (I) of the above aspect [1], -L 1 More preferably, the compound according to the following table: [Table 5-1] [Table 5-2] (In each formula, the areas outside the dashed lines at both ends are not included.) (However, when carrying out the crosslinking reaction, -L 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 Among these, the linkers (L1-1-1), (L1-2a-1), (L1-2b-1), (L1-11-1) and (L1-12-p2) are excluded from the more preferred embodiments.

[0077] [1-1-3] In the formula (I) of the above aspect [1], -L 1 More preferably, [Table 6-1] [Table 6-2] (In each formula, the areas outside the dashed lines at both ends are not included.) (However, when carrying out the crosslinking reaction, -L 2When 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 Among these, the linkers (L1-1-X), (L1-2-X), (L1-11-X) and (L1-12-X) are excluded from further preferred embodiments.

[0078] [1-2-1] In the formula (I) of the above aspect [1], Akn is preferably any one of the following tables: [Table 7] [In each formula, the right side of the dashed line is not included].

[0079] [1-2-2] In the formula (I) of the above aspect [1], Akn is more preferably any one of the following tables: [Table 8] [In each formula, the right side of the dashed line is not included].

[0080] [1-2-3] In the formula (I) of the above aspect [1], Akn is more preferably any one of the following tables: [Table 9] [In each formula, the right side of the dashed line is not included].

[0081] [1-3-1] In the formula (II) of the above aspect [1], -L 2 is preferably as shown in the following table: [Table 10] (In each formula, the areas outside the dashed lines at both ends are not included.) (However, when carrying out the crosslinking reaction, -L1 In the case where an alginic acid derivative represented by formula (I) is used, -L in the above table is any one of the linkers selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11), (L1-12), (L1-1-1), (L1-2a-1), (L1-2b-1), (L1-11-1), (L1-12-p1), (L1-1-X), (L1-2-X), (L1-11-X) and (L1-12-X). 2 -Of these, the linker (L2-10-p1) is excluded from the preferred embodiments.

[0082] [1-3-2] In the formula (II) of the above aspect [1], -L 2 More preferably, the compound according to the following table: [Table 11] (In each formula, the areas outside the dashed lines at both ends are not included.) (However, when carrying out the crosslinking reaction, -L 1 In the case where an alginic acid derivative represented by formula (I) is used, -L in the above table is any one of the linkers selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11), (L1-12), (L1-1-1), (L1-2a-1), (L1-2b-1), (L1-11-1), (L1-12-p1), (L1-1-X), (L1-2-X), (L1-11-X) and (L1-12-X). 2 -Of these, the linker (L2-10-p2) is excluded from the more preferred embodiment.

[0083] [1-3-3] In the formula (II) of the above aspect [1], -L 2 More preferably, [Table 12] (In each formula, the areas outside the dashed lines at both ends are not included.) (However, when carrying out the crosslinking reaction, -L 1In the case where an alginic acid derivative represented by formula (I) is used, -L in the above table is any one of the linkers selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11), (L1-12), (L1-1-1), (L1-2a-1), (L1-2b-1), (L1-11-1), (L1-12-p1), (L1-1-X), (L1-2-X), (L1-11-X) and (L1-12-X). 2 Of these, the linker (L2-10-X) is excluded from the more preferred embodiments.

[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 2When 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 (L1-1-1), (L1-2a-1), (L1-2b-1), (L1-11-1) and (L1-12-p1) are excluded from the more preferred embodiments.

[0086] [1-4-3] In the formula (I) of the above aspect [1], Akn and -L 1 - more preferably the combination of the formula [Table 15] 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 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-X), (L1-2-X), (L1-11-X) and (L1-12-X) are excluded from further preferred embodiments.

[0087] [1-4-4] In the formula (I) of the above aspect [1], Akn and -L 2 - is particularly preferably a combination of the following partial structural formula: [ka] (However, when the crosslinking reaction is performed, -L 2 In the case where 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, the alginic acid derivative represented by formula (II) has the following partial structural formula: [ka] (partial structures selected from the group: are excluded from particularly preferred embodiments).

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

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

[0090] [1-Ib] The embodiment of 1-Ib is as follows: An alginic acid derivative represented by formula (I) according to the embodiment [1], which has a weight-average molecular weight of about 100,000 Da to about 3,000,000 Da as measured by gel filtration chromatography.

[0091] [1-Ib-1] In the above aspect [1-Ib], the weight-average molecular weight of the alginic acid derivative measured by gel filtration chromatography is preferably about 300,000 Da to about 2.5 million Da, more preferably about 500,000 Da to about 1 million Da.

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

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

[0094] [1-Id] The embodiment of 1-Id is as follows: An alginic acid derivative represented by formula (II) according to the embodiment [1], which has a weight-average molecular weight of about 100,000 Da to about 3,000,000 Da as measured by gel filtration chromatography.

[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 about 300,000 Da to about 2.5 million Da, more preferably about 500,000 Da to about 1 million Da.

[0096] A preferred embodiment of the above aspect [1], further Akn, -L 1 - and -L 2 By appropriately combining the definitions of -, any preferred embodiment of the cross-linked alginic acid of the above embodiment [1] can be formed.

[0097] [2] The second embodiment is as follows: An amide bond and a divalent linker (-L) are bonded to any one or more carboxyl groups of alginic acid. 1 A cyclic alkyne group (Akn) is introduced via a cyclic alkyne group (Akn) represented by the following formula (I): [ka] [In formula (I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 1 -See the table below: [Table 16-1] [Table 16-2] [In each formula, the areas outside the dashed lines at both ends are not included]; Akn is shown in the table below: [Table 17] An alginic acid derivative represented by the partial structural formula (I) shown below (each formula does not include the right side of the dashed line), which represents a cyclic alkyne group selected from the group consisting of:

[0098] [2-1] In the formula (I) of the above aspect [2], -L 1 is preferably as shown in the following table: [Table 18-1] [Table 18-2] [In each formula, the areas outside the dashed lines at both ends are not included]; More preferably, the following table: [Table 19-1] [Table 19-2] [In each formula, the areas outside the dashed lines at both ends are not included]; More preferably, the following table: [Table 20-1] [Table 20-2] [In each formula, the areas outside the dashed lines at both ends are not included].

[0099] [2-2] In the formula (I) of the above aspect [2], Akn is preferably any one of the following tables: [Table 21] [In each formula, the right side of the dashed line is not included], and More preferably, [Table 22] [In each formula, the right side of the dashed line is not included], and More preferably, the following table: [Table 23] [In each formula, the right side of the dashed line is not included].

[0100] [2-3] In the formula (I) of the above aspect [2], Akn and -L 1 - the combination is preferably as shown in the following table: [Table 24] (Akn, -L in the table) 1 - each formula is as described in embodiment [2] above); More preferably, Akn-L 1 -The combination is as shown in the table below: [Table 25] (Akn, -L in the table) 1 - each formula is as described in the above embodiments [2-1] and [2-2]; More preferably, Akn-L 1 -The combination is as shown in the table below: [Table 26] (Akn, -L in the table) 1 - each formula is as described in the above embodiments [2-1] and [2-2]; Particularly preferably, Akn-L 1 The combination of - is represented by the following partial structural formula: [ka] The structure is as shown by a partial structure selected from the group consisting of:

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

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

[0103] [3-1] In the above aspect [3], Akn-L 1 The introduction rate of -NH2 groups 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 formula (I) according to the above aspect [2], which has a weight-average molecular weight of about 100,000 Da to about 3,000,000 Da as measured by gel filtration chromatography.

[0105] [4-1] In the above aspect [4], the weight-average molecular weight of the alginic acid derivative measured by gel filtration chromatography 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 embodiment is as follows: An amide bond and a divalent linker (-L 2 -), an azide group is introduced via the following formula (II): [ka] [In formula (II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 2 -See the table below: [Table 27-1] [Table 27-2] (wherein each formula does not include the areas outside the dashed lines at both ends)

[0107] [5-1] In the alginic acid derivative of the formula (II) according to the above aspect [5], -L 2 is preferably as shown in the following table: [Table 28] [In each formula, the areas outside the dashed lines at both ends are not included]; More preferably, the following table: [Table 29] [In each formula, the areas outside the dashed lines at both ends are not included]; More preferably, the following table: [Table 30] [In each formula, the areas outside the dashed lines at both ends are not included].

[0108] [5a] The embodiment of 5a is as follows: An amide bond and a divalent linker (-L) are bonded to any one or more carboxyl groups of alginic acid. 2 -), an azide group is introduced via the following formula (II): [ka] [In formula (II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 2 -See the table below: [Table 31-1] [Table 31-2] (wherein each formula does not include the areas outside the dashed lines at both ends)

[0109] [5a-1] In the alginic acid derivative of the above formula (II) according to the above aspect [5a], -L 2 is preferably as shown in the following table: [Table 32] [In each formula, the areas outside the dashed lines at both ends are not included]; More preferably, the following table: [Table 33] [In each formula, the areas outside the dashed lines at both ends are not included]; More preferably, the compound according to the following table: [Table 34] [In each formula, the areas outside the dashed lines at both ends are not included].

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

[0111] [6-1] In the above aspect [6], N3-L 2 The introduction rate of -NH2 groups 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) according to the above aspect [5] or aspect [5a], wherein the weight-average molecular weight of the alginic acid derivative measured by gel filtration chromatography is about 100,000 Da to about 3,000,000 Da.

[0113] [7-1] In the above aspect [7], the weight-average molecular weight of the alginic acid derivative of formula (II) measured by gel filtration chromatography 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 represented by the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -L 1 - is as defined in the above embodiment [1]; -L 2 - is as defined in the above embodiment [1]; X is as shown in the table below: [Table 35-1] [Table 35-2] The crosslinked alginic acid according to the above aspect [1], wherein the alginic acid is bonded via a cyclic group selected from the group of partial structural formulas shown in the following formula (excluding the areas outside the dashed lines at both ends of each formula). (However, in formula (III-L), -L 1When - is any one linker selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11) and (L1-12), the corresponding -L 2 - (excluding the linker (L2-10)).

[0115] [8a] The embodiment of 8a 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): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -L 1 - is as defined in the above embodiment [1]; -L 2 - is as defined in the above embodiment [1]; X is as defined in the above embodiment [8]. Cross-linked alginate attached via. (However, in formula (III-L), -L 1 When - is any one linker selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11) and (L1-12), the corresponding -L 2 - (excluding the linker (L2-10)).

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

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

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

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

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

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

[0122] [8-3-1] In the formula (III-L) of the above-mentioned embodiment [8] or embodiment [8a], X is preferably a cyclic group selected from the group consisting of partial structural formulae (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-mentioned embodiment [8].

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

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

[0125] [8-4-1] In the formula (III-L) of the above embodiment [8] or embodiment [8a], preferably, -L 2 -XL 1 - The combination is as shown in the table below: [Table 36-1] [Table 36-2] As shown by the partial structure selected from the group of (-L in the table) 1 - and -L 2 - is as described in the above embodiment [1]; -X- is as described in the above embodiment [8] (wherein, in the above table, -L 1 When - is any one linker selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11), or (L1-12), the corresponding -L 2 - (excluding the linker of (L2-10-p1)).

[0126] [8-4-2] In the formula (III-L) of the above embodiment [8] or embodiment [8a], -L 2 -XL 1 - The combination is as shown in the table below: [Table 37-1] [Table 37-2] As shown by the partial structure selected from the group of (-L in the table) 1 - and -L 2 - is as described in the above embodiment [1]; -X- is as described in the above embodiment [8] (wherein, in the above table, -L 1 When - is any one linker selected from the group consisting of (L1-1-1), (L1-2a-1), (L1-2b-1), (L1-11-1), or (L1-12-p1), the corresponding -L 2 - (excluding the linker of (L2-10-p2)).

[0127] [8-4-3] In the formula (III-L) of the above embodiment [8] or embodiment [8a], -L 2 -XL 1 -The combination is as shown in the table below: [Table 38] As shown by the partial structure selected from the group of (-L in the table) 1 - and -L 2 - is as described in the above embodiment [1]; -X- is as described in the above embodiment [8] (wherein, in the above table, -L 1 When - is any one linker selected from the group consisting of (L1-1-X), (L1-2-X), (L1-11-X) and (L1-12-X), the corresponding -L 2 - (excluding the linker of (L2-10-X)).

[0128] [8-4-4] In the formula (III-L) of the above embodiment [8] or embodiment [8a], -L 2 -XL 1 The combination of - is represented by the following partial structural formula (wherein the areas outside the dashed lines at both ends are not included): [ka] The structure is as shown by a partial structure selected from the group consisting of:

[0129] [8-5-1] In the crosslinked alginic acid according to the above aspect [1] or aspect [8a], the crosslinks are chemical crosslinks formed by triazole rings via the Huisgen reaction and ionic crosslinks partially formed by divalent metal ions.

[0130] [8-5-2] In the crosslinked alginic acid according to the above aspect [1] or aspect [8a], the crosslinking is a chemical crosslinking via 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, and zinc ion; more preferably, calcium ion or barium ion; and even more preferably, calcium ion.

[0132] [8-5-4] In the above aspect [8-5-1], the divalent metal ion used for forming the ionic crosslinks can be preferably 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, the aqueous calcium chloride solution or the aqueous barium chloride solution; and even more preferably, the aqueous calcium chloride solution.

[0133] A preferred embodiment of the embodiment [8], further -L 1 -, -L 2 By appropriately combining the definitions of - and X, any preferred embodiment of the cross-linked alginic acid of the above embodiment [8] can be formed. A preferred embodiment of embodiment [8a], further -L 1 -, -L 2 By appropriately combining the definitions of - and X, any preferred embodiment of the cross-linked alginic acid of the above embodiment [8a] can be formed.

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

[0135] [9-1] The embodiment of the 9-1 is as follows: A method for producing a crosslinked alginic acid, comprising adding a solution of an alginic acid derivative represented by formula (I) described in the embodiment [1] to a solution of an alginic acid derivative represented by formula (II) described in the embodiment [1] to perform a crosslinking reaction (Huisgen reaction) to obtain the crosslinked alginic acid described in the embodiment [1] or embodiment [8a].

[0136] [9-2] The embodiment of the 9-2 is as follows: A method for producing a crosslinked alginic acid, comprising adding a solution of an alginic acid derivative represented by formula (II) described in the embodiment [1] to a solution of an alginic acid derivative represented by formula (I) described in the embodiment [1] to perform a crosslinking reaction (Huisgen reaction) to obtain the crosslinked alginic acid described in the embodiment [1] or embodiment [8a].

[0137]

[10] The chemical crosslink formed by carrying out a Huisgen reaction (crosslinking reaction) using the alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to the above aspect [1] is represented by the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends, X, -L 1 - and -L 2 A method for producing a cross-linked alginic acid described in embodiment [1] or embodiment [8a], wherein the structure is the same as defined in embodiment [8].

[0138]

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

[0139] [11-1] The embodiment of item 11-1 is as follows: A crosslinked alginate structure obtained by dropping a solution of an alginate derivative represented by formula (I) described in any one of the above items [1] into a solution containing a divalent metal ion to obtain a gel, and adding the gel to a solution of an alginate derivative represented by formula (II) described in the above item [1] to carry out a crosslinking reaction.

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

[0141] [12-1] The embodiment of the ...

[0142] [12-2] The embodiment of the ...

[0143] [12-3-1] In the above 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, and zinc ion; more preferably, calcium ion or barium ion; and even more preferably, calcium ion.

[0144] [12-3-2] In the above aspect [12-1], the divalent metal ion used for forming the ionic crosslinks can be preferably 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, the aqueous calcium chloride solution or the aqueous barium chloride solution; and even more preferably, the aqueous calcium chloride solution.

[0145]

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

[0146] [13-1-1] In the above 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, and zinc ion; more preferably, calcium ion or barium ion; and even more preferably, calcium ion.

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

[13] , the divalent metal ion used for forming the ionic crosslinks can be preferably 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, the aqueous calcium chloride solution or the aqueous barium chloride solution; and even more preferably, the aqueous calcium chloride solution.

[0148]

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

[11] to

[13] , wherein - is as defined in the above aspect [8].

[0149]

[15] The fifteenth aspect is as follows: A method for producing a crosslinked alginate structure, comprising dropping a mixed solution of the alginate derivative represented by formula (I) and the alginate derivative represented by formula (II) described in the above aspect [1] into a solution containing a divalent metal ion to carry out a crosslinking reaction.

[0150] [15-1] The embodiment of the 15-1 is as follows: A method for producing a crosslinked alginic acid structure, which is obtained by adding a solution of the alginic acid derivative represented by formula (I) described in the embodiment [1] above to a solution containing a divalent metal ion to obtain a gel, and then adding the gel to a solution of the alginic acid derivative represented by formula (II) described in the embodiment [1] above to carry out a crosslinking reaction.

[0151] [15-2] The embodiment of the 15-2 is as follows: A method for producing a crosslinked alginate structure, which is obtained by adding a solution of the alginate derivative represented by formula (II) described in the embodiment [1] above to a solution containing a divalent metal ion, to obtain a gel, and then adding the gel to a solution of the alginate derivative represented by formula (I) described in the embodiment [1] above to carry out a crosslinking reaction.

[0152] [16-1] The embodiment of the 16-1 is as follows: A method for producing a crosslinked alginate structure according to any one of the above embodiments

[15] to [15-2], comprising chemical crosslinking by triazole rings formed by the Huisgen reaction and ionic crosslinking partially formed by divalent metal ions as crosslinks.

[0153] [16-2] The embodiment of the 16-2 is as follows: A method for producing a crosslinked alginate structure according to any one of the above embodiments

[15] to [15-2], wherein the crosslinking is a chemical crosslinking via a triazole ring formed by a Huisgen reaction.

[0154] [16-3-1] In the above 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, and zinc ion; more preferably, calcium ion or barium ion; and even more preferably, calcium ion.

[0155] [16-3-2] In the above aspect [16-1], the divalent metal ion used for forming the ionic crosslinks can be preferably 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, the aqueous calcium chloride solution or the aqueous barium chloride solution; and even more preferably, the aqueous calcium chloride solution.

[0156]

[17] The seventeenth aspect is as follows. The chemical crosslink formed by the Huisgen reaction using the alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) described in the above aspect [1] is represented by the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends, X, -L 1 - and -L 2 A method for producing a crosslinked alginate structure according to any one of the above aspects

[15] to [16-2], wherein the structure is the same as defined in the above aspect [8].

[0157]

[18] The eighteenth aspect is as follows: The crosslinked alginate structure according to any one of the above aspects

[11] to

[14] , which is in the form of beads or a substantially spherical gel.

[0158]

[19] The nineteenth aspect is as follows: A medical material comprising the crosslinked alginic acid structure according to any one of the above aspects

[11] to

[14] .

[0159]

[20] The twentieth aspect is as follows: The medical material according to the above aspect

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

[0160]

[21] The twenty-first aspect is as follows: A biocompatible crosslinked alginic acid according to the aspect [1] or aspect [8a], an alginic acid derivative according to the aspect [2] or aspect [5], or a crosslinked alginic acid structure according to any one of the aspects

[11] to

[14] .

[0161]

[22] The twenty-second aspect is as follows: The following formula (AM-1): [ka] [In formula (AM-1), -L 1 The definitions of -, and Akn are the same as those in the above embodiment [2], or a pharma- ceutically acceptable salt thereof, or a solvate thereof, provided that the following table: [Table 39] or a pharma- ceutically acceptable salt thereof, or a solvate thereof.

[0162] [22-1] The compound represented by formula (AM-1) in the above aspect

[22] is preferably -L 1 - is the preferred -L described in the embodiment [2-1]. 1 - is the same as the definition of -, and Akn is the same as the preferred definition of Akn described in the embodiment [2-2], or a pharma- ceutically acceptable salt thereof, or a solvate thereof. However, in the following table: [Table 40] or a pharma- ceutically acceptable salt thereof, or a solvate thereof.

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

[22] is more preferably -L 1 - is more preferably -L as described in the embodiment [2-1]. 1 - is the same as the definition of -, and Akn is the same as the more preferred definition of Akn described in the above embodiment [2-2], or a pharma- ceutically acceptable salt thereof, or a solvate thereof. However, in the following table: [Table 41] or a pharma- ceutically acceptable salt thereof, or a solvate thereof.

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

[22] is more preferably -L 1 - is more preferably -L described in the embodiment [2-1]. 1 - is the same as the definition of -, and Akn is the same as the more preferred definition of Akn described in the above embodiment [2-2], or a pharma- ceutically acceptable salt thereof, or a solvate thereof. However, in the following table: [Table 42] or a pharma- ceutically acceptable salt thereof, or a solvate thereof.

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

[22] is particularly preferably represented by the following formula: [ka] or a pharma- ceutically acceptable salt thereof, or a solvate thereof.

[0166]

[23] The 23rd aspect is as follows: The following formula (AM-2): [ka] [In formula (AM-2), -L 2 The definition of - is the same as the definition of (L2-2a), (L2-2b-A), (L2-3), (L2-4), (L2-5a), (L2-5b), (L2-6a), (L2-6b), (L2-7a), (L2-7b), (L2-8a), (L2-8b), (L2-9a) and (L2-9b) described in the above embodiment [5], or a pharma- ceutically acceptable salt thereof, or a solvate thereof, provided that, in the following table: [Table 43] or a pharma- ceutically acceptable salt thereof, or a solvate thereof.

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

[23] is preferably -L 2 - is an amino compound, or a pharma- ceutically acceptable salt thereof, or a solvate thereof, in which (L2-2a), (L2-2b-A), (L2-3), (L2-4-p1), (L2-5a-p1), and (L2-5b-p1) are defined as those in the above embodiment [5-1], provided that, in the following table: [Table 44] or a pharma- ceutically acceptable salt thereof, or a solvate thereof.

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

[23] is more preferably -L 2 - is an amino compound having the same definition as that of (L2-2a-1), (L2-2b-B), (L2-3-1), (L2-4-p2), (L2-5a-p2), and (L2-5b-p2) described in the above embodiment [5-1], or a pharma- ceutically acceptable salt thereof, or a solvate thereof, provided that, as shown in the following table: [Table 45] or a pharma- ceutically acceptable salt thereof, or a solvate thereof.

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

[23] is more preferably represented by the following formula: [ka] or a pharma- ceutically acceptable salt thereof, or a solvate thereof. In this specification, unless otherwise specified, when a higher-level embodiment is cited, the lower-level embodiment of the embodiment is also included. For example, when embodiment [1] is cited, the lower-level embodiment of embodiment [1] is also included.

[0170] Each aspect will be described in more detail below.

[0171] 1. Alginic acid In the present specification, the term alginic acid refers to at least one type of alginic acid (sometimes referred to as "alginic acid derivatives") selected from the group consisting of alginic acid, alginic acid esters, and salts thereof (e.g., sodium alginate). The alginic acid used may be natural or synthetic, but is preferably natural. The alginic acid derivatives preferably used are bioabsorbable polysaccharides extracted from brown algae such as Lessonia, Macrocystis, Laminaria, Ascophyllum, Durvillea, Sculpin, Eisenia bifida, and Laminae, and are linear polymers of two types of uronic acid, D-mannuronic acid (M) and L-guluronic acid (G). More specifically, they are block copolymers 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 bonded.

[0172] Alginic acid is a type of natural polysaccharide extracted and purified from brown seaweed, and is a polymer of D-mannuronic acid (M) and L-guluronic acid (G). The composition ratio (M / G ratio) of D-mannuronic acid and L-guluronic acid in alginic acid, i.e., gel strength, varies mainly depending on the type of organism from which the seaweed is derived, and is also influenced by the place of growth and season of the organism, ranging 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 of alginic acid and the arrangement of M and G, and the preferred uses may differ. The gelling ability of alginic acids and the properties of the gel produced are affected by the M / G ratio, and it is generally known that a higher G ratio results in a higher gel strength. The M / G ratio also affects the hardness, brittleness, water absorption, flexibility, etc. of the gel. Therefore, the alginic acid used in the present invention should have an appropriate M / G ratio and appropriate viscosity depending on the final use.

[0173] There are two industrial methods for producing alginic acid: the acid method and the calcium method. In the present invention, alginic acid produced by either method can be used. The HPLC method quantitative value is preferably within the range of 80 to 120% by mass, more preferably within the range of 90 to 110% by mass, and even more preferably within the range of 95 to 105% by mass, as determined by purification. In the present invention, the HPLC method quantitative value is within the above range and is called high-purity alginic acid. The alginic acid or its salt used in the present invention is preferably high-purity alginic acid. As a commercially available product, for example, the Kimica Algin series sold by Kimica Co., Ltd., preferably a high-purity food and pharmaceutical grade product can be purchased and used. It is also possible to further purify the commercially available product as appropriate and use it. For example, it is preferable to perform low-endotoxin treatment. As the purification method or low-endotoxin treatment method, for example, the method described in JP-A-2007-75425 can be adopted.

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

[0175] In this specification, alginic acid may be expressed as (ALG)-COOH, where alginic acid is (ALG) and any one of the carboxyl groups of alginic acid is -COOH.

[0176] The alginic acid used in the present invention should have an appropriate weight-average molecular weight depending on the final use. For example, it is preferable to use alginic acid with a weight-average molecular weight of 10,000 to 10 million, more preferably 100,000 to 5 million, and even more preferably 150,000 to 3 million.

[0177] In some embodiments, the alginic acid is sodium alginate. Commercially available sodium alginate can be used as the sodium alginate. Here, in the examples described below, sodium alginate A-1, A-2, A-3, B-1, B-2, and B-3 (sold by Mochida Pharmaceutical Co., Ltd.) are used as the sodium alginate. The viscosity, weight average molecular weight, and M / G value of a 1 w / w% aqueous solution of each sodium alginate are shown in the table below.

[0178] [Table 46]

[0179] The physical properties of the sodium alginates A-1, A-2, A-3, B-1, B-2, and B-3 were measured by the following methods. The measurement methods are not limited to these methods, but the physical properties may differ from those described above depending on the measurement method.

[0180] [Viscosity measurement of sodium alginate] Measurements were performed using a rotational viscometer (cone-plate type rotational viscometer) according to the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). Specific measurement conditions are as follows. Sample solutions were prepared using MilliQ water. The measurement equipment used was a cone-plate type rotational viscometer (viscosity and viscoelasticity measuring device Rheostress RS600 (Thermo Haake GmbH) sensor: 35 / 1). The rotation speed was 1 rpm when measuring 1 w / w% sodium alginate solution. The reading time was 2 minutes, and the average value from 1 minute to 2 minutes was used. The average value of three measurements was used as the measured value. The measurement temperature was 20°C.

[0181] [Weight-average molecular weight measurement of sodium alginate] The measurements were performed using two methods: (1) gel permeation chromatography (GPC) and (2) GPC-MALS. The measurement conditions were as follows:

[0182] [Pretreatment method] The sample was dissolved in an eluent and then filtered through a 0.45 μm membrane filter to prepare a measurement solution.

[0183] (1) Gel permeation chromatography (GPC) measurement [Measurement conditions (relative molecular weight distribution measurement)] Column: TSKgel GMPW-XL x 2 + G2500PW-XL (7.8 mm ID x 300 mm x 3) Eluent: 200mM sodium nitrate aqueous solution Flow rate: 1.0mL / min Concentration: 0.05% Detector: RI detector Column temperature: 40℃ Injection volume: 200μL Molecular weight standards: standard pullulan, glucose

[0184] (2)GPC-MALS measurement [Refractive index increment (dn / dc) measurement (measurement conditions)] Differential refractometer: Optilab T-rEX Measurement wavelength: 658nm Measurement temperature: 40℃ Solvent: 200mM sodium nitrate aqueous solution Sample concentration: 0.5-2.5mg / mL (5 concentrations)

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

[0186] In this specification, the molecular weight of alginic acid, alginic acid derivatives, crosslinked alginic acid, and crosslinked alginic acid may be expressed in units of Da (dalton).

[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 seaweed is derived, and is also influenced by the growing place and season of the organism, and ranges 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 gelling ability of alginic acids and the properties of the gel formed are affected by the M / G ratio, and it is generally known that a high G ratio leads to high gel strength. The M / G ratio also affects the hardness, brittleness, water absorption, flexibility, etc. of the gel. The M / G ratio of the alginic acids and / or salts thereof used is usually 0.1 to 4.0, in one embodiment, 0.1 to 3.0, in one embodiment, 0.1 to 2.0, in one embodiment, 0.5 to 1.8, and in one embodiment, 0.8 to 1.2. In another embodiment, it is 0.1 to 0.5.

[0188] In addition, the alginic acid used in the present invention should have an appropriate viscosity and an appropriate M / G ratio depending on the final use.

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

[0190] The term "alginic acid ester" and "alginate salt" used herein is not particularly limited, but it is necessary that they do not have a functional group that inhibits the crosslinking reaction in order to react with a crosslinking agent. Preferred examples of alginic acid esters include propylene glycol alginate.

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

[0192] Alginic acid is a polymeric polysaccharide, and although it is difficult to accurately determine its molecular weight, its weight-average molecular weight is generally in the range of 1,000 to 10,000,000, preferably 10,000 to 8,000,000, and more preferably 20,000 to 3,000,000. It is known that when measuring the molecular weight of polymeric substances derived from natural products, the values ​​can vary depending on the measurement method.

[0193] In the present specification, when the molecular weight of the alginic acid derivative or alginic acid or its salt of the present invention is specified, it is the weight average molecular weight calculated by size exclusion chromatography (SEC) unless otherwise specified. It is desirable to use alginic acid or its salt used in the present invention with an appropriate molecular weight distribution depending on the final use.

[0194] For example, under the measurement conditions of gel permeation chromatography (GPC) or gel filtration chromatography (collectively referred to as size exclusion chromatography (SEC)) described in the Examples below, it is preferably 100,000 to 5 million, more preferably 150,000 to 3 million. In one embodiment, it is in the range of 500,000 to 3 million, more preferably 1 million to 2.5 million, and even more preferably 1 million to 2 million.

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

[0196] Usually, when the molecular weight of a polymeric polysaccharide is calculated by the above-mentioned method using SEC or SEC-MALS, a measurement error of about 10% to about 30% may occur. For example, if the molecular weight is 500,000, the value may vary within a range of about 350,000 to 650,000, and if the molecular weight is 1,000,000, the value may vary within a range of about 700,000 to 1,300,000. In this specification, when the word "about" is used in the description of molecular weight measurement, it may include a value of up to ±10% of the numerical value, and in some embodiments, a value of up to ±20% of the numerical value.

[0197] Generally, polymeric substances derived from natural products do not have a single molecular weight, but are an aggregate of molecules with various molecular weights, and are therefore measured as a molecular weight distribution with a certain range. A typical measurement method is gel filtration chromatography. Typical information on molecular weight distribution obtained by gel filtration chromatography includes weight average molecular weight (Mw), number average molecular weight (Mn), and polydispersity ratio (Mw / Mn).

[0198] The weight average molecular weight is determined by considering the contribution of large molecular weight polymers to the average molecular weight, and is expressed 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 polymers 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 polymers, Wi is the weight of the i-th polymer, Mi is the molecular weight at the i-th elution time, Ni is the number of molecules with molecular weight Mi, and Hi is the height at the i-th elution time.

[0201] It is known that the molecular weight of naturally derived polymeric substances 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 is a document that describes a method of calculating from intrinsic viscosity and a method of calculating 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 calculated by a standard method such as that shown in the above document, for example, by measuring the molecular weight by size exclusion chromatography (SEC) and using a calibration curve with pullulan as a standard substance. In the present invention, the weight average molecular weight can be the absolute molecular weight measured by a conventional method such as size exclusion chromatography (SEC)-MALS, as described in the above-mentioned literature.

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

[0203] In the present specification, when the molecular weight of alginic acid or a salt thereof is specified, it is the weight average molecular weight calculated by gel filtration chromatography unless otherwise specified. When gel filtration chromatography is used for molecular weight measurement, the typical conditions can be, for example, the conditions of the present embodiment described later. For example, a Superose6 Increase10 / 300 GL column (GE Healthcare Sciences) can be used as the column, and for example, a 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl can be used as the developing solvent, and blue dextran, thyroglobulin, ferritin, aldolase, conalbumin, ovalbumin, ribonuclease A and aprotinin can be used as the molecular weight standard.

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

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

[0206] When alginic acids are first extracted from brown algae, they have a large molecular weight and a high viscosity, but the molecular weight decreases and the viscosity decreases during processes such as drying by heat and purification. Alginic acids with different molecular weights can be produced by controlling conditions such as temperature during the production process, selecting the brown algae used as raw material, and molecular weight fractionation during the production process. Furthermore, it is possible to produce alginic acids with the desired molecular weight by mixing with other lots of alginic acids with different molecular weights or viscosities.

[0207] The alginic acid used herein is, in some embodiments, alginic acid that has not been treated with low endotoxins, or, in other embodiments, alginic acid that has been treated with low endotoxins. Low endotoxins means that the endotoxin level is low enough to not substantially cause inflammation or fever. More preferably, alginic acids that have been treated with low endotoxins are desirable.

[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 of endotoxin treatment used is not particularly limited, but as a result, the endotoxin content of 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 particularly preferably 30 EU / g or less, when endotoxin is measured using a 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-mentioned numerical range. Sodium alginate that has been treated to reduce endotoxins is, for example, Sea Matrix (registered trademark) (Mochida Pharmaceutical Co., Ltd.), PRONOVA TM It is available commercially as a product such as UP LVG (FMCBioPolymer).

[0211] 2. Alginic acid derivatives The present specification provides a novel alginic acid derivative, in which a reactive group in the Huisgen reaction or a complementary reactive group to the reactive group is introduced to any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker. More specifically, the compound represented by the following formula (I): [ka] [In formula (I), (ALG), -L 1 The definitions of -, Akn and -NH-CO- are the same as those in the first embodiment above], and an alginic acid derivative represented by the following formula (II): [ka] [In formula (II), (ALG), -L 2 The definitions of - and -NH-CO- are the same as those in the fourth aspect above.

[0212] The bivalent linker (-L 1 -or-L 2Any straight-chain group can be used as long as it does not inhibit the reaction between the reactive group and a complementary reactive group. Specifically, for example, a straight-chain alkylene group (-(CH2) n -, n=1 to 30) (-CH2- in the group may be replaced by a plurality of groups (for example, 1 to 10 or 1 to 5) such as -C(=O)-, -CONH-, -O-, -NH-, -S-, a benzene ring, or a heterocycle (a 5- to 6-membered aromatic heterocycle or a 5- to 6-membered non-aromatic heterocycle such as a pyridine ring, a piperidine ring, or a piperazine ring), and the hydrogen atom of the -CH2- may be replaced by an oxo group (=O), 1-6 Examples of the alkyl group include, but are not limited to, a methyl group, an ethyl group, an n-propyl group, an iso-propyl group, and the like, a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, and the like), a hydroxyl group (-OH), and the like.

[0213] In the -NH-CO- group of the alginic acid derivative represented by the above formula (I) or (II), the hydrogen atom of the imino group (-NH-) can be substituted with a methyl group to form a -N(Me)-CO- group. In the alginic acid derivative represented by the formula (I) or (II), 1 -, -L 2 The bond between the alkyl group and alginic acid may be an -NH-CO- bond or an -N(Me)-CO- bond; preferably, the bond is an -NH-CO- bond.

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

[0215] [ka]

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

[0217] In the present specification, Akn-L of formula (I) 1 The -NH- group does not necessarily have to be bonded to all the carboxyl groups of the alginic acid building block, and the N3-L 2 It is not necessary for the --NH-- group to be attached to every carboxyl group of the alginic acid building block.

[0218] In the present specification, Akn-L of formula (I) 1 When the -NH- group is referred to as a reactive group, N3-L in formula (II) 2 The -NH- group serves as a complementary reactive group. Conversely, the N3-L 2 When the -NH- group is referred to as a reactive group, Akn-L of formula (I) 1 The -NH- group serves as 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%, and more preferably 3% to 10%.

[0220] The introduction rate of the reactive group or the complementary reactive group is the number of uronic acid monosaccharide units into which each reactive group is introduced, expressed as a percentage, among the uronic acid monosaccharide units, which are the repeating units of alginic acids. In this specification, unless otherwise specified, % 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 Huisgen reaction, thereby forming a bridge.

[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 unnecessary by-products are generated. Although it is thought that the reaction may produce a 1,4- or 1,5-disubstituted triazole ring, it is possible to obtain the triazole ring regioselectively by using a copper catalyst.

[0223] [ka]

[0224] In addition, the Huisgen reaction without using a copper catalyst has been reported by Wittig and Krebs. In other words, this reaction produces a cycloadduct by simply mixing cyclooctyne and phenyl azide (see the following formula, R 3 = phenyl). In this reaction, the triple bond of cyclooctyne is significantly distorted, so the reaction with phenyl azide removes this distortion, and the reaction proceeds spontaneously, making a catalyst unnecessary.

[0225] [ka]

[0226] As described above, the Huisgen reaction can use azide compounds having substituted primary azides, secondary azides, tertiary azides, aromatic azides, etc., and compounds having terminal or cyclic alkyne groups, which are complementary reactive groups to the azide group. In addition, since the Huisgen reaction reacts almost exclusively with azide groups and alkyne groups, it is possible to substitute various functional groups (e.g., ester groups, carboxyl groups, alkenyl groups, hydroxyl groups, amino groups, etc.) in the reaction substrate.

[0227] In some embodiments, in order to form crosslinks between alginic acid molecules via 1,2,3-triazole rings quickly, easily, and efficiently without using a copper catalyst to avoid the production of undesirable by-products and to avoid the cytotoxicity of copper catalysts, a cyclic alkyne group (cyclooctyl group) as described in the above embodiment [1] is used as the alkyne group in the Huisgen reaction.

[0228] In the preferred embodiment, the cross-linking method of alginic acid derivatives hardly forms any undesirable by-products in the reaction (Huisgen reaction), which allows the creation of novel forms of biocompatible materials using alginic acid and the incorporation of various biologically active molecules in the formation of alginic acid hydrogels, as well as the incorporation of cellular materials in alginic acid hydrogels for reconstructive surgery or gene therapy.

[0229] 4. Cross-linked Alginate Crosslinked alginates can be classified into those (i) via divalent metal ion bonds, (ii) via chemical bonds, or (iii) via both divalent metal ion bonds and chemical bonds. All crosslinked alginates have the property of forming gel-like to semi-solid, and in some cases sponge-like, morphologies.

[0230] In crosslinked alginic acid via divalent metal ion bonds, the reaction proceeds at an ultrahigh speed and is reversible, whereas in crosslinked alginic acid via chemical bonds, the reaction proceeds slowly under relatively mild conditions and is irreversible. The physical properties of crosslinked alginic acid can be adjusted, for example, by changing the concentration of the aqueous solution containing the divalent metal ion (e.g., calcium chloride aqueous solution) used or the introduction rate of reactive groups introduced into alginic acid.

[0231] By utilizing the above-mentioned crosslinking reaction, it is possible to prepare various alginate structures. For example, a specific structure can be instantly prepared from an alginate solution by ionic crosslinking reaction, and a crosslinking reaction by chemical bonds can be used to strengthen the structure (e.g., to obtain long-term stability, etc.). In addition, for example, in a crosslinked alginate structure via both divalent metal ion bonds and chemical bonds, it is possible to reversibly release the divalent metal ions incorporated by ionic crosslinking, thereby preparing a structure in which only crosslinks by chemical bonds remain.

[0232] A crosslinked alginic acid in one embodiment can be obtained by mixing the alginic acid derivatives of the formula (I) and the formula (II) and carrying out a Huisgen reaction.

[0233] In one embodiment, the crosslinked alginic acid forms a three-dimensional network structure through chemical crosslinking (crosslinking by a triazole ring formed from an alkyne group and an azide group). A preferred alginic acid derivative is one that has improved stability after crosslinking.

[0234] In some embodiments, the crosslinked alginic acid has a structure represented by the following formula (III-L) between any carboxyl group of a first alginic acid and any carboxyl group of a second alginic acid: [ka] [In formula (III-L), -CONH- and -NHCO- at both ends represent amide bonds via any carboxyl group of alginic acid; 1 -, -L 2 -, and X is as defined in the eighth embodiment above.

[0235] 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, for example, 1 to 1.5:1, preferably 1.2 to 1.5:1, or 1 to 1.2:1, and more preferably 1:1, in terms of weight ratio of the derivative of formula (I) to the derivative of formula (II).

[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, 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, and more preferably 1:1, in terms of weight ratio of the derivative of formula (II) to the derivative of formula (I).

[0237] In some embodiments, the mixing ratio of the alginic acid derivative of formula (I) to the alginic acid derivative of formula (II) when preparing crosslinked alginic acid is, more preferably, the ratio of the introduction rate (mol%) of the reactive group of the alginic acid derivative of formula (I) to the alginic acid derivative of formula (II) is, 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, the mixing ratio of the alginic acid derivative of formula (II) to the alginic acid derivative of formula (I) when preparing crosslinked alginic acid is, more preferably, the ratio of the introduction rate (mol%) of the reactive group 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] In the above mixing ratio, the alginic acid derivative of formula (I) can be replaced with the alginic acid derivative of formula (II), and the alginic acid derivative of formula (II) can be replaced with the derivative of formula (I).

[0240] In the crosslinked alginic acid, it is not necessary that all carboxyl groups of the constituent units of alginic acid have the crosslink of the above formula (III-L). The introduction rate of the crosslink represented by the above formula (III-L) in the crosslinked alginic acid (also called the crosslinking rate) 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 to obtain crosslinked alginic acid is usually in the range of about 1 to about 500 mg / mL, and preferably about 5 to about 100 mg / mL.

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

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

[0244] The reaction solvent or reaction solution used in the Huisgen reaction is not particularly limited, but 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 saline, with ultrapure water being preferred.

[0245] The cross-linked alginate in some embodiments is a cross-linked alginate that includes chemical cross-links via triazole rings formed by the Huisgen reaction as cross-links, and ionic cross-links formed in part by divalent metal ions.

[0246] 5. Cross-linked Alginate Constructs The crosslinked alginic acid structure can be obtained by a method comprising carrying out a crosslinking reaction using the alginic acid derivative of the formula (I) and the alginic acid derivative of the formula (II).

[0247] In this specification, "subjecting to a crosslinking reaction" or "carrying out a crosslinking reaction" means that a Huisgen reaction is carried out using an alginic acid derivative of formula (I) and an alginic acid derivative of formula (II) to form a chemical crosslink (chemical bond) between the alginic acid derivative of formula (I) and the alginic acid derivative of formula (II), or that ionic crosslinks (ionic bonds) are formed between the alginic acid derivative of formula (I) and / or the alginic acid derivative of formula (II) by causing a divalent metal ion to coexist with the alginic acid derivative of formula (I) and the alginic acid derivative of formula (II), or that both a chemical crosslink by the Huisgen reaction and an ionic crosslink by a divalent metal ion are formed.

[0248] The cross-linked alginate structure can be prepared, for example, by the following methods, but is not limited thereto.

[0249] [Mixing method] By dropping a mixed solution of alginic acid derivatives obtained by mixing the alginic acid derivative of the formula (I) and the alginic acid derivative of the formula (II) into a solution containing divalent metal ions, it is possible to obtain a cross-linked alginic acid structure, which is a specific structure in which chemical cross-links (cross-links by triazole rings formed from alkyne groups and azide groups by the Huisgen reaction) and ionic cross-links (cross-links partially formed by divalent metal ions) are formed.

[0250] [Coating method] A solution containing the alginic acid derivative of formula (I) is dropped into a solution containing a divalent metal ion to obtain a specific partially crosslinked structure. The structure obtained above, such as a gel, is added to a solution containing the alginic acid derivative of formula (II) to perform a further crosslinking reaction (Huisgen reaction) on the surface of the structure, thereby obtaining a crosslinked alginic acid structure. This method can also be carried out by replacing the alginic acid derivative of formula (I) with the alginic acid derivative of formula (II) and replacing the alginic acid derivative of formula (II) with the alginic acid derivative of formula (I).

[0251] The divalent metal ion used in the method is not particularly limited, but may be, for example, a divalent metal ion selected from the group consisting of calcium ion, magnesium ion, barium ion, strontium ion, zinc ion, etc.; preferably, calcium ion or barium ion; and more preferably, calcium ion.

[0252] The solution containing a divalent metal ion used in the method is not particularly limited, and may be, for example, 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. An aqueous calcium chloride solution or an aqueous barium chloride solution is preferable; and an aqueous calcium chloride solution is more preferable.

[0253] The divalent metal ion concentration of the solution containing the divalent metal ion used in the above method is not particularly limited, but may be, for example, about 1 mM to about 1 M, preferably about 5 mM to about 500 mM, and more preferably about 10 mM to about 300 mM.

[0254] The solvent or solution used in the method 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 saline, with ultrapure water being preferred.

[0255] Specific examples of the crosslinked alginate structure include fibrous structures, fibers, beads, gels, and roughly spherical gels. A preferred crosslinked alginate structure has improved stability. The crosslinked alginate structure may also have the ability to retain contents therein (content retention).

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

[0257] 6. Biocompatibility of alginate derivatives and cross-linked alginate structures In the present specification, the alginic acid derivative, crosslinked alginic acid, or crosslinked alginic acid structure has biocompatibility. In the present specification, biocompatibility means a property of not inducing an interaction between a biomaterial (here, referring to an alginic acid derivative represented by formula (I) or formula (II), and a crosslinked alginic acid or a crosslinked alginic acid structure produced using both of the alginic acid derivatives) and a living body, a local reaction of tissue adjacent to the biomaterial, or a systemic reaction.

[0258] In this specification, the biocompatibility of the alginic acid derivatives, crosslinked alginic acid, and crosslinked alginic acid structures will be confirmed in the examples regarding biocompatibility described later.

[0259] 7. Stability of cross-linked alginate structures The stability of the crosslinked alginate structure can be confirmed, for example, by measuring the gel stability, and the permeability can be confirmed by measuring the gel permeability.

[0260] [Method for measuring gel stability] Phosphate buffered saline (PBS) is added to the crosslinked alginate structure gel placed in a container, and the concentration (μg / mL) of alginate dissolved in PBS is measured. The amount of dissolved alginate is calculated from the measured alginate concentration, and the disintegration rate is calculated by dividing the amount by the total amount of alginate calculated from the total alginate concentration obtained by decomposing the crosslinked alginate structure gel. Specifically, gel stability can be determined by the method described in the Examples below.

[0261] In this specification, the gel collapse rate of the crosslinked alginate structure is preferably 0% to about 90%, more preferably 0% to about 70%, and even more preferably 0% to about 50%. The lower the concentration of alginate leaked into the aqueous solution, i.e., the lower the gel collapse rate, the higher the stability of the crosslinked alginate structure.

[0262] [Method for measuring gel permeability] A crosslinked alginate structure gel containing fluorescein isothiocyanate-dextran is prepared, and physiological saline is added to the gel placed in a container, and the concentration of dextran leaked into the physiological saline is measured. The amount of dextran calculated from the measured concentration of dextran is divided by the total amount of dextran calculated from the total dextran concentration obtained by decomposing the fluorescein isothiocyanate-dextran-containing crosslinked alginate structure gel, and the value expressed as a percentage is the gel permeability. Specifically, the gel permeability can be determined by the method described in the Examples below.

[0263] The gel permeability of the crosslinked alginate 24 hours after addition of physiological saline is preferably 0% to about 90%, more preferably 0% to about 70%, and even more preferably 0% to about 50% when it contains dextran with a molecular weight of about 2 million. When it contains dextran with a molecular weight of about 150,000, for example, if the intended use of the crosslinked alginate structure gel is for the release or 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%. When the intended use is as an immune barrier, it is preferably 0% to about 90%, more preferably 0% to about 70%, and even more preferably 0% to about 50%.

[0264] Regarding the permeability of the cross-linked alginate structure, a lower permeability indicates a lower permeability to the contents and substances outside the gel, and a higher permeability indicates a higher permeability to the contents and substances outside the gel.

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

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

[0267] (1) A solution of the alginic acid derivative represented by formula (I) is mixed with a fluorescein isothiocyanate-dextran solution. (2) The mixed solution obtained in (1) is mixed with a solution of the alginic acid derivative represented by formula (II). (When formula (I) in (1) is changed to formula (II), formula (II) in (2) is changed to formula (I).) (3) The mixed solution obtained in (2) is dropped into a solution containing calcium ions, and the resulting gel forms chemical and ionic crosslinks in the solution, thereby producing a crosslinked alginate structure gel containing fluorescein isothiocyanate-dextran.

[0268] In the present specification, unless otherwise specified, the term "about" includes values ​​up to ±20% of the numerical value, preferably up to ±10% of the numerical value.

[0269] 8. Method for synthesizing alginic acid derivatives In the present specification, the alginic acid derivatives represented by formula (I) and formula (II) are each H2N-L 1 -Akn(in the formula, L 1 and Akn are the same as defined in the above embodiment [1]), or H2N-L 2 -N3 (in the formula, L 2 is the same as defined in the above embodiment [4]), can be produced by subjecting any carboxyl group of alginic acid to a condensation reaction using a condensing agent.

[0270] [ka]

[0271] [Method for producing alginic acid derivative of formula (I)] Using a 0.5% to 1% by weight aqueous solution of alginic acid and the amine represented by formula (AM-1), 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 ( The alginic acid derivative of formula (I) can be produced by carrying out a condensation reaction at a temperature between 0°C and 50°C in the presence of a condensing agent selected from 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), etc., in a mixed solvent of water and a solvent selected from ether solvents such as tetrahydrofuran and 1,4-dioxane, alcohol solvents such as methanol, ethanol, 2-propanol, and polar solvents such as N,N-dimethylformamide, to such an extent that alginic acid does not precipitate, in the presence or absence of an inorganic base such as sodium bicarbonate or sodium carbonate, or an organic base such as triethylamine or pyridine.

[0272] [Method for producing alginic acid derivative of formula (II)] An alginic acid derivative of formula (II) can be produced by reacting a 0.5% to 1% by weight aqueous solution of alginic acid and an amine represented by formula (AM-2) in accordance with the above-mentioned [Method for producing an alginic acid derivative of formula (I)].

[0273] In the above-mentioned method for producing the alginic acid derivative of formula (I) or the alginic acid derivative of formula (II), the introduction rate of the amine of formula (AM-1) or formula (AM-2) can be adjusted by appropriately selecting and combining the following reaction conditions (i) to (v), taking into consideration the properties of the amine, etc.: (i) increasing or decreasing the equivalent amount of the condensing agent, (ii) increasing or decreasing the reaction temperature, (iii) extending or shortening the reaction time, (iv) adjusting the concentration of the alginic acid as the reaction substrate, (v) adding a water-miscible organic solvent to increase the solubility of the amine of formula (AM-1) or formula (AM-2), etc.

[0274] A more specific method for producing the amine represented by formula (AM-1) or formula (AM-2) will be described below.

[0275] In addition, among the following manufacturing methods, m1, n1, m2a, n2a, p2a, m2b, n2b, p2b, m3, n3, p3, m4a, n4a, m4b, n4b, m5a, n5a, p5a, q5a, m5b, n5b, p5b, q 5b, m6a, n6a, p6a, m6b, n6b, p6b, m7, n7, m8a, n8a, m8b, n8b, m9a, n9a, p9a, m9b, n9b, p9b, m10, n10, p10, m11, m12, n12, The definitions of 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 those described in the above embodiment [1]; 1 is a protecting group for an amino group selected from a -C(O)O-tertBu group, a -C(O)O-Bn group, a -C(O)CH3 group, a -C(O)CF3 group, a -SOPh, a -SOPhMe group, a -SOPh(NO2) group, and the like; E is a leaving group such as a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, and the like), an -OTs group, a -OMs group, and the like.

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

[0277] [Production Method AM-A] Production method of amine represented by formula (AM-1-B1): [ka] 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 a production method known in the literature] are used in a condensation reaction similar to that in the above [Production method of alginic acid derivative of formula (I)], followed by the removal of the protecting group P 1 By deprotecting the group, an amine compound represented by formula (AM-1-B1) or a salt thereof can be produced.

[0278] [Production Method AM-B] Production method of amines represented by formula (AM-1-B2a) and formula (AM-1-B2b): [ka]

[0279] The amine compound represented by formula (AM-1-B2a) or its salt can be produced by reacting 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 produced from commercially available compounds by a production method known in the literature] in the same manner as in [Production method AM-A]. Similarly, the amine compound represented by formula (AM-1-B2b) or its salt can be produced by reacting 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 produced from commercially available compounds by a production method known in the literature] in the same manner as in [Production method AM-A].

[0280] [Production Method AM-C] Production method of amine represented by formula (AM-1-B3): [ka]

[0281] The amine compound represented by formula (AM-1-B3) or a salt thereof can be produced by carrying out a reaction according to the above synthesis scheme (each step corresponds to the reaction described in [Production Method AM-A]) using a compound of formula (SM-B3) [the compound of formula (SM-B3) 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]. 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 produced from a commercially available compound by a production method known in the literature.

[0282] [Preparation Method AM-D] Preparation method of amines represented by formula (AM-1-B5a) and formula (AM-1-B5b): [ka]

[0283] By carrying out the reaction according to the above synthesis scheme (the reaction in each step is similar to the reaction described in [Production Method AM-A]), the amine compounds represented by formula (AM-1-B5a) and formula (AM-1-B5b), or salts 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 a production method known in the literature.

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

[0285] By carrying out the reaction according to the above synthesis scheme (the reaction in each step is similar to the reaction described in [Production Method AM-A]), the amine compounds represented by formula (AM-1-B6a) and formula (AM-1-B6b), or salts 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 a production method known in the literature.

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

[0287] By carrying out the reaction according to the above synthesis scheme (the reaction in each step is similar to the reaction 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 formulae (SM-B10), (RG-B10), (RG-B10-1), and (RG-B10-2) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known in the literature.

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

[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 a production method known in the literature], in accordance with a method known in the literature, for example, a method described in "Journal of the American Chemical Society, 126(46), pp. 15046-15047, 2004", etc., <Step 1a> a compound of formula (IM-B4a-1) is obtained by reacting in a reaction-inert solvent such as toluene or dichloromethane in the presence of a reagent such as silver trifluoromethanesulfonate or AgClO4, <Step 2a> a compound of formula (IM-B4a-2) is obtained by subsequently carrying out a debromination reaction using a base such as sodium hydride or NaOMe, and <Step 3a> a protecting group P 1 By deprotecting the group, an amine compound represented by formula (AM-1-B4a) or a salt thereof can be produced. Similarly, an amine compound represented by formula (AM-1-B4b) or a salt thereof can be produced by carrying out a reaction according to the above scheme using formula (RG-B4b) instead of formula (RG-B4a).

[0290] [Preparation Method AM-H] Preparation method of amines represented by formula (AM-1-B8a) and formula (AM-1-B8b): [ka]

[0291] By carrying out the reaction according to the above synthesis scheme (the reaction in each step is similar to the reaction described in [Production Method AM-A]), it is possible to produce the amine compounds represented by formula (AM-1-B8a) and formula (AM-1-B8b), or salts thereof. In the above scheme, formula (SM-B8a) or formula (SM-B8b) is a commercially available compound or a compound that can be produced according to the reaction described in [Production Method AM-G], and 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] Production method of amine represented by formula (AM-1-B7): [ka]

[0293] 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] are used in a condensation reaction similar to that described above in [Production method of alginic acid derivative of formula (I)], followed by the removal of the protecting group P 1 By deprotecting the compound, an amine compound represented by formula (AM-1-B7) or a salt thereof can be produced.

[0294] [Production Method AM-J-2] Production method of amine represented by formula (AM-1-B7): [ka]

[0295] 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] are subjected to a condensation reaction similar to that described above in [Production method of alginic acid derivative of formula (I)] (<Step 1> and <Step 2>), followed by the removal of the protecting group P 1is deprotected, and then a condensation reaction similar to that in the above <Step 1> is carried out 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 in the literature), followed by removing the protecting group P 1 By deprotecting the compound, an amine compound represented by formula (AM-1-B7) or a salt thereof can be produced.

[0296] [Production Method AM-K] Production method of amines represented by formula (AM-1-B9a) and formula (AM-1-B9b): [ka]

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

[0298] [Production Method AM-L] Production method of amine represented by formula (AM-2-Z1): [ka]

[0299] The 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 in the literature] is reacted with NaN3 in a reaction-inert solvent such as dimethyl sulfoxide to introduce an azide group, in accordance with a method known in the literature, for example, the method described in "Organometallics, 29(23), p6619-6622; 2010", and then a protecting group P 1 By deprotecting the group, an amine compound represented by formula (AM-2-Z1) or a salt thereof can be produced. Incidentally, the amine compound represented by the formula (AM-2-Z1) or a salt thereof may be available as a commercially available compound.

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

[0301] The compound of formula (SM-Z2a) or the 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 in the literature] are reacted with NaN3 in the same manner as in [Production method AM-L] to introduce an azide group, and then a protecting group P 1 The amine compound represented by formula (AM-2-Z2a) or formula (AM-2-Z2b), or a salt thereof, can be produced by deprotecting the amine compound represented by formula (AM-2-Z2a) or formula (AM-2-Z2b), or a salt thereof. Some of the amine compounds represented by formula (AM-2-Z2a) or formula (AM-2-Z2b), or a salt thereof, are commercially available.

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

[0303] The compound of formula (SM-Z3) and the 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 in the literature] are used in a condensation reaction similar to that described above in [Production method of alginic acid derivative of formula (I)], followed by the removal of the protecting group P 1 By deprotecting the group, an amine compound represented by the formula (AM-2-Z3) or a salt thereof can be produced.

[0304] [Production Method AM-O] Production method of amine represented by formula (AM-2-Z4): [ka]

[0305] 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 in the literature] are subjected to a condensation reaction similar to that described above in [Production method of alginic acid derivative of formula (I)], followed by the removal of the protecting group P 1 By deprotecting the group, an amine compound represented by the formula (AM-2-Z4) or a salt thereof can be produced.

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

[0307] A compound having a side chain introduced therein is obtained by reacting a compound of formula (SM-Z5a) and a 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 in 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, dimethylsulfoxide, etc. Then, a protecting group P 1 By deprotecting the compound, an amine compound represented by formula (AM-2-Z5a) or a salt thereof can be produced. Similarly, the amine compound represented by formula (AM-2-Z5b) or a salt thereof can be produced by reacting the compound represented by formula (SM-Z5b) and the compound represented by formula (RG-Z5b) [the compound represented by formula (SM-Z5b) and the compound represented by formula (RG-Z5b) are commercially available compounds or compounds that can be produced from commercially available compounds by production methods known in the literature] in a similar manner.

[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 method shown in the following scheme in accordance with the above [Production Methods AM-A] to [Production Methods AM-P].

[0309] [ka]

[0310] [ka]

[0311] [ka]

[0312] [ka]

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

[0314] <Step 1> Using a compound of formula (SM-Q) [the compound of formula (SM-Q) 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] and a compound of formula (RG-Q1) [the compound of formula (RG-Q1) 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], in accordance with a method known in the literature, for example, a method described in “European Journal of Organic Chemistry, 2014(6), p1280-1286; 2014”, (i) in the presence of PPh3 and N2(CO2CHMe2)2 reagents, in a reaction inert solvent such as tetrahydrofuran, and subsequently in the presence of a base such as sodium hydroxide, in a reaction inert solvent such as methanol, ethanol, tetrahydrofuran, water, or a mixture thereof, a compound represented by formula (IM-Q1) can be produced.

[0315] <Step 2> [Production method AM-Q] The compound of formula (IM-Q1) obtained in <Step 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 produced from a commercially available compound by a production method known in the literature] are used in a condensation reaction similar to that in the above [Production method of alginic acid derivative of formula (I)] to obtain a condensate, and then the protecting group P 1 By deprotecting the group, an amine compound represented by formula (AM-2-Z10) or a salt thereof can be produced.

[0316] [Production Method AM-R] Production method of amine represented by formula (AM-1-B10): [ka]

[0317] <Step 1> Using a compound of formula (SM-R) [a 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 in the literature], in accordance with a method known in the literature, for example, a method described in "Faming Zhuanli Shenqing, 104529898, 22 Apr 2015", (i) in the presence of a base such as pyridine, in a reaction inert solvent such as ethanol, H2NOH-HCl is reacted to form an oxime, (ii) in P2O5, methanesulfonic acid, diphosphorus pentoxide is reacted to form an 8-membered ring lactam by Beckmann rearrangement, and (iii) in a reaction inert solvent such as diethyl ether, the amide group is reduced using a reducing agent such as BH3 or LiAlH4.

[0318] <Step 2> [Production method AM-R] The compound of formula (IM-R1) obtained by <Step 1> and the compound of formula (RG-R1) [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 in the literature] are used in a condensation reaction similar to that in the above [Production method of alginic acid derivative of formula (I)] to obtain a condensation product. Then, bromine is added, and a debromination reaction is performed using tert-BuOK to form an alkyne group, and then a protecting group P is removed. 1 By deprotecting the group, an amine compound represented by formula (AM-1-B10) or a salt thereof can be produced.

[0319] [Production Method AM-S] Production method of amine represented by formula (AM-2-Z11): [ka]

[0320] 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 a production method known in the literature] are subjected to a condensation reaction similar to that described above in [Production method of alginic acid derivative of formula (I)], followed by the removal of the protecting group P 1By deprotecting the compound represented by the formula (AM-2-Z11) or a salt thereof, a compound represented by the formula (AM-2-Z11) or a salt thereof can be produced.

[0321] [Manufacturing method AM-T] Method for preparing amines represented by formula (AM-1-T1) and formula (AM-1-T2): [ka]

[0322] <Step 1> Using the compound of formula (SM-T) and the 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 production methods known in the literature], a Grignard reaction is carried out in accordance with a method known in the literature, for example, a method described in WO2004 / 035017, etc., followed by an oxidation reaction 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) (e.g., an acetal group, etc.), bromine is added to the cyclooctene ring, followed by debromination reaction using a base such as tert-BuOK, followed by deprotection of the carbonyl group and the protecting group P 1 is deprotected to produce an amine represented by formula (AM-1-T1) or a salt thereof.

[0324] <Step 3> A condensation reaction is carried out 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 a commercially available compound by a production method known in the literature] to remove a protecting group P 1 is deprotected to produce an amine represented by formula (AM-1-T2) or a salt thereof.

[0325] [Manufacturing method AM-U] Method for preparing amines represented by formula (AM-1-U1) and formula (AM-1-U2): [ka]

[0326] In the above [Production Method AM-T], the compound of formula (SM-T) is replaced 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 production method known in the literature], and a reaction is carried out in accordance with the method described in [Production Method AM-T] to produce amines represented by formula (AM-1-U1) and formula (AM-1-U2), or salts thereof.

[0327] The aldehyde used in <Step 1> of the above [Production Method AM-T] and [Production Method AM-U] is replaced with an 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 production method known in the literature], thereby producing an amine having a corresponding linker, or a salt thereof. [ka] [Manufacturing method AM-V] Method for preparing amines represented by formula (AM-1-V1) and formula (AM-1-V2): [ka]

[0328] <Step 1> 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 in the literature, for example, the method described in Bioorganic & Medicinal Chemistry, 23(22), p. 7150-7157, 2015, etc.] is converted into an acid chloride in a conventional manner, and then a Grignard reaction is carried out 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 production method known in the literature], followed by removing a protecting group P 1 is deprotected to produce an amine represented by formula (AM-1-V1) or a salt thereof.

[0329] <Step 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) to form a protecting group P 1 is deprotected to produce an amine represented by formula (AM-1-V2) or a salt thereof.

[0330] The compound used in <Step 1> of the above [Production Method AM-V] is replaced 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 in the literature] and a reaction is carried out to produce an amine having a corresponding linker, or a salt thereof. [ka]

[0331] [Manufacturing method AM-W] Method for preparing amines represented by formula (AM-1-W1) and formula (AM-1-W2): [ka]

[0332] In the above [Production Method AM-V], the compound of formula (SM-V) is replaced with the 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 in the literature], and a reaction is carried out in accordance with the method described in [Production Method AM-V] to produce amines represented by formula (AM-1-W1) and formula (AM-1-W2), or salts thereof.

[0333] The compound used in <Step 1> of the above [Production Method AM-W] is replaced 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 in the literature], and a reaction is carried out to produce an amine having a corresponding linker, or a salt thereof. [ka]

[0334] An amine having an alkyne group (Akn-L) used for producing an alginic acid derivative represented by formula (I) or formula (II) 1 -NH2) or azide-group-introduced amine (N3-L 2 With regard to —NH2), the desired amine can be produced by appropriately combining the reactions described in the above [Production Methods AM-A] to [Production Methods AM-P] and the like, and methods known in the literature, for example, the methods described in “Experimental Chemistry Lectures, 5th Edition, various books, 2007, Maruzen”, “Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 3rd Edition (Edited by Richard C. Larock), 2018”, and “Strategic Applications of Named Reactions in Organic Synthesis, (Edited by Laszlo Kurti, Barbara Czako), Academic Press, 2005”, and the like.

[0335] In the present specification, the amine compounds represented by formula (AM-1) or formula (AM-2) (including subformulae of each formula) may form pharmaceutically acceptable salts (e.g., acid addition salts). Such salts are not particularly limited as long as they are pharmaceutically acceptable salts, and examples thereof include salts with inorganic acids, salts with organic acids, and salts with acidic amino acids. Suitable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Suitable examples of salts with organic acids include salts 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, and mandelic acid; salts with aliphatic dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, and tartaric acid; salts with aliphatic tricarboxylic acids such as citric acid; salts with aromatic monocarboxylic acids such as benzoic acid and salicylic acid; salts with aromatic dicarboxylic acids such as phthalic acid; salts with organic carboxylic acids such as cinnamic acid, glycolic acid, pyruvic acid, oxylic acid, salicylic acid, and N-acetylcysteine; salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid; and acid addition salts with acidic amino acids such as aspartic acid and glutamic acid. Suitable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, etc. Among these, pharma- ceutically acceptable salts are preferred.

[0336] The salts can be obtained by a conventional method, for example, by mixing the compound of the present invention with a solution containing an appropriate amount of acid or base to form the desired salt, and then separating and filtering the salt or by distilling off the mixed solvent. A general description of salts is available in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Stahl & Wermuth (Wiley-VCH, 2002), which provides a detailed description.

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

[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 means that it can be substituted on any of the carbon atoms i, ii, iii, iv, or v in the formula A. [ka]

[0339] In the present specification, the linker (-L) in the chemically modified alginic acid derivative represented by formula (I) or formula (II) is 1 -or-L 2 When an asymmetric carbon is present in --), each of the optical isomers is also included.

[0340] For example, -L in formula (I) 1 - is the formula (L1-8a), m8a = 2, n8a = 1, R 1 The following formula (L1-8a-M) when =Me (the formula does not include the areas outside the dashed lines): [ka] If R 1 The following formula (L1-8a-MS) has an S-configuration at the carbon substituted with a benzyl group, and the following formula (L1-8a-MR) has an R-configuration at the carbon substituted with a benzyl group (both formulas do not include the areas outside the dashed lines): [ka] This means that the linker represented by

[0341] The linker (-L) in the chemically modified alginic acid derivative represented by formula (I) or formula (II) 1 -or-L 2When an asymmetric carbon is present in formula (-) (when it is an optically active substance), in the process of synthesizing an amine derivative (AM-1) corresponding to formula (I) or formula (II), it is possible to separate each optically active substance from the racemic mixture by a conventional optical resolution means (separation method). Also, in the process of synthesizing the amine derivative of formula (AM-1) or formula (AM-2) 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. Applications of alginate derivatives and cross-linked alginate structures Alginic acid derivatives can be used in place of conventional alginic acid in a wide range of fields, including food, medicine, cosmetics, textiles, papermaking, etc. Specific preferred applications of alginic acid derivatives or photocrosslinked alginic acid structures include medical materials such as wound dressings, postoperative adhesion barriers, drug sustained release substrates, cell culture substrates, and cell transplant substrates.

[0343] When used as a medical material, the shape of the cross-linked alginate structure may be tubular, fibrous, fiber, beads, gel, or approximately spherical gel, etc., and is preferably in the form of beads, gel, or approximately spherical gel, and more preferably in the form of an approximately spherical gel.

[0344] Furthermore, all literature and publications mentioned in this specification are incorporated herein by reference in their entirety, regardless of purpose.

[0345] In addition, the objects, features, advantages, and ideas of the present invention will be 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 are shown to show preferred embodiments of the present invention, and are shown for illustrative or explanatory purposes, and do not limit the present invention thereto. It is clear to those skilled in the art that various modifications can be made based on the description of this specification within the intent and scope of the present invention disclosed in this specification. EXAMPLES

[0346] Next, examples and test examples will be given in order to explain the present invention in more detail. However, these examples are merely examples and test examples and do not limit the present invention. In addition, the examples may be modified within the scope of the present invention.

[0347] Nuclear magnetic resonance spectroscopy (NMR) was measured using a JEOL JNM-ECX400 FT-NMR (JEOL). Liquid chromatography-mass spectrometry (LC-Mass) was measured using the following method. [UPLC] A Waters AQUITY UPLC system and a BEH C18 column (2.1 mm x 50 mm, 1.7 μm) (Waters) were used, with a 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).

[0348] 1 In the H-NMR data, the NMR signal pattern is: s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, CDCl3: deuterated chloroform, DMSO-d6: deuterated dimethyl sulfoxide, and D2O: deuterated water. 1 In the H-NMR data, signals that cannot be confirmed due to broadbands, such as protons of the hydroxyl group (OH), amino group (NH2), and carboxyl group (COOH), are not included in the data.

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

[0350] In the examples, "room temperature" generally refers to a temperature of about 0°C to about 35°C. The reactive substituent introduction rate (mol%) in the examples is 1This indicates the ratio of the number of moles of the introduced reactive substituents to the number of moles of the monosaccharide units (guluronic acid and mannuronic acid) constituting alginic acid calculated from H-NMR (D2O).

[0351] In the examples, sodium alginate having the physical properties shown in Table 46 was used as the sodium alginate before the reactive group or the complementary reactive group was introduced.

[0352] Table 48 shows the physical properties (specifically, reactive group introduction rate (mol%), molecular weight, and weight average molecular weight (10,000 Da)) of the alginic acid derivatives into which reactive groups had been introduced, obtained in (Example 1) to (Example 20). Tables 49-1 to 49-5 show the 1H-NMR of the intermediates in (Example 1) to (Example 20), and Table 50 shows the LCM-Mass of the intermediates in (Example 1) to (Example 20).

[0353] Example 1 Synthesis of 3-azidopropylamino-functionalized alginic acid (EX1-A2): [ka]

[0354] To a 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (20 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (56 mg), a solution of commercially available 3-azidopropylamine [CAS REGISTRY NO.: 88192-19-2] (1-1, 5.1 mg) in ethanol (2 mL), and 1 molar 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 added in sequence and stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The resulting solid was dissolved in water and then freeze-dried to obtain the title compound EX1-A2 (187 mg) as a white solid.

[0355] Example 2 Synthesis of 2-(2-(2-azidoethoxy)ethoxy)ethane-1-amino group-introduced alginic acid (EX2-A2): [ka]

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

[0357] Example 3 Synthesis of 2-amino-N-(3-azidopropyl)acetamide-functionalized alginic acid (EX3-A2): [ka]

[0358] <Process 1> Synthesis of tert-butyl (2-(3-azidopropyl)amino)-2-oxoethyl)carbamate (3-2): [ka] 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), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (197 mg) was added and stirred at room temperature for 18 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated saline in that order. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting oily substance was dissolved in methyl-tert-butyl ether (10 mL) and washed with saturated sodium bicarbonate water, water, and saturated saline in that order. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain the title compound 3-2 (95 mg) as a colorless oil.

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

[0360] To the compound (3-2, 95 mg) obtained in <Step 1> of (Example 3), 4N hydrogen chloride / 1,4-dioxane (665 μL) was added under ice-water cooling, and the mixture was stirred at room temperature for 1 hour. Diisopropyl ether (2.0 mL) was added to the reaction solution, and the mixture 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 3-3 (62 mg) as a colorless gum.

[0361] <Process 3> Synthesis of 2-amino-N-(3-azidopropyl)acetamide-functionalized alginic acid (EX3-A2): [ka]

[0362] To an aqueous solution (20 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) prepared to 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 (Example 3) <Step 2> in ethanol (2 mL), and 1 molar sodium bicarbonate water (76 μL) were added. After stirring at 30° C. for 3 hours, sodium chloride (0.2 g) and ethanol (40 mL) were added in that order, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting 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): [ka]

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

[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), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (146 mg) was added and stirred at room temperature for 18 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated saline in that order. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the title compound 4-2 (124 mg) as a white wax.

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

[0367] To the compound (4-2, 124 mg) obtained in <Step 1> of Example 4, 4N hydrogen chloride / 1,4-dioxane (868 μL) was added under ice-water cooling, and the mixture was stirred at room temperature for 1 hour. Diisopropyl ether (2.6 mL) was added to the reaction solution, and the mixture 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.

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

[0369] To an aqueous solution (20 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) prepared to 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 (Example 4) <Step 2> in ethanol (2 mL), and 1 molar concentration sodium bicarbonate water (76 μL) were added. After stirring at 30° C. for 3 hours, sodium chloride (0.2 g) and ethanol (40 mL) were added in that order, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting 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): [ka]

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

[0372] A solution of azidoacetic acid chloride (1.0 mL) prepared from commercially available 2-azidoacetic acid [CAS REGISTRY NO.: 18523-48-3] (1-1, 41 μL) in methylene chloride (1.0 mL) was added to a solution of commercially available 1-(N-tert-butyloxycarbonyl-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-water cooling and stirred at room temperature for 2.5 hours. Ethyl acetate (20 mL) and water (5 mL) were added to the reaction solution, and after separation, the organic layer was washed successively with water, saturated sodium bicarbonate water, water, and saturated saline. The insoluble matter was removed by filtration, and 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 obtain the title compound 5-2 (91 mg) as a light beige solid.

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

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

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

[0376] 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (70 mg), the compound (5-3, 16.1 mg) obtained in (Example 5) <Step 2>, and 1 molar sodium bicarbonate water (95 μL) were added to a 1 wt% aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.). After stirring for 3 hours at 30°C, sodium chloride (0.25 g) and ethanol (50 mL) were added in that order, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting 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): [ka]

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

[0379] To 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), potassium carbonate (0.61 g) was added 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. The crude product was dissolved in methyl tert-butyl ether (20 mL), washed twice with 1N aqueous sodium hydroxide solution (5 mL), twice with water (5 mL), and then with saturated saline (5 mL), and dried over anhydrous sodium sulfate. The organic layer was filtered and concentrated under reduced pressure to give the title compound 6-3 (0.411 g) as a purple oil.

[0380] <Process 2> Synthesis of 2-(4-azidophenoxy)ethan-1-amine hydrochloride (6-4): [ka]

[0381] To a mixture of the compound (6-3, 0.41 g) obtained in (Example 6) <Step 1> and 1,4-dioxane (2.87 mL), 4N-hydrogen chloride / 1,4-dioxane (2.87 mL) was added under stirring with water cooling, and 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 collected solid was dried under reduced pressure to obtain the title compound 6-4 (0.2834 g) as a pale purple solid.

[0382] <Process 3> Synthesis of 2-(4-azidophenoxy)ethane-1-amino group-introduced alginic acid (EX6-A2): [ka] 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (91.52 mg) and 1 molar sodium bicarbonate solution (68.63 μL) were added to a 1% by weight aqueous solution (29.66 mL) of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.). Then, a solution of the compound (6-4, 14.73 mg) obtained in (Example 6) <Step 2> 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 added in sequence, 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 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): [ka]

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

[0385] Ethyl trifluoroacetate (2.24 mL) was added dropwise 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). The reaction mixture was stirred at room temperature for 14.5 hours. The reaction mixture was concentrated under reduced pressure, and the residue was triturated with tert-butyl methyl ether (5 mL) and heptane (25 mL). 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] <Process 2> Synthesis of N-(2-aminoethyl)-2,2,2-trifluoroacetamide hydrochloride (7-3): [ka]

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

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

[0389] A solution of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (1.09 g), compound 7-3 (380 mg) obtained in (Example 7) <Step 2>, and triethylamine (321 μL) were added to an ethanol (2 mL) solution of a carboxylic acid (7-4, 300 mg) synthesized according to a method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), and the mixture was stirred at 30 ° C for 3 hours, and then triethylamine (229 μL) was added and 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, the mixture was separated, and 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 saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. To the residue was added tert-butyl methyl ether, insoluble matter was removed by filtration, and 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): [ka]

[0391] To a solution of compound 7-5 (322 mg) obtained in (Example 7) <Step 3> in methanol (4.8 mL), a solution of potassium carbonate (278 mg) in water (1.6 mL) was added and 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 x 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] <Process 5> Synthesis of N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide-group-introduced alginic acid (EX7-B2): [ka]

[0393] To a 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (120 mL), 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 (Example 7) <Step 4> in ethanol (12 mL), and 1 molar concentration sodium bicarbonate water (303 μL) were added in that order under stirring at room temperature, and the mixture was stirred at 30 degrees for 3 hours. Sodium chloride (1.2 g) was added to the reaction solution, followed by addition of ethanol (240 mL), and the mixture was stirred for 1.5 hours. The resulting precipitate was collected by filtration, washed with ethanol (20 mL×5), and then dried under reduced pressure. The resulting 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-yn-1-yloxy)acetamide-group-introduced alginic acid (EX8-A2): [ka]

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

[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), ethyl trifluoroacetate (0.6 mL) was added dropwise. 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] <Process 2> Synthesis of N-(2-(2-aminoethoxy)ethyl)-2,2,2-trifluoroacetamide hydrochloride (8-3): [ka]

[0398] Compound 8-2 (1.5 g) obtained in (Example 8) <Step 1> was added with 4N hydrogen chloride / 1,4-dioxane solution (10.3 mL) under ice-water cooling, and stirred at room temperature for 1 hour. Diisopropyl ether (30 mL) was added to the reaction solution, and stirred at room temperature for 30 minutes. The solvent was distilled off under reduced pressure, and the mixture was 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] <Process 3> Synthesis of N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)ethyl)-2,2,2-trifluoroacetamide (8-4): [ka]

[0400] Carboxylic acid (7-4, 300 mg) synthesized according to a method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110) and compound 8-3 (443 mg) obtained in (Example 8) <Step 2> 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 stirred at room temperature for 2.5 hours. Ethyl acetate (20 mL) and water (10 mL) were added to the reaction solution, and the mixture was separated. The organic layer was washed successively with water (10 mL) and saturated saline (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 give the title compound 8-4 (469 mg) as a colorless gum.

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

[0402] To a solution of compound 8-4 (220 mg) obtained in (Example 8) <Step 3> in methanol (3.0 mL), a solution of potassium carbonate (103 mg) in water (0.99 mL) was added and stirred at room temperature for 4.5 hours. Methanol was distilled off under reduced pressure, water (2 mL) was added, and the mixture was saturated with sodium chloride. Extraction was performed with ethyl acetate (15 mL, 10 mL x 4), and the mixture was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was dissolved in ethyl acetate (10 mL), and insoluble matter was removed by filtration, and the mixture was concentrated under reduced pressure to obtain the title crude compound 8-5 (140 mg) as a pale yellow gum.

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

[0404] To a 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (40 mL), 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 (Example 8) <Step 4> in ethanol (4.0 mL), and 1 molar concentration sodium bicarbonate water (101 μL) were added in sequence at room temperature under stirring, and the mixture was stirred at 30° C. for 3 hours. Sodium chloride (0.4 g) was added to the reaction solution, followed by ethanol (80 mL) and stirring for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting solid was dissolved in water and freeze-dried to obtain the title compound EX8-A2 (410 mg) as a white solid.

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

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

[0407] N-(tert-butoxycarbonyl)glycine (91 mg, [CAS REGISTRY NO.: 4530-20-5]) and the compound (7-3, 100 mg) obtained in (Example 7) <Step 2> were 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 stirred at room temperature for 3.5 hours. Ethyl acetate (15 mL) and water (5 mL) were added to the reaction solution, and after separation, the organic layer was washed with water and saturated saline in that order. 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 light beige amorphous.

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

[0409] To the compound (9-1, 180 mg) obtained in <Step 1> of (Example 9), 4N hydrogen chloride / 1,4-dioxane (1.2 mL) was added under ice-water cooling, and the mixture was stirred at room temperature for 0.8 hours. To the reaction solution, diisopropyl ether (3.6 mL) was added, 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] <Process 3> Synthesis of N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)acetamido)ethyl)-2,2,2-trifluoroacetamide (9-3): [ka]

[0411] A carboxylic acid (7-4, 80 mg) synthesized according to a method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), and a compound (9-2, 110 mg) obtained in (Example 9) <Step 2> 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. Water (3.2 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes, after which the solid was filtered and washed with water. Ethyl acetate / ethanol (1 / 1, 10 mL) was added to the obtained solid, and insoluble matter was removed by filtration. 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)acetamide)acetamide (9-4): [ka]

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

[0414] <Process 5-1> Synthesis of N-(2-(aminoethyl)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)acetamido group-introduced alginic acid (EX9a-A2): [ka]

[0415] To an aqueous solution (38 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) 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 (9-4, 30.3 mg) obtained in (Example 9) <Step 4> in ethanol (3.8 mL), and 1 molar sodium bicarbonate water (96 μL) were added. After stirring at 30° C. for 3.2 hours, sodium chloride (0.38 g) and ethanol (76 mL) were added in that order, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting 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 N-(2-(aminoethyl)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)acetamido group-introduced alginic acid (EX9b-B2): [ka]

[0417] 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 (Example 9) <Step 4>, and 1 molar sodium bicarbonate solution (58 μL) were added to a 1 wt% aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.). After stirring for 3.2 hours at 30°C, sodium chloride (0.38 g) and ethanol (76 mL) were added in that order, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting 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-yn-1-yloxy)acetamido)propanamide group-introduced alginic acid (EX10-A2): [ka]

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

[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 (Example 7) <Step 2> 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 stirred at room temperature for 3 hours. Ethyl acetate (15 mL) and water (5 mL) were added to the reaction solution, and after separation, the organic layer was washed with water and saturated saline solution in that order. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and 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 with 1N citric acid, water, and saturated saline, successively, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was triturated with tert-butyl methyl ether (10 mL), and the solid was collected by filtration to obtain the title compound 10-1 (80 mg) as a white solid.

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

[0422] To the compound (10-1, 80 mg) obtained in (Example 10) <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] <Process 3> Synthesis of 3-(2-(cyclooct-2-yn-1-yloxy)acetamido)-N-(2-(2,2,2-trifluoroacetamido)ethyl)propanamide (10-3): [ka]

[0424] A carboxylic acid (7-4, 44 mg) synthesized according to a method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), and a compound (10-2, 61 mg) obtained in (Example 10) <Step 2> 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 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 with water and saturated saline solution in turn, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained solid was added with tert-butyl methyl ether (10 mL), 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-yn-1-yloxy)acetamido)propanamide (10-4): [ka]

[0426] To a solution of the compound (10-3, 60 mg) obtained in (Example 10) <Step 3> in methanol (3.0 mL), a solution of potassium carbonate (42 mg) in water (0.3 mL) was added, and the mixture was stirred at room temperature for 3 hours. A solution of potassium carbonate (42 mg) in water (0.3 mL) was then added, and the mixture was stirred at room temperature for 16.5 hours. The reaction solution was concentrated under reduced pressure, and then saturated saline (2 mL) was added, and the mixture was further saturated with sodium chloride. The mixture was extracted with ethyl acetate (15 mL, 10 mL x 4), and 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 removed by filtration. The obtained filtrate was concentrated under reduced pressure to obtain the title compound 10-4 (31 mg) as a colorless oil.

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

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

[0429] (Examples 11a and 11b) Synthesis of 2-(cyclooct-2-yn-1-yloxy)ethane-1-amino group-introduced alginic acid (EX11a-A2, EX11b-B2): [ka]

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

[0431] Dichloromethane (2 mL) was added at room temperature to a mixture of dibromo compound (11-1, 1 g) synthesized according to a method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110) and alcohol compound (11-2, 5.28 g) synthesized according to a method known in the literature (International Publication No. WO 2015 / 140807). The internal temperature was kept at room temperature, and the reaction vessel was wrapped in aluminum foil to protect it from light. Then, 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 saline (5 mL) was added under ice cooling, the precipitated silver salt was removed by celite filtration, 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). The mixture was then 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) to obtain a fraction containing compound 11-3 (0.46 g).

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

[0433] (Example 11) To a mixture of a fraction containing 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 stirring while cooling with water, 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 the crude product of the title compound 11-4 (0.196 g) as a brown oil.

[0434] <Process 3-1> Synthesis of 2-(cyclooct-2-yn-1-yloxy)ethane-1-amino group-introduced alginic acid (EX11a-A2): [ka]

[0435] 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (213.55 mg) was added to an aqueous solution (69.2 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) prepared at 1% by weight at room temperature. Then, a solution of the compound (11-4, 26.78 mg) obtained in (Example 11) <Step 2> in water (1 mL) and ethanol (1 mL) was added at room temperature, and the mixture was stirred at the same temperature for 24 hours. Then, sodium chloride (700 mg) and ethanol (138.4 mL) were added in sequence, 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] <Process 3-2> Synthesis of 2-(cyclooct-2-yn-1-yloxy)ethane-1-amino group-introduced alginic acid (EX11b-B2): [ka]

[0437] Using a 1 wt% aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (70.1 mL), 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 (Example 11) <Step 2>, the same procedure as in (Example 11) <Step 3-1> was carried out to obtain the titled compound EX11b-B2 (648 mg) as a white solid.

[0438] Example 12 Synthesis of 2-(2-(cyclooct-2-yn-1-yloxy)ethoxy)ethane-1-amino group-introduced alginic acid (EX12-A2): [ka]

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

[0440] 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), 2,2,2-ethyl trifluoroacetate (2.5 mL) was added dropwise over 5 minutes, and the mixture was stirred at room temperature for 20 hours. The reaction solution was concentrated under reduced pressure, and then ethyl acetate (30 mL) and water (10 mL) were added and the mixture was separated. The aqueous layer was extracted with ethyl acetate (10 mL), and the combined organic layer was washed with water and saturated saline solution in that order. 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] <Process 2> Synthesis of (E)-N-(2-(2-((2-bromocyclooct-2-en-1-yl)oxy)ethoxy)ethyl)-2,2,2-trifluoroacetamide (12-3): [ka]

[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 the compound (12-2, 1.86 g) obtained in (Example 12) <Step 1> and silver trifluoromethanesulfonate (0.52 g) were added under light shielding with aluminum foil. After stirring at room temperature for 1.5 hours under light shielding, saturated sodium bicarbonate water (2.0 mL) and saturated saline (3.0 mL) were added sequentially to the reaction solution under ice water cooling. The solid was removed by filtration through Celite and washed with tert-butyl methyl ether (10 mL × 3). The filtrate was separated, and the organic layer was washed sequentially with water and saturated saline. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain the title compound 12-3 (424 mg) as a light brown oil.

[0443] <Process 3> Synthesis of N-(2-(2-(cyclooct-2-yn-1-yloxy)ethoxy)ethyl)-2,2,2-trifluoroacetamide (12-4): [ka]

[0444] The compound (12-3, 100 mg) obtained in (Example 12) <Step 2> was dissolved in tetrahydrofuran (0.7 mL) and N,N-dimethylformamide (0.7 mL). 60% sodium hydride (21 mg) was added under ice water and stirred at the same temperature for 3 hours. 60% sodium hydride (10 mg) was added and stirred at room temperature for 1 hour, and 60% sodium hydride (10 mg) was further added and stirred at room temperature for 20 hours. Water (3 mL) was added, and the mixture was extracted with ethyl acetate (15 mL, 10 mL), and the organic layer was washed with water and saturated saline in that order. The organic layer was dried over anhydrous sodium sulfate and then 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 oil.

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

[0446] To a solution of the compound (12-4, 37 mg) obtained in (Example 12) <Step 3> in methanol (555 μL), a solution of potassium carbonate (50 mg) in water (185 μL) was added and stirred at room temperature for 17 hours. The reaction solution was concentrated under reduced pressure, and then water (1 mL) was added and saturated with sodium chloride. Extraction was performed 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 insoluble matter was removed by filtration. The obtained filtrate was concentrated under reduced pressure to obtain the title compound 12-5 (30 mg) as a colorless oil.

[0447] <Process 5> Synthesis of 2-(2-(cyclooct-2-yn-1-yloxy)ethoxy)ethane-1-amino group-introduced alginic acid (EX12-A2): [ka]

[0448] To a 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (52 mL), 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 (Example 12) <Step 4> in ethanol (5.2 mL), and 1 molar sodium bicarbonate water (131 μL) were added. After stirring at 30° C. for 3.2 hours, sodium chloride (0.52 g) and ethanol (104 mL) were added in that order, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting 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 3-amino-N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)ethyl)propanamide group-introduced alginic acid (EX13-A2): [ka]

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

[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. 2,2,2-trifluoroacetic acid (3.4 mL) was added dropwise over 5 minutes under water cooling, 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 1N hydrochloric acid. Extraction was performed with ethyl acetate (100 mL x 2, 50 mL), and the organic layer was washed with saturated saline. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain the title compound 13-2 (2.9 g) as a white solid.

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

[0453] To a solution of the compound (13-2, 400 mg) obtained in (Example 13) <Step 1>, tert-butyl (2-aminoethyl) carbamate (8-1, 441 mg, [CAS REGISTRY NO.: 57260-73-8]) in ethanol (4.0 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (897 mg) was added and stirred for 3.5 hours. Water (5 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL, 10 mL), and the organic layer was washed with water and saturated saline in that order. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and 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] <Process 3> Synthesis of N-(2-(2-aminoethoxy)ethyl)-3-(2,2,2-trifluoroacetamido)propanamide hydrochloride (13-4): [ka]

[0455] To the compound (13-3, 451 mg) obtained in (Example 13) <Step 2>, 4N hydrogen chloride / 1,4-dioxane (3.16 mL) was added under ice-water cooling, and the mixture was stirred at room temperature for 3 hours. To the reaction solution, diisopropyl ether (6.4 mL) was added, 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): [ka]

[0457] To 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 (Example 13) <Step 3>, 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) were added and stirred at room temperature for 21 hours. Water (5 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (15 mL). The organic layer was washed successively with water and saturated saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting 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] <Process 5> Synthesis of 3-amino-N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)ethyl)propanamide (13-6): [ka]

[0459] To a solution of the compound (13-5, 35 mg) obtained in (Example 13) <Step 4> in methanol (700 μL), a solution of potassium carbonate (33 mg) in water (175 μL) was added, and the mixture was stirred at room temperature for 16.5 hours. The reaction solution was concentrated under reduced pressure, and then water (2 mL) was added and saturated with sodium chloride. Extraction was performed with ethyl acetate (10 mL×5), 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 removed by filtration. The obtained filtrate was concentrated under reduced pressure to obtain the title compound 13-6 (24 mg) as a colorless gum.

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

[0461] To a 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (28 mL), 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 (Example 13) <Step 5> in ethanol (2.8 mL), and 1 molar sodium bicarbonate water (71 μL) were added. After stirring at 30° C. for 3.5 hours, sodium chloride (0.28 g) and ethanol (56 mL) were added in that order, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting solid was dissolved in water and 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-yn-1-yloxy)acetamide group-introduced alginic acid (EX14a-A2, EX14b-B2): [ka]

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

[0464] To a solution of commercially available 2-bromoethylamine hydrobromide [CAS REGISTRY NO.: 2576-47-8] (14-1, 3 g) in methanol (30 mL), triethylamine (4.29 mL) was added under stirring while cooling on ice. 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 the reaction was completed, the reaction solution was concentrated under reduced pressure, and water (10 mL) was added. Extraction was performed three times with ethyl acetate (10 mL), and the organic layer was washed successively with water (5 mL) and saturated saline (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] <Process 2> Synthesis of tert-butyl (4-(2-(2,2,2-trifluoroacetamido)ethoxy)benzyl)carbamate (14-4): [ka]

[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 (14-2, 0.46 g) obtained in (Example 14) <Step 1>, potassium iodide (0.35 g) and N-methylpyrrolidone (3.6 mL), potassium carbonate (0.45 g) was added at room temperature and stirred at 140°C for 5 hours. After the reaction was completed, the 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 washed twice with 1N sodium hydroxide aqueous solution (5 mL), water (5 mL), and saturated saline (5 mL) in that order, and dried over anhydrous sodium sulfate. The organic layer was filtered and then 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 substance.

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

[0468] The compound (14-4, 0.2 g) obtained in (Example 14) <Step 2> was used to carry out the same procedure as in (Example 6) <Step 2> 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): [ka]

[0470] A mixture of carboxylic acid (7-4, 50 mg) synthesized according to a method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), compound (14-5, 81.96 mg) synthesized in (Example 14) <Step 3>, and ethanol was added with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (137.22 mg) and triethylamine (38.25 μL) under ice-cooling and stirring, and stirred at room temperature for 1 hour and 30 minutes. After the reaction was completed, 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 saline (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] <Process 5> Synthesis of N-(4-(2-aminoethoxy)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (14-7): [ka]

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

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

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

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

[0476] Using 1 wt% sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) aqueous solution (40.08 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (123.66 mg) and the compound (14-7, 30.64 mg) obtained in (Example 14) <Step 5>, the same procedure as in (Example 11) <Step 3-1> was carried out to obtain the titled 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): [ka]

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

[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 stirred at room temperature for 4.5 hours. Ethyl acetate (15 mL) and water (5 mL) were added to the reaction solution, and after separation, the organic layer was washed successively with water and saturated saline. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain the title compound 15-2 (63 mg) as a light beige amorphous solid.

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

[0481] A solution of piperidine (56 μL) in N,N-dimethylformamide (315 μL) was added to the compound (15-2, 63 mg) obtained in (Example 15) <Step 1>, 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, and the mixture was separated. The organic layer was washed with water and saturated saline in that order. The organic layer was dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. Tert-butyl methyl ether (5 mL) was added to the obtained solid, and the mixture was triturated and filtered to obtain the title compound 15-3 (10 mg) as a light beige solid. The title compound 15-3 (11 mg) was additionally obtained as a pale yellow gum by recovering it from the filtrate.

[0482] <Process 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): [ka]

[0483] To an aqueous solution (19 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) 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 (Example 15) <Step 2> 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 added in that order, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting 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): [ka]

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

[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 stirred at room temperature for 23 hours. After the reaction was completed, 1N hydrochloric acid (20 mL) was gradually added until the pH became 2, and the mixture was extracted three times with ethyl acetate (10 mL), and washed successively with water (5 mL) and saturated saline (5 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a pale yellow oil. The obtained oil 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 substance.

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

[0488] To a mixture of compound 11-4 (80 mg) and the compound (16-2, 81.83 mg) obtained in (Example 16) <Step 1>, 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 and 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 saline (5 mL), and dried over anhydrous sodium sulfate. The organic layer was filtered and concentrated under reduced pressure to obtain a crude product. 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] <Process 3> Synthesis of 2-amino-N-(2-(cyclooct-2-yn-1-yloxy)ethyl)acetamide (16-4): [ka]

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

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

[0492] Using a 1 wt% aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (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 (Example 16) <Step 3-1>, the title compound EX16-A2 (279 mg) was obtained as a white solid.

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

[0494] <Process 1> Synthesis of (2,2,2-trifluoroacetyl)-L-phenylalanine (17-2): [ka]

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

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

[0497] To a mixture of compound 11-4 (60 mg) and compound (17-2, 93.7 mg) obtained in (Example 17) <Step 1>, 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 saline (5 mL), and dried over anhydrous sodium sulfate. The organic layer was filtered and 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 substance.

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

[0499] The compound (17-3, 57 mg) obtained in (Example 17) <Step 2>, methanol (855 μL), potassium carbonate (38.39 mg) and water (285 μL) were used to carry out the same operation as in (Example 14) <Step 5>, and the title compound 17-4 (35 mg) was obtained as a pale yellow oil.

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

[0501] Using a 1 wt% aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (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 (Example 17) <Step 3-1>, the title compound EX17-A2 (383 mg) was obtained as a white solid.

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

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

[0504] A solution of diethyl azodicarboxylate (40% toluene solution, 1.92 mL) was added to a solution of triphenylphosphine (0.96 g) in tetrahydrofuran (2.59 mL) under ice-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 twice with 1N sodium hydroxide aqueous solution (5 mL) and then with saturated saline (5 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was then distilled off under reduced pressure to obtain the title compound 18-2 (0.45 g) as a pink oily substance.

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

[0506] Lithium hydroxide monohydrate (0.25 g) was added to a methanol (4.4 mL) solution of compound 18-2 (0.44 g) obtained in (Example 18) <Step 1>, and the mixture was stirred at 60°C for 3 hours and 30 minutes. 1N-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 washed successively with water (5 mL) and saturated saline (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, N,N-diisopropylethylamine (0.52 mL) was added under ice-cooling and stirring, and the mixture was stirred at room temperature for 5 hours. Water (10 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (15 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain a fraction containing compound 18-3 (0.71 g).

[0507] To the fraction containing compound 18-3 (0.71 g), 4N hydrogen chloride / 1,4-dioxane (4.9 mL) was added and 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] <Process 3> Synthesis of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group-introduced alginic acid (compound EX18-A2): [ka]

[0509] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (19.6 mL) was cooled on ice and stirred, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (50.19 mg), compound 18-4 (54.37 mg) obtained in (Example 18) <Step 2>, and 1 molar sodium bicarbonate water (181.4 μL) were used to carry out the same operation as in (Example 11) <Step 3-1>, to obtain the titled compound EX18-A2 (198 mg) as a white solid.

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

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

[0512] (Example 20) Synthesis of N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX20-B2): [ka]

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

[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 extracted three times with ethyl acetate (10 mL). The collected organic layer was washed with saturated saline (5 mL) and dried over anhydrous sodium sulfate. The dried organic layer was filtered and concentrated to obtain the title crude compound 20-2 (0.67 g) as a pale yellow amorphous substance.

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

[0516] To the 1,4-dioxane solution (3.5 mL) of compound 20-2 (0.5 g) obtained in (Example 20) <Step 1>, 4N-hydrogen chloride / 1,4-dioxane (3.5 mL) was added under stirring in water cooling, 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] <Process 3> Synthesis of N-(4-((2-(cyclooct-2-yn-1-yloxy)acetamido)methyl)benzyl)-2,2,2-trifluoroacetamide (compound 20-4): [ka]

[0518] A carboxylic acid (7-4, 0.17 g) synthesized according to a method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.26 g) were added dropwise to an acetonitrile (1.7 mL) solution of the compound 20-3 (0.26 g) obtained in (Example 20) <Step 2> and N,N-diisopropylethylamine (0.51 mL) under ice-cooling and stirring, 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 saline (3 mL) and then dried over anhydrous sodium sulfate. The dried organic layer was filtered, and 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-yn-1-yloxy)acetamide (compound 20-5): [ka]

[0520] To a mixture of compound 20-4 (0.18 g) obtained in (Example 20) <Step 3> and methanol (1.8 mL), potassium carbonate (0.13 g) aqueous solution (0.9 mL) was added dropwise under ice-cooling and stirring, and the mixture was stirred at room temperature for 17 hours and 30 minutes. Methanol was distilled off under reduced pressure, and the mixture was extracted three times with ethyl acetate (5 mL). The organic layer was washed with saturated saline (5 mL) and then dried over anhydrous sodium sulfate. The organic layer was filtered, and 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] <Process 5> Synthesis of N-(4-(aminoethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX20-B2): [ka]

[0522] Using a 1 wt% aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (50.9 mL), 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 (Example 20) <Step 4> in ethanol (3 mL), the same procedure as in (Example 11) <Step 3-1> was carried out to obtain the titled compound EX20-B2 (521 mg) as a white solid.

[0523] (Examples P1 to P7) The alginic acid derivatives shown in the table below (Example P1) to (Example P7) are produced in accordance with the method shown in the above examples using the corresponding amino compound (which may be a pharma- ceutically acceptable salt thereof or a solvate thereof) and alginic acid. [Table 47]

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

[0525] NMR data of intermediate compounds [Table 49-1] [Table 49-2] [Table 49-3] [Table 49-4] [Table 49-5]

[0526] LC-Mass data of intermediate compounds [Table 50]

[0527] [Measurement of introduction rate of reactive group or complementary reactive group] The introduction rate of the reactive group or the complementary reactive group means the number of reactive groups or the complementary reactive groups introduced per uronic acid monosaccharide unit, which is a repeating unit of alginic acid, expressed as a percentage. In this embodiment, the introduction rate (mol%) of the reactive group or the complementary reactive group is 1 The amount of alginic acid required to calculate the introduction rate was 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 an absorbance measurement method using a calibration curve.

[0528] [Molecular weight measurement] The alginic acid solid obtained in the examples into which reactive groups or complementary reactive groups have been introduced was dissolved in 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl to prepare a 0.1% or 0.2% solution, which was then passed through a polyethersulfone filter (Minisart High Flow Filter, Sartorius) with a pore size of 0.22 μm to remove insoluble matter, and used as a sample for gel filtration. The spectrum of each sample was measured using a spectrophotometer DU-800 (Beckman-Coulter), and the measurement wavelength in gel filtration for each compound was determined. For compounds that did not have a specific absorption wavelength, a differential refractometer was used.

[0529] 200 μL of the gel filtration sample was applied to a Superose6 Increase10 / 300 GL column (GE Healthcare Sciences). Gel filtration was performed at room temperature with a flow rate of 0.8 mL / min using an AKTA Explorer 10S chromatograph and 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl as the developing solvent. The elution profile of the sample was created by monitoring the absorption at the wavelength determined for each compound. The obtained chromatogram was analyzed with Unicorn5.31 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 for alginic acid into which a reactive group or a complementary reactive group has been introduced, using blue dextran (molecular weight 2 million Da, SIGMA), thyroglobulin (molecular weight 669,000 Da, GE Healthcare Science), ferritin (molecular weight 440,000 Da, GE Healthcare Science), aldolase (molecular weight 158,000 Da, GE Healthcare Science), conalbumin (molecular weight 75,000 Da, GE Healthcare Science), ovalbumin (molecular weight 44,000 Da, GE Healthcare Science), ribonuclease A (molecular weight 13,700 Da, GE Healthcare Science), and aprotinin (molecular weight 6500 Da, GE Healthcare Science) as standards, and 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, respectively, and linear regression was performed to create a calibration curve. Two calibration curves were prepared, one from blue dextran to ferritin and the other from ferritin to aprotinin.

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

[0532]

number

[0533] [Measurement of gel stability] (Gel stability measurement (1)): Stability in PBS The alginic acid 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 aqueous alginic acid 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), respectively, and placed in a syringe equipped with an 18-gauge needle, and the syringe was placed in a syringe pump set to 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 alginic acid gel. This gel was washed once with 10 mL of PBS, and left to stand in PBS at 37°C for 10 minutes to perform chemical crosslinking, obtaining a chemically crosslinked alginic acid gel. Cross-linked alginate gels (beads) made with (Ex18-A2) / (Ex19-A2) were also prepared in the same manner. 19.5 mL of PBS was added to this gel, and the gel was shaken at 37°C to recover the aqueous solution over time, and the same amount of PBS as that recovered was replenished. After the test was completed, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and the gel was completely disintegrated by shaking at 37°C for more than 3 hours, and the aqueous solution was recovered. The alginate concentration in the recovered 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 times and the alginate concentration after the test was completed, and the value expressed as a percentage was used as the disintegration rate, which was used as an index of gel stability.

[0534] The results shown in Figure 1 were obtained. The cross-linked alginate gel (beads) did not collapse even after 96 hours, confirming the stability of the gel. In other words, it was suggested that the (bead) structure produced was able to maintain its structure for a long period of time due to the formation of chemical cross-links by the Huisgen reaction. The cross-linked alginate gel (beads) produced by (Ex18-A2) / (Ex19-A2) was the control for this test.

[0535] (Gel stability measurement (2)): Stability under EDTA The alginic acid aqueous solutions obtained in (Gel Stability Measurement (1)) were mixed in equal amounts in combinations of (1-1) and (19-1), (4-1) and (19-1), and (5-1) and (19-1), respectively, and placed in a syringe equipped with an 18-gauge needle. The syringe was placed in a syringe pump set to a flow rate of 1 mL / min, and the mixture was dropped into a calcium chloride solution with a concentration of 30 mmol / L for 30 seconds and stirred for 5 minutes to obtain an alginic acid gel. Crosslinked alginic acid gels (beads) made with (Ex18-A2) / (Ex19-A2) were also prepared in the same manner. The gels were washed once with 10 mL of saline, and left to stand in saline at 37°C for 10 minutes to perform chemical crosslinking, obtaining chemically crosslinked alginic acid gels. To this gel, 19.5 mL of 5 mM ethylenediaminetetraacetic acid dipotassium salt dihydrate (EDTA·2K) / physiological saline was added, and the gel was shaken at 37°C. The aqueous solution was collected over time, and the same amount of 5 mM EDTA·2K / physiological saline was replenished. After the test was completed, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and the gel was completely collapsed by shaking at 37°C for more than 3 hours, 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 alginic acid dissolved up to each time point was divided by the total amount of alginic acid calculated from the alginic acid concentration at all times and the alginic acid concentration after the test was completed. The value expressed as a percentage was used as an index of gel stability.

[0536] The results are shown in Figure 2. The cross-linked alginate gel (beads) had a disintegration rate of 2% or less after 24 hours. This suggests that cross-linking by the Huisgen reaction allows the fabricated structure to maintain its structure even in a solution without calcium ions (below the physiological concentration for the body). The cross-linked alginate gel (beads) fabricated by (Ex18-A2) / (Ex19-A2) was used as a control for this test.

[0537] (Gel stability measurement (3)): Stability in PBS The alginic acid 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 aqueous alginic acid 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), respectively, and placed in a syringe equipped with an 18-gauge needle. The syringe was placed in a syringe pump set to a flow rate of 1 mL / min, and the mixture was 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 left to stand in PBS at 37°C for 10 minutes to perform chemical crosslinking, obtaining a chemically crosslinked alginate gel. Crosslinked alginate gels (beads) made with (Ex18-A2) / (Ex19-A2) were also prepared in the same manner. 19.5 mL of PBS was added to this gel, and the gel was shaken at 37°C to collect the aqueous solution over time, and the same amount of PBS 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 the gel was completely disintegrated by shaking at 37°C for more than 3 hours, 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 alginic acid dissolved up to each time point was divided 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 was completed. The value expressed as a percentage was used as the disintegration rate, which was used as an index of gel stability.

[0538] The results shown in FIG. 3 were obtained. The cross-linked alginate gel (beads) did not collapse even after 96 hours, confirming the stability of the gel. In other words, it was suggested that the (bead) structure produced was able to maintain its structure for a long period of time due to the formation of chemical cross-links by the Huisgen reaction. The cross-linked alginate gel (beads) produced by (Ex18-A2) / (Ex19-A2) was used as a control in this test.

[0539] (Gel stability measurement (4)): Stability under EDTA The alginic acid aqueous solution obtained in (Gel Stability Measurement (3)) was mixed in equal amounts with the combinations of (6-1) and (9-1), (3-1) and (10-1), (5-1) and (10-1), and (18-1) and (12-1), respectively, and placed in a syringe equipped with an 18-gauge needle. The syringe was placed in a syringe pump set to a flow rate of 1 mL / min, and the mixture was dropped into a calcium chloride solution with a concentration of 30 mmol / L for 30 seconds and stirred for 5 minutes to obtain an alginic acid gel. The gel was washed once with 10 mL of physiological saline, and left to stand in physiological saline at 37°C for 10 minutes to perform chemical crosslinking, obtaining a chemically crosslinked alginic acid gel. Crosslinked alginic acid gels (beads) made by (Ex18-A2) / (Ex19-A2) were also prepared in the same manner. To this gel, 19.5 mL of 5 mM ethylenediaminetetraacetic acid dipotassium salt dihydrate (EDTA·2K) / physiological saline was added, and the gel was shaken at 37°C. The aqueous solution was collected over time, and the same amount of 5 mM EDTA·2K / physiological saline was replenished. After the test was completed, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and the gel was completely collapsed by shaking at 37°C for more than 3 hours, 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 alginic acid dissolved up to each time point was divided by the total amount of alginic acid calculated from the alginic acid concentration at all times and the alginic acid concentration after the test was completed. The value expressed as a percentage was used as an index of gel stability.

[0540] The results are shown in Figure 4. The cross-linked alginate gel (beads) had a disintegration rate of 4% or less after 24 hours. This suggests that cross-linking by the Huisgen reaction allows the fabricated structure to maintain its structure even in a solution without calcium ions (below the physiological concentration for the body). The cross-linked alginate gel (beads) fabricated with Ex18-A2 / Ex19-A2 was used as a control in this test.

[0541] (Gel stability measurement (5)): Stability in PBS The alginic acid derivatives (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 aqueous alginic acid solutions (4-1), (9-1), (16-1), (18-1) and (20-1), respectively. These were mixed in equal amounts in combinations of (4-1) and (20-1), (18-1) and (9-1), and (18-1) and (16-1), respectively, and placed in a syringe equipped with an 18-gauge needle. The syringe was placed in a syringe pump set to a flow rate of 1 mL / min, and the mixture was dropped into a calcium chloride solution with a concentration of 30 mmol / L for 30 seconds and stirred for 5 minutes to obtain an alginic acid gel. This gel was washed once with 10 mL of PBS, and left to stand in PBS at 37°C for 10 minutes to perform chemical crosslinking, obtaining a chemically crosslinked alginate gel. Crosslinked alginate gels (beads) made with (Ex18-A2) / (Ex19-A2) were also prepared in the same manner. 19.5 mL of PBS was added to this gel, and the gel was shaken at 37°C to collect the aqueous solution over time, and the same amount of PBS 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 the gel was completely disintegrated by shaking at 37°C for 3 hours or more to collect the aqueous solution. 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 times and the alginate concentration after the test was completed, and the value expressed as a percentage was used as an index of gel stability.

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

[0543] (Gel stability measurement (6)): Stability under EDTA The alginic acid aqueous solution obtained in (Gel Stability Measurement (5)) was mixed in equal amounts with the combinations of (4-1) and (20-1), (18-1) and (9-1), and (18-1) and (16-1), respectively, and placed in a syringe equipped with an 18-gauge needle. The syringe was placed in a syringe pump set to a flow rate of 1 mL / min, and the mixture was dropped into a calcium chloride solution with a concentration of 30 mmol / L for 30 seconds and stirred for 5 minutes to obtain an alginic acid gel. The gel was washed once with 10 mL of physiological saline, and left to stand in physiological saline at 37°C for 10 minutes to perform chemical crosslinking, obtaining a chemically crosslinked alginic acid gel. Crosslinked alginic acid gels (beads) made by (Ex18-A2) / (Ex19-A2) were also prepared in the same manner. To this gel, 19.5 mL of 5 mM ethylenediaminetetraacetic acid dipotassium salt dihydrate (EDTA·2K) / physiological saline was added, and the gel was shaken at 37°C. The aqueous solution was collected over time, and the same amount of 5 mM EDTA·2K / physiological saline was replenished. After the test was completed, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and the gel was completely collapsed by shaking at 37°C for more than 3 hours, 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 alginic acid dissolved up to each time point was divided by the total amount of alginic acid calculated from the alginic acid concentration at all times and the alginic acid concentration after the test was completed. The value expressed as a percentage was used as an index of gel stability.

[0544] The results shown in FIG. 6 were obtained. The cross-linked alginate gel (beads) had a disintegration rate of 18% or less after 24 hours. This suggests that the cross-linking formed by the Huisgen reaction allows the fabricated structure to maintain its structure even in a solution without calcium ions (below the physiological concentration for the body). The cross-linked alginate gel (beads) fabricated with Ex18-A2 / Ex19-A2 was the control for this test.

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

[0546] Furthermore, each of the alginic acid derivatives (Ex10-A2), (Ex12-A2) and (Ex19-A2) obtained in Examples 10, 12 and 19 was 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 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), and (18-2) and (12-1), respectively, and 40 mL of a 30 mmol / L calcium chloride solution was added and stirred for 5 minutes to obtain an alginate gel. This gel was washed once with 10 mL of saline, and left to stand in saline at 37°C for 10 minutes to perform chemical crosslinking, obtaining a fluorescein isothiocyanate-dextran-encapsulated chemically crosslinked alginate gel. The fluorescein isothiocyanate-dextran-encapsulated chemically crosslinked alginate gel made of (Ex18-A2) / (Ex19-A2) was also prepared in the same manner. 19.5 mL of physiological saline was added to this gel, and the gel was shaken at 37°C to recover the aqueous solution over time, and the same amount of physiological saline was replenished. After the test was completed, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and the gel was completely disintegrated by shaking at 37°C for more than 3 hours, and the aqueous solution was recovered. The dextran concentration in the recovered aqueous solution was measured by fluorometry (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, expressed as a percentage, was taken as the transmittance.

[0548] The results shown in Figure 7 were obtained. The transmittance after 24 hours was in the range of 25% to 40%. The fluorescein isothiocyanate-dextran-encapsulated chemically cross-linked alginate gels prepared in (Ex18-A2) / (Ex19-A2) were used as controls in this study.

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

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

[0551] These were mixed in equal amounts in combinations of (4-2) and (20-1), (5-2) and (10-1), (6-2) and (9-1), (18-2) and (9-1), and (18-2) and (16-1), respectively, and 40 mL of a 30 mmol / L calcium chloride solution was added and stirred for 5 minutes to obtain an alginate gel. This gel was washed once with 10 mL of saline, and left to stand in saline at 37°C for 10 minutes to perform chemical crosslinking, obtaining a fluorescein isothiocyanate-dextran-encapsulated chemically crosslinked alginate gel. The fluorescein isothiocyanate-dextran-encapsulated chemically crosslinked alginate gels made with (Ex18-A2) / (Ex19-A2) were also prepared in the same manner. 19.5 mL of saline was added to this gel, and the gel was shaken at 37°C to collect the aqueous solution over time, and the same amount of saline was replenished as the collected amount. After the test was completed, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and the gel was completely broken down by shaking at 37°C for more than 3 hours, and the aqueous solution was collected. The dextran concentration in the collected aqueous solution was measured by fluorimetry (excitation light: 485 nm, fluorescence: 535 nm), and the amount of dextran up to each time point was divided by the total amount of dextran after the test was completed, and expressed as a percentage to determine the transmittance.

[0552] The results shown in Figure 8 were obtained. The transmittance after 24 hours was in the range of 25% to 30%. The fluorescein isothiocyanate-dextran-encapsulated chemically cross-linked alginate gel prepared by Ex18-A2 / Ex19-A2 was the control for this study.

[0553] [Biocompatibility evaluation of cross-linked alginate derivatives (gels)] The alginic acid 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 were dissolved in water to prepare reactive group-introduced alginic acid solutions. This was sterilized by filtration using a Mini Sart High Flow (Sartorius, 16532GUK), and then a 1.0% reactive group-introduced alginic acid / physiological saline solution was prepared. The cell concentration was 5×10 3HeLa cells were seeded onto a 96-well plate at 1.0x the number of cells / well and then cultured for one day. A 1.0% reactive group-introduced alginate / saline solution was added to the cells in the following combinations: (EX18-A2) and (EX19-A2), (Ex5-A2) and (Ex19-A2), (Ex4-A2) and (Ex20-B2), (Ex18-A2) and (Ex12-A2), or (Ex16-A2) at a final concentration of 0.1%. After one day of culture, ATP activity was evaluated as an indicator of cytotoxicity using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571).

[0554] The results shown in Figure 9 were obtained. ATP activity was confirmed in all cross-linked alginate gels evaluated by the above method, suggesting that the cross-linked alginate gels have no cytotoxicity and that the alginate structures (beads) with chemical cross-links formed by the Huisgen reaction have biocompatibility.

Claims

1. An amide bond and a divalent linker (-L １ and 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 ２ A crosslinked alginic acid obtained by carrying out a crosslinking reaction using an alginic acid derivative represented by the following formula (II) having an azide group introduced therein via an alkyl group (R-), [Alginic acid derivative represented by formula (I)] The following formula (I): 【Chemistry 164】 [In formula (I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L １ - Refer to the table below: 【Table 51-1】 【Table 51-2】 [In each formula, the areas outside the dashed lines at both ends are not included]; Akn is shown in the following table: 【Table 52】 an alginic acid derivative represented by the partial structural formula (I) shown in the following formula (I): [Alginic acid derivative represented by formula (II)] The following formula (II): 【Chemistry 165】 In formula (II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L ２ - Refer to the table below: 【Table 53-1】 【Table 53-2】 [In each formula, the outside of the broken lines at both ends is not included]] (provided that in the alginic acid derivative represented by formula (I), -L １ a derivative in which - is any one linker selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11) and (L1-12); and an alginic acid derivative represented by formula (II), in which -L ２ (Excluding cross-linked alginic acid obtained by carrying out a cross-linking reaction using a derivative in which - is the linker of (L2-10)).

2. An amide bond and a divalent linker (-L １ A cyclic alkyne group (Akn) is introduced via a cyclic alkyne group (Akn) represented by the following formula (I): 【Chemistry 166】 [In formula (I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L １ - Refer to the table below: 【Table 54-1】 【Table 54-2】 [In each formula, the areas outside the dashed lines at both ends are not included]; Akn is shown in the following table: 【Table 55】 An alginic acid derivative represented by the partial structural formula (I) below, wherein the cyclic alkyne group is selected from the group consisting of the partial structural formula (I) below, excluding the right side of the dashed line.

3. Akn-L １ -NH ２ Groups (Akn and -L １ 3. The alginic acid derivative of formula (I) according to claim 2, wherein the introduction rate of the alkyl group (- is the same as defined in claim 2) is 0.1% to 30%.

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

5. An amide bond and a divalent linker (-L ２ -), an azide group is introduced via the following formula (II): 【Chemistry 167】 [In formula (II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L ２ - Refer to the table below: 【Table 56-1】 【Table 56-2】 An alginic acid derivative represented by the partial structural formula (I) shown below, wherein the linker is selected from the group consisting of the partial structural formula (I) shown below, wherein the parts outside the dashed lines at both ends are not included.

6. N ３ -L ２ -NH ２ Group (-L ２ 6. The alginic acid derivative of formula (II) according to claim 5, wherein the introduction rate of the alkyl group (- is the same as that in claim 5) is 0.1% to 30%.

7. The alginic acid derivative of formula (II) according to claim 5, wherein the weight average molecular weight of the alginic acid derivative measured by gel filtration chromatography is 100,000 Da to 3,000,000 Da.

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): 【Chemistry 168】 [In formula (III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -L １ - is as defined in claim 1; -L ２ - is as defined in claim 1; X is as shown in the following table: 【Table 57-1】 【Table 57-2】 The crosslinked alginic acid according to claim 1, wherein the alginic acid is bonded via a cyclic group selected from the group of partial structural formulas shown in the following formula (excluding the areas outside the dashed lines at both ends): １ When - is any one linker selected from the group consisting of (L1-1), (L1-2a), (L1-2b), (L1-11) and (L1-12), the corresponding -L ２ - (excluding the linker of (L2-10)).

9. A method for producing cross-linked alginic acid, comprising mixing an alginic acid derivative represented by formula (I) and an alginic acid derivative represented by formula (II) described in claim 1 and carrying out a cross-linking reaction (Huisgen reaction) to obtain the cross-linked alginic acid described in claim 1.

10. The chemical crosslink formed by subjecting the alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to claim 1 to a Huisgen reaction is represented by the following formula (III-L): 【Chemistry 169】 [In formula (III-L), -CONH- and -NHCO- at both ends, X, -L １ -, and -L ２ A method for producing the cross-linked alginic acid described in claim 1, wherein the cross-linked alginic acid has the structure:

11. A crosslinked alginate structure obtained by dropping a mixed solution of an alginate derivative represented by formula (I) and an alginate derivative represented by formula (II) according to claim 1 into a solution containing a divalent metal ion.

12. The crosslinked alginate structure according to claim 11, comprising chemical crosslinks via triazole rings formed by the Huisgen reaction and ionic crosslinks partially formed by divalent metal ions as crosslinks.

13. A crosslinked alginate structure having the ability to retain contents, obtained by ionic crosslinking with divalent metal ions and chemical crosslinking by the Huisgen reaction of an alginate derivative represented by formula (I) and an alginate derivative represented by formula (II) described in claim 1.

14. The chemical crosslink formed by carrying out the Huisgen reaction using the alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to claim 1 is represented by the following formula (III-L): 【Chemistry 170】 [In formula (III-L), -CONH- and -NHCO- at both ends, X, -L １ -, and -L ２ The crosslinked alginate structure according to any one of claims 11 to 13, having a structure: - is as defined in claim 8.

15. A method for producing a crosslinked alginate structure, comprising dropping a mixed solution of an alginate derivative represented by formula (I) and an alginate derivative represented by formula (II) according to claim 1 into a solution containing a divalent metal ion, and carrying out a crosslinking reaction.

16. A method for producing the crosslinked alginate structure described in claim 15, comprising chemical crosslinking via triazole rings formed by the Huisgen reaction and ionic crosslinking partially formed by divalent metal ions as crosslinks.

17. The chemical crosslink formed by carrying out the Huisgen reaction using the alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to claim 1 is represented by the following formula (III-L): 【Chemistry 171】 [In formula (III-L), -CONH- and -NHCO- at both ends, X, -L １ -, and -L ２ A method for producing a crosslinked alginate structure according to claim 15 or 16, wherein the structure is:

18. 15. The crosslinked alginate structure according to any one of claims 11 to 14, which is in the form of beads or an approximately spherical gel.

19. A medical material comprising the crosslinked alginate structure according to any one of claims 11 to 14.

20. 20. The medical material according to claim 19, which is a bead or an approximately spherical gel.

21. 15. The cross-linked alginate of claim 1, the alginate derivative of claim 2 or claim 5, or the cross-linked alginate structure of any one of claims 11 to 14, which is biocompatible.

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