Novel cross-linked alginate

Abstract

To provide a novel alginic acid derivative or a novel crosslinked alginic acid, and production methods therefor.SOLUTION: The present invention provides alginic acid derivatives represented by formula (I) and formula (II), and a novel crosslinked alginic acid obtained through a Huisgen reaction using the alginic acid derivative of formula (I) and the alginic acid derivative of formula (II). This can provide a novel alginic acid derivative and a novel crosslinked alginic acid.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a novel alginic acid derivative, a novel crosslinked alginic acid, and a method for producing the same. Regarding. [Background technology]

[0002] Alginic acid is used in Lessonia, Macrocystis, Laminaria, Ascophyllum, and Derby A polymeric acidic polysaccharide extracted from the cell walls of natural brown algae such as sculpin, sculpin, Eisenia bicolor, and kelp. The molecule is composed 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) in which the two are bonded 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. Glycic acid is 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, etc.) are water-soluble, but divalent salts of alkaline earth metal alginates (e.g., alginic acid Calcium, etc.) have the property of being cross-linked by metal ions and becoming gelled (insoluble). Attempts have been made to utilize these properties to modify or mold them into materials suitable for various applications.

[0004] Polysaccharides (e.g., hyaluronic acid, chondroitin sulfate, alginic acid, etc.) for various materials To explore the possibility of modifying or shaping it 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 crosslinked polysaccharides include: (1) aldehyde crosslinking with formaldehyde or the like; A crosslinking method using a crosslinking 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. 1999 / 023636) (3) Homo-bifunctional crosslinking agents (diepoxides, divinyl sulfones, diamines, or dihydrazides) or heterobifunctional crosslinkers (epihalohydrides, etc.) A crosslinking method using phosphorus, etc. (Patent Document 3: International Publication No. 2009 / 073437) It is known that

[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 A crosslinking method in which the polymer is irradiated with light after introducing coumarin or the like (Patent Documents 4 and 5: International Patent Publication No. WO 2005 / 023363) Publication No. 2005 / 026214, JP-A-09-87236, and (5) A crosslinking method in which polysaccharides to which thiol groups have been introduced are crosslinked with each other via disulfide bonds and a thio The Michael addition reaction was carried out using polysaccharides with aryl groups and polysaccharides with maleimide groups. A crosslinking method by causing the polymer to undergo crosslinking (Patent Document 6: International Publication No. 2008 / 071058) etc. are known.

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

[0008] A crosslinked polysaccharide obtained by crosslinking polysaccharides by the Huisgen reaction is disclosed in (i) International Publication No. 2008 / 0 (ii) International Publication No. 2012 / 165462 (iii) International Publication No. 2015 / 020206 (iv) Chinese Patent Publication No. 106140040 ( This is disclosed in Patent Document 10, etc.

[0009] However, (i) Patent Document 7 discloses a method for preparing a first polysaccharide from hyaluronic acid and a second polysaccharide from chondroitin. As a polysaccharide selected from among glycerin, sulfated dermatan, alginic acid or a salt thereof, each polysaccharide is provided with a linker. The chain-like alkyne and azide groups introduced via the anchor were reacted with the Huisg The cross-linked polysaccharide obtained by the en reaction is a novel cross-linked alginic acid described below. has not been disclosed.

[0010] In addition, (ii) Patent Document 8 discloses a method for preparing a first polysaccharide and a second polysaccharide by mixing hyaluronic acid, carboxymethylcellulose, and the like. a polysaccharide selected from ethyl dextran, a cellulose derivative, and chitosan (the first polysaccharide and Each polysaccharide is provided with a linker (a linker between the polysaccharide and the second polysaccharide, which may be the same or different). The cyclic alkyne group and azide group were introduced via an ester bond (an ester linker) into Hui This is related to the cross-linked polysaccharide obtained by the sgen reaction, but the new cross-linked alkanol described below Glycic acid is not disclosed.

[0011] In addition, (iii) Patent Document 9 discloses a method for preparing a hyaluronic acid-based saccharide comprising the first polysaccharide and chondroitin. The cyclic alkyne group and azide group introduced into each polysaccharide via a linker were used as the carboxylate sulfate. This is related to the cross-linked polysaccharide obtained by the isgen reaction, but the new cross-linked Luggic acid is not disclosed.

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

[0013] (v) Patent Document 11 describes a method for derivatizing sugars by binding eight-membered cycloalkyne groups to the sugars. The present invention relates to a method for the preparation of sugars from unnatural sugars (Streptococcus agalactiae). Capsular saccharide derived from alginic acid, not alginic acid, and an 8-membered cycloalkyne group to be attached The terminal of the saccharide is not amide-bonded to the carboxyl group of the saccharide. The novel alginic acid derivative and its manufacturing method are different.

[0014] (vi) Non-Patent Document 1 discloses a branched alginate having a cyclooctyne side chain introduced into the side chain ( bAlg-DBCO) has been described, but it is a combination of alginate and branched polyethylene glycol (4-arm P Branched alginic acid (bAlg) synthesized from EG-NH2 was aminated This was obtained by reacting cyclooctyne (DBCO-PEG-amine) with the new arginine described below. It has a different structure from phosphate derivatives and has a different purpose of use. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] International Publication No. 2011 / 028031 Brochure [Patent Document 2] International Publication No. 1989 / 10941 Pamphlet [Patent Document 3] International Publication No. 2009 / 073437 Brochure [Patent Document 4] International Publication No. 2005 / 026214 Brochure [Patent Document 5] Japanese Patent Application Publication No. 09-87236 [Patent Document 6] International Publication No. 2008 / 071058 Brochure [Patent Document 7] International Publication No. 2008 / 031525 Brochure [Patent Document 8] International Publication No. 2012 / 165462 Brochure [Patent Document 9] International Publication No. 2015 / 020206 Brochure [Patent Document 10] China Patent Application Publication No. 106140040 [Patent Document 11] International Publication No. 2014 / 111344 Brochure [Non-patent literature]

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

[0017] In the above circumstances, there is a need for a novel alginic acid derivative or a novel crosslinked alginic acid. There was also a demand for a method for producing them. [Means for solving the problem]

[0018] As a result of intensive research to solve the above problems, the present inventors have discovered a compound represented by formula (I) or formula (I Furthermore, the present inventors have discovered novel alginic acid derivatives represented by the formula (I) and the formula (II). A novel cross-linked alginate derivative was 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, compared with conventional gels, have a higher permeability according to the purpose. The present invention has been completed based on the discovery 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 carried out, for example, by the Huisgen reaction (1,3-dipolar cycloaddition reaction). For example, the crosslinking reaction is carried out between alginic acid derivatives of formula (I) and formula (II). or, for example, between the alginic acid derivative of formula (I) and another molecule having an azide group. or by reacting an alginic acid derivative of formula (II) with another molecule having an alkyne group. It may be carried out between

[0021] The Huisgen reaction with terminal alkyne and azide groups generally proceeds at temperatures above 100°C. This reaction requires heating, making it unsuitable for chemical modification of biomolecules. However, by using a copper catalyst (e.g., Cu(I)) in the reaction, Reaction conditions were found under which the cycloadduct (triazole ring) was formed in almost 100% yield at room temperature. (Angew. Chem. Int. Ed. Engl., 14, p2596-2599 ,2002;J.Org.Chem.,9,p3057-3064,2002), raw On the other hand, the copper catalyst Huisg When cross-linked alginic acid is obtained by the en reaction, a trace amount of copper catalyst is added to the cross-linked alginic acid. There is a possibility that copper-derived alginate may remain in the cross-linked alginate or in the cross-linked alginate structure. There is concern that cytotoxicity may occur.

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

[0023] Here, any one or more carboxyl groups of the alginic acid shown in the following embodiment 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 an alginic acid derivative of formula (II) A novel bridged aryl group obtained by the Huisgen reaction (1,3-dipolar cycloaddition reaction) Methods for producing alginic acid, and each of said alginic acid derivatives and cross-linked alginic acid are provided. That is, exemplary embodiments may be as follows: [1] to

[17] .

[0024] [1] An amide bond and a divalent linker ( -L 1 A cyclic alkyne group (Akn) is introduced via a cyclic alkyne group (Akn) represented by the following formula (I): [ka] [In formula (I), (ALG), -L 1 -, Akn is defined as in the first embodiment described below. An alginic acid derivative represented by the formula:

[0025] [2] Akn-L 1 -NH2 group (Akn, and -L 1 - is defined in the first aspect below. The introduction rate of the compound represented by formula (I) described in [1] is 0.1% to 30%. An alginic acid derivative.

[0026] [3] Weight-average molecular weight of alginic acid derivatives measured by gel filtration chromatography The alginic acid represented by formula (I) according to [1] has a molecular weight of 100,000 Da to 3,000,000 Da. derivative.

[0027] [4] 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 fourth aspect described below. An alginic acid derivative represented by the formula:

[0028] [5] N3-L 2 -NH2 group(-L 2- has the same definition as in the fourth aspect described below. The introduction rate of the alginate represented by formula (II) according to [4] is 0.1% to 30%. Acid derivatives.

[0029] [6] Weight-average molecular weight of alginic acid derivatives measured by gel filtration chromatography The molecular weight of the alginate represented by the formula (II) in [4] is 100,000 Da to 3,000,000 Da. Acid derivatives.

[0030] [7] Any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid The syl group is represented by the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO- at both ends represent any carboxyl group of alginic acid. represents an amide bond via a carboxyl group; -L 1 -, -L 2 - and X are the following 7. Cross-linked alginate linked via the same definition as in embodiment 7.

[0031] [8] A method for producing an alginic acid derivative represented by formula (I) according to any one of [1] to [3] above, and a method for producing an alginic acid derivative represented by formula (I) according to any one of [1] to [3] above, [4] to [6], and the alginic acid derivative of formula (II) according to any one of [1] to [5] is mixed with the alginic acid derivative of formula (II) according to [1] to [5]. The cross-linked alginic acid according to the present invention is obtained by carrying out an sgen reaction. A method for producing arginic acid.

[0032] [8-1] Chemical bridges using triazole rings formed by the Huisgen reaction Cross-linked alginate, including bridges and ionic bridges formed in part by calcium ions .

[0033] [9] The alginic acid derivative of formula (I) according to any one of [1] to [3] above and the [4] to [6] are mixed with an alginic acid derivative of formula (II) A cross-linked alginate is obtained by dropping a mixed solution of phosphate derivatives into a calcium chloride solution. acid structure.

[0034]

[10] Chemical cross-linking using triazole rings formed by the Huisgen reaction and the ionic bridge partially formed by calcium ions, Cross-linked alginate constructs.

[0035]

[11] A method for producing an alginic acid derivative represented by formula (I) according to any one of [1] to [3] above, [4] to [6] are mixed with an alginic acid derivative of formula (II) The mixed solution of the phosphoric acid derivative is dropped into the calcium chloride solution to obtain the above [9] or

[10] . A method for producing a crosslinked alginate structure comprising obtaining the crosslinked alginate structure described above.

[0036]

[12] The crosslinked alginate according to [9] or

[10] above, which is in the form of beads or a roughly spherical gel. acid structure.

[0037]

[13] A medical material comprising the crosslinked alginate structure described in [9] or

[10] above.

[0038]

[14] The medical material according to

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

[0039]

[15] The alginic acid derivative according to any one of [1] to [6] above, which is biocompatible. The crosslinked alginic acid according to the above [7] or [8-1], and the above [9],

[10] and The crosslinked alginate structure according to any one of

[12] and

[13] .

[0040]

[16] The following formula (AM-1): [ka] [In formula (AM-1), -L 1 The definitions of - and Akn are the same as those in the 16th aspect described below. or a pharma- ceutically acceptable salt thereof, or Solvate.

[0041]

[17] The following formula (AM-2): [ka] [In formula (II), -L 2 The definition of - is the same as that in the seventeenth aspect described below. or a pharma- ceutically acceptable salt thereof, or a solvate thereof. Effect of the Invention

[0042] The present invention relates to novel alginic acid derivatives, novel We provide cross-linked alginic acid and other materials. Preferably, the alginic acid derivative is one in which a reactive group not present in a living body is introduced, and the unreacted group is Even if it remains, there is no risk of cross-linking reactions with biological components such as cells. It is expected to be safe. In addition, preferably, the crosslinking reaction uses 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 can be prepared by the Huisgen reaction (1,3-dipolar cycloaddition reaction). Chemical crosslinking and divalent cations using, for example, calcium ions It can be used in combination with crosslinking using metallic gold ions, and the reaction conditions can be adjusted. and preferably the stability of the non-crosslinked alginate or non-chemically crosslinked alginate (e.g. This is an improvement over cross-linked alginate (cross-linked with calcium ions). In addition, it is preferable that the gel properties of the crosslinked body can be adjusted, and the substance permeability can be adjusted. It is also possible. The present invention has at least one or more of these advantages. [Brief description of the drawings]

[0043] [Figure 1] FIG. 1 shows the evaluation of gel stability of cross-linked alginate structures. [Diagram 2] FIG. 1 shows the evaluation of the gel transmittance of a cross-linked alginate structure. [Diagram 3] FIG. 1 shows the evaluation of the gel transmittance of a cross-linked alginate structure. [Figure 4] FIG. 1 shows the evaluation of gel stability of cross-linked alginate structures. [Diagram 5] FIG. 1 shows the evaluation of the gel transmittance of a cross-linked alginate structure. [Figure 6] FIG. 1 shows the evaluation of the gel transmittance of a cross-linked alginate structure. [Figure 7] FIG. 1 shows an evaluation of the biocompatibility of a gel of a cross-linked alginic acid derivative. [Figure 8] FIG. 1 shows the evaluation of gel stability of cross-linked alginate structures. [Figure 9] FIG. 13. Evaluation of gel stability under EDTA of cross-linked alginate constructs. [Figure 10] FIG. 1 shows the evaluation of gel stability of cross-linked alginate structures. [Figure 11] FIG. 1 shows the evaluation of gel stability under cross-linked alginate construct EDTA. [Figure 12] FIG. 1 shows the evaluation of gel stability of cross-linked alginate structures. [Figure 13] Figure 1 shows the evaluation of gel stability under cross-linked alginate structure EDTA. [Figure 14] FIG. 1 shows the evaluation of the gel transmittance of a cross-linked alginate structure. [Figure 15] FIG. 1 shows the evaluation of the gel transmittance of a cross-linked alginate structure. [Figure 16] FIG. 1 shows the evaluation of the gel transmittance of a cross-linked alginate structure. [Figure 17] FIG. 1 shows an evaluation of the biocompatibility of a gel of a cross-linked alginic acid derivative. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] [Specific aspects] The following aspects [1] to

[17] may be included. [1] The first aspect is as follows: At least one carboxyl group of alginic acid is Amide bond and divalent linker (-L 1 -) to introduce a cyclic alkyne group (Akn). The compound represented by the following formula (I): [ka] [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 represented by the following partial structural formula: [Does not include the areas outside the wavy lines at either end]: [ka] represents a bivalent linker selected from the group consisting of: Akn has the following partial structural formula (each formula does not include the part to the right of the wavy line): [ka] wherein the asterisk represents a chiral center. Lungic acid derivatives.

[0045] [1-1] In the alginic acid derivative of the above-mentioned formula (I) of the above-mentioned aspect [1], -L 1 -I like Preferably, the compound has the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group More preferably, the compound has the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group More preferably, the compound has the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group consisting of:

[0046] [1-2] In the alginic acid derivative of the formula (I) according to the aspect [1], Akn is preferably represented by the following partial structural formula (in each formula, the part to the right of the wavy line is not included): [ka] is a cyclic alkyne group selected from the group consisting of: More preferably, the compound has the following partial structural formula (each formula does not include the part to the right of the wavy line): [ka] is a cyclic alkyne group selected from the group consisting of:

[0047] [1-3] In the alginic acid derivative of the formula (I) of the aspect [1], Akn and -L 1 The combination of - is preferably represented by the following partial structural formula: [Not Included]: [ka] [ka] is as represented by a group selected from the group More preferably, the compound has the following partial structural formula (in each formula, the right side of the wavy line (the imino group side) is not included): [ka] is as represented by a group selected from the group More preferably, the partial structural formula is as follows (each formula does not include the right side of the wavy line (the imino group side)): [ka] The group is as shown by a group selected from the following:

[0048] [1-1a] In the alginic acid derivative of the formula (I) according to the embodiment [1], -L 1 -teeth, Preferably, the compound has the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group More preferably, the compound has the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group More preferably, the compound has the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group consisting of:

[0049] [1-2a] In the alginic acid derivative of the formula (I) of the aspect [1], Akn is preferably Preferably, the compound has the following partial structural formula (each formula does not include the part to the right of the wavy line): [ka] is a cyclic alkyne group selected from the group consisting of: More preferably, the compound has the following partial structural formula (each formula does not include the part to the right of the wavy line): [ka] is a cyclic alkyne group selected from the group consisting of:

[0050] [1-3a] In the alginic acid derivative of the formula (I) according to the aspect [1], Akn and - L 1 The combination of - is preferably represented by the following partial structural formula: Does not include: [ka] [ka] is as represented by a group selected from the group More preferably, the compound has the following partial structural formula (in each formula, the right side of the wavy line (the imino group side) is not included): [ka] [ka] is as represented by a group selected from the group More preferably, the partial structural formula is as follows (each formula does not include the right side of the wavy line (the imino group side)): [ka] The group is as shown by a group selected from the following:

[0051] [1-1b] In the alginic acid derivative of the formula (I) according to the aspect [1], -L 1 -teeth, Preferably, the compound has the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group More preferably, the compound has the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group More preferably, the compound has the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group consisting of:

[0052] [1-2b] In the alginic acid derivative of the formula (I) of the aspect [1], Akn is preferably Preferably, the compound has the following partial structural formula (each formula does not include the part to the right of the wavy line): [ka] is a cyclic alkyne group selected from the group consisting of: More preferably, the compound has the following partial structural formula (each formula does not include the part to the right of the wavy line): [ka] is a cyclic alkyne group selected from the group consisting of:

[0053] [1-3b] In the alginic acid derivative of the formula (I) according to the aspect [1], Akn and - L 1 - the combination is preferably as shown in the table below: [Table 1] Any combination of (-L in the table) 1 - or Akn are the above-mentioned embodiments [1], [1-1 ], [1-1a], [1-2], [1-2a], and [1-1b]) or a group selected from the group of the following formulas (in each formula, the right side of the wavy line (imino group side) is not included): As shown; [ka]

[0054] More preferably, the following table: [Table 2] Any combination of (-L in the table) 1 - or Akn are the above-mentioned embodiments [1], [1-1 ], [1-1a], [1-2], [1-2a], and [1-1b]) As shown in

[0055] More preferably, the following table: [Table 3] Any combination of (-L in the table) 1 - or Akn are the above-mentioned embodiments [1], [1-1 ], [1-1a], [1-2], [1-2a], and [1-1b]) As shown in

[0056] Particularly preferred are the following partial structural formulas (in each formula, the right side of the wavy line (the imino group side) is not included): [ka] The group is as shown by a group selected from the following:

[0057] A preferred embodiment of the above aspect [1], 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 [1] is The shape can be arbitrarily formed.

[0058] [2] The second aspect is as follows: Akn-L 1 -NH2 group (Akn, and -L 1 - is the same as defined in the above aspect [1]) is introduced at a rate of 0.1% to 30%. An alginic acid derivative of formula (I) according to embodiment [1].

[0059] [2-1] In the above aspect [2], Akn-L 1 The introduction rate of -NH2 groups is preferably It is 2% to 20%; more preferably, it is 3% to 10%.

[0060] [2-1a] In the above aspect [2], Akn-L 1 The introduction rate of -NH2 groups is preferably , 0.3% to 20%; more preferably, 0.5% to 10%.

[0061] [3] The third aspect is as follows: Gel filtration chromatography of alginic acid derivatives The weight average molecular weight measured by the above method is 100,000 Da to 3,000,000 Da. 1. An alginic acid derivative of formula (I) according to claim 1

[0062] [3-1] In the above aspect [3], the method for the gel filtration chromatography of an alginic acid derivative The weight average molecular weight measured by the above method is preferably 300,000 Da to 2,500,000 Da, and more preferably Preferably between 500,000 and 2 million Da.

[0063] [3-1a] In the above aspect [3], the gel filtration chromatography of the alginic acid derivative The weight average molecular weight measured by the method is preferably 300,000 Da to 2,500,000 Da, and more preferably Preferably, it is 1 million Da to 2 million Da.

[0064] [4] The fourth aspect is as follows: Amide bond and divalent linker (-L 2 An azide group is introduced via an azide group represented by the following formula ( II): [ka] In formula (II), (ALG) represents alginic acid; -NHCO- represents any one of alginic acid. represents an amide bond via any carboxyl group;-L 2 - is represented by the following partial structural formula: , not including the wavy lines on either end]: [ka] The alginic acid derivative is represented by the following formula:

[0065] [4-1] In the alginic acid derivative of the formula (II) according to the above aspect [4], -L 2 -teeth, Preferably, the compound has the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.) More preferably, the compound has the following partial structural formula: [ka] is a linker selected from the group:

[0066] [4-1a] In the alginic acid derivative of the formula (II) according to the aspect [4], -L 2 -teeth Preferably, the compound has the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.) More preferably, the compound has the following partial structural formula: [ka] is a linker selected from the group: [4-1b] In the alginic acid derivative of the formula (II) according to the aspect [4], -L 2 -teeth Preferably, the compound has the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.) More preferably, the compound has the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.)

[0067] A preferred embodiment of the above aspect [4], further comprising an azide group and -L 2 -Combine the definitions as appropriate By this, the preferred alginic acid derivative represented by the above formula (II) of the above aspect [4] can be obtained. Any desired embodiment can be formed.

[0068] [5] The fifth aspect is as follows: N3-L 2 -NH2 group(-L 2 - is the above-mentioned aspect [ The introduction rate of the aryl group (which is the same as the definition in [4]) is 0.1% to 30%. The alginic acid derivative of formula (II) above.

[0069] [5-1] In the above aspect [5], N3-L 2 The introduction rate of -NH2 groups is preferably 2 % to 20%; more preferably, 3 to 10%.

[0070] [5-1a] In the above aspect [5], N3-L 2The introduction rate of -NH2 groups is preferably It is 0.3% to 20%; more preferably, it is 0.5% to 15%.

[0071] [6] The sixth aspect is as follows: Gel filtration chromatography of alginic acid derivatives The weight average molecular weight measured by the above method is 100,000 Da to 3,000,000 Da. An alginic acid derivative of formula (II) according to the invention.

[0072] [6-1] In the above aspect [6], the gel filtration chromatography of the alginic acid derivative of formula (II) The weight average molecular weight measured by the fluorochemical method is preferably 300,000 Da to 2,500,000 Da. and more preferably 500,000 Da to 2,000,000 Da.

[0073] [6-1a] In the above aspect [6], the gel filtration chromatography of the alginic acid derivative of formula (II) The weight average molecular weight measured by a tomography method is preferably 300,000 Da to 2,500,000 Da. and more preferably 1 million Da to 2 million Da.

[0074] [7] The seventh 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 carboxyl group of alginic acid. represents an amide bond via a carboxyl group; -L 1 - is as defined in the above embodiment [1]; -L 2 - is as defined in the above embodiment [4]; X is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.) [center represents cross-linked alginate bonded via].

[0075] [7-1] In the formula (III-L) of the embodiment [7], preferably, -L 1 -teeth, The partial structural formula is as follows (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group -L 2 - is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.) X is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included).

[0076] [7-2] In the formula (III-L) of the embodiment [7], -L 1 - is the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group -L 2 - is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.) X is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included).

[0077] [7-3] In the formula (III-L) of the embodiment [7], -L 1 - has the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group -L 2 - is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included); X is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.)

[0078] [7-3-1] In the formula (III-L) of the embodiment [7], -L 1 - is the following partial structural formula: [ka] (wherein the formula does not include the areas outside the wavy lines at both ends); -L 2 - is the following partial structural formula: [ka] (wherein the formula does not include the areas outside the wavy lines at both ends); X is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.)

[0079] [7-4] In the formula (III-L) of the embodiment [7], preferably, -L 2 -X- L 1 The combination of - is represented by the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is as represented by a substructure selected from the group consisting of: More preferably, -L 2 -XL 1 The combination of - is represented by the following partial structural formula: [Does not include the area outside the wavy line]: [ka] or

[0080] [7-1a] In the formula (III-L) of the embodiment [7], preferably, -L 1 -teeth , the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group -L 2 - is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.) X is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included).

[0081] [7-2a] In the formula (III-L) of the embodiment [7], -L 1 - is the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group -L 2 - is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.) X is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included).

[0082] [7-3a] In the formula (III-L) of the embodiment [7], -L 1 - is the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group -L 2 - is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included); X is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.)

[0083] [7-3a-1] In the formula (III-L) of the embodiment [7], particularly preferably, - L 1 - is the following partial structural formula: [ka] (wherein the outside of the wavy lines at both ends is not included); -L 2 - is the following partial structural formula: [ka] (wherein the outside of the wavy lines at both ends is not included); X is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.)

[0084] [7-4a] In the formula (III-L) of the embodiment [7], preferably, -L 2 -X -L 1 -The combination is as shown in the table below: [Table 4] (-L in the table) 1 -, -L 2 -or-X - Each formula is the same as the above embodiments [1], [1-1], [1-1a], [1-1b], [4], and [4- 1], [4-1a], [4-1b], [7], [7-1], [7-2], [7-3], [ 7-3-1], [7-1a], [7-2a], [7-3a], and [7-3a-1] (As set forth above); More preferably, -L 2 -XL 1 The combination of - is represented by the following partial structural formula: [Does not include the area outside the wavy line]: [ka] The structure is as shown by a partial structure selected from the group consisting of:

[0085] [7-1b] In the formula (III-L) of the embodiment [7], preferably, -L 1 -teeth , the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group -L 2 - is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.) X is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included).

[0086] [7-2b] In the formula (III-L) of the aspect [7], -L 1 - is the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group -L 2 - is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.) X is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included).

[0087] [7-3b] In the formula (III-L) of the embodiment [7], -L 1 - is the following partial structural formula (each formula does not include the areas outside the wavy lines at both ends): [ka] is a bivalent linker selected from the group -L 2 - is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included); X is the following partial structural formula: [ka] (In each formula, the areas outside the wavy lines at both ends are not included.) [7-4b] In the formula (III-L) of the embodiment [7], preferably, -L 1 -X -L 2 -The combination is as shown in the table below: [Table 5-1] [Table 5-2] [Table 5-3] (-L in the table) 1 -, -L 2 Each of the formulas - or -X- is the same as that of the above-mentioned embodiments [1], [1-1], [1-1a], [1-1b], and [1 -1b], [4], [4-1], [4-1a], [4-1b], [7][7-1], [7 -2], [7-3], [7-3-1], [7-1a], [7-2a], [7-3a], [ (As described in [7-3a-1], [7-1b], [7-2b], and [7-3b]) ; More preferably, -L 2 -XL 1 The combination of - is represented by the following partial structural formula: [Does not include the area outside the wavy line]: [ka] [ka] The structure is as shown by a partial structure selected from the group consisting of:

[0088] A preferred embodiment of the embodiment [7], further -L 1 -, -L 2 - and the definition of X can be combined as appropriate. By this, the cross-linked alginic acid derivative according to the preferred embodiment of the above-mentioned embodiment [7] can be arbitrarily formed. obtain.

[0089] [8] The eighth aspect is as follows: The alginic acid derivative of formula (I) according to the above aspect [1]. The conductor is mixed with the alginic acid derivative of formula (II) described in the above embodiment [4] to form a Huisge The crosslinked alginic acid according to the embodiment [7] is obtained by carrying out the crosslinking alginic acid reaction. A method for producing arginic acid.

[0090] [8-1] The embodiment of No. 8-1 is as follows. Chemical bridges formed by triazole rings and calcium ions partially form Cross-linked alginate, including ionic cross-links.

[0091] [9] The ninth aspect is as follows: The alginic acid derivative of formula (I) according to the above aspect [1]. An alginic acid derivative obtained by mixing an alginic acid derivative of the formula (II) described in the above aspect [4] and A cross-linked alginate structure is obtained by dropping a mixed conductor solution into a calcium chloride solution. body.

[0092]

[10] The tenth embodiment is as follows: The chemical bridges are formed by triazole rings and calcium ions form part of the iridium complex. The crosslinked alginate structure according to the above aspect [9], which comprises on-crosslinking.

[0093]

[11] The eleventh embodiment is as follows: The arginine of formula (I) according to the above embodiment [1]. The alginic acid derivative of the formula (II) described in the above aspect [4] is mixed with the alginic acid derivative of the formula (II). The mixed solution of the acid derivative is dropped into the calcium chloride solution, and the above-mentioned embodiment [9] or

[10] is performed. A method for producing a crosslinked alginate structure, comprising obtaining the crosslinked alginate structure according to claim 1. .

[0094]

[12] The twelfth embodiment is as follows. The above embodiment [ 9] or

[10] .

[0095]

[13] The thirteenth aspect is as follows. A medical material comprising the crosslinked alginate structure according to any one of claims 1 to 4.

[0096]

[14] The fourteenth aspect is as follows: The medical material according to the above aspect

[0013] , which is a bead or a roughly spherical gel.

[0097]

[15] The fifteenth aspect is as follows: The above aspects [1] to [6] are biocompatible. The alginic acid derivative according to any one of claims 1 to 7, Alginic acid and the crosslinker according to any one of the above aspects [9],

[10] and

[12] Alginate structure.

[0098]

[16] The sixteenth aspect is as follows: The following formula (AM-1): [ka] [In formula (AM-1), -L 1 - and Akn combinations are shown in the table below: [Table 6-1] [Table 6-2] (Each formula is the same as the definition of the above embodiment [1]) or a pharma- ceutically acceptable salt thereof, or a solvate thereof.

[0099] [16-1] In the formula (AM-1) of the aspect

[16] , preferably, Akn-L 1 -The combinations are shown in the table below: [Table 7] (Each formula is the above-mentioned embodiment [1-1], [1-2], [1-1a]) , [1-2a], [1-1b], and [1-2b]); More preferably, the following table: [Table 8] (Each formula is the above-mentioned embodiment [1-1], [1-2], [1-1a]) , [1-2a], [1-1b], and [1-2b]); More preferably, the following table: [Table 9] (Each formula is the above-mentioned embodiment [1-1], [1-2], [1-1a]) , [1-2a], [1-1b], and [1-2b]); For example, the following structural formula: [ka] The compound is as represented by any one of the structural formulas in the above.

[0100] A preferred embodiment of the embodiment

[16] , further Akn, and -L 1 -Combine the definitions as appropriate By this, the cross-linked alginic acid derivative according to the preferred embodiment of the above-mentioned embodiment

[16] can be arbitrarily formed. do.

[0101]

[17] The seventeenth aspect is as follows: The following formula (AM-2): [ka] [In formula (II), -L 2 - is a group represented by the formula (LK-1) (wherein the substitution pattern of the phenyl ring is p-position) (except for n1=1 and n2=3), formula (LK-2), formula (LK-3), formula (LK -4) (wherein the substitution pattern of the phenyl ring is m substitution, n7=3, and The ring substitution pattern is p-substitution, and n7=2, 3, 4, 6 is excluded), formula (LK-5) (wherein wherein the substitution pattern of the phenyl ring is p-substitution, except for n8=1 and n9=2), 6), and formula (LK-7) [each formula is the same as defined in the above embodiment [4]] or a pharma- ceutically acceptable salt thereof, or a solvate thereof.

[0102] [17-1] In the formula (AM-2) of the aspect

[17] , preferably, -L 2 -teeth , Formula (LK-1-1) (excluding n1=1 and n2=3 in the formula), Formula (LK-2-1) , (LK-3-1), (LK-4-1) (except for n7=2, 3, 4, 6), (LK-5-1) (excluding n8=1 and n9=2 in the formula), formula (LK-6-1), and and formula (LK-7-1) [each formula is defined as in the above embodiment [4-1], [4-1a] or [4-1b]] is the same as righteousness; More preferably, the formula (LK-1-1-a), the formula (LK-2-1-a), the formula (LK-3- 1-a), formula (LK-5-1-a), formula (LK-6-1-a), formula (LK-7-1-a) and formula (LK-7-1-b) [each formula is the above-mentioned embodiment [4-1], [4-1a] or [4-1b ] is the same as the definition of ].

[0103] A preferred embodiment of embodiment

[17] , further comprising an azide group and -L 2 -Combine the definitions as appropriate By this, the cross-linked alginic acid derivative according to the preferred embodiment of the above-mentioned embodiment

[17] can be arbitrarily formed. do.

[0104] Each aspect will be described in more detail below.

[0105] 1. Alginic acid In this specification, the term "alginic acid" refers to alginic acid, alginic acid esters, and alginic acid esters thereof. At least one alginate selected from the group consisting of salts thereof (e.g., sodium alginate). The alginic acid used is: They may be of natural or synthetic origin, but are preferably of natural origin. The alginates used are Lessonia, Macrocystis, Laminaria, Ascophyllum, Darmstadt, and others. 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 homopolymer fraction of D-mannuronic acid A block copolymer in which randomly arranged L-guluronic acid fractions (M / G fractions) are arbitrarily linked. It is a combination.

[0106] In the present specification, alginic acid refers to alginic acid (ALG) and any calcination of alginic acid. One of the carboxyl groups may be represented as -COOH, as in (ALG)-COOH.

[0107] In some embodiments, the alginic acid is sodium alginate. The alginate may be a commercially available sodium alginate. Sodium alginate is A-1, A-2, A-3, B-1, B-2 as shown in the table below. The sodium alginate B-3 (distributor: Mochida Pharmaceutical Co., Ltd.) was used. The viscosity, weight average molecular weight and M / G ratio of a 1 w / w% aqueous solution of sodium alginate are as follows: As shown in the table.

[0108] [Table 10]

[0109] 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.

[0110] [Viscosity measurement of sodium alginate] According to 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 measurements were performed using MilliQ water. The measurement equipment was a cone-plate type rotational viscometer (viscosity and viscoelasticity measurement equipment). The RheoStress RS600 (Thermo Haake GmbH) sensor: 35 / 1) was used. The speed was 1 rpm when measuring 1 w / w% sodium alginate solution. The reading time was 2 minutes. The average value from 1 minute to 2 minutes after the start was taken. The average value of three measurements was taken as the measured value. The constant temperature was 20°C.

[0111] [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 performed according to a conventional method under the following measurement conditions.

[0112] [Pretreatment method] The sample is dissolved in the eluent, then filtered through a 0.45 μm membrane filter. The solution was made into a liquid. (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

[0113] (2)GPC-MALS measurement [Refractive index increment (dn / dc) measurement (measurement conditions)] Differential refractometer: Optilab T-rEX Measurement wavelength: 658nm Measurement temperature: 40℃ Solvent: 200mM sodium nitrate aqueous solution Sample concentration: 0.5-2.5mg / mL (5 concentrations)

[0114] [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

[0115] In the present specification, the terms "alginic acid, alginic acid derivatives, cross-linked alginic acid, and cross-linked alginic acid" refer to In molecular weights, Da (daltons) may be added as a unit.

[0116] The composition ratio (M / G ratio) of alginic acids, D-mannuronic acid and L-guluronic acid, 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 gels formed were 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 In addition, it also affects the hardness, brittleness, water absorption, flexibility, etc. of the gel. The M / G ratio of the phosphate and / or its salt is usually 0.2 to 4.0, more preferably is preferably 0.4 to 3.0, and more preferably 0.5 to 3.0.

[0117] In this specification, the numerical range indicated using "~" refers to the numerical range written before and after "~". The ranges are shown as minimum and maximum values, respectively.

[0118] In the present specification, the term "alginic acid ester" and "alginate salt" 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.

[0119] In the present specification, the alginate salt includes, for example, a monovalent salt of alginic acid, a divalent salt of alginic acid, The monovalent salt of alginic acid is preferably sodium alginate. Alginate, potassium alginate, ammonium alginate, etc. are preferred. Sodium alginate or potassium alginate is particularly preferred. As the divalent salt of alginic acid, calcium alginate, alginic acid, Examples of alginate include magnesium alginate, barium alginate, and strontium alginate. do.

[0120] Alginic acid is a polymeric 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 natural polymers is in the range of 20,000 to 3 million. It is known that values ​​can vary.

[0121] For example, gel permeation chromatography (GPC) or gel filtration chromatography (GPC) The weight average molecular weight measured by size exclusion chromatography (also known as size exclusion chromatography) is preferably 100,000 or more, more preferably 500,000 or more, and more preferably 500,000 or more. The preferred range is 100,000 to 5 million. and more preferably 150,000 to 3,000,000.

[0122] In addition, for example, the absolute weight average molecular weight can be measured by the GPC-MALS method. The weight average molecular weight (absolute molecular weight) measured by the GPC-MALS method is preferably 1 10,000 or more, more preferably 50,000 or more, even more preferably 60,000 or more, and preferably 1 million or less, more preferably 800,000 or less, even more preferably 700,000 or less, and particularly preferably The preferred range is 10,000 to 1,000,000, and the more preferred range is 10,000 to 1,000,000. 50,000 to 800,000, more preferably 60,000 to 700,000, and particularly preferably 60,000 to 500,000 It is.

[0123] Usually, when calculating the molecular weight of high molecular weight polysaccharides using the above method, 10% to 20% of the measurement For example, if the number is 400,000, it may be 320,000 to 480,000, and if the number is 500,000, it may be 400,000 to 600,000. If it is 0 million or 1 million, the value can fluctuate within a range of about 800,000 to 1.2 million.

[0124] The molecular weight of alginic acids can be measured according to a conventional method.

[0125] Representative conditions for using gel filtration chromatography for molecular weight measurement are as described below. The column is, for example, a Superose6 Incr An ease10 / 300 GL column (GE Healthcare Sciences) can be used. For example, a 10 mmol / L phosphorus solution containing 0.15 mol / L NaCl is used as the developing solvent. Acid buffer (pH 7.4) can be used, and blue dextran can be used as a molecular weight standard. Thyroglobulin, ferritin, aldolase, conalbumin, ovalbumin, ribonuclease Crease A and aprotinin can be used.

[0126] The viscosity of the alginic acid used in this specification is not particularly limited, but is preferably 1 w / w% alginic acid. When the viscosity is measured as an aqueous solution of acetic acid, it is preferably 10 mPa·s to 1000 mPa·s. Pa·s, and more preferably, 50 mPa·s to 800 mPa·s.

[0127] 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 type rotational viscometer (cone-plate type viscometer), etc. It is preferable to follow the viscosity measurement method in the Japanese Pharmacopoeia (16th edition). More preferably, a cone-plate type viscometer is used.

[0128] 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 concentration, selecting the brown algae used as raw materials, and molecular weight fractionation during the manufacturing process. It is possible to produce alginic acids having different molecular weights. By mixing with another lot of alginic acid with different viscosity, it is possible to obtain a polymer having the desired molecular weight. Alginic acids may also be used.

[0129] As used herein, alginic acid is, in some embodiments, low endotoxin. With untreated alginate or, in some alternative embodiments, with low endotoxin Low endotoxin means that it does not substantially cause inflammation or fever. More preferably, low endotoxin is used. Toxin-treated alginates are preferred.

[0130] 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. 9-32526) 4001, etc.), the method of purifying β1,3-glucan by Yoshida et al. (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 WO 93 / 131) for purifying polysaccharides; 36 pamphlet, etc.), the method of Lewis et al. (e.g., 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, etc.), hydrophobic substances, Adsorption to 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 above-mentioned reaction can be carried out by any of the known methods, such as those described above, or by a suitable combination of 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

[0131] The endotoxin level can be confirmed by a known method, for example, using the Limulus reagent ( The method was performed using Endospecy (registered trademark) ES-24S set (Seikagaku Corporation). It can be measured by methods such as using a company.

[0132] The method for treating endotoxin used is not particularly limited, but as a result, The endotoxin content of phosphates was measured using the Limulus Ammonia (LAL) reagent. When the test is performed, it is preferable that the endotoxin concentration is 500 or less (EU) / g. Most preferably, it is 100 EU / g or less, particularly preferably, it is 50 EU / g or less, and particularly preferably, it is 100 EU / g or less. Or, it is 30EU / g or less. Sodium alginate that has been treated with low endotoxin is For example, Sea Matrix® (Mochida Pharmaceutical Co., Ltd.), PRONOVA T M It is available commercially as a product such as UP LVG (FMCBioPolymer).

[0133] 2. Alginic acid derivatives Herein, a novel alginic acid derivative is provided. The method is to bond alginic acid to one or more carboxyl groups of the alginic acid with 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 -, Akn is defined as in the first embodiment above. and an alginic acid derivative represented by the following formula (II): [ka] [In formula (II), (ALG), -L 2 The definition of - is the same as that in the fourth aspect above. It is an alginic acid derivative represented by the formula:

[0134] The bivalent linker (-L 1 -or-L 2 -) is a group complementary to the reactive group Any linear group may be used as long as it does not inhibit the reaction with the reactive group. , a straight chain alkylene group (-(CH2) n -, n = 1 to 30) (-CH2- in the group is -C(=O)-, -CONH-, -O-, -NH-, -S-, benzene ring, heterocycle (pyridine 5- to 6-membered aromatic heterocyclic rings such as benzene ring, piperidine ring, piperazine ring, etc., or 5- to 6-membered non-aromatic Heterocycles) and the like may be substituted multiple times (for example, 1 to 10 or 1 to 5 times). The hydrogen atom of the -CH2- is an oxo group (=O), 1-6 Alkyl groups (e.g., methyl halogen atoms (e.g., ethyl, n-propyl, iso-propyl, etc.); For example, fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), hydroxyl group (-OH), etc. may be substituted with a plurality (for example, 1 to 10 or 1 to 5) of groups selected from the group Examples include:

[0135] The novel alginic acid derivatives described herein are represented by formula (I) and formula (II) Alginic acid derivatives can be produced, for example, by the method shown below (for details, see the general production method described below). It is possible to manufacture

[0136] [ka]

[0137] 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.

[0138] In the present specification, Akn-L of formula (I) 1 The -NH- groups are located in all the calcium phosphate units of the alginate building blocks. It is not necessary that the N3-L of formula (II) is bonded to a carboxyl group. 2 The -NH- group is It is not necessary for all carboxyl groups of the carboxylic acid building block to be bonded.

[0139] In the present specification, Akn-L of formula (I) 1 When the -NH- group is referred to as a reactive group, the —NH- group in the formula (II) N3-L 2 The -NH- group serves as a complementary reactive group. Conversely, the N3-L 2 -N When the H-group is referred to as a reactive group, Akn-L of formula (I) 1 The -NH- group is a complementary reactive group. become.

[0140] 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.

[0141] The introduction rate of the reactive group or the complementary reactive group is determined by the ratio of the repeating units of alginic acids, i.e., .alpha.-(.alpha.-methyl)- ... 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 percentages used for the introduction rates of reactive groups or complementary reactive groups in each of the above formulas are in mol%. The introduction rate of each reactive group or complementary reactive group is determined by the method described in the Examples below. It can be done.

[0142] 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, which then forms the bridge.

[0143] 3. Huisgen reaction The Huisgen reaction (1,3-dipolar cycloaddition reaction) is carried out by reacting terminal aryl groups with aryl groups as shown in the following formula. This is a condensation reaction between compounds having a dihydro 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 believed 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.

[0144] [ka]

[0145] 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. Since the bond is highly distorted, the reaction with phenyl azide is the driving force for the removal of the distortion. The reaction proceeds spontaneously, making a catalyst unnecessary.

[0146] [ka]

[0147] As described above, the Huisgen reaction can be carried out using substituted primary azides, secondary azides, and tertiary azides. azide compounds having a terminal group which is a complementary reactive group to the azide group, such as an aromatic azide, 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, ester groups, carboxyl groups, alkenyl groups, hydroxyl groups, amino groups, etc.) It is possible.

[0148] In some embodiments, the method avoids copper-catalyzed cytotoxicity without producing undesirable by-products. To avoid this, a copper catalyst is not used, and the 1,2,3-triazolium chloride can be easily and efficiently synthesized in a short time. In order to form crosslinks between alginic acid molecules by alkyl rings, the alkyne group of the Huisgen reaction is For example, the cyclic alkyne group (cyclooctyl group) described in the above embodiment [1] can be used as the cyclic alkyne group. There are.

[0149] In a preferred embodiment of the method for crosslinking an alginic acid derivative, the reaction (Huisgen reaction) In this case, the alginic acid reaction hardly forms any undesirable by-products. 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.

[0150] 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. Both cross-linked alginates have a gel-like to semi-solid, sometimes sponge-like, morphology. It has the property of forming.

[0151] 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 divalent metal ion used, for example. The concentration of an aqueous solution containing calcium chloride (e.g., calcium chloride solution) or an aqueous solution containing alginic acid This can be adjusted by, for example, changing the introduction rate of the reactive group introduced.

[0152] 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 an alginate solution by ionic crosslinking. In order to strengthen the structure (e.g., to obtain long-term stability, etc.), chemical bonds are formed. In addition, for example, divalent metal ion bonds and chemical bonds can be used. In the cross-linked alginate structure, divalent metals were incorporated by ionic cross-linking. The ions can be reversibly released to create structures that only remain cross-linked by chemical bonds. It is.

[0153] 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 to obtain the desired product.

[0154] In one embodiment, the crosslinked alginate is chemically crosslinked (a trialkyl group formed from an alkyne group and an azide group). It forms a three-dimensional network structure through cross-linking by azole rings. The present invention relates to cross-linked alginate with improved stability after cross-linking.

[0155] In some embodiments, the crosslinked alginate is a crosslinked alginate that is formed by crosslinking any carboxyl group of a first alginate with any carboxyl group of a 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 carboxyl group of alginic acid. represents an amide bond via a carboxyl group; -L 1 -, -L 2 -, and X is the seventh The alginic acid is cross-linked via an amide bond, which is the same as the definition in the embodiment.

[0156] In some embodiments, the alginic acid derivative of formula (I) is used in preparing crosslinked alginic acid. The mixing ratio of the alginic acid derivative of formula (II) is the weight 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.

[0157] In some embodiments, in preparing crosslinked alginic acid, an alginic acid derivative of formula (II) The mixing ratio of the alginic acid derivative of formula (I) is the weight ratio of the derivative of formula (II) to the derivative of formula (I). The ratio of the amounts is, for example, 1 to 4.0:1, preferably 1.5 to 4.0:1, or 1.2 to 1. .5:1, or 1 to 1.2:1, more preferably 1:1.

[0158] In some embodiments, the alginic acid derivative of formula (I) is used in preparing crosslinked alginic acid. The mixing ratio of the alginic acid derivative of formula (II) is preferably The ratio of the introduction rate (mol%) of the reactive group of the alginic acid derivative of the formula (II) to that of the carboxylate of the alginic acid derivative of the 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.

[0159] In some embodiments, in preparing crosslinked alginic acid, an alginic acid derivative of formula (II) The mixing ratio of the alginic acid derivative of formula (I) is preferably the alginic acid derivative of formula (II). The ratio of the introduction rate (mol%) of the reactive group of the conductor to that of the alginic acid derivative of formula (I) is, for example, 1 ~4.0:1, preferably 1.5~4.0:1, or 1.2~1.5:1, or 1~ 1.2:1, more preferably 1:1.

[0160] 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), It is possible.

[0161] In crosslinked alginic acid, all of the carboxyl groups of the constituent units of alginic acid are represented by the above formula (III- In the cross-linked alginic acid, it is not necessary to have a cross-link of the above formula (III-L). The crosslinking introduction rate (also referred to as the crosslinking rate) is, for example, 0.1 to 80%, 0.3 to 60%, %, 0.5-30%, or 1.0-10%.

[0162] 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 1 to 500 mg / mL, preferably 5 to 100 mg / mL. / mL.

[0163] The reaction temperature of the Huisgen reaction is usually 4 to 60°C, preferably 15 The range is ~40°C.

[0164] The stirring time for forming cross-linked alginate (hydrogel) is, for example, from a few seconds to 24 hours. , a few seconds to 12 hours, a few seconds to 30 minutes, or a few seconds to 10 minutes.

[0165] 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.

[0166] In some embodiments, the crosslinked alginate is formed by the Huisgen reaction as the crosslinks. Chemical bridges formed by triazole rings and calcium ions in part It is cross-linked alginate, including cross-links.

[0167] 5. Cross-linked Alginate Constructs The crosslinked alginate structure can be produced by a method including subjecting the alginate derivative to a crosslinking reaction. For example, the preparation can be performed by the following methods, but is not limited to these. It is not something that can be determined.

[0168] [Mixing method] An alginic acid derivative of formula (I) and an alginic acid derivative of formula (II) are mixed to obtain an alginic acid derivative of formula (I) and an alginic acid derivative of formula (II). Chemical crosslinking was achieved by dropping a mixed solution of arginic acid derivatives into a solution containing divalent metal ions. (The triazole ring formed from the alkyne group and the azide group by the Huisgen reaction) The formation of cross-links (cross-links formed partially by divalent metal ions) and ionic cross-links (cross-links formed partially by divalent metal ions) Thus, a specific structure, a cross-linked alginate structure, can be obtained.

[0169] [Coating method] A solution containing the alginic acid derivative of formula (I) is dropped into a solution containing a divalent metal ion, The resulting structure, such as a gel, can be partially crosslinked. The compound having the structure (II) is added to a solution containing the alginic acid derivative of the formula (II), By carrying out a further cross-linking reaction (Huisgen reaction) on the surface of the body, cross-linked alginates are formed. In this method, the alginic acid derivative of formula (I) can be converted into the alginic acid structure of formula (II ) an alginic acid derivative of formula (II) and an alginic acid derivative of formula (I) It is also possible to replace the above with the above.

[0170] The divalent metal ion used in the above method is not particularly limited, but for example, calcium Mu ion, magnesium ion, barium ion, strontium ion, zinc ion, etc. Of these, calcium ion is preferred.

[0171] The solution containing calcium ions used in the method is not particularly limited, but may be, for example, For example, calcium chloride solution, calcium carbonate solution, calcium gluconate solution, etc. An example is an aqueous solution of calcium chloride, preferably an aqueous solution of calcium chloride.

[0172] The calcium ion concentration of the solution containing calcium ions used in the method is not particularly limited. However, the concentration is, for example, 1 mM to 1M, and preferably 5 mM to 500 mM. , and more preferably 10 mM to 300 mM.

[0173] The solvent or solution used in the above method is not particularly limited, but may be, for example, 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), and saline, and preferably ultrapure water. be.

[0174] Specific crosslinked alginate structures include, for example, fibrous structures, fibers, beads, The cross-linked alginate structures are preferably gels, roughly spherical gels, etc., and have improved stability. In addition, the cross-linked alginate structure has the ability to retain contents inside (content retention). The material may have a property of retaining the substance.

[0175] 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.

[0176] 6. Biocompatibility of alginate derivatives and photocrosslinked alginate derivatives In the present specification, the alginic acid derivative or the photocrosslinked alginic acid structure has biocompatibility. In the present specification, biocompatibility refers to a biomaterial (herein, represented by formula (I) Alginic acid derivative having a photoreactive group introduced therein, and a method for producing the same using the alginic acid derivative The interaction between the biomaterial and the living body, The property of not causing local or systemic reactions in the tissues it comes into contact with is called biocompatibility. It is said to have biocompatibility.

[0177] In the present specification, the biocompatibility of alginic acid derivatives or photocrosslinked alginic acid structures is This will be confirmed in the examples relating to biocompatibility described below.

[0178] 7. Stability of cross-linked alginate structures The stability of the crosslinked alginate structure can be measured, for example, by measuring gel stability and gel permeability. This can be confirmed by measuring the transmittance, for example.

[0179] [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 leaked into PBS. The percentage of the cross-linked alginate gel was calculated by dividing the concentration of the cross-linked alginate gel by the total alginate concentration obtained by decomposing the cross-linked alginate gel. The value expressed as a percentage is the disintegration rate. The gel stability was specifically measured according to the method described in the Examples below. It may be required by law.

[0180] In the present specification, the gel collapse rate of the crosslinked alginate structure is preferably 0% to 90%, More preferably, it is 0% to 70%, and even more preferably, it is 0% to 50%. The stability of the acid structure is improved as the concentration of alginic acid leaking into the aqueous solution decreases, i.e., the gel collapses more rapidly. The lower the fracture rate, the higher the stability.

[0181] [Method for measuring gel permeability] Cross-linked alginate structures containing fluorescein isothiocyanate-dextran A gel was prepared, and physiological saline was added to the gel placed in a container. The dextran concentration was measured. The measured dextran concentration was measured using fluorescein isothiocyanate. Decomposition of the cross-linked alginate gel containing dextran acetate The gel permeability is calculated by dividing the value by the concentration of ethylene glycol and expressed as a percentage. It can be determined by the method described in the Examples below.

[0182] The gel permeability of cross-linked alginate after 24 hours of addition of physiological saline is, for example, When dextran is encapsulated, the dextran content is preferably 0% to 90%, and more preferably 0% to 70%, and more preferably 0% to 50%. Also, dextran having a molecular weight of 150,000 is When the cross-linked alginate structure gel is used for the purpose of binding proteins or antibodies, If it is release or production, it is preferably 1% to 100%, more preferably 10% to 1 00%, and more preferably 30% to 100%. If so, it is preferably 0% to 90%, more preferably 0% to 70%, and even more preferably Or it is 0% to 50%.

[0183] The permeability of the cross-linked alginate structure is such that the lower the permeability, 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.

[0184] The permeability of the gel depends on the molecular weight and concentration of the alginic acid used, and the number of cross-linking groups introduced into the alginic acid. The type and introduction rate, the type and concentration of divalent metal ions used for gelation, or a combination of these It is possible to adjust the amount of heat generated by the heat generation.

[0185] [Method for preparing cross-linked alginate structure gel containing ingredients] For example, crosslinking agents containing fluorescein isothiocyanate-dextran as the content The bridge alginate structure gel can be prepared in the following manner.

[0186] (1) A solution of the alginic acid derivative represented by formula (I) and fluorescein isothiocyanate Mix the to-dextran solution. (2) Add a solution of the alginic acid derivative represented by formula (II) to the mixed solution obtained in (1). Mix. (When formula (I) in (1) is changed to formula (II), formula (II) in (2) is changed to formula (I) (To be (3) The mixed solution obtained in (2) was dropped into a solution containing calcium ions. In solution, fluorescein isothiocyanate is linked to the fluorescein isothiocyanate by forming chemical and ionic crosslinks. A cross-linked alginate structure gel containing ocyanate-dextran is obtained.

[0187] 8. Method for synthesizing alginic acid derivatives In the present specification, the alginic acid derivatives represented by formula (I) and formula (II) are each H2N-L 1 -Akn(in the formula, L 1 and Akn are the same as defined in the above embodiment [1]. ), or H2N-L 2 -N3 (in the formula, L 2 teeth, The amine derivative (AM-2) represented by the same definition as in the above embodiment [4] is It can be produced by condensation reaction of any carboxyl group of carboxylic acids using a condensing agent. Cut.

[0188] [ka]

[0189] [Method for producing alginic acid derivative of formula (I)] An aqueous solution of 0.5% by weight to 1% by weight of alginic acid and an amine represented by formula (AM-1) Using the method known in the literature, for example, "Experimental Chemistry Course, 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 above, 1,3-dicyclohexylcarbodiimide (DCC), 1-ethyl -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-triazolidinediamine dimethylmorpholinium chloride (DMTF-MM), etc. In the presence of a condensing agent, tetrahydrofuran, 1,4-dihydrofuran, etc., to such an extent that alginic acid does not precipitate, Ether solvents such as xanthene, alcohols such as methanol, ethanol, and 2-propanol, Mixtures of water and a solvent selected from the group consisting of alkane-based 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 temperatures between 0°C and 50°C in the presence or absence of an organic base such as pyridine. By carrying out the above procedure, the alginic acid derivative of formula (I) can be produced.

[0190] [Method for producing alginic acid derivative of formula (II)] An aqueous solution of 0.5% by weight to 1% by weight of alginic acid and an amine represented by formula (AM-2) and reacting according to the above-mentioned [Method for producing an alginic acid derivative represented by formula (I)]. Thus, the alginic acid derivative of formula (II) can be produced.

[0191] In the method for producing the alginic acid derivative of formula (I) or the alginic acid derivative of formula (II), The introduction rate of the amine of formula (AM-1) or formula (AM-2) 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) Adjustment of the concentration of alginic acid as the reaction substrate; (v) Formula (AM- 1) or by adding a water-miscible organic solvent to increase the solubility of the amine of formula (AM-2) , etc.

[0192] More specific examples of the amines represented by formula (AM-1) or formula (AM-2) are given below. The method for producing an amine is shown.

[0193] In addition, in the following manufacturing methods, R A C = methyl, ethyl, etc. 1~6 is an alkyl group ;P 1 is a -C(O)O-tertBu group, a -C(O)O-Bn group, a -C(O)CH3 group, -C(O)CF3 group, etc.; 2 -C(O)O -tertBu group, -C(O)O-Bn group, -C(O)CH3 group, -C(O)CF3 group, an amine selected from -SO2Ph, -SO2PhMe, -SO2Ph(NO2) and the like; E = halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom) , etc.), -OTs group, -OMs group, etc.

[0194] In addition, in each of the following production methods, the protecting group P 1 and P 2 The protection and deprotection of the above can be carried out by methods known in the literature, e.g. For example, Protective Groups in Organic Synthesis tive Groups in Organic Synthesis 4thEdit ion) 4th edition, 2007, John Wiley & Sons The deprotection method described in the book by Greene et al. The protection and deprotection can be carried out according to the above.

[0195] [Manufacturing method A] Method for preparing an amine represented by formula (AM-OL-1): [ka]

[0196] Compounds of formula (SM-1) [Compounds of formula (SM-1) are commercially available compounds or are prepared by synthesis from commercially available compounds. Compounds of formula (RG-1) [which can be produced by known production methods] and compounds of formula (RG-2) [compounds of formula (R Compound G-1) is a commercially available compound or can be produced from a commercially available compound by a production method known in the literature. and m1 is an integer of 2 to 6], and the compound is used to prepare a compound having a structure similar to that described in the literature, for example, rbohydrate Polymers, 169, p332-340, 2017” etc. (i) in the presence of AgO3SCF3 、 Participates in reactions with toluene, etc. (RG-1) in a solvent free of bromine, followed by (ii) debromination using DBU to form an alkyne group, and (iii) to form a protecting group P 1 By deprotecting the -OL-1), or a salt of the formula (AM-OL-1). This can be done.

[0197] [Manufacturing method B] Method for preparing an amine represented by formula (AM-OL-2): [ka]

[0198] <Process 1> Compounds of formula (SM-2) [Compounds of formula (SM-2) are commercially available compounds or can be prepared by synthesis from commercially available compounds. Compounds of formula (RG-2) [which can be produced by known production methods] 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. The compound of the present invention may be used in a method known in the art, for example, in the European Journal of l of Organic Chemistry, 2014(6), p1280-1 (i) PPh3 and N2(CO In the presence of the reagent 2CHMe2)2, in a solvent that does not participate in the reaction such as tetrahydrofuran, The reaction is then carried out in the presence of a base such as sodium hydroxide, followed by (ii) In a solvent that does not participate in the reaction, such as tetrahydrofuran or water, or a mixture of these solvents, By carrying out decomposition, the compound represented by formula (IM-1) can be produced.

[0199] <Process 2> [Production Method B] Compound of formula (IM-1) obtained by <Step 1> and compound of formula (RG-3) [The compound of formula (RG-3) is a commercially available compound or is prepared from a commercially available compound by a preparation method known in the literature. and m5=an integer of 2 to 6; A condensation reaction similar to that of the method for producing an alginic acid derivative of formula (I) is carried out, followed by (iv) removing a protecting group. P 1 By deprotecting the compound represented by formula (AM-OL-2), or M-OL-2) can be prepared as a salt thereof.

[0200] [Manufacturing method C] Method for preparing an amine represented by formula (AM-OL-3): [ka]

[0201] <Process 1> The compound of formula (SM-1) and the compound of formula (RG-4) [the compound of formula (RG-4) is commercially available] A compound that can be produced from a commercially available compound by a production method known in the literature; m8=1 1 to 6], according to a method known in the literature, for example, e American Chemical Society, 126(46), p150 (i) the presence of AgClO4, according to the method described in In a solvent that does not participate in the reaction, such as toluene, the compound of formula (RG-4) is substituted, followed by (ii) forming an alkyne group by debromination using NaOMe; i) In the presence of a base such as lithium hydroxide or sodium hydroxide, methanol, ethanol, tetrahydrofuran Hydrolysis is carried out in a solvent that does not participate in the reaction, such as dihydrofuran or water, or in a mixture of these solvents. By carrying out the above reaction, a compound represented by formula (IM-2) can be produced.

[0202] <Process 2> [Production Method C] Compound of formula (IM-2) obtained by <Step 1> and compound of formula (RG-5) The compound of formula (RG-5) is a commercially available compound or can be prepared by a method known in the art from a commercially available compound. and m9 is an integer of 2 to 6], and the compound represented by the formula (I) The same condensation reaction as in the method for producing alginic acid derivatives of the formula (1) is carried out, followed by the removal of the protecting group P 1 By deprotecting and an amine compound represented by the formula (AM-OL-3) or a salt of the formula (AM-OL-3) It can be produced as:

[0203] [Manufacturing method D] Method for preparing an amine represented by formula (AM-OL-5): [ka]

[0204] <Process 1> Compounds of formula (SM-3) [Compounds of formula (SM-3) are commercially available compounds or are prepared by synthesis from commercially available compounds. The compound can be produced by a method known in the art, for example, 『Faming Zhuanli Shenqing, 104529898, 22 According to the method described in the "April 2015" and other publications, (i) ethylenediaminetetraacetate was added in the presence of a base such as pyridine. In a non-reactive solvent such as ethanol, H2NOH-HCl is reacted to form an oxime. Then, (ii) P2O5 was reacted with diphosphorus pentoxide in methanesulfonic acid to obtain Beck's (iii) diethyl ether to form an eight-membered lactam by Mann rearrangement; In a solvent that does not react with the tail, the amide group is reduced using a reducing agent such as BH3 or LiAlH4. The compound represented by formula (IM-3) can be produced by reduction of the above compound.

[0205] <Process 2> [Production method D] Formula (IM-3) and Formula (RG-6) [Formula (R Compound G-6) is a commercially available compound or can be produced from a commercially available compound by a production method known in the literature. and m3=an integer of 1 to 6], (iv) using the compound represented by the formula (I) The same condensation reaction as in the method for producing alginic acid derivatives of (v) is carried out to obtain a condensation product. After addition of bromine, the alkyne is debrominated with tert-BuOK to give the desired aryl group. followed by (vi) forming a protecting group P 1 By deprotecting the compound represented by formula (AM-OL-5), The amine compound represented by the formula (AM-OL-5) can be prepared as a salt thereof.

[0206] [Manufacturing method E] Method for preparing amines represented by formula (AM-OL-6) and formula (AM-OL-7): [ka]

[0207] <Process 1> [Production Method D] The compound of formula (IM-4) obtained in (ii) of <Step 1> and the compound of formula (RG Compounds of formula (RG-7) are commercially available compounds or compounds prepared by known methods using commercially available compounds. and m2' is an integer of 2 to 6. Methods of knowledge, e.g., Synthesis, 46(5), p669-677, 2014 In accordance with the method described in the "Research Report on the Chemical Reaction of 1,2-Dimethylphenylacetamide (PET)," a base such as sodium hydroxide and tetrabutylammonium The reaction is carried out in the presence of a phase transfer catalyst such as methyl bromide in a solvent that does not participate in the reaction such as toluene. By this, the compound represented by formula (IM-5) can be produced.

[0208] <Process 2> [Production Method E] Bromine is added to the compound of formula (IM-5) obtained in <Step 1>, and then , and the alkyne group is formed by debromination using a base such as tert-BuOK. Then, the protecting group P 2 By deprotecting the compound represented by formula (AM-OL-6), The compound of formula (AM-OL-6) can be prepared as a compound, a salt thereof, or a salt of formula (AM-OL-6).

[0209] <Process 3> [Production Method E] The compound of formula (IM-5) obtained in <Step 1> is used to produce [Production Method D ] By carrying out a reaction according to the reduction method of (iii) in <Step 1>, a compound of the formula (IM-6) can be manufactured.

[0210] <Step 4> [Production Method E] Using the compound of formula (IM-6) obtained in <Step 3>, [Production Method E] By carrying out the reaction in the same manner as in <Step 2>, an amine compound represented by the formula (AM-OL-7) is obtained. or as a salt of formula (AM-OL-7).

[0211] [Manufacturing method F] Method for preparing an amine represented by formula (AM-OL-8): [ka]

[0212] <Process 1> Compounds of formula (SM-4) [Compounds of formula (SM-4) are commercially available compounds or are prepared by synthesis from commercially available compounds. The compound can be produced by a method known in the art, for example, Synthesis, (9), p1191-1194; 2002, etc. Following the method described above, bromine was added and then debromination was carried out using tert-BuOK. By forming an alkyne group by the above reaction, a compound represented by formula (IM-7) is produced. It is possible.

[0213] <Process 2> [Production Method F] Compound of formula (IM-7) obtained in <Step 1> and compound of formula (RG-8) The compound of formula (RG-8) is a commercially available compound or a compound prepared from a commercially available compound by a method known in the art. (For details, see Production Method H described later); m6 is an integer of 1 to 6. and m7 is an integer of 2 to 6], and the resulting mixture was subjected to a method known in the art, for example, the method described in "Journal .American.Chemical.Society.,126, p15046-1 5047, 2004" or "Chem. Ber., 94, p3260-3275, 19 According to the method described in "61", etc., the Huisgen reaction was carried out, followed by the addition of the protecting group P 1 of By deprotection, an amine compound represented by formula (AM-OL-8) or an amine compound represented by formula (AM-O L-8) salt.

[0214] [Manufacturing method G] Method for preparing an amine represented by formula (AM-OL-9): [ka]

[0215] Compounds of formula (SM-5) [Compounds of formula (SM-5) are commercially available compounds or are prepared by synthesis from commercially available compounds. The compound can be produced by a method known in the art, for example, According to the method described in U.S. Patent Application Publication No. 2013-0137861, In a solvent that does not participate in the reaction, such as dichloromethane, in the presence or absence of a base, such as pyridine, The carbonate form is obtained by reacting with p-nitrophenyl phthalate. In the presence of triethylamine, in N,N-dimethylformamide solvent, [The compound of formula (RG-9) is a commercially available compound or is prepared from a commercially available compound by a preparation method known in the literature. m4 is an integer of 1 to 6, Furthermore, the protecting group P 1 By deprotecting the compound represented by the formula (AM-OL-9), It can be prepared as an amine compound or as a salt of formula (AM-OL-9).

[0216] [Manufacturing method H] A method for producing an amine represented by formula (AM-LK-1) [n The p-substituted amines with 1=1 and n2=3 are described in WO 2016 / 152980. It can also be produced according to the method described in, for example, [ka]

[0217] <Process 1> Compounds of formula (SM-6) [Compounds of formula (SM-6) are commercially available compounds or are prepared by synthesis from commercially available compounds. The compound can be produced by a known production method; n1=an integer of 1 to 6] and the formula ( Compounds of formula (RG-10) [Compounds of formula (RG-10) are commercially available compounds or compounds extracted from the literature a compound which can be produced by a known production method; n2=an integer of 2 to 6] By carrying out a condensation reaction similar to that of the above [Method for producing an alginic acid derivative represented by formula (I)], -8) can be manufactured.

[0218] <Process 2> [Production Method H] Using the compound of formula (IM-8) obtained in <Step 1>, For example, see Organometallics, 29(23), p6619-6622 In accordance with the method described in the "2010" and other publications, dimethyl sulfoxide and other substances that do not participate in the reaction were used. In a cold solvent, NaN3 is reacted to introduce the azide group, and then the protecting group P 1 By deprotecting As an amine compound represented by the formula (AM-LK-1) or a salt of the formula (AM-LK-1), It can be manufactured.

[0219] [Manufacturing method J] Method for preparing an amine represented by formula (AM-LK-2): [ka]

[0220] <Process 1> Compound of formula (SM-7) [Compound of formula (SM-7) is a commercially available compound or can be prepared by synthesis from a commercially available compound. The compound of formula (RG-11) [which can be produced by a known production method] and the compound of formula (RG-12) [the compound of formula ( The compound RG-11) is a commercially available compound or can be produced from a commercially available compound by a production method known in the literature. and n4 is an integer of 2 to 6], and then in step 1 of [Production Method B] The Mitsunobu reaction is then carried out in the presence of a base such as sodium hydroxide, followed by addition of methanol or ethanol. In a solvent that does not participate in the reaction, such as ethanol, tetrahydrofuran, water, or a mixture of these solvents, Hydrolysis of the ester group produces a compound represented by formula (IM-9). can be done.

[0221] <Process 2> [Production Method J] Compound of formula (IM-9) obtained in <Step 1> and compound of formula (RG-12) [ The compound of formula (RG-12) is a commercially available compound or can be prepared from a commercially available compound by a method known in the art. and n3 is an integer of 2 to 6], and the compound represented by the formula (I) can be produced by using the compound represented by the formula (I) A condensation product is obtained by carrying out a condensation reaction similar to that of the alginic acid derivative of the above method. Protecting group P 1 by deprotection to obtain an amine compound represented by formula (AM-LK-2), or It can be prepared as a salt of the formula (AM-LK-2).

[0222] [Manufacturing method K] Method for preparing an amine represented by formula (AM-LK-3): [ka]

[0223] <Process 1> [Production Method J] <Step 1> Compound of formula (SM-7) and compound of formula (RG-13) [ The compound of formula (RG-13) is a commercially available compound or can be prepared from a commercially available compound by a method known in the art. and n6 is an integer of 2 to 6. The Mitsunobu reaction is then carried out in the presence of a base such as sodium hydroxide, followed by addition of methanol, ethanol, etc. In a solvent that does not participate in the reaction, such as alcohol, tetrahydrofuran, water, etc., or a mixture of these solvents Then, the ester group is hydrolyzed to produce a compound represented by formula (IM-10). It is possible.

[0224] <Process 2> [Production Method K] Compound of formula (IM-10) obtained in <Step 1> and compound of formula (RG-14) [The compound of formula (RG-14) is a commercially available compound or a preparation method known in the art from a commercially available compound. and n5=an integer of 1 to 6] is used to produce a compound represented by the formula (I A condensation product is obtained by carrying out a condensation reaction similar to that described in the method for producing alginic acid derivatives of Protecting group P 1 by deprotection to obtain an amine compound represented by formula (AM-LK-3), can be prepared as a salt of formula (AM-LK-3).

[0225] [Manufacturing method L] Method for preparing an amine represented by formula (AM-OL-4): [ka]

[0226] <Process 1> Compounds of formula (SM-8) [Compounds of formula (SM-8) are commercially available compounds or are prepared by synthesis from commercially available compounds. The compound can be produced by a method known in the art, for example, According to the method described in "International Publication No. 2009 / 067663 Pamphlet" etc., After adding bromine, debromination with LiN(i-Pr)2 gives the formula (IM-11 ) can be prepared.

[0227] <Process 2> [Production Method L] Compound of formula (IM-11) obtained in <Step 1> and compound of formula (RG-15) The compound represented by formula (RG-15) is a commercially available compound or a compound obtained by subjecting a commercially available compound to a literature publication. and m1 is an integer of 2 to 6; React in the presence of a base such as sodium chloride in a solvent that does not participate in the reaction such as tetrahydrofuran. The compound with the side chain is obtained by removing the protecting group P. 1 Deprotection of An amine compound represented by the formula (AM-OL-4) or a salt of the formula (AM-OL-4) It can be produced as:

[0228] [Manufacturing method M] Method for preparing an amine represented by formula (AM-LK-4): [ka]

[0229] <Process 1> Compound of formula (SM-M) and compound of formula (RG-M-1) [Compound of formula (SM-M) and The compound of formula (RG-M-1) is a commercially available compound or can be prepared from a commercially available compound by a method known in the art. and n7 is an integer of 2 to 6], and the compound represented by the formula (I) can be produced by the above process. By carrying out a condensation reaction similar to that described in the method for producing arginic acid derivatives, the compound represented by the formula (IM-M-1) is obtained. It is possible to prepare compounds that can be

[0230] The carboxylic acid represented by the formula (SM-M) can be synthesized by a method known in the literature, for example, Lecture Series 5th Edition 16, Carboxylic Acids and Derivatives, Acid Halides, Acid Anhydrides, 99-11 According to the method described in "Maruzen, Vol. 8, 2007", etc., The compound of formula (RG-M-1) is used to convert the amine to a base such as triethylamine or pyridine. In the presence of halogenated solvents such as dichloromethane and chloroform, diethyl ether, tetrahydrofuran Ether solvents such as hydrofuran, aromatic hydrocarbon solvents such as toluene and benzene, N, In a solvent selected from polar solvents such as N-dimethylformamide, the solvent refluxes from 0°C. The compound of formula (IM-M-1) can be similarly produced by reacting at a temperature Cut. <Process 2> [Production Method M] Using the compound of formula (IM-M-1) obtained in <Step 1>, For example, Greene et al.'s Protective Groups in Organic Synthesis (Protective Groups in Organ ic Synthesis) 4th Edition, 2007, John Wiley & Sons The method described in the "J.D.F.S. (John Wiley & Sons)" manual determines the type of protecting group. By selecting a more appropriate deprotection method and carrying out the reaction, the compound represented by formula (AM-LK-4) can be obtained. or a salt of formula (AM-LK-4).

[0231] [Manufacturing method N] Method for preparing an amine represented by formula (AM-OL-17): [ka]

[0232] <Process 1> Compounds of formula (SM-N) and compounds of formula (RG-N-1) [Compounds of formula (SM-N) and The compound of formula (RG-N-1) is a commercially available compound or can be prepared from a commercially available compound by a method known in the art. m10=1 to 4, m11=1 to 6, m12=1 to 6; is used to carry out a condensation reaction similar to that in <Step 1> of the above-mentioned [Production Method M] to obtain The compound represented by formula (IM-N-1) can be prepared.

[0233] <Process 2> [Production Method N] Using the compound of formula (IM-N-1) obtained in <Step 1>, For example, Greene et al.'s Protective Groups in Organic Synthesis (Protective Groups in Organ ic Synthesis) 4th Edition, 2007, John Wiley & Sons The method described in the "J.D.F.S. (John Wiley & Sons)" manual determines the type of protecting group. By selecting a more appropriate deprotection method and carrying out the reaction, the compound represented by formula (AM-OL-17) can be obtained. The compound of formula (AM-OL-17) can be prepared as a compound, a salt thereof, or a salt of formula (AM-OL-17).

[0234] [Manufacturing method P] A method for producing an amine represented by formula (AM-OL-18) The amines having m13=1 and m14=2 are described in WO 2015 / 143092. It can also be produced according to the method described in the document, etc. [ka]

[0235] <Process 1> Compound of formula (SM-P) and compound of formula (RG-P-1) [Compound of formula (SM-P) and The compound of formula (RG-P-1) is a commercially available compound or can be prepared from a commercially available compound by a method known in the art. m13=an integer of 1 to 4, and m14=an integer of 2 to 6], By carrying out a condensation reaction similar to that in <Step 1> of the above [Production Method M], The compounds represented can be prepared.

[0236] <Process 2> [Production Method P] Using the compound of formula (IM-P-1) obtained in <Step 1>, For example, Greene et al.'s Protective Groups in Organic Synthesis (Protective Groups in Organ ic Synthesis) 4th Edition, 2007, John Wiley & Sons The method described in the "J.D.F.S. (John Wiley & Sons)" manual determines the type of protecting group. By selecting a more appropriate deprotection method and carrying out the reaction, the compound represented by formula (AM-OL-18) can be obtained. The compound of formula (AM-OL-18) can be prepared as a compound, a mixture, or a salt of formula (AM-OL-18).

[0237] [Manufacturing method Q] Method for preparing an amine represented by formula (AM-OL-19): [ka]

[0238] <Process 1> Compound of formula (SM-Q) and compound of formula (RG-Q-1) [Compound of formula (SM-Q) and The compound of formula (RG-Q-1) is a commercially available compound or can be prepared from a commercially available compound by a method known in the art. m15=an integer of 1 to 4, and m16=an integer of 1 to 6], By carrying out a condensation reaction similar to that in <Step 1> of the above [Production Method M], The compounds represented can be prepared.

[0239] <Process 2> [Production Method Q] Using the compound of formula (IM-Q-1) obtained in <Step 1>, For example, Greene et al.'s Protective Groups in Organic Synthesis (Protective Groups in Organ ic Synthesis) 4th Edition, 2007, John Wiley & Sons The method described in the "J.D.F.S. (John Wiley & Sons)" manual determines the type of protecting group. By selecting a more appropriate deprotection method and carrying out the reaction, the compound represented by formula (AM-OL-19) can be obtained. The compound of formula (AM-OL-19) can be prepared as a compound, a mixture, or a salt of formula (AM-OL-19).

[0240] [Manufacturing method R] A method for producing an amine represented by formula (AM-LK-5) [n The amine with n8=1 and n9=2 is described in the pamphlet of International Publication No. 2016 / 152980. It can also be prepared according to the method described in the above. [ka]

[0241] <Process 1> Compounds of formula (SM-R) [Compounds of formula (SM-R) are commercially available compounds or are prepared by synthesis from commercially available compounds. The compound can be produced by a known production method; n8=an integer of 1 to 4] and the formula ( Compound of formula (RG-R-1) [Compound of formula (RG-R-1) is a commercially available compound or a compound synthesized from a commercially available compound. It is a compound that can be produced by a production method known in the literature; n9=an integer of 1 to 6. and carrying out a condensation reaction similar to that in <Step 1> of the above-mentioned [Production Method M] to obtain a compound of the formula (IM-R-1 ) can be prepared.

[0242] <Process 2> [Production Method R] Using the compound of formula (IM-R-1) obtained in <Step 1>, In the same manner as in step 2 of the synthesis method H, NaN3 is reacted to introduce an azide group, and then the protecting group P 1 of By deprotection, an amine compound represented by formula (AM-LK-5) or an amine compound represented by formula (AM-L K-5) can be prepared as a salt thereof.

[0243] [Manufacturing method S] Method for preparing an amine represented by formula (AM-LK-6): [ka]

[0244] <Process 1> [When E=OTs or OMs group]: Compounds of formula (SM-S) [Compounds of formula (SM-S) are commercially available compounds or are prepared by synthesis from commercially available compounds. The compound can be produced by a known production method; n10=an integer of 1 to 4; and Using reagents such as ethanesulfonic acid chloride, tosylic acid chloride, and tosylic anhydride, According to a known method, for example, the method described in Journal of the American Chemical Society, 136(29), p. According to the method described in "-10459, 2014" and others, triethylamine, N,N-diisopropyl In the presence of a base such as ethylamine or pyridine, a halogen atom such as dichloromethane or chloroform is used. solvents, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, 1,4 - Ether solvents such as dioxane, aromatic hydrocarbon solvents such as benzene and toluene, etc. Using a solvent that does not participate in the reaction or a mixture of these solvents or without a solvent, from -78℃ to The reaction is carried out at a temperature at which the solvent refluxes to produce a compound represented by formula (IM-S-1). can be done.

[0245] [E = halogen (chlorine, bromine, iodine)]: The compound of formula (SM-S) is used in a method known in the literature, for example, 19. Organic Synthesis I, Hydrocarbons - Halogen Compounds, pp. 363 - 482, 1992, Maruzen According to the methods described in etc., various halogenating agents (chlorinating agents, brominating agents, iodinating agents) shown below and a solvent that does not participate in the reaction are appropriately selected, and the reaction is carried out at a temperature from 0 °C to the reflux temperature of the solvent, to produce a halogenated compound represented by formula (IM - S - 1) (E = chlorine, bromine, iodine). <When E = chlorine> As the chlorinating agent, by using reagents such as hydrogen chloride / zinc chloride (HCl / ZnCl2), hydrogen chloride / hexamethylphosphoric triamide (HCl / HMPA), thionyl chloride (SOCl2), carbon tetrachloride / triphenylphosphine (CCl4 / PPh3), triphosgene / triphenylphosphine ((CCl3)2CO / PPh3), triphosgene / N,N - dimethylformamide ( POCl3 / DMF), etc., the desired chloride can be produced. <When X = bromine> As the brominating agent, by using reagents such as 48% hydrobromic acid (48% HBr), 48% hydrobromic acid / sulfuric acid (48% HBr / H2SO4), hydrogen bromide / lithium bromide (HBr / LiBr), sodium bromide / sulfuric acid (NaBr / H2SO4), phosphorus tribromide (PBr3), etc., the desired chloride can be produced. Also, in formula (IM - S - 1), by reacting a compound where E = OTs or OMs with sodium bromide (NaBr), the desired bromide can also be produced. <When X = iodine> As the iodinating agent, by using reagents such as hydroiodic acid (HI), iodine / triphenylphosphine (I2 / PPh3), etc., the desired iodide can be produced. Also, ​​​​​​​​​​In the formula (IM-S-1), a compound in which E=OTs or OMs is added with sodium iodide ( The desired iodide can also be produced by reacting with NaI.

[0246] <Process 2> [Production Method S] Using the compound of formula (IM-S-1) obtained in <Step 1>, Method H] By reacting NaN3 in the same manner as in <Step 2>, the compound of formula (IM-S-2) is obtained. can be manufactured. <Process 3> [Production Method S] Using the compound of formula (IM-S-2) obtained in <Step 2>, Method B] By carrying out hydrolysis in the same manner as the hydrolysis reaction of the ester group in <Step 1>, A compound of formula (IM-S-3) can be prepared.

[0247] <Step 4> [Production Method S] Reaction of Compound (IM-S-3) and Compound (RG-S-1) Obtained in <Step 3> The compound of formula (RG-S-1) is a commercially available compound or a compound produced from a commercially available compound by a method known in the art. and n11 is an integer of 1 to 6], M] A compound represented by the formula (IM-S-4) can be obtained by carrying out a condensation reaction similar to that in <Step 1>. You can manufacture things. <Process 5> [Production Method S] Protecting group P of the compound of formula (IM-S-4) obtained in <Step 4> 1 Deprotection to obtain an amine compound represented by formula (AM-LK-6) or ) salt.

[0248] [Manufacturing method T] Method for preparing an amine represented by formula (AM-LK-7): [ka]

[0249] <Process 1> Compound of formula (SM-M) and compound of formula (RG-T-1) [Compound of formula (SM-M) and The compound of formula (RG-T-1) is a commercially available compound or can be prepared from a commercially available compound by a method known in the art. and n12=an integer of 1 to 6], ] by carrying out a condensation reaction similar to that in <Step 1>, a compound represented by formula (IM-T-1) can be manufactured.

[0250] The carboxylic acid represented by the formula (SM-M) can be synthesized by a method known in the literature, for example, Lecture Series 5th Edition 16, Carboxylic Acids and Derivatives, Acid Halides, Acid Anhydrides, 99-11 According to the method described in "Maruzen, Vol. 8, 2007", etc., The compound of formula (RG-T-1) is used to convert the amine to a base such as triethylamine or pyridine. In the presence of halogenated solvents such as dichloromethane and chloroform, diethyl ether, tetrahydrofuran Ether solvents such as hydrofuran, aromatic hydrocarbon solvents such as toluene and benzene, N, In a solvent selected from polar solvents such as N-dimethylformamide, the solvent refluxes from 0°C. The compound of formula (IM-T-1) can be similarly prepared by reacting at a temperature Cut. <Process 2> [Production Method T] Using the compound of formula (IM-T-1) obtained in <Step 1>, For example, Greene et al.'s Protective Groups in Organic Synthesis (Protective Groups in Organ ic Synthesis) 4th Edition, 2007, John Wiley & Sons The method described in the "J.D.F.S. (John Wiley & Sons)" manual determines the type of protecting group. By selecting a more appropriate deprotection method and carrying out the reaction, the compound represented by formula (AM-LK-7) can be obtained. or a salt of formula (AM-LK-7).

[0251] The alginic acid derivative represented by formula (I) or (II) is produced by the method of the present invention. Amines with alkynyl groups (Akn-L 1 -NH2) or amine with azide group (N3-L 2 -NH2) are described in the above [Production Method A] to [Production Method N] and [Production Method Each reaction described in [Method P] to [Production Method T], the method known in the literature, for example, 5th edition, various books, 2007, Maruzen, "Comprehensive Organic Transformations, AG uide to Functional Group Preparations, 3rd Edition (Edited by Richard C. Larock) , 2018”, “Strategic Applications of Named Reactions in Organic Synthesis, (E The method described in "The International Journal of Clinical Chemistry," edited by Laszlo Kurti and Barbara Czako, Academic Press, 2005, is By appropriately combining them, the desired amine can be produced. The amines can also be prepared by the methods described in the prior art documents listed in the table. can be done.

[0252] [Table 11]

[0253] In the present specification, the amine compound represented by formula (AM-1) or formula (AM-2) (each of the formulas (including the sub-formulae) may form pharma- ceutically acceptable salts (e.g., acid addition salts). Such salts are not particularly limited as long as they are pharma- ceutically acceptable salts, but examples 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 with 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 with aromatic monocarboxylic acids such as phthalic acid, salts with 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, glutamic acid, and glutamic acid. Among these, pharma- ceutically acceptable salts are preferred.

[0254] 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, and then separated by filtration or the mixed solvent is distilled. 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.

[0255] In the present specification, the amine compound represented by formula (AM-1) or formula (AM-2) (each of the formulas or a salt thereof may be solvated with a solvent such as water, ethanol, or glycerol. It can be formed.

[0256] 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]

[0257] 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. It can be used instead of alginic acid. Alginic acid derivatives or photocrosslinked alginic acid structures Preferred applications for the body include wound dressings, postoperative adhesion barriers, and drug sustained release substrates. Examples of the materials include medical materials such as substrates for cell culture and substrates for cell transplantation.

[0258] 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 roughly spherical gels. It is preferable to form a gel having a shape of about 100 μm, and it is more preferable to form a gel having a shape of about 100 μm.

[0259] All literature and publications mentioned in this specification are incorporated herein by reference for all purposes. It is incorporated herein in its entirety.

[0260] Moreover, 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 obvious to those skilled in the art that the present invention can be easily implemented by those skilled in the art from the description of the present specification. The best mode for carrying out the invention and specific examples are preferred embodiments of the present invention. These are illustrative and are presented for purposes of illustration and description 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 disclosure herein. EXAMPLES

[0261] 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.

[0262] The nuclear magnetic resonance (NMR) spectrum was measured 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) was used, 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.

[0263] 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) , amino group (NH2), carboxyl group (COOH) protons, etc. Signals that cannot be confirmed due to the nature of the data are not included in the data.

[0264] In the LC-Mass data, M is the molecular weight, RT is the retention time, [M+H] + ,[M+Na] + means the molecular ion peak.

[0265] 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 of the number of moles of monosaccharide (guluronic acid and mannuronic acid) units that compose alginic acid The molar ratio of reactive substituents to the total amount of the reacted group is intended to indicate the ratio of the moles of the reacted group to the total amount of the reacted group.

[0266] In the examples, sodium alginate before the reactive groups or complementary reactive groups are introduced is The material used was sodium alginate having the physical properties shown in Table 10 above.

[0267] Table 12 shows the results of the arginines to which reactive groups were introduced, obtained in (Example 1) to (Example 15). Acid derivatives (Example 1a, Example 1b, Example 1c, Example 1d, Example 1e, Example 1 f, Example 2, Example 3a, Example 3b, Example 3c, Example 3d, Example 3e, Example 3f, Example 4, Example 5a, Example 5b, Example 6, Example 7a, Example 7b, Example 8, Example 9a, Example 9b, Example 9c, Example 10, Example 11, Example 12, The physical properties of Examples 13, 14, and 15 (specifically, the reactive group introduction rate (mol%), The molecular weight, molecular weight, and weight average molecular weight (10,000 Da) are shown.

[0268] Example 1 Dibenzocyclooctyne-amine group-introduced alginic acid (Example 1a, Example 1b, Example Synthesis of Example 1c, Example 1d, Example 1e, Example 1f, and Example 1g): [ka]

[0269] (Example 1a) Dibenzocyclooctyne-amine group-introduced alginic acid (EX1-(I)-A -2) Synthesis: A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (4 3.6 mL) of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (111.65 mg), 1 molar concentration Aqueous sodium bicarbonate solution (403.5 μL) was added to this solution. -Amine [CAS: 1255942-06-3] (EX1-SM, 83.62 mg) An ethanol solution (2 mL) was added dropwise, and the mixture was stirred at room temperature for 18 hours. 0 mg) was added, followed by addition of ethanol (87.2 mL) and stirring at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound EX1-(I )-A-2 (376 mg) was obtained as a pale yellow solid.

[0270] The introduction rate of reactive substituents (dibenzocyclooctyne-amino groups) was 6.9 mol% (NM R integral ratio).

[0271] (Example 1b) Dibenzocyclooctyne-amine group-introduced alginic acid (EX1-(I)-A -1) Synthesis:

[0272] A 1% by weight aqueous solution of sodium alginate (A-1, manufactured by Mochida Pharmaceutical Co., Ltd.) (1 9.32 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl) -4-Methylmorpholinium chloride (DMT-MM) (49.47 mg), 1 molar concentration Aqueous sodium bicarbonate solution (178.8 μL) was added to this solution. -amine [CAS:1255942-06-3] (EX1-SM, 37.05 mg) An ethanol solution (4 mL) was added dropwise, and the mixture was stirred at room temperature for 20 hours. After adding ethanol (38.64 mL), 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 to obtain the title compound EX1-( I)-A-1 (184 mg) was obtained as a pale yellow solid.

[0273] The introduction rate of reactive substituents (dibenzocyclooctyne-amino groups) was 6.5 mol% (NM R integral ratio).

[0274] (Example 1c) Dibenzocyclooctyne-amine group-introduced alginic acid (EX1-(I)-A -3) Synthesis: A 1% by weight aqueous solution of sodium alginate (A-3, manufactured by Mochida Pharmaceutical Co., Ltd.) (1 5.06 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl) -4-Methylmorpholinium chloride (DMT-MM) (38.57 mg), 1 molar concentration Aqueous sodium bicarbonate solution (139.4 μL) was added to this solution. -Amine [CAS: 1255942-06-3] (EX1-SM, 28.88 mg) An ethanol solution (2 mL) was added dropwise and the mixture was stirred at room temperature for 23 hours. After adding ethanol (60.24 mL), 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 to obtain the title compound EX1-( I)-A-3 (164 mg) was obtained as a pale yellow solid.

[0275] The introduction rate of reactive substituents (dibenzocyclooctyne-amino groups) was 6.6 mol% (NM R integral ratio).

[0276] (Example 1d) Dibenzocyclooctyne-amine group-introduced alginic acid (EX1-(I)-B Synthesis of -2a): A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (5 3.0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (111.0 mg), dibenzothiophene Clooctyne-amine [CAS:1255942-06-3] (EX1-SM, 36.9 mg) in ethanol (5.3 mL), 1 molar sodium bicarbonate solution (113.7 μL) The mixture was stirred at 30°C for 3 hours. Sodium chloride (530 mg) was added, and then ethanol was added. The resulting precipitate was collected by filtration and diluted with ethanol (101 mL). After washing with methanol and drying under reduced pressure, the title compound EX-(I)-B-2a (465 mg) was obtained. Obtained as a coloured solid.

[0277] The introduction rate of reactive substituents (dibenzocyclooctyne-amino groups) was 4.9 mol% (NM R integral ratio).

[0278] (Example 1e) Dibenzocyclooctyne-amine group-introduced alginic acid (EX1-(I)-B Synthesis of -2b): A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (3 5.0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (14.7 mg), dibenzocyclohexane Looctin-amine [CAS:1255942-06-3] (EX1-SM, 4.9 m g), 1 molar sodium bicarbonate solution (17.7 μL), and ethanol (3.5 mL) were added. The mixture was stirred at 0°C for 3.5 hours. Sodium chloride (350 mg) was added, and then ethanol ( 70 mL) was added and stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration and washed with ethanol. After washing, it was dried under reduced pressure to obtain the title compound EX-(I)-B-2 (329 mg) as a white solid. I got it.

[0279] The introduction rate of reactive substituents (dibenzocyclooctyne-amino groups) was 0.8 mol% (NM R integral ratio).

[0280] (Example 1f) Dibenzocyclooctyne-amine group-introduced alginic acid (EX1-(I)-B Synthesis of -2c): A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (6 0.0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (67.0 mg), dibenzocyclohexane Looctin-amine [CAS:1255942-06-3] (EX1-SM, 16.7 mg), 1 molar sodium bicarbonate solution (60.5 μL), and ethanol (6.0 mL) were added. The mixture was stirred at 30°C for 3 hours. Sodium chloride (600 mg) was added, followed by ethanol (1 20 mL) was added and stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration and washed with ethanol. After washing, it was dried under reduced pressure to obtain the title compound EX-(I)-B-2c (558 mg) as a white solid. And obtained.

[0281] The introduction rate of reactive substituents (dibenzocyclooctyne-amino groups) was 1.9 mol% (NM R integral ratio).

[0282] (Example 1g) Dibenzocyclooctyne-amine group-introduced alginic acid (EX1-(I)-A Synthesis of -2b): In the same manner as in Example 1a, the introduction rate of reactive substituents (NMR integral ratio) was 4.9 m % of the title compound (EX1-(I)-A-2b) was obtained.

[0283] (Example 2) N-(1R,8S,9s)-bicyclo[6.1.0]non-4-yne-9- Arginine containing 1,8-ylmethoxycarbonyl-1,8-diamino-3,6-dioxaoctane group Synthesis of the acid (compound EX2-(I)-A-2): [ka]

[0284] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (1 0.9 mL) at room temperature, Dimethylmorpholinium chloride (DMT-MM) (27.91 mg), 1 A molarity-based sodium bicarbonate solution (100.9 μL) was added. ,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl-1, 8-Diamino-3,6-dioxaoctane [CAS1263166-93-3] (EX- A solution of 2-SM (24.54 mg) in ethanol (2 mL) and water (1 mL) was The mixture was stirred at the same temperature for 21 hours. Sodium chloride (100 mg) was added, and then Ethanol (21.8 mL) was added and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration. After washing with ethanol, the mixture was dried under reduced pressure to give the title compound EX2-(I)-A-2 (100 ml g) was obtained as a pale yellow solid.

[0285] Reactive Substituent (N-(1R,8S,9s)-bicyclo[6.1.0]non-4-yne Introduction of 9-ylmethoxycarbonyl-1,8-diamino-3,6-dioxaoctane group The conversion was 5.8 mol% (NMR integral ratio).

[0286] Example 3: 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide Alginic acid with functional groups (Example 3a, Example 3b, Example 3c, Example 3d, Example 3e, Synthesis of Example 3f and Example 3g): [ka]

[0287] <Step 1> Methyl 4-(2-((tert-butoxycarbonyl)amino)ethoxy) Synthesis of benzoate (compound EX3-IM-1): [ka]

[0288] Dissolve triphenylphosphine (0.96 g) in tetrahydrofuran (2.59 mL). Add diethyl azodicarboxylate (40% toluene solution, 1.92 mL) to the solution under ice cooling and stirring. The solution was added and stirred at room temperature for 20 minutes. Methyl benzoate [CAS:99-76-3] (compound EX3-SM, 0.37 g ) and 2-(tert-butoxycarbonyl)ethanolamine [CAS:26690- A solution of 80-2 (0.39 g) in tetrahydrofuran (1.1 mL) was added and the mixture was stirred at room temperature. The reaction mixture was stirred for 17 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography. - (5% ethyl acetate / n-heptane to 40% ethyl acetate / n-heptane) A mixture of Compound 1 and Compound 2 was obtained. This mixture was dissolved in methyl tert-butyl ether ( The solution was dissolved in 20 mL of 1N sodium hydroxide solution (5 mL) twice and saturated The organic layer was washed with brine (5 mL), dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The solvent was removed by distillation, and compound EX3-IM-1 (0.45 g) was obtained as a pink oily substance. And obtained.

[0289] NMR data (CDCl3)(δ:ppm):7.98 (2H, d, J = 8. 8 Hz), 6.90 (2H, d, J = 8.8 Hz), 4.97 (1 H, br s), 4.07 (2H, t, J = 5.2 Hz), 3.88 (3H, s), 3.56 (2H, q, J = 5.2 Hz), 1.45 (9H, s)

[0290] <Step 2> 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide hydrochloride Synthesis of salt (compound EX3-IM-3): [ka]

[0291] (Example 3) Compound EX3-IM-1 (0.44 g) obtained in <Step 1> was mixed with ethanol to obtain Lithium hydroxide monohydrate (0.25 g) was added to a solution of ethanol (4.4 mL) and heated at 60 °C. The mixture was stirred for 3 hours and 30 minutes. 1N hydrochloric acid (5 mL) was added to the reaction mixture, and ethyl acetate (10 The organic layer was washed with water (5 mL) and saturated saline (5 mL) in that order. The solvent was removed under reduced pressure. The residue was dissolved in acetonitrile ( 4.4 mL) and dissolved in 3-azidopropan-1-amine [CAS:88192-1 9-2] (0.15 g) and O-(7-azabenzotriazol-1-yl)-N,N, N',N'-tetramethyluronium hexafluorophosphate (0.57 g) was added. Next, N,N-diisopropylethylamine (0.52 mL) was added under ice cooling and stirring. The mixture was stirred at room temperature for 5 hours. Water (10 mL) was added to the reaction mixture, and ethyl acetate (15 mL) was added. The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane ~ 10 0% ethyl acetate) and the fraction containing compound EX3-IM-2 (0.71 g) was Got it.

[0292] The fraction containing compound EX3-IM-2 (0.71 g) was diluted with 4N-hydrogen chloride / 1, 4-Dioxane (4.9 mL) was added and the mixture was stirred at room temperature for 20 minutes. After adding pyroether, the precipitate was filtered to obtain the title compound EX3-IM-3 (0. 49 g) was obtained as a white solid.

[0293] NMR data (CDCl3)(δ:ppm):7.60 (2H, d, J = 8. 8 Hz), 6.93 (2H, d, J = 8.8 Hz), 4.19 (2 H, t, J = 4.8 Hz), 3.31-3.29 (6H, m), 1. 77-1.71 (2H, m).LC-MS:M(free amine)=263, RT=0.54(min), [M+H] + =264

[0294] (Example 3a) 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide Synthesis of doped alginic acid (compound EX3-(II)-A-2): [ka]

[0295] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (1 9.6 mL) and add 4-(4,6-dimethoxy-1,3,5-triazine- 2-yl)-4-methylmorpholinium chloride (DMT-MM) (50.19 mg) Compound EX3-IM-3 (54.37 mg) obtained in Step 2 of Example 3, Molar concentration-sodium bicarbonate solution (181.4 μL) was added and stirred at room temperature for 5 hours. (200 mg) was added, followed by ethanol (39.2 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 to obtain the title compound EX3. -(II)-A-2 (198 mg) was obtained as a white solid.

[0296] Reactive Substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide The introduction rate of the aryl group was 6.1 mol% (NMR integral ratio).

[0297] Example 3b: 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide Synthesis of doped alginic acid (compound EX3-(II)-A-1): [ka]

[0298] A 1% by weight aqueous solution of sodium alginate (A-1, manufactured by Mochida Pharmaceutical Co., Ltd.) (1 9.32 mL) and 4-(4,6-dimethoxy-1,3,5-triazine -2-yl)-4-methylmorpholinium chloride (DMT-MM) (49.47 mg ), Compound EX3-IM-3 (53.39 mg) obtained in (Example 3) <Step 2>, 1M sodium bicarbonate solution (178.8 μL) was added and stirred at room temperature for 20 hours. After adding ethanol (38.64 mL), the mixture was incubated at room temperature for 30 minutes. The mixture was stirred at room temperature. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain compound EX. -(II)-A-1 (221 mg) was obtained as a white solid.

[0299] Reactive Substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide The introduction rate of the aryl group was 9.4 mol% (NMR integral ratio).

[0300] Example 3c 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide Synthesis of doped alginic acid (compound EX3-(II)-A-3): [ka]

[0301] A 1% by weight aqueous solution of sodium alginate (A-3, manufactured by Mochida Pharmaceutical Co., Ltd.) (1 5.06 mL) and add 4-(4,6-dimethoxy-1,3,5-triazine under ice-cooling and stirring. -2-yl)-4-methylmorpholinium chloride (DMT-MM) (38.57 mg ), Compound EX3-IM-3 (41.78 mg) obtained in (Example 3) <Step 2>, 1M sodium bicarbonate solution (139.4 μL) was added and stirred at room temperature for 5 hours. After adding ethanol (60.24 mL), the mixture was incubated at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain compound EX3. -(II)-A-3 (155 mg) was obtained as a white solid.

[0302] Reactive Substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide The introduction rate of the aryl group was 6.9 mol% (NMR integral ratio).

[0303] (Example 3d) 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide Synthesis of doped alginic acid (compound EX3-(II)-B-2a): [ka]

[0304] A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (6 0.0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (125.6 mg), (Example 3 ) Compound EX3-IM-3 (45.4 mg) obtained in <Step 2>, 1 molar concentration of sodium bicarbonate Water (211.8 μL) was added and the mixture was stirred at 30° C. for 3 hours. g) was added, followed by addition of ethanol (120 mL), and the mixture was stirred at room temperature for 30 minutes. The precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound EX3-(II)-A. -2 (553 mg) was obtained as a white solid.

[0305] Reactive Substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide The introduction rate of the aryl group was 3.7 mol% (NMR integral ratio).

[0306] (Example 3e) 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide Synthesis of doped alginic acid (compound EX3-(II)-B-2b): [ka]

[0307] A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (3 5.0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (14.7 mg), (Example 3) Compound EX3-IM-3 (5.3 mg) obtained in <Step 2> and 1 molar sodium bicarbonate solution ( 26.5 μL) was added and stirred at 30° C. for 3.5 hours. After adding ethanol (70 mL), the mixture was stirred at room temperature for 30 minutes. The precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound EX3-(II)-A-2. (304 mg) was obtained as a white solid.

[0308] Reactive Substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide The introduction rate of the aryl group was 0.6 mol % (NMR integral ratio).

[0309] (Example 3f) 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide Synthesis of doped alginic acid (compound EX3-(II)-B-2c): [ka]

[0310] A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (6 0.0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (67.0 mg), (Example 3) Compound EX3-IM-3 (18.1 mg) obtained in <Step 2>, 1 molar sodium bicarbonate solution (90.8 μL) was added and the mixture was stirred at 30° C. for 3 hours. Sodium chloride (600 mg) After adding ethanol (120 mL), the mixture was stirred at room temperature for 30 minutes. The precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound EX3-(II)-A-2. (568 mg) was obtained as a white solid.

[0311] Reactive Substituent (4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide The introduction rate of the aryl group was 1.5 mol % (NMR integral ratio).

[0312] Example 3g: 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide Synthesis of functionalized alginic acid (EX3-(II)-A-2b): In the same manner as in Example 3a, the introduction rate of reactive substituents (NMR integral ratio) was 4.3. The title compound (EX3-(II)-A-2b) was obtained in mol %.

[0313] Example 4: 4-(3-aminopropoxy)-N-(2-(2-azidoethoxy) )Ethoxy)ethyl)benzamide group-introduced alginic acid (compound EX4-(II)-A-2 ) synthesis: [ka]

[0314] <Step 1> 4-(3-((tert-butoxycarbonyl)amino)propoxy)benzoic acid Synthesis of aromatic acid (compound EX4-IM-2): [ka]

[0315] A solution of triphenylphosphine (2.07 g) in tetrahydrofuran (7 mL) was Diisopropyl azodicarboxylate (40% toluene solution, 4.15 mL) was added and the mixture was precipitated. The mixture was stirred until a precipitate formed. After stirring for an additional hour, commercially available tert-butyl(3-hydroxypropyl) (xypropyl)carbamate [CAS: 58885-58-8] (1.15 g) and 4 -Hydroxybenzoic acid methyl ester [CAS:99-76-3] (compound EX4-SM A solution of 1 g) in tetrahydrofuran (3 mL) was added and stirred for 3 hours. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (8% ethyl acetate / n-heptane The product was purified by elution with 20% ethyl acetate / n-heptane (66% ethyl acetate / n-heptane). Dissolve in t-butyl ether (20 mL) and add 1N sodium hydroxide solution (5 The organic layer was washed with saturated saline (5 mL) twice, and then with anhydrous sodium sulfate (1 mL). After drying, the solvent was removed under reduced pressure to obtain compound EX4-IM-1 (2.94 g). The fraction was obtained as a white solid.

[0316] Fractions containing compound EX4-IM-1 (2.94 g) in methanol (15.6 mL) Lithium hydroxide monohydrate (1.06 g) was added to the solution under stirring at room temperature, and the mixture was heated to 60°C. The mixture was stirred for 3 hours. After cooling to room temperature, the solvent was distilled off under reduced pressure. The mixture was extracted twice with methyl tert-butyl ether (20 mL). The layer was acidified with 1N hydrochloric acid (25 mL) and extracted three times with ethyl acetate (20 mL). The organic layer was washed with water (10 mL) and saturated saline (10 mL) in that order. After drying over sodium, the solvent was removed under reduced pressure. Add diethyl ether (30 mL) and 1N aqueous sodium hydroxide solution (20 mL) and The aqueous layer was extracted twice with 1N hydrochloric acid ( The mixture was acidified with ethyl acetate (20 mL) and extracted twice with ethyl acetate (20 mL). After drying over sodium sulfate, the solvent was removed under reduced pressure to give compound 4-3 (1.4 g). Obtained as a white solid.

[0317] NMR data (CDCl3)(δ:ppm):8.03 (2H, d, J = 7. 6 Hz), 6.92 (2H, d, J = 8.8 Hz), 4.73 (1 H, br s), 4.09 (2H, t, J = 6.0 Hz), 3.34 (2H, q, J = 6.3 Hz), 2.05-1.98 (2H, m), 1.45 (9H, s)

[0318] <Step 2> 4-(3-aminopropoxy)-N-(2-(2-azidoethoxy) Synthesis of Ethoxy)ethyl)benzamide hydrochloride (compound EX4-IM-4): [ka]

[0319] (Example 4) Compound EX4-IM-2 (1 g) obtained in <Step 1>, commercially available 2-( 2-(2-Azidoethoxy)ethoxy)ethan-1-amine [CAS:166388-5 7-4] (0.62 g) and O-(7-azabenzotriazol-1-yl)-N,N Acetate of ,N',N'-tetramethyluronium hexafluorophosphate (1.35 g) A solution of N,N-diisopropylethylamine was added to a solution of 20 mL of triisopropylamine under ice cooling and stirring. The reaction mixture was stirred at room temperature for 1 hour. The mixture was extracted three times with ethyl acetate (20 mL), and then diluted with water (10 mL) and saturated saline (1 The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane The extract was purified by hexane to 100% ethyl acetate (hexane to 100% ethyl acetate) to give compound EX4-IM-3 (1.37 g). A fraction containing 1,000 mercaptoethanol was obtained.

[0320] The fraction containing compound EX4-IM-3 (1.37 g) was diluted with 1,4-dioxane (9 To this solution, 4N-hydrogen chloride / 1,4-dichloromethane was added under stirring while cooling with water. C. hexane (9.58 mL) was added and the mixture was stirred at room temperature for 1 hour. After adding ether (100 mL), the suspension was stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure. The residue was dissolved in ethyl acetate (20 mL) and methyl tert-butyl ether (10 The resulting solid was filtered and dried under reduced pressure to give the title compound. Compound EX4-IM-4 (1.23 g) was obtained as a white solid.

[0321] NMR data (D2O) (δ: ppm): 7.66-7.64 (2H, m), 6. 98-6.94 (2H, m), 4.12 (2H, t, J = 5.6 Hz ), 3.66-3.57 (6H, m), 3.57-3.52 (2H, m), 3.47 (2H, t, J = 5.2 Hz), 3.29 (2H, t, J = 4.8 Hz), 3.12 (2H, t, J = 7.2 Hz), 2 .10-2.04 (2H, m), LC-MS:M(free amine)=351 , RT=0.57(min), [M+H] + =352

[0322] <Step 3> 4-(3-aminopropoxy)-N-(2-(2-azidoethoxy) Ethoxy)ethyl)benzamide group-introduced alginic acid (compound EX4-(II)-A-2) Synthesis of: [ka]

[0323] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (1 9.6 mL) and add 4-(4,6-dimethoxy-1,3,5-triazine- 2-yl)-4-methylmorpholinium chloride (DMT-MM) (50.19 mg) Compound EX4-IM-4 (70.35 mg) obtained in Step 2 of Example 4, Molar concentration-sodium bicarbonate solution (181.4 μL) was added and stirred at room temperature for 5 hours. (200 mg) was added, followed by ethanol (39.2 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 to obtain the title compound EX4. -(II)-A-2 (199 mg) was obtained as a white solid.

[0324] Reactive Substituent (4-(3-aminopropoxy)-N-(2-(2-azidoethoxy) The introduction rate of the (ethoxy)ethyl)benzamide group was 4.3 mol% (NMR integral ratio). there were.

[0325] (Example 5) N-(2-aminoethyl)-4-(azidomethyl)benzamide group-introduced alkanol Synthesis of acetic acid (Example 5a, 5b and 5c): [ka]

[0326] <Step 1> tert-Butyl(2-(4-(chloromethyl)benzamido)ethyl)carbamate Synthesis of bamate (compound EX5-IM-1): [ka]

[0327] EX5-SM (4-(chloromethyl)benzoyl chloride, [CAS:876-08 -4] (2.0 g) was dissolved in tetrahydrofuran (10.0 mL) and tert- Tyl(2-aminoethyl)carbamate [CAS:57260-73-8](1.7 g ) and N,N'-diisopropylethylamine (3.7 mL) in tetrahydrofuran (1 A solution of ethyl acetate (0.0 mL) was added dropwise to the reaction mixture while cooling with ice water, and the mixture was stirred at room temperature for 1.5 hours. Chilled water (30 mL) and water (10 mL) were added and the layers were separated. The organic layer was diluted with half-saturated sodium bicarbonate water (10 The mixture was washed with water (10 mL), saturated saline (5 mL), and then with sodium sulfate anhydride. The residue was triturated with tert-butyl methyl ether, and the mixture was concentrated under reduced pressure. The resulting solid was then filtered and washed with tert-butyl methyl ether to give the title compound. EX5-IM-1 (2.9 g) was obtained as a white solid.

[0328] NMR data (CDCl3) (δ:ppm): 7.81 (2H, d, J = 8 Hz ), 7.44(2H, d, J = 8 Hz), 7.24(1H, brs), 4.96( 1H, brs), 4.60(2H, s), 3.56(2H, q, J = 5 Hz), 3 .45-3.38(2H, m), 1.43(9H, s)

[0329] <Step 2> tert-Butyl(2-(4-(azidomethyl)benzamido)ethyl)carbamate Synthesis of bamate (EX-IM-2): [ka]

[0330] Dissolve sodium azide (100 mg) in dimethyl sulfoxide (6.0 mL). Compound EX5-IM-1 (400 mg) obtained in (Example 5) <Step 1> was added, The mixture was stirred at room temperature for 2.5 hours. Water (12 mL) was added to the reaction mixture under ice-water cooling, and the precipitated solid was The solid was filtered and washed with water. The obtained solid was dried under reduced pressure at 50° C. to obtain the title compound EX5-I. M-2 (380 mg) was obtained as a white solid.

[0331] NMR data (CDCl3) (δ:ppm): 7.84 (2H, d, J = 8 Hz ), 7.37(2H, d, J = 8 Hz), 7.22(1H, brs), 4.95( 1H, brs), 4.39(2H, s), 3.56(2H, q, J = 5 Hz), 3 .45-3.38(2H, m), 1.43(9H, s)

[0332] <Step 3> N-(2-aminoethyl)-4-(azidomethyl)benzamide hydrochloride ( Synthesis of compound EX5-IM-3): [ka]

[0333] (Example 5) Compound EX5-IM-2 (250 mg) obtained in <Step 2> was added to ice water. Add 4N hydrogen chloride / 1,4-dioxane (1.75 mL) under cooling and let the mixture stand at room temperature for 1 hour. The reaction mixture was stirred for 1 h. Diisopropyl ether (5.25 mL) was added to the reaction mixture, and the resulting precipitate was The compound was filtered, washed with diisopropyl ether, and dried under reduced pressure to obtain the title compound EX5-IM- Obtained 3 (192 mg) as a white solid.

[0334] NMR data (DMSO-d6) (δ: ppm): 8.68 (1H, t, J = 6 Hz), 7.91 (2H, d, J = 8 Hz), 7.80 (3H, brs), 7.4 7(2H, d, J = 8 Hz), 4.53(2H, s), 3.51(2H, q, J = 6 Hz), 2.98(2H, t, J = 6 Hz), LC-MS:M(free amine)=219, RT=0.56(min), [M+H] + =220

[0335] <Step 4-1> (Example 5a) N-(2-aminoethyl)-4-(azidomethyl)benz Synthesis of amido-alginic acid (compound EX5-(II)-A-2): [ka]

[0336] 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) (84 mg), (Example 5) < Step 3 Compound EX5-IM-3 (52 mg) obtained in step 1 and 1 molar sodium bicarbonate solution (252 μL) were The mixture was stirred at 30°C for 3 hours. Sodium chloride (200 mg) was added, and then ethanol was added. Ethanol (40 mL) was added and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration and diluted with ethanol. After washing with alcohol, the mixture was dried under reduced pressure to give the title compound EX5-(II)-A-2 (185 mg). was obtained as a white solid.

[0337] Reactive Substituent (N-(2-aminoethyl)-4-(azidomethyl)benzamide group) The introduction rate was 9.4 mol% (NMR integral ratio).

[0338] <Step 4-2> (Example 5b) N-(2-aminoethyl)-4-(azidomethyl)benzene Synthesis of alginic acid with azamido group (compound EX5-(II)-B-2): [ka]

[0339] A 1% by weight aqueous solution of sodium alginate (B-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) (84 mg), (Example 5) < Step 3 Compound EX5-IM-3 (26 mg) obtained in step 1 and 1 molar sodium bicarbonate solution (151 μL) were The mixture was stirred at 30°C for 3 hours. Sodium chloride (200 mg) was added, and then ethanol was added. Ethanol (40 mL) was added and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration and diluted with ethanol. After washing with alcohol, the mixture was dried under reduced pressure to give the title compound EX5-(II)-B-2 (187 mg). was obtained as a white solid.

[0340] Introduction of reactive group (N-(2-aminoethyl)-4-(azidomethyl)benzamide group) The conversion was 11 mol% (NMR integral ratio).

[0341] (Example 5c) N-(2-aminoethyl)-4-(azidomethyl)benzamide group introduction Synthesis of arginic acid (EX5-(II)-A-2b): In the same manner as in Example 5a, the introduction rate of reactive substituents (NMR integral ratio) was 4.9. The title compound (EX5-(II)-A-2b) was obtained in mol %.

[0342] Example 6 N-(3-aminopentynyl)-5,6-dihydro-11,12-didehy Alginic acid with dolodibenzo[b,f]azocine group (ADIBO-C3-amine) (compound Synthesis of product EX6-(I)-B-2): [ka]

[0343] <Step 1> N-trifluoroacetyl-5-aminopentanoic acid (EX6-IM-1) Synthesis: [ka]

[0344] 5-Aminopentanoic acid (EX6-SM1, [CAS:660-88-8] 2.0 g) , trifluoroacetic acid ethyl ester (3.1 mL), triethylamine (3.6 mL ) was dissolved in methanol (90.0 mL) and stirred at 40° C. for 5 hours. The reaction solution was concentrated under reduced pressure. The residue was concentrated under reduced pressure and ethanol (10 mL) was added to the residue, which was then concentrated twice. Dissolve in ethyl acetate (200 mL) and add 0.1 molar sodium dihydrogen phosphate aqueous solution. The organic layer was washed three times with saturated aqueous sodium chloride (50 mL) and then with anhydrous sodium sulfate. The extract was dried over an aqueous solution of 1.8 g of the title compound EX6-IM-1. was obtained as a white solid.

[0345] NMR data (DMSO-d6) (δ: ppm): 12.04 (1H, brs), 9. 43(1H, brs), 3.17(2H, q, J = 6 Hz), 2.22(2H, H , tt, J = 7, 2 Hz), 1.51-1.46(4H, m)

[0346] <Step 2> (Z)-N-(5-(dibenzo[b,f]azocin-5(6H)-yl)- Synthesis of 5-oxopentyl-trifluoroacetamide (compound EX6-IM-2): [ka]

[0347] (Example 6) Compound EX6-IM-1 (617 mg) obtained in <Step 1> was added with chloride. Add thionyl (440 μL) and N,N-dimethyl sulfoxide (2 μL) and heat at 80°C. The mixture was stirred at rt for 1.5 hours, and the reaction mixture was concentrated under reduced pressure. According to the method described in [Production Method D] <Step 1>, Compound EX6-SM2 [CAS: 23294-93-6] synthesized from 22-33-5] (500 mg), pyridine (585 μL) in methylene chloride (5.0 mL) on ice. The reaction mixture was added under cooling with water and stirred at room temperature for 30 minutes. The mixture was diluted with 1N hydrochloric acid (10 mL), water (10 mL), 1N hydrochloric acid (10 mL), water (10 The mixture was washed with saturated aqueous sodium chloride (5 mL), dried over anhydrous sodium sulfate, and then The residue was purified by silica gel column chromatography (heptane to 60% After purification with ethyl acetate / heptane, the obtained solid was The solid was filtered and washed with heptane to give the title compound. The product EX6-IM-2 (840 mg) was obtained as a white solid.

[0348] NMR data (CDCl3) (δ:ppm): 7.37-7.27(4H, m), 7. 22-7.14(4H, m), 7.08(1H, brs), 6.76(1H, d, J = 13 Hz), 6.57(1H, d, J = 13 Hz), 5.43(1H, d, J = 15 Hz), 4.17(1H, d, J = 15 Hz), 3.22(1H, d t, J = 13, 6 Hz), 2.83(1H, dt, J = 13, 6 Hz), 2 .22-2.12(1H, m), 1.87(1H, dq, J = 16, 5 Hz), 1 .68-1.58(1H, m), 1.52-1.36(2H, m), 1.28-1.16 (1H,m), LC-MS:M=402, RT=1.05(min), [M+H] + =403

[0349] <Step 3> N-(5-(11,12-dibromo-11,12-dihydrodibenzo[b, f]Azocin-5(6H)-yl)-5-oxopentyl-trifluoroacetamide (chemical Synthesis of compound EX6-IM-3): [ka]

[0350] (Example 6) Compound EX6-IM-2 (700 mg) obtained in <Step 2> was dissolved in methylene chloride. Pyridinium bromide perbromide (612 mg) was added to a solution of 2.8 mL of toluene on ice. The mixture was added under cooling with water, and stirred at room temperature for 1.5 hours. After that, pyridinium bromide perbromide (11 The reaction mixture was diluted with ethyl acetate (20 mL) and stirred at room temperature for 1 hour. The mixture was diluted and washed with 2N hydrochloric acid (10 mL) and saturated saline (5 mL) in that order. After drying over anhydrous sodium sulfate, the mixture was concentrated under reduced pressure to give the crude title compound EX6-IM-3 (1. 03 g) was obtained as a yellow amorphous solid.

[0351] LC-MS: M=562, RT=1.10 (min), [M+H] + =563(561:56 3:565=1:2:1)

[0352] <Step 4> N-(3-aminopentynyl)-5,6-dihydro-11,12-didehydro Synthesis of dibenzo[b,f]azocine-trifluoroacetamide (EX6-IM-4): [ka]

[0353] (Example 6) Tetracycline synthesis of crude compound EX6-IM-3 (100 mg) obtained in <Step 3> In a solution of trihydrofuran (1.5 mL) potassium tert-butoxide (100 mg) The reaction mixture was diluted with ethyl acetate (15 mL) and added in small portions over a period of 8 hours. The organic layer was diluted and washed with water (3 mL) and saturated saline (2 mL) in that order. After drying over sodium hydroxide, the mixture was concentrated under reduced pressure to give the crude compound EX6-IM-4 (58 mg). Obtained as a pale brown gum.

[0354] NMR data (CDCl3)(δ:ppm): 7.69(1H, d, J = 7 Hz ), 7.44-7.23(7H, m), 5.17(1H, d, J = 14 Hz), 3 .70(1H, d, J = 14 Hz), 3.21(1H, dt, J = 13, 6 Hz), 2.57(1H, dq, J = 19, 5 Hz), 2.36-2.28(1H , m), 1.82 (1H, dq, J = 16, 5 Hz), 1.46-1.34 (2H , m), 1.29-1.24(1H,m), 1.15-1.05(1H,m), LC-M S:M=400, RT=1.08(min), [M+H] + =401, [M+Na] + =42 3

[0355] <Step 5> N-(3-aminopentynyl)-5,6-dihydro-11,12-didehyde Synthesis of rhodibenzo[b,f]azocine (compound EX6-IM-5): [ka]

[0356] (Example 6) Methanolysis of crude compound EX6-IM-4 (58 mg) obtained in <Step 4> In a solution of 1.2 mL of ethanol, add 40 mg of potassium carbonate in 0.25 mL of water. The reaction mixture was concentrated, and then diluted with ethyl acetate (10 mL), methylene chloride (1 mL), and stirred at room temperature for 23 hours. Ethylene (1 mL) and half-saturated saline (2 mL) were added and the layers were separated. The organic layer was extracted with anhydrous sodium sulfate. The gum was purified by silica gel chromatography (acetic acid The title compound EX6-IM-5 (2 2 mg) was obtained as a colorless gum.

[0357] NMR data (CDCl3)(δ:ppm): 7.70(1H, d, J = 8 Hz ), 7.43-7.23(7H, m), 5.18(1H, d, J = 14 Hz), 3 .65(1H, d, J = 14 Hz), 2.45(2H, t, J = 7 Hz), 2.24-2.16(1H, m), 1.96-1.89(1H, m), 1.48-1.3 8(2H,m), 1.21-1.10(2H,m), LC-MS:M=304, RT=0 .76(min), [M+H] + =305

[0358] <Step 6> N-(3-aminopentynyl)-5,6-dihydro-11,12-didehyde Lodibenzo[b,f]azocine (ADIBO-C3-amino) group-introduced alginate (EX6 Synthesis of -(I)-B-2): [ka]

[0359] A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (2 8.5 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (60 mg), (Example 6) Compound EX6-IM-5 (22 mg) obtained in step 5 was dissolved in ethanol (2.9 mL). The solution was added with 1 molar sodium bicarbonate solution (72 μL) and stirred at 30° C. for 3 hours. Add methanol (285 mg), then add ethanol (57 mL), and stir at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain the title compound EX5. -(II)-B-2 (277 mg) was obtained as a white solid.

[0360] Reactive group (N-(3-aminopentynyl)-5,6-dihydro-11,12-didehyde The introduction rate of the rhodibizo[b,f]azocine (ADIBO-C3-amino) group was 2.7 The ratio was expressed as mol% (NMR integral ratio).

[0361] (Example 7) N-(2-aminoethyl)-4-azidobenzamide group-introduced alginic acid ( Synthesis of Examples 7a, 7b, and 7c): [ka]

[0362] <Step 1> tert-Butyl (2-(4-azidobenzamido)ethyl)carbamate ( Synthesis of compound EX7-IM-1): [ka]

[0363] 4-Azidobenzoic acid (EX7-SM, [CAS:6427-66-3] 700 mg) Add thionyl chloride (783 μL) and N,N-dimethyl sulfoxide (3 μL) to the solution. The mixture was stirred at 70° C. for 1 hour. The reaction mixture was concentrated under reduced pressure, and methylene chloride (1 mL) was added to the residue. ert-Butyl (2-aminoethyl) carbamate [CAS: 57260-73-8] ( 825 mg), pyridine (1.04 mL) in methylene chloride (7.0 mL) on ice. The reaction mixture was added under cooling with water and stirred at room temperature for 1 hour. (30 mL), water (10 mL), saturated sodium bicarbonate water (5 mL), 0.5 N-Q The organic layer was washed successively with phosphate (5 mL twice), water (5 mL), and saturated saline (5 mL). The extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Triturate with ether / heptane, filter the solid, and then add tert-butyl methyl The title compound EX7-IM-1 (1.1 g) was obtained as a white solid by washing with diethyl ether / heptane. Got it as a body.

[0364] NMR data (CDCl3) (δ:ppm): 7.83 (2H, d, J = 8 Hz ), 7.26(1H, brs), 7.05(2H, d, J = 8 Hz), 4.97( 1H, brs), 3.55(2H, q, J = 5 Hz), 3.45-3.37(2H , m), 1.43 (9H, s), LC-MS: M = 305, RT = 0.90 (min), [M +H] + =306, [M+Na] + =328

[0365] <Step 2> N-(2-aminoethyl)-4-azidobenzamide hydrochloride (compound EX7- Synthesis of IM-2): [ka]

[0366] (Example 7) The compound obtained in <Step 1> (EX7-IM-1, 500 mg) was The mixture was suspended in 4-dioxane (1.5 mL) under ice-water cooling. The reaction mixture was stirred at room temperature for 1 hour. (10.5 mL) was added and stirred at room temperature for 50 minutes. The solid was filtered and diluted with diisopropyl ether. After washing with ether, the mixture was dried under reduced pressure to give the title compound EX7-IM-2 (365 mg). Obtained as a beige solid.

[0367] NMR data (DMSO-d6) (δ: ppm): 8.68 (1H, t, J = 6 Hz), 7.93(2H, d, J = 9 Hz), 7.82(1H, brs), 7.2 2(2H, d, J = 9 Hz), 3.49(2H, q, J = 6 Hz), 2.9 7(2H, t, J = 6 Hz), LC-MS: M(free amine) = 205 , RT=0.56(min), [M+H] + =206

[0368] <Step 3-1> (Example 7a) N-(2-aminoethyl)-4-azidobenzamide derivative Synthesis of alginic acid (EX7-(II)-B-2a): [ka]

[0369] A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (3 0.0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (63 mg), (Example 7) Compound EX7-IM-2 (18 mg) obtained in step 2, 1 molar sodium bicarbonate solution (114 After adding sodium chloride (300 mg), the mixture was stirred at 30°C for 3 hours. Ethanol (60 mL) was added and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration. After washing with ethanol, the product was dried under reduced pressure to obtain the title compound EX7-(II)-B-2a (282 mg) as a white solid.

[0370] The introduction rate of the reactive group (N-(2-aminoethyl)-4-azidobenzamide group) was 5.1 The ratio was expressed as mol% (NMR integral ratio).

[0371] <Step 3-2> (Example 7b) N-(2-aminoethyl)-4-azidobenzamide derivative Synthesis of alginic acid (EX7-(II)-B-2b): [ka]

[0372] A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (6 0.0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (67 mg), (Example 7) Compound EX7-IM-2 (15 mg) obtained in step 2, 1 molar sodium bicarbonate solution (91 After adding sodium chloride (600 mg), the mixture was stirred at 30° C. for 3 hours. Ethanol (120 mL) was added and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration. After washing with ethanol and drying under reduced pressure, the title compound EX7-(II)-B-2b (560 mg) as a white solid.

[0373] The introduction rate of the reactive group (N-(2-aminoethyl)-4-azidobenzamide group) was 2.0 The ratio was expressed as mol% (NMR integral ratio).

[0374] (Example 7c) N-(2-aminoethyl)-4-azidobenzamide group-introduced alginic acid ( Synthesis of EX7-(II)-A-2): The introduction rate of reactive substituents ( As a result, the title compound (EX7-(II)-A-2) was obtained with a molecular weight of 5.0 mol% (NMR integral ratio).

[0375] (Example 8) N-(4-(aminoethyl)benzyl)-2-(cyclooct-2-yne Synthesis of 1-(1-hydroxy)acetamide group-introduced alginic acid (EX8-(I)-B-2): [ka]

[0376] <Step 1> tert-Butyl (4-(4((2,2,2-trifluoroacetamide) Synthesis of methyl)benzyl)carbamate (compound EX8-IM-1): [ka]

[0377] According to the method known in the literature (Bioorganic & Medicinal Chemistry y(2003)11:4189-4206) and 1,4-bis(aminomethyl) benzyl tert-Butyl (4-(aminoethyl)benzene [CAS: 539-48-0] synthesized Benzyl)carbamate [CAS:108468-80-4] (EX8-SM1,0 .67 g), triethylamine (0.39 mL) and methanol (6.67 mL). To the mixture was added dropwise ethyl trifluoroacetate (0.44 mL) under stirring and ice cooling. The reaction mixture was warmed to room temperature and stirred at the same temperature for 5 hours. The reaction was quenched with water (10 mL) and The mixture was extracted three times with ethyl acetate (10 mL). The collected organic layer was washed with saturated saline (5 mL). The organic layer was filtered, concentrated, and the crude product was extracted with 1,000 ml of ethyl acetate. Compound EX8-IM-1 (0.671 g) was obtained as a pale yellow amorphous substance.

[0378] NMR data (CDCl3)(δ:ppm):δ: 7.29 (2H, d, J = 8.4 Hz), 7.25 (2H, d, J = 7.6 Hz), 6.51 (1H, br s), 4.86 (1H, br s), 4.51 (2H, d , J = 5.2 Hz), 4.31 (2H, d, J = 6.0 Hz), 1.46 (9H, s).LC-MS:M=332,RT=0.97(min),[M+ Na]+=355.

[0379] <Step 2> N-(4-(aminoethyl)benzyl)-2,2,2-trifluoroacetoacetate Synthesis of amide hydrochloride (compound EX8-IM-2): [ka]

[0380] (Example 8) Compound EX8-IM-1 (0.5 g) obtained in <Step 1>, 1,4- To a dioxane solution (3.5 mL), 4N-hydrogen chloride / 1,4-dioxane was added under stirring in a water-cooled bath. Xanthene (3.5 mL) was added and the mixture was stirred at room temperature for 3 hours. After adding ethanol (40 mL), the precipitate was filtered to obtain the title compound EX8-IM-2( 0.36 g) was obtained as a white solid.

[0381] NMR data (D2O)(δ:ppm):δ: 7.29 (2H, d, J = 8 .0 Hz), 7.25 (2H, d, J = 8.4 Hz), 4.38 ( 2H, s), 4.02 (2H, s).LC-MS:M(free amine) =232,RT=0.53(min),[M+H]+=233.

[0382] <Step 3> N-(4-((2-(cyclooct-2-yn-1-yloxy)acetamide (methyl)benzyl)-2,2,2-trifluoroacetamide (compound EX8-IM -3) Synthesis: [ka]

[0383] A method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110) was synthesized from cycloheptene [CAS: 628-92-2]. Carboxylic acid [CAS: 917756-42-4] (EX8-SM2, 0.17 g) and O -(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurethane A solution of 0.26 g of hexafluorophosphate in 1.7 mL of acetonitrile EX8-IM-2 (0.26 ml) obtained in step 2 of Example 8 was added to the mixture under ice-cooling and stirring. g) and N,N-diisopropylethylamine (0.51 mL) were added dropwise, and the mixture was stirred at room temperature for 1 hour. The mixture was stirred for 30 minutes. Water (5 mL) was added to quench the reaction, and then ethyl acetate (5 mL) was added. The organic layer was washed with saturated saline (3 mL) and then extracted with anhydrous sodium sulfate. The organic layer was dried and filtered, and the solvent was removed under reduced pressure. The residue was concentrated with silica gel. Column chromatography (12% ethyl acetate / n-heptane to 100% ethyl acetate) The title compound EX8-IM-3 (0.189 g) was obtained as a white amorphous Got it.

[0384] NMR data (CDCl3)(δ:ppm):δ: 7.31 (2H, d, J = 8.4 Hz), 7.26 (2H, d, J = 8.0 Hz, Overl apped with solvent peak.), 6.84 (1H, br s), 6.52 (1H, br s), 4.52 (2H, d, J = 6. 0 Hz), 4.49 (2H, d, J = 6.4 Hz), 4.26-4. 23 (1H, m), 4.11 (1H, d, J = 15.2 Hz), 3 .94 (1H, d, J = 15.2 Hz), 2.26-2.09 (3H, m), 2.00-1.58 (6H, m), 1.48-1.44 (1H, m ).LC-MS:M=396,RT=0.99(min),[M+H]+=397.

[0385] <Step 4> N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yne Synthesis of 1-(1-phenyloxy)acetamide (compound EX8-IM-4): [ka]

[0386] (Example 8) Compound EX8-IM-3 (0.18 g) obtained in <Step 3> and meta A mixture of ethanol (1.8 mL) and potassium carbonate (0.126 g) was added to the mixture under ice-cooling and stirring. Aqueous solution of 1,2-dichlorophenyl ether (0.9 mL) was added dropwise, and the mixture was stirred at room temperature for 17 hours and 30 minutes. The organic layer was extracted three times with ethyl acetate (5 mL). The organic layer was filtered, and the solvent was removed under reduced pressure. The crude title compound EX8-IM-4 (0.13 g) was obtained as a pale yellow oil.

[0387] NMR data (CDCl3)(δ:ppm):δ: 7.28-7.28 (4H, m ), 6.80 (1H, br s), 4.48 (2H, d, J = 6.0 Hz), 4.26-4.21 (1H, m), 4.11 (1H, d, J = 15.2 Hz), 3.93 (1H, d, J = 15.2 Hz), 3 .86 (2H, s), 2.28-2.07 (3H, m), 1.99-1.4 0 (7H, m, Overlapped with solvent peak.) .LC-MS:M=300,RT=0.68(min),[M+H]+=301.

[0388] <Step 5> N-(4-(aminoethyl)benzyl)-2-(cyclooct-2-yne Synthesis of 1-(1-hydroxy)acetamide group-introduced alginic acid (EX8-(I)-B-2): [ka]

[0389] A 1% by weight aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (5 0.86 mL) and 4-(4,6-dimethoxy-1,3,5-triazine at room temperature with stirring. -2-yl)-4-methylmorpholinium chloride (DMT-MM) (0.118 g) Then, the compound EX8-IM-4 (0.0 A solution of 35 g of ethanol (3 mL) was added dropwise at the same temperature, and the mixture was stirred at 40°C for 4 hours. After cooling to room temperature, sodium chloride (500 mg) was added, followed by ethanol (101.72 The resulting precipitate was collected by filtration and diluted with ethanol (2 mL) for 30 minutes. After washing twice, the mixture was dried under reduced pressure to give the title compound EX8-(I)-B-2 (521 mg) as a white solid. Got it as a body.

[0390] Reactive Substituent (N-(4-(aminoethyl)benzyl)-2-(cyclooct-2-yl) The introduction rate of the (1-phenyloxy)acetamide group was 4.46 mol% (NMR integral ratio). I did.

[0391] (Example 8b) N-(4-(aminoethyl)benzyl)-2-(cyclooct-2-yne) Synthesis of (1-(1-yloxy)acetamide group-introduced alginic acid (EX8-(I)-A-2): The introduction rate of reactive substituents ( As a result, the title compound (EX8-(I)-A-2) was obtained with an NMR integral ratio of 4.4 mol%.

[0392] (Example 9) N-(2-aminoethyl)-2-(cyclooct-2-yn-1-oxy) Synthesis of acetamido-functionalized alginic acid (9a, 9b, 9c): [ka]

[0393] (Example 9a) N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yl) (i) Synthesis of acetamide-group-introduced alginic acid (EX9-(I)-A-2):

[0394] <Step 1> tert-Butyl (2-(2,2,2-trifluoroacetamido)carba Synthesis of Mate (EX9-IM-1): [ka]

[0395] Commercially available tert-butyl (2-aminoethyl) carbamate (EX9-SM1, 3.0 0 g, [CAS:57260-73-8]) in tetrahydrofuran (12.0 mL) Ethyl trifluoroacetate (2.24 mL) was added dropwise to the solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was stirred for 14.5 hours. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with tert-butyl methyl ether (5 mL) and heptane (25 mL) were added and triturated. The solid was filtered and then The title compound EX9-IM-1 (4.36 g) was obtained as a white solid. .

[0396] NMR data (CDCl3) (δ: ppm): 7.80 (1H, brs), 4.93 ( 1H, brs), 3.45 (2H, q, J = 5 Hz), 3.41-3.34 (2H ,m), 1.44(9H,s)

[0397] <Step 2> N-(2-aminoethyl)-2,2,2-trifluoroacetamide hydrochloride Synthesis of salt (EX9-IM-2): [ka]

[0398] (Example 9a) Compound EX9-IM-1 (0.50 g) obtained in <Step 1> was added to gi Acid (3.1 mL) was added and the mixture was stirred at room temperature for 22.5 hours. Formic acid was removed by distillation, and the mixture was co-evaporated with toluene. The resulting oil was mixed with hydrogen chloride / methanol and concentrated under reduced pressure. After sequential azeotropic distillation with ert-butyl methyl ether and drying under reduced pressure, the crude title compound EX9- IM-2 (0.35 g) was obtained as a colorless oil.

[0399] NMR data (DMSO-d6) (δ:ppm): 3.42 (2H, d, J = 6 Hz), 2.92 (2H, d, J = 6 Hz)

[0400] <Step 3> N-(2-(2-(cyclooct-2-yn-1-yloxy)acetamide Synthesis of ethyl)-2,2,2-trifluoroacetamide (EX9-IM-3): [ka]

[0401] A method known in the literature (Org. Process Res. Dev. (2018) 22: The carboxylic acid (EX8-SM2, 100 mg) synthesized according to the method described in (108-110) was added to ethanol. (1.0 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl )-4-methylmorpholinium chloride (DMT-MM) (304 mg), (Example 9 a) Compound EX9-IM-2 (159 mg) obtained in <Step 2>, triethylamine (153 μL) was added and the mixture was stirred at room temperature for 3.5 hours. Water (4 mL) was added and ethyl acetate was added. The organic layer was extracted with 0.5N citric acid (5 mL). The mixture was washed with water (5 mL × 2) and saturated saline (3 mL) in that order, and then dried over anhydrous sodium sulfate. After drying, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (10% ethyl acetate). The title compound EX9 was obtained by purifying the compound in ethyl acetate / n-heptane to 60% ethyl acetate / n-heptane. -IM-3 (103 mg) was obtained as a white solid.

[0402] NMR data (DMSO-d6) (δ: ppm): 9.42 (1H, brs), 7.8 3(1H, brs), 4.29-4.24(1H, m), 3.87(2H, d, J = 15 Hz), 3.73 (1H, d, J = 15 Hz), 3.28-3.20 (4H , m), 2.27-2.04(3H, m), 1.96-1.70(4H, m), 1.6 7-1.50(2H, m), 1.43-1.34(1H, m)

[0403] <Step 4> N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy) ) Synthesis of acetamide (EX9-IM-4): [ka]

[0404] (Example 9a) Metathesis of compound EX9-IM-3 (103 mg) obtained in <Step 3> In a solution of ethanol (1.55 mL), potassium carbonate (89 mg) was dissolved in water (515 μL). The mixture was stirred at room temperature for 6 hours. Methanol was removed under reduced pressure, and water (2 mL) was added. After that, it was saturated with sodium chloride and extracted with ethyl acetate (15 mL, 10 mL x 5). After drying over anhydrous sodium sulfate, the solvent was removed under reduced pressure to obtain the title compound EX9- IM-4 (75 mg) was obtained as a colorless oil.

[0405] NMR data (CDCl3) (δ: ppm): 6.83 (1H, brs), 4.28- 4.22(1H, m), 4.06(1H, d, J = 15 Hz), 3.90(1H, d, J = 15 Hz), 3.42-3.30(2H, m), 2.86(2H, t, J = 6 Hz), 2.31-2.12(3H,m), 2.04-1.78(4H,m) , 1.75-1.57(2H, m), 1.51-1.43(1H, m)

[0406] <Step 5> N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy) ) Synthesis of acetamide-group-introduced alginic acid (EX9-(I)-A-2): [ka]

[0407] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (3 0 mL) was added to 4-(4,6-dimethoxy-1,3,5-triazine-2- yl)-4-methylmorpholinium chloride (DMT-MM) (84 mg), (Example 9a) Compound EX9-IM-4 (17 mg) obtained in <Step 4> was dissolved in ethanol (3 A 1 mol% sodium bicarbonate solution (76 μL) was added in that order, and the mixture was stirred at 30°C for 3 hours. Sodium chloride (0.3 g) was added to the solution, followed by ethanol (60 mL). The mixture was stirred for 1.5 hours. The resulting precipitate was collected by filtration, washed with ethanol (10 mL x 5), and then After drying under reduced pressure, the title compound EX9-(I)-A-2 (290 mg) was obtained as a white solid. .

[0408] Reactive Substituent (N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yl) The introduction rate of the hydroxyacetamide group was 4.3 mol% (NMR integral ratio).

[0409] (Example 9b) N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yl) (i) Synthesis of acetamide group-introduced alginic acid (EX9-(I)-B-2a): <Step 1> N-(2-aminoethyl)-2,2,2-trifluoroacetamide hydrochloride Synthesis of salt (EX9-IM-2): [ka]

[0410] (Example 9a) Compound EX9-IM-1 (0.50 g) obtained in <Step 1> was mixed with 1 The mixture was suspended in 1,4-dioxane (3.0 mL). Dioxane (7.0 mL) was added and the mixture was stirred at room temperature for 3 hours. Ether (30.0 mL) was added and the mixture was stirred at room temperature for 50 minutes. The solid was collected by filtration and diluted with diisopropyl After washing with propyl ether, the mixture was dried under reduced pressure to give the title compound EX9-IM-2 (0.70 g). was obtained as a white solid.

[0411] NMR data (DMSO-d6) (δ: ppm): 9.56 (1H, brs), 8.0 0(3H, brs), 3.45(2H, d, J = 6 Hz), 2.95(2H, d, J = 6 Hz)

[0412] <Step 2> N-(2-(2-(cyclooct-2-yn-1-yloxy)acetamide Synthesis of ethyl)-2,2,2-trifluoroacetamide (EX9-IM-3): [ka]

[0413] A method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110) and (EX8-SM2, 300 mg) Example 9b) Compound EX9-IM-2 (380 mg) obtained in <Step 1> was used (execution The same procedure as in Example 9a) <Step 3> was carried out to obtain the title compound EX9-IM-3 (322 mg). was obtained as a white solid.

[0414] NMR data (CDCl3) (δ: ppm): 7.95 (1H, brs), 6.95 ( 1H, brs), 4.28-4.23(1H, m), 4.08(2H, d, J = 15 Hz), 3.91(1H, d, J = 15 Hz), 3.56-3.50(4H, m), 2.31-2.12(3H, m), 2.03-1.78(4H, m), 1.75- 1.61(2H, m), 1.52-1.42(1H, m)

[0415] <Step 3> N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy) ) Synthesis of acetamide (EX9-IM-4): [ka]

[0416] (Example 9b) Compound EX9-IM-3 (322 mg) obtained in <Step 2> was The same procedure as in Example 9b) <Step 4> was carried out to obtain the title compound EX9-IM-4 (238 mg ) was obtained as a colorless oil.

[0417] NMR data (CDCl3) (δ: ppm): 6.82 (1H, brs), 4.28- 4.22(1H, m), 4.06(1H, d, J = 15 Hz), 3.90(1H, d, J = 15 Hz), 3.40-3.31(2H, m), 2.86(2H, t, J = 6 Hz), 2.31-2.12(3H,m), 2.02-1.78(4H,m) , 1.75-1.57(2H, m), 1.52-1.41(1H, m)

[0418] <Step 4> N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy) ) Synthesis of acetamide-group-introduced alginic acid (EX9-(I)-B-2a): [ka]

[0419] Sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) aqueous solution (1 20 mL) and 4-(4,6-dimethoxy-1,3,5-triazine-2 -yl)-4-methylmorpholinium chloride (DMT-MM) (335 mg), ( Example 9b) Compound EX9-IM-4 (68 mg) obtained in <Step 3> was dissolved in ethanol ( 12 mL) solution, then 1 mol% sodium bicarbonate water (303 μL) were added in that order, and the mixture was stirred at 30°C for 3 hours. Sodium chloride (1.2 g) was added to the reaction mixture, and then ethanol (240 mL) was added. The resulting precipitate was collected by filtration and diluted with ethanol (20 mL x 5). After washing, the product was dried under reduced pressure to obtain the title compound EX9-(I)-B-2a (1.16 g) as a white solid. Got it as a body.

[0420] Reactive Substituent (N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yl) The introduction rate of the hydroxyacetamide group was 4.2 mol% (NMR integral ratio).

[0421] (Example 9c) N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yl) (i) Synthesis of acetamide-group-introduced alginic acid (EX9-(I)-B-2b): [ka]

[0422] Sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) aqueous solution (1 20 mL) and 4-(4,6-dimethoxy-1,3,5-triazine-2 -yl)-4-methylmorpholinium chloride (DMT-MM) (167 mg), ( Example 9b) Compound EX9-IM-4 (34 mg) obtained in <Step 3> was dissolved in ethanol ( 12 mL) solution, 1 mol% sodium bicarbonate water (151 μL) were added in that order, and the mixture was stirred at 30°C for 3 hours. Sodium chloride (1.2 g) was added to the reaction mixture, and then ethanol (240 mL) was added. The resulting precipitate was collected by filtration and diluted with ethanol (20 mL x 5). After washing, the product was dried under reduced pressure to obtain the title compound EX9-(I)-B-2b (1.12 g) as a white solid. Got it as a body.

[0423] Reactive Substituent (N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yl) The introduction rate of the hydroxyacetamide group was 2.1 mol% (NMR integral ratio).

[0424] (Example 10) N-(2-(2-aminoethoxy)ethyl)-4-(azidomethyl)benzene Synthesis of alginic acid with azamido group (EX10-(II)-A-2): [ka]

[0425] <Step 1> tert-Butyl (2-(2-(4-(chloromethyl)benzamido)ethoxy Synthesis of ethyl carbamate (EX10-IM-1): [ka]

[0426] EX5-SM (4-(chloromethyl)benzoyl chloride, 0.50 g) was added to tetrahydrofuran Dissolve in hydrofuran (5.0 mL) and tert-butyl(2-(2-aminoethoxy) )ethyl)carbamate (0.54 g, [CAS:127828-22-2]) and di A solution of isopropylethylamine (0.92 mL) in tetrahydrofuran (5.0 mL) The reaction mixture was stirred at room temperature for 3 hours. The organic layer was washed with water (5 mL) and saturated saline (5 mL) in that order. The mixture was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The residue was extracted with tert-butyl methyl ether After trituration with a mixed solvent of n-heptane and n-heptane, the resulting solid was filtered and The extract was washed with ethyl acetate to obtain the title compound EX10-IM-1 (0.79 g) as a white solid.

[0427] NMR data (CDCl3) (δ:ppm): 7.79 (2H, d, J = 8 Hz ), 7.46(2H, d, J = 8 Hz), 6.62(1H, brs), 4.83( 1H, brs), 4.61(2H, s), 3.68-3.62(4H, m), 3.55( 2H, t, J = 5 Hz), 3.33(2H, t, J = 5 Hz), 1.42( 9H, s)

[0428] <Step 2> tert-Butyl(2-(2-(4-(azidomethyl)benzamido)ethoxy Synthesis of ethyl carbamate (EX10-IM-2): [ka]

[0429] Dissolve sodium azide (109 mg) in dimethyl sulfoxide (7.5 mL). Compound EX10-IM-1 (500 mg) obtained in (Example 10) <Step 1> was added. The mixture was stirred at room temperature for 3 hours. Water (15 mL) was added to the reaction mixture under ice-water cooling, and the precipitated solid was The solid was filtered and washed with water. The obtained solid was dried to obtain the title compound EX10-IM-2 (47 8 mg) was obtained as a white solid.

[0430] NMR data (CDCl3) (δ:ppm): 7.82 (2H, d, J = 8 Hz ), 7.39(2H, d, J = 8 Hz), 6.63(1H, brs), 4.83( 1H, brs), 4.40(2H, s), 3.68-3.62(4H, m), 3.55( 2H, t, J = 5 Hz), 3.33(2H, q, J = 5 Hz), 1.42( 9H, s)

[0431] <Step 3> N-(2-(2-aminoethoxy)ethyl)-4-(azidomethyl)benzaldehyde Synthesis of IMido hydrochloride (EX10-IM-3): [ka]

[0432] (Example 10) Compound EX10-IM-2 (400 mg) obtained in <Step 2> was added Add 4N hydrogen chloride / 1,4-dioxane (2.8 mL) under ice-water cooling, and mix at room temperature for 1 The mixture was stirred for 75 hours. Diisopropyl ether (8.4 mL) was added to the reaction mixture to give a gum-like mixture. The supernatant was removed by decantation, and the residue was washed with diisopropyl ether and then dried under reduced pressure. Drying afforded the title compound EX10-IM-3 (298 mg) as a beige solid.

[0433] NMR data (DMSO-d6) (δ: ppm): 8.60 (1H, t, J = 6 Hz), 7.89(2H, d, J = 8 Hz), 7.90(3H, brs), 7.4 5(2H, d, J = 8 Hz), 4.52(2H, s), 3.62(2H, t, J = 5 Hz), 3.58(2H, t, J = 6 Hz), 3.47(2H, q, J = 6 Hz), 2.98 (2H, t, J = 5 Hz), LC-MS (free a mine):RT=0.58(min), [M+H] + =264

[0434] <Step 4> N-(2-(2-aminoethoxy)ethyl)-4-(azidomethyl)benzaldehyde Synthesis of amido-functionalized alginic acid (EX10-(II)-A-2): [ka]

[0435] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (4 0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (112 mg), (Example 10) Compound EX10-IM-3 (30 mg) obtained in step 3, 1 molar sodium bicarbonate solution (15 1 μL) was added and stirred at 30° C. for 3 hours. Sodium chloride (400 mg) was added. Then, ethanol (80 mL) was added and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration. After washing with ethanol, the mixture was dried under reduced pressure to obtain the title compound EX10-(II)-A-2 (408 mg) as a white solid.

[0436] Reactive Substituent (N-(2-(2-aminoethoxy)ethyl)-4-(azidomethyl)benzene The introduction rate of the amide group was 4.7 mol% (NMR integral ratio).

[0437] (Example 11) N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(aminoethoxy)ethyl Synthesis of alginic acid containing dimethylbenzamide group (EX11-(II)-A-2): [ka]

[0438] <Step 1> tert-Butyl (2-(2-(2-(4-(chloromethyl)benzamide) ethyl ester Synthesis of (ethoxy)ethoxy)ethyl)carbamate (EX11-IM-1): [ka]

[0439] EX5-SM (4-(chloromethyl)benzoyl chloride, 0.50 g) was added to tetrahydrofuran Dissolve in hydrofuran (5.0 mL) and tert-butyl (2-(2-aminoethyl) (0.66 g) diisopropyl ethyl amine A solution of 1.0 mL of tetrahydrofuran (5.0 mL) was added dropwise to the mixture, and the mixture was stirred at room temperature for 4. The mixture was stirred for 7 hours. Ethyl acetate (25 mL) and water (10 mL) were added to the reaction mixture, and the mixture was separated. The organic layer was diluted with half-saturated sodium bicarbonate water (10 mL), water (10 mL), and saturated saline (5 mL). The mixture was washed with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Methyl ether was added, and the solid was removed by filtration. The filtrate was concentrated under reduced pressure and then washed with silica gel. Purify by column chromatography (20% ethyl acetate / n-heptane to ethyl acetate) The title compound EX11-IM-1 (0.82 g) was obtained as a colorless oil.

[0440] NMR data (CDCl3) (δ:ppm): 7.79 (2H, d, J = 8 Hz ), 7.45(2H, d, J = 8 Hz), 6.71(1H, brs), 4.97( 1H, brs), 4.60(2H, s), 3.70-3.60(8H, m), 3.55( 2H, t, J = 5 Hz), 3.31(2H, q, J = 6 Hz), 1.43( 9H, s)

[0441] <Step 2> tert-Butyl (2-(2-(2-(4-(azidomethyl)benzamido)ethyl) Synthesis of (ethoxy)ethoxy)ethyl)carbamate (EX11-IM-2): [ka]

[0442] (Example 11) Dimethylation of Compound EX11-IM-1 (0.82 g) Obtained in <Step 1> To a solution of methyl sulfoxide (11.7 mL), sodium azide (152 mg) was added. The mixture was stirred at room temperature for 3.5 hours. Water (23 mL) was added to the reaction mixture under ice-water cooling, and the mixture was then cooled to the same temperature. The mixture was stirred for 30 minutes. The mixture was extracted with ethyl acetate (30 mL, 10 mL), and the organic layer was washed with water (10 The organic layer was washed with saturated aqueous sodium chloride (5 mL) and then with anhydrous sodium sulfate. After drying, the precipitated solid was filtered and dried under reduced pressure to obtain the title compound EX11-IM-2 (0.8 0 g) was obtained as a colorless oil.

[0443] NMR data (CDCl3) (δ:ppm): 7.81 (2H, d, J = 8 Hz ), 7.39(2H, d, J = 8 Hz), 6.73(1H, brs), 4.97( 1H, brs), 4.40(2H, s), 3.72-3.60(8H, m), 3.55( 2H, t, J = 5 Hz), 3.31(2H, q, J = 5 Hz), 1.43( 9H, s)

[0444] <Step 3> N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(azide Synthesis of (trimethyl)benzamide hydrochloride (EX11-IM-3): [ka]

[0445] (Example 11) Compound EX11-IM-2 (0.80 g) obtained in <Step 2> was added Add 4N hydrogen chloride / 1,4-dioxane (5.3 mL) under ice-water cooling, and mix at room temperature for 1 The mixture was stirred for 30.75 hours. Diisopropyl ether (16.0 mL) was added to the reaction mixture, and the mixture was stirred for 30. The mixture was stirred for 1 min, the solvent was removed by decantation, and the residue was washed with diisopropyl ether. The resulting residue was dried under reduced pressure to give the title compound EX11-IM-3 (0.73 g) as a colorless The product was obtained as a globular solid.

[0446] NMR data (DMSO-d6) (δ: ppm): 8.62 (1H, t, J = 6 Hz), 7.95 (3H, brs), 7.88 (2H, d, J = 8 Hz), 7.4 5(2H, d, J = 8 Hz), 4.52(2H, s), 3.62-3.52(8H , m), 3.43 (2H, q, J = 6 Hz), 2.98-2.89 (2H, m), LC-MS (free amine): RT=0.59 (min), [M+H] + =308

[0447] <Step 4> N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(azide Synthesis of (trimethyl)benzamide group-introduced alginic acid (EX11-(II)-A-2): [ka]

[0448] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (4 0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (112 mg), (Example 11) Compound EX11-IM-3 (38 mg) obtained in step 3 was dissolved in ethanol (4.0 mL ) solution, 1 molar sodium bicarbonate solution (151 μL) was added, and the mixture was stirred at 30° C. for 3 hours. Thorium (0.4 g) was added, followed by ethanol (80 mL), and the mixture was incubated at room temperature for 30 min. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain the title compound EX. 11-(II)-A-2 (416 mg) was obtained as a white solid.

[0449] The reactive substituent (N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-( The introduction rate of azidomethylbenzamide groups was 4.2 mol% (NMR integral ratio).

[0450] (Example 12) N-(2-(2-aminoethoxy)ethyl)-6-(azidomethyl)nicotinic acid Synthesis of tinamide-group-introduced alginic acid (EX12-(II)-A-2): [ka]

[0451] <Step 1> Synthesis of methyl 6-(azidomethyl)nicotinate (EX12-IM-1) : [ka]

[0452] According to a method known in the literature (Angew. Chem. Int. Ed. (2012) 51:5 852-5856), commercially available methyl 6-(hydroxymethyl)nicotinate [ CAS:56026-36-9] (EX12-SM1, 0.5 g) and tetrahydrofuran To a mixture of orchid (5 mL) was added p-toluenesulfonyl chloride (0. 68 g) and triethylamine (0.63 mL) were added. The reaction mixture was stirred at room temperature for 1 hour. After stirring for 20 hours and 30 minutes, sodium azide (0.29 g) was added at the same temperature, and the mixture was stirred at room temperature. The mixture was stirred for 4 hours. After the reaction was completed, ethyl acetate (10 mL) and water (10 mL) were added. After diluting the reaction solution, the aqueous layer was extracted three times with ethyl acetate (10 mL). The extract was washed with water (5 mL) and saturated saline (5 mL) in that order, and then dried over anhydrous sodium sulfate. The organic layer was filtered and concentrated under reduced pressure to obtain a crude product. By purifying with gel column chromatography (n-heptane / ethyl acetate), the title compound was obtained. Compound EX12-IM-1 (0.34 g) was obtained as a pale yellow amorphous substance.

[0453] NMR data (CDCl3) (δ:ppm): 9.18 (1H, d, J = 2.0H z), 8.33(1H, dd, J = 8.0, 2.0 Hz), 7.45(1H, d, J = 8.0 Hz), 4.57(2H, s), 3.96(3H, s).LC-MS: M=192, RT=0.78(min), [M+H]+=193.

[0454] <Step 2> Synthesis of 6-(azidomethyl)nicotinic acid (EX12-IM-2): [ka]

[0455] (Example 12) Compound EX12-IM-1 (0.342 g) obtained in <Step 1> and and methanol (6.84 mL) to give a 1 molar concentration of lithium hydroxide monohydrate. The reaction mixture was stirred at the same temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography. The compound was purified by solvent (n-heptane / ethyl acetate to ethyl acetate / methanol) to give the title compound E. X12-IM-2 (0.28 g) was obtained as a pale yellow amorphous substance.

[0456] NMR data (CD3OD) (δ:ppm): 9.09 (1H, d, J = 2.4H z), 8.38(1H, dd, J = 8.0, 2.4 Hz), 7.56(1H, d, J = 8.0 Hz), 4.57(2H, s).LC-MS: M=178, RT=0. 60(min),[M+H]+=179.

[0457] <Step 3> tert-Butyl (2-(2-(6-(azidomethyl)nicotinamide) Synthesis of ethoxy)ethyl)carbamate (EX12-IM-3): [ka]

[0458] (Example 12) Compound 12-IM-2 (100 mg) obtained in <Step 2>, commercially available N-(tert-butoxycarbonyl)-2-(2-aminoethoxy)ethylamine [C AS:127828-22-2] (108.07 μL) and acetonitrile (2000 To the mixture of 1 μL of O-(7-azabenzotriazol-1-yl) -N,N,N',N'-Tetramethyluronium hexafluorophosphate (213.43 mg) and triethylamine (156.48 μL), and stirred at room temperature for 1 hour and 45 minutes. After that, N-(tert-butoxycarbonyl)-2-(2-amino) Ethoxy)ethylamine (54 μL) and O-(7-azabenzotriazole-1-yl) 106. The mixture was stirred at the same temperature for 17 hours. Water (5 mL) was added to quench the reaction. Ethyl acetate (10 mL) was added. The aqueous layer was extracted three times with ethyl acetate (10 mL). The organic layer was filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (n-heptane / ethyl acetate). After purification, the title compound EX12-IM-3 (187 mg) was obtained as a colorless oily compound.

[0459] NMR data (CDCl3) (δ:ppm): 9.00 (1H, d, J = 2.0H z), 8.18(1H, dd, J = 8.0, 2.0 Hz), 7.43(1H, d , J = 8.0 Hz), 6.83(1H, br s), 4.82(1H, br s) , 4.55(2H, s), 3.69-3.65(4H, m), 3.56(2H, t, J = 5.2 Hz), 3.34(1H, d, J = 5.6 Hz), 3.32(1H, d, J = 5.6 Hz), 1.41(9H, s).LC-MS:M=364,RT= 0.78(min),[M+H]+=365.

[0460] <Step 4> N-(2-(2-aminoethoxy)ethyl)6-(azidomethyl)nicotine Synthesis of amide dihydrochloride (EX12-IM-4): [ka]

[0461] (Example 12) Compound EX12-IM-3 (0.187 g) obtained in <Step 3> and A mixture of 1,4-dioxane solution (1.31 mL) and 4N-sodium chloride was added to the mixture under stirring in a water-cooled bath. Hydrogen chloride / 1,4-dioxane (1.31 mL) was added and the mixture was stirred at room temperature for 3 hours. Diisopropyl ether (20 mL) was added to the mixture, and the precipitate was filtered to obtain the title compound. Compound EX12-IM-4 (0.16 g) was obtained as an off-white solid.

[0462] NMR data (DMSO-d6) (δ: ppm): 9.02-9.02 (1H, m), 8 .80(1H, br s), 8.27-8.25(1H, m), 7.89(3H, br s), 7.54(1H, d, J = 8.4 Hz), 4.59(2H, s), 3.64 -3.57(4H, m), 3.51-3.47(2H, m), 3.01-2.97(2H ,m).LC-MS(free amine):M=264,RT=0.49(min),[ M+H]+=265.

[0463] <Step 5> N-(2-(2-aminoethoxy)ethyl)-6-(azidomethyl)nicotinic acid Synthesis of amide-group-introduced alginic acid (EX12-(II)-A-2): [ka]

[0464] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (3 9.55 mL) and 4-(4,6-dimethoxy-1,3,5-triazine were added at room temperature with stirring. -2-yl)-4-methylmorpholinium chloride (DMT-MM) (91.52 mg ) and 1 molar sodium bicarbonate solution (183 μL) were added. Compound EX12-IM-4 (30 mg) obtained in step 1, water (1 mL) and ethanol (1 mL) was added at the same temperature and stirred at 40°C for 4 hours. After adding ethanol (79.1 mL), 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 to obtain the title compound EX12-(I I)-A-2 (378 mg) was obtained as a white solid.

[0465] Reactive group N-(2-(2-aminoethoxy)ethyl)-6-(azidomethyl)nicotine The introduction rate of the amide group was 4.8 mol% (NMR integral ratio).

[0466] (Example 13) N-(2-(2-aminoethoxy)ethyl)-4-azidobenzamide group Synthesis of introduced alginic acid (EX13-(II)-A-2): [ka]

[0467] <Step 1> tert-Butyl(2-(2-(4-azidobenzamido)ethoxy)ethyl) Synthesis of carbamate (EX13-IM-1): [ka]

[0468] 4-Azidobenzoic acid (EX7-SM, 300 mg), tert-butyl (2-(2- Aminoethoxy)ethyl)carbamate [CAS:127828-22-2] (376 mg) was dissolved in acetonitrile (6.0 mL). (1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.77 g) and diisopropylethylamine (707 μL) were added, and the mixture was incubated at room temperature for 16 hours. The reaction mixture was stirred for 1 hour. Ethyl acetate (20 mL) and water (10 mL) were added to the reaction mixture, and the mixture was separated. The organic layer was washed with water (10 mL) and saturated saline (5 mL), and then with anhydrous sodium sulfate. The residue was purified by silica gel column chromatography (20% acetic acid). Ethyl acetate / n-heptane to 70% ethyl acetate / n-heptane) to obtain the title compound EX. 13-IM-1 (673 mg) was obtained as a pale yellow gum.

[0469] NMR data (CDCl3) (δ:ppm): 7.83 (2H, d, J = 9 Hz ), 7.08(2H, d, J = 9 Hz), 6.61(1H, brs), 4.84( 1H, brs), 3.68-3.64(4H, m), 3.56(2H, t, J = 5 Hz), 3.34 (2H, q, J = 5 Hz), 1.44 (9H, s)

[0470] <Step 2> N-(2-(2-aminoethoxy)ethyl)-4-azidobenzamide hydrochloride Synthesis of salt (EX13-IM-2): [ka]

[0471] (Example 13) Compound EX13-IM-1 (670 mg) obtained in <Step 1> Add 4-HCl / 1,4-dioxane (4.7 mL) under ice-water cooling, and incubate at room temperature for 2 hours. Diisopropyl ether (14.0 mL) was added to the reaction mixture and stirred for 30 minutes. The resulting solid was collected by filtration, washed with diisopropyl ether, and then dried under reduced pressure to give the title compound. Compound EX13-IM-2 (604 mg) was obtained as a pale beige solid.

[0472] NMR data (DMSO-d6) (δ: ppm): 8.61 (1H, t, J = 6 Hz), 7.95 (3H, brs), 7.93 (2H, d, J = 9 Hz), 7.2 0(2H, d, J = 9 Hz), 3.62(2H, t, J = 5 Hz), 3.5 7(2H, t, J = 6 Hz), 3.46(2H, q, J = 6 Hz), 3.0 2-2.93(2H, m), LC-MS(free amine): RT=0.57(min ), [M+H] + =250

[0473] <Step 3> Introduction of N-(2-(2-aminoethoxy)ethyl)-4-azidobenzamide group Synthesis of alginic acid (EX13-(II)-A-2): [ka]

[0474] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (4 0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (112 mg), (Example 13) Compound EX13-IM-2 (31 mg) obtained in step 2, 1 molar sodium bicarbonate solution (15 1 μL) was added and stirred at 30°C for 3 hours. After adding sodium chloride (0.4 g), Ethanol (80 mL) was added and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration. After washing with ethanol, the mixture was dried under reduced pressure to obtain the title compound EX13-(II)-A-2 (400 mg) as a white solid.

[0475] Reactive group (N-(2-(2-aminoethoxy)ethyl)-4-azidobenzamide group) The introduction rate was 3.9 mol% (NMR integral ratio).

[0476] (Example 14) N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-azide Synthesis of dobenzamide-group-introduced alginic acid (EX14-(II)-A-2): [ka]

[0477] <Step 1> tert-Butyl(2-(2-(2-(4-azidobenzamido)ethoxy) Synthesis of ethoxy)ethyl)carbamate (EX14-IM-1): [ka]

[0478] 4-Azidobenzoic acid (EX7-SM, 300 mg), tert-butyl (2-(2- (2-Aminoethoxy)ethoxy)ethyl)carbamate (457 mg) was added to acetonite The mixture was dissolved in 1.0 mL of ethyl acetate (6.0 mL). ,N,N',N'-Tetramethyluronium hexafluorophosphate (0.77 g), Diisopropylethylamine (707 μL) was added, and the mixture was stirred at room temperature for 16 hours. Ethyl acetate (20 mL) and water (10 mL) were added to the mixture, and the layers were separated. The organic layer was diluted with water (1 The extract was washed successively with saturated aqueous sodium chloride (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (40% ethyl acetate / n-heptane The title compound EX14-IM-1 (6 03 mg) was obtained as a pale yellow oil.

[0479] NMR data (DMSO-d6) (δ: ppm): 8.53 (1H, t, J = 6 Hz), 7.89(2H, d, J = 9 Hz), 7.19(2H, d, J = 9 Hz), 6.76 (1H, t, J = 5 Hz), 3.55-3.47 (6H, m), 3.42-3.33(4H, m), 3.04(2H, q, J = 6 Hz), 1.36 (9H, s)

[0480] <Step 2> N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-azide Synthesis of benzamide hydrochloride (EX14-IM-2): [ka]

[0481] (Example 14) To the compound obtained in <Step 1> (EX14-IM-1, 600 mg), Add 4-HCl / 1,4-dioxane solution (4.2 mL) under ice-water cooling, and then add at room temperature. The reaction mixture was stirred at room temperature for 2 hours. Diisopropyl ether (12.0 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The mixture was stirred for 30 minutes, the solvent was removed by decantation, and the residue was washed with diisopropyl ether. The resulting residue was dried under reduced pressure to give the title compound EX14-IM-2 (596 mg). This was obtained as a beige gum.

[0482] NMR data (DMSO-d6) (δ: ppm): 8.62 (1H, t, J = 6 Hz), 7.97(3H, brs), 7.91(2H, d, J = 9 Hz), 7.2 0(2H, d, J = 9 Hz), 3.62-3.51(8H, m), 3.42(2H , q, J = 6 Hz), 2.97-2.89(2H, m), LC-MS(free amine):RT=0.58(min), [M+H] + =294

[0483] <Step 3> N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-azide Synthesis of benzamide-group-introduced alginic acid (EX14-(II)-A-2): [ka]

[0484] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (4 0 mL) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (112 mg), (Example 14) Compound EX14-IM-2 (45 mg) obtained in step 2 was dissolved in ethanol (4.0 mL ) solution, 1 molar sodium bicarbonate solution (151 μL) was added, and the mixture was stirred at 30° C. for 3 hours. Thorium (0.4 g) was added, followed by ethanol (80 mL), and the mixture was incubated at room temperature for 30 min. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain the title compound EX. 14-(II)-A-2 (408 mg) was obtained as a white solid.

[0485] Reactive group (N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-azide The introduction rate of the benzamide group was 4.2 mol% (NMR integral ratio).

[0486] (Example 15) N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2- Synthesis of (1-(1-oxy)acetamide-group-introduced alginic acid (EX15-(I)-A-2) : [ka]

[0487] <Step 1> tert-Butyl (2-(2-(2,2,2-trifluoroacetamide) Synthesis of ethoxy)ethyl carbamate (EX15-IM-1): [ka]

[0488] tert-Butyl (2-aminoethyl)carbamate (EX9-SM1, 1.0 g, [CAS:57260-73-8]) in tetrahydrofuran (4.0 mL), Ethyl trifluoroacetate (0.6 mL) was added dropwise, and the reaction mixture was stirred at room temperature for 3.5 hours. The reaction mixture was concentrated under reduced pressure to give the crude title compound EX15-IM-1 (1.5 g). Obtained as a colorless oil.

[0489] NMR data (CDCl3) (δ: ppm): 7.01 (1H, brs), 4.84 (1H,brs), 3.62-3.51(6H,m), 3.31(2H,q,J = 5 Hz), 1.45(9H,s)

[0490] <Step 2> N-(2-(2-aminoethoxy)ethyl)-2,2,2-trifluoroacetate Synthesis of cetoamide hydrochloride (EX15-IM-2): [ka]

[0491] (Example 15) Compound EX15-IM-1 (1.5 g) obtained in <Step 1> was added to ice Add 4-HCl / 1,4-dioxane solution (10.3 mL) under cooling with water, and then add at room temperature. The mixture was stirred for 1 hour. Diisopropyl ether (30 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. The solvent was removed by distillation under reduced pressure, and the residue was subjected to azeotropic distillation with diisopropyl ether and then dried under reduced pressure. As a result, the title compound EX15-IM-2 (1.3 g) was obtained as a colorless oil.

[0492] NMR data (DMSO-d6) (δ: ppm): 9.55 (1H, brs), 8.0 5(3H, brs), 3.61(2H, d, J = 5 Hz), 3.54(2H, t, J = 6 Hz), 3.39(2H, q, J = 6Hz), 3.00-2.91(2 H, m)

[0493] <Step 3> N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetate Amido)ethoxy)ethyl)-2,2,2-trifluoroacetamide (EX15-IM -3) Synthesis: [ka]

[0494] A method known in the literature (Org. Process Res. Dev. (2018) 22: 108-110), carboxylic acid (EX8-SM2, 300 mg), Example 14) The compound obtained in <Step 2> (443 mg) was dissolved in acetonitrile (6.0 mL ) was dissolved in O-(7-azabenzotriazol-1-yl)-N,N,N',N'- Tetramethyluronium hexafluorophosphate (0.75 g), diisopropyl ethyl Dimethylamine (920 μL) was added and the mixture was stirred at room temperature for 2.5 hours. The organic layer was separated by adding water (10 mL), saturated aqueous sodium chloride (NaCl), and ethanol. The mixture was washed with brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Silica gel column chromatography (50% ethyl acetate / n-heptane to 70% ethyl acetate The title compound EX15-IM-3 (469 mg) was obtained as a colorless Obtained as a gum.

[0495] NMR data (DMSO-d6) (δ: ppm): 9.45 (1H, brs), 7.6 1(1H, t, J = 6 Hz), 4.29-4.25(1H, m), 3.87(2H , d, J = 15 Hz), 3.75(1H, d, J = 15 Hz), 3.50( 2H, t, J = 6 Hz), 3.43(2H, t, J = 6 Hz), 3.37- 3.31 (2H, m), 3.24 (2H, q, J = 6 Hz), 2.27-2.03 (3H, m), 1.96-1.69(4H, m), 1.67-1.50(2H, m), 1 .43-1.35(1H,m)

[0496] <Step 4> N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2-yl) Synthesis of (1-phenyloxy)acetamide (EX15-IM-4): [ka]

[0497] (Example 15) Compound EX15-IM-3 (220 mg) obtained in <Step 3> A solution of potassium carbonate (103 mg) in ethanol (3.0 mL) was added to water (0.99 mL ) solution was added and stirred at room temperature for 4.5 hours. Methanol was distilled off under reduced pressure, and water (2 mL ) was added, and the mixture was saturated with sodium chloride. It was extracted with ethyl acetate (15 mL, 10 mL x 4), After drying over anhydrous sodium sulfate, the solvent was removed under reduced pressure. The residue was diluted with ethyl acetate (10 The mixture was dissolved in 1 mL of ethyl acetate, filtered to remove insoluble matter, and concentrated under reduced pressure to give the crude compound EX15- IM-4 (140 mg) was obtained as a pale yellow gum.

[0498] NMR data (CDCl3) (δ: ppm): 6.89 (1H, brs), 4.27- 4.22(1H, m), 4.07(1H, d, J = 15 Hz), 3.88(1H, d, J = 15 Hz), 3.58-3.47(6H, m), 2.87(2H, t, J = 5 Hz), 2.31-2.10(3H,m), 2.03-1.77(4H,m) , 1.73-1.59(2H, m), 1.51-1.43(1H, m), LC-MS:R T=0.60(min), [M+H] + =269

[0499] <Step 5> N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2-yl) Synthesis of alginic acid containing (1-phenyloxy)acetamide group (EX15-(I)-A-2) : [ka]

[0500] A 1% by weight aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (4 0 mL) was added to 4-(4,6-dimethoxy-1,3,5-triazine-2- Dimethylmorpholinium chloride (DMT-MM) (112 mg), Example 15) Compound EX15-IM-4 (30 mg) obtained in <Step 4> was dissolved in ethanol ( Add 4.0 mL) solution and 1 mol% sodium bicarbonate water (101 μL) in that order, and stir at 30°C for 3 hours. Sodium chloride (0.4 g) was added to the reaction mixture, and then ethanol (80 mL) was added. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The title compound EX15-(I)-A-2 (410 mg) was obtained as a white solid.

[0501] Reactive Substituent (N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2 The introduction rate of (1-yn-1-yloxy)acetamide group was 3.2 mol% (NMR integral ratio). there were.

[0502] [Table 12]

[0503] [Measurement of introduction rate of reactive group or complementary reactive group] The introduction rate of reactive groups or complementary reactive groups is determined by the ratio of uronic acid units, which are the repeating units of alginic acid. The percentage represents the number of reactive groups or complementary reactive groups introduced per sugar unit. do. In this embodiment, 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 an absorbance measurement method utilizing the above method.

[0504] [Molecular weight measurement] The alginic acid solid having the reactive group or the complementary reactive group introduced therein obtained in the examples was added at 0.1 Dissolve 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 prepared for gel filtration. The spectrum of each sample was The concentration of each compound was measured using a spectrophotometer DU-800 (Beckman-Coulter). The measurement wavelengths for the gel filtration of the compounds were determined. A differential refractometer was used.

[0505] 200μL of sample for gel filtration was added to Superose6 Increase10 / 30 The gel was subjected to 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. The solution was diluted with 10 mmol / L phosphate buffer (pH 7.4) containing 10 mmol / L NaCl at room temperature at a flow rate of The elution profile of each compound was determined at 0.8 mL / min. The absorption of the wavelengths was monitored and created. The chromatogram obtained was created using Unicorn5. The data was analyzed using 31 software (GE Healthcare Sciences) to determine the peak range.

[0506] The molecular weight of alginate having a reactive group or a complementary reactive group introduced therein was determined by using Blue Dextrose. Thyroglobulin (molecular weight 669,000 Da, SIGMA) Ferritin (molecular weight 440,000 Da, GE Healthcare Sciences) 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, 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 the 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 standard curve was created by linear regression from blue dextran to ferritin. We created two types, ranging from ferritin to aprotinin.

[0507] 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.

[0508]

number

[0509] [Gel stability measurement] (Measurement of gel stability (1)) The alginic acid derivative (EX1-(I)-A-2) obtained in (Example 1a), and The alginic acid derivative (EX3-(II)-A-2) obtained in Example 3a) was The solution was dissolved in water to a concentration of 1% by weight and used as (alginic acid aqueous solution 1-1) and (alginic acid aqueous solution 2 (Alginic acid aqueous solution 1-1) and (Alginic acid aqueous solution 2-1) were mixed in equal amounts. The mixed solution was then poured into a syringe equipped with an 18-gauge needle, and the syringe was then flushed. The syringe pump was set to a flow rate of 1 mL / min, and the solution was filled with potassium chloride at a concentration of 30 mmol / L. The solution was then dropped into the calcium solution for 30 seconds and stirred for 5 minutes to obtain an alginate gel. After washing once with 1 mL of phosphate-buffered saline (PBS), the cells were incubated in PBS at 37°C for 10 min. The mixture was allowed to stand for a period of time to allow chemical cross-linking to occur, yielding a chemically and ionically cross-linked alginate gel. 20 mL of PBS was added to the gel, and the gel was shaken at 37°C. The aqueous solution was then collected over time and then washed. The same amount of PBS as that of the collected solution was replenished. After the test was completed, the test solution was diluted with alginate lyase (Ni Add 5 μL of PONGENE (319-08261) and shake at 37°C for 2 hours to form the gel. The alginic acid concentration in the collected aqueous solution was determined by the carbazole sulfuric acid concentration. The alginic acid concentration in the aqueous solution at each time point was measured using the acid method, and was corrected with the concentration of alginic acid already collected. The corrected value was calculated from the alginic acid concentration at all time points and the alginic acid concentration after the end of the test. The value obtained by dividing the value by the alginic acid concentration and expressing it as a percentage was taken as the disintegration rate, which was used as an index of gel stability. Similarly, as controls, alginic acid (A-2), (EX1-(I)-A-2) and (A- 2), (A-2) and (EX3-(II)-A-2) were used to crosslink alginate gel (B The disintegration rate of each was measured.

[0510] The results are shown in Figure 1. (A-2), (EX1-(I)-A-2) and (A-2), (A-2) and (EX3-(II)-A-2) were used to prepare cross-linked alginates. The phosphate gel was almost completely dissolved in 4 hours, whereas (EX1-(I)-A-2) and (EX3- The cross-linked alginic acid obtained using each alginic acid derivative (II)-A-2) was The gel did not collapse even after a certain period of time, and its stability was improved. The resulting structure is then in a calcium ion-free solution. It has been shown that the structure is maintained for a long period of time even under aqueous conditions (below physiological concentrations for the body). I was tempted.

[0511] The collapse rate on the vertical axis of FIG. 1 means the relative collapse rate (%). (A-2) alone (actual value after 8 hours) was corrected to 100%, The collapse rate of each point is expressed as a relative value to the maximum value.

[0512] (Measurement of gel stability (2)) The alginic acid derivative (EX1-(I)-B-2a) obtained in (Example 1d), The alginic acid derivative (EX3-(II)-B-2a) obtained in (Example 7a) The alginic acid derivative (EX7-(II)-B-2a) obtained in Example 8 The acid derivative (EX8-(I)-B-2) was dissolved in water to a concentration of 1.0 w / w%. Thus, the alginic acid aqueous solutions (1d-1), (3d-1), (7a-1), and (8a-1) Next, equal amounts of the alginic acid aqueous solution (1d-1) and the alginic acid aqueous solution (3d-1) were Mixture of equal amounts of the mixed solution, alginic acid aqueous solution (1d-1) and alginic acid aqueous solution (7a-1) Solution, an equal mixture of an aqueous solution of alginic acid (8a-1) and an aqueous solution of alginic acid (3d-1) , and a mixture of equal amounts of an aqueous solution of alginic acid (8a-1) and an aqueous solution of alginic acid (7a-1) Using each mixed solution, chemically prepare alginate gel (beads) according to the following alginate gel (beads) preparation method. Cross-linked and ionically cross-linked alginate gels (beads) (A1, B1, C1, D1) were obtained. (See Table 13).

[0513] [How to make alginate gel (beads)] The mixed solution prepared above was placed in a syringe equipped with an 18-gauge needle, and the syringe was was placed on a syringe pump set at a flow rate of 1 mL / min, and a salt solution with a concentration of 30 mmol / L was Add dropwise to the calcium chloride solution for 30 seconds and stir for 5 minutes to obtain alginate gel (beads). The gel was washed once with 10 mL of phosphate-buffered saline (PBS) and then soaked in pure water for 3 h. The mixture was left to stand at 7°C for 10 minutes to perform chemical cross-linking, and the resulting alginate gel was chemically and ionically cross-linked. Obtain beads.

[0514] [Table 13]

[0515] [Stability measurement of alginate gels (A1-D1)] The above-obtained chemically crosslinked and ionically crosslinked alginate gels (beads) (A1 ~D1) Add 19.5 mL of PBS, shake at 37℃, and repeat steps 1, 2, 4, 8, and 24. After 48, 72, and 144 hours, the aqueous solution was collected and replenished with the same amount of PBS as the collected amount. After the test, the test solution was diluted with alginate lyase (Creative Enzymes, NA TE-1563) was added to the mixture, and the mixture was shaken overnight at 37°C to completely disintegrate the gel. The solution was collected. The concentration of alginic acid in the collected aqueous solution was measured by the carbazole-sulfuric acid method. The alginic acid concentration in the aqueous solution at each time point was measured. The disintegration rate was calculated by dividing the ginic acid concentration by the total alginic acid concentration and expressing the result as a percentage. This was used as an index of gel stability. As a control, alginic acid (B-2) without reactive groups was used. Using the above, alginate gel (beads) (REF) was prepared according to the above method, and the disintegration rate was measured. It was determined.

[0516] The results are shown in Figure 4. The control alginic acid (B- 2) The alginate gel (Gel REF) made using this method collapsed by more than 90% in 144 hours. In contrast, each alginic acid derivative (EX1-(I)-B-2a, EX3-(II)-B-2a, The crosslinked alkenyl compounds obtained using EX7-(II)-B-2a and EX8-(I)-B-2) The acetic acid gel (beads) (A1 to D1) did not collapse even after 144 hours, and the gel was stable. In other words, the cross-linking caused by the Huisgen reaction resulted in The fabricated structure was created in a solution without calcium ions (below the physiological concentration for the body). It was suggested that the structure would be maintained for a long time even under these conditions.

[0517] (Measurement of gel stability (3): Measurement of gel stability in the presence of EDTA) The gel stability was measured by the method (2) for each of the chemically crosslinked and ionically crosslinked alginates. Add 19.5 mL of 5 mM ethylenediaminetetraacetic acid to the acetate gel (beads) (A1-D1). Add EDTA dipotassium salt dihydrate (2K) / PBS solution and shake at 37°C for 2 h. After 4 hours, the aqueous solution was collected and the EDTA-treated cross-linked alginate gel (beads) (A2-D After the test, the test solution was diluted with alginate lyase (Creative Enzyme es, NATE-1563) was added and the gel was shaken overnight at 37°C to completely The alginic acid concentration in the collected aqueous solution was measured using the carbazole-sulfuric acid method. The alginic acid concentration in the aqueous solution after 24 hours was measured by The percentage is calculated by dividing the total alginic acid concentration by the alginic acid concentration after the test. The value was taken as the disintegration rate and was used as an index of gel stability. Using unmodified alginic acid (B-2), we made alginate gel (beads) (REF2). The disintegration rate was measured.

[0518] [Table 14]

[0519] The results in Table 14 were obtained. As a control, alginic acid without reactive groups ( B-2) was used to prepare cross-linked alginate gels (B-3) treated with EDTA. The alginate derivatives (EX1) (REF2) were completely destroyed in 24 hours. -(I)-B-2a, EX3-(II)-B-2a, EX7-(II)-B-2a, and EX8 -(I)-B-2) to obtain the crosslinked alginate gel (beads) (A1 to D1). The EDTA-treated cross-linked alginate gels (beads) (A2 to D2) showed The gel did not collapse even after 10 min, and the stability of the gel was improved. By forming the bridge, the (bead) structure created was able to absorb calcium ions. It was suggested that the structure was maintained for a long period of time even in the absence of on-crosslinks.

[0520] (Measurement of gel stability (4)) The reactive substituent-introduced alginic acid (EX1-(I)-A-2b) obtained in (Example 1g), ( The reactive substituent-introduced alginic acid (EX3-(II)-A-2b) obtained in Example 3g) (Example The reactive substituent-introduced alginic acid (EX5-(II)-A-2b) obtained in Example 5c) (Example The reactive substituent-introduced alginic acid (EX7-(II)-A-2) obtained in Example 7c) and The reactive substituent-introduced alginic acid (EX9-(I)-A-2) obtained in Example 9a) was dissolved in water. Alginic acid solutions with a concentration of 1.0% (1g-1), (3g-1), (5c-1), (7c The aqueous alginic acid solution was mixed with (9a-1) and (5c- 1), (9a-1) and (7c-1), (9a-1) and (3g-1), (1g-1) and (3 g-1) were mixed in equal amounts, and each mixed solution was injected into a separate 18-gauge syringe. The syringes were then placed in syringe pumps set to a flow rate of 1 mL / min. , and then dropwise added to a 30 mmol / L calcium chloride solution for 30 seconds, stirred for 5 minutes, and Alginate gels were obtained. Each gel was washed once with 10 mL of PBS and then diluted with PBS. The mixture was allowed to stand at 37°C for 10 minutes to carry out chemical crosslinking, yielding a chemically crosslinked alginate gel. 19.5 mL of PBS was added to the mixture, and the mixture was shaken at 37°C to collect the aqueous solution over time. After the test, the test solution was supplemented with the same amount of PBS as that of the sample. Add 10 μL of 100% glycerol (Chem., 319-08261) and shake overnight at 37°C to completely coat the gel. The alginic acid concentration in the collected aqueous solution was measured using carbazole sulfate. The alginic acid concentration in the aqueous solution at each time point was corrected by the concentration of alginic acid already collected. The total alginate concentration was calculated from the alginate concentration at all time points and at the end of the test. The value obtained by dividing the value by the concentration of glutamic acid was expressed as a percentage to obtain the disintegration rate, which was used as an index of gel stability.

[0521] The results are shown in Figure 8. The stability of each of the cross-linked alginate gels (beads) after 96 hours was The range of the crystal structure was 0.3 to 4.8%, suggesting that the structure was maintained for a long period of time (Example 1g). ) and (Example 3g) were used as controls. .

[0522] Measurement of gel stability (5): Measurement of gel stability in the presence of EDTA The reactive substituent-introduced alginic acid (EX1-(I)-A-2b) obtained in (Example 1g), ( The reactive substituent-introduced alginic acid (EX3-(II)-A-2b) obtained in Example 3g) (Example The reactive substituent-introduced alginic acid (EX5-(II)-A-2b) obtained in Example 5c) (Example The reactive substituent-introduced alginic acid (EX7-(II)-A-2) obtained in Example 7c) and The reactive substituent-introduced alginic acid (EX9-(I)-A-2) obtained in Example 9a) was dissolved in water. Alginic acid solutions with a concentration of 1.0% (1g-1), (3g-1), (5c-1), (7c The aqueous alginic acid solution was mixed with (9a-1) and (5c- 1), (9a-1) and (7c-1), (9a-1) and (3g-1), (1g-1) and (3 g-1) were mixed in equal amounts, and each mixed solution was injected into a separate 18-gauge syringe. The syringes were then placed in syringe pumps set to a flow rate of 1 mL / min. , and then dropwise added to a 30 mmol / L calcium chloride solution for 30 seconds, stirred for 5 minutes, and Alginate gels were obtained. Each gel was washed once with 10 mL of PBS and then diluted with PBS. The mixture was allowed to stand at 37°C for 10 minutes to carry out chemical crosslinking, yielding a chemically crosslinked alginate gel. Add 19.5 mL of 5 mM ethylenediaminetetraacetic acid dipotassium salt dihydrate ( EDTA 2K) / physiological saline was added, the mixture was shaken at 37°C, and the aqueous solution was collected over time. The collected amount was replenished with 5 mM EDTA·2K / physiological saline. After 4 hours, the test solution was added with alginate lyase (Nippon Gene, 319-08261). 30 μL of the solution was added, and the mixture was shaken overnight at 37°C to completely disintegrate the gel, and the aqueous solution was collected. The alginic acid concentration in the collected aqueous solution was measured by the carbazole-sulfuric acid method. The alginate concentration was corrected by the concentration of alginate already collected, and the alginate concentration at each time point was calculated. The percentage was calculated by dividing the alginic acid concentration after the test by the total alginic acid concentration. The value shown was taken as the disintegration rate and was used as an index of gel stability.

[0523] The results shown in FIG. 9 were obtained. The cross-linked alginate gel (beads) showed no changes even after 24 hours. The gel did not collapse, and its stability was confirmed. In other words, the chemical crosslinking by the Huisgen reaction was As a result, the (bead) structure created will maintain its structure for a long period of time. It was suggested that this is the case.

[0524] (Measurement of gel stability (6)) The reactive substituent-introduced alginic acid (EX1-(I)-A-2b) obtained in (Example 1g), ( The reactive substituent-introduced alginic acid (EX3-(II)-A-2b) obtained in Example 3g) (Example The reactive substituent-introduced alginic acid (EX5-(II)-A-2b) obtained in Example 5c) (Example The reactive substituent-introduced alginic acid (EX7-(II)-A-2) obtained in Example 7c) and The reactive substituent-introduced alginic acid (EX8-(I)-A-2) obtained in Example 8b) was dissolved in water. 1.0% concentration alginate aqueous solution (1g-1), (3g-1), (5c-1), (7c- The alginic acid aqueous solution was used to prepare (1g-1) and (5c-1). ), (1g-1) and (7c-1), (8b-1) and (5c-1), (8b-1) and (7c -1), (8b-1) and (3g-1), and (1g-1) and (3g-1) in equal amounts. Mix the solutions together, place each solution in a separate syringe equipped with an 18-gauge needle, and inject each solution into the syringe. The combined solutions were placed in separate syringes fitted with 18-gauge needles, and the syringes were then pumped at a flow rate of 1 mL / min. The syringes were set to a syringe pump set to 300 µL, and each syringe was filled with calcium chloride at a concentration of 30 mmol / L. The solution was added dropwise for 30 seconds and stirred for 5 minutes to obtain each alginate gel. The plates were washed once with 1 mL of PBS, and then left to stand in PBS at 37°C for 10 minutes to perform chemical crosslinking. A chemically crosslinked alginate gel was obtained. 19.5 mL of PBS was added to the gel, and the gel was shaken at 37°C. The aqueous solution was collected over time and replenished with the same amount of PBS as that collected. Add 10 μL of alginate lyase (Nippon Gene, 319-08261) to the test solution. The gel was completely broken down by shaking at 37°C overnight, and the aqueous solution was recovered. The concentration of alginic acid in the solution was measured by the carbazole-sulfuric acid method, and the amount of alginic acid dissolved at each time point was was calculated from the alginate concentration at all time points and at the end of the test. The value divided by the amount of acid was expressed as a percentage to determine the disintegration rate, which was used as an index of gel stability.

[0525] The results are shown in Figure 10. The stability of each of the cross-linked alginate gels (beads) after 96 hours The percentage of endothelial cells in the control group was approximately 20%, suggesting that the structure was maintained for a long period of time ( The stability of the gels prepared in Example 1g and Example 3g was 20.4% over 96 hours. %.

[0526] (Measurement of gel stability (7): Measurement of gel stability in the presence of EDTA) The reactive substituent-introduced alginic acid (EX1-(I)-A-2b) obtained in (Example 1g), ( The reactive substituent-introduced alginic acid (EX3-(II)-A-2b) obtained in Example 3g) (Example The reactive substituent-introduced alginic acid (EX5-(II)-A-2b) obtained in Example 5c) (Example The reactive substituent-introduced alginic acid (EX7-(II)-A-2) obtained in Example 7c) and The reactive substituent-introduced alginic acid (EX8-(I)-A-2) obtained in Example 8b) was dissolved in water. 1.0% concentration alginate aqueous solution (1g-1), (3g-1), (5c-1), (7c- The alginic acid aqueous solution was used to prepare (1g-1) and (5c-1). ), (1g-1) and (7c-1), (8b-1) and (5c-1), (8b-1) and (7c -1), (8b-1) and (3g-1), and (1g-1) and (3g-1) in equal amounts. Mix the solutions together, place each solution in a separate syringe equipped with an 18-gauge needle, and inject each solution into the syringe. The combined solutions were placed in separate syringes fitted with 18-gauge needles, and the syringes were then pumped at a flow rate of 1 mL / min. The syringes were set to a syringe pump set to 300 µL, and each syringe was filled with calcium chloride at a concentration of 30 mmol / L. The solution was added dropwise for 30 seconds and stirred for 5 minutes to obtain each alginate gel. mL of 5 mM ethylenediaminetetraacetic acid dipotassium salt dihydrate (EDTA 2K) / saline Saline was added, and the mixture was shaken at 37°C. The aqueous solution was collected over time, and the same amount of 5 After the test was completed (24 hours later), the test solution was replenished with 2 mM EDTA·2K / physiological saline. Add 30 μL of alginate lyase (Nippon Gene, 319-08261) and incubate for 37 The gel was completely broken down by shaking at 50° C. overnight, and the aqueous solution was recovered. The alginic acid concentration was measured by the carbazole-sulfuric acid method, and the amount of alginic acid dissolved at each time point was calculated. The total amount of alginic acid was calculated from the alginic acid concentration at the end of the test and the alginic acid concentration at the end of the test. The value obtained was expressed as a percentage to obtain the disintegration rate, which was used as an index of gel stability.

[0527] The results shown in FIG. 11 were obtained. The cross-linked alginate gel (beads) showed The gel did not collapse even after 10 min, confirming its stability. Due to the formation of the beads, the structure of the beads is maintained for a long period of time. It was suggested that this could be done.

[0528] (Measurement of gel stability (8)) The reactive substituent-introduced alginic acid (EX1-(I)-A-2b) obtained in (Example 1g), ( The reactive substituent-introduced alginic acid (EX3-(II)-A-2b) obtained in Example 3g) (Example The reactive substituent-introduced alginic acid (EX10-(II)-A-2) obtained in Example 10, The reactive substituent-introduced alginic acid (EX11-(II)-A-2) obtained in Example 11, 12), the reactive substituent-introduced alginic acid (EX12-(II)-A-2) obtained in (Example 1 3) the reactive substituent-introduced alginic acid (EX13-(II)-A-2) obtained in (Example 14 ) and the reactive substituent-introduced alginic acid (EX14-(II)-A-2) obtained by (Example 1 The reactive substituent-introduced alginic acid (EX15-(I)-A-2) obtained in 5) was dissolved in water for 1 0% concentration alginic acid aqueous solution (1g-1), (3g-1), (10-1), (11-1 ), (12-1), (13-1), (14-1), and (15-1) were prepared. Aqueous solutions of arginic acid were prepared as follows: (3g-1) and (15-1), (1g-1) and (10-1), (1g- 1) and (11-1), (1g-1) and (12-1), (1g-1) and (13-1), (1 g-1) and (14-1), and (1g-1) and (3g-1) were mixed in equal amounts. The mixed solutions were placed in separate syringes equipped with 18-gauge needles, and each mixed solution was The syringe was then placed into a syringe barrel fitted with an 18-gauge needle and the syringe barrel was set to a flow rate of 1 mL / min. The syringe pump was set up and each was immersed in a 30 mmol / L calcium chloride solution for 30 seconds. The gel was then diluted with 10 mL of PBS for 10 min and stirred for 5 min to obtain each alginate gel. The gel was washed once and then left to stand in PBS at 37°C for 10 minutes to perform chemical crosslinking. 19.5 mL of PBS was added to this gel, and the gel was shaken at 37°C to obtain a water-soluble gel. The solution was collected and replenished with the same amount of PBS as that collected. Add 10 μL of acid lyase (Nippon Gene, 319-08261) and incubate at 37°C overnight. The gel was completely broken down by shaking, and the aqueous solution was recovered. The amount of alginic acid dissolved at each time point was measured by the carbazole-sulfuric acid method, and the amount of alginic acid dissolved at each time point was calculated. The percentile was calculated by dividing the total amount of alginic acid calculated from the total amount of alginic acid and the total amount of alginic acid after the test. The value expressed as a percentage was taken as the disintegration rate and was used as an index of gel stability.

[0529] The results are shown in Figure 12. The stability of each of the cross-linked alginate gels (beads) after 96 hours The morphology was less than 0.5%, suggesting that the structure was maintained for a long period of time (control The stability of the gels prepared in (Example 1g) and (Example 3g) was 0.3% after 96 hours. (It is.)

[0530] (Measurement of gel stability (9): Measurement of gel stability in the presence of EDTA The reactive substituent-introduced alginic acid (EX1-(I)-A-2b) obtained in (Example 1g), ( The reactive substituent-introduced alginic acid (EX3-(II)-A-2b) obtained in Example 3g) (Example The reactive substituent-introduced alginic acid (EX10-(II)-A-2) obtained in Example 10, The reactive substituent-introduced alginic acid (EX11-(II)-A-2) obtained in Example 11, 12), the reactive substituent-introduced alginic acid (EX12-(II)-A-2) obtained in (Example 1 3) the reactive substituent-introduced alginic acid (EX13-(II)-A-2) obtained in (Example 14 ) and the reactive substituent-introduced alginic acid (EX14-(II)-A-2) obtained by (Example 1 The reactive substituent-introduced alginic acid (EX15-(I)-A-2) obtained in 5) was dissolved in water for 1 0% concentration alginic acid aqueous solution (1g-1), (3g-1), (10-1), (11-1 ), (12-1), (13-1), (14-1), and (15-1) were prepared. Aqueous solutions of arginic acid were prepared as follows: (3g-1) and (15-1), (1g-1) and (10-1), (1g- 1) and (11-1), (1g-1) and (12-1), (1g-1) and (13-1), (1 g-1) and (14-1), and (1g-1) and (3g-1) were mixed in equal amounts. The mixed solutions were placed in separate syringes equipped with 18-gauge needles, and each mixed solution was The syringe was then placed into a syringe barrel fitted with an 18-gauge needle and the syringe barrel was set to a flow rate of 1 mL / min. The syringe pump was set up and each was immersed in a 30 mmol / L calcium chloride solution for 30 seconds. The mixture was added dropwise for 5 minutes and stirred for 5 minutes to obtain each alginate gel. Add 19.5 mL of 5 mM ethylenediaminetetraacetic acid dipotassium salt dihydrate (E DTA 2K) / physiological saline was added, and the mixture was shaken at 37°C. The aqueous solution was then collected over time and circulated. The collected solution was replenished with 5 mM EDTA·2K / physiological saline in the same amount as that collected. After 3 hours, alginate lyase (Nippon Gene, 319-08261) was added to the test solution. 0 μL was added, and the mixture was shaken overnight at 37° C. to completely disintegrate the gel, and the aqueous solution was collected. The concentration of alginic acid in the aqueous solution was measured by the carbazole-sulfuric acid method, and the amount of eluted alginic acid was measured at each time point. The amount of alginic acid was calculated from the alginic acid concentration at all time points and the alginic acid concentration after the end of the test. The value obtained by dividing the value by the total amount of alginic acid was expressed as a percentage to obtain the disintegration rate, which was used as an index of gel stability.

[0531] The results shown in FIG. 13 were obtained. The cross-linked alginate gel (beads) showed The gel did not collapse even after 10 min, confirming its stability. Due to the formation of the beads, the structure of the beads is maintained for a long period of time. It was suggested that this could be done.

[0532] [Gel permeability measurement] (Measurement of gel permeability (1)) The alginic acid derivative (EX1-(I)-A-2) obtained in (Example 1a), and The alginic acid derivative (EX3-(II)-A-2) obtained in Example 3a) was diluted to a concentration of 2%. The alginic acid solution was dissolved in water to obtain (alginic acid solution 1-2) and (alginic acid solution 2-2). In addition, (alginic acid aqueous solution 1-2) was added with 1 mg / mL of 2 million molecular weight alginate. Fluorescein isothiocyanate-dextran (Sigma-Aldrich, FD2000S ) or fluorescein isothiocyanate-dextran with a molecular weight of 150,000 (Sigma-Aldrich) Add an equal amount of (Alginic acid aqueous solution 3) or (Alginic acid aqueous solution Liquid 4) was obtained. In addition, an equal amount of physiological saline was added to (alginic acid aqueous solution 2-2), and (alginic acid An aqueous solution 5) was obtained.

[0533] Mix equal amounts of (alginic acid aqueous solution 3) and (alginic acid aqueous solution 5), and dissolve the resulting aqueous solution. The solution was placed in a syringe fitted with an 18-gauge needle and the syringe was set to a flow rate of 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 for about 20 minutes and stirred to obtain an alginate gel. Wash once with water and fluorescein isothiocyanate (molecular weight 2 million)-dextran In addition, (alginic acid aqueous solution 4) and (alginic acid aqueous solution 5) were obtained. 5) and fluorescein isothiocyanate in the same manner as above. Chemically cross-linked alginate gel (beads) encapsulating 150,000-dextran (molecular weight: 150,000) was obtained.

[0534] 20 mL of physiological saline was added to the obtained gel (beads), and the gel was shaken at 37°C and then allowed to stand for 1 hour. After the test was completed, the aqueous solution was collected. Add 10 μL of 319-08261) and shake at 37°C for 2 hours to completely disintegrate the gel. The dextran concentration in the collected aqueous solution was measured using a fluorescence quantification method (excitation light: 4 The dextran concentration at each time point was measured after the test was completed. The value divided by the dextran concentration and expressed as a percentage was taken as the transmittance.

[0535] The gel obtained by mixing equal amounts of (alginic acid aqueous solution 3) and (alginic acid aqueous solution 5) (E X1-(I)-A-2 / EX3-(II)-A-2 / dextran (molecular weight: 2 million) The results are shown in Figure 2. 6.4% permeation was observed in 24 hours.

[0536] The gel obtained by mixing equal amounts of (alginic acid aqueous solution 4) and (alginic acid aqueous solution 5) (E X1-(I)-A-2 / EX3-(II)-A-2 / dextran (molecular weight: 150,000) The results are shown in Figure 3. A leakage of 23.6% was observed in 3 hours and 31.9% in 24 hours. It was.

[0537] (Measurement of gel permeability (2)) The alginic acid derivative (EX1-(I)-B-2a) obtained in (Example 1d), The alginic acid derivative (EX3-(II)-B-2a) obtained in (Example 7a) The alginic acid derivative (EX7-(II)-B-2a) obtained in Example 8 The acid derivative (EX8-(I)-B-2) was dissolved in water to a concentration of 1.5 w / w%. Thus, the alginic acid aqueous solutions (1d-2), (3d-2), (7a-2), and (8a-2) Furthermore, (alginic acid aqueous solution 3d-2) was added with 1 mg / mL of 200 10,000 μg of fluorescein isothiocyanate-dextran (Sigma-Aldrich, FD20 00S) or fluorescein isothiocyanate-dextran (Si) with a molecular weight of 150,000 (Alginic acid aqueous solution 3d-2-A) or (A Similarly, 1 ml of (alginic acid aqueous solution 7a-2) was added to obtain an alginic acid aqueous solution 3d-2-B). Fluorescein isothiocyanate-dextran with a molecular weight of 2 million, adjusted to g / mL (Sigma-Aldrich, FD2000S) or fluorescein isothiocyanate with a molecular weight of 150,000. Add ocyanate-dextran (Sigma-Aldrich, FD150S) and (arginine An aqueous acid solution 7a-2-A) or an aqueous alginic acid solution 7a-2-B) was obtained.

[0538] These aqueous solutions were prepared in the combinations shown in Table 15, with a final alginic acid concentration of 1.0 w / w%, full The final concentration of olescein isothiocyanate-dextran was 100 μg / mL. The mixed solution was placed in a syringe equipped with an 18-gauge needle, and the solution was injected. 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 The solution was dropped into calcium chloride solution for 30 seconds and stirred for 5 minutes to obtain an alginate gel. After washing once with 10 mL of phosphate-buffered saline (PBS), the plate was soaked in pure water at 37°C for 1 The plate was left to stand for 10 minutes to perform chemical cross-linking, followed by fluorescein isothiocyanate (molecular weight 2 million). -Dextran-encapsulated chemically cross-linked alginate gel (beads) and fluorescein isothiocyanate Alginate (molecular weight 150,000) - dextran-encapsulated chemically cross-linked alginate gel (beads) (gel a ~h) was obtained.

[0539] [Table 15]

[0540] Add 19.5 mL of physiological saline to each of gels a to h, shake at 37°C, and repeat the procedure for 3. After 24 hours, the aqueous solution was collected and replenished with the same amount of physiological saline as that collected. The test solution contained alginate lyase (Creative Enzymes, NATE-1563 ) was added, and the mixture was shaken overnight at 37°C to completely disintegrate the gel, and the aqueous solution was collected. The dextran concentration in the collected aqueous solution was measured by fluorescence quantification (excitation light: 485 nm, fluorescence: 53 The dextran concentration at each time point was measured using a 5 nm (5 nm) fluoroscopy. The dextran concentration after the end of the experiment was divided by the total dextran concentration, and the value was expressed as a percentage. The obtained value was regarded as the transmittance.

[0541] For (Alginic acid aqueous solution 3d-2-A) and (Alginic acid aqueous solution 7a-2-A), Alginic acid aqueous solution 1d-2) and (alginic acid aqueous solution 8a-2) were mixed together. The results of the gels (gel a, gel b, gel c, and gel d in Table 15) are shown in Figure 5. The transmittance after 24 hours was 0%. and (alginic acid aqueous solution 7a-2-B), (alginic acid aqueous solution 1d-2), (alginic acid aqueous solution The gels obtained by mixing each of the aqueous solutions of phosphate phosphate 8a-2) (Gels e, f, and g in Table 15) The results of gels g and h are shown in Figure 6. The transmittance after 3 hours was 2.5% to 4.6%, and after 24 hours it was 2.5% to 4.6%. The transmittance at time was 6.8%-8.6%.

[0542] (Measurement of gel permeability (3)) The reactive substituent-introduced alginic acid (EX3-(II)-A-2b) obtained in (Example 3g), The reactive substituent-introduced alginic acid (EX5-(II)-A-2b) obtained in (Example 5c), and The reactive substituent-introduced alginic acid (EX7-(II)-A-2) obtained in Example 7c was An aqueous solution of alginic acid was prepared by dissolving it in water to a concentration of 2.0%. Fluorescein isothiocyanate (150,000 molecular weight) was added to the solution at 2 / 5 volume of 1 mg / mL. Add 100 ml of ethyl acetate-dextran (Sigma-Aldrich, FD150S) and 3 / 5 volume of water. 0.2 mg / mL fluorescein isothiocyanate-dextran containing 1.0% Aqueous solutions of alginic acid (3g-2), (5c-2), and (7c-2) were prepared. The reactive substituent-introduced alginic acid (EX1-(I)-A-2b) obtained in (Example 1g), and The reactive substituent-introduced alginic acid (EX9-(I)-A-2) obtained in (Example 9a) was added to a solution of 100 ml of 10 ... The alginic acid solution (1g-1) and (9a- 1) was prepared. The above-mentioned aqueous alginic acid solution was mixed with (9a-1) and (5c-2), and (9a-1) and Combinations of (7c-2), (9a-1) and (3g-2), and (1g-1) and (3g-2) Mix equal amounts of 100 mL of calcium chloride solution at 30 mmol / L, add 40 mL of calcium chloride solution at 5 After stirring for 10 minutes, an alginate gel was obtained. The gel was washed once with 10 mL of physiological saline. Chemical crosslinking was performed by leaving the membrane in physiological saline at 37°C for 10 minutes, and fluorescein isothiocyanate was added. The resulting alginate gels were then encapsulated in dextran-containing alginate. 100 ml of physiological saline was added, the mixture was shaken at 37°C, and the aqueous solution was collected over time. After the test was completed (24 hours later), the test solution was supplemented with alginate lyase ( Add 10 μL of Nippon Gene Co., Ltd., 319-08261) and shake at 37°C for at least 3 hours. The gel was completely disintegrated by stirring, and the aqueous solution was recovered. The dextran concentration in the recovered aqueous solution was measured using a fluorescent The amount of dextran was measured at each time point by photometric quantification (excitation light: 485 nm, fluorescence: 535 nm). The transmittance was calculated by dividing the dextran concentration by the dextran concentration after the test and expressing the result as a percentage.

[0543] The results shown in Figure 14 were obtained. The transmittance after 3 hours was about 30% in all cases. The transmittance after 4 hours was approximately 40% in all cases.

[0544] (Measurement of gel permeability (4)) The reactive substituent-introduced alginic acid (EX3-(II)-A-2b) obtained in (Example 3g), The reactive substituent-introduced alginic acid (EX5-(II)-A-2b) obtained in (Example 5c), and The reactive substituent-introduced alginic acid (EX7-(II)-A-2) obtained in Example 7c was An aqueous solution of alginic acid was prepared by dissolving it in water to a concentration of 2.0%. Fluorescein isothiocyanate (150,000 molecular weight) was added to the solution at 2 / 5 volume of 1 mg / mL. Add 100 ml of ethyl acetate-dextran (Sigma-Aldrich, FD150S) and 3 / 5 volume of water. 0.2 mg / mL fluorescein isothiocyanate-dextran containing 1.0% Aqueous solutions of alginic acid (3g-2), (5c-2), and (7c-2) were prepared. The reactive substituent-introduced alginic acid (EX1-(I)-A-2b) obtained in (Example 1g), and The reactive substituent-introduced alginic acid (EX8-(I)-A-2) obtained in (Example 8b) was added to a solution of 100 ml of 10 ... The alginic acid solution (1g-1) and (8b- 1) were prepared. These were then mixed with (1g-1) and (5c-2), and (1g-1) and (7 c-2), (8b‐1) and (5c-2), (8b‐1) and (7c-2), (8b‐1) and (3g-2), (1g-1) and (3g-2) were mixed in equal amounts to a concentration of 30 μM. Add 40 mL of 100 mol / L calcium chloride solution and stir for 5 minutes to form an alginate gel. The gel was washed once with 10 mL of physiological saline and incubated in physiological saline at 37° C. for 10 minutes. The mixture was left to stand for a period of time to perform chemical crosslinking, and then fluorescein isothiocyanate-dextran encapsulation chemical A cross-linked alginate gel was obtained. 19.5 mL of physiological saline was added to each gel and incubated at 37°C. The aqueous solution was collected over time and replenished with an equal amount of physiological saline. After 24 hours, the test solution was diluted with alginate lyase (Nippon Gene, 319-08 Add 10 μL of 261) and shake 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 measured by fluorescence quantification (excitation light: 485 nm The dextran concentration at each time point was measured using a fluorescent spectrometry (fluorescence: 535 nm) and the dextran concentration at each time point was The value divided by the orchid concentration was expressed as a percentage to determine the transmittance.

[0545] The results shown in Figure 15 were obtained. The transmittance after 3 hours was about 35% in all cases. The transmittance after 4 hours was about 45% in all cases.

[0546] (Measurement of gel permeability (5)) The reactive substituent-introduced alginic acid (EX3-(II)-A-2b) obtained in (Example 3g), The reactive substituent-introduced alginic acid obtained in Example 10 (EX10-(II)-A-2), ( The reactive substituent-introduced alginic acid (EX11-(II)-A-2) obtained in Example 11, The reactive substituent-introduced alginic acid (EX12-(II)-A-2) obtained in Example 12, The reactive substituent-introduced alginic acid (EX13-(II)-A-2) obtained in Example 13 and The reactive substituent-introduced alginic acid (EX14-(II)-A-2) obtained in Example 14 was concentrated An aqueous solution of alginic acid was prepared by dissolving it in water to a concentration of 2.0%. Fluorescein isothiocyanate with a molecular weight of 150,000 was prepared at 1 mg / mL in 2 / 5 volume of 100 ml ... Add 100 ml of dimethyl ether (Sigma-Aldrich, FD150S) and 3 / 5 volume of water. , 0.2 mg / mL fluorescein isothiocyanate-dextran containing 1.0% Aqueous solution of arginic acid (3g-2), (10-2), (11-2), (12-2), (13-2 ), and (14-2) were prepared. Furthermore, the reactive substituent-introduced arylene glycol obtained in (Example 1g) was used to prepare the above-mentioned aryl group. carboxylic acid (EX1-(I)-A-2b) and the reactive substituent-introduced alkenyl group obtained in (Example 15) Dissolve acetic acid (EX15-(I)-A-2) in water to a concentration of 1.0%. Then, aqueous solutions of alginic acid (1g-1) and (15-1) were prepared. 15-1) and (3g-2), (1g-1) and (10-2), (1g-1) and (11-2) , (1g-1) and (12-2), (1g-1) and (13-2), (1g-1) and (14- 2) (1g-1) and (3g-2) were mixed in equal amounts to a concentration of 30 mmol / L. 40 mL of calcium chloride solution was added and stirred for 5 minutes to obtain an alginate gel. The gel was washed once with 10 mL of physiological saline and left to stand in physiological saline at 37°C for 10 minutes. Chemical crosslinking was performed using fluorescein isothiocyanate-dextran-encapsulated chemically crosslinked alginate. To each gel, 19.5 mL of physiological saline was added and the gel was shaken at 37°C. The aqueous solution was collected over time and the same amount of saline was added as the collected amount. After 24 hours, the test solution was added with alginate lyase (Nippon Gene, 319-08261). Add 10 μL of the solution, shake at 37°C for 3 hours or more to completely disintegrate the gel, and collect the aqueous solution. The dextran concentration in the collected aqueous solution was measured by fluorometric analysis (excitation light: 485 nm, fluorescence: 535 nm) and the dextran concentration at each time point was calculated as the dextran concentration at the end of the test. The value obtained by dividing the measured value by the measured value and expressed as a percentage was taken as the transmittance.

[0547] The results shown in FIG. 16 were obtained. The transmittance after 3 hours was about 17 to about 28%. The transmittance after 24 hours was about 25 to about 35%.

[0548] [Biocompatibility evaluation of cross-linked alginate derivatives (gels)] Produced in the same manner as in Example 1d, with an introduction rate (NMR integral ratio) of 0.9 mol% Alginic acid derivative (EX1-(I)-B-2-L), prepared in a similar manner to Example 3d The alginic acid derivative (EX3-(II)-B) with an introduction rate (NMR integral ratio) of 0.6 mol% -2-L), (Example 7a) was prepared in the same manner as above. Introduction rate (NMR integral ratio) = 0.9 mol% alginic acid derivative (EX7-(II)-B-2-L), in the same manner as in Example 8 The alginic acid derivative (EX8-) with an introduction rate (NMR integral ratio) of 0.3 mol% was prepared by the method. (I)-B-2-L) were dissolved in physiological saline to a concentration of 1.5 w / w%. Alginic acid aqueous solutions (1dL), (3dL), (7aL), and (8aL) were obtained. In addition, 1.5×1 0 7 Add the PBS suspension of CHO cells adjusted to 100 cells / mL (Arg As a result, aqueous phosphonic acid solution 3d-LC or aqueous alginic acid solution 7a-LC was obtained.

[0549] These aqueous solutions were prepared in the combinations shown in Table 16, with a final alginic acid concentration of 1.0 w / w%, CH Final cell concentration: 5.0 x 10 6 The mixture was mixed so that the concentration was 100 cells / mL. The aqueous solution was placed in a syringe fitted with an 18-gauge needle, and the syringe was set at a flow rate of 1 mL / min. The syringe pump was set up and the solution was filled with 50 mmol / L calcium chloride solution. After stirring for 5 minutes, add 10 mL of phosphate buffered saline (PBS) once. Wash and prepare CHO cell-encapsulated alginate gels (beads) (Gel CHO-1 to Gel CHO-4) obtained.

[0550] [Table 16]

[0551] (Gel CHO-1) to (Gel CHO-4) were placed in a 6-well plate (FALCON, Ca The medium with the composition shown in Table 17 below was added at 5 mL / well. After the gel was impregnated, it was shaken at 125 rpm in a 37°C, 5% CO2 incubator. After the test, the gel was collected and re-immersed in 5 mL of fresh medium. After that, alginate lyase (Creative Enzymes, NATE-1563 ) was added, and the mixture was shaken at 37°C for 1 hour to completely disintegrate the gel, and the medium was collected. The number of live and dead cells in the collected medium was counted by trypan blue staining, and the number of live cells was calculated as the viable cell count. The percentage obtained by dividing the number of cells by the sum of the number of dead cells and the number of cells in the culture was used as the cell viability, and was used as an index of the biocompatibility of the gel. Similarly, alginic acid (B-2) without reactive groups was used as a control. We prepared CHO cell-encapsulated alginate gel (beads) (REF-CHO) using the REF-CHO antibody and confirmed cell viability. The rate was measured.

[0552] [Table 17]

[0553] The results are shown in Figure 7. The results obtained with the combination of each alginic acid derivative shown in Table 16 The cell viability in alginate gels (Gel CHO-1 to Gel CHO-4) was 87. The results were as follows: 0.2% to 89.0%. In addition, the arylene glycol without reactive groups, which was used as a control, Cell viability in alginate gel (REF-CHO) prepared using alginic acid (B-2) The result showed that the alginic acid derivatives with reactive groups and H The alginate structure (beads) on which chemical crosslinks have been formed by the Uisgen reaction is It was suggested that the biocompatibility of the alginic acid was as high as that of the unincorporated alginic acid. .

[0554] [Biocompatibility evaluation of cross-linked alginate derivatives (gels) (2)] The reactive substituent-introduced alginic acid (EX1-(I)-A-2b) obtained in (Example 1g), ( The reactive substituent-introduced alginic acid (EX3-(II)-A-2b) obtained in Example 3g) (Example The reactive substituent-introduced alginic acid (EX5-(II)-A-2b) obtained in Example 5c) (Example The reactive substituent-introduced alginic acid (EX7-(II)-A-2) obtained in Example 7c) and The reactive substituent-introduced alginic acid (EX8-(I)-A-2) obtained in Example 8b) (Example 9a The reactive substituent-introduced alginic acid (EX9-(I)-A-2) obtained in (Example 10) The reactive substituent-introduced alginic acid (EX10-(II)-A-2) obtained in Example 11 Reactive substituent-introduced alginic acid (EX11-(II)-A-2), the reaction mixture obtained in Example 12 Reactive Substituent-Introduced Alginic Acid (EX12-(II)-A-2), Reaction Obtained in Example 13 Alginic acid with introduced reactive substituents (EX13-(II)-A-2) (Example 14) Substituent-introduced alginic acid (EX14-(II)-A-2) and the reaction obtained in (Example 15) The cross-linked alginic acid (EX15-(I)-A-2) was dissolved in water to prepare the cross-linked alginic acid. This was filtered through a Mini Sart High Flow (Sartorius, 16532GUK). After over-sterilization, a 1.0% cross-linked alginic acid / physiological saline solution was prepared. Cell concentration 5 ×10^3 cells / well in a 96-well plate and then cultured for 1 day. The cultured HeLa cells were then treated with 1.0% cross-linked alginate / physiological saline solution ( Example 1g), (Example 8b), (Example 9a), or a combination of (Example 15) and (Example 3g) Combination of (Example 1g) with (Example 5c), (Example 7c), (Example 10), Combination of (Example 11), (Example 12), (Example 13), or (Example 14) After one day of culture, ATP activity was measured as an index of cytotoxicity. llTiter-Glo Luminescent Cell Viability A The results were evaluated using a PCR assay (Promega, G7571).

[0555] The results shown in Figure 17 were obtained. ATP activity was confirmed in all of the cross-linked alginate gels. These results suggest that cross-linked alginate gels are not cytotoxic. The alginate structures (beads) formed by chemical cross-linking through the EN reaction are biocompatible. It was suggested that there is.

Claims

[Claim 1] The following formula: 【Chemistry 1】 or a salt thereof, or a solvate thereof.

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