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 This document relates to structures and their manufacturing methods. [Background technology]

[0002] Alginate is used in Lessonia, Macrocystis, Laminaria, Ascophyllum, and Derby A high molecular weight acidic polysaccharide extracted from the cell walls of natural brown algae such as Ria, Ecklonia cava, Eisenia bicolor, and Laminaria kombu. It is a molecule consisting of β-D-mannuronic acid (M component) and its C-5 epimer, α-L-glucuronic acid. It is a linear heteropolymer of two types of uronic acid (G component) linked together in a 1-4 bond. Specifically, its chemical structure is a homopolymer block of mannuronic acid (MM), guluronic acid The homopolymer block (GG) and the randomly arranged mannuronic acid and guluronic acid It is a block copolymer in which blocks (MG) are complexly bonded in any order and ratio. Acids are widely used in the fields of medicine, biotechnology, cosmetics, textiles, paper, food, etc. It is widely used.

[0003] Alginic acid alkali metal salts of monovalent salts of alginic acid (e.g., sodium alginate, Alginic acid alkaline earth metal salts (e.g., alginic acid calcium, etc.) have the property of being cross-linked by metal ions and gelling (becoming insoluble). Attempts are being made to utilize these properties to modify or mold them into materials suitable for various uses.

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

[0005] Specific methods for obtaining cross-linked polysaccharides include: (1) aldehyde cross-linking with formaldehyde or the like; a crosslinking method using an agent (Patent Document 1: International Publication No. 2011 / 028031); (2) Self-crosslinking method using carboxyl and hydroxyl groups in polysaccharides (Patent Document 2: International Publication No. 89 / 10941 pamphlet), (3) homobifunctional crosslinking agents (diepoxides, divinylsulfonyl sulfone, diamine, or dihydrazide, etc.) or heterobifunctional crosslinkers (epihalohydrins Crosslinking method using a crosslinker (Patent Document 3: International Publication No. 2009 / 073437) is known.

[0006] Also, (4) photoreactive groups (cinnamic acid, substituted cinnamic acid, acrylic acid, maleic acid, fumaric acid) , furyl acrylic acid, thiophene acrylic acid, cinnamylidene acetic acid, sorbic acid, thymine and then irradiating the resulting polymer with light (Patent Documents 4 and 5: International Publication No. 2005 / 026214, Japanese Patent Application Laid-Open No. 9-87236, and (5) A cross-linking method in which polysaccharides to which thiol groups have been introduced are cross-linked with disulfide bonds, and a thio The Michael addition reaction was carried out using a polysaccharide having an aryl group and a polysaccharide having a maleimide group. A crosslinking method by allowing the polymer to crosslink (Patent Document 6: International Publication No. 2008 / 071058) (To), etc. are known.

[0007] Furthermore, as a method for cross-linking polysaccharides by covalent bonds, (6) polysaccharides containing alkyne groups have been reported. The Huisgen reaction (1,3-dipolar addition) was carried out using saccharides and azide-containing polysaccharides. A crosslinking method using a cyclization reaction is known.

[0008] A cross-linked polysaccharide obtained by cross-linking polysaccharides by the Huisgen reaction is disclosed in (i) International Publication No. 2008 / 0 31525 (Patent Document 7), (ii) International Publication No. 2012 / 165462 (iii) International Publication No. 2015 / 020206 (iv) Chinese Patent Application Publication No. 106140040 (Patent Reference 10), and (v) International Publication No. 2019 / 240219 (Patent Document 1 3), etc.

[0009] However, (i) Patent Document 7 discloses a method in which the first polysaccharide is hyaluronic acid and the second polysaccharide is chondroitin. The polysaccharide is selected from the group consisting of cellulose, sulfated dermatan, alginic acid and its salts, and each polysaccharide is linked to The chain-like alkyne and azide groups introduced via the linker were converted to the Huisg The present invention relates to a cross-linked polysaccharide obtained by the en reaction, and a novel cross-linked alginate as described below. The acid is not disclosed.

[0010] In addition, (ii) Patent Document 8 discloses a method for preparing a first polysaccharide and a second polysaccharide by the use of hyaluronic acid, carboxymethylcellulose, and the like. a polysaccharide selected from ethyl dextran, a cellulose derivative, and chitosan (the first polysaccharide and Each polysaccharide is linked to a linker (the second polysaccharide may be the same or different). The cyclic alkyne group and azide group introduced via an ester bond were This paper is about cross-linked polysaccharides obtained by the s-gen reaction, but the novel cross-linked Lactic acid is not disclosed.

[0011] Furthermore, (iii) Patent Document 9 discloses a method for preparing a hydroxybenzoate comprising using hyaluronic acid as the first polysaccharide and chondroitin as the second polysaccharide. The cyclic alkyne group and azide group introduced into each polysaccharide via a linker were used as the hydroxybenzoate. This paper is about cross-linked polysaccharides obtained by the Isgen reaction, but the novel cross-linking Alginate is not disclosed.

[0012] Furthermore, (iv) Patent Document 10 discloses a method for preparing a polysaccharide containing chitosan as the first polysaccharide and sodium alginate as the second polysaccharide. As a group, each polysaccharide is introduced via a linker (the linker is an ester bond between the polysaccharide and the linker). The cross-linked polysaccharide was obtained by subjecting the cyclic alkyne group and azide group to a Huisgen reaction. However, the novel cross-linked alginic acid described below is not disclosed.

[0013] Also, International Publication No. 2016 / 019391 (Patent Document 11) and International Publication In the pamphlet of 2017 / 165389 (Patent Document 12), an azide group is introduced into the side chain. Alginic acid with an alkyne group in the side chain has been described. The cross-linked alginate structure formed is not disclosed, and its intended use is different from that of the present invention. become.

[0014] Furthermore, Non-Patent Document 1 discloses a branched alginate (bA) in which a cyclooctyne side chain is introduced into the side chain. lg-DBCO) has been described, but alginate and branched polyethylene glycol (4-arm PEG Branched alginic acid (bAlg) synthesized from α-NH2 was then aminated. It was obtained by reacting clooctyne (DBCO-PEG-amine) and is a novel arginine. It has a different structure from phosphoric acid derivatives 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 Pamphlet [Patent Document 4] International Publication No. 2005 / 026214 Pamphlet [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] Chinese 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 situation, a novel alginic acid derivative, a compound formed from the novel alginic acid derivative, Therefore, there is a need for novel crosslinked alginates, crosslinked alginate structures, and methods for producing the same. was. [Means for solving the problem]

[0018] As a result of extensive research to solve the above problems, the present inventors have discovered a compound represented by formula (I) or formula (I Furthermore, novel alginic acid derivatives represented by formula (I) and formula (II) have been discovered. A novel cross-linked alginate derivative obtained by subjecting the novel alginate derivative of Using alginic acid, beads (pigment-containing beads), which are one type of cross-linked alginic acid structure, are formed. The results showed that the beads have high stability and are more suitable for various purposes than conventional gels. The present invention has been completed based on the findings that the gel can be adjusted to have a desired transmittance.

[0019] The novel alginic acid derivatives (formula (I) and formula (II)) provided herein can be used, for example, as a chemical It can be used for chemical crosslinking, i.e., it can be used for chemical crosslinking. A reactive group having a complementary reactive group to the reactive group is introduced.

[0020] The chemical crosslinking can be achieved, for example, by the Huisgen reaction (1,3-dipolar cycloaddition reaction). For example, a crosslinking reaction is carried out between alginic acid derivatives of formula (I) and formula (II). Alternatively, for example, an alginic acid derivative of formula (I) may be used in combination with another molecule having an azide group. Alternatively, the alginic acid derivative of formula (II) may be reacted with other molecules having an alkyne group. It may be done between.

[0021] The Huisgen reaction with terminal alkyne and azide groups generally proceeds at temperatures above 100°C. However, this reaction is not suitable for chemical modification of biomolecules because it requires high heat. However, by adding a copper catalyst (e.g., Cu(I)) to the reaction, Reaction conditions were found that allowed the cycloadduct (triazole ring) to be formed in almost 100% yield at room temperature. (Angew. Chem. Int. Ed. Engl., 41, p2596-2599 ,2002;J.Org.Chem.,67,p3057-3064,2002), It has become possible to use it for chemical modification of biomolecules. When cross-linked alginic acid is obtained by the gen reaction, traces of copper catalyst are present in the cross-linked alginic acid. The catalyst may remain, and copper-derived This raises concerns about future cytotoxicity.

[0022] In a preferred embodiment, the cross-linked alginate is free of copper catalyst to avoid copper-derived cytotoxicity. Cross-linked alginic acid is obtained by using the Huisgen reaction without the need for a catalyst. The alkyne group introduced into the acid derivative is a cyclooctyne derivative (a highly strained cyclic alkyne group). By using this, the reaction can be carried out without the need for high temperature conditions of 100°C or above or a copper catalyst. Therefore, the novel cross-linked alginic acid of a preferred embodiment does not contain a copper catalyst. Even when the final product (cross-linked alginate structure) is formed, copper-derived toxicity is still present. It is also superior in that it does not require

[0023] Here, any one or more carboxyl groups of alginic acid shown in the following embodiments are added with alginic acid. Formula (I) in which a cyclic alkyne group or azide group is introduced via an amido bond and a divalent linker or an alginic acid derivative of formula (II), an alginic acid derivative of formula (I) and formula (II) Novel bridged alkylenes obtained by the Huisgen reaction (1,3-dipolar cycloaddition reaction) Alginic acid, cross-linked alginic acid structures, and the above alginic acid derivatives, cross-linked alginic acid, and A method for producing a cross-linked alginate structure is provided. That is, an exemplary embodiment includes the following steps: It can 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 compound of the following formula (I): [ka] [In formula (I), Akn, -L 1 -, -NHCO-, and (ALG) are the same as those in the first embodiment described below. The alginic acid derivatives represented by the formula (I) are the same as those defined in the formula (I), and any one or more alginic acid derivatives. The carboxyl group on the 2 -) through the azide group The following formula (II) was introduced: [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 polymer. Cross-linked alginate.

[0025] [1-Ia] Akn-L 1 -NH2 group (Akn, and -L 1 - in the first aspect described below The introduction rate of the formula (I ) is an alginic acid derivative represented by the formula:

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

[0027] [1-IIa]N3-L 2 -NH2 group(-L 2 - is the same as the definition in the first aspect described below. The introduction rate of the compound represented by formula (II) described in [1] above is 0.1% to 30%. Alginic acid derivative.

[0028] [1-IIb] Weight average values ​​of alginic acid derivatives determined by gel filtration chromatography The average molecular weight of the polymer represented by formula (II) in [1] is 100,000 Da to 3,000,000 Da. An alginic acid derivative.

[0029] [2] An amide bond and a divalent linker ( -L 1 A compound represented by the following formula (I): [ka] [In formula (I), (ALG), -L 1 -, the definition of Akn is the same as the definition in the second aspect described below. The alginic acid derivative represented by the formula [is the same as].

[0030] [3] Akn-L 1 -NH2 group (Akn, and -L 1 - is defined in the third aspect below. (which is the same as the formula (I)) is introduced at a rate of 0.1% to 30%. A commonly used alginic acid derivative.

[0031] [4] Weight-average molecular weight of alginic acid derivatives measured by gel filtration chromatography The alkyl group represented by formula (I) described in [2] above has a molecular weight of 100,000 Da to 3,000,000 Da. Phosphoric acid derivatives.

[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 aspect described below. An alginic acid derivative represented by the formula:

[0033] [6] N3-L 2 -NH2 group(-L 2 - has the same definition as in the sixth aspect described below) The introduction rate of the arsenic compound represented by formula (II) according to [5] above is 0.1% to 30%. Glycic acid derivatives.

[0034] [7] Weight-average molecular weight of alginic acid derivatives measured by gel filtration chromatography The arsenic represented by the formula (II) described in [5] above has a molecular weight of 100,000 Da to 3,000,000 Da. Glycic acid derivatives.

[0035] [8] Any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid The sil group is represented by the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends represent any carbon atom of alginic acid. represents an amide bond via a carboxyl group; -L 1 -, -L 2 - and X are the The cross-linked alginic acid according to [1] above, which is bound via the same as the definition in embodiment 8. .

[0036] [8a] Any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid The xyl group is represented by the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends represent any carbon atom of alginic acid. represents an amide bond via a carboxyl group; -L 1 -, -L 2 - and X are the 8a] as defined in embodiment 8a.

[0037] [8-1] Chemical bridges using triazole rings formed by the Huisgen reaction [1] or [2], wherein the bridge and the ionic bridge partially formed by a divalent metal ion are included. 8a). [8-2] Chemical bridges using triazole rings formed by the Huisgen reaction The cross-linked alginate according to [1] or [8a] above, which contains a bridge.

[0038] [8-3-1] In the above [8-1], the divalent metal ions are calcium ions, magnesium ions, etc. from the group consisting of sodium ions, barium ions, strontium ions, and zinc ions is the ion of choice.

[0039] [8-3-2] In the above [8-1], calcium chloride is used as a source of divalent metal ions. Aqueous solution of calcium carbonate, aqueous solution of calcium gluconate, aqueous solution of barium chloride , and the like.

[0040] [9] The alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) described in [1] above By mixing the alginate derivative with the prepolymer and carrying out a cross-linking reaction (Huisgen reaction), A method for producing crosslinked alginic acid, comprising obtaining the crosslinked alginic acid according to [1] or [8a]. How to do it.

[0041] [9-1] A solution of the alginic acid derivative represented by formula (I) described in [1] above is added to the 1] is added to a solution of the alginic acid derivative represented by formula (II) described above, and a crosslinking reaction (Hui The cross-linked alginic acid described in [1] or [8a] can be obtained by carrying out a cross-linking reaction (s-gen reaction). A method for producing cross-linked alginate, comprising:

[0042] [9-2] A solution of the alginic acid derivative represented by formula (II) described in [1] above is added to the Adding the alginic acid derivative represented by formula (I) described in [1] to a solution to cause a crosslinking reaction (Hui The cross-linked alginic acid described in [1] or [8a] can be obtained by carrying out a cross-linking reaction (s-gen reaction). A method for producing cross-linked alginate, comprising:

[0043]

[10] The alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to [1] above The alginic acid derivative is used to form the Huisgen reaction (cross-linking reaction). The chemical crosslink formed is represented by the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends, -L 1 - and -L 2 - is the same as defined in the tenth embodiment described below]. a) a method for producing a cross-linked alginic acid, the method comprising obtaining the cross-linked alginic acid according to the method of claim 1.

[0044]

[11] The alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to [1] above The mixed solution of alginic acid derivatives containing divalent metal ions was A cross-linked alginate structure can be obtained by dropping the alginate into a solution containing the compound.

[0045] [11-1] A solution of the alginic acid derivative represented by formula (I) described in [1] above is added to a bivalent The gel obtained by dropping the compound into a solution containing metal ions is represented by the formula (II) described in [1] above. Cross-linked alginic acid is obtained by adding it to a solution of alginic acid derivatives and carrying out a cross-linking reaction. Glycine structure.

[0046] [11-2] A solution of the alginic acid derivative represented by formula (II) described in [1] above is added to a solution of 2 The gel obtained by dropping the compound represented by formula (I) described in [1] above into a solution containing a valent metal ion is Cross-linked alginic acid is obtained by adding it to a solution of alginic acid derivatives and carrying out a cross-linking reaction. Glycine structure.

[0047] [12-1] Chemistry with triazole rings formed by Huisgen reaction as bridges

[11] to

[13] , including crosslinks and ionic crosslinks partially formed by divalent metal ions. [11-2] The crosslinked alginate structure according to any one of the above items.

[0048] [12-2] Chemistry with triazole rings formed by Huisgen reaction as bridges The crosslinked alginate structure according to any one of

[11] to [11-2] above, which comprises crosslinking. .

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

[0050] [12-3-2] In the above [12-1], potassium chloride is used as a source of divalent metal ions. Calcium aqueous solution, calcium carbonate aqueous solution, calcium gluconate aqueous solution, barium chloride aqueous solution The aqueous solution is selected from the group consisting of a solution, a water solution, and the like.

[0051] [13-1] The alginic acid derivative represented by formula (I) and formula (II) The alginic acid derivative represented by the formula (I) is crosslinked with divalent metal ions and subjected to the Huisgen reaction. A cross-linked alginate structure having the ability to retain contents, obtained by chemical cross-linking through a reaction.

[0052] [13-2] The alginic acid derivative represented by formula (I) and formula (II) The alginic acid derivative represented by the formula (I) is chemically crosslinked by the Huisgen reaction. Cross-linked alginate structures with material retention properties.

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

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

[0055]

[14] The alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) described in [1] above The compound formed by the Huisgen reaction using the alginic acid derivative The chemical bridge has the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends, -L 1 - and -L 2 - is the same as defined in the fourteenth embodiment described below]. The crosslinked alginate structure according to [13-2].

[0056]

[15] The alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to [1] above The mixed solution of alginic acid derivatives containing divalent metal ions was A cross-linked alginate structure was prepared by dropping the alginate into a solution containing the alginate. How to make it.

[0057] [15-1] A solution of the alginic acid derivative represented by formula (I) described in [1] above is dissolved in a divalent hydroxyl group. The gel obtained by dropping the compound into a solution containing metal ions is represented by the formula (II) described in [1] above. Cross-linked alginic acid is obtained by adding it to a solution of alginic acid derivatives and carrying out a cross-linking reaction. A method for producing a ginic acid structure.

[0058] [15-2] A solution of the alginic acid derivative represented by formula (II) described in [1] above is added to a solution of 2 The gel obtained by dropping the compound represented by formula (I) described in [1] above into a solution containing a valent metal ion is Cross-linked alginic acid is obtained by adding it to a solution of alginic acid derivatives and carrying out a cross-linking reaction. A method for producing a ginic acid structure.

[0059] [16-1] Chemistry with Triazole Rings Formed by Huisgen Reaction as Bridges

[15] to

[16] , including crosslinks and ionic crosslinks partially formed by divalent metal ions. [15-2] A method for producing a crosslinked alginate structure according to any one of the above items.

[0060] [16-2] Chemistry with Triazole Rings Formed by Huisgen Reaction as Bridges The crosslinked alginate structure according to any one of

[15] to [15-2] above, which comprises crosslinking. A method for manufacturing the above.

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

[0062] [16-3-2] In the above [16-1], the source of divalent metal ions is potassium chloride. Calcium aqueous solution, calcium carbonate aqueous solution, calcium gluconate aqueous solution, barium chloride aqueous solution The aqueous solution is selected from the group consisting of a solution, a water solution, and the like.

[0063]

[17] An alginic acid derivative represented by formula (I) according to any one of [1] above, and By carrying out the Huisgen reaction using the alginic acid derivative represented by formula (II), The chemical crosslink formed by the above reaction is represented by the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends, -L 1 - and -L 2 - is the same as defined in the seventeenth embodiment described below]. 6-2).

[0064]

[18] The gel according to any one of

[11] to

[14] , which is a bead or a roughly spherical gel. Cross-linked alginate structures.

[0065]

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

[11] to

[14] . Medical materials.

[0066]

[20] The medical material according to

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

[0067]

[21] The cross-linked alginic acid according to [1] or [8a], which is biocompatible, or the alginic acid derivative according to [5] above, or any one of

[11] to

[14] above. Item 1. The crosslinked alginate structure according to item 1.

[0068]

[22] The following formula (AM-1): [ka] [In formula (AM-1), -L 1 The definitions of - and Akn are the same as those in the 22nd aspect described below. or a pharmaceutically acceptable salt thereof, or Solvate.

[0069]

[23] The following formula (AM-2): [ka] [In formula (II), -L 2 The definition of - is -L in the 23rd aspect described below. 2 -Select from definitions or a pharmaceutically acceptable salt thereof, or a solvent thereof. Japanese food. [Effects of the Invention]

[0070] The present invention relates to novel alginic acid derivatives, novel alginic acid derivatives which can be used for chemical crosslinking, The present invention provides novel cross-linked alginates, novel cross-linked alginate structures, and the like. Preferably, the alginic acid derivative is one into which a reactive group not present in a living body is introduced, and the unreacted group is Even if they remain, there is no risk of cross-linking reactions with biological components such as cells. It is expected to be safe. Preferably, the crosslinking reaction is carried out using a metal catalyst. Since the reaction is completed at room temperature without any need for a solvent, it is safe and easy to use. Some embodiments of the cross-linked alginate are prepared by the Huisgen reaction (1,3-dipolar cycloaddition reaction). The cross-linking is a combination of chemical cross-linking and divalent metal gold ions (e.g., cations). It can be used in combination with ionic crosslinking using calcium ions, and the reaction conditions can be adjusted By adjusting the stability, it is preferable to use non-crosslinked alginic acid or non-chemically crosslinked alginic acid. This is an improvement over conventional alginic acid (e.g., calcium ion cross-linked alginic acid). Preferably, the gel properties of the crosslinked body can be adjusted, and the substance permeability can be adjusted. The present invention has at least one or more of these effects. [Brief explanation of the drawings]

[0071] [Figure 1]FIG. 1 shows evaluation of gel stability of cross-linked alginate structures. [Figure 2] FIG. 1 shows evaluation of gel stability of cross-linked alginate constructs under EDTA. [Figure 3] FIG. 1 shows evaluation of gel stability of cross-linked alginate structures. [Figure 4] FIG. 1 shows evaluation of gel stability of cross-linked alginate constructs under EDTA. [Figure 5] FIG. 1 shows evaluation of gel stability of cross-linked alginate structures. [Figure 6] FIG. 1 shows evaluation of gel stability of cross-linked alginate constructs under EDTA. [Figure 7] FIG. 1 shows the evaluation of the transmittance of the gel of a cross-linked alginate structure. [Figure 8] FIG. 1 shows the evaluation of the transmittance of the gel of a cross-linked alginate structure. [Figure 9] FIG. 1 shows the biocompatibility evaluation of gels of cross-linked alginic acid derivatives. DETAILED DESCRIPTION OF THE INVENTION

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

[23] may be included. [1] The first aspect is as follows: At any one or more carboxyl groups of alginic acid, Amide bond and bivalent linker (-L 1 -) to introduce a cyclic alkyne group (Akn). and an alginic acid derivative represented by the following formula (I), and any one or more of alginic acid. The carboxyl group is connected to an amide bond and a divalent linker (-L 2 -) to introduce an azide group By carrying out a crosslinking reaction using the alginic acid derivative represented by the following formula (II): The resulting cross-linked alginate.

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

[0074] [Alginic acid derivative represented by formula (II)] The following formula (II): [ka] (In formula (II), (ALG) represents alginic acid; -NHCO- represents any one of alginic acid. represents an amide bond via a carboxyl group; -L 2 -See the table below: [Table 3-1] [Table 3-2] [Each formula does not include the areas outside the dashed lines at both ends] The alginic acid derivative represented by the formula (I) represents a linker In phosphoric acid derivatives, -L 1 - is (L1-1), (L1-2a), (L1-2b), (L (L1-11) or (L1-12) In the alginic acid derivative represented by formula (II), -L 2 - (L2-10) (Excluding cross-linked alginic acid obtained by cross-linking using derivatives of alginic acid.) .

[0075] [1-1-1] In the formula (I) of the above aspect [1], -L 1 is preferably selected from the group consisting of the following: [Table 4-1] [Table 4-2] [Each formula does not include the areas outside the dashed lines at both ends] (However, when performing the crosslinking reaction, -L 2 - (L2-10), (L2-1 Any one selected from the group consisting of (L2-10-p1), (L2-10-p2) or (L2-10-X) When an alginic acid derivative represented by formula (II) having one linker is used, -L in the table 1 -Of which, (L1-1), (L1-2a), (L1-2b), (L1-11 ) and (L1-12-p1) are excluded from preferred embodiments).

[0076] [1-1-2] In the formula (I) of the above aspect [1], -L 1 is more preferably table: [Table 5-1] [Table 5-2] [Each formula does not include the areas outside the dashed lines at both ends] (However, when performing the crosslinking reaction, -L 2 - (L2-10), (L2-1 Any one selected from the group consisting of (L2-10-p1), (L2-10-p2) or (L2-10-X) When an alginic acid derivative represented by formula (II) having one linker is used, -L in the table 1 -Of which, (L1-1-1), (L1-2a-1), (L1-2b-1), The linkers (L1-11-1) and (L1-12-p2) are, in a more preferred embodiment, except).

[0077] [1-1-3] In the formula (I) of the above aspect [1], -L 1 More preferably, table: [Table 6-1] [Table 6-2] [Each formula does not include the areas outside the dashed lines at both ends] (However, when performing the crosslinking reaction, -L 2 - (L2-10), (L2-1 Any one selected from the group consisting of (L2-10-p1), (L2-10-p2) or (L2-10-X) When an alginic acid derivative represented by formula (II) having one linker is used, -L in the table 1 -Of which, (L1-1-X), (L1-2-X), (L1-11-X) and (Linkers that are 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 selected from the group consisting of the following: [Table 7] [In each formula, the right side of the dashed line is not included] It is an alkyne group.

[0079] [1-2-2] In the formula (I) of the above aspect [1], Akn is more preferably selected from the group consisting of the groups shown in the following table. : [Table 8] [In each formula, the right side of the dashed line is not included] It is an alkyne group.

[0080] [1-2-3] In the formula (I) of the above aspect [1], Akn is more preferably selected from the group consisting of the following compounds: : [Table 9] [In each formula, the right side of the dashed line is not included] It is an alkyne group.

[0081] [1-3-1] In the formula (II) of the above aspect [1], -L 2 - is preferably as shown in the table below : [Table 10] [Each formula does not include the areas outside the dashed lines at both ends] (However, when performing the crosslinking reaction, -L 1 - (L1-1), (L1-2a ), (L1-2b), (L1-11), (L1-12), (L1-1-1), (L1-2 a-1), (L1-2b-1), (L1-11-1), (L1-12-p1), (L1- Selected from the group consisting of (L1-1-X), (L1-2-X), (L1-11-X) or (L1-12-X) The alginic acid derivative represented by formula (I) having any one of the linkers In this case, -L in the table 2 Among these, the linker (L2-10-p1) is a preferred embodiment. (excluding from).

[0082] [1-3-2] In the formula (II) of the above aspect [1], -L 2 - is more preferably Notation: [Table 11] [Each formula does not include the areas outside the dashed lines at both ends] (However, when performing the crosslinking reaction, -L 1 - (L1-1), (L1-2a ), (L1-2b), (L1-11), (L1-12), (L1-1-1), (L1-2 a-1), (L1-2b-1), (L1-11-1), (L1-12-p1), (L1- Selected from the group consisting of (L1-1-X), (L1-2-X), (L1-11-X) or (L1-12-X) The alginic acid derivative represented by formula (I) having any one of the linkers In this case, -L in the table 2 Among these, the linker (L2-10-p2) is more preferred. (Excluding the above aspects).

[0083] [1-3-3] In the formula (II) of the above aspect [1], -L 2 More preferably, Notation: [Table 12] [Each formula does not include the areas outside the dashed lines at both ends] (However, when performing the crosslinking reaction, -L 1 - (L1-1), (L1-2a ), (L1-2b), (L1-11), (L1-12), (L1-1-1), (L1-2 a-1), (L1-2b-1), (L1-11-1), (L1-12-p1), (L1- Selected from the group consisting of (L1-1-X), (L1-2-X), (L1-11-X) or (L1-12-X) The alginic acid derivative represented by formula (I) having any one of the linkers In this case, -L in the table 2 Among these, the linker (L2-10-X) has a more preferred form. (Excluding from sama).

[0084] [1-4-1] In the formula (I) of the above aspect [1], Akn and -L 1 -The combination , preferably of the formula in the table below: [Table 13] (Akn, -L in the table) 1 -Each expression is As described in the embodiment [1]) (However, when performing the crosslinking reaction, -L 2 - is (L2-10), (L2-10-p1), (L2-10-p2) or (L2- 10-X) group, When an alginic acid derivative is used, -L in the above table 1 -Of which, (L1-1), (L1- The linkers (L1-2a), (L1-2b), (L1-11) and (L1-12) are preferably (Excluding from sama).

[0085] [1-4-2] In the formula (I) of the above aspect [1], Akn and -L 1 -The combination , and more preferably, the formula of the table below: [Table 14] (Akn, -L in the table) 1 -Each expression is As described in the embodiment [1-1]) (However, when carrying out the crosslinking reaction, -L 2 - (L2 -10), (L2-10-p1), (L2-10-p2) or (L2-10-X) Alginic acid derivatives represented by formula (II) having any one linker selected from the following: When using -L in the table above 1 -Of which, (L1-1-1), (L1-2a-1), The linkers (L1-2b-1), (L1-11-1) and (L1-12-p1) are more (Excluding the preferred embodiment).

[0086] [1-4-3] In the formula (I) of the above aspect [1], Akn and -L 1 -The combination and more preferably of the formula in the table below: [Table 15] (Akn, -L in the table) 1 -Each expression is As described in the embodiment [1-1]) (However, when carrying out the crosslinking reaction, -L 2 - (L2 -10), (L2-10-p1), (L2-10-p2) or (L2-10-X) Alginic acid derivatives represented by formula (II) having any one linker selected from the following: When using -L in the table above 1-Of which, (L1-1-X), (L1-2-X), ( The linkers (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 -The combination Particularly preferably, the compound has the following partial structural formula: [ka] (However, when carrying out the crosslinking reaction, -L 2 - is (L2-10), (L2-10-p1), (L2-10-p2) or (L2-10- X) a linker selected from the group consisting of an arginine represented by formula (II) and a hydroxyl group; When a phosphoric acid derivative is used, 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 - is the same as defined in the above embodiment [1]) is introduced at a rate of about 0.1% to about 3 The alginic acid derivative represented by formula (I) according to the above aspect [1], wherein the alginic acid derivative has a solubility of 0%.

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

[0090] [1-Ib] The embodiment of 1-Ib is as follows: Gel filtration chromatography of alginic acid derivatives The weight average molecular weight measured by the lithography method is about 100,000 Da to about 3,000,000 Da. An alginic acid derivative represented by formula (I) according to the above aspect [1].

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

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

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

[0094] [1-Id] The embodiment of 1-Id is as follows: Gel filtration chromatography of alginic acid derivatives The weight average molecular weight measured by the lithography method is about 100,000 Da to about 3,000,000 Da. An alginic acid derivative represented by formula (II) according to the above aspect [1].

[0095] [1-Id-1] In the above aspect [1-Id], a gel of the alginic acid derivative of formula (II) The weight average molecular weight measured by filtration chromatography is preferably about 300,000 Da to It is about 2.5 million Da, and 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 -Definitions are appropriately combined By combining these, the preferred embodiment of the cross-linked alginic acid of the above embodiment [1] can be arbitrarily formed. obtain.

[0097] [2] The second aspect is as follows: At any one or more carboxyl groups of alginic acid, Amide bond and bivalent linker (-L 1 -) to introduce a cyclic alkyne group (Akn). The compound of formula (I): [ka] [In formula (I), (ALG) represents alginic acid; -NHCO- represents any of the groups of alginic acid. represents an amide bond via a carboxyl group; -L 1 -See the table below: [Table 16-1] [Table 16-2] [Each formula does not include the areas outside the dashed lines at both ends] represents a linker; Akn is shown in the table below: [Table 17] [In each formula, the right side of the dashed line is not included] An alginic acid derivative represented by the formula:

[0098] [2-1] In the formula (I) of the above aspect [2], -L 1 is preferably selected from the group consisting of the following: [Table 18-1] [Table 18-2] [Each formula does not include the areas outside the dashed lines at both ends] a linker; More preferably, the following table: [Table 19-1] [Table 19-2] [Each formula does not include the areas outside the dashed lines at both ends] a linker; More preferably, the following table: [Table 20-1] [Table 20-2] [Each formula does not include the areas outside the dashed lines at both ends] It is a linker.

[0099] [2-2] In the formula (I) of the above aspect [2], Akn is preferably selected from the group consisting of the following: [Table 21] [In each formula, the right side of the dashed line is not included] is an alkyne group; More preferably, [Table 22] [In each formula, the right side of the dashed line is not included] is an alkyne group; More preferably, the following table: [Table 23] [In each formula, the right side of the dashed line is not included] It is an alkyne group.

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

[0101] A preferred embodiment of the above embodiment [2], further Akn, and -L 1-Combine the definitions as appropriate As a result, the preferred embodiment of the alginic acid derivative represented by the formula (I) in the embodiment [2] is The shape can be arbitrarily formed.

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

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

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

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

[0106] [5] The fifth aspect is as follows: Any one or more carboxyl groups of alginic acid are Amide bond and bivalent linker (-L 2 -), an azide group is introduced via the following formula ( II): [ka] In formula (II), (ALG) represents alginic acid; -NHCO- represents any of the groups of alginic acid. represents an amide bond via any carboxyl group; -L 2 -See the table below: [Table 27-1] [Table 27-2] [Each formula does not include the areas outside the dashed lines at both ends] An alginic acid derivative represented by the formula (I) (representing a linker).

[0107] [5-1] In the alginic acid derivative of the formula (II) according to the aspect [5], -L 2 -teeth, Preferably, the following table: [Table 28] [Each formula does not include the areas outside the dashed lines at both ends] a linker; More preferably, the following table: [Table 29] [Each formula does not include the areas outside the dashed lines at both ends] a linker; More preferably, the following table: [Table 30] [Each formula does not include the areas outside the dashed lines at both ends] It is a linker.

[0108] [5a] Embodiment 5a is as follows: Any one or more carboxyl groups of alginic acid The group has an amide bond and a divalent linker (-L 2-), an azide group was introduced via the following Formula (II): [ka] In formula (II), (ALG) represents alginic acid; -NHCO- represents any of the groups of alginic acid. represents an amide bond via any carboxyl group; -L 2 -See the table below: [Table 31-1] [Table 31-2] [Each formula does not include the areas outside the dashed lines at both ends] An alginic acid derivative represented by the formula (I) (representing a linker).

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

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

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

[0112] [7] The seventh aspect is as follows: Gel filtration chromatography of alginic acid derivatives The weight average molecular weight measured by the method is about 100,000 Da to about 3,000,000 Da. 5] or an alginic acid derivative of formula (II) according to embodiment [5a].

[0113] [7-1] In the above aspect [7], the gel filtration chromatography of the alginic acid derivative of formula (II) The weight average molecular weight measured by the lithography method is preferably about 300,000 Da to about 2,500,000 Da. a, and 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 alginic acid of 2 may be represented by the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends represent any carbon atom of alginic acid. represents an amide bond via a carboxyl group; -L 1 - is the same as defined in the above aspect [1]; -L 2- is the same as defined in the above aspect [1]; X is shown in the table below: [Table 35-1] [Table 35-2] (In each formula, the areas outside the dashed lines at both ends are included.) The cross-linked alginic acid according to the above aspect [1], wherein the alginic acid is bonded via a group represented by the formula (I) II-L), -L 1 - is (L1-1), (L1-2a), (L1-2b), (L (L1-11) or (L1-12), 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 is represented by the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends represent any carbon atom of alginic acid. represents an amide bond via a carboxyl group; -L 1 - is the same as defined in the above aspect [1]; -L 2 - is the same as defined in the above aspect [1]; X is the same as defined in the above embodiment [8]. Cross-linked alginate bonded via (However, in formula (III-L), -L 1 - (L1-1), (L1-2a), (L1 -2b), (L1-11) or (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], preferred , -L 1 - is -L described in the above embodiment [1-1-1] 1 -From the group consisting of expressions Same as the selected linker.

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

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

[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 selected from the group consisting of formulas This is the same as the linker selected.

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

[0121] [8-2-3] In the formula (III-L) of the above embodiment [8] or embodiment [8a], it is further preferred that Shii-L 2 - is -L described in the above embodiment [1-2-3] 2 - is a group consisting of expressions The linker is the same as the linker selected.

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

[0123] [8-3-2] In the formula (III-L) of the above embodiment [8] or embodiment [8a], X is preferably More preferably, the partial structural formula (TZ-2), the formula (TZ-3), the formula (TZ-6), the formula (TZ- 10), formula (TZ-2-r), formula (TZ-3-r), formula (TZ-6-r), and formula (TZ -10-r).

[0124] [8-3-3] In the formula (III-L) of the embodiment [8] or embodiment [8a], X further Partial structural formula (TZ-2), formula (TZ-6), formula (TZ-2-r), and formula (TZ-6-r) is a cyclic group selected from the group consisting of

[0125] [8-4-1] In the formula (III-L) of the embodiment [8] or embodiment [8a], preferably -L 2 -XL 1-The combinations are as shown in the table below: [Table 36-1] [Table 36-2] (-L in the table) 1 - and -L 2 - is the above As described in the embodiment [1]; -X- is as described in the embodiment [8]) (provided that the above-mentioned Medium, -L 1 - is (L1-1), (L1-2a), (L1-2b), (L1-11), or (L1-12), the corresponding -L 2 - (excluding the linker (L2-10-p1)).

[0126] [8-4-2] In the formula (III-L) of the above embodiment [8] or embodiment [8a], more preferred Or, -L 2 -XL 1 -The combinations are as shown in the table below: [Table 37-1] [Table 37-2] (-L in the table) 1 - and -L 2 - is the above As described in the embodiment [1]; -X- is as described in the embodiment [8]) (provided that the above-mentioned Medium, -L 1 - is (L1-1-1), (L1-2a-1), (L1-2b-1), (L1- 11-1), or (L1-12-p1), If so, the corresponding -L 2- (excluding the linker (L2-10-p2)).

[0127] [8-4-3] In the formula (III-L) of the above embodiment [8] or embodiment [8a], it is further preferred that Or, -L 2 -XL 1 -The combinations are as shown in the table below: [Table 38] (-L in the table) 1 - and -L 2 - is the above As described in the embodiment [1]; -X- is as described in the embodiment [8]) (provided that the above-mentioned Medium, -L 1 - is (L1-1-X), (L1-2-X), (L1-11-X) or (L1- 12-X), the corresponding -L 2 -to (excluding the linker (L2-10-X)).

[0128] [8-4-4] In the formula (III-L) of the embodiment [8] or embodiment [8a], particularly preferred Or, -L 2 -XL 1 The combination of - is represented by the following partial structural formula: Not include: [ka] as represented by a partial structure selected from the group:

[0129] [8-5-1] In the cross-linked alginate according to the above aspect [1] or aspect [8a], cross-linking The chemical crosslinking of the triazole ring formed by the Huisgen reaction and the divalent metal ion It is an ionic bridge formed in part by ions.

[0130] [8-5-2] In the cross-linked alginate according to the above aspect [1] or aspect [8a], cross-linking is a chemical bridge formed by a triazole ring via the Huisgen reaction.

[0131] [8-5-3] In the above aspect [8-5-1], the divalent metal ion is preferably calcium ion. Sodium ion, magnesium ion, barium ion, strontium ion or zinc ion and more preferably, calcium ion or It is preferably a barium ion; more preferably a calcium ion.

[0132] [8-5-4] In the above aspect [8-5-1], the divalent metal used for forming ionic crosslinks The ions are preferably calcium chloride aqueous solution, calcium carbonate aqueous solution, calcium gluconate aqueous solution, The aqueous solution is selected from the group consisting of an aqueous sodium chloride solution and an aqueous barium chloride solution. more preferably, an aqueous solution of calcium chloride or barium chloride; A particularly preferred solution is an aqueous calcium chloride solution.

[0133] A preferred embodiment of the embodiment [8], further -L 1 -, -L 2 - and X definitions can be combined as appropriate. By doing so, the preferred embodiment of the cross-linked alginic acid of the above embodiment [8] can be formed arbitrarily. A preferred embodiment of embodiment [8a], further -L 1 -, -L 2 - and the definition of X can be combined appropriately. By combining the above, the preferred embodiment of the cross-linked alginic acid of the above embodiment [8a] can be arbitrarily formed. do.

[0134] [9] The ninth embodiment is as follows: The arylene glycol compound represented by formula (I) according to the above embodiment [1]. The ginic acid derivative and the alginic acid derivative represented by formula (II) are mixed to cause a crosslinking reaction (Hu By carrying out a cross-linked alginate reaction (Isgen reaction), the cross-linked alginate according to the above aspect [1] or aspect [8a] can be obtained. 1. A method for producing crosslinked alginic acid, comprising obtaining the acid.

[0135] [9-1] The 9-1st embodiment is as follows: The solution of the alginic acid derivative is added to the alginic acid derivative represented by the formula (II) in the above aspect [1]. By adding the compound to a solution of a carboxylic acid derivative and carrying out a cross-linking reaction (Huisgen reaction), the above-mentioned embodiment [ 1] or aspect [8a], How to do it.

[0136] [9-2] The 9-2nd embodiment is as follows: The solution of the alginic acid derivative is added to the alginic acid derivative represented by formula (I) in the above embodiment [1]. By adding the compound to a solution of a carboxylic acid derivative and carrying out a cross-linking reaction (Huisgen reaction), the above-mentioned embodiment [ 1] or aspect [8a], How to do it.

[0137]

[10] The alginic acid derivative represented by formula (I) according to the above aspect [1] and the alginic acid derivative represented by formula (II) By carrying out the Huisgen reaction (crosslinking reaction) using an alginic acid derivative represented by The chemical crosslink formed by the above reaction 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 - is the same as defined in the embodiment [8]. A method for producing cross-linked alginic acid according to [8a].

[0138]

[11] The eleventh aspect is as follows: A compound represented by formula (I) described in the above [1]. Alginic acid derivatives and alginic acid derivatives represented by formula (II) Cross-linked alginate obtained by dropping a mixed solution of the body into a solution containing divalent metal ions structure.

[0139] [11-1] The aspect of the 11-1 is as follows: A solution of the alginic acid derivative represented by formula (I) is added dropwise to a solution containing a divalent metal ion. The gel obtained by the above procedure is dissolved in a solution of the alginic acid derivative represented by formula (II) described in [1]. A cross-linked alginate structure obtained by subjecting the above to a cross-linking reaction.

[0140] [11-2] The embodiment 11-2 is as follows: The gel obtained by dropping a solution of the alginic acid derivative containing the divalent metal ions into the solution containing the divalent metal ions is The alginic acid derivative represented by formula (I) in [1] above is added to the solution to carry out the crosslinking reaction. A cross-linked alginate structure obtained by subjecting the alginate to a cross-linking reaction.

[0141] [12-1] The embodiment of the 12-1 is as follows: Chemical cross-linking by triazole rings formed by cations and partially formed by divalent metal ions The crosslinked adhesive composition according to any one of the above aspects

[11] to [11-2], which contains ionic crosslinking. Luggic acid structure.

[0142] [12-2] The embodiment of No. 12-2 is as follows: The above-mentioned embodiments

[11] to [11-2] include chemical crosslinking by a triazole ring formed by the above-mentioned The crosslinked alginate structure according to any one of claims 1 to 4.

[0143] [12-3-1] In the above aspect [12-1], the divalent metal ion is preferably calcium ion. Sodium ion, magnesium ion, barium ion, strontium ion or zinc ion and more preferably, calcium ion or It is preferably a barium ion; more preferably a calcium ion.

[0144] [12-3-2] In the above aspect [12-1], the divalent metal used for forming ionic crosslinks The ions are preferably calcium chloride aqueous solution, calcium carbonate aqueous solution, calcium gluconate aqueous solution, The aqueous solution is selected from the group consisting of an aqueous sodium chloride solution and an aqueous barium chloride solution. more preferably, an aqueous solution of calcium chloride or barium chloride; A particularly preferred solution is an aqueous calcium chloride solution.

[0145]

[13] The thirteenth embodiment is as follows: and an alginic acid derivative represented by formula (II) treated with a divalent metal ion. Content retention achieved by ionic cross-linking via the Huisgen reaction and / or chemical cross-linking via the Huisgen reaction Cross-linked alginate structure with high bioactivity.

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

[13] , the divalent metal ion is preferably calcium. mu ion, magnesium ion, barium ion, strontium ion or zinc ion and more preferably, calcium ion or valence ion. more preferably, calcium ions.

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

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

[0148]

[14] The fourteenth aspect is as follows: Using the alginic acid derivative represented by the formula (II), Huisg The chemical crosslink formed by the en reaction 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

[0013] , wherein - is the same as defined in the above aspect [8].

[0149]

[15] The fifteenth embodiment is as follows: and an alginic acid mixture of the alginic acid derivative represented by formula (II). The mixed solution of the derivatives is dropped into a solution containing divalent metal ions to cause a crosslinking reaction. and a method for producing a cross-linked alginate structure obtained by the method.

[0150] [15-1] The embodiment of the 15-1 is as follows: The solution of the alginic acid derivative represented by the formula (I) is dropped into a solution containing divalent metal ions. The gel is added to a solution of the alginic acid derivative represented by formula (II) described in the above embodiment [1]. and subjecting the resulting alginate structure to a crosslinking reaction.

[0151] [15-2] The embodiment of the 15-2 is as follows: Formula (II) according to the embodiment [1] A solution of an alginic acid derivative represented by the formula: The gel is added to a solution of the alginic acid derivative represented by formula (I) described in the above embodiment [1]. and subjecting the resulting alginate structure to a crosslinking reaction.

[0152] [16-1] The embodiment of the 16-1 is as follows: Chemical cross-linking by triazole rings formed by cations and partially formed by divalent metal ions The crosslinked adhesive composition according to any one of the above aspects

[15] to [15-2], which contains ionic crosslinking. Method for producing glutinic acid structures.

[0153] [16-2] The embodiment of No. 16-2 is as follows: The above-mentioned embodiments

[15] to [15-2] include chemical crosslinking by a triazole ring formed by the above-mentioned A method for producing the crosslinked alginate structure described in any one of claims 1 to 4.

[0154] [16-3-1] In the above aspect [16-1], the divalent metal ion is preferably calcium ion. Sodium ion, magnesium ion, barium ion, strontium ion or zinc ion and more preferably, calcium ion or It is preferably a barium ion; more preferably a calcium ion.

[0155] [16-3-2] In the above aspect [16-1], the divalent metal used for forming ionic crosslinks The ions are preferably calcium chloride aqueous solution, calcium carbonate aqueous solution, calcium gluconate aqueous solution, The aqueous solution is selected from the group consisting of an aqueous sodium chloride solution and an aqueous barium chloride solution. more preferably, an aqueous solution of calcium chloride or barium chloride; A particularly preferred solution is an aqueous calcium chloride solution.

[0156]

[17] The seventeenth embodiment is as follows: Using the alginic acid derivative represented by the formula (II), Huisg The chemical crosslink formed by the en reaction 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 - is the same as defined in the embodiment [8]]. 16-2].

[0157]

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

[0011] to

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

[0158]

[19] The nineteenth aspect is as follows: Any one of the above aspects

[11] to

[14] . A medical material comprising the crosslinked alginate structure according to claim 1.

[0159]

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

[0019] , which is a bead or a substantially spherical gel.

[0160]

[21] The 21st aspect is as follows: The above-mentioned aspect [1] or aspect [1] is biocompatible. [8a], the cross-linked alginic acid according to the above aspect [2] or the above aspect [5] The cross-linked alginic acid structure according to any one of the above aspects

[11] to

[14] . body.

[0161]

[22] The 22nd 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 described in the above aspect [2]. or a pharmaceutically acceptable salt thereof, or It is a solvate, except as shown in the following table: [Table 39] or a pharmaceutically 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 Akn, An amino compound or compound having the same definition as the preferred Akn described in the embodiment [2-2]. A pharmaceutically acceptable salt thereof, or a solvate thereof, provided that the following table: [Table 40] or a pharmaceutically acceptable salt thereof, or a solvate thereof.

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

[22] is more preferably -L 1 - is more preferably -L described in the above embodiment [2-1] 1 - is the same as the definition of A kn is the same as the more preferred definition of Akn described in the embodiment [2-2]. or a pharmaceutically acceptable salt thereof, or a solvate thereof, provided that the following table: [Table 41] or a pharmaceutically 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 above embodiment [2-1] 1 - is the same as the definition of A amination, wherein kn is the same as the more preferred definition of Akn described in the embodiment [2-2]; or a pharmaceutically acceptable salt thereof, or a solvate thereof, provided that the following table: [Table 42] or a pharmaceutically 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 is expressed by the following formula: [ka] or a pharmaceutically acceptable salt thereof, or is a solvate of

[0166]

[23] The 23rd aspect is as follows: Formula (AM-2): [ka] [In formula (AM-2), -L 2 The definition of - is (L2-2a), (L2-2b) and (L2-2c) described in the above embodiment [5]. L2-2b-A), (L2-3), (L2-4), (L2-5a), (L2-5b), ( L2-6a), (L2-6b), (L2-7a), (L2-7b), (L2-8a), ( The definitions of (L2-8b), (L2-9a) and (L2-9b) are the same as those of or a pharmaceutically acceptable salt thereof, or a solvate thereof, provided that: table: [Table 43] or a pharmaceutically 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 - but (L2-2a), (L2-2b-A), (L2-3), (L2 -4-p1), (L2-5a-p1), and (L2-5b-p1), an amino compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof, provided that: The table below: [Table 44] or a pharmaceutically acceptable salt thereof, or a solvate thereof.

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

[23] is more preferably -L 2- (L2-2a-1), (L2-2b-B) described in the above aspect [5-1], (L2-3-1), (L2-4-p2), (L2-5a-p2), and (L2-5b-p 2) an amino compound having the same definition as that of the compound 2), or a pharmaceutically acceptable salt thereof, or It is a solvate, except as shown in the following table: [Table 45] or a pharmaceutically 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 is expressed by the following formula: [ka] or a pharmaceutically acceptable salt thereof, or is a solvate of In this specification, unless otherwise specified, when a higher-level embodiment is cited, it also includes lower-level embodiments of that embodiment. For example, if the embodiment [1] is cited, the sub-embodiments of the embodiment [1] are also included. It shall be.

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

[0171] 1. Alginate In this specification, the term "alginic acid" refers to alginic acid, alginate esters, and the like. at least one alginate selected from the group consisting of salts thereof (e.g., sodium alginate); The alginic acid used is They may be naturally derived or synthetic, but are preferably naturally derived. The alginates used are Lessonia, Macrocystis, Laminaria, Ascophyllum, and Darwinia. It is a bioabsorbable polysaccharide extracted from brown algae such as birria, sculpin, Eisenia bicolor, and kelp. Two types of uronic acid, D-mannuronic acid (M) and L-guluronic acid (G), are linearly linked. More specifically, the homopolymer fraction of D-mannuronic acid (M M fraction), homopolymer fraction of L-guluronic acid (GG fraction), and D-mannuronic acid Block copolymers consisting of randomly arranged L-guluronic acid fragments (M / G fragments) It is a combination.

[0172] Alginic acid is a type of natural polysaccharide extracted from brown seaweed and purified. Algin is a polymer of D-mannuronic acid (M) and L-guluronic acid (G). The ratio of D-mannuronic acid to L-guluronic acid (M / G ratio), i.e., gel strength, is determined by the The quality of seaweed varies depending on the type of organism from which it is derived, as well as the location and season of the organism. The influence of the M / G ratio 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 physicochemical properties of alginate vary depending on the M / G ratio of alginate, the arrangement of M and G, etc. The gelling ability and bioavailability of alginates may vary. The properties of the gel formed are affected by the M / G ratio. Generally, the gel is more stable when the G ratio is high. It is known that the gel strength increases. The M / G ratio also influences the hardness, brittleness, and It also affects the water absorption, flexibility, etc. Therefore, the alginic acid used in the present invention Depending on the final use, it is advisable to use a material with an appropriate M / G ratio and viscosity.

[0173] There are two industrial methods for producing alginic acid: the acid method and the calcium method. Products produced by either method can be used. Purification allows for quantitative determination by HPLC. The amount is preferably in the range of 80 to 120 mass%, more preferably in the range of 90 to 110 mass%. It is more preferable that the content of the hydroxybenzoate is in the range of 95 to 105 mass %. In the present invention, a substance whose quantitative value by HPLC falls within the above range is considered to be of high purity. The alginic acid or its salt used in the present invention is a highly purified alginic acid. As commercially available products, for example, the Chimica Algin series is available from Purchase high-purity food and pharmaceutical grade products, preferably those sold by Kimika. Commercially available products can also be used after further purification. For example, it is preferable to carry out a low-endotoxin treatment. For example, the method described in Japanese Patent Application Laid-Open No. 2007-75425 can be used. Cut.

[0174] The salt of alginic acid in the "alginic acid" used in the present invention includes "alginic acid 1 valent metal salts of D-mannuronic acid or L-guluronic acid carboxylic acids of alginic acid A salt made by ion-exchanging hydrogen ions with monovalent metal ions such as Na+ and K+. Specific examples of monovalent metal salts of alginic acid include sodium alginate, algin Examples of suitable alginates include potassium alginate, but sodium alginate is particularly preferred.

[0175] In this specification, alginic acid refers to alginic acid (ALG) and any calorific value of alginic acid. One of the carboxyl groups may be represented as -COOH, and the resulting formula may be (ALG)-COOH.

[0176] The alginic acid used in the present invention has an appropriate weight average molecular weight depending on its final use. For example, it is recommended to use a polymer with a weight average molecular weight of 10,000 to 10,000,000. is preferable, more preferably 100,000 or more and 5,000,000 or less, and further preferably 150,000 or more and 30,000,000 or less. It is less than 0 million.

[0177] In some embodiments, the alginate is sodium alginate. Commercially available sodium alginate can be used as the alginate. Sodium alginate is A-1, A-2, A-3, B-1, B-2 listed in the table below. and B-3 sodium alginate (distributor: Mochida Pharmaceutical Co., Ltd.). The viscosity, weight average molecular weight and M / G ratio of a 1 w / w% aqueous solution of sodium glutamate are as follows: As shown in the table.

[0178] [Table 46]

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

[0180] [Viscosity measurement of sodium alginate] In accordance with the viscosity measurement method of the Japanese Pharmacopoeia (16th edition), the rotational viscometer method (cone-plate type rotational viscometer) was used. The specific measurement conditions are as follows: The sample solution was prepared as follows: The measurement was carried out using MilliQ water. The measuring equipment was a cone-plate type rotational viscometer (viscosity and viscoelasticity measuring device). A RheoStress RS600 (Thermo Haake GmbH) sensor (35 / 1) was used. When measuring 1 w / w% sodium alginate solution, the speed was 1 rpm. The reading time was 2 minutes. The average value from 1 minute to 2 minutes after the start of measurement was taken as the measured value. The constant temperature was 20°C.

[0181] [Weight-average molecular weight measurement of sodium alginate] Two types of measurements are used: (1) gel permeation chromatography (GPC) and (2) GPC-MALS. The measurement was carried out by a standard method under the following conditions.

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

[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 I .D.×300mm×3 pieces) 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: 200 mM sodium nitrate aqueous solution Sample concentration: 0.5 to 2.5 mg / mL (5 concentrations)

[0185] [Measurement conditions (absolute molecular weight distribution measurement)] Column: TSKgel GMPW-XL x 2 + G2500PW-XL (7.8 mm I .D.×300mm×3 pieces) 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 the present specification, the terms "alginic acid, alginic acid derivatives, cross-linked alginic acid, and cross-linked alginic acid" refer to alginic acid derivatives, cross-linked alginic acid derivatives, and cross-linked alginic acid derivatives. Molecular weights 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 is mainly found in seaweed. These vary depending on the type of organism from which they are derived, and are also influenced by the habitat and season of the organism. These range from a high G type with an M / G ratio of approximately 0.2 to a high M type with an M / G ratio of approximately 5. The gelling ability of alginates and the properties of the resulting gels are affected by the M / G ratio. It is generally known that a higher G ratio leads to higher gel strength. The M / G ratio is It also affects the hardness, brittleness, water absorption, flexibility, etc. of the gel. The M / G ratio of the carboxylic acids and / or salts thereof is usually 0.1 to 4.0. , 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, 0.1 to 0.5 is.

[0188] The alginic acid used in the present invention has an appropriate viscosity and a suitable viscosity depending on the final use. It is advisable to use a material with an appropriate M / G ratio.

[0189] In this specification, numerical ranges indicated using "~" refer to the numerical values ​​before and after "~" as follows. The ranges are shown as minimum and maximum values, respectively.

[0190] The terms "alginic acid ester" and "alginate salt" used herein are not particularly limited. However, since it reacts with the crosslinking agent, it is necessary that it does not have any functional groups that inhibit the crosslinking reaction. The alginate ester is preferably propylene glycol alginate. , etc.

[0191] In the present specification, examples of alginate salts include monovalent salts of alginic acid, divalent salts of alginic acid, and the like. The monovalent salt of alginic acid is preferably sodium alginate. Examples of suitable alginates include potassium alginate, ammonium alginate, and the like. Sodium alginate or potassium alginate is particularly preferred. The divalent salt of alginic acid is preferably calcium alginate. Examples include magnesium alginate, barium alginate, and strontium alginate. do.

[0192] Alginic acid is a high molecular weight polysaccharide, and it is difficult to determine its molecular weight accurately. Generally, the weight average molecular weight is 1,000 to 10,000,000, preferably 10,000 to 8,000,000, and more preferably The molecular weight of naturally occurring polymeric substances is in the range of 20,000 to 3,000,000. It is known that values ​​may differ.

[0193] In the present specification, the molecular weight of the alginic acid derivative or alginic acid or its salt of the present invention is specifically defined. Unless otherwise specified, size exclusion chromatography (SEC) was used for the determination of The weight average molecular weight is calculated from the above formula. The alginic acid or salt thereof used in the present invention is It is desirable to use a polymer having an appropriate molecular weight distribution depending on the final use.

[0194] For example, gel permeation chromatography (GPC) or gel filtration chromatography, as described in the Examples below, may be used. Chromatography (collectively known as size exclusion chromatography (SEC)) ) under the measurement conditions, the molecular weight is preferably 100,000 to 5,000,000, and more preferably 150,000 to 30 In one embodiment, it is in the range of 500,000 to 3,000,000, and more preferably 1 The molecular weight is preferably in the range of 1 million to 2.5 million, and more preferably in the range of 1 million to 2 million.

[0195] In addition, for example, according to the GPC-MALS (SEC-MALS) method, the absolute weight average The weight average molecular weight (absolute molecular weight) measured by the GPC-MALS method can be measured. The molecular weight is preferably 10,000 or more, more preferably 50,000 or more, and even more preferably 60,000 or more. and preferably 1 million or less, more preferably 800,000 or less, and even more preferably It is preferably 700,000 or less, and more preferably 500,000 or less. The preferred range is 10,000 to 100,000. Ten thousand, more preferably 50,000 to 800,000, and even more preferably 60,000 to 500,000.

[0196] Usually, the molecular weight of high molecular weight polysaccharides is determined by the above-mentioned SEC and SEC-MALS methods. When calculating, there may be a measurement error of about 10% to about 30%. If it is 1 million, the value may fluctuate in the range of 700,000 to 1.3 million. In the specification, when the word "approximately" is used in describing molecular weight measurements, the value is within ±10% of the value in question. In some embodiments, values ​​up to ±20% of the numerical value may be included.

[0197] Generally, naturally occurring polymeric substances do not have a single molecular weight, but consist of various molecules. Because it is an aggregate of molecules with different molecular weights, it is measured as a molecular weight distribution with a certain range. A typical measurement method is gel filtration chromatography. Representative information on molecular weight distribution obtained by this method is the weight average molecular weight (Mw), number average molecular weight (Mw), These include molecular weight (Mn) and dispersion ratio (Mw / Mn).

[0198] The weight-average molecular weight emphasizes the contribution of large molecular weight polymers to the average molecular weight. It is expressed in 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) where W is the total weight of polymers, Wi is the weight of the i-th polymer, and Mi is the i-th elution time. where Ni is the number of molecules of molecular weight Mi, and Hi is the height at the i-th elution time. .

[0201] When measuring the molecular weight of polymeric substances derived from natural products, the values ​​can differ depending on the measurement method. It is known (example of hyaluronic acid: Chikako YOMOTA et.al. Bu ll.Natl.Health Sci., Vol.117, pp135-139( (1999), Chikako YOMOTA et.al. Bull.Natl.In st. Health Sci., Vol.121, pp30-33(2003)) The molecular weight of alginic acid was measured by intrinsic viscosity ( ty), SEC-MALLS (Size Exclusion Chr omatography with Multiple Angle Laser Li A method for calculating the light scattering detection (FDS) was described. There are references (ASTM F2064-00(2006), ASTM International In the present invention, the weight average molecular weight is determined by the conventional method as shown in the above document. The molecular weight is measured by, for example, size exclusion chromatography (SEC), and pullulan is can be a value calculated from a calibration curve using a standard substance. In the present invention, the weight average molecular weight can be determined by a conventional method such as that shown in the above-mentioned literature. For example, absolute molecular weight measured by size exclusion chromatography (SEC)-MALS It is possible.

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

[0203] In the present specification, when specifying the molecular weight of alginic acid or a salt thereof, unless otherwise specified, Unless otherwise specified, the molecular weight is the weight average molecular weight calculated by gel filtration chromatography. Typical conditions for using gel filtration chromatography for measurement are, for example, the conditions described below. The conditions in the examples can be adopted. The column is, for example, Superose6 Inc. A rease10 / 300 GL column (GE Healthcare Sciences) can be used. For example, a 10 mmol / L solution containing 0.15 mol / L NaCl is used as the developing solvent. Phosphate buffer (pH 7.4) can be used, and blue dextran can be used as a molecular weight standard. , thyroglobulin, ferritin, aldolase, conalbumin, ovalbumin, ribo Nuclease A and aprotinin can be used.

[0204] The viscosity of the alginic acid used in this specification is not particularly limited, but is When the viscosity of the aqueous solution of carboxylic acids is measured, it is preferably 10 mPa·s to 1000 mPa·s. Pa·s, and more preferably 50 mPa·s to 800 mPa·s.

[0205] The viscosity of the aqueous solution of alginic acid can be measured by a conventional method. Viscometer method: Coaxial double cylinder rotational viscometer, single cylinder rotational viscometer (Brookfield type) Viscometer), cone-plate rotational viscometer (cone-plate type viscometer), etc. Preferably, the viscosity measurement method in the Japanese Pharmacopoeia (16th edition) should be followed. 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 high viscosity. During the drying and refining process, the molecular weight becomes smaller and the viscosity becomes lower. The method involves controlling conditions such as the temperature, selecting the brown algae used as raw materials, and fractionating the molecular weight during the manufacturing process. It is possible to produce alginic acids with different molecular weights. By mixing with a different batch of alginic acid with different viscosity, it is possible to obtain a polymer with the desired molecular weight. Alginic acids may also be used.

[0207] As used herein, alginate is, in some embodiments, low endotoxin. untreated alginate, or in some other embodiments, low endotoxin Low endotoxin means that it does not substantially cause inflammation or fever. More preferably, low endotoxin is used. Preferably, the alginates are treated with doxorubicin.

[0208] The endotoxin reduction treatment can be carried out by a known method or a method similar thereto. For example, the method of purifying sodium hyaluronate by Kan et al. (see, for example, Japanese Patent Application Laid-Open No. 9-327626) 4001, etc.), the method of purifying β1,3-glucan by Yoshida et al. (see, for example, Patent Publication No. (See, for example, JP-A-8-269102) and biopolymer salts such as alginate and gellan gum. Purification by the method of Williams et al. (see, for example, JP-A-2002-530440) , the method of James et al. (see, e.g., International Publication No. WO 93 / 131) for purifying polysaccharides; No. 36 pamphlet), the method of Lewis et al. (see, for example, U.S. Pat. No. 5,589,591 (see the specification, etc.), the method of purifying alginate by Herman Frank et al. (see, for example, Ap pl Microbiol Biotechnol(1994)40:638-643 Low endotoxin Treatments include, but are not limited to, washing, filtering (endotoxin removal filters and charged filters) Filtration using filters, ultrafiltration, columns (endotoxin adsorption affinity columns, etc.) Purification using columns (e.g., gel filtration columns, ion exchange resin columns), hydrophobic substances, Adsorption onto resin or activated carbon, organic solvent treatment (extraction with organic solvent, addition of organic solvent) precipitation, sedimentation, etc.), surfactant treatment (for example, JP 2005-036036 A, etc. The method can be carried out by known methods such as those described above, or by appropriately combining these methods. These treatment steps may be appropriately combined with known methods such as centrifugation. It is desirable to select an appropriate one according to the type of

[0209] The endotoxin level can be confirmed by known methods, for example, using a Limulus reagent ( LAL (Lysine Alcohol-Like Leukemia) method, Endospecy (registered trademark) ES-24S Set (Seikagaku Corporation) It can be measured by methods such as using the

[0210] The method of treating endotoxin used is not particularly limited, but as a result, The endotoxin content of phosphates is measured using the Limulus Aqueous Lysate (LAL) reagent. When the test is carried out, it is preferable that the endotoxin concentration is 500 endotoxin units (EU) / g or less, and more preferably Preferably, it is 100 EU / g or less, more preferably 50 EU / g or less, and particularly preferably In the present invention, "substantially endotoxin-free" refers to a food product containing "substantially no endotoxin." "The endotoxin value measured by the Japanese Pharmacopoeia Endotoxin Test is within the above-mentioned range. Low-endotoxin treated sodium alginate is, for example, S ea Matrix (registered trademark) (Mochida Pharmaceutical Co., Ltd.), PRONOVA TM UP L It is available as a commercial product such as VG (FMCBioPolymer).

[0211] 2. Alginic acid derivatives Herein, a novel alginic acid derivative is provided. The alginate is bonded to one or more carboxyl groups of the alginic acid via an amide bond and a divalent linker. The reactive group in the Huisgen reaction or the complementary reactive group of the reactive group is It has been introduced. 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 as set forth 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. It is an alginic acid derivative represented by the formula [which is the same as the definition in

[0212] The bivalent linker (-L 1 -or-L 2 -) is a group complementary to the reactive group. Any linear group can be used as long as it does not inhibit the reaction with the reactive group. , for example, a straight chain alkylene group (-(CH2) n -, n = 1 to 30) (-CH 2- is -C(=O)-, -CONH-, -O-, -NH-, -S-, benzene ring, heterocyclic ring (pyridine ring, piperidine ring, piperazine ring, etc. 5- to 6-membered aromatic heterocyclic ring or 5- to 6-membered non-aromatic heterocycle), etc., and The hydrogen atom of the -CH2- may be replaced by an oxo group (=O), C 1-6 Alkyl groups (e.g. groups such as methyl, ethyl, n-propyl, isopropyl, etc.), halogen atoms (e.g., fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), hydroxyl group (-OH), and the like, and is substituted with a plurality of (for example, 1 to 10, or 1 to 5) groups selected from the group (also suitable), but are not limited to these.

[0213] In the —NH—CO— group of the alginic acid derivative represented by the formula (I) or (II), The hydrogen atom of the imino group (-NH-) is replaced with a methyl group to form the -N(Me)-CO- group. It is possible to do this. In the alginic acid derivative represented by the formula (I) or (II), the linker (-L 1 -, -L 2 The bond between α- and alginic acid is -NH-CO- or -N(Me)- and preferably, the —NH—CO— bond.

[0214] The novel alginic acid derivatives described herein are represented by formula (I) and formula (II) Alginic acid derivatives can be prepared, for example, by the following method (for details, see the general production method described below): It is possible to manufacture more.

[0215] [ka]

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

[0217] As used herein, Akn-L of formula (I) 1 The -NH- groups are located in all the carbon atoms of the alginate building blocks. It is not necessary for the N3-L of formula (II) to be bonded to a carboxylic acid group. 2 The -NH- group is It is not necessary for the bond to be with all the carboxyl groups of the carboxylic acid building blocks.

[0218] As used herein, Akn-L of formula (I) 1 When the —NH— group is referred to as a reactive group, the —NH— group in formula (II) N3-L 2 The -NH- group serves as a complementary reactive group. 2 -N When the H-group is referred to as a reactive group, Akn-L in formula (I) 1 The -NH- group is a complementary reactive group. become.

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

[0220] The introduction rate of the reactive group or the complementary reactive group is determined by the ratio of the repeating unit of alginic acid to the total number of alginic acid groups. The number of uronic acid monosaccharide units to which each reactive group was introduced was expressed as a percentage. In this specification, unless otherwise specified, the alginic acid derivative (formula (I) or formula (II) The percentage used for the introduction rate of the reactive group or the complementary reactive group in )) means mol%. The introduction rate of each reactive group or complementary reactive group can be determined by the method described in the Examples below. It can be done.

[0221] In the present specification, the cyclic alkyne group (Akn) in formula (I) and the azide group in formula (II) are The Huisgen reaction forms a triazole ring, thereby forming a bridge.

[0222] 3. Huisgen reaction The Huisgen reaction (1,3-dipolar cycloaddition reaction) is carried out by reacting terminal alkyl groups with aryl groups as shown in the following formula: This is a condensation reaction between compounds containing a dihydrogen group and a terminal alkyne group. The reaction results in the formation of disubstituted 1, The 2,3-triazole ring is obtained in good yield and no unnecessary by-products are produced. This reaction is thought to produce a 1,4- or 1,5-disubstituted triazole ring. However, 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 was reported by Wittig and Krebs. In other words, a cycloadduct can be obtained simply by mixing cyclooctyne and phenyl azide. This is a reaction in which 3 = phenyl). This reaction is a triple cyclooctyne reaction. Because the bond is highly distorted, the reaction with phenyl azide is the driving force for the removal of the distortion. The reaction proceeds spontaneously, eliminating the need for a catalyst.

[0225] [ka]

[0226] As mentioned above, the Huisgen reaction can be carried out using substituted primary azides, secondary azides, and tertiary azides. azide compounds having an aromatic azide, etc., and a terminal group which is a complementary reactive group of the azide group Alternatively, a compound having a cyclic alkyne group can be used. Since almost only azide and alkyne groups react, various functional groups (e.g., For example, an ester group, a carboxyl group, an alkenyl group, a hydroxyl group, an amino group, etc.) It is possible to do this.

[0227] In some embodiments, copper-catalyzed cytotoxicity is reversed without the production of undesirable by-products. To avoid this, 1,2,3-triazole can be easily and efficiently synthesized in a short time without using a copper catalyst. In order to form cross-links between alginic acid molecules by alkyl rings, the alkyne group in the Huisgen reaction For example, the cyclic alkyne group (cyclooctyl group) described in the above embodiment [1] is used as the compound. There are.

[0228] In a preferred embodiment of the method for crosslinking an alginic acid derivative, the reaction (Huisgen reaction) In this case, alginate is used. In the creation of novel biocompatible materials and the formation of alginate hydrogels, various Incorporation of bioactive molecules into alginate hydrogels for reconstructive surgery or gene therapy This allows for the uptake of cellular material.

[0229] 4. Cross-linked Alginate Cross-linked alginate is either (i) via divalent metal ion bonds or (ii) via chemical bonds. or (iii) via both a divalent metal ion bond and a chemical bond. All cross-linked alginates have a gel-like to semi-solid, sometimes sponge-like, morphology. It has the property of forming.

[0230] Cross-linked alginate via divalent metal ion bonds undergoes ultrafast and reversible reactions. In contrast, cross-linked alginate via chemical bonds reacts slowly under relatively mild conditions. The physical properties of cross-linked alginic acid vary depending on the type of divalent metal ion used, for example. The concentration of an aqueous solution containing calcium chloride (for example, calcium chloride solution) or an aqueous solution containing alginic acid This can be adjusted by changing the introduction rate of the reactive groups introduced.

[0231] By utilizing the above-mentioned cross-linking reaction, it is possible to create various alginate structures. For example, specific structures can be instantly created from alginate solutions using ionic crosslinking reactions. In order to strengthen the structure (for example, to obtain long-term stability), chemical bonding is used. In addition, for example, divalent metal ion bonds and chemical bonds can be used. In the cross-linked alginate structure via both ionic cross-linking and The ions can be reversibly released, allowing the creation of structures in which only cross-linking by chemical bonds remains. is.

[0232] In one embodiment, the crosslinked alginic acid is an alginic acid derivative represented by the formula (I) or the formula (II). The resulting mixture can be mixed and subjected to a Huisgen reaction.

[0233] In one embodiment, the cross-linked alginate is chemically cross-linked (trimethylsilyl group formed from an alkyne group and an azide group). Forms a three-dimensional network structure through cross-linking via azole rings. Preferred alginate derivatives The body is a cross-linked alginate with improved stability after cross-linking.

[0234] In some embodiments, the cross-linked alginate is a cross-linked alginate formed by cross-linking any carboxyl group of the first alginate with any carboxyl group of the second alginate. Any carboxyl group of alginic acid is represented by the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends represent any carbon atom of alginic acid. represents an amide bond via a carboxyl group; -L 1 -, -L 2 -, and X is the eighth The alginic acid is cross-linked via an amide bond, which is the same as the definition in the embodiment.

[0235] In some embodiments, the alginic acid derivative of formula (I) is used in preparing cross-linked alginic acid. The mixing ratio of the alginic acid derivative of formula (II) is the ratio of the derivative of formula (I) to the derivative of formula (II). The ratio of amounts 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.

[0236] In some embodiments, when preparing cross-linked alginic acid, an alginic acid derivative of formula (II) The mixing ratio of the alginic acid derivative of formula (I) is the ratio of the derivative of formula (II) to the derivative of formula (I). The ratio of the amount is, for example, 1 to 4.0:1, preferably 1.5 to 4.0:1, or 1.2 to 1 0.5:1, or 1 to 1.2:1, more preferably 1:1.

[0237] In some embodiments, the alginic acid derivative of formula (I) is used in preparing cross-linked alginic acid. The mixing ratio of the alginic acid derivative of formula (II) is more preferably The ratio of the introduction rate (mol%) of the reactive group of the compound to the alginic acid derivative of formula (II) is, for example, 1 ~1.5:1, preferably 1.2-1.5:1, or 1-1.2:1, more preferably is 1:1.

[0238] In some embodiments, when preparing cross-linked alginic acid, an alginic acid derivative of formula (II) The mixing ratio of the alginic acid derivative of formula (I) is more preferably the alginic acid derivative of formula (II). The ratio of the introduction rate (mol%) of the reactive group of the conductor to the alginic acid derivative of formula (I) is, for example, 1 ~4.0:1, preferably 1.5 to 4.0:1, or 1.2 to 1.5:1, or 1 to It is 1.2:1, more preferably 1:1.

[0239] In the above mixing ratio, the alginic acid derivative of formula (I) is mixed with the alginic acid derivative of formula (II). Alternatively, the alginic acid derivative of formula (II) may be replaced by the derivative of formula (I), respectively. It is possible.

[0240] Crosslinked alginic acid is a compound in which all the carboxyl groups of the constituent units of alginic acid are represented by the above formula (III- It is not necessary to have crosslinks of the formula (III-L) in the crosslinked alginic acid. The crosslinking rate (also referred to as the crosslinking rate) is, for example, about 0.1 to about 80%, about 0.3 The range is about 0.5 to about 60%, about 0.5 to about 30%, or about 1.0 to about 10%.

[0241] The alginate of formula (I) or (II) in the Huisgen reaction to obtain crosslinked alginic acid The concentration of the arginic acid derivative is usually about 1 to about 500 mg / mL, preferably about 5 to about 1 The range is 00mg / mL.

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

[0243] The stirring time for forming cross-linked alginate (hydrogel) is, for example, from a few seconds to about 24 hours. The time is 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. For example, , tap water, pure water (e.g., distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultra-pure water Examples include water, cell culture medium, phosphate buffered saline (PBS), and saline. Ultrapure water is preferred.

[0245] In some embodiments, the cross-linked alginate is formed by the Huisgen reaction as the cross-link. Chemical bridges formed by triazole rings and partially formed by divalent metal ions Cross-linked alginate, including on-cross-linked alginate.

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

[0247] In the present specification, the term "to subject to a crosslinking reaction" or "to carry out a crosslinking reaction" refers to the reaction of the alginate of the formula (I) The Huisgen reaction is carried out using the alginic acid derivative of formula (II) and the alginic acid derivative of formula (II). By this, a reaction between the alginic acid derivative of formula (I) and the alginic acid derivative of formula (II) can be achieved. The alginic acid derivative of formula (I) and the alginic acid derivative of formula (I) are formed. By making the alginic acid derivative of formula (II) coexist with a divalent metal ion, the alginic acid derivative of formula (I) and / or the alginic acid derivative of formula (II) (ionic bond) is formed, or chemical crosslinking and bivalent This means that both the metal ions and the ionic bridges are formed.

[0248] The cross-linked alginate structure can be prepared, for example, by the following method. It is not limited to.

[0249] [Mixing method] The alginic acid derivative of the formula (I) and the alginic acid derivative of the formula (II) are mixed together to obtain The mixed solution of the alginic acid derivatives is dropped into a solution containing divalent metal ions, Chemical crosslinking (triazolidine formed from alkyne and azide groups via the Huisgen reaction) The cross-linking is formed by the divalent metal ion. A specific structure, a cross-linked alginate structure, can be obtained.

[0250] [Coating method] A solution containing the alginic acid derivative of formula (I) is added dropwise to a solution containing a divalent metal ion. A specific structure that is partially crosslinked can be obtained by, for example, crosslinking the gel obtained above. The structure is added to a solution containing the alginic acid derivative of formula (II), By carrying out a further cross-linking reaction (Huisgen reaction) on the surface of the structure, cross-linked alginate is formed. In this method, the alginic acid derivative of formula (I) can be converted into the alginic acid structure of formula (I The alginic acid derivative of formula (I) is added to the alginic acid derivative of formula (II) It is also possible to replace the above with the body.

[0251] The divalent metal ion used in the method is not particularly limited, but for example, calcium Mu ion, magnesium ion, barium ion, strontium ion, zinc ion, and the like; preferably, calcium ion or It is preferably a barium ion; more preferably a calcium ion.

[0252] The solution containing divalent metal ions used in the method is not particularly limited, but may be, for example, , calcium chloride aqueous solution, calcium carbonate aqueous solution, calcium gluconate aqueous solution, or salt The aqueous solution may be selected from the group consisting of an aqueous solution of calcium chloride, an aqueous solution of barium chloride, and the like. and more preferably, an aqueous solution of calcium chloride. .

[0253] The divalent metal ion concentration of the divalent metal ion-containing solution used in the method is not particularly limited. However, for example, the concentration may be about 1 mM to about 1M, and 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, but examples thereof include tap water, pure water (e.g., For example, distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water, cell culture medium phosphate buffered saline (PBS), physiological saline, etc., and preferably ultrapure water. be.

[0255] Specific cross-linked alginate structures include, for example, fibrous structures, fibers, beads, The cross-linked alginate structure has improved stability. In addition, the cross-linked alginate structure has the ability to retain contents inside (content retention). It may also have the property of retaining material.

[0256] The physical properties of alginate gel are determined by physical properties such as hardness, elasticity, resilience, rupture strength, and stress at break. It is possible to adjust it.

[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 refers to As used herein, biocompatibility refers to a biomaterial (herein, the formula ( and an alginic acid derivative represented by formula (I) or formula (II), and a method using both of the alginic acid derivatives. Interaction between the produced cross-linked alginate or cross-linked alginate structure) and the living body does not cause a reaction, such as a local reaction in tissue adjacent to the biomaterial or a systemic reaction. Such a property is said to be biocompatible.

[0258] In the present specification, the term "alginic acid derivatives, crosslinked alginic acids, or crosslinked alginic acid structures" refers to Biocompatibility will be confirmed in the examples relating to biocompatibility described below.

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

[0260] [Method for measuring gel stability] Phosphate buffered saline (PBS) was added to the cross-linked alginate structure gel placed in a container. Measure the concentration (μg / mL) of alginic acid dissolved in PBS. The amount of eluted alginate was calculated from the degree of elution, and the total amount of alginate obtained by decomposing the cross-linked alginate structure gel was calculated. The disintegration rate was calculated by dividing the total amount of alginic acid by the alginic acid concentration and expressing the result as a percentage. Specifically, the gel stability can be determined by the method described in the Examples below.

[0261] In the present specification, the gel collapse rate of the crosslinked alginate structure is preferably 0% to about 90%. The crosslinking ratio is more preferably 0% to about 70%, and even more preferably 0% to about 50%. The stability of the alginate structure increases as the concentration of alginate leaking into the aqueous solution decreases, i.e. A lower gel collapse rate means higher stability.

[0262] [Method for measuring gel permeability] Cross-linked alginate gel containing fluorescein isothiocyanate-dextran A container was prepared, and physiological saline was added to the gel. The dextran concentration was measured. The amount of dextran calculated from the measured dextran concentration was Degradation of fluorescein isothiocyanate-dextran-encapsulated cross-linked alginate gel structures The total dextran concentration obtained by dividing it by the total dextran amount calculated is expressed as a percentage. The value shown is the gel permeability. The gel permeability can be measured by the method described in the Examples below. You can ask for more.

[0263] The gel permeability of cross-linked alginate after 24 hours of addition of physiological saline was When encapsulating 10,000 dextran, the content is preferably 0% to about 90%, more preferably 0 % to about 70%, and more preferably 0% to about 50%. When the cross-linked alginate structure gel is used for the purpose of protein synthesis, If the release or production of proteins or antibodies is desired, the concentration is preferably about 1% to about 100%, more preferably about 1% to about 100%. It is usually about 10% to about 100%, and more preferably about 30% to about 100%. If the purpose is to form an immune barrier, the concentration is preferably 0% to about 90%, more preferably 0%. The range is preferably from about 70% to about 70%, and more preferably from 0% to about 50%.

[0264] The lower the permeability of the cross-linked alginate structure, the less permeability of the contents and substances outside the gel. The higher the permeability, the higher the permeability of the contents and substances outside the gel. do.

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

[0266] [Method for preparing cross-linked alginate structure gel containing ingredients] For example, crosslinked fluorescein isothiocyanate-dextran isoforms are included as the contents. The bridge alginate structure gel can be prepared in the following manner.

[0267] (1) A solution of the alginic acid derivative represented by formula (I) and fluorescein isothiocyanate Mix the acetone-dextran solution. (2) To the mixed solution obtained in (1), a solution of the alginic acid derivative represented by formula (II) is added. Mix. (When formula (I) in (1) is changed to formula (II), formula (II) in (2) is changed to formula (I) (This will be (3) The mixed solution obtained in (2) was dropped into a solution containing calcium ions. In solution, fluorescein isothiocyanate is bonded to the polymer by forming chemical and ionic crosslinks. A cross-linked alginate structure gel containing ocyanate-dextran is obtained.

[0268] In the present specification, when the word "about" is used, unless otherwise specified, the numerical value The range may also include values ​​up to ±20% of the stated value, preferably up to ±10% of the stated value.

[0269] 8. Method for synthesizing alginic acid derivatives In the present specification, the alginic acid derivatives represented by formula (I) or formula (II) are each H2N-L 1 -Akn(in the formula, L 1 and Akn are the same as defined in the above aspect [1] ) or H2N-L 2 -N3 (in the formula, L 2 teeth, The amine derivative (AM-2) represented by the formula (1) is the same as that in the embodiment [4] above, It can be produced by a condensation reaction of any carboxyl group of carboxylic acids with a condensing agent. Cut.

[0270] [ka]

[0271] [Method for producing alginic acid derivative of formula (I)] 0.5% by weight to 1% by weight of an aqueous solution of alginic acid and an amine represented by formula (AM-1) Using the method known in the literature, for example, "Experimental Chemistry Lectures, 5th Edition, 16, Synthesis of Organic Compounds I" V. Carboxylic acids and derivatives, esters, p35-70, acid amides and acid imides, p 118-154, Amino Acids and Peptides, p258-283, 2007, Maruzen, etc. According to the method described in -3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC·HCl), 1-isotriazolyloxytris(dimethylamino)phosphonium hexafluoro Phosphate (BOP reagent), Bis(2-oxo-3-oxazolidinyl)phosphine BOP-Cl, 2-chloro-1,3-dimethylimidazolinium hexafluoride Fluorophosphate (CIP), or 4-(4,6-dimethoxy-1,3,5-triazol-1,4-dione dimethyl-4-methylmorpholinium chloride (DMT-MM), etc. In the presence of a condensing agent, tetrahydrofuran, 1,4-dihydrofuran, to such an extent that alginic acid does not precipitate, ether solvents such as xanthane, alcohols such as methanol, ethanol, and 2-propanol, Mixtures of water and solvents selected from the group consisting of alcoholic solvents, polar solvents such as N,N-dimethylformamide, etc. In the combined solvent, an inorganic base such as sodium bicarbonate or sodium carbonate, or triethylamine, The condensation reaction is carried out at a temperature between 0°C and 50°C in the presence or absence of an organic base such as pyridine. By carrying out the above steps, the alginic acid derivative of formula (I) can be produced.

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

[0273] In the method for producing an alginic acid derivative of formula (I) or an alginic acid derivative of formula (II), The introduction rate of the amine of formula (AM-1) or formula (AM-2) is determined by taking into consideration the properties of the amine. By appropriately selecting and combining the reaction conditions such as the following (i) to (v), (i) Increasing or decreasing the equivalent amount of condensing agent, (ii) Increasing or decreasing the reaction temperature, (iii) (iv) adjusting the concentration of alginic acid as a reaction substrate; (v) adjusting the reaction time; 1) or adding a water-miscible organic solvent to increase the solubility of the amine of formula (AM-2) , etc.

[0274] More specific examples of the amines represented by formula (AM-1) or formula (AM-2) are listed below. A method for producing an amine is shown.

[0275] In addition, among the following manufacturing methods, m1, n1, m2a, n2a, p2a, m2b, n2b, p 2b, m3, n3, p3, m4a, n4a, m4b, n4b, m5a, n5a, p5a, q5a, m5b, n5b, p5b, q5b, m6a, n6a, p6a, m6b, n6b, p6b, m7, n7, m8a, n8a, m8b, n8b, m9a, n9a, p9a, m9 b, n9b, p9b, m10, n10, p10, m11, m12, n12, p12, x1 , x2, x2a, y2a, x2b, y2b, x3, y3, x4, y4, z4, x5a, y 5a, x5b, y5b, x6a, y6a, z6a, v6a, x6b, y6b, z6b, v 6b, x7a, y7a, z7a, x7b, y7b, z7b, x8a, y8a, z8a, x 8b, y8b, z8b, x9a, y9a, z9a, x9b, y9b, z9b, x10 and The definition of y10 is the same as that in the above embodiment [1]; P 1 -C(O)O-ter tBu group, -C(O)O-Bn group, -C(O)CH3 group, -C(O)CF3 group, -SO2 -Ph, -SO2PhMe group, -SO2Ph(NO2) group, etc. E = halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), Leaving groups include -OTs and -OMs groups.

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

[0277] [Production Method AM-A] Production method of amine represented by formula (AM-1-B1): [ka] Compound of formula (SM-B1) and compound of formula (RG-B1) [Compound of formula (SM-B1) The compound of formula (RG-B1) is a commercially available compound or can be prepared from a commercially available compound by a known method. The compound can be produced by the same method as in the above [Method for producing an alginic acid derivative of formula (I)]. A similar condensation reaction is then carried out to form the protecting group P 1 By deprotecting the compound represented by formula (AM-1-B1), The amine compound represented by the formula (I) or a salt thereof can be prepared.

[0278] [Production Method AM-B] A compound represented by formula (AM-1-B2a) and formula (AM-1-B2b) How Minh is made: [ka]

[0279] Compound of formula (SM-B2a) and compound of formula (RG-B2a) [Compound of formula (SM-B2a)] The compounds and compounds of formula (RG-B2b) are commercially available compounds or preparations known in the literature from commercially available compounds. [This compound can be produced by the method] and reacted in the same manner as in [Production method AM-A]. to produce an amine compound represented by formula (AM-1-B2a) or a salt thereof. Similarly, the compound of formula (SM-B2b) and the compound of formula (RG-B2b) can be The compounds of formula (SM-B2b) and formula (RG-B2b) are commercially available compounds or The compound can be produced from the compound by a known method in the literature. By doing so, an amine compound represented by formula (AM-1-B2b) or a salt thereof can be produced. can be done.

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

[0281] The compound of formula (SM-B3) [the compound of formula (SM-B3) is a commercially available compound or [This compound can be produced by a known production method from the literature], and By carrying out the reaction (the reaction in each step is similar to the reaction described in [Production Method AM-A]), The amine compound represented by formula (AM-1-B3) or a salt thereof can be produced. In the scheme, the compounds of formula (RG-B3), formula (RG-B3-1), and formula (RG-B3-2) The compound 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. .

[0282] [Production Method AM-D] A compound represented by formula (AM-1-B5a) and formula (AM-1-B5b) How Minh is made: [ka]

[0283] By carrying out the reaction according to the above synthesis scheme (the reaction of each step is described in [Manufacturing Method AM-A]), (According to the reaction described above), the compounds represented by formula (AM-1-B5a) and formula (AM-1-B5b) In the above scheme, the amine compound represented by formula (SM-B5a) or a salt thereof can be produced. , 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) The compounds are commercially available compounds or compounds that can be produced from commercially available compounds by known production methods. be.

[0284] [Production Method AM-E] A compound represented by formula (AM-1-B6a) and formula (AM-1-B6b) How Minh is made: [ka]

[0285] By carrying out the reaction according to the above synthesis scheme (the reaction of each step is described in [Manufacturing Method AM-A]), (According to the reaction described above), the compounds represented by formula (AM-1-B6a) and formula (AM-1-B6b) In the above scheme, the amine compound represented by formula (SM-B6a) or a salt thereof can be produced. The compounds of formula (RG-B6a), formula (SM-B6b), and formula (RG-B6b) are commercially available. These compounds can be prepared from commercially available compounds or compounds by known preparation methods.

[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 of each step is described in [Manufacturing Method AM-A]), According to the reaction method described above, an amine compound represented by formula (AM-1-B10) or a salt thereof is prepared. In the above scheme, the formula (SM-B10), the formula (RG-B10), the formula ( The compounds of formula (RG-B10-1) and formula (RG-B10-2) are commercially available compounds or This compound can be produced by a production method known in the literature.

[0288] [Production Method AM-G] A compound represented by formula (AM-1-B4a) and formula (AM-1-B4b) How Minh is made: [ka]

[0289] Compound of formula (SM-B4) and compound of formula (RG-B4a) [Compound of formula (SM-B4)] The compound and the compound of formula (RG-B4a) are commercially available compounds or can be prepared from commercially available compounds by a method known in the literature. and the compound can be prepared by a method known in the literature, for example, of the American Chemical Society, 126(46 ), pp. 15046-15047, 2004, etc., > In the presence of reagents such as silver trifluoromethanesulfonate and AgClO4, toluene, dichloromethane By reacting in a solvent that does not participate in the reaction, such as methane, a compound of formula (IM-B4a-1) can be obtained. This is then debrominated using a base such as sodium hydride or NaOMe. By carrying out the reaction, a compound of formula (IM-B4a-2) is obtained, and in <Step 3a>, further protection base P 1 by deprotecting the compound, an amine compound represented by formula (AM-1-B4a), or The salt can be prepared as follows: Similarly, the above formula (RG-B4b) is used instead of formula (RG-B4a). By carrying out a reaction according to the procedure, an amine compound represented by formula (AM-1-B4b) can be obtained, or The salts can be prepared.

[0290] [Production Method AM-H] A compound represented by formula (AM-1-B8a) and formula (AM-1-B8b) How Minh is made: [ka]

[0291] By carrying out the reaction according to the above synthesis scheme (the reaction of each step is described in [Manufacturing Method AM-A]), (According to the reaction described above), the compounds represented by formula (AM-1-B8a) and formula (AM-1-B8b) In the above scheme, the amine compound represented by formula (SM-B8a) or a salt thereof can be produced. Formula (SM-B8b) is a commercially available compound or can be prepared according to the reaction described in [Production Method AM-G]. The compounds of formula (RG-B8a) or formula (RG-B8b) are commercially available. These compounds are compounds that can be prepared from compounds listed in the literature or commercially available compounds by known preparation methods.

[0292] [Production Method AM-J] Production method of amine represented by formula (AM-1-B7): [ka]

[0293] Compound of formula (SM-B7) and compound of formula (RG-B7) [Compound of formula (SM-B7) The compound of formula (RG-B7) is a commercially available compound or can be prepared from a commercially available compound by a known method. The compound can be produced by the same method as in the above [Method for producing an alginic acid derivative of formula (I)]. A similar condensation reaction is then carried out to form the protecting group P 1 By deprotecting the compound represented by formula (AM-1-B7), The compound can be prepared as an amine compound represented by the following formula: or a salt thereof.

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

[0295] Compound of formula (SM-B7) and compound of formula (RG-B7-2) [Compound of formula (SM-B7)] The compound and the compound of formula (RG-B7-2) are commercially available compounds or compounds prepared from commercially available compounds by known methods in the literature. The alginic acid derivative of formula (I) is a compound that can be produced by the method. The same condensation reaction as in the method [<Step 1> and <Step 2>] is carried out, followed by the addition of the protecting group P 1 Deprotection Then, a compound of formula (RG-B7-3) (a commercially available compound or a compound known in the literature from a commercially available compound) is obtained. A condensation reaction similar to that in the above <Step 1> is carried out using the compound (which can be produced by the production method). followed by the addition of the protecting group P 1 by deprotection to obtain an amine represented by formula (AM-1-B7). It can be prepared as a compound or a salt thereof.

[0296] [Production Method AM-K] A compound represented by formula (AM-1-B9a) and formula (AM-1-B9b) How Minh is made: [ka]

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

[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 It is a compound that can be produced by a production method known in the literature from For example, Organometallics, 29(23), p6619-6622;201 In accordance with the method described in "The Journal of Chemical Engineering, Vol. 1, No. 10, pp. 111-112, 1999," in a solvent that does not participate in the reaction, such as dimethyl sulfoxide, After the azide group was introduced by reaction with NaN3, the protecting group P 1 By deprotecting the An amine compound represented by M-2-Z1) or a salt thereof can be produced. The amine compound represented by formula (AM-2-Z1) or a salt thereof is commercially available. Some are available.

[0300] [Production Method AM-M] A compound represented by the formula (AM-2-Z2a) and the formula (AM-2-Z2b) How Minh is made: [ka]

[0301] A compound of formula (SM-Z2a) or a compound of formula (SM-Z2b) [a compound of formula (SM-Z2a)] The compound and the compound of formula (SM-Z2b) are commercially available compounds or are prepared from commercially available compounds by methods known in the literature. This compound can be produced by the same method as in [Production Method AM-L]. After reacting N3 to introduce an azide group, the protecting group P 1 By deprotecting the compound of formula (AM-2 and producing an amine compound represented by formula (AM-2-Z2a) or (AM-2-Z2b), or a salt thereof. The amination compound represented by formula (AM-2-Z2a) or (AM-2-Z2b) can be Some of the compounds or salts thereof are commercially available.

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

[0303] Compounds of formula (SM-Z3) and compounds of formula (RG-Z3) [Compounds of formula (SM-Z3) The compound of formula (RG-Z3) is a commercially available compound or can be prepared from a commercially available compound by a known method. The compound of formula (I) can be prepared by the method of the present invention. Then, a condensation reaction similar to that of the above method is carried out, followed by the removal of the protecting group P 1 by deprotecting the compound of 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] Compounds of formula (SM-Z4) and compounds of formula (RG-Z4) [Compounds of formula (SM-Z4) The compound of formula (RG-Z4) is a commercially available compound or can be prepared from a commercially available compound by a known method. The compound can be produced by the same method as in the above [Method for producing an alginic acid derivative of formula (I)]. A similar condensation reaction is then carried out to form the protecting group P 1 By deprotecting the compound of formula (AM-2-Z4), The amine compound represented by the formula (I) or a salt thereof can be prepared.

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

[0307] Compounds of formula (SM-Z5a) and compounds of formula (RG-Z5a) [compounds of formula (SM-Z5a)] The compounds of formula (RG-Z5a) are commercially available compounds or are prepared from commercially available compounds by methods known in the literature. It is a compound that can be produced by the method described above. In the presence of tetrahydrofuran, N,N-dimethylformamide, N-methylpyrrolidone, By reacting in a solvent that does not participate in the reaction, such as dimethyl sulfoxide, a side chain is introduced. The compound is then protected by the protecting group P 1 By deprotecting the compound of formula (AM-2-Z5 The amine compound represented by formula (a) or a salt thereof can be produced. Similarly, the compound of formula (SM-Z5b) and the compound of formula (RG-Z5b) [formula (SM The compounds of formula (RG-Z5b) and (RG-Z5b) are commercially available compounds or are synthesized from commercially available compounds. This compound can be produced by a known production method. An amine compound represented by (AM-2-Z5b) or a salt thereof can be produced.

[0308] 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 amino compounds represented by formula (AM-2-Z9b), or The salt can be prepared according to the above-mentioned [Production Methods AM-A] to [Production Methods AM-P] as shown in the following scheme: It can be produced by a production method.

[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> Compound of formula (SM-Q) [Compound of formula (SM-Q) is a commercially available compound or and a compound of formula (RG-Q1) which can be produced from the compound by a production method known in the literature. Compounds of formula (RG-Q1) are commercially available compounds or can be prepared from commercially available compounds by methods known in the literature. The compound can be produced by a method known in the literature, for example, Journal of Organic Chemistry, 2014(6), (i) PPh3, In the presence of N2(CO2CHMe2)2 reagents, tetrahydrofuran and other The Mitsunobu reaction is carried out in a solvent containing 1,000 methyltrimethylsilyl methylisothiazolinone, and then the reaction is carried out in the presence of a base such as sodium hydroxide, methanol, ethanol, or the like. In a solvent that does not participate in the reaction, such as alcohol, tetrahydrofuran, or water, or a mixture of these solvents Then, the ester group is hydrolyzed to produce a compound represented by formula (IM-Q1). It is possible.

[0315] <Step 2> [Production Method AM-Q] Compound of formula (IM-Q1) obtained in <Step 1> and compound of formula (IM-Q2) Compound (RG-Q2) [Compound (RG-Q2) is a commercially available compound or a compound synthesized from a commercially available compound. The compound of formula (I) can be prepared by a known method. A condensation product is obtained by carrying out a condensation reaction similar to that in the method for producing alginic acid derivatives, and then the product is stored. Mamoru P 1 by deprotecting the compound represented by formula (AM-2-Z10), The salts can be prepared.

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

[0317] <Step 1> Compound of formula (SM-R) [Compound of formula (SM-R) is a commercially available compound or a compound which can be produced from a compound by a production method known in the literature] using a method known in the literature , for example, "Faming Zhuanli Shenqing, 104529898 , 22 Apr 2015, etc., (i) a base such as pyridine In the presence of hydroxybenzoates, H2NOH-HCl is reacted with hydroxybenzoates in a solvent that does not participate in the reaction, such as ethanol. (ii) P2O5, followed by reaction with diphosphorus pentoxide in methanesulfonic acid. and (iii) forming an eight-membered lactam by Beckmann rearrangement. In a solvent that does not participate in the reaction, such as ethyl ether, a reducing agent such as BH3 or LiAlH4 is used. The compound represented by formula (IM-R1) can be produced by reducing the amide group using the can.

[0318] <Step 2> [Production Method AM-R] Compound of Formula (IM-R1) Obtained by <Step 1> and a compound of formula (RG-R1) [the compound of formula (RG-R1) is a commercially available compound or a commercially available compound The compound of formula (I) can be prepared by a known method from the compound of formula (I) A condensation reaction similar to that in the preparation of hydroxypropyltrimonials is carried out to obtain a condensate. After this, an alkyne group is formed by debromination using tert-BuOK. , followed by a protecting group P 1 by deprotection to obtain an amination compound represented by formula (AM-1-B10). A compound or a salt thereof can be produced.

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

[0320] Compounds of formula (SM-S) and compounds of formula (RG-S1) [Compounds of formula (SM-S) and compounds of formula The compound (RG-S1) is a commercially available compound or is prepared from a commercially available compound by a preparation method known in the literature. The compound is a compound capable of being produced by the same condensation as in the above [Method for producing an alginic acid derivative of formula (I)]. The coupling reaction is then carried out, followed by the addition of the protecting group P 1 By deprotecting the compound represented by formula (AM-2-Z11), The compound or a salt thereof can be prepared.

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

[0322] <Step 1> Reacting a compound of formula (SM-T) and a compound of formula (RG-T-1) [formula (SM-T ) and the compound of formula (RG-T-1) are commercially available compounds or compounds known in the literature from commercially available compounds. [This compound can be produced by the method described above], and then, by a method known in the literature, for example, WO2 Grignard reaction was carried out according to the method described in JP-A-004 / 035017, followed by acid By carrying out a conversion reaction, a compound of formula (IM-T-1) is obtained.

[0323] <Step 2> After protecting the carbonyl group of the compound of formula (IM-T-1) (for example, acetone), After adding bromine to the cyclooctene ring, a base such as tert-BuOK is used. Then, the protecting group of the carbonyl group and the protecting group P 1 Deprotecting the An amine represented by formula (AM-1-T1) or a salt thereof is produced by the above procedure.

[0324] <Step 3> Reacting an amine of formula (AM-1-T1) or a salt thereof with a compound of formula (RG-T-2) [ The compound of formula (RG-T-2) is a commercially available compound or can be obtained from a commercially available compound by a known method. A condensation reaction is carried out using a compound that can be produced by the method of the present invention, and a protecting group P 1 By deprotecting Thus, an amine represented by the formula (AM-1-T2) or a salt thereof is produced.

[0325] [Manufacturing method AM-U] Method for producing 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 converted into the compound of formula (SM-U) [The compound of formula (SM-U) is a commercially available compound or can be prepared from a commercially available compound by a preparation method known in the literature.] [This compound can be produced by the method described in [Production Method AM-T]]. by carrying out the above steps, an amine represented by formula (AM-1-U1) and formula (AM-1-U2), or a salt thereof.

[0327] The aldehyde used in step 1 of the above [Production Method AM-T] and [Production Method AM-U] , an aldehyde of the following formula (RG-T-3) or formula (RG-T-4) [formula (RG-T-3) The compounds of formula (RG-T-4) are commercially available compounds or compounds known in the literature that can be prepared from commercially available compounds. By replacing the compound with the corresponding linker, An amine having the formula: or a salt thereof is prepared. [ka] [Manufacturing method AM-V] Method for producing amines represented by formula (AM-1-V1) and formula (AM-1-V2): [ka]

[0328] <Step 1> Compound of formula (SM-V) [Compound of formula (SM-V) is a commercially available compound or a compound published in the literature] Methods of knowledge, e.g., Bioorganic & Medicinal Chemistr y,23(22),pp.7150-7157,2015, etc.

[0043] can be used to convert the compound into an acid chloride according to a conventional method, and then the compound of formula (RG-V-1) [ The compound of formula (RG-V-1) is a commercially available compound or can be obtained from a commercially available compound by a known method. The compound can be prepared by Grignard reaction using the compound, followed by the addition of a protecting group P 1 Deprotection This produces an amine represented by formula (AM-1-V1) or a salt thereof.

[0329] <Step 2> Reacting an amine of formula (AM-1-V1) or a salt thereof with a compound of formula (RG-T-1) A condensation reaction is carried out using the protecting group P 1 By deprotecting the compound represented by formula (AM-1-V2), The amine represented by the formula (I) or a salt thereof is prepared.

[0330] The compound used in <Step 1> of the above [Production Method AM-V] is a compound of the following formula [each compound is a commercially available compound or a compound that can be prepared from a commercially available compound by a method known in the literature] and reacting to prepare an amine having a corresponding linker or a salt thereof. [ka]

[0331] [Manufacturing method AM-W] Method for producing 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 converted into the compound of formula (SM-W) [The compound of formula (SM-W) is a commercially available compound or can be prepared from a commercially available compound by a preparation method known in the literature.] [This compound can be produced by the method described in [Production Method AM-V]]. by carrying out the above steps, an amine represented by formula (AM-1-W1) and formula (AM-1-W2), or a salt thereof.

[0333] The compound used in <Step 1> of the above [Production Method AM-W] is a compound of the following formula [each compound is a commercially available compound or a compound that can be prepared from a commercially available compound by a method known in the literature] and reacting to prepare an amine having a corresponding linker or a salt thereof. [ka]

[0334] Alginic acid derivatives represented by formula (I) or (II) are used to produce Amine with a hydroxyl group (Akn-L 1 -NH2) or amine with azide group (N3-L 2 -NH2) are described in the above [Production Method AM-A] to [Production Method AM-P]. The reactions described in the literature, methods known in the literature, for example, "Experimental Chemistry Lectures, 5th Edition, each book, 20 2007, Maruzen, "Comprehensive Organic Transformations, A Guide to Functional G roup Preparations, 3rd Edition (Edited by Richard C. Larock), 2018”, “Strate gic Applications of Named Reactions in Organic Synthesis, (Edited by Laszlo Kurt i, Barbara Czako), Academic Press, 2005, etc. The desired amine can be produced by

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

[0336] The salts can be prepared in a conventional manner, for example, by dissolving the compound of the present invention in a solution containing an appropriate amount of acid or base. The desired salt is formed by mixing the two components together, and then the salt is separated and filtered off, or the mixed solvent is evaporated. For a general overview of salt, see Handbook of Pharmaceutical Salts:Properties, Selecti on, and Use, Stahl & Wermuth (Wiley-VCH, 2002 ) has been published and is described in detail in this book.

[0337] In the present specification, the amine compound represented by formula (AM-1) or formula (AM-2) (each formula or a salt thereof may be solvated with a solvent such as water, ethanol, or glycerol. It can be formed.

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

[0339] In the present specification, the phosphorus in the chemically modified alginic acid derivative represented by formula (I) or formula (II) Car (-L 1 -or-L 2 -) if there is an asymmetric carbon, each optical isomer thereof This also means that

[0340] For example, -L in formula (I) 1- is equation (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) is used, in which the configuration of the carbon substituted by the group is S-configuration. ) and the following formula (L1-8a-MR) in which the configuration of the carbon substituted with the benzyl group is R-configuration (In all formulas, the areas outside the dashed lines are not included): [ka] This means that a linker represented by

[0341] The linker (-L) in the chemically modified alginic acid derivative represented by formula (I) or formula (II) 1 -or-L 2 -) has an asymmetric carbon (if 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 the racemic compound into each optically active compound by conventional optical resolution (separation method). and the amine derivatives of formula (AM-1) or (AM-2) corresponding to formula (I) In the process of synthesizing the compound, one of the optical isomers can be selectively synthesized by using asymmetric synthesis. It is possible to synthesize each optically active substance.

[0342] 9. Applications of alginate derivatives and cross-linked alginate structures Alginic acid derivatives have been widely used in a wide range of fields, including food, medicine, cosmetics, textiles, and paper manufacturing. Alginic acid derivatives or photocrosslinked alginic acid structures can be used instead of alginic acid. Specific preferred uses for the body include wound dressings, postoperative adhesion barriers, and drug sustained release substrates. Examples of such materials include medical materials such as substrates for cell culture and substrates for cell transplantation.

[0343] When used as a medical material, the cross-linked alginate structure may be in the form of a tube or fiber. Examples of the particles include beads, gels, and approximately spherical gels. It is preferable to form a gel having a shape of approximately sphere, and more preferable to form a gel having a shape of approximately sphere.

[0344] All literature and publications mentioned herein are hereby incorporated by reference for all purposes. The entire contents of which are incorporated herein by reference.

[0345] Furthermore, the objects, features, advantages, and ideas of the present invention will become apparent to those skilled in the art through the description of this specification. It is clear to those skilled in the art that the present invention can be easily implemented by those skilled in the art from the description of this specification. The best mode for carrying out the invention and specific examples are preferred embodiments of the present invention. These are illustrative of the various embodiments shown and are shown for purposes of illustration and description only and should not be construed as limiting the invention. Within the spirit and scope of the invention disclosed herein, It will be apparent to those skilled in the art that various modifications can be made based on the description in the specification. [Example]

[0346] Next, examples and test examples will be given to explain the present invention in more detail. These are merely examples and test examples, and do not limit the present invention, nor do they depart from the scope of the present invention. It may be changed within a range that does not deviate from the normal range.

[0347] Nuclear magnetic resonance (NMR) spectroscopy was performed using a JEOL JNM-ECX400 FT -NMR (JEOL) was used. Liquid chromatography-mass spectrometry (LC- Mass was measured using the following method: [UPLC] Waters AQUITY UPL C system and a BEH C18 column (2.1 mm × 50 mm, 1.7 μm) (Wat ers), and acetonitrile: 0.05% trifluoroacetic acid aqueous solution = 5:95 (0 min ) to 95:5 (1.0 min) to 95:5 (1.6 min) to 5:95 (2.0 min) mobile phase and and gradient conditions were used.

[0348] 1 In the H-NMR data, s indicates a singlet and d indicates a doublet in the NMR signal pattern. t is triplet, q is quartet, m is multiplet, br is broad, J is Coupling constants, Hz is hertz, CDCl3 is deuterated chloroform, DMSO-d6 is deuterated dichloroform Methyl sulfoxide, D2O means heavy water. 1 In H-NMR data, hydroxyl group (OH) broadband, such as the protons of the amino group (NH2) and carboxyl group (COOH). Therefore, signals that cannot be confirmed 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 1 Calculated from H-NMR (DO) Introduction to the number of moles of monosaccharide (guluronic acid and mannuronic acid) units that compose alginic acid The molar ratio of reactive substituents is intended to indicate the ratio of the number of moles of reactive substituents.

[0351] In the examples, sodium alginate before the reactive groups or complementary reactive groups are introduced is The emulsion used was sodium alginate having the physical properties shown in Table 46 above.

[0352] Table 48 shows the results of the alginates to which reactive groups were introduced, obtained in (Example 1) to (Example 20). The physical properties of the phosphoric acid derivatives (specifically, the reactive group introduction rate (mol%), molecular weight, and weight average The molecular weight (10,000 Da) is shown. Tables 49-1 to 49-5 show the 1H-NM intermediates in Examples 1 to 20. R is the intermediate LCM-Mass in (Example 1) to (Example 20). .

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

[0354] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (2 0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (56 mg), commercially available 3-azidopropanol Pyramine [CAS REGISTRY NO.:88192-19-2] (1-1, 5 Add 1 molar sodium bicarbonate solution (50 μL) of ethanol (2 mL) containing 0.1 mg of ethanol. After stirring at 30°C for 3 hours, sodium chloride (0.2 g), ethanol (40 ml) L) was added successively and stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration and washed with ethanol. The resulting solid was dissolved in water and then freeze-dried to give the title compound EX1-A2 (1 87 mg) was obtained as a white solid.

[0355] Example 2 2-(2-(2-azidoethoxy)ethoxy)ethane-1-amino group-introduced alginic acid (E Synthesis of X2-A2): [ka]

[0356] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (1 0.9 mL) and add 4-(4,6-dimethoxy-1,3,5-triazine- 2-yl)-4-methylmorpholinium chloride (DMT-MM) (55.83 mg) and 1 molar sodium bicarbonate solution (252.17 μL) were added. 2-Azidoethoxy)ethoxy)ethan-1-amine [CAS REGISTRY NO. .:166388-57-4] (2-1, 26.36 mg) in ethanol (1 mL) and water (1 mL) solution was added, and the mixture was stirred at room temperature for 15 hours. mg) and ethanol (21.8 mL) were added successively, and the mixture was stirred at room temperature for 30 minutes. The precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain the title compound EX2-A2 (99 mg ) was obtained as a white solid.

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

[0358] <Process 1> tert-Butyl(2-(3-azidopropyl)amino)-2-oxoethyl)carba Synthesis of Mate (3-2): [ka] Commercially available 3-azidopropylamine [CAS REGISTRY NO.: 88192- 19-2] (1-1, 41 μL), N-(tert-butoxycarbonyl)glycine [ CAS REGISTRY NO.: 4530-20-5] (3-1, 100 mg) In an ethanol (2 mL) solution, 4-(4,6-dimethoxy-1,3,5-triazine- 2-yl)-4-methylmorpholinium chloride (DMT-MM) (197 mg) was added. The reaction mixture was added with water and extracted with ethyl acetate, and the organic layer was washed with water, saturated The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The oil was dissolved in methyl tert-butyl ether (10 mL) and added to saturated sodium bicarbonate water and water. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The title compound 3-2 (95 mg) was obtained as a colorless oil.

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

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

[0361] <Process 3> 2-Amino-N-(3-azidopropyl)acetamide group-introduced alginic acid (EX3-A2 ) synthesis: [ka]

[0362] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (2 0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (56 mg), (Example 3) <Step 2 A solution of the compound (3-3, 10.6 mg) obtained in step 1 in ethanol (2 mL) was Concentration - Sodium bicarbonate solution (76 μL) was added. After stirring at 30°C for 3 hours, sodium chloride (0 0.2 g) and ethanol (40 mL) were added successively, and the mixture was stirred at room temperature for 30 minutes. The precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting solid was dissolved in water and freeze-dried. As a result, the title compound EX3-A2 (207 mg) was obtained as a white solid.

[0363] Example 4 3-Amino-N-(3-azidopropyl)propanamide group-introduced alginic acid (EX4-A 2) Synthesis of: [ka]

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

[0365] Commercially available 3-azidopropylamine [CAS REGISTRY NO.: 88192- 19-2](1-1, 38 μL), N-(tert-butoxycarbonyl)-β-ara Nin [CAS REGISTRY NO.:3303-84-2] (4-1, 100 m g) in ethanol (2 mL), Dimethyl-2-methyl-4-methylmorpholinium chloride (DMT-MM) (146 mg ) was added and stirred at room temperature for 18 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The title compound 4-2 (124 mg) was obtained as a white wax.

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

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

[0368] <Process 3> 3-Amino-N-(3-azidopropyl)propanamide group-introduced alginic acid (EX4-A 2) Synthesis of: [ka]

[0369] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (2 0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (56 mg), (Example 4) <Step 2 A solution of the compound (4-3, 12.6 mg) obtained in step 1 in ethanol (2 mL) was Concentration - Sodium bicarbonate solution (76 μL) was added. After stirring at 30°C for 3 hours, sodium chloride (0 0.2 g) and ethanol (40 mL) were added successively, and the mixture was stirred at room temperature for 30 minutes. The precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting solid was dissolved in water and freeze-dried. As a result, the title compound EX4-A2 (211 mg) was obtained as a white solid.

[0370] Example 5 N-(4-(aminomethyl)benzyl)-2-azidoacetamide group-introduced alginate (EX Synthesis of 5-A2): [ka]

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

[0372] Commercially available 2-azidoacetic acid [CAS REGISTRY NO.: 18523-48-3] (1-1, 41 μL), Organic Letters (2017), 19 (23), 6400-6403. A solution of commercially available 1-(N-tert-butyloxycarbonyl- 4-(aminomethyl)benzene [CAS REGISTRY NO.: 108468-00-4] (5-1, 100 mg), 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 mixture, and after separation, the organic layer was washed with water and The mixture was washed with saturated sodium bicarbonate water, water, and saturated saline in that order. Insoluble matter was removed by filtration, and the filtrate was diluted with anhydrous sodium sulfate. The residue was extracted with methyl tert-butyl ether / n-heptane. The resulting solid was collected by filtration, and the title compound 5-2 (91 mg) was dissolved in a thin The product was obtained as a gel solid.

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

[0374] (Example 5) The compound (5-2, 91 mg) obtained in <Step 1> was added to 4N water under ice-water cooling. -Hydrogen chloride / 1,4-dioxane (637 μL) was added, followed by 1,4-dioxane (6 After adding 27 μL of diisopropyl ether, the reaction mixture was stirred at room temperature for 3.5 hours. Ethanol (3.8 mL) was added and stirred for 10 minutes. The resulting solid was filtered to give the title compound. 5-3 (62 mg) was obtained as a beige solid.

[0375] <Process 3> N-(4-(aminomethyl)benzyl)-2-azidoacetamide group-introduced alginate (EX Synthesis of 5-A2): [ka]

[0376] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (2 5 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (70 mg), (Example 5) <Step 2 The compound obtained in step (5-3, 16.1 mg) and 1 molar sodium bicarbonate solution (95 μL) were added. After stirring at 30°C for 3 hours, sodium chloride (0.25 g), ethanol (5 0 mL) was added successively, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration and washed with ethanol. After washing, the solid was dried under reduced pressure. The obtained solid was dissolved in water and then freeze-dried to obtain the title compound EX5-A. 2 (239 mg) was obtained as a white solid.

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

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

[0379] Commercially available 4-azidophenol [CAS REGISTRY NO.: 24541-43 -3] (6-1, 0.3 g), commercially available tert-butyl (2-bromoethyl)carbamate Mate [CAS REGISTRY NO.:39684-80-5] (6-2, 0.6 A mixture of 1000 mg of methylpyrrolidone (3 mL) and potassium carbonate (0 mL) 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 Then, water (10 mL) and methyl tert-butyl ether (20 mL) were added. The resulting suspension was filtered through Celite, and the residue was extracted 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) and diluted with 1N hydroxybenzoate. The solution was diluted twice with sodium chloride solution (5 mL), twice with water (5 mL), and twice with saturated saline solution (5 mL). The organic layer was filtered and then concentrated under reduced pressure. This gave 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] (Example 6) Compound (6-3, 0.41 g) obtained in <Step 1> and 1,4-dihydro- A mixture of 4N-hydrogen chloride / 1,4-dichloromethane (2.87 mL) was added to the mixture under stirring in a water bath. After adding oxane (2.87 mL), the mixture was stirred at room temperature for 18 hours. Dipropyl ether (40 mL) was added and the suspension was stirred at room temperature for 30 minutes. The collected solid was dried under reduced pressure to give the title compound 6-4 (0.2834 g) as a pale purple solid. was obtained as.

[0382] <Process 3> 2-(4-Azidophenoxy)ethane-1-amino group-introduced alginic acid (EX6-A2) Synthesis of: [ka] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (2 9.66 mL) at room temperature. (methyl)-4-methylmorpholinium chloride (DMT-MM) (91.52 mg) and 1 molar sodium bicarbonate solution (68.63 μL) was added. The obtained compound (6-4, 14.73 mg) was dissolved in water (1 mL) and ethanol (1 mL). L) solution was added at room temperature, and the mixture was stirred at the same temperature for 42 hours. Then, ethanol (59.3 mL) was added successively and the mixture was stirred at room temperature for 30 minutes. The solid was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting solid was dissolved in water and freeze-dried. The title compound EX6-A2 (269 mg) was obtained as a pink solid.

[0383] Example 7 N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamido Synthesis of alkylated alginic acid (EX7-B2): [ka]

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

[0385] Commercially available tert-butyl(2-aminoethyl)carbamate (7-1, 3.00 g, Tetrahydrofuran [CAS REGISTRY NO.:57260-73-8] To the (12.0 mL) solution was added dropwise ethyl trifluoroacetate (2.24 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 treated with tert-butyl ether. Cetyl methyl ether (5 mL) and heptane (25 mL) were added and triturated. The solid was collected by filtration and washed with heptane to give the title compound 7-2 (4.36 g) as a white solid. And got it.

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

[0387] (Example 7) Compound 7-2 (0.50 g) obtained in <Step 1> was treated with 1,4-dioxa- The mixture was suspended in 4N hydrogen chloride / 1,4-dioxane ( 7.0 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 3 hours. 0.0 mL) was added and stirred at room temperature for 50 minutes. After washing with ethyl acetate, the extract was dried under reduced pressure to obtain the title compound 7-3 (0.70 g) as a white solid.

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

[0389] A method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110) was dissolved in ethanol (2 mL) solution of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4 -methylmorpholinium chloride (DMT-MM) (1.09 g), (Example 7) Compound 7-3 (380 mg) obtained in step 2 and triethylamine (321 μL) were added. After adding the mixture and stirring at 30°C for 3 hours, triethylamine (229 μL) was added and the mixture was stirred at the same temperature for 1 hour. After stirring at room temperature for an additional 15.5 hours, water (10 mL) and ethyl acetate (50 mL) were added. The aqueous layer was extracted with ethyl acetate (10 mL). The extract was washed with citric acid, water, and saturated saline in that order, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. To the residue was added tert-butyl methyl ether, and the insoluble matter was removed by filtration. The filtrate was concentrated and then Silica gel column chromatography (10% ethyl acetate / n-heptane to 40% ethyl acetate / n-heptane) The title compound 7-5 (322 mg) was obtained as a white solid by filtration using hexane / n-heptane. Got it.

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

[0391] (Example 7) Compound 7-5 (322 mg) obtained in <Step 3> was dissolved in methanol (4. To the solution, add a solution of potassium carbonate (278 mg) in water (1.6 mL) and The reaction mixture was concentrated under reduced pressure, and water (3 mL) was added, followed by stirring at room temperature for 7.5 hours. The aqueous layer was extracted with ethyl acetate (30 mL, 10 mL x 3) and diluted with anhydrous sulfuric acid. After drying over sodium hydroxide, the mixture was concentrated under reduced pressure to give the title compound 7-6 (238 mg) as a colorless oil. was obtained as.

[0392] <Process 5> N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamido Synthesis of alkylated alginic acid (EX7-B2): [ka]

[0393] A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) was prepared. 20 mL) at room temperature with stirring. -yl)-4-methylmorpholinium chloride (DMT-MM) (335 mg), Example 7) Compound 7-6 (68 mg) obtained in <Step 4> in ethanol (12 mL) The solution and 1 molar sodium bicarbonate solution (303 μL) were added sequentially, and the mixture was stirred at 30°C for 3 hours. Sodium chloride (1.2 g) was added to the solution, followed by ethanol (240 mL). The mixture was stirred for 1.5 hours, and the resulting precipitate was collected by filtration, washed with ethanol (20 mL × 5), and then The solid obtained was dissolved in water and freeze-dried to give the title compound EX7-B2 (1 0.16 g) as a white solid.

[0394] Example 8 N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2-yn-1-yl) Synthesis of (hydroxy)acetamido-functionalized alginic acid (EX8-A2): [ka]

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

[0396] tert-Butyl (2-aminoethyl) carbamate (8-1, 1.0 g, [CAS REGISTRY NO.:57260-73-8) of tetrahydrofuran (4.0 Ethyl trifluoroacetate (0.6 mL) was added dropwise to the solution. The mixture was stirred at room temperature for 3.5 hours, and then concentrated under reduced pressure to give crude compound 8-2 (1.5 g) as a colorless oil. Obtained as a material object.

[0397] <Process 2> N-(2-(2-aminoethoxy)ethyl)-2,2,2-trifluoroacetamide salt Synthesis of acid salt (8-3): [ka]

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

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

[0400] A method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), carboxylic acid (7-4, 300 mg) synthesized according to (Example 8) Compound 8-3 (443 mg) obtained in step 2 was dissolved in acetonitrile (6.0 mL). Dissolved. O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetrahydrobenzotriazol-1-yl Tetramethyluronium hexafluorophosphate (0.75 g), N,N-diisopropyl Ethylamine (920 μL) was added, and the mixture was stirred at room temperature for 2.5 hours. The organic layer was separated with water (10 mL), saturated sodium hydroxide solution (20 mL), and water (10 mL). The residue was washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography (50% ethyl acetate / n-heptane to 70% acetic acid). The title compound 8-4 (469 mg) was obtained as a colorless gum by elution with ethyl acetate / n-heptane. was obtained as.

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

[0402] (Example 8) Compound 8-4 (220 mg) obtained in <Step 3> was dissolved in methanol (3. To the solution, add a solution of potassium carbonate (103 mg) in water (0.99 mL), The mixture was stirred at room temperature for 4.5 hours, and the methanol was removed under reduced pressure. Water (2 mL) was added, and then The mixture was saturated with sodium chloride, extracted with ethyl acetate (15 mL, 10 mL x 4), and then washed with anhydrous sodium sulfate. After drying over methanol, the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (10 mL). After removing the insoluble matter by filtration, the mixture was concentrated under reduced pressure to give crude compound 8-5 (140 mg). Obtained as a pale yellow gum.

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

[0404] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (4 0 mL), 4-(4,6-dimethoxy-1,3,5-triazine-2- (DMT-MM) (112 mg), Example 8) Compound 8-5 (30 mg) obtained in <Step 4> in ethanol (4.0 mL) The solution and 1 molar sodium bicarbonate solution (101 μL) were added successively, and the mixture was stirred at 30°C for 3 hours. Sodium chloride (0.4 g) was added to the solution, followed by ethanol (80 mL). The mixture was stirred for 10 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The solid was dissolved in water and freeze-dried to obtain the title compound EX8-A2 (410 mg) as a white solid. was obtained as.

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

[0406] <Process 1> tert-Butyl (2-oxo-2-((2-(2,2,2-trifluoroacetamide) Synthesis of ethylaminoethyl carbamate (9-1): [ka]

[0407] N-(tert-butoxycarbonyl)glycine (91 mg, [CAS REGIS TRY NO.: 4530-20-5]), the compound obtained in (Example 7) <Step 2> ( 7-3 (100 mg) was dissolved in acetonitrile (3.0 mL). Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexa Fluorophosphate (217 mg), N,N-diisopropylethylamine (281 μ The reaction mixture was added with ethyl acetate (15 mL), water (5 mL), and stirred at room temperature for 3.5 hours. After separation, the organic layer was washed with water and saturated brine. The residue was purified by column chromatography (eluent: 40 % ethyl acetate / n-heptane → ethyl acetate) to obtain the title compound 9-1 (180 mg ) was obtained as a pale beige amorphous solid.

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

[0409] (Example 9) The compound (9-1, 180 mg) obtained in <Step 1> was added to 4 mL of 10 ... After adding hydrogen chloride / 1,4-dioxane (1.2 mL), the mixture was stirred at room temperature for 0.8 hours. To the reaction mixture was added diisopropyl ether (3.6 mL), and the mixture was stirred for 30 minutes. The resulting solid was filtered to give the title compound 9-2 (114 mg) as a white solid.

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

[0411] A method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), carboxylic acid (7-4, 80 mg) synthesized according to (Example 9) The compound obtained in step 2 (9-2, 110 mg) was added to ethanol (1.6 mL), -(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin Dimethylmethacrylate (DMT-MM) (219 mg) and triethylamine (67 μL) were added. The reaction mixture was stirred at room temperature for 3 hours, and water (3.2 mL) was added to the reaction mixture, followed by stirring at room temperature for 30 minutes. After that, the solid was filtered and washed with water. The obtained solid was diluted with ethyl acetate / ethanol (1 / 1, 10 mL) was added, and the insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure to give the title compound 9-3( 101 mg) was obtained as a white solid.

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

[0413] (Example 9) The compound (9-3, 60 mg) obtained in <Step 3> was dissolved in methanol (1. To the solution of 100 mg of potassium carbonate (59 mg) in water (0.3 mL), add the solution and heat at room temperature. The reaction mixture was concentrated under reduced pressure, and then water (2 mL) was added and saturated with sodium chloride. The mixture was extracted 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 the insoluble material was removed by filtration. The filtrate was concentrated under reduced pressure to give the title compound 9-4 (49 mg) as a colorless gum. .

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

[0415] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (3 8 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (106 mg), (Example 9) <Step A solution of the compound (9-4, 30.3 mg) obtained in 4> in ethanol (3.8 mL), 1 molar sodium bicarbonate solution (96 μL) was added. After stirring at 30°C for 3.2 hours, sodium chloride was added. Sodium (0.38 g) and ethanol (76 mL) were added successively, 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. After thawing, the mixture was freeze-dried to obtain the title compound EX9a-A2 (381 mg) as a white solid.

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

[0417] A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (3 8 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (64 mg), (Example 9) <Step 4 The compound obtained in step (9-4, 18.2 mg) and 1 molar sodium bicarbonate solution (58 μL) were After stirring at 30°C for 3.2 hours, sodium chloride (0.38 g), ethanol (76 mL) was added successively, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration and The resulting solid was dissolved in water and then freeze-dried to give the title compound EX9. b-B2 (366 mg) was obtained as a white solid.

[0418] Example 10 N-(2-aminoethyl)-3-(2-(cyclooct-2-yn-1-yloxy)a Synthesis of alginic acid (EX10-A2) incorporating acetamidopropanamide groups: [ka]

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

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

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

[0422] (Example 10) The compound (10-1, 80 mg) obtained in <Step 1> was added to 4 After adding normal hydrogen chloride / 1,4-dioxane (1.1 mL), the mixture was stirred at room temperature for 2 hours. To the reaction mixture was added diisopropyl ether (3.4 mL), and the mixture was stirred for 1.5 hours. The resulting solid was filtered to give the title compound 10-2 (61 mg) as a white solid.

[0423] <Process 3> 3-(2-(cyclooct-2-yn-1-yloxy)acetamide)-N-(2-(2, Synthesis of 2,2-trifluoroacetamido)ethyl)propanamide (10-3): [ka]

[0424] A method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), carboxylic acid (7-4, 44 mg) synthesized according to (Example 10) The compound (10-2, 61 mg) obtained in step 2 was added to ethanol (1.2 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholino Dimethylammonium chloride (DMT-MM) (115 mg), triethylamine (39 μL) The reaction mixture was added with water (3.7 mL) and stirred at room temperature for 2 hours. The organic layer was washed with water and saturated brine, and then extracted with anhydrous sodium sulfate. The resulting solid was dried over sodium and then concentrated under reduced pressure. 0 mL), triturated, and filtered. The resulting solid was purified by column chromatography. (80% ethyl acetate / n-heptane → ethyl acetate → 20% methanol / ethyl acetate) to give the title compound 10-3 (60 mg) as a pale yellow solid.

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

[0426] (Example 10) The compound (10-3, 60 mg) obtained in <Step 3> was dissolved in methanol ( 3.0 mL) solution, add potassium carbonate (42 mg) in water (0.3 mL), After stirring at room temperature for 3 hours, a solution of potassium carbonate (42 mg) in water (0.3 mL) was added. The reaction mixture was concentrated under reduced pressure, and saturated saline (2 mL) was added. The mixture was then saturated with sodium chloride. The extract layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. mL) and a few drops of methanol were added, and the insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure to give the target The title compound 10-4 (31 mg) was obtained as a colorless oil.

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

[0428] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (4 1 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (114 mg), (Example 10) The compound obtained in step 4 (10-4, 30.5 mg) was dissolved in ethanol (4.1 mL). After stirring at 30°C for 3 hours, sodium chloride was added to the solution. Add thorium (0.41 g) and ethanol (82 mL) in that order, and stir at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. After dissolution, the mixture was lyophilized to give the title compound EX10-A2 (406 mg) as a white solid. .

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

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

[0431] A method known in the literature (Org. Process Res. Dev. (2018) 22: The dibromo compound (11-1, 1 g) synthesized according to the method described in the literature ( The alcohol derivative (11) was synthesized according to the procedure of International Publication No. 2015 / 140807. To a mixture of 1,2-dimethyl-2, 5.28 g, dichloromethane (2 mL) was added at room temperature. The reaction vessel was wrapped in aluminum foil to protect it from light while maintaining the temperature at room temperature. Add silver trifluoromethanesulfonate (1.92 g) at once and stir at the same temperature for 1 hour. After stirring, saturated saline (5 mL) was added under ice cooling, and the precipitated silver salt was filtered through Celite. The residue was washed with methyl tert-butyl ether (10 mL). The solution was separated, and the organic layer was washed twice with water (5 mL), then dried over anhydrous sodium sulfate. The crude product was dried, filtered, and concentrated under reduced pressure to obtain a crude product. The compound was purified by column chromatography (n-heptane / ethyl acetate) to give compound 11-3 (0. A fraction containing 46 g of methylcellulose was obtained.

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

[0433] (Example 11) Fractions containing the compound (11-3, 0.46 g) obtained in <Step 1> and A mixture of 28% sodium hydroxide and dimethyl sulfoxide (1.38 mL) was added to the mixture under stirring in a water bath. A solution of ammonium methoxide in methanol (1.82 mL) was added, and the mixture was stirred at room temperature for 16 hours. The reaction was quenched by adding methanol (10 mL), and the methanol was concentrated under reduced pressure. The organic layer was extracted three times with ethyl tert-butyl ether (10 mL). The mixture was dried over sodium, filtered, and concentrated under reduced pressure to give the title compound 11-4 (0.196%). The crude product of g) was obtained as a brown oil.

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

[0435] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (6 9.2 mL) at room temperature. Dimethyl-4-methylmorpholinium chloride (DMT-MM) (213.55 mg) was added. Subsequently, the compound (11-4, 26.78 m) obtained in (Example 11) <Step 2> was g) in water (1 mL) and ethanol (1 mL) at room temperature, and the mixture was left at the same temperature for 24 hours. After stirring, sodium chloride (700 mg) and ethanol (138.4 mL) were added in that order. 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 EX11a-A2 (661 mg) was obtained as a white solid.

[0436] <Process 3-2> 2-(cyclooct-2-yn-1-yloxy)ethane-1-amino group-introduced alginic acid ( Synthesis of EX11b-B2): [ka]

[0437] A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (7 0.1 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4 -methylmorpholinium chloride (DMT-MM) (216.4 mg) and (Example 1 1) The compound (11-4, 27.14 mg) obtained in <Step 2> was used (Example 11 ) The same procedure as in <Step 3-1> was carried out to obtain the title compound EX11b-B2 (648 mg). Obtained as a white solid.

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

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

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

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

[0442] A method known in the literature (Org. Process Res. Dev. (2018) 22: The dibromo compound (11-1, 0.30 g) synthesized according to the procedure described in (108-110) was dissolved in methylene chloride. (0.54 mL), and the solution was placed under aluminum foil to protect from light. Compound (12-2, 1.86 g), silver trifluoromethanesulfonate (0.52 g), After stirring for 1.5 hours at room temperature under light protection, the reaction mixture was cooled with ice water and saturated aqueous sodium bicarbonate (2. The solid was removed by filtration through Celite, and the The filtrate was washed with ert-butyl methyl ether (10 mL x 3). The organic layer was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The title compound 12-3 (424 mg) was obtained as a pale brown oil.

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

[0444] (Example 12) The compound (12-3, 100 mg) obtained in <Step 2> was added to tetrahydrofuran. Dissolved in 0.7 mL of toluene and 0.7 mL of N,N-dimethylformamide 60% Sodium hydride (21 mg) was added under ice water, and the mixture was stirred at the same temperature for 3 hours. 60% sodium hydride (10 mg) was added, and the mixture was stirred at room temperature for 1 hour, followed by 60% sodium hydride. Sodium (10 mg) was added and the mixture was stirred at room temperature for 20 hours. Water (3 mL) was added and ethyl acetate was added. The mixture was extracted with chilled water (15 mL, 10 mL), and the organic layer was washed with water and saturated brine successively. 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 give the title compound 12-4 (3 7 mg) was obtained as a colorless oil.

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

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

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

[0448] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (5 2 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (145 mg), (Example 12) A solution of the compound (12-5, 29 mg) obtained in step 4 in ethanol (5.2 mL), 1 molar sodium bicarbonate solution (131 μL) was added. After stirring at 30°C for 3.2 hours, sodium chloride was added. Add thorium (0.52 g) and ethanol (104 mL) in that order, and stir at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting solution was dissolved in 100 ml of ethyl acetate and then lyophilized to give the title compound EX12-A2 (522 mg) as a white solid. Ta.

[0449] Example 13 3-amino-N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetate Synthesis of amido)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. 2,2,2-trifluoroacetic acid (3.4 mL) was added to the mixture under ice-cooling for 5 minutes. After adding dropwise thereto, the mixture was stirred at room temperature for 20.5 hours. The reaction mixture was concentrated under reduced pressure, and water (20 mL) was added. The pH was adjusted to 4 with 1N hydrochloric acid. Extraction was performed with ethyl acetate (100 mL x 2, 50 mL). The organic layer was extracted, washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The title compound 13-2 (2.9 g) was obtained as a white solid.

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

[0453] (Example 13) Compound (13-2, 400 mg) obtained in <Step 1>, tert- Butyl (2-aminoethyl)carbamate (8-1, 441 mg, [CAS REG. STRY NO.: 57260-73-8) in ethanol (4.0 mL), -(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin Dimethylmethacrylate (DMT-MM) (897 mg) was added and the mixture was stirred for 3.5 hours. After adding water (5 mL) and extracting with ethyl acetate (20 mL, 10 mL), the organic layer was The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The product was purified by column chromatography (30% ethyl acetate / n-heptane → ethyl acetate). The title compound 13-3 (451 mg) was obtained as a colorless oil.

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

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

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

[0457] A method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), carboxylic acid (7-4, 111 mg) (Example 13) The compound (13-4, 215 mg) obtained in <Step 3> was added to ethanol (1.7 mL ), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl mol Dimethicone chloride (DMT-MM) (253 mg), triethylamine (102 μ Water (5 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 21 hours. The organic layer was washed with water and saturated brine, and then with anhydrous sodium sulfate. 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) was obtained as a colorless oil.

[0458] <Process 5> 3-amino-N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetate Synthesis of (13-6)(mido)ethoxy)ethyl)propanamide: [ka]

[0459] (Example 13) The compound (13-5, 35 mg) obtained in <Step 4> was dissolved in methanol ( To the solution (700 μL), add a solution of potassium carbonate (33 mg) in water (175 μL), The reaction mixture was stirred at room temperature for 16.5 hours. After concentrating the reaction mixture under reduced pressure, water (2 mL) was added and sodium chloride was added. The extract was saturated with sodium thorium. The residue was dried over sodium and concentrated under reduced pressure. The resulting filtrate was concentrated under reduced pressure to give the title compound 13-6 (24 ml). g) was obtained as a colorless gum.

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

[0461] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (2 8 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (78 mg), (Example 13) <Step A solution of the compound (13-6, 24 mg) obtained in 5> in ethanol (2.8 mL), Molar concentration of sodium bicarbonate solution (71 μL) was added. After stirring at 30°C for 3.5 hours, sodium chloride was added. C. (0.28 g) and ethanol (56 mL) were added successively, 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. This was followed by freeze-drying to obtain the title compound EX13-A2 (272 mg) as a white solid.

[0462] Example 14 N-(4-(2-aminoethoxy)benzyl)-2-(cyclooct-2-yn-1-yl) Synthesis of (hydroxy)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] Commercially available 2-bromoethylamine hydrobromide [CAS REGISTRY NO.: 2 576-47-8] (14-1, 3 g) in methanol (30 mL) was cooled on ice. Triethylamine (4.29 mL) was added under stirring. Ethyl fluoroacetate (1.92 mL) was slowly added, and the mixture was stirred at room temperature for 42 hours. After completion, the reaction mixture was concentrated under reduced pressure, and water (10 mL) was added. The organic layer was washed with water (5 mL) and saturated brine (5 mL) in that order, and then extracted with anhydrous After drying over sodium sulfate, filtration, and concentration under reduced pressure, the title compound 14-2 (2 0.457 g) as a pale brown solid.

[0465] <Process 2> tert-Butyl (4-(2-(2,2,2-trifluoroacetamido)ethoxy)benzyl Synthesis of benzoyl)carbamate (14-4): [ka]

[0466] Commercially available tert-butyl (4-hydroxybenzyl)carbamate [CAS REG ISTRY NO.: 149505-94-2] (14-3, 0.36 g), (Example 14) The compound (14-2, 0.46 g) obtained in <Step 1> and potassium iodide (0. A mixture of potassium carbonate (35 g) and N-methylpyrrolidone (3.6 mL) was added at room temperature. After adding ammonium hydroxide (0.45 g), the mixture was stirred at 140°C for 5 hours. After the reaction was completed, the mixture was cooled to room temperature. The mixture was diluted with water (10 mL). The organic layer was extracted twice with 1N aqueous sodium hydroxide solution (5 mL) and twice with water (5 mL). The organic layer was washed with saturated aqueous sodium chloride (5 mL) and then dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure to obtain a crude product. The product was purified by column chromatography (n-heptane / ethyl acetate) to give the title compound 14-4 (0 0.202 g) was obtained as a white amorphous solid.

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

[0468] (Example 14) The compound (14-4, 0.2 g) obtained in <Step 2> was used. By carrying out the same procedure as in Example 6) <Step 2>, the title compound 14-5 (0.147 g) was obtained. ) was obtained as a white solid.

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

[0470] A method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), carboxylic acid (7-4, 50 mg) synthesized according to (Example 14) To a mixture of the compound synthesized in step 3 (14-5, 81.96 mg) and ethanol, Then, under ice-cooling and stirring, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4 -methylmorpholinium chloride (DMT-MM) (137.22 mg) and triethyl After the reaction was completed, the mixture was stirred at room temperature for 1 hour and 30 minutes. 2 mL) was added, the suspension was stirred, and methyl tert-butyl ether (0.5 mL The separated aqueous layer was extracted twice with methyl tert-butyl ether (5 mL). The mixture was washed with water (5 mL) and saturated brine (5 mL) in that order, and then 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. The title compound 14-6 (99 ml) was obtained by chromatography (n-heptane / ethyl acetate). g) was obtained as a white amorphous substance.

[0471] <Process 5> N-(4-(2-aminoethoxy)benzyl)-2-(cyclooct-2-yn-1-yl) Synthesis of (hydroxy)acetamide (14-7): [ka]

[0472] (Example 14) The compound obtained in <Step 4> (14-6, 99 mg) and methanol To the mixture (1485 μL) was added potassium carbonate (64.17 mg) and After the reaction was completed, methanol was removed under reduced pressure. The mixture was concentrated under reduced pressure, and the resulting aqueous layer was extracted three times with ethyl acetate (5 mL). The organic layer was washed with water (5 mL). The extract was washed successively with saturated aqueous sodium chloride (5 mL) and brine (5 mL), and then dried over anhydrous sodium sulfate. The organic layer was filtered and concentrated under reduced pressure to give the title compound 14-7 (68 mg). The crude product was obtained as a yellow oil.

[0473] <Process 6-1> N-(4-(2-aminoethoxy)benzyl)-2-(cyclooct-2-yn-1-yl) Synthesis of (hydroxy)acetamido-functionalized alginic acid (EX14a-A2): [ka]

[0474] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (4 9.44 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (152.54 mg) and ( Example 14) The compound (14-7, 37.79 mg) obtained in <Step 5> was used (Example 11) The same procedure as in <Step 3-1> was carried out to obtain the title compound EX14a-A2 (479 mg ) was obtained as a white solid.

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

[0476] A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (4 0.08 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (123.66 mg) and ( Example 14) The compound (14-7, 30.64 mg) obtained in <Step 5> was used (Example 11) The same procedure as in <Step 3-1> was carried out to obtain the title compound EX14b-B2 (356 mg ) was obtained as a white solid.

[0477] Example 15 2-Amino-N-[3-(11,12-didehydrodibenz[b,f]azocine-5(6 H)-yl)-3-oxopropyl]acetamide group-introduced alginic acid (EX15-A2) Synthesis: [ka]

[0478] <Process 1> (9H-Fluoren-9-yl)methyl-N-[3-(11,12-didehydrodibenzyl)methyl] [b,f]Azocin-5(6H)-yl)-3-oxopropyl]acetamido-2-carboxamide Synthesis of the bamate group (15-2): [ka]

[0479] Commercially available 3-amino-1-(11,12-didehydrodibenz[b,f]azocine-5( 6H)-Il)-1-propanone [CAS REGISTRY NO.:1255942 -06-3](15-1, 50 mg), N-[(9H-fluoren-9-ylmethoxy )Carbonyl]glycine [CAS REGISTRY NO.:29022-11-5] (54 mg) was dissolved in acetonitrile (1.5 mL). (triazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluoro Add phosphate (76 mg) and N,N-diisopropylethylamine (70 μL) The mixture was stirred at room temperature for 4.5 hours, and then ethyl acetate (15 mL) and water (5 mL) were added to the reaction mixture. 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. After drying, the mixture was concentrated under reduced pressure and purified by column chromatography to give the title compound 15-2 (63 mg) was obtained as a pale beige amorphous substance.

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

[0481] (Example 15) The compound (15-2, 63 mg) obtained in <Step 1> was treated with piperidine Add 56 μL of N,N-dimethylformamide (315 μL) and let stand at room temperature for 3 The reaction mixture was stirred for 10 minutes. Ethyl acetate (15 mL) and water (5 mL) were added to the reaction mixture, and the mixture was separated. The organic layer was washed with water and saturated brine in that order. The organic layer was dried over anhydrous sodium sulfate and then The mixture was concentrated under reduced pressure. To the obtained solid, tert-butyl methyl ether (5 mL) was added, and After retentivity, the title compound 15-3 (10 mg) was collected by filtration as a light beige solid. The title compound 15-3 (11 mg) was recovered from the filtrate as a pale yellow gum. Obtained as a material object.

[0482] <Process 3> 2-Amino-N-[3-(11,12-didehydrodibenz[b,f]azocine-5(6 H)-yl)-3-oxopropyl]acetamide group-introduced alginic acid (EX15-A2) Synthesis: [ka]

[0483] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (1 9 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (106 mg), (Example 15) A solution of the compound (15-3, 21 mg) obtained in step 2 in ethanol (1.9 mL), 1 molar sodium bicarbonate solution (48 μL) was added. After stirring at 30°C for 3 hours, sodium chloride was added. To the mixture were added ethanol (38 mL) 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 Lyophilization gave the title compound EX15-A2 (188 mg) as a white solid.

[0484] Example 16 2-Amino-N-(2-(cyclooct-2-yn-1-yloxy)ethyl)acetamide Synthesis of functionalized alginate (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. After the reaction was completed, 1N hydrochloric acid (20 mL) was added to the reaction mixture and stirred at room temperature for 23 hours. ) was added slowly until the pH reached 2, and the mixture was extracted three times with ethyl acetate (10 mL), and then with water (5 mL). The organic layer was washed with water (5 mL) and saturated brine (5 mL) in that order. The organic layer was dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure to give a pale yellow oil. The solution was dissolved in n-heptane (10 mL) and concentrated under reduced pressure. The title compound 16-2 (3.22 g) was obtained as a white amorphous substance.

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

[0488] Compound 11-4 (80 mg) and the compound (16) obtained in (Example 16) <Step 1> To the mixture of 2, 81.83 mg of ethanol (1600 μL) was added under ice-cooling and stirring. and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl mol Add folinium chloride (DMT-MM) (239.21 mg) and stir at room temperature for 3 hours. The reaction was stopped by adding water (2 mL), and methyl tert-butyl ether (5 mL) was added. The organic layer was washed with water (5 mL) and saturated brine (5 mL) in that order. The organic layer was filtered and concentrated under reduced pressure to give the crude product. This crude product was triturated with n-heptane (10 mL), filtered, and evaporated. Drying under reduced pressure gave the title compound 16-3 (95.1 mg) as a white solid.

[0489] <Process 3> 2-Amino-N-(2-(cyclooct-2-yn-1-yloxy)ethyl)acetamide Synthesis of (16-4): [ka]

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

[0491] <Step 4> 2-Amino-N-(2-(cyclooct-2-yn-1-yloxy)ethyl)acetamide Synthesis of functionalized alginate (EX16-A2): [ka]

[0492] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (2 9.66 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (91.52 mg) and (Example 16) The compound (16-4, 15 mg) obtained in <Step 3> was used to prepare the compound (16-4) of (Example 11) The same procedure as in step 3-1 was carried out to obtain the title compound EX16-A2 (279 mg) as a white solid. Got it as a body.

[0493] Example 17 (2S)-2-amino-N-(2-(cyclooct-2-yn-1-yloxy)ethyl) Synthesis of 3-phenylpropanamide-functionalized 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 2 g) was dissolved in methanol (10 mL) and cooled to 4°C. Ethyl trifluoroacetate (1.59 mL) and triethylamine (1.69 mL) were added at room temperature. After the reaction was completed, 1N hydrochloric acid (10 mL) was added to the reaction mixture and stirred at room temperature for 16 hours. The suspension was gradually added until the concentration reached H1, and the suspension was stirred for 30 minutes. Drying under reduced pressure gave the title compound 17-2 (2.53 g) as a white solid.

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

[0497] Compound 11-4 (60 mg) and the compound (17) obtained in (Example 17) <Step 1> A mixture of 4-2 (93.7 mg) was added to ethanol (1200 μL) and 4-2 (93.7 mg) under ice cooling. -(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin Dimethylmethacrylate (DMT-MM) (179.41 mg) was added thereto, and the mixture was stirred at room temperature for 3 hours. The reaction was quenched by adding water (2 mL) and methyl tert-butyl ether (5 mL) The organic layer was washed with water (5 mL) and saturated brine (5 mL) in that order, and then extracted with anhydrous The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to obtain the crude product. The product was purified by silica gel column chromatography (n-heptane / ethyl acetate). The title compound 17-3 (57 mg) was obtained as a white amorphous substance.

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

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

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

[0501] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (4 7.46 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (146.44 mg) and ( Example 17) The compound (17-4, 34.53 mg) obtained in <Step 3> was used (Example 11) The same procedure as in <Step 3-1> was carried out to obtain the title compound EX17-A2 (383 mg). was obtained as a white solid.

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

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

[0504] Dissolve triphenylphosphine (0.96 g) in tetrahydrofuran (2.59 mL) To the solution, diethyl azodicarboxylate (40% toluene solution, 1.92 mL) was added under ice cooling and stirring. The solution was added with the commercially available 4-hydroxybenzoate under ice-cooling and stirring, and the mixture was stirred at room temperature for 20 minutes. 2-(tert-butoxybenzoic acid) (compound 18-1, 0.37 g) and 2-(tert-butoxycarbonyl) Add a solution of ethanolamine (0.39 g) in tetrahydrofuran (1.1 mL). The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography. by chromatograph (5% ethyl acetate / n-heptane to 40% ethyl acetate / n-heptane). This mixture was purified by methyl te Dissolve in rt-butyl ether (20 mL) and add 1N sodium hydroxide solution (5 The organic layer was washed with saturated aqueous sodium chloride (5 mL) twice and then with saturated aqueous sodium chloride (5 mL). After drying, the solvent was distilled off under reduced pressure to give the title compound 18-2 (0.45 g). It was obtained as a colored oil.

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

[0506] (Example 18) Compound 18-2 (0.44 g) obtained in <Step 1> was dissolved in methanol ( Lithium hydroxide monohydrate (0.25 g) was added to the solution (4.4 mL) and heated at 60°C for 3 hours. After stirring for 30 minutes, 1N hydrochloric acid (5 mL) was added to the reaction mixture, and ethyl acetate (10 mL) was added. The organic layer was washed with water (5 mL) and saturated brine (5 mL) in that order, and then extracted with anhydrous The residue was dried over sodium sulfate and the solvent was removed under reduced pressure. mL) and 3-azidopropan-1-amine (0.15 g) and O-(7-azidopropan-1-amine (benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexahydrate Then, N,N-diisopropyl phosphate (0.57 g) was added to the mixture under ice-cooling and stirring. To the reaction mixture was added propylethylamine (0.52 mL), and the mixture was stirred at room temperature for 5 hours. (10 mL) was added, and the mixture was extracted three times with ethyl acetate (15 mL). The organic layer was then washed with anhydrous sodium sulfate. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography. Compound 1 was purified by filtration (16% ethyl acetate / n-heptane to 100% ethyl acetate). A fraction containing 8-3 (0.71 g) was obtained.

[0507] The fraction containing compound 18-3 (0.71 g) was treated with 4N-HCl / 1,4-dihydrochloride. Xanthan Gum (4.9 mL) was added and the mixture was stirred at room temperature for 20 minutes. After adding ether, the precipitate was filtered to obtain the title compound 18-4 (0.49 g) as a white solid. Obtained as a solid.

[0508] <Process 3> 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group-introduced alginate Synthesis of acid (compound EX18-A2): [ka]

[0509] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (1 9.6 mL) and 4-(4,6-dimethoxy-1,3,5-triazine- 2-yl)-4-methylmorpholinium chloride (DMT-MM) (50.19 mg) Compound 18-4 (54.37 mg) obtained in (Example 18) <Step 2>, 1 molar Using sodium bicarbonate solution (181.4 μL), the same procedure as in (Example 11) <Step 3-1> was carried out. The title compound EX18-A2 (198 mg) was obtained as a white solid.

[0510] Example 19 3-Amino-1-(11,12-didehydrodibenz[b,f]azocine-5(6H)- Synthesis of (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.) (4 3.6 mL) with 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), commercially available 3-amino-1-(11,12-didehydrodibenzyl) Benz[b,f]azocin-5(6H)-yl-1-propanone [CAS REGIS TRY NO.: 1255942-06-3] (19-1, 83.6 mg) was used. The same procedure as in Example 11, <Step 3-1> was carried out to obtain the title compound EX19-A2 (376 mg) was obtained as a pale yellow solid.

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

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

[0514] Methods known in the literature (Bioorganic & Medicinal Chemistry) y(2003)11:4189-4206) was synthesized based on the tert-butyl (4-( Aminoethyl)benzyl)carbamate (20-1, 0.67 g), triethylamine A mixture of 100 mL of ethanol (0.39 mL) and methanol (6.67 mL) was added to the flask under ice-cooling and stirring. Ethyl trifluoroacetate (0.44 mL) was added dropwise, and the reaction mixture was allowed to warm to room temperature. The mixture was stirred at rt for 5 h. The reaction was quenched with water (10 mL) and extracted three times with ethyl acetate (10 mL). The collected organic layer was washed with saturated brine (5 mL) and dried over anhydrous sodium sulfate. The dried organic layer was filtered and concentrated to give crude compound 20-2 (0.67 g). It was obtained as a pale yellow amorphous substance.

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

[0516] (Example 20) 1,4-dioxo-2 of Compound 20-2 (0.5 g) obtained in <Step 1> To the San solution (3.5 mL), 4N hydrogen chloride / 1,4-dioxane was added under stirring in water. (3.5 mL) was added and stirred at room temperature for 3 hours. After adding 100 mL of HCl, the precipitate was filtered to give the title compound 20-3 (0.4 g). Obtained as a white solid.

[0517] <Process 3> N-(4-((2-(cyclooct-2-yn-1-yloxy)acetamido)methyl)benzyl Synthesis of benzyl-2,2,2-trifluoroacetamide (compound 20-4): [ka]

[0518] A method known in the literature (Org. Process Res. Dev. (2018) 22: The carboxylic acid (7-4, 0.17 g) and O-(7-a)- (benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexahydrate To a solution of 0.26 g of tetrafluorophosphate in 1.7 mL of acetonitrile, Compound 20-3 (0.26 g) obtained in Step 2 of Example 20 and N ,N-Diisopropylethylamine (0.51 mL) was added dropwise and stirred at room temperature for 1 hour and 30 minutes. The reaction was stopped by adding water (5 mL), and the mixture was extracted three times with ethyl acetate (5 mL). The organic layer was washed with saturated brine (3 mL) and then dried over anhydrous sodium sulfate. The dried organic layer was filtered, and the solvent was removed under reduced pressure. Purification was performed by chromatography (12% ethyl acetate / n-heptane to 100% ethyl acetate). The title compound 20-4 (0.19 g) was obtained as a white amorphous substance.

[0519] <Step 4> N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy) ) Synthesis of acetamide (compound 20-5): [ka]

[0520] (Example 20) Compound 20-4 (0.18 g) obtained in <Step 3> and methanol (1.8 mL) was added to an aqueous solution of potassium carbonate (0.13 g) under ice-cooling and stirring. (0.9 mL) was added dropwise, and the mixture was stirred at room temperature for 17 hours and 30 minutes. Methanol was evaporated under reduced pressure. The organic layer was washed with saturated brine (5 mL) and extracted three times with ethyl acetate (5 mL). The organic layer was filtered, and the solvent was removed under reduced pressure to obtain the crude product. Compound 20-5 (0.13 g) was obtained as a pale yellow oil.

[0521] <Process 5> N-(4-(aminoethyl)benzyl)-2-(cyclooct-2-yn-1-yl) (i) Synthesis of acetamide group-introduced alginic acid (EX20-B2): [ka]

[0522] A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (5 0.9 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4 -methylmorpholinium chloride (DMT-MM) (0.12 g), (Example 20) Using a solution of compound 20-5 (35 mg) obtained in step 4 in ethanol (3 mL), The same procedure as in (Example 11) <Step 3-1> was carried out to obtain the title compound EX20-B2 (52 1 mg) was obtained as a white solid.

[0523] (Examples P1 to P7) The alginic acid derivatives of (Example P1) to (Example P7) shown in the table below were used in the above examples. According to the method shown, the corresponding amino compound (a pharmaceutically acceptable salt thereof, or It is prepared using a compound (which may be 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 is determined by the ratio of the uronic acid unit, which is the repeating unit of alginic acid, to the total number of alginic acid units. It means the number of reactive groups or complementary reactive groups introduced per sugar unit expressed as a percentage. do. In this example, the introduction rate (mol%) of the reactive group or the complementary reactive group is 1 HN The amount of alginic acid required to calculate the introduction rate was calculated using the calibration curve. The amount of reactive groups or complementary reactive groups was measured by the carbazole-sulfuric acid method using a calibration curve. It can also be measured by absorbance measurement using the method.

[0528] [Molecular weight measurement] The alginic acid solid into which the reactive group obtained in the examples or the complementary reactive group was introduced was added in an amount of 0.1 Dissolved in 10 mmol / L phosphate buffer (pH 7.4) containing 5 mol / L NaCl Prepare a 0.1% or 0.2% solution and filter it through a polyethersulfone filter with a pore size of 0.22 μm. Filter (Minisart High Flow Filter, Sartoriu After removing insoluble matter, the sample was used for gel filtration. The concentration of each compound was measured using a spectrophotometer DU-800 (Beckman-Coulter). The measurement wavelengths for the compounds in gel filtration were determined. A differential refractometer was used.

[0529] Add 200 μL of the sample for gel filtration to Superose 6 Increase 10 / 30 The gel filtration was performed using a 0.0 GL column (GE Healthcare Sciences). The AKTA Explorer 10S was used as the apparatus, and 0.15 mol / L was used as the developing solvent. A 10 mmol / L phosphate buffer solution (pH 7.4) containing 10 mmol / L NaCl was used at room temperature with a flow rate of 100 µL / L. The elution profile of each sample was determined for each compound. The chromatogram was created by monitoring the absorption of the selected wavelengths. The data was analyzed using 31 software (GE Healthcare Sciences) to determine the peak range.

[0530] The molecular weight of alginate to which reactive groups or complementary reactive groups have been introduced is determined by Blue Dextrose. Thyroglobulin (molecular weight 669,000 Da, SIGMA) GE Healthcare Science) Ferritin (molecular weight 440,000 Da, GE Healthcare Science) Aldolase (molecular weight 158,000 Da, GE Healthcare Sciences), Conal Ovalbumin (molecular weight 75,000 Da, GE Healthcare Sciences), ovalbumin (molecular weight 44,000 Da, GE Healthcare Sciences), ribonuclease A (molecular weight 13,700 Da, GE Healthcare Sciences) and aprotinin (molecular weight 6500 Da, GE Healthcare (Scare Science Co., Ltd.) was used as a standard product, and reactive groups or complementary reactive groups were introduced. Gel filtration was performed under the same conditions as for alginic acid, and the elution volume of each component was calculated using Unicorn software. The elution volume of each component was plotted on the horizontal axis and the logarithmic value of the molecular weight on the vertical axis. The calibration curve was created by linear regression of blue dextran to ferritin. We created two types ranging from ferritin to aprotinin.

[0531] Using this calibration curve, the molecular weight (Mi ) was calculated. Next, the absorbance at elution time i was read and taken as Hi. The weight average molecular weight (Mw) was calculated from the data using the following formula.

[0532]

number

[0533] [Gel stability measurement] (Gel stability measurement (1)): Stability in PBS (Ex1-A2), (Ex4-A2), (Ex Each of the alginic acid derivatives (Ex19-A2) and (Ex19-A2) was dissolved in water to a concentration of 1.0%. and dissolved them in alginic acid aqueous solutions (1-1), (4-1), (5-1), and (19-1) ) were obtained. These were respectively (1-1) and (19-1), (4-1) and (19-1), Equal amounts of (5-1) and (19-1) were mixed and injected into an 18-gauge syringe. The syringe was placed in a syringe pump set at a flow rate of 1 mL / min. Add dropwise to 30 mmol / L calcium chloride solution for 30 seconds and stir for 5 minutes to form alginate. The gel was washed once with 10 mL of PBS and then soaked in PBS at 37°C for 10 minutes. The mixture was left to stand to undergo chemical cross-linking, yielding a chemically cross-linked alginate gel. The cross-linked alginate gel (beads) prepared in 19-A2) was also prepared in the same manner. 19.5 mL of PBS was added to the gel, and the mixture was shaken at 37°C, and the aqueous solution was collected over time. After the test, the test solution was replenished with the same amount of PBS as the collected amount. Add 10 μL of Nippon Gene (319-08261) and shake at 37°C for at least 3 hours. The gel was completely broken down by stirring, and the aqueous solution was recovered. The amount of alginic acid dissolved at each time point was measured by the Bazol sulfuric acid method, and the alginic acid concentration at all times was calculated. The value was divided by the total amount of alginic acid calculated from the alginic acid concentration at the end of the test and expressed as a percentage. The value shown was taken as the disintegration rate and was used as an index of gel stability.

[0534] The results shown in Figure 1 were obtained. The cross-linked alginate gel (beads) remained stable even after 96 hours. The gel did not collapse, confirming its stability. By forming the bead structure, the structure can be maintained for a long period of time. It was suggested that the cross-linked alginate prepared by (Ex18-A2) / (Ex19-A2) Phosphoric acid gel (beads) served as the control in this study.

[0535] (Gel stability measurement (2)): Stability under EDTA The alginic acid aqueous solution obtained in (Gel Stability Measurement (1)) was used as (1-1) and (19 -1), (4-1) and (19-1), (5-1) and (19-1) in equal amounts. The syringe was then fitted with an 18-gauge needle and the flow rate set to 1 mL / min. The syringe was placed in a syringe pump and immersed in a 30 mmol / L calcium chloride solution for 30 seconds. The mixture was added dropwise and stirred for 5 minutes to obtain an alginate gel. The cross-linked alginate gel (beads) prepared in 2) was also prepared in the same manner. Wash once with 10 mL of saline, then leave in saline at 37°C for 10 minutes to crosslink. The crosslinking was performed to obtain a chemically crosslinked alginate gel. Dipotassium EDTA dihydrate (EDTA·2K) / physiological saline was added, and the The mixture was shaken at 5°C and the aqueous solution was collected over time. The same amount of 5 mM EDTA 2K was added. After the test, the test solution was replenished with saline. Add 10 μL of the HCl solution (319-08261) and shake at 37°C for at least 3 hours to completely disintegrate the gel. The alginic acid concentration in the collected aqueous solution was determined by the carbazole sulfuric acid method. The amount of alginic acid dissolved at each time point was measured, and the alginic acid concentration at all time points and at the end of the test were The value calculated from the alginic acid concentration after the treatment is divided by the total amount of alginic acid, and the value expressed as a percentage is the disintegration rate. and was used as an index of gel stability.

[0536] The results are shown in Figure 2. The cross-linked alginate gel (beads) showed The disintegration rate was 2% or less. This means that cross-linking was formed by the Huisgen reaction. By doing so, the fabricated structure is in a solution without calcium ions (a physiological solution for the living body). It was suggested that the structure is maintained even under conditions of (Ex18-A2) / (E The cross-linked alginate gel (beads) prepared by the method (x19-A2) was used as a control in this study. be.

[0537] (Gel stability measurement (3)): Stability in PBS (Ex3-A2), (Ex 5-A2), (Ex6-A2), (Ex9a-A2), (Ex10-A2), (Ex12-A 2) and (Ex18-A2) were dissolved in water to a concentration of 1.0%. The resulting solutions were alginic acid solutions (3-1), (5-1), (6-1), (9-1), and ( 10-1), (12-1), and (18-1). These are then divided into (6-1) and (9 -1), (3-1) and (10-1), (5-1) and (10-1), (18-1) and (12 -1) and mix them in equal amounts, place them in a syringe equipped with an 18-gauge needle, and inject this mixture. The syringe was placed on a syringe pump set at a flow rate of 1 mL / min and a concentration of 30 mmol / L. The solution was added dropwise to the calcium chloride solution for 30 seconds and stirred for 5 minutes to obtain an alginate gel. The tube was washed once with 10 mL of PBS and then left to stand in PBS at 37°C for 10 minutes to allow chemical cross-linking. Chemically crosslinked alginate gels were obtained. A cross-linked alginate gel (beads) was also prepared in the same manner. 1 mL of PBS was added, and the mixture was shaken at 37°C. The aqueous solution was collected over time, and the same amount as the collected amount was After the test, the test solution was supplemented with alginate lyase (Nippon Gene, 3 Add 10 μL of 19-08261) and shake at 37°C for more than 3 hours to completely disintegrate the gel. The concentration of alginic acid in the collected aqueous solution was determined by the carbazole sulfuric acid method. The amount of alginic acid dissolved at each time point was measured, and the alginic acid concentration at all time points and after the test were The value obtained by dividing the total amount of alginic acid calculated from the alginic acid concentration in the sample by the total amount of alginic acid was expressed as a percentage and used as the disintegration rate. This was used as an index of gel stability.

[0538] The results shown in Figure 3 were obtained. The cross-linked alginate gel (beads) remained stable even after 96 hours. The gel did not collapse, confirming its stability. By forming the bead structure, the structure can be maintained for a long period of time. It was suggested that the cross-linked polymer prepared by (Ex18-A2) / (Ex19-A2) Alginate gel (beads) served as the control in this study.

[0539] (Gel stability measurement (4)): Stability under EDTA The alginic acid aqueous solution obtained in (Measurement of gel stability (3)) was used as (6-1) and (9- 1), (3-1) and (10-1), (5-1) and (10-1), (18-1) and (12- Mix equal amounts of the combination of 1) and place in a syringe equipped with an 18-gauge needle. The tube was placed on a syringe pump set at a flow rate of 1 mL / min, and a concentration of 30 mmol / L was added. The solution was added dropwise to calcium chloride solution for 30 seconds and stirred for 5 minutes to obtain an alginate gel. The tissue was washed once with 10 mL of saline, and then left to stand in saline at 37°C for 10 minutes. Crosslinking was performed to obtain chemically crosslinked alginate gels. (Ex18-A2) / (Ex19-A2) The cross-linked alginate gel (beads) prepared in the same manner as above was also prepared. 0.5 mL of 5 mM ethylenediaminetetraacetic acid dipotassium salt dihydrate (EDTA·2K) Physiological saline was added, and the mixture was shaken at 37°C. The aqueous solution was collected over time, and the same amount as the collected amount was collected. After the test, the test solution was replenished with 5 mM EDTA·2K / physiological saline. Add 10 μL of acid lyase (Nippon Gene, 319-08261) and incubate at 37°C for 3 hours. The gel was completely broken down by further shaking, and the aqueous solution was recovered. The concentration was measured by the carbazole-sulfuric acid method, and the amount of alginic acid dissolved up to each time point was calculated as the total amount of alginic acid dissolved at all time points. The value calculated from the alginic acid concentration and the alginic acid concentration after the test divided by the total amount of alginic acid The percentage of disintegration was used as an index of gel stability.

[0540] The results are shown in Figure 4. The cross-linked alginate gel (beads) showed The disintegration rate was 4% or less. This means that cross-linking was formed by the Huisgen reaction. By doing so, the fabricated structure is in a solution without calcium ions (a physiological solution for the living body). It was suggested that the structure is maintained even under conditions of low or high concentrations. The cross-linked alginate gel (beads) prepared in -A2 served as the control for this study.

[0541] (Gel stability measurement (5)): Stability in PBS (Ex4-A2), (EX9a-A2) obtained in Examples 4, 9, 16, 18 and 20 ), (Ex16-A2), (Ex18-A2) and (Ex20-B2) alginate-induced The body was dissolved in water to a concentration of 1.0% and prepared into aqueous solutions of alginic acid (4-1) and ( 9-1), (16-1), (18-1), and (20-1). -1) and (20-1), (18-1) and (9-1), (18-1) and (16-1) Mix equal amounts of the two together and place them in a syringe equipped with an 18-gauge needle. A syringe pump set to mL / min was used to deliver a solution of calcium chloride at a concentration of 30 mmol / L. The solution was added dropwise for 30 seconds and stirred for 5 minutes to obtain an alginate gel. Wash once with 1 L of PBS, then leave in PBS at 37°C for 10 minutes to perform chemical crosslinking. Cross-linked alginate gels were obtained. The cellulose gel (beads) was prepared in the same manner. 19.5 mL of PBS was added to the gel. The mixture was shaken at 37°C, and the aqueous solution was collected over time. The same amount of PBS was added to the collected solution. After the test, the test solution was diluted with alginate lyase (Nippon Gene, 319-082 61) was added to the solution, and the mixture was shaken at 37°C for 3 hours or more to completely disintegrate the gel. The alginic acid concentration in the collected aqueous solution was measured by the carbazole sulfuric acid method. The amount of alginic acid dissolved up to the test point was compared with the alginic acid concentration at all time points and the alginic acid concentration after the test was completed. The value calculated from the gel strength was divided by the total amount of alginic acid, and the value expressed as a percentage was used as the disintegration rate. was used as an indicator.

[0542] The results are shown in Figure 5. The cross-linked alginate prepared by the above method remained intact even after 96 hours. The disintegration rate was approximately 12% or less. This means that chemical cross-linking was formed by the Huisgen reaction. This suggests that the structure of the fabricated (bead) structure is maintained. The cross-linked alginate gel (beads) made with Ex18-A2 / Ex19-A2 was This is the control for the study.

[0543] (Gel stability measurement (6)): Stability under EDTA The alginic acid aqueous solution obtained in (Measurement of gel stability (5)) was used as (4-1) and (20 -1), (18-1) and (9-1), (18-1) and (16-1) in equal amounts. The solution was mixed with the solution and placed in a syringe fitted with an 18-gauge needle. The syringe was set to a flow rate of 1 mL / min. The syringe was placed in a syringe pump with a fixed concentration of 30 mmol / L and 30 The alginate gel was obtained by adding 10 mL of saline solution to the solution. The cells were washed once with saline, and then left to stand in physiological saline at 37°C for 10 minutes to perform chemical crosslinking. Cross-linked alginate gels were obtained using (Ex18-A2) / (Ex19-A2). Acid gel (beads) was prepared in the same manner. 19.5 mL of 5 mM ethanol was added to this gel. Dipotassium dihydrate diamine tetraacetic acid (EDTA·2K) / physiological saline was added, and the The mixture was shaken at 7°C and the aqueous solution was collected over time. The same amount of 5 mM EDTA 2 After the test, the test solution was supplemented with alginate lyase (Nippon G Add 10 μL of ethanol (319-08261) and shake at 37°C for at least 3 hours to completely dissolve the gel. The alginic acid concentration in the collected aqueous solution was measured using the carbazole sulfuric acid method. The amount of alginic acid dissolved at each time point was measured, and the alginic acid concentration at all time points and at the end of the test were The value calculated from the alginic acid concentration after the treatment was divided by the total amount of alginic acid, and the value expressed as a percentage was used as the decay rate. The rate was used as an index of gel stability.

[0544] The results shown in Figure 6 were obtained. After 24 hours, the cross-linked alginate gel (beads) The disintegration rate was 18% or less. In other words, cross-linking by the Huisgen reaction did not occur. By doing so, the fabricated structure is in a solution without calcium ions (a biological solution for the living body). It was suggested that the structure is maintained even under conditions of (subphysical concentration). Cross-linked alginate gel (beads) prepared by 19-A2 served as a control for this study. .

[0545] [Gel permeability measurement] (Gel Permeability Measurement (1)) (Ex1-A2), (Ex3-A2), (Ex4-A3), (Ex5-A4), (Ex6-A5), (Ex7-A6), (Ex8-A7), (Ex9-A8), (Ex10-A9), (Ex11-A10), (Ex12-A11), (Ex13-A12), (Ex14-A13), (Ex15-A14), (Ex16-A15), (Ex17-A16), Each alginic acid derivative (Ex4-A2), (Ex5-A2) and (Ex18-A2) was added at a concentration of The alginic acid solution was prepared by dissolving it in water to a concentration of 2.0%. Fluorescein isothiocyanate with a molecular weight of 150,000, prepared at 1 mg / mL in 2 / 5 volumes Add iso-dextran (Sigma-Aldrich, FD150S) and 3 / 5 volume of water. 0.2 mg / mL fluorescein isothiocyanate-dextran containing 1.0% aluminum Aqueous solutions of acetic acid (1-2), (3-2), (4-2), (5-2), and (18-2) were obtained.

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

[0547] These are (1-2) and (19-1), (4-2) and (19-1), (5-2) ) and (19-1), (3-2) and (10-1), (18-2) and (12-1) Mix equal amounts of the two together, add 40 mL of 30 mmol / L calcium chloride solution, After stirring for 5 minutes, an alginate gel was obtained. The gel was washed once with 10 mL of physiological saline. The cells were then left to stand in saline at 37°C for 10 minutes to perform chemical crosslinking. Chemically cross-linked alginate gels containing anhydrous dextran were obtained. (Ex18-A2) / (E Fluorescein isothiocyanate-dextran inclusion chemistry prepared with x19-A2 Cross-linked alginate gel was also prepared in the same manner. 19.5 mL of saline was added to this gel. The mixture was shaken at 37°C and the aqueous solution was collected over time. The same amount of physiological saline was added to the collected solution. After the test, alginate lyase (Nippon Gene, 319-0 Add 10 μL of 8261) and shake at 37°C for 3 hours or more to completely disintegrate the gel. The solution was collected. The dextran concentration in the collected aqueous solution was determined by fluorometric quantification (excitation light: 485nm). The amount of dextran measured at each time point was the total amount of dextran measured at the end of the test. The value divided by the amount of xanthan was expressed as a percentage and used 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%. Fluorescein isothiocyanate prepared by (Ex18-A2) / (Ex19-A2) The hydroxydextran-encapsulated chemically cross-linked alginate gel served as a control in this study.

[0549] (Gel permeability measurement (2)) (Ex4-A2), (Ex5-A2), (Ex Each of the alginic acid derivatives (Ex18-A2) and (Ex18-A2) was dissolved in water to a concentration of 2.0%. To this alginic acid solution, add 2 / 5 of the volume of 1 mg / Fluorescein isothiocyanate-dextran (Sig) with a molecular weight of 150,000 was prepared in mL. Add 0.2 mg / mL full-strength HCl (Aldrich, FD150S) and 3 / 5 volume of water. Olescein isothiocyanate-dextran containing 1.0% alginate solution (4-2) , (5-2), (6-2), (18-2).

[0550] Furthermore, (EX9a-A2), (Ex10-A3), (EX11-A4), (EX12-A5), (EX13-A6), (EX14-A7), (EX15-A8), (EX16-A9), (EX17-A10), (EX18-A11), (EX19-A22), (EX19-A33), (EX19-A44), (EX19-A12), (EX19-A1 A2), (Ex16-A2) and (Ex20-B2) alginic acid derivatives were added at a concentration of 1. 0% to obtain aqueous solutions of alginic acid (9-1), (10-1), and (1 6-1), (20-1).

[0551] These are (4-2) and (20-1), (5-2) and (10-1), (6-2) ) and (9-1), (18-2) and (9-1), (18-2) and (16-1) Mix equal amounts of 100 ml of 100 ml of 100 ml of calcium chloride solution at a concentration of 30 mmol / L, add 40 mL of 100 ml of 100 ml of calcium chloride solution at a concentration of 50 mmol / L, and After stirring for 1 minute, an alginate gel was obtained. The gel was washed once with 10 mL of physiological saline. Chemical crosslinking was carried out by leaving the membrane in physiological saline at 37°C for 10 minutes, and fluorescein isothiocyanate was added. Chemically cross-linked alginate gels containing hydroxybenzoates and dextran were obtained. Fluorescein isothiocyanate-dextran encapsulated chemical bridge prepared by 19-A2) A bridge alginate gel was prepared in the same manner. 19.5 mL of saline was added to this gel. The mixture was shaken at 37°C, and the aqueous solution was collected over time. The same amount of physiological saline was added. After the test, alginate lyase (Nippon Gene, 319-08 261) was added, and the mixture was shaken at 37°C for more than 3 hours to completely disintegrate the gel and dissolve it in water. The dextran concentration in the collected aqueous solution was determined by fluorometric quantification (excitation light: 485 nm The amount of dextran up to each time point was measured using a fluorescent microscope (fluorescence: 535 nm), and the total amount of dextran at the end of the test was calculated. The value divided by the amount of strand was expressed as a percentage and used as the transmittance.

[0552] The results shown in Figure 8 were obtained. The transmittance after 24 hours was in the range of 25% to 30%. Fluorescein isothiocyanate-dextrin prepared with 18-A2 / Ex19-A2 Orchid-encapsulated chemically cross-linked alginate gel served as a control in this study.

[0553] [Biocompatibility evaluation of cross-linked alginate derivatives (gels)] (EX4-A2), (EX4-A3), (EX4-A4), (EX4-A5), (EX4-A6), (EX4-A7), (EX4-A8), (EX4-A9), (EX4-B10), (EX4-B11), (EX4-B12), (EX4-B13), (EX4-B14), (EX4-B15), (EX4-B16), (EX4-B17), (EX4-B18 EX5-A2), (EX12-A2), (EX16-A2), (EX18-A2), (E Each alginic acid derivative (X19-A2) and (EX20-B2) was dissolved in water to form a reactive group. This was used as the alginate solution for introduction. After sterilization by filtration using a filter (UK), a 1.0% reactive group-introduced alginate / physiological saline solution was prepared. . Cell concentration 5×10 3 Cells were seeded onto a 96-well plate at 100 cells / well. After 1 day of incubation, HeLa cells were incubated with 1.0% reactive group-introduced alginate / physiological saline. The aqueous solutions were (EX18-A2) and (EX19-A2), (Ex5-A2) and (Ex19-A 2), (Ex4-A2) and (Ex20-B2), (Ex18-A2) and (Ex12-A2 ) or (Ex16-A2) in combination to a final concentration of 0.1%, and cultured for 1 day. After incubation, ATP activity was measured using CellTiter-Glo Lumines as an indicator of cytotoxicity. cent Cell Viability Assay (Promega, G7571) was evaluated.

[0554] The results shown in Figure 9 were obtained. In all cross-linked alginate gels evaluated by the above method, AT The P activity was confirmed, suggesting that the cross-linked alginate gel is not cytotoxic. The alginate structure (beads) formed by chemical cross-linking through the Huisgen reaction is biocompatible. It was suggested that it was compatible.

Claims

1. 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): Alginic acid derivatives and alginic acid derivatives having an amide bond and a bivalent bond at any one or more carboxyl groups of the alginic acid The linker (-L ２ -), an azide group is introduced via the following formula (II): Cross-linked alginic acid obtained by cross-linking reaction using alginic acid derivatives: [Alginic acid derivative represented by formula (I)] The following formula (I): 【Chemical 164】 [In formula (I), (ALG) represents alginic acid; -NHCO- represents any one of alginic acid. represents an amide bond via a carboxyl group; -L １ - is the table below: 【Table 51-1】 【Table 51-2】 [Each formula does not include the areas outside the dashed lines at both ends] represents a linker; Akn is shown in the table below: Table 52 [In each formula, the right side of the dashed line is not included] an alginic acid derivative represented by the formula (I) wherein R represents an alkyne group; [Alginic acid derivative represented by formula (II)] The following formula (II): 【Chemistry 165】 (In formula (II), (ALG) represents alginic acid; -NHCO- represents any of the groups of alginic acid. represents an amide bond via a carboxyl group; -L ２ - is the table below: 【Table 53-1】 【Table 53-2】 [Each formula does not include the areas outside the dashed lines at both ends] The alginic acid derivative represented by the formula (I) represents a linker In phosphoric acid derivatives, -L １ - is (L1-1), (L1-2a), (L1-2b), (L1 and a derivative which is any one linker selected from the group consisting of (L1-11) or (L1-12). In the alginic acid derivative represented by formula (II), -L ２ - is the linker of (L2-10) (Excluding cross-linked alginic acid obtained by cross-linking using a derivative of alginic acid.)

2. 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): 【166】 [In formula (I), (ALG) represents alginic acid; -NHCO- represents any one of alginic acid. represents an amide bond via a carboxyl group; -L １ - is the table below: 【Table 54-1】 【Table 54-2】 [Each formula does not include the areas outside the dashed lines at both ends] represents a linker; Akn is shown in the table below: Table 55 [In each formula, the right side of the dashed line is not included] An alginic acid derivative represented by the formula: wherein R represents an alkyne group.

3. Akn-L １ -NH ２ Groups (Akn, and -L １ - is the same as the definition in claim 2 The alginic acid derivative of formula (I) according to claim 2, wherein the introduction rate of body.

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

5. 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): 【167】 [In formula (II), (ALG) represents alginic acid; -NHCO- represents any of the groups of alginic acid. represents an amide bond via any carboxyl group; -L ２ - is the table below: 【Table 56-1】 【Table 56-2】 [Each formula does not include the areas outside the dashed lines at both ends] The alginic acid derivative is represented by the formula (I), wherein R represents a linker.

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

7. The weight average molecular weight of the alginic acid derivative measured by gel filtration chromatography is: The alginic acid derivative of formula (II) according to claim 5, which has a molecular weight of 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 The group is represented by the following formula (III-L): 【168】 [In formula (III-L), -CONH- and -NHCO- at both ends represent any carbon atom of alginic acid. represents an amide bond via a carboxyl group; -L １ - is as defined in claim 1; -L ２ - is as defined in claim 1; X is as shown in the table below: 【Table 57-1】 【Table 57-2】 (In each formula, the areas outside the dashed lines at both ends are included.) The cross-linked alginic acid of claim 1 (wherein the formula (III-L) is not included) is bound via a cross-linked alginic acid. ) in which -L １ - is (L1-1), (L1-2a), (L1-2b), (L1-11 ) or (L1-12), the corresponding -L ２ - (excluding the linker (L2-10)).

9. The alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to claim 1 By mixing the cellulose acetate copolymer with an alginic acid derivative and carrying out a crosslinking reaction (Huisgen reaction), the cellulose acetate copolymer can be obtained as claimed in claim 1.

10. A method for producing cross-linked alginate, comprising obtaining the cross-linked alginate according to claim 1.

10. The alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to claim 1 The chemical crosslink formed by the Huisgen reaction of an alginic acid derivative is represented by the following formula: (III-L): 【169】 [In formula (III-L), -CONH- and -NHCO- at both ends, X, -L １ -,and- L ２ The crosslinked alginate of claim 1, wherein the crosslinked alginate has the same structure as in claim 8. A method for producing phosphoric acid.

11. The alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to claim 1 The mixed solution of the alginic acid derivatives was mixed with a solution containing divalent metal ions. Cross-linked alginate structure obtained by dropping into

12. Chemical crosslinking with triazole rings formed by the Huisgen reaction as crosslinks, and The crosslinked adhesive of claim 11, comprising ionic crosslinks formed in part by divalent metal ions. Luggic acid structure.

13. The alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to claim 1 Alginic acid derivatives were ionically crosslinked with divalent metal ions and chemically synthesized by the Huisgen reaction. A cross-linked alginate structure obtained by cross-linking, which has the ability to retain contents.

14. The alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to claim 1 Chemical crosslinking formed by the Huisgen reaction using alginic acid derivatives , the following formula (III-L): 【Chemistry 170】 [In formula (III-L), -CONH- and -NHCO- at both ends, X, -L １ -,and- L ２ - is the same as defined in claim 8]. The crosslinked alginate structure according to any one of claims 1 to 10.

15. The alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to claim 1 The mixed solution of the alginic acid derivatives was mixed with a solution containing divalent metal ions. and subjecting the mixture to a crosslinking reaction to produce a crosslinked alginate structure. Law.

16. Chemical crosslinking with triazole rings formed by the Huisgen reaction as crosslinks, and 16. The crosslinked adhesive of claim 15, comprising ionic crosslinks formed in part by divalent metal ions. Method for producing glutinic acid structures.

17. The alginic acid derivative represented by formula (I) and the alginic acid derivative represented by formula (II) according to claim 1 Chemical crosslinking formed by the Huisgen reaction using alginic acid derivatives , the following formula (III-L): 【171】 [In formula (III-L), -CONH- and -NHCO- at both ends, X, -L １ -,and- L ２ - is as defined in claim 8]. Method for producing crosslinked alginate structures.

18. The crosslinked adhesive according to any one of claims 11 to 14, which is a bead or a roughly spherical gel. Luggic acid structure.

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. The cross-linked alginate of claim 1, claim 2 or claim 5, which is biocompatible. An alginic acid derivative or a crosslinked alginic acid structure according to any one of claims 11 to 14. Structure.

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