Novel cross-linked alginic acid

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to a novel alginate derivative, a novel crosslinked alginate, and a novel crosslinked alginate structure. This relates to the construction of bodies and the methods for manufacturing them. [Background technology]

[0002] Alginic acid is found in Lessonia, Macrocystis, Laminaria, Ascophyllum, and Derby. High molecular weight acidic polysaccharides extracted from the cell walls of natural brown algae such as Rhiza, Kajime, Arame, and Kombu. It is a molecule consisting of β-D-mannuronic acid (component M) and its C-5 epimer, α-L-glucan. It is a linear heteropolymer in which two types of uronic acid (component G) are linked in a 1-4 bond. Specifically, its chemical structure is a homopolymer block (MM) of mannuronic acid, guluronic acid Homopolymer blocks (GG) of mannuronic acid and guluronic acid arranged randomly It is a block copolymer in which blocks (MG) are complexly bonded in any permutation and proportion. Ginic acid has a wide range of applications in fields such as medicine, biotechnology, cosmetics, textiles, papermaking, and food. It is widely used.

[0003] Alkali metal salts of monovalent alginic acid (e.g., sodium alginate) While divalent alginate alkaline earth metal salts (for example, alginic acid) are water-soluble, divalent alginate alkaline earth metal salts (for example, alginic acid) Calcium, etc., has the property of being crosslinked and gelled (insoluble) by metal ions. Attempts are being made to modify or mold these materials to suit various applications by utilizing their properties.

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

[0005] Specifically, as a method for obtaining cross-linked polysaccharides, (1) aldehyde cross-linking such as formaldehyde Crosslinking method using an agent (Patent Document 1: International Publication No. 2011 / 028031 pamphlet), (2) Self-crosslinking method using carboxyl groups and hydroxyl groups in polysaccharides (Patent Document 2: International Publication No. 89) (Pamphlet No. 10941), (3) Homobifunctional crosslinking agents (diepoxide, divinyl (e.g., rufon, diamine, or dihydrazide) or heterobifunctional crosslinking agent (epihalohydrin) Crosslinking method using (etc.) (Patent Document 3: International Publication No. 2009 / 073437 pamphlet) 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, cinnamyridene acetate, sorbic acid, thymine A crosslinking method (Patent Documents 4, 5: Japan) involves introducing substances such as coumarin and then irradiating them with light. International Publication No. 2005 / 026214 (Japanese Patent Publication No. 9-87236), and (5) A crosslinking method and thio Michael addition reaction using polysaccharides to which a 'L' group has been introduced and polysaccharides to which a maleimide group has been introduced. Crosslinking method by causing (Patent Document 6: International Publication No. 2008 / 071058 pamphlet) (T), etc. are known.

[0007] Furthermore, as a method of crosslinking polysaccharides by covalent bonding, (6) a polysaccharide in which an alkyne group is introduced Huisgen reaction (1,3-dipolar addition) using sugars and polysaccharides to which azide groups have been introduced A crosslinking method using cyclization reactions is known.

[0008] Crosslinked polysaccharides obtained by crosslinking polysaccharides via the Huisgen reaction, (i) International Publication No. 2008 / 0 Pamphlet No. 31525 (Patent Document 7), (ii) International Publication No. 2012 / 165462 (iii) Pamphlet No. 2015 / 020206 (Patent Document 8), (iii) Pamphlet International Publication No. 2015 / 020206 Lett (Patent Document 9), (iv) Specification of Chinese Patent Application Publication No. 106140040 (Patent Reference 10), and (v) International Publication No. 2019 / 240219 (Patent Document 1) 3) Disclosed in, etc.

[0009] However, (i) Patent Document 7 states that the first polysaccharide is hyaluronic acid and the second polysaccharide is chondroitin. A polysaccharide selected from , sulfated dermatan, alginic acid or its salts, etc., is added to each polysaccharide. The chain-like alkyne and azide groups introduced via a carbide converter are subjected to Huisg in the presence of a copper catalyst. This relates to cross-linked polysaccharides obtained by EN reaction, and to the novel cross-linked algin described later. The acid is not disclosed.

[0010] Furthermore, (ii) Patent Document 8 describes a first polysaccharide and a second polysaccharide as hyaluronic acid, carboxymethyl A polysaccharide selected from tildextran, cellulose derivatives, and chitosan (the first polysaccharide and The second polysaccharide may be of the same or different species) and each polysaccharide has a linker (polysaccharide and linker) The linker is an ester bond) through which the cyclic alkyne group and azide group are introduced. This relates to cross-linked polysaccharides obtained by SGEN reaction, but a novel cross-linked a Luginic acid is not disclosed.

[0011] Furthermore, (iii) Patent Document 9 describes a first polysaccharide as hyaluronic acid and a second polysaccharide as chondroitin. As sulfuric acid, the cyclic alkyne group and azide group introduced to each polysaccharide via a linker are Hu This relates to crosslinked polysaccharides obtained by isgen reaction, but the novel crosslinking described later... Alginate is not disclosed.

[0012] Furthermore, (iv) Patent Document 10 describes a first polysaccharide as chitosan and a second polysaccharide as sodium alginate. As a result, linkers are introduced into each polysaccharide via an ester bond (the relationship between polysaccharides and linkers is ester bonds). A cross-linked polysaccharide obtained by Huisgen reaction of the cyclic alkyne group and azide group is However, the novel cross-linked alginic acid described later is not disclosed.

[0013] Also, see International Publication No. 2016 / 019391 (Patent Document 11) and International Publication Pamphlet No. 2017 / 165389 (Patent Document 12) describes the introduction of an azide group into the side chain. Alginic acid is described, but alginic acid in which an alkyne group is introduced into the side chain is also described. The cross-linked alginate structure formed is not disclosed, and its intended use is different from that of the present invention. Yes.

[0014] Furthermore, Non-Patent Document 1 describes branched alginate (bA) in which a cyclooctin side chain is introduced into the side chain. Although lg-DBCO) is listed, alginic acid and branched polyethylene glycol (4-arm PEG) are also listed. Branched alginic acid (bAlg) synthesized from -NH2 is aminated This was obtained by reacting with chlorooctin (DBCO-PEG-amine), and is a novel argy that will be described later. It differs in structure from nic acid derivatives, and therefore also in its intended use. [Prior art documents] [Patent Documents]

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

[0016] [Non-Patent Document 1] Nat Commun.9(1), p2195-, 2018. [Overview of the project] [Problems that the invention aims to solve]

[0017] In the aforementioned situation, a novel alginate derivative, formed from the novel alginate derivative Novel cross-linked alginic acid, cross-linked alginic acid structures, and methods for producing them are needed. He was. [Means for solving the problem]

[0018] The inventors of this invention have conducted extensive research to solve the above problems, and as a result, have obtained formula (I) or formula (I Novel alginate derivatives represented by formula (I) were discovered. Furthermore, formulas (I) and (II) were also discovered. A novel crosslinked alginate derivative obtained by subjecting it to the Huisgen reaction Using ginic acid, beads (pigment-containing beads), which are one type of cross-linked alginic acid structure, are formed. Upon investigation, it was found that the beads have high stability and, compared to conventional gels, are suitable for the intended purpose. We discovered that the gel can be adjusted to have permeability, and thus completed the present invention.

[0019] The novel alginate derivatives (formulas (I) and (II)) provided herein are, for example, chemically formulated. It can be used for chemical crosslinking, that is, it can be used for chemical crosslinking. A reactive group or a reactive group complementary to the said reactive group has been introduced.

[0020] The aforementioned chemical crosslinking is carried out, for example, by the Huisgen reaction (1,3-dipole cycloaddition reaction). This is carried out by a crosslinking reaction, for example, between alginic acid derivatives of formula (I) and formula (II). It may also be done, or, for example, between the alginate derivative of formula (I) and other molecules having an azide group. This may also be done with an alginate derivative of formula (II) and another molecule having an alkyne group. It can be done in between.

[0021] Huisgen reactions involving terminal alkyne and terminal azide groups generally occur at temperatures above 100°C. Because this reaction requires heating, it was not suitable for the chemical modification of biomolecules. However, by introducing a copper catalyst (e.g., Cu(I)) into the reaction, Reaction conditions have been found in which a cyclization adduct (triazole ring) is formed at temperature with nearly 100% yield. (Angew.Chem.Int.Ed.Engl.,41,p2596-2599) ,2002;J.Org.Chem.,67,p3057-3064,2002), It has become possible to use it for the chemical modification of biomolecules. On the other hand, Huis in the presence of the copper catalyst When attempting to obtain cross-linked alginate by the GEN reaction, a trace amount of copper hydroxide may be present in the cross-linked alginate. There is a possibility that the medium may remain, and copper is present in the cross-linked alginate or cross-linked alginate structure. There is a concern that cytotoxicity may develop in the future.

[0022] In a preferred embodiment, in order to avoid the expression of copper-derived cytotoxicity in crosslinked alginate, copper Crosslinked alginate is obtained using the Huisgen reaction, which does not require a medium. Specifically, alginate A cyclooctyne derivative (a highly strained cyclic alkyne group) is introduced into the alkyne group of an acid derivative. By using this method, the reaction can be carried out without requiring high temperatures of 100°C or higher, or a copper catalyst. Therefore, a novel crosslinked alginic acid in a preferred embodiment does not contain a copper catalyst. Even when molded into the final form (cross-linked alginate structure), copper-derived toxicity does not manifest. It is also superior in that it does not have to do anything.

[0023] Here, any one or more carboxyl groups of alginic acid as shown in the following embodiment are Formula (I) is a compound in which a cyclic alkyne group or azide group is introduced via a mido bond and a divalent linker. Alternatively, using an alginate derivative of formula (II), or alginate derivatives of formula (I) and formula (II) A novel crosslinking reaction can be obtained by performing the Huisgen reaction (1,3-dipolar cycloaddition reaction). Alginic acid, crosslinked alginic acid structures, and each of the aforementioned alginic acid derivatives, crosslinked alginic acid, and A method for producing a crosslinked alginate structure is provided. That is, an exemplary embodiment is as follows: It can be as follows: 1) to 23).

[0024] [1] An amide bond and a divalent linker to any one or more carboxyl groups of alginic acid ( -L 1 The following formula (I) is formed by introducing a cyclic alkyne group (Akn) via -): [ka] [In formula (I), Akn, -L 1 -, -NHCO- and (ALG) are the first aspect described below. Alginic acid derivatives represented by [the same definition as in the middle], and any one of alginic acid The carboxyl group above has an amide bond and a divalent linker (-L 2 -) via the azide group The following formula (II) was introduced: [ka] [In formula (II), -L 2 -, -NHCO- and (ALG) are defined in the first aspect described later. It is obtained by performing a crosslinking reaction using an alginic acid derivative represented as [same as]. Cross-linked alginic acid.

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

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

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

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

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

Chemical formula

[0030] 〔3〕The Akn-L 1 -NH2 group (Akn and -L 1 - are the definitions in the third aspect described below The adoption rate of (which is the same as) is 0.1% to 30%, expressed by formula (I) described in [2] above. Alginic acid derivatives that are used.

[0031] [4] Weight-average molecular weight of alginate derivatives measured by gel filtration chromatography However, the argy expressed by formula (I) described in [2] above is between 100,000 Da and 3,000,000 Da. Acid derivative.

[0032] [5] An amide bond and a divalent linker to any one or more carboxyl groups of alginic acid ( -L 2 -) through which an azide group is introduced, as shown in formula (II): [ka] [In formula (II), (ALG), -L 2 The definition of - is the same as the definition in the fifth aspect described later. Alginic acid derivatives represented as [a certain]

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

[0034] [7] Weight-average molecular weight of alginate derivatives measured by gel filtration chromatography However, the range is 100,000 Da to 3,000,000 Da, which is expressed by formula (II) described in [5] above. Ginate derivative.

[0035] [8] Any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid The sil group is represented by the following formula (III-L): [ka] [In formula (III-L), the -CONH- and -NHCO- at both ends are any of the components of alginic acid. Represents an amide bond via a ruboxyl group; -L 1 -, -L 2 -, and X are described below. The cross-linked alginic acid described in [1] above, bonded via the same definition as in the embodiment of 8. .

[0036] [8a] Any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid The xyl group is represented by the following formula (III-L): [ka] [In formula (III-L), the -CONH- and -NHCO- at both ends are any of the components of alginic acid. Represents an amide bond via a ruboxyl group; -L 1 -, -L 2 -, and X are described below. Crosslinked alginic acid bonded via the same method as defined in aspect 8a.

[0037] [8-1] Chemical bridges formed by triazole rings formed by the Huisgen reaction as crosslinks The above [1] or [ Cross-linked alginic acid as described in [8a]. [8-2] Chemical bridges formed by triazole rings formed by the Huisgen reaction as crosslinks. Crosslinked alginic acid as described in [1] or [8a] above, including a bridge.

[0038] [8-3-1] In the above [8-1], the divalent metal ions are calcium ions, magnesium ions. From the group consisting of cium ions, barium ions, strontium ions, and zinc ions It is the selected ion.

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

[0040] [9] Alginic acid derivatives represented by formula (I) and formula (II) described in [1] above By mixing the alginic acid derivative with the resulting crosslinking reaction (Huisgen reaction), the A method for producing crosslinked alginic acid, including obtaining the crosslinked alginic acid described in [1] or [8a]. How to do it.

[0041] [9-1] A solution of the alginate derivative represented by formula (I) described in [1] above is used as described above. Add the solution of the alginic acid derivative represented by formula (II) described in [1] to the crosslinking reaction (Hui By performing the SGEN reaction, the crosslinked alginic acid described in [1] or [8a] above can be obtained. A method for producing cross-linked alginic acid, which includes [the specified element].

[0042] [9-2] A solution of the alginic acid derivative represented by formula (II) described in [1] above, Add the solution of the alginic acid derivative represented by formula (I) described in [1] to the crosslinking reaction (Hui By performing the SGEN reaction, the crosslinked alginic acid described in [1] or [8a] above can be obtained. A method for producing cross-linked alginic acid, which includes [the specified element].

[0043]

[10] Alginic acid derivatives represented by formula (I) and those represented by formula (II) as described in [1] above By performing the Huisgen reaction (crosslinking reaction) using the alginic acid derivative, the shape is formed. The chemical crosslinking that is formed is shown in the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO-, -L at both ends] 1 -, and -L 2 - is the same as the definition in the 10th aspect described later], which has the structure of the above [1] or [8 A method for producing crosslinked alginic acid, comprising obtaining the crosslinked alginic acid described in (a).

[0044]

[11] Alginic acid derivatives represented by formula (I) and those represented by formula (II) as described in [1] above. A mixed solution of alginate derivatives, which is a mixture of alginate derivatives, is used to remove divalent metal ions. A cross-linked alginate structure obtained by dropping it into a solution containing it.

[0045] [11-1] A solution of the alginate derivative represented by formula (I) described in [1] above is divalent. The gel obtained by dropping it into a solution containing metal ions is expressed using formula (II) described in [1] above. A cross-linked alginate is obtained by adding a cross-linking reaction to a solution of the alginate derivative to the cross-linked alginate. Ginate structure.

[0046] [11-2] A solution of the alginic acid derivative represented by formula (II) described in [1] above, 2 The gel obtained by dropping it into a solution containing valence metal ions is expressed using formula (I) described in [1] above. A cross-linked alginate is obtained by adding a cross-linking reaction to a solution of the alginate derivative to the cross-linked alginate. Ginate structure.

[0047] [12-1] Chemical crosslinking by triazole rings formed by the Huisgen reaction The above

[11] ~ includes crosslinking and ionic crosslinking partially formed by divalent metal ions. A cross-linked alginate structure as described in any one of the items in [11-2].

[0048] [12-2] Chemical reactions by triazole rings formed by the Huisgen reaction as crosslinks A crosslinked alginate structure according to any one of the above items

[11] to [11-2], including crosslinking .

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

[0050] [12-3-2] In [12-1] above, the source of divalent metal ions is chloride Calcium aqueous solution, calcium carbonate aqueous solution, calcium gluconate aqueous solution, barium chloride aqueous solution It is an aqueous solution selected from the group consisting of solutions, etc.

[0051] [13-1] Alginate derivatives represented by formula (I) and formula (II) described in [1] above Alginic acid derivatives represented by are subjected to ion crosslinking with divalent metal ions and Huisgen reaction. A cross-linked alginate structure having content retention properties, obtained by chemical cross-linking according to the application.

[0052] [13-2] Alginate derivatives represented by formula (I) and formula (II) described in [1] above The alginic acid derivative represented by is obtained by chemical crosslinking via the Huisgen reaction. A cross-linked alginate structure that has the ability to retain substances.

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

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

[0055]

[14] Alginic acid derivatives represented by formula (I) and formula (II) as described in [1] above The chemical compound formed by performing the Huisgen reaction using the alginate derivative is formed. The bridge is formed by the following equation (III-L): [ka] [In formula (III-L), -CONH- and -NHCO-, -L at both ends] 1 -, and -L 2 - is the same as the definition in the 14th aspect described later], the structure of the above

[11] to The cross-linked alginate structure described in [13-2].

[0056]

[15] Alginic acid derivatives represented by formula (I) and those represented by formula (II) as described in [1] above A mixed solution of alginate derivatives, which is a mixture of alginate derivatives, is used to remove divalent metal ions. A cross-linked alginate structure is obtained by adding it dropwise to a solution containing it and carrying out a cross-linking reaction. Method of making.

[0057] [15-1] A solution of the alginate derivative represented by formula (I) described in [1] above, divalent The gel obtained by dropping it into a solution containing metal ions is expressed using formula (II) described in [1] above. A cross-linked alginate is obtained by adding a cross-linking reaction to a solution of the alginate derivative to the cross-linked alginate. A method for producing a ginate structure.

[0058] [15-2] A solution of the alginic acid derivative represented by formula (II) described in [1] above, 2 The gel obtained by dropping it into a solution containing valence metal ions is expressed using formula (I) described in [1] above. A cross-linked alginate is obtained by adding a cross-linking reaction to a solution of the alginate derivative to the cross-linked alginate. A method for producing a ginate structure.

[0059] [16-1] Chemical crosslinking by triazole rings formed by the Huisgen reaction The above

[15] ~ includes crosslinking and ionic crosslinking partially formed by divalent metal ions. A method for producing a crosslinked alginate structure as described in any one of the items in [15-2].

[0060] [16-2] Chemical crosslinking by triazole rings formed by the Huisgen reaction A crosslinked alginate structure according to any one of the above items

[15] to [15-2], including crosslinking A method for manufacturing.

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

[0062] [16-3-2] In [16-1] above, the source of divalent metal ions is chloride Calcium aqueous solution, calcium carbonate aqueous solution, calcium gluconate aqueous solution, barium chloride aqueous solution It is an aqueous solution selected from the group consisting of solutions, etc.

[0063]

[17] Alginic acid derivatives represented by formula (I) as described in any one of the items in [1] above and By performing the Huisgen reaction using an alginate derivative represented by formula (II), The chemical crosslinks formed are of the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO-, -L at both ends] 1 -, and -L 2 - is the same as the definition in the 17th aspect described later], the structure of the above

[15] ~[1 A method for producing a crosslinked alginate structure as described in any one of the items in 6-2.

[0064]

[18] Beads or a roughly spherical gel as described in any one of the above items

[11] to

[14] . A cross-linked alginate structure.

[0065]

[19] A medical device containing a cross-linked alginate structure as described in any one of the above items

[11] to

[14] . Medical materials.

[0066]

[20] The medical material described in

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

[0067]

[21] A crosslinked alginic acid as described in [1] or [8a] above, which is biocompatible, [2 ) or the alginic acid derivative described in [5] above, or any one of

[11] to

[14] above The cross-linked alginate structure described in the section.

[0068]

[22] The following formula (AM-1): [ka] [In formula (AM-1), -L 1 -, and the definition of Akn are the same as the definition in the 22nd aspect described later. Amino compounds represented as [the same], or pharmaceutically acceptable salts thereof, or those Solvate.

[0069]

[23] The following formula (AM-2): [ka] [In formula (II), -L 2 - is defined as -L in the 23rd aspect described later. 2 Select from the definition of - Amino compounds represented by [ ], or pharmaceutically acceptable salts thereof, or solvents thereof Japanese items. [Effects of the Invention]

[0070] The present invention relates to a novel alginic acid derivative that can be used, for example, for chemical crosslinking, The present invention provides cross-linked alginic acid, novel cross-linked alginic acid structures, and the like. Preferably, the alginate derivative is one in which a reactive group not present in the body is introduced, and the unreacted group For living organisms where remaining substances do not pose a risk of cross-linking reactions with biological components such as cells, Safety is expected. Furthermore, preferably, the crosslinking reaction uses a metal catalyst. Since the reaction is completed at room temperature, it is safe and easy to use. Cross-linked alginates in several embodiments undergo the Huisgen reaction (1,3-dipolar cycloaddition reaction). It is chemically crosslinked using (). The crosslinking is achieved by chemical crosslinking and divalent metal gold ions (for example, It can be used in combination with ion crosslinking using calcium ions, and the reaction conditions can be adjusted. By adjusting the stability, preferably the stability of non-crosslinked alginic acid or non-chemically crosslinked alginic acid is achieved. This is an improvement compared to acids (for example, cross-linked alginic acid with calcium ions). Furthermore, preferably, the gel properties of the crosslinked material can be adjusted, and the permeability of the material can be adjusted. It is also possible. The present invention has at least one of these effects. [Brief explanation of the drawing]

[0071] [Figure 1]This figure shows the evaluation of the gel stability of the cross-linked alginate structure. [Figure 2] This figure shows the evaluation of the gel stability of a cross-linked alginate structure under EDTA. [Figure 3] This figure shows the evaluation of the gel stability of the cross-linked alginate structure. [Figure 4] This figure shows the evaluation of the gel stability of a cross-linked alginate structure under EDTA. [Figure 5] This figure shows the evaluation of the gel stability of the cross-linked alginate structure. [Figure 6] This figure shows the evaluation of the gel stability of a cross-linked alginate structure under EDTA. [Figure 7] This figure shows the evaluation of the permeability of a gel with a cross-linked alginate structure. [Figure 8] This figure shows the evaluation of the permeability of a gel with a cross-linked alginate structure. [Figure 9] This figure shows the biocompatibility evaluation of cross-linked alginate derivative gels. [Modes for carrying out the invention]

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

[23] may be included. [1] The first embodiment is as follows: one or more carboxyl groups of alginic acid Amide bonds and divalent linkers (-L 1 A cyclic alkyne group (Akn) is introduced via -). Alginic acid derivatives represented by the following formula (I), and any one or more alginic acid derivatives. A carboxyl group with an amide bond and a divalent linker (-L 2 -) via which an azide group is introduced By performing a crosslinking reaction using the alginate derivative represented by the following formula (II), The resulting cross-linked alginic acid.

[0073] [Alginate derivatives represented by formula (I)] The following formula (I): [ka] [In formula (I), (ALG) represents alginic acid; -NHCO- represents any of alginic acid. Represents an amide bond via the carboxyl group; -L 1 - is shown in the table below: [Table 1-1] [Table 1-2] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It represents the linker; Akn is shown in the table below: [Table 2] A ring selected from the group consisting of the substructure formulas described [excluding the part to the right of the dashed line in each formula]. Alginic acid derivatives represented by [alkyne group].

[0074] [Alginate derivatives represented by formula (II)] Formula (II) below: [ka] (In formula (II), (ALG) represents alginic acid; -NHCO- represents any of alginic acid.) Represents an amide bond via the carboxyl group; -L 2 - is shown in the table below: [Table 3-1] [Table 3-2] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] Alginic acid derivatives represented by [a linker represented by formula (I)] (however, the alginic acid derivative represented by formula (I)) In acid derivatives, -L 1 - is (L1-1), (L1-2a), (L1-2b), (L A derivative which is any one linker selected from the group (1-11) or (L1-12). And, in the alginate derivative represented by formula (II), -L 2 - (L2-10) (Excluding crosslinked alginic acid obtained by crosslinking using a derivative that acts as a linker.) .

[0075] [1-1-1] In equation (I) of the above embodiment [1], -L 1 - Preferably, see the table below: [Table 4-1] [Table 4-2] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It is a linker (however, when the crosslinking reaction is carried out, -L 2 -ga (L2-10), (L2-1 Select one of the groups (0-p1), (L2-10-p2), or (L2-10-X). When using an alginate derivative represented by formula (II) having a single linker, -L in the table 1 -Of these, (L1-1), (L1-2a), (L1-2b), (L1-11 Linkers that are (L1-12-p1) and (L1-12-p1) are excluded from the preferred embodiment.

[0076] [1-1-2] In equation (I) of the above embodiment [1], -L 1 - is more preferably the following table: [Table 5-1] [Table 5-2] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It is a linker (however, when the crosslinking reaction is carried out, -L 2 -ga (L2-10), (L2-1 Select one of the groups (0-p1), (L2-10-p2), or (L2-10-X). When using an alginate derivative represented by formula (II) having a single linker, -L in the table 1 -Of these, (L1-1-1), (L1-2a-1), (L1-2b-1), Linkers (L1-11-1) and (L1-12-p2) are, in a more preferred embodiment, except).

[0077] [1-1-3] In equation (I) of the above embodiment [1], -L 1 - is more preferably the following table: [Table 6-1] [Table 6-2] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It is a linker (however, when the crosslinking reaction is carried out, -L 2 -ga (L2-10), (L2-1 Select one of the groups (0-p1), (L2-10-p2), or (L2-10-X). When using an alginate derivative represented by formula (II) having a single linker, -L in the table 1 -Of these, (L1-1-X), (L1-2-X), (L1-11-X) and Linkers that are (L1-12-X) are excluded from a more preferred embodiment.

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

Table 7

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

Table 8

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

Table 9

[0081] [1-3-1] In the formula (II) of the above aspect [1], -L 2 - is preferably one of the following tables :

Table 10

[0082] [1-3-2] In equation (II) of the above embodiment [1], -L 2 - is more, below Note: [Table 11] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It is a linker (however, when the crosslinking reaction is carried out, -L 1 -ga (L1-1), (L1-2a ), (L1-2b), (L1-11), (L1-12), (L1-1-1), (L1-2 a-1), (L1-2b-1), (L1-11-1), (L1-12-p1), (L1- Select from the groups (1-X), (L1-2-X), (L1-11-X), or (L1-12-X). An alginate derivative represented by formula (I) having one of the following linkers is used. In that case, -L in the table above. 2 -Of these, the linker (L2-10-p2) is more preferred. (Excluded from the description).

[0083] [1-3-3] In equation (II) of the above embodiment [1], -L 2 - is more preferably below Note: [Table 12] Selected from the group consisting of the partial structural formulas described [where the outside of the dashed lines at both ends is not included] is a linker (however, when performing the crosslinking reaction, -L 1 - is (L1-1), (L1-2a ), (L1-2b), (L1-11), (L1-12), (L1-1-1), (L1-2 a-1), (L1-2b-1), (L1-11-1), (L1-12-p1), (L1- 1-X), (L1-2-X), (L1-11-X) or (L1-12-X), and the alginate derivative represented by formula (I) having a linker selected from any one of the groups is used In this case, among the -L - in the table, the linker of (L2-10-X) is excluded from the more preferred form). 2 ).

[0084] [1-4-1] In formula (I) of the above aspect [1], the combination of Akn and -L 1 - is , preferably, the formula in the following table:

Table 13

[0085] ​​​[1-4-2] In equation (I) of the above embodiment [1], Akn and -L 1 - combinations More preferably, the formula in the table below: [Table 14] As shown by the substructure selected from the group (Akn, -L in the table) 1 Each of the expressions marked with - is before As described in [1-1] (however, when performing the crosslinking reaction, -L 2 -ga(L2 -10), (L2-10-p1), (L2-10-p2), or (L2-10-X) Alginic acid derivatives represented by formula (II) having a linker selected from any one of the above. When using -L in the table above, 1 -Of these, (L1-1-1), (L1-2a-1), Linkers (L1-2b-1), (L1-11-1), and (L1-12-p1) are more (Excluded from preferred embodiments).

[0086] [1-4-3] In the equation (I) of the above embodiment [1], Akn and -L 1 - combinations More preferably, the formula in the table below: [Table 15] As shown by the substructure selected from the group (Akn, -L in the table) 1 Each of the expressions marked with - is before As described in [1-1] (however, when performing the crosslinking reaction, -L 2 -ga(L2 -10), (L2-10-p1), (L2-10-p2), or (L2-10-X) Alginic acid derivatives represented by formula (II) having a linker selected from any one of the above. When using -L in the table above, 1-Of these, (L1-1-X), (L1-2-X), ( Linkers L1-11-X) and L1-12-X are excluded from a more preferred embodiment.

[0087] [1-4-4] In equation (I) of the above embodiment [1], Akn and -L 2 - combinations Particularly preferred is the following partial structural formula: [ka] As shown by the substructure selected from the group (however, when crosslinking, -L 2 -ga(L2-10),(L2-10-p1),(L2-10-p2) or(L2-10- The argy represented by formula (II) has a linker selected from any one of the group X) When using an acid derivative, the following partial structural formula is used: [ka] Substructures selected from this group are excluded from particularly preferred embodiments.

[0088] [1-Ia] The aspect of 1-Ia is as follows: Akn-L 1 -NH2 group (Akn, and -L 1 - is the same as the definition in the above embodiment [1]) The adoption rate is approximately 0.1% to approximately 3% An alginate derivative represented by formula (I) as described in the above embodiment [1], which is 0%.

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

[0090] [1-Ib] The embodiment of 1-Ib is as follows: Gel filtration chromatography of alginate derivatives The weight-average molecular weight measured by tography ranges from approximately 100,000 Da to approximately 3,000,000 Da. , an alginate derivative represented by formula (I) as described in the above embodiment [1].

[0091] [1-Ib-1] In the above embodiment [1-Ib], gel filtration chromatography of alginate derivatives The weight-average molecular weight measured by the Graph method is preferably about 300,000 Da to about 2,500,000 Da. It is a, and more preferably about 500,000 Da to about 1,000,000 Da.

[0092] [1-Ic] The aspect of 1-Ic is as follows: N3-L 2 -NH2 group(-L 2 -teeth The adoption rate (which is the same as the definition in the above embodiment [1]) is approximately 0.1% to approximately 30%, An alginate derivative represented by formula (II) as described in [1].

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

[0094] [1-Id] The embodiment of 1-Id is as follows: Gel filtration chromatography of alginate derivatives The weight-average molecular weight measured by tography ranges from approximately 100,000 Da to approximately 3,000,000 Da. , an alginate derivative represented by formula (II) as described in the above embodiment [1].

[0095] [1-Id-1] In the above embodiment [1-Id], a gel of the alginate derivative of formula (II) The weight-average molecular weight measured by filtration chromatography is preferably about 300,000 Da. It is approximately 2.5 million Da, and more preferably approximately 500,000 Da to 1 million Da.

[0096] A preferred embodiment of the above embodiment [1], and furthermore, Akn, -L 1 - and -L 2 - Combine the definitions as appropriate By combining them, a preferred form of the crosslinked alginic acid of the above embodiment [1] can be arbitrarily formed. obtain.

[0097] [2] The second embodiment is as follows: any one or more carboxyl groups of alginic acid Amide bonds and divalent linkers (-L 1 A cyclic alkyne group (Akn) is introduced via -). The following equation (I): [ka] [In formula (I), (ALG) represents alginic acid; -NHCO- represents any of alginic acid. Represents an amide bond via the carboxyl group; -L 1 - is shown in the table below: [Table 16-1] [Table 16-2] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It represents the linker; Akn is shown in the table below: [Table 17] A ring selected from the group consisting of the substructure formulas described [excluding the part to the right of the dashed line in each formula]. Alginic acid derivatives represented by [alkyne group].

[0098] [2-1] In equation (I) of the above embodiment [2], -L 1 - Preferably, see the table below: [Table 18-1] [Table 18-2] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It is a linker; For more details, see the table below: [Table 19-1] [Table 19-2] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It is a linker; More preferably, see the table below: [Table 20-1] [Table 20-2] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It is a linker.

[0099] [2-2] In the formula (I) of the above embodiment [2], Akn is preferably as shown in the table below: [Table 21] A ring selected from the group consisting of the substructure formulas described [excluding the part to the right of the dashed line in each formula]. It is an alkyne group; More precisely, see the table below. [Table 22] A ring selected from the group consisting of the substructure formulas described [excluding the part to the right of the dashed line in each formula]. It is an alkyne group; More preferably, see the table below: [Table 23] A ring selected from the group consisting of the substructure formulas described [excluding the part to the right of the dashed line in each formula]. It is an alkyne group.

[0100] [2-3] In equation (I) of the above embodiment [2], Akn and -L 1 - The combination is good For more information, see the table below: [Table 24] As shown by the substructure selected from the group (Akn, -L in the table) 1 Each of the expressions marked with - is before As described in the description [2]; More preferably, Akn-L 1 - The combinations are shown in the formula in the table below: [Table 25] As shown by the substructure selected from the group (Akn, -L in the table) 1 Each of the expressions marked with - is before As described in the notation methods [2-1] and [2-2]); More preferably, Akn-L 1 - The combinations are shown in the formula in the table below: [Table 26] As shown by the substructure selected from the group (Akn, -L in the table) 1 Each of the expressions marked with - is before As described in the notation methods [2-1] and [2-2]); Particularly preferred is Akn-L 1 - The combination is shown in the following substructure formula: [ka] This is as shown by the substructure selected from the group.

[0101] A preferred embodiment of the above embodiment [2], and further Akn, and -L 1Appropriately combine the definitions of "-" By doing so, the preferred forms of the alginic acid derivative represented by the formula (I) in the above aspect [2] Can be arbitrarily formed.

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

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

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

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

[0106] [5] The fifth aspect is as follows. To any one or more carboxyl groups of alginic acid An azide group is introduced via an amide bond and a divalent linker (-L 2 -), the following formula ( II): [Chemical formula] [In formula (II), (ALG) represents alginic acid; -NHCO- represents alginic acid. Represents an amide bond via a carboxyl group; -L 2 - is shown in the table below: [Table 27-1] [Table 27-2] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] Alginic acid derivatives represented by the linker.

[0107] [5-1] In the alginic acid derivative of formula (II) of the above embodiment [5], -L 2 -teeth, Preferably, see the table below: [Table 28] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It is a linker; For more details, see the table below: [Table 29] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It is a linker; More preferably, see the table below: [Table 30] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It is a linker.

[0108] [5a] The aspect of 5a is as follows: any one or more carboxyl groups of alginic acid The base has an amide bond and a divalent linker (-L 2-) via which an azide group is introduced, as follows Formula (II): [ka] [In formula (II), (ALG) represents alginic acid; -NHCO- represents alginic acid. Represents an amide bond via a carboxyl group; -L 2 - is shown in the table below: [Table 31-1] [Table 31-2] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] Alginic acid derivatives represented by the linker.

[0109] [5a-1] In the alginic acid derivative of formula (II) of the above embodiment [5a], -L 2 - Preferably, the following table: [Table 32] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It is a linker; For more details, see the table below: [Table 33] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It is a linker; More preferably, see the table below: [Table 34] Selected from the group consisting of the substructure formulas described [excluding the area outside the dashed lines at both ends of each formula] It is a linker.

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

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

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

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

[0114] [8] The eighth aspect is as follows. Any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (III-L):

Chemical formula

[0115] [8a] The aspect of 8a is as follows: an arbitrary carboxyl group of the first alginic acid and , any carboxyl group of the second alginic acid is given by the following formula (III-L): [ka] [In formula (III-L), the -CONH- and -NHCO- at both ends are any of the components of alginic acid. Represents an amide bond via a ruboxyl group; -L 1 - is the same as the definition in the above embodiment [1]; -L 2 - is the same as the definition in the above embodiment [1]; X is the same as the definition in the above embodiment [8]. Cross-linked alginic acid bonded via a mediated linkage. (However, in equation (III-L), -L 1 -ga (L1-1), (L1-2a), (L1 -2b), one linker selected from the group (L1-11) or (L1-12) - If so, the corresponding -L 2 - (excluding the linker at L2-10).

[0116] [8-1-1] In the formula (III-L) of the above embodiment [8] or embodiment [8a], preferred , -L 1 - is -L as described in the above embodiment [1-1-1]. 1 From the group consisting of expressions representing - It is the same linker that is selected.

[0117] [8-1-2] In the formula (III-L) of the above embodiment [8] or embodiment [8a], more preferably Shii, -L 1 - is -L as described in the above embodiment [1-1-2]. 1 A group consisting of expressions representing - It is the same as the more selectable linker.

[0118] [8-1-3] In the above embodiment [8] or [8a], formula (III-L) is more preferable. -L 1 - is -L as described in the above embodiment [1-1-3]. 1 Select from the group consisting of expressions representing - It is the same linker that will be selected.

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

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

[0121] [8-2-3] In the formula (III-L) of the above embodiment [8] or embodiment [8a], more preferably Shii-L 2 - is -L as described in the above embodiment [1-2-3]. 2 A group consisting of expressions representing - It is the same linker that is selected.

[0122] [8-3-1] In the formula (III-L) of the above embodiment [8] or embodiment [8a], X is favorable. The substructure formulas (TZ-1), (TZ-2), and ( TZ-3), formula (TZ-4), formula (TZ-5), formula (TZ-6), formula (TZ-10), formula (TZ-1-r), formula (TZ-2-r), formula (TZ-3-r), formula (TZ-4-r), formula Select from the group consisting of (TZ-5-r), formula (TZ-6-r), and formula (TZ-10-r). It is a cyclic group.

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

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

[0125] [8-4-1] In the formula (III-L) of the above embodiment [8] or embodiment [8a], preferably is, -L 2 -XL 1- The combinations are shown in the formula in the table below: [Table 36-1] [Table 36-2] As shown by the substructure selected from the group (in the table, -L 1 - and -L 2 - is the above As described in Embodiment [1]; -X- is as described in Embodiment [8]) (however, the above table Medium, -L 1 - (L1-1), (L1-2a), (L1-2b), (L1-11), or If it is one of the linkers selected from the group (L1-12), then the corresponding -L 2 - (excluding the linker L2-10-p1).

[0126] [8-4-2] In the formula (III-L) of the above embodiment [8] or embodiment [8a], more preferably For more information, see -L 2 -XL 1 - The combinations are shown in the formula in the table below: [Table 37-1] [Table 37-2] As shown by the substructure selected from the group (in the table, -L 1 - and -L 2 - is the above As described in Embodiment [1]; -X- is as described in Embodiment [8]) (however, the above table Medium, -L 1 - is (L1-1-1), (L1-2a-1), (L1-2b-1), (L1- One linker selected from the group (L1-1) or (L1-12-p1) In that case, the corresponding -L 2- (excluding the linker L2-10-p2).

[0127] [8-4-3] In the formula (III-L) of the above embodiment [8] or embodiment [8a], more preferably For more information, see -L 2 -XL 1 - The combinations are shown in the formula in the table below: [Table 38] As shown by the substructure selected from the group (in the table, -L 1 - and -L 2 - is the above As described in Embodiment [1]; -X- is as described in Embodiment [8]) (however, the above table Medium, -L 1 - is (L1-1-X), (L1-2-X), (L1-11-X) or (L1- If it is any one linker selected from the group 12-X), then the corresponding -L 2 - (Excluding the linker (L2-10-X)).

[0128] [8-4-4] In the formula (III-L) of the above embodiment [8] or embodiment [8a], particularly preferred For more information, see -L 2 -XL 1 - The combination is shown in the following substructure formula [In the formula, the area outside the dashed lines at both ends is [Does not include]: [ka] This is as shown by the substructure selected from the group.

[0129] [8-5-1] In the crosslinked alginic acid described in embodiment [1] or embodiment [8a], This involves chemical crosslinking by triazole rings formed by the Huisgen reaction and divalent metal i This is an ionic crosslink that is partially formed by ON.

[0130] [8-5-2] In the crosslinked alginic acid described in embodiment [1] or embodiment [8a], This is a chemical crosslink formed by a triazole ring through the Huisgen reaction.

[0131] [8-5-3] In the above embodiment [8-5-1], the divalent metal ion is preferably, Cium ions, magnesium ions, barium ions, strontium ions, or zinc ions A divalent metal ion selected from the group of ON; more preferably, a calcium ion or It is a barium ion; more preferably, a calcium ion.

[0132] [8-5-4] In the above embodiment [8-5-1], the divalent metal used for ion crosslinking The ions are preferably an aqueous solution of calcium chloride, an aqueous solution of calcium carbonate, or calcium gluconate. The source of the aqueous solution is selected from the group consisting of sodium aqueous solution and barium chloride aqueous solution. It can be; more preferably, an aqueous solution of calcium chloride or an aqueous solution of barium chloride; further Preferably, it is an aqueous solution of calcium chloride.

[0133] A preferred embodiment of the embodiment [8], and furthermore, -L 1 -, -L 2 - and the definition of X are combined as appropriate. By doing so, any preferred embodiment of the crosslinked alginic acid of the above embodiment [8] can be arbitrarily formed. A preferred embodiment of embodiment [8a], and furthermore, -L 1 -, -L 2 - and the definition of X are combined as appropriate. By combining these, preferred embodiments of the crosslinked alginic acid of the above embodiment [8a] can be arbitrarily formed. ru.

[0134] [9] The ninth aspect is as follows: Al represented by formula (I) described in the above aspect [1] A crosslinking reaction is carried out by mixing a ginate derivative and an alginate derivative represented by formula (II) (Hu By performing the isgen reaction, the crosslinked algin described in embodiment [1] or embodiment [8a] is produced. A method for producing crosslinked alginic acid, including obtaining an acid.

[0135] [9-1] The 9-1 aspect is as follows: Represented by formula (I) described in the above aspect [1] A solution of the alginic acid derivative is given by the alginic acid represented by formula (II) as described in embodiment [1] above. By adding a solution of ginate derivative and performing a crosslinking reaction (Huisgen reaction), the above embodiment [ The production of crosslinked alginic acid, which includes obtaining the crosslinked alginic acid described in [1] or embodiment [8a]. How to do it.

[0136] [9-2] The 9-2 aspect is as follows: The expression is given by formula (II) as described in the above aspect [1]. A solution of the alginic acid derivative is prepared using the formula (I) described in the above embodiment [1]. By adding a solution of ginate derivative and performing a crosslinking reaction (Huisgen reaction), the above embodiment [ The production of crosslinked alginic acid, which includes obtaining the crosslinked alginic acid described in [1] or embodiment [8a]. How to do it.

[0137]

[10] Alginate derivatives represented by formula (I) and formula (II) described in the above embodiment [1] By performing the Huisgen reaction (crosslinking reaction) using an alginic acid derivative represented by , The chemical crosslinks formed are of the following formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO-, X, and -L at both ends. 1 -,and- L 2 - is the same as the definition in the above embodiment [8]], which has the structure of the above embodiment [1] or state A method for producing the crosslinked alginic acid described in [8a].

[0138]

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

[0139] [11-1] The aspects of 11-1 are as follows: as described in any one of the items in [1] above A solution of the alginate derivative represented by formula (I) is added dropwise to a solution containing divalent metal ions. The resulting gel is then used as a solution of the alginic acid derivative represented by formula (II) described in [1] above. A cross-linked alginate structure obtained by applying a cross-linking reaction in addition to the above.

[0140] [11-2] The 11-2 aspect is as follows: Expressed in formula (II) described in [1] above A solution of the alginate derivative is dropped dropwise into a solution containing divalent metal ions to obtain a gel Add the solution of the alginic acid derivative represented by formula (I) described in [1] above to crosslink the reaction A cross-linked alginate structure obtained by applying a treatment.

[0141] [12-1] The aspect of 12-1 is as follows: Crosslinking by Huisgen reaction Chemical crosslinking by the triazole ring formed therein, and partially formed by divalent metal ions Crosslinking according to any one of the above embodiments

[11] to [11-2], including ion crosslinking Luginate structure.

[0142] [12-2] The aspect of 12-2 is as follows: Crosslinking by Huisgen reaction The embodiments

[11] to [11-2] include chemical crosslinking by a triazole ring formed therein A cross-linked alginate structure as described in any one of the items.

[0143] [12-3-1] In the above embodiment [12-1], the divalent metal ion is preferably a calc Cium ions, magnesium ions, barium ions, strontium ions, or zinc ions A divalent metal ion selected from the group of ON; more preferably, a calcium ion or It is a barium ion; more preferably, a calcium ion.

[0144] [12-3-2] In the above embodiment [12-1], the divalent metal used for ion crosslinking The ions are preferably an aqueous solution of calcium chloride, an aqueous solution of calcium carbonate, or calcium gluconate. The source of the aqueous solution is selected from the group consisting of sodium aqueous solution and barium chloride aqueous solution. It can be; more preferably, an aqueous solution of calcium chloride or an aqueous solution of barium chloride; further Preferably, it is an aqueous solution of calcium chloride.

[0145]

[13] The thirteenth aspect is as follows: Represented by formula (I) as described in the above aspect [1] Alginate derivatives and alginate derivatives represented by formula (II) are used with divalent metal ions. The contents are retained by ionic crosslinking and / or chemical crosslinking by the Huisgen reaction. A cross-linked alginate structure possessing properties.

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

[13] , the divalent metal ion is preferably calcium Mu ions, magnesium ions, barium ions, strontium ions, or zinc ions A divalent metal ion selected from the group; more preferably, a calcium ion or a variol. It is a um ion; more preferably, a calcium ion.

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

[13] , the divalent metal ions used for ion crosslinking The solution is preferably an aqueous solution of calcium chloride, an aqueous solution of calcium carbonate, or calcium gluconate. The source of the supply is an aqueous solution selected from the group consisting of aqueous solutions of humic acid and aqueous solutions of barium chloride. This can be done; more preferably, it is an aqueous solution of calcium chloride or an aqueous solution of barium chloride; even more preferably It is a calcium chloride solution.

[0148]

[14] The fourteenth aspect is as follows: Represented by formula (I) as described in the above aspect [1] Using alginic acid derivatives and alginic acid derivatives represented by formula (II), Huisg The chemical crosslink formed by the EN reaction is shown in formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO-, X, and -L at both ends. 1 -,and- L 2 - is the same as the definition in the above embodiment [8], a crosslinked alginate structure according to any one of the above embodiments

[11] to

[0013] .

[0149]

[15] The 15th aspect is as follows: Represented by formula (I) as described in aspect [1] above Alginic acid obtained by mixing alginic acid derivatives and alginic acid derivatives represented by formula (II) By adding a mixed solution of derivatives dropwise into a solution containing divalent metal ions and carrying out a crosslinking reaction, A method for producing a cross-linked alginate structure.

[0150] [15-1] The aspect of 15-1 is as follows: In the formula (I) described in the above aspect [1] The resulting alginate derivative is obtained by dropwise adding a solution of the alginate derivative to a solution containing divalent metal ions. Add the gel to the solution of the alginate derivative represented by formula (II) described in embodiment [1] above. A method for producing a crosslinked alginate structure obtained by subjecting it to a crosslinking reaction.

[0151] [15-2] The 15-2 aspect is as follows: Formula (II) as described in the above aspect [1] It is obtained by dropping a solution of an alginate derivative represented by into a solution containing divalent metal ions. Add the gel to the solution of the alginate derivative represented by formula (I) described in embodiment [1] above. A method for producing a crosslinked alginate structure obtained by subjecting it to a crosslinking reaction.

[0152] [16-1] The aspect of 16-1 is as follows: Crosslinking by Huisgen reaction Chemical crosslinking by the triazole ring formed therein, and partially formed by divalent metal ions Crosslinking according to any one of the above embodiments

[15] to [15-2], including ion crosslinking A method for producing a luginate structure.

[0153] [16-2] The aspect of 16-2 is as follows: Crosslinking by Huisgen reaction The embodiments

[15] to [15-2] include chemical crosslinking by a triazole ring formed therein A method for producing a cross-linked alginate structure as described in any one of the items.

[0154] [16-3-1] In the above embodiment [16-1], the divalent metal ion is preferably a cal Cium ions, magnesium ions, barium ions, strontium ions, or zinc ions A divalent metal ion selected from the group of ON; more preferably, a calcium ion or It is a barium ion; more preferably, a calcium ion.

[0155] [16-3-2] In the above embodiment [16-1], the divalent metal used for ion crosslinking The ions are preferably an aqueous solution of calcium chloride, an aqueous solution of calcium carbonate, or calcium gluconate. The source of the aqueous solution is selected from the group consisting of sodium aqueous solution and barium chloride aqueous solution. It can be; more preferably, an aqueous solution of calcium chloride or an aqueous solution of barium chloride; further Preferably, it is an aqueous solution of calcium chloride.

[0156]

[17] The seventeenth aspect is as follows: Represented by formula (I) as described in aspect [1] above Using alginic acid derivatives and alginic acid derivatives represented by formula (II), Huisg The chemical crosslink formed by the EN reaction is shown in formula (III-L): [ka] [In formula (III-L), -CONH- and -NHCO-, X, and -L at both ends. 1 -,and- L 2 - is the same as the definition in the above embodiment [8]], the above embodiment

[15] ~[ A method for producing a crosslinked alginate structure as described in any one of the items in [16-2].

[0157]

[18] The 18th aspect is as follows: a crosslinked alginate structure according to any one of the embodiments

[0011] to

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

[0158]

[19] The 19th aspect is as follows: any one of the above aspects

[11] to

[14] Medical materials containing the cross-linked alginate structure described in [reference].

[0159]

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

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

[0160]

[21] The 21st aspect is as follows: a biocompatible aspect [1] or aspect Crosslinked alginic acid as described in [8a], algin as described in embodiment [2] or embodiment [5] Acid derivatives, or the crosslinked alginate structure described in any one of the embodiments

[11] to

[14] above. body.

[0161]

[22] The 22nd aspect is as follows: Formula (AM-1): [ka] [In formula (AM-1), -L 1 -, and the definition of Akn are the same as the definitions described in the above embodiment [2]. Amino compounds represented as [the same], or pharmaceutically acceptable salts thereof, or those It is a solvate. However, see the table below: [Table 39] This excludes amino compounds of or pharmaceutically acceptable salts thereof, or solvates thereof.

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

[22] is preferably -L 1 -However, the preferred -L described in the description [2-1] 1 - is the same as the definition, and Akn is, An amino compound, or manufactured product, which is the same as the definition of preferred Akn described in [2-2]. A pharmaceutically acceptable salt thereof, or a solvate thereof. However, see the table below: [Table 40] This excludes amino compounds of or pharmaceutically acceptable salts thereof, or solvates thereof.

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

[22] is more preferably, -L 1 -However, the more preferable -L described in the description [2-1] 1 - is the same as the definition of A kn is the same as the more preferred definition of Akn described in [2-2], amination Compounds, pharmaceutically acceptable salts thereof, or solvates thereof. However, see the table below: [Table 41] This excludes amino compounds of or pharmaceutically acceptable salts thereof, or solvates thereof.

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

[22] is more preferably, -L 1 -However, a more preferable -L described in the description [2-1] 1 - is the same as the definition of A kn is the same as the more preferred definition of Akn described in [2-2], amination Compounds, pharmaceutically acceptable salts thereof, or solvates thereof. However, see the table below: [Table 42] This excludes amino compounds of or pharmaceutically acceptable salts thereof, or solvates thereof.

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

[22] is particularly preferred The formula is as follows: [ka] An amino compound selected from the group consisting of, or a pharmaceutically acceptable salt thereof, or the same It is the solvate of [the substance].

[0166]

[23] The 23rd aspect is as follows: Formula (AM-2): [ka] [In formula (AM-2), -L 2 - is defined as (L2-2a) in the above embodiment [5], ( L2-2b-A), (L2-3), (L2-4), (L2-5a), (L2-5b), ( L2-6a), (L2-6b), (L2-7a), (L2-7b), (L2-8a), ( The definition of (L2-8b), (L2-9a), and (L2-9b) is the same as the definition of (L2-9b). The compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof. However, the following table: [Table 43] This excludes amino compounds of or pharmaceutically acceptable salts thereof, or solvates thereof.

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

[23] is preferably -L 2 -but, (L2-2a), (L2-2b-A), (L2-3), (L2 This is the same as the definitions of (L2-5a-p1), (L2-5a-p1), and (L2-5b-p1). Amino compounds, or pharmaceutically acceptable salts thereof, or solvates thereof. However, See table below: [Table 44] This excludes amino compounds of or pharmaceutically acceptable salts thereof, or solvates thereof.

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

[23] is more preferably, -L 2-However, (L2-2a-1), (L2-2b-B) described in the above embodiment [5-1], (L2-3-1), (L2-4-p2), (L2-5a-p2), and (L2-5b-p 2) Amino compounds that are the same as defined therein, or pharmaceutically acceptable salts thereof, or those It is a solvate. However, see the table below: [Table 45] This excludes amino compounds of or pharmaceutically acceptable salts thereof, or solvates thereof.

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

[23] is more preferably The formula is as follows: [ka] An amino compound selected from the group consisting of, or a pharmaceutically acceptable salt thereof, or the same It is the solvate of [the substance]. In this specification, unless otherwise specified, when a higher-level embodiment is cited, the lower-level embodiments of that embodiment are also included. This shall be the case. For example, if a description [1] is cited, then the subordinate descriptions of description [1] shall also be included. It shall be considered as such.

[0170] The following provides a more detailed explanation of each aspect.

[0171] 1. Alginic acid In this specification, when referring to alginic acid, it refers to alginic acid, alginic acid esters, and the same At least one of the salts (e.g., sodium alginate) selected from the group consisting of these This refers to alginic acid (sometimes called "alginic acid compounds"). The alginic acid used is: It may be of natural origin or synthetic origin, but it is preferable that it be of natural origin. Preferably used Alginic acid compounds include Lessonia, Macrocystis, Laminaria, Ascophyllum, and Dar. It is a bioabsorbable polysaccharide extracted from brown algae such as Bilia, Kajika, Arame, and Kombu. That is, two types of uronic acid, D-mannuronic acid (M) and L-guluronic acid (G), are in a linear chain. It is a polymer polymerized to [a specific form]. More specifically, the homopolymer fraction of D-mannuronic acid (M M fraction), L-glucuronic acid homopolymer fraction (GG fraction), and D-mannuronic acid Block copolymers formed by the arbitrary linkage of fractions (M / G fractions) in which L-glucuronic acid is randomly arranged. It's a fusion.

[0172] Alginic acid is a type of natural polysaccharide that is extracted and purified from brown algae. Algin is a polymer formed by the polymerization of D-mannuronic acid (M) and L-guluronic acid (G). The ratio of D-mannuronic acid to L-guluronic acid in the acid (M / G ratio), i.e., the gel strength, is primarily It varies depending on the type of organism from which the seaweed originates, as well as the habitat and season of that organism. Due to this influence, the M / G ratio ranges widely from high-G type with an M / G ratio of approximately 0.2 to high-M type with an M / G ratio of approximately 5. The physicochemical properties of alginate depend on the M / G ratio of alginate, the arrangement of M and G, etc. The properties may differ, and the preferred applications may also differ. The gelling ability and bioavailability of alginates. The properties of the resulting gel are influenced by the M / G ratio, and generally, when the G ratio is high, the gel It is known that the strength increases. The M / G ratio also affects the hardness and brittleness of the gel. It also affects water absorption, flexibility, etc. Therefore, the alginic acid used in this invention is Depending on the end-use, it is best to use a product with an appropriate M / G ratio and viscosity.

[0173] Industrial methods for producing alginic acid include the acid method and the calcium method, but in this invention... Products manufactured using a different method can also be used. Purification by HPLC method is also possible. Preferably, the quantity falls within the range of 80 to 120% by mass, and is in the range of 90 to 110% by mass. It is more preferable that it is contained in the range of 95-105% by mass, and even more preferable that it is contained in the range of 95-105% by mass. In this invention, high purity is defined as a substance whose quantitative value obtained by HPLC falls within the above range. This is referred to as alginic acid. The alginic acid or salt thereof used in this invention is high-purity alginic acid. It is preferable to have it. As a commercially available product, for example, the Kimika Algin series is available from (Co., Ltd.) Purchase products sold by Kimika, preferably high-purity food / pharmaceutical grade products. It can be used after being added to the water. It is also possible to use commercially available products after further purification as needed. For example, treatment with low endotoxins is preferable. Purification method or low endotoxin treatment method The law may adopt, for example, the method described in Japanese Patent Publication No. 2007-75425. Cut.

[0174] In the present invention, the alginic acid salt used in "alginic acid" is "1 alginic acid It is a "valence metal salt," and is a carboxylic acid of D-mannuronic acid or L-guluronic acid of alginic acid. Salts are formed by ion exchange of hydrogen ions with monovalent metal ions such as Na+ and K+. Yes, there are. Specifically, monovalent metal salts of alginate include sodium alginate and alginate. Potassium acid and other similar substances can be mentioned, but sodium alginate is particularly preferred.

[0175] In this specification, alginic acid is referred to as (ALG), and any calorific value of alginic acid. Sometimes, one of the boxyl groups is designated as -COOH, and the notation is written as (ALG)-COOH.

[0176] The alginic acid used in this invention has an appropriate weight-average molecular weight depending on its final intended use. It is best to use materials with a weight-average molecular weight of 10,000 to 10 million. Preferably, 100,000 to 5,000,000, and even more preferably 150,000 to 30 It is less than 0.

[0177] In some embodiments, alginic acid is sodium alginate. For the sodium, commercially available sodium alginate can be used. Here, in the examples described later... Sodium alginate is listed in the table below as A-1, A-2, A-3, B-1, B-2 , and B-3 sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.) are used. The viscosity, weight-average molecular weight, and M / G lamp of a 1 w / w% aqueous solution of sodium luginate are as follows: This is shown in the table.

[0178] [Table 46]

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

[0180] [Viscosity measurement of sodium alginate] According to the viscosity measurement method of the Japanese Pharmacopoeia (16th edition), rotational viscometer method (cone plate type rotational viscometer) The measurement was performed using a thermometer. The specific measurement conditions are as follows: The sample solution was prepared as follows: The test was performed using MilliQ water. The measuring instrument used was a cone-plate type rotational viscometer (viscoelasticity measuring device). A Rheostress RS600 (Thermo Haake GmbH) sensor (35 / 1) was used. The rotational speed was: When measuring a 1 w / w% sodium alginate solution, the speed was set to 1 rpm. The reading time was 2 minutes. Measurements were taken, and the average value from the first 1 to 2 minutes was used. The average of three measurements was used as the measured value. The constant temperature was set at 20°C.

[0181] [Weight-average molecular weight measurement of sodium alginate] (1) Gel permeation chromatography (GPC), and (2) GPC-MALS, two types of measurement. Measurements were taken using standard methods. The measurement conditions are as follows:

[0182] [Pre-treatment method] After dissolving the sample with the eluent, the sample was filtered through a 0.45 μm membrane filter and measured. It was made into a liquid.

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

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

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

[0186] In this specification, alginic acid, alginic acid derivatives, crosslinked alginic acid, and crosslinked alginic acid In molecular weight measurements, the unit Da (dalton) is sometimes included.

[0187] The composition ratio (M / G ratio) of D-mannuronic acid and L-guluronic acid in alginic acid is mainly found in seaweed. This varies depending on the type of organism from which the odor originates, and is also influenced by the organism's habitat and the season. As a result, the M / G ratio ranges widely from high-G type with an M / G ratio of approximately 0.2 to high-M type with an M / G ratio of approximately 5. The gelling ability of alginates and the properties of the resulting gels are influenced by the M / G ratio. Generally, it is known that a higher G ratio results in higher gel strength. The M / G ratio is, In addition, it also affects the hardness, brittleness, water absorption, and flexibility of the gel. The M / G ratio of nitrates and / or their salts is typically 0.1 to 4.0, and in some embodiments , 0.1~3.0, in some embodiments 0.1~2.0, and in some embodiments 0.5~ It is 1.8, and in one embodiment it is 0.8 to 1.2. In another embodiment it is 0.1 to 0.5. That is the case.

[0188] Furthermore, the alginic acid used in this invention has an appropriate viscosity and suitable properties depending on its final intended use. It is best to use one with a precise M / G ratio.

[0189] In this specification, numerical ranges indicated using "~" refer to the numerical values ​​written before and after "~". This indicates the range in which the minimum and maximum values ​​are included.

[0190] In this specification, the terms "alginate ester" and "alginate salt" are used without particular limitation. However, since it reacts with the crosslinking agent, it is essential that it does not have functional groups that inhibit the crosslinking reaction. This is essential. Preferably, the alginate ester is propylene glycol alginate. Examples include, etc.

[0191] In this specification, alginates include, for example, monovalent salts of alginic acid and divalent salts of alginic acid. Examples include monovalent salts. Preferably, a monovalent salt of alginate is sodium alginate. Examples include potassium alginate, ammonium alginate, etc., and more preferably, Sodium alginate or potassium alginate, and particularly preferably sodium alginate. It is lium. Preferably, the divalent salt of alginate is calcium alginate, a Examples include magnesium alginate, barium alginate, and strontium alginate. ru.

[0192] Alginic acid is a high molecular weight polysaccharide, and it is difficult to determine its molecular weight precisely, but generally The weight-average molecular weight is 10 million to 10 million, preferably 10,000 to 8 million, more preferably The range is 20,000 to 3,000,000. In measuring the molecular weight of polymeric substances derived from natural products, the measurement method is It is known that differences in values ​​can occur.

[0193] In this specification, the molecular weight of the alginic acid derivative or alginic acid or salt thereof of the present invention is specified. If determined, unless otherwise specified, size exclusion chromatography (SEC) will be used. This is the weight-average molecular weight calculated from the above. It also refers to alginic acid or its salts used in this invention. Therefore, it is desirable to use a material with an appropriate molecular weight distribution depending on its end use.

[0194] For example, gel permeation chromatography (GPC) or gel filtration as described in the examples below. Chromatography (these are collectively also called size exclusion chromatography (SEC)) Under the measurement conditions of ), the value is preferably 100,000 to 5,000,000, and more preferably 150,000 to 30 It is 0 million. In some embodiments, it is in the range of 500,000 to 3,000,000, and more preferably 1 The range is between 0 million and 2.5 million, and more preferably between 1 million and 2 million.

[0195] Furthermore, for example, according to the GPC-MALS (SEC-MALS) method, the absolute weight average is The amount of molecules can be measured. Weight-average molecular weight (absolute) measured by GPC-MALS method. The molecular weight is preferably 10,000 or more, more preferably 50,000 or more, and even more preferably 60,000 or more. Preferably, 1 million or less, more preferably 800,000 or less, and even more preferably It is 700,000 or less, and particularly preferably 500,000 or less. The preferred range is 10,000 to 1,000 It is 10,000, more preferably 50,000 to 800,000, and even more preferably 60,000 to 500,000.

[0196] Typically, the molecular weight of high-molecular-weight polysaccharides is determined using methods such as SEC and SEC-MALS as described above. When calculating, a measurement error of approximately 10% to 30% can occur. For example, if the value is 500,000, then 35 If the initial value is between 10,000 and 650,000, the value can fluctuate within a range of approximately 700,000 to 1,300,000. In the detailed specifications, when "approximately" is used in the description of molecular weight measurement, it means that the value may be within ±10% of the stated value. Depending on the configuration, values ​​up to ±20% of the given value may also be included.

[0197] Here, generally speaking, polymeric substances derived from natural products do not have a single molecular weight, but rather various molecular weights. Because it is an aggregate of molecules with molecular weight, it is measured as a molecular weight distribution with a certain width. A typical measurement method is gel filtration chromatography. Typical information on molecular weight distribution obtained by this method includes weight-average molecular weight (Mw) and number-average molecular weight. Examples include the amount of molecules (Mn) and the variance ratio (Mw / Mn).

[0198] Weight-average molecular weight emphasizes the contribution of large molecular weight polymers to the average molecular weight, and below It is expressed in notation.

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

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

[0201] In measuring the molecular weight of naturally derived polymeric substances, the values ​​may differ depending on the measurement method. Known example of hyaluronic acid: Chikako YOMOTA et.al. Bu ll.Natl.Health Sci., Vol.117, pp135-139( (1999), Chikako YOMOTA et.al. Bull.Natl.In st. Health Sci., Vol.121, pp30-33(2003)) Regarding the molecular weight measurement of alginate, intrinsic viscosity (Intrinsic viscosity) is used. Methods for calculating from ty, SEC-MALLS (Size Exclusion Chr omatography with Multiple Angle Laser Li The method for calculating it using ght Scattering Detection is described. There is literature available (ASTM F2064-00(2006), ASTM International). (Published by online). In this invention, the weight-average molecular weight is determined by the conventional method as shown in the above-mentioned literature. For example, the molecular weight is measured by size exclusion chromatography (SEC), and pullulan The value can be calculated using a calibration curve that uses the standard substance. Furthermore, in this invention, the weight-average molecular weight is determined by a conventional method as shown in the above-mentioned literature, for example The absolute molecular weight is determined by size exclusion chromatography (SEC)-MALS. It is possible.

[0202] The molecular weight of alginates can be measured according to conventional methods.

[0203] In this specification, when specifying the molecular weight of alginic acid or its salts, unless otherwise specified, Unless otherwise specified, the weight-average molecular weight is calculated by gel filtration chromatography. Typical conditions when using gel filtration chromatography for measurement include, for example, the following: The conditions of the example can be adopted. The column is, for example, Superose6 Inc. You can use the rease10 / 300 GL column (GE Healthcare Sciences). For example, as the developing solvent, a 10 mmol / L solution containing 0.15 mol / L NaCl is used. Acid buffer (pH 7.4) can be used, and blue dextran can be used as the molecular weight standard. thyroglobulin, ferritin, aldolase, conalbumin, obalbumin, ribo Nuclease A and aprotinin can be used.

[0204] The viscosity of alginic acid used herein is not particularly limited, but is 1 w / w% alginic acid. When the viscosity of an aqueous solution of ginic acid is measured, it is preferably 10 mPa·s to 1000 m The pressure range is Pa·s, more preferably 50 mPa·s to 800 mPa·s.

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

[0206] Alginic acid compounds, initially extracted from brown algae, have a large molecular weight and high viscosity, but when heated... During processes such as drying and purification, the molecular weight decreases, resulting in lower viscosity. This involves controlling conditions such as temperature, selecting brown algae as raw materials, and techniques such as molecular weight fractionation in the manufacturing process. Alginic acid derivatives with different molecular weights can be produced. Furthermore, different molecular weights or By mixing it with a different batch of alginic acid with viscosity, it can obtain the desired molecular weight. It is also possible to use alginic acid derivatives.

[0207] Alginic acid as used herein is, in some embodiments, low endotoxin Untreated alginate, or in some other embodiments, low endotoxin It is cin-treated alginate. Low endotoxin means virtually no inflammation or fever. This refers to an endotoxin level that is low enough not to cause an allergic reaction. More preferably, low endotoxin levels. Dotoxin-treated alginates are preferable.

[0208] Low-endotoxin treatment can be carried out by known methods or similar methods. For example, the method of purifying sodium hyaluronate, as described by Suga et al. (for example, Japanese Patent Publication No. 9-32) (See Publication No. 4001, etc.), the method of Yoshida et al. for purifying β1,3-glucan (for example, special (See Publication No. 8-269102, etc.), biopolymer salts such as alginates and gellan gum Purification by the method of William et al. (see, for example, Japanese Patent Publication No. 2002-530440). , the method of purifying polysaccharides, James et al. (e.g., International Publication No. 93 / 131) (See Pamphlet No. 36, etc.), Lewis et al.'s method (for example, U.S. Patent No. 5589591). (See specifications, etc.), purifying alginates, Herman Frank et al.'s method (e.g., Ap pl Microbiol Biotechnol(1994)40:638-643 This can be carried out by methods such as (see reference) or similar methods. Low endotoxin The process is not limited to those, but also includes washing, filtering (endotoxin removal filters and static charge removal filters) Filtration by filters, ultrafiltration, column (endotoxin adsorption affinity filter) Purification using rams, gel filtration columns, ion exchange resin columns, etc., hydrophobic substances, Adsorption onto resin or activated carbon, or treatment with organic solvents (extraction with organic solvents, addition of organic solvents) (Precipitation, sedimentation, etc.), surfactant treatment (for example, Japanese Patent Publication No. 2005-036036, etc.) This can be done by known methods (see reference, etc.) or by combining them as appropriate. These processing steps may be combined with known methods such as centrifugation as appropriate. (Alginic acid) It is best to select the appropriate option based on the type of product.

[0209] Endotoxin levels can be confirmed by known methods, for example, Limulus reagent ( LAL) method, Endospecy (registered trademark) ES-24S set (Biochemical Industries Ltd.) It can be measured by methods such as those using a company.

[0210] The method of treating the endotoxin used is not particularly limited, but as a result, The endotoxin content of nic acid compounds is measured using Limulus reagent (LAL). When this is done, it is preferable that the endotoxin levels are 500 EU units / g or less, and further Preferably 100 EU / g or less, particularly preferably 50 EU / g or less, especially preferred Or, it is 30 EU / g or less. In this invention, "substantially contains no endotoxins" "i" means that the endotoxin value measured by the Japanese Pharmacopoeia endotoxin test falls within the aforementioned numerical range. It means something. Low-endotoxin treated sodium alginate is, for example, S ea Matrix® (registered trademark) (Mochida Pharmaceutical Co., Ltd.), PRONOVA TM UP L It is available commercially as VG (FMCBioPolymer), etc.

[0211] 2. Alginic acid derivatives Novel alginate derivatives are provided herein. For example, an amide bond and a divalent linker are attached to any one or more carboxyl groups of alginic acid. Through this, the reactive group in the Huisgen reaction or a reactive group complementary to said reactive group It was introduced. More specifically, see formula (I): [ka] [In formula (I), (ALG), -L 1 -, Akn and -NH-CO- are defined as in the first above. Alginic acid derivatives represented by the same definition as in the embodiment, and the following formula (II): [ka] [In formula (II), (ALG), -L 2 - and -NH-CO- are defined in the fourth aspect described above. It is an alginic acid derivative that is defined as having the same definition as the one inside.

[0212] The aforementioned divalent linker (-L 1 - or -L 2 -) indicates a reactive group and a reactive group that is complementary to that reactive group. Any linear group can be used as long as it does not inhibit the reaction with reactive groups. Specifically, For example, a linear alkylene group (-(CH2) n -, n=1~30) (-CH in the group) 2- represents -C(=O)-, -CONH-, -O-, -NH-, -S-, benzene ring, complex Rings (such as pyridine rings, piperidine rings, piperazine rings, etc., 5-6 member aromatic heterocycles or 5-6 member rings) Multiple groups (e.g., 1 to 10 or 1 to 5) are replaced by non-aromatic heterocycle groups. It is also possible that the hydrogen atom of the -CH2- is an oxo group (=O), C 1-6 Alkyl(for example) (For example, groups such as methyl group, ethyl group, n-propyl group, iso-propyl group, etc.), halogenated groups Atom (for example, fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), hydroxyl group (-OH), Multiple substitutions (for example, 1 to 10 or 1 to 5) are made by groups selected from such groups. Other examples include (which are also good), but are not limited to these.

[0213] In the -NH-CO- group of the alginate derivative represented by formula (I) or formula (II) above By substituting the hydrogen atom of the imino group (-NH-) with a methyl group, a -N(Me)-CO- group is formed. This is possible. In the alginate derivative represented by formula (I) or formula (II) above, the linker (-L 1 -, -L 2 -) The bonding mode between and alginate is a -NH-CO- bond, or -N(Me)- It has a CO- bond; preferably an -NH-CO- bond.

[0214] The novel alginate derivatives described herein are represented by formulas (I) and (II). Alginate derivatives can be produced, for example, by the method shown in the following formula (see the general manufacturing method described later for details). It is possible to manufacture more.

[0215] [ka]

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

[0217] In this specification, Akn-L of formula (I) 1 -NH- groups are all the structural units of alginate. It does not need to be bonded to a boxyl group, and also the N3-L of formula (II) 2 -NH- group is a It is not necessary for the carboxyl groups of the ginic acid constituent units to be bonded to all of them.

[0218] In this specification, Akn-L of formula (I) 1 When the -NH- group is called a reactive group, formula (II) N3-L 2 The -NH- group acts as a complementary reactive group. Conversely, the N3-L group in formula (II) 2 -N When the H- group is called a reactive group, the Akn-L in formula (I) 1 -NH- group is a complementary reactive group Yes.

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

[0220] The rate of introduction of the aforementioned reactive group or complementary reactive group is determined by the repeating unit of alginic acid. This represents the percentage of uronic acid monosaccharide units into which each reactive group has been introduced. This is the value. In this specification, unless otherwise specified, alginate derivatives (formula (I) or formula (II) In the formula, the percentage used for the introduction rate of a reactive group or complementary reactive group means mol%. The introduction rate of each reactive group or complementary reactive group is determined by the method described in the examples below. It is possible to do so.

[0221] In this 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 in turn creates a crosslink.

[0222] 3. Huisgen reaction The Huisgen reaction (1,3-dipolar cycloaddition reaction) is as shown in the following equation, with terminal a This is a condensation reaction between compounds having a zide group and a terminal alkyne group. As a result of the reaction, disubstituted 1, It has the advantage of yielding the 2,3-triazole ring in good yield and not producing any unwanted byproducts. It is thought that this reaction can produce a 1,4- or 1,5-disubstituted triazole ring. However, by using a copper catalyst, it is possible to obtain a triazole ring regioselectively.

[0223] [ka]

[0224] Furthermore, a Huisgen reaction without a copper catalyst was reported by Wittig and Krebs. It is done. That is, a cycloadduct is obtained simply by mixing cyclooctin and phenylazide. This is a reaction (in the following formula, R 3 =phenyl). This reaction involves the triple cyclooctin. Because the bond is greatly distorted, the reaction with phenyl azide to relieve the distortion becomes the driving force. The reaction proceeded spontaneously, eliminating the need for a catalyst.

[0225] [ka]

[0226] As described above, the Huisgen reaction involves the substituted primary, secondary, and tertiary azides. Azide compounds having aromatic azides, etc., and terminal reactive groups that are complementary to the azide group. Alternatively, compounds having a cyclic alkyne group can be used. In addition, in the Huisgen reaction... Since almost only azide groups and alkyne groups react, various functional groups (e.g.) are present in the reaction substrate. For example, by substituting ester groups, carboxyl groups, alkenyl groups, hydroxyl groups, amino groups, etc. It is possible to do so.

[0227] In some embodiments, the cytotoxicity induced by the copper catalyst is reduced without producing undesirable byproducts. To avoid this, a copper catalyst is not used, and 1,2,3-triazole can be produced quickly, easily, and efficiently. To form a crosslink between alginic acid molecules by the ring, the alkyne group of the Huisgen reaction For example, using the cyclic alkyne group (cyclooctyl group) described in the above embodiment [1]. Yes, they are.

[0228] In a preferred embodiment of the method for crosslinking alginic acid derivatives, the reaction (Huisgen reaction) In this case, alginate is used. In the fabrication of novel forms of biocompatible materials and the formation of alginate hydrogels, various The incorporation of bioactive molecules, and the use of alginate hydrogels for reconstructive surgery or gene therapy. This makes it possible to take in cellular substances.

[0229] 4. Cross-linked alginic acid Cross-linked alginic acid has (i) divalent metal ion bonds and (ii) chemical bonds. (iii) via both divalent metal ion bonds and chemical bonds Yes. Cross-linked alginates can take on a gel-like, semi-solid, or even sponge-like form. It possesses the characteristic of forming.

[0230] Cross-linked alginate via divalent metal ion bonding proceeds at an ultrafast rate and is reversible. In contrast, cross-linked alginic acid reacts slowly under relatively mild conditions. The process is irreversible. The physical properties of cross-linked alginate are, for example, the divalent metal ions used. The concentration of an aqueous solution containing (for example, an aqueous solution of calcium chloride), or the amount of alginate. The reaction can be adjusted by methods such as changing the rate at which the reactive groups are introduced.

[0231] By utilizing the aforementioned crosslinking reaction, it becomes possible to create various alginate structures. For example, by ionic crosslinking, a specific structure can be instantly created from an alginic acid solution. This can be achieved, and in order to strengthen the structure of the structure (for example, to achieve long-term stability, etc.), chemical bonding is used. It is possible to utilize crosslinking reactions. Also, for example, divalent metal ion bonds and chemical bonds In a crosslinked alginate structure via both of the above, the divalent metal incorporated by ionic crosslinking is The ions can be reversibly released, creating a structure where only the cross-linking due to chemical bonding remains. That is the case.

[0232] In one embodiment, crosslinked alginic acid is a derivative of alginic acid of formula (I) and formula (II). It can be obtained by mixing and carrying out the Huisgen reaction.

[0233] In one embodiment, crosslinked alginic acid is a chemical crosslink (formed from alkyne groups and azide groups). It forms a three-dimensional network structure via crosslinking by an azole ring. Preferred alginate derivative The body shows improved stability of cross-linked alginate after cross-linking.

[0234] Crosslinked alginic acid in several embodiments involves any carboxyl group of a first alginic acid and a second The following formula (III-L) applies to any carboxyl groups of alginic acid: [ka] [In formula (III-L), the -CONH- and -NHCO- at both ends are any of the components of alginic acid. Represents an amide bond via a ruboxyl group; -L 1 -, -L 2 -, and X are the 8th It is a crosslinked alginic acid bonded via an amide linkage [which is the same as the definition in the embodiment].

[0235] In some embodiments, when preparing crosslinked alginic acid, the alginic acid derivative of formula (I) and The mixing ratio of the alginate derivative of formula (II) is the weight of the derivative of formula (I) and the derivative of formula (II). In terms of quantity ratio, for example, 1 to 1.5:1, preferably 1.2 to 1.5:1, or 1 to 1. The ratio is 2:1, more preferably 1:1.

[0236] Alginic acid derivative of formula (II) when preparing crosslinked alginic acid in several embodiments The mixing ratio of the alginate derivative of formula (I) is the weight of the derivative of formula (II) and the derivative of formula (I). In terms of quantity ratio, for example, 1 to 4.0:1, preferably 1.5 to 4.0:1, or 1.2 to 1 The ratio is 0.5:1, or 1 to 1.2:1, more preferably 1:1.

[0237] In some embodiments, when preparing crosslinked alginic acid, the alginic acid derivative of formula (I) and The mixing ratio of the alginate derivative of formula (II) is more preferably the alginate derivative of formula (I). The ratio of the rate of introduction of reactive groups (mol%) between the isomer and the alginic acid derivative of formula (II) is, for example, 1 ~1.5:1, preferably 1.2~1.5:1, or 1~1.2:1, more preferably The ratio is 1:1.

[0238] Alginic acid derivative of formula (II) when preparing crosslinked alginic acid in several embodiments The mixing ratio of the alginate derivative of formula (I) is, more preferably, the alginate derivative of formula (II). The ratio of the rate of introduction of reactive groups (mol%) between the conductor and the alginate 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~ The ratio is 1.2:1, more preferably 1:1.

[0239] Furthermore, in the above mixing ratio, the alginate derivative of formula (I) is used as the alginate derivative of formula (II). In the body, the alginate derivative of formula (II) can be replaced with the derivative of formula (I). It is possible.

[0240] Crosslinked alginic acid is formed when all carboxyl groups of the constituent units of alginic acid are of the above formula (III- It is not necessary to have crosslinking of L). In crosslinked alginic acid, the above formula (III-L) The cross-linking rate (also called the cross-linking ratio) expressed is, for example, approximately 0.1 to approximately 80%, or approximately 0.3 The range is approximately 60%, 0.5-30%, or 1.0-10%.

[0241] In the Huisgen reaction to obtain cross-linked alginate, the a of formula (I) or formula (II) The concentration of the luginate derivative is usually about 1 to about 500 mg / mL, preferably about 5 to about 1 The range is 00 mg / mL.

[0242] The reaction temperature for the Huisgen reaction is typically around 4 to 60°C ambient temperature, preferably ambient temperature The temperature range is approximately 15 to 40 degrees Celsius.

[0243] The stirring time required to form cross-linked alginate (hydrogel) ranges from a few seconds to approximately 24 hours. The interval can range from a few seconds to approximately 12 hours, a few seconds to approximately 30 minutes, or a few seconds to approximately 10 minutes.

[0244] The reaction solvent or reaction solution used in the Huisgen reaction is not particularly limited, but for example, Tap water, purified water (e.g., distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure Examples include water, cell culture medium, phosphate-buffered saline (PBS), and physiological saline. Preferably, it is ultrapure water.

[0245] In some embodiments, crosslinked alginic acid is formed as a crosslink by the Huisgen reaction. Chemical crosslinking by triazole rings and partial formation by divalent metal ions This is a cross-linked alginic acid containing on-crosslinking.

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

[0247] In this specification, "to perform a crosslinking reaction" or "to carry out a crosslinking reaction" refers to the argy of formula (I) above. The Huisgen reaction is carried out using an alginic acid derivative and the alginic acid derivative of formula (II) described above. As a result, chemical reactions occur between the alginate derivative of formula (I) and the alginate derivative of formula (II). The formation of a chemical crosslink (chemical bond), or the alginic acid derivative of formula (I) and the By coexisting a divalent metal ion with the alginate derivative of formula (II), the same formula (I) is obtained. Ion crosslinking between each derivative of the alginate derivative of formula (II) and / or the alginate derivative of formula (II). (Ionic bond) formation, or chemical crosslinking and divalent ionization by the Huisgen reaction. This means that both ionic crosslinks by metal ions are formed.

[0248] Cross-linked alginate structures can be prepared, for example, by the following methods, but these It is not limited to that.

[0249] [Mixing method] The alginate derivative of formula (I) and the alginate derivative of formula (II) are mixed to obtain By dropping a mixed solution of alginic acid derivatives into a solution containing divalent metal ions, Chemical crosslinking (triazole formed from alkyne and azide groups by Huisgen reaction) Crosslinking by rings and ionic crosslinking (crosslinking partially formed by divalent metal ions) A specific structure, a cross-linked alginate structure, can be obtained.

[0250] [Coating Method] A solution containing the alginate derivative of formula (I) is added dropwise to a solution containing divalent metal ions. A specific structure that is partially crosslinked is obtained by doing so, for example, a gel. By adding the structure to a solution containing the alginic acid derivative of formula (II), the structure By applying a further cross-linking reaction (Huisgen reaction) to the surface of the structure, cross-linked algebra An alginate structure can be obtained. This method involves using an alginate derivative of formula (I) I) Alginate derivative of formula (II) Alginate derivative of formula (I) It is also possible to substitute different parts of the body and perform the exercise accordingly.

[0251] The divalent metal ions used in the above method are not particularly limited, but for example, calcium Mu ions, magnesium ions, barium ions, strontium ions, zinc ions, Examples include divalent metal ions selected from the group such as; preferably, calcium ions or It is a barium ion; more preferably, a calcium ion.

[0252] The solution containing divalent metal ions used in the above method is not particularly limited, but for example, , calcium chloride aqueous solution, calcium carbonate aqueous solution, calcium gluconate aqueous solution, or salt Examples include aqueous solutions selected from the group such as barium chloride aqueous solution, and preferably calcium chloride aqueous solution. It is an aqueous solution of citric acid or an aqueous solution of barium chloride; more preferably, an aqueous solution of calcium chloride. .

[0253] The concentration of divalent metal ions in the solution containing divalent metal ions used in the above method is not particularly limited. However, for example, a range of approximately 1 mM to 1 M is possible, preferably approximately 5 mM to 500 mM. Yes, and more preferably, about 10 mM to about 300 mM.

[0254] The solvent or solution used in the above method is not particularly limited, but for example, tap water, pure water (e.g.) Examples include distilled water, ion-exchanged water, RO water, RO-EDI water, etc., ultrapure water, and cell culture media. Examples include phosphate-buffered saline (PBS) and physiological saline, preferably with ultrapure water. be.

[0255] Examples of specific cross-linked alginate structures include fibrous structures, fibers, beads, Examples include gels, approximately spherical gels, etc. Preferred crosslinked alginate structures have improved stability. Furthermore, the cross-linked alginate structure has the ability to hold contents inside (contents It may have (object-holding properties).

[0256] The physical properties of alginate gel are determined by physical properties such as hardness, elasticity, resilience, fracture force, and stress at fracture. It can be adjusted.

[0257] 6. Biocompatibility of alginate derivatives and cross-linked alginate structures In this specification, alginic acid derivatives, crosslinked alginic acid, or crosslinked alginic acid structures are defined as follows: It is biocompatible. In this specification, biocompatibility means a biomaterial (here, formula ( I) Alginic acid derivatives represented by formula (II), and using both alginic acid derivatives Interactions between manufactured cross-linked alginate or cross-linked alginate structures and living organisms , without causing local or systemic reactions in tissues adjacent to the biomaterial. The property of having biocompatibility is called biocompatibility.

[0258] In this specification, alginic acid derivatives, crosslinked alginic acid, or crosslinked alginic acid structures are used. Biocompatibility will be confirmed in the biocompatible examples described later.

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

[0260] [Method for measuring gel stability] Add phosphate-buffered saline (PBS) to the cross-linked alginate structure gel in the container. The concentration (μg / mL) of alginate eluted into PBS is measured. The amount of eluted alginate was calculated from the degree, and the total amount obtained by decomposing the cross-linked alginate structure gel was obtained. The disintegration rate is defined as the value obtained by dividing the alginate concentration by the total amount of alginate, expressed as a percentage. The gel stability can be determined specifically by the method described in the examples below.

[0261] In this specification, the gel decay rate of the crosslinked alginate structure is preferably 0% to about 90%. More preferably 0% to about 70%, and even more preferably 0% to about 50%. The stability of the alginate structure is such that the lower the concentration of alginate leaking into the aqueous solution, the better. A lower gel decay rate indicates higher stability.

[0262] [Method for measuring gel permeability] Cross-linked alginate structure containing fluorescein isothiocyanate-dextran Prepare a gel, add physiological saline to the gel placed in a container, and remove the condensate that leaked into the physiological saline. Measure the dextran concentration. Calculate the amount of dextran from the measured dextran concentration. Degradation of fluorescein isothiocyanate-dextran-encapsulated cross-linked alginate structure gel. The value obtained by dividing the total dextran concentration by the total amount of dextran is expressed as a percentage. The value shown is the gel permeability. Specifically, the gel permeability is determined by the method described in the examples below. It can be obtained more.

[0263] The gel permeability of cross-linked alginic acid after 24 hours of saline addition is, for example, that of a molecular weight of approximately 200. When 10,000 dextran is included, the concentration is preferably 0% to about 90%, and more preferably 0% The molecular weight is approximately 70%, and more preferably 0% to approximately 50%. If strands are included, for example, if the purpose of use of the cross-linked alginate structure gel is protein If we are talking about quality or antibody release / production, it is preferably about 1% to about 100%, and more preferably The percentage is approximately 10% to 100%, and more preferably approximately 30% to 100%. If the intended use is for immune septamentation, then preferably 0% to about 90%, and more preferably 0%. It is approximately 70%, and more preferably 0% to approximately 50%.

[0264] The permeability of a cross-linked alginate structure is such that the lower the permeability, the more easily the contents and substances outside the gel can be permeated. A low transmittance indicates that the contents or substances outside the gel are more permeable. ru.

[0265] The permeability of the gel depends on the molecular weight and concentration of the alginic acid used, and the reactive groups introduced into the alginic acid. The type and introduction rate of divalent metal ions used for gelation, the type and concentration of these, or combinations thereof. It can be adjusted by [this method].

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

[0267] (1) Solution of alginate derivative represented by formula (I) and fluorescein isothiocyanate Mix the todextran solution. (2) Add the solution of the alginic acid derivative represented by formula (II) to the mixed solution obtained in (1) Mix them together. (If equation (I) in (1) is changed to equation (II), then equation (II) in (2) is changed to equation (I). (This will happen) (3) The mixed solution obtained in (2) was added dropwise to a solution containing calcium ions to obtain the gel In solution, fluorescein isothiocyanate forms chemical and ionic crosslinks. A cross-linked alginate structure gel containing ocyanate-dextran is obtained.

[0268] Where the term "approximately" is used in this specification, unless otherwise specified, the numerical value refers to the given figure. This may include values ​​up to ±20% of the given value, preferably up to ±10% of the given value.

[0269] 8. Method for synthesizing alginic acid derivatives In this specification, alginate derivatives represented by formula (I) or formula (II) are, respectively, H2N-L 1 -Akn(in the formula, L 1 And Akn are the same as defined in the above embodiment [1]. Amine derivatives represented by (AM-1), or H2N-L 2 -N3 (in the formula, L 2 teeth, An amine derivative (AM-2) represented by the same definition as in the above embodiment [4] is given by A It can be produced by a condensation reaction using a condensing agent with any carboxyl group of ginic acid compounds. Cut.

[0270] [ka]

[0271] [Method for producing alginate derivatives of formula (I)] A 0.5% to 1% by weight aqueous solution of alginic acid and an amine represented by formula (AM-1) Using methods known from 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, pp. 258-283, 2007, Maruzen, etc. According to the established method, 1,3-dicyclohexylcarbodiimide (DCC), 1-ethyl -3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC·HCl), Ben Zotriazole-1-yloxytris(dimethylamino)phosphonium hexafluoro Phosphate (BOP reagent), bis(2-oxo-3-oxazolidinyl) phosphate Cuchloride (BOP-Cl), 2-chloro-1,3-dimethylimidazolinium hexaf Luolophosphate (CIP), or 4-(4,6-dimethoxy-1,3,5-triazi Selected from (2-yl)-4-methylmorpholinium chloride (DMT-MM), etc. In the presence of a condensing agent, tetrahydrofuran, 1,4-dioxide, to an extent that does not precipitate alginic acid. Ether-based solvents such as xanes, alcohols such as methanol, ethanol, and 2-propanol. Mixing a solvent selected from polar solvents such as sulfur-based solvents and N,N-dimethylformamide with water. In the solvent, an inorganic base such as sodium bicarbonate or sodium carbonate, or triethylamine, Condensation reactions are carried out at temperatures between 0°C and 50°C, with or without the presence of organic bases such as pyridine. By doing so, an alginate derivative of formula (I) can be produced.

[0272] [Method for producing alginate derivatives of formula (II)] A 0.5% to 1% by weight aqueous solution of alginic acid and an amine represented by formula (AM-2) Using this, the reaction is carried out in accordance with the aforementioned [Method for producing alginate derivative of formula (I)]. This allows for the production of alginate derivatives of formula (II).

[0273] In the method for producing the alginate derivative of formula (I) or the alginate derivative of formula (II) described above, The introduction rate of the amine of formula (AM-1) or formula (AM-2) shall be determined considering the properties of the amine, etc. By doing so, the reaction conditions such as (i) to (v) below can be appropriately selected and combined to adjust the reaction. This becomes possible: (i) increasing or decreasing the amount of condensing agent by the same amount, (ii) increasing or decreasing the reaction temperature, (iii) (iv) Adjustment of the concentration of the reaction substrate alginate, (v) Formula (AM- 1) Add an organic solvent that is miscible with water to increase the solubility of the amine of formula (AM-2). , etc.

[0274] Below are some more specific examples of amines represented by formula (AM-1) or formula (AM-2). The method for producing amines is shown.

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

[0276] Furthermore, in each of the following manufacturing methods, protective group P 1 The protection and deprotection of are methods known from the literature, for example, Protective Groups in Organic Synthesis Groups in Organic Synthesis 4th Edition) 4th edition, 2007, John Wiley & Sons Following the method of deprotection described in the book by Sons, Greene, et al. It can perform protection and deprotection.

[0277] [Manufacturing Method AM-A] Method for producing the amine represented by formula (AM-1-B1): [ka] Compounds of formula (SM-B1) and compounds of formula (RG-B1) [Compounds of formula (SM-B1) And the compound of formula (RG-B1) is a commercially available compound or can be produced from a commercially available compound by a method known from the literature. Using a compound that can be manufactured in the same manner as described above [method for producing alginic acid derivative of formula (I)], A condensation reaction is carried out, followed by the addition of a protecting group P. 1 By deprotecting, equation (AM-1-B1) The amine compounds represented, or salts thereof, can be produced.

[0278] [Manufacturing method AM-B] Represented by formulas (AM-1-B2a) and (AM-1-B2b) Min manufacturing method: [ka]

[0279] Compounds of formula (SM-B2a) and compounds of formula (RG-B2a) [of formula (SM-B2a) The compound and the compound of formula (RG-B2b) are commercially available compounds or manufactured from commercially available compounds in a manner known from the literature. This compound can be produced by the method, and using it, in the same manner as in [production method AM-A], By reacting with the product, an amine compound represented by formula (AM-1-B2a) or a salt thereof can be produced. This can be done. Similarly, the compounds of formula (SM-B2b) and the compounds of formula (RG-B2b) Compounds of formula (SM-B2b) and (RG-B2b) are commercially available compounds or commercially available compounds. This compound can be produced from the compound by a manufacturing method known in the literature, and the reaction is carried out similarly using this compound. This involves producing an amine compound represented by formula (AM-1-B2b), or a salt thereof. It is possible.

[0280] [Manufacturing Method AM-C] Method for producing amines represented by formula (AM-1-B3): [ka]

[0281] Compound of formula (SM-B3) [Compound of formula (SM-B3) is a commercially available compound or is a commercially available compound? Using a compound that can be produced by a manufacturing method known from the literature, according to the above synthesis scheme, By carrying out the reaction (the reaction in each step is in accordance with the reaction described in [Manufacturing Method AM-A]), the formula An amine compound represented by (AM-1-B3) or a salt thereof can be produced. In the scheme, equations (RG-B3), (RG-B3-1), and (RG-B3-2) The compound is a commercially available compound or a compound that can be produced from a commercially available compound by a manufacturing method known in the literature. .

[0282] [Manufacturing method AM-D] Represented by formulas (AM-1-B5a) and (AM-1-B5b) Min manufacturing method: [ka]

[0283] The reaction is carried out according to the above synthesis scheme (the reaction for each step is described in [Manufacturing Method AM-A]). (According to the reaction described above), represented by formulas (AM-1-B5a) and (AM-1-B5b) A mine compound or a salt thereof can be produced. In the above scheme, formula (SM-B5a) , formula (RG-B5a), formula (RG-B5a-1), formula (RG-B5a-2), formula (SM- B5b), formula (RG-B5b), formula (RG-B5b-1), and formula (RG-B5b-2) The compound is a commercially available compound or a compound that can be produced from a commercially available compound by a manufacturing method known in the literature. be.

[0284] [Manufacturing method AM-E] Represented by formulas (AM-1-B6a) and (AM-1-B6b) Min manufacturing method: [ka]

[0285] The reaction is carried out according to the above synthesis scheme (the reaction for each step is described in [Manufacturing Method AM-A]). (According to the reaction described above), represented by formulas (AM-1-B6a) and (AM-1-B6b) A mine compound or a salt thereof can be produced. In the above scheme, formula (SM-B6a) Compounds of formula (RG-B6a), formula (SM-B6b), and formula (RG-B6b) are commercially available. This compound can be produced from a compound or a commercially available compound by a manufacturing method known from the literature.

[0286] [Manufacturing Method AM-F] Method for producing the amine represented by formula (AM-1-B10): [ka]

[0287] The reaction is carried out according to the above synthesis scheme (the reaction for each step is described in [Manufacturing Method AM-A]). (According to the reaction described above), an amine compound represented by formula (AM-1-B10), or a salt thereof, is prepared. It can be manufactured. In the above scheme, formula (SM-B10), formula (RG-B10), formula ( Compounds of formula (RG-B10-1) and (RG-B10-2) are commercially available compounds or commercially available compounds This compound can be produced by manufacturing methods known from the literature.

[0288] [Manufacturing method AM-G] Represented by formulas (AM-1-B4a) and (AM-1-B4b) Min manufacturing method: [ka]

[0289] Compound of formula (SM-B4) and compound of formula (RG-B4a) [compound of formula (SM-B4) The substance and compound of formula (RG-B4a) are commercially available compounds or manufactured by methods known from the literature using commercially available compounds. This compound can be produced by using methods known in the literature, for example, in the Journal of the American Chemical Society, 126(46 ), pp. 15046-15047, 2004, etc., in accordance with the method described, <Step 1a >In the presence of reagents such as silver trifluoromethanesulfonate and AgClO4, toluene and dichloro By reacting in a solvent that does not participate in the reaction, such as methane, the compound of formula (IM-B4a-1) can be obtained. <Step 2a> Subsequently, debromination is performed using a base such as sodium hydride or NaOMe. By carrying out the reaction, a compound of formula (IM-B4a-2) is obtained, and <Step 3a> further protection base P 1 By deprotecting the compound, an amine compound represented by formula (AM-1-B4a) is obtained, or It is possible to produce salt. Similarly, using equation (RG-B4b) instead of equation (RG-B4a), the above ski By carrying out the reaction according to the formula, an amine compound represented by formula (AM-1-B4b) or It is possible to manufacture that salt.

[0290] [Manufacturing method AM-H] Represented by formulas (AM-1-B8a) and (AM-1-B8b) Min manufacturing method: [ka]

[0291] The reaction is carried out according to the above synthesis scheme (the reaction for each step is described in [Manufacturing Method AM-A]). (According to the reaction described above), represented by formulas (AM-1-B8a) and (AM-1-B8b) A mine compound or a salt thereof can be produced. In the above scheme, formula (SM-B8a) Alternatively, formula (SM-B8b) may be obtained using a commercially available compound or by following the reaction described in [Manufacturing Method AM-G]. The compound can be manufactured, and the compound of formula (RG-B8a) or formula (RG-B8b) is commercially available. This is a compound that can be produced from a compound or a commercially available compound by a manufacturing method known from the literature.

[0292] [Manufacturing Method AM-J] Method for producing amines represented by formula (AM-1-B7): [ka]

[0293] Compounds of formula (SM-B7) and compounds of formula (RG-B7) [Compounds of formula (SM-B7) And the compound of formula (RG-B7) is a commercially available compound or can be produced from a commercially available compound by a method known from the literature. Using a compound that can be manufactured in the same manner as described above [method for producing alginic acid derivative of formula (I)], A condensation reaction is carried out, followed by the addition of a protecting group P. 1 By deprotecting, equation (AM-1-B7) It can be produced as the amine compound shown, or as a salt thereof.

[0294] [Manufacturing Method AM-J-2] Method for producing the amine represented by formula (AM-1-B7): [ka]

[0295] Compound of formula (SM-B7) and compound of formula (RG-B7-2) [Chemical formula of (SM-B7) The compound and the compound of formula (RG-B7-2) are commercially available compounds or manufactured from commercially available compounds in a manner known from the literature. The compound can be produced by the method, and the above-mentioned [production of the alginic acid derivative of formula (I)] A condensation reaction similar to the method is carried out (<Step 1> and <Step 2>), followed by the protection group P 1 Deprotect Next, the compound of formula (RG-B7-3) (a commercially available compound or a commercially available compound known from literature) Using a compound that can be produced by the manufacturing method, a condensation reaction similar to that in <Step 1> is carried out. This is followed by the protection base P 1 Deprotecting the amine results in the amine represented by formula (AM-1-B7). It can be produced as a compound or a salt thereof.

[0296] [Manufacturing method AM-K] represented by formulas (AM-1-B9a) and (AM-1-B9b) Min manufacturing method: [ka]

[0297] The reaction is carried out according to the above synthesis scheme (the reaction for each step is described in [Manufacturing Method AM-J]). (According to the reaction described above), represented by formulas (AM-1-B9a) and (AM-1-B9b) A mine compound or a salt thereof can be produced. In the above scheme, formula (SM-B7), Compounds of formula (RG-B9a) or (RG-B9b) are commercially available compounds or compounds derived from commercially available compounds. This compound can be produced by a manufacturing method that is publicly known.

[0298] [Manufacturing Method AM-L] Method for producing the amine represented by formula (AM-2-Z1): [ka]

[0299] Compound of formula (SM-Z1) [Compound of formula (SM-Z1) is a commercially available compound or is a commercially available compound? [These compounds can be produced by methods known in the literature, for example] , Organometallics, 29(23), p6619-6622;201 In accordance with the method described in "0 Years," etc., in a solvent that does not participate in the reaction, such as dimethyl sulfoxide. After reacting with NaN3 to introduce an azide group, a protecting group P 1 By deprotecting equation (A An amine compound represented by M-2-Z1) or a salt thereof can be produced. Furthermore, the amine compound represented by formula (AM-2-Z1), or its salt, is a commercially available compound. Some are available.

[0300] [Manufacturing method AM-M] Represented by formulas (AM-2-Z2a) and (AM-2-Z2b) Min manufacturing method: [ka]

[0301] Compound of formula (SM-Z2a) or compound of formula (SM-Z2b) [Chemical formula (SM-Z2a) The compound and the compound of formula (SM-Z2b) are commercially available compounds or manufactured by commercially available compounds according to known methods in the literature. Using a compound that can be manufactured by the method, Na is produced in the same manner as in [Manufacturing Method AM-L]. After reacting with N3 to introduce an azide group, a protecting group P 1 By removing protection, equation (AM-2 The production of an amine compound represented by formula (AM-2-Z2b) or formula (AM-2-Z2b), or a salt thereof. This can be done. Furthermore, the amined form represented by formula (AM-2-Z2a) or formula (AM-2-Z2b) Compounds, or salts thereof, are sometimes available as commercially produced compounds.

[0302] [Manufacturing Method AM-N] Method for producing the amine represented by formula (AM-2-Z3): [ka]

[0303] Compounds of formula (SM-Z3) and compounds of formula (RG-Z3) [Compounds of formula (SM-Z3) And the compound of formula (RG-Z3) is a commercially available compound or can be produced from a commercially available compound by a method known from the literature. Using the compound that can be manufactured, the above-mentioned [production of the alginic acid derivative of formula (I)] A condensation reaction similar to the [method] is carried out, followed by the protection group P 1 By deprotecting the equation (AM-2- An amine compound represented by Z3) or a salt thereof can be produced.

[0304] [Manufacturing Method AM-O] Method for producing the amine represented by formula (AM-2-Z4): [ka]

[0305] Compounds of formula (SM-Z4) and compounds of formula (RG-Z4) [Compounds of formula (SM-Z4) And the compound of formula (RG-Z4) is a commercially available compound or can be produced from a commercially available compound by a method known from the literature. Using a compound that can be manufactured in the same manner as described above [method for producing alginic acid derivative of formula (I)], A condensation reaction is carried out, followed by the addition of a protecting group P. 1 By removing protection, the formula (AM-2-Z4) The amine compounds represented, or salts thereof, can be produced.

[0306] [Manufacturing method AM-P] The mesh represented by formulas (AM-2-Z5a) and (AM-2-Z5b) Manufacturing method: [ka]

[0307] Compound of formula (SM-Z5a) and compound of formula (RG-Z5a) [Chemical formula (SM-Z5a) The compound and the compound of formula (RG-Z5a) are commercially available compounds or manufactured by commercially available compounds according to known methods in the literature. [A compound that can be manufactured by law] is used as a base such as sodium hydride or potassium carbonate. In the presence of tetrahydrofuran, N,N-dimethylformamide, N-methylpyrrolidone, By carrying out the reaction in a solvent that does not participate in the reaction, such as dimethyl sulfoxide, the side chain is introduced. A compound is obtained. Next, the protecting group P 1 By removing the protection, the formula (AM-2-Z5 an amine compound represented by a), or a salt thereof, can be produced. Similarly, compounds of formula (SM-Z5b) and compounds of formula (RG-Z5b) [formula (SM Compounds of formula (RG-Z5b) and compounds of formula (RG-Z5b) are commercially available compounds or compounds derived from commercially available compounds. This compound can be produced by a known manufacturing method, and by carrying out a similar reaction using the formula, An amine compound represented by (AM-2-Z5b) or a salt thereof can be produced.

[0308] Formula (AM-2-Z6a), Formula (AM-2-Z6b), Formula (AM-2-Z7a), Formula (AM-2-Z7b), Formula (AM-2-Z8a), Formula (AM-2-Z8b), Formula (AM amino compounds represented by formula (AM-2-Z9a) and formula (AM-2-Z9b), or the same The salt is prepared according to the above-mentioned [Manufacturing Method AM-A] to [Manufacturing Method AM-P] as shown in the scheme below. It can be manufactured by the manufacturing method.

[0309] [ka]

[0310] [ka]

[0311] [ka]

[0312] [ka]

[0313] [Manufacturing Method AM-Q] Method for producing the amine represented by formula (AM-2-Z10): [ka]

[0314] <Step 1> Compound of formula (SM-Q) [The compound of formula (SM-Q) is a commercially available compound or commercially available compound. [A compound that can be produced from the compound by a manufacturing method known in the literature] and the chemical formula (RG-Q1) Compound [Formula (RG-Q1) is a commercially available compound or manufactured from a commercially available compound using a method known from the literature] Using compounds that can be produced more easily, by methods known in the literature, for example, 'European Journal of Organic Chemistry, 2014(6), In accordance with the method described on pages 1280-1286; 2014, etc., (i) PPh3, And in the presence of the reagent N2(CO2CHMe2)2, it is not involved in the reaction of tetrahydrofuran, etc. The Mitsunobu reaction is carried out in a solvent, followed by methanol and ethanol in the presence of a base such as sodium hydroxide. In solvents that do not participate in the reaction, such as ol, tetrahydrofuran, and water, or mixtures thereof. By hydrolyzing the ester group, a compound represented by formula (IM-Q1) is produced. It is possible.

[0315] <Step 2> [Manufacturing Method AM-Q] Compound and formula (IM-Q1) obtained in <Step 1> Compound (RG-Q2) [The compound of formula (RG-Q2) is a commercially available compound or derived from a commercially available compound. Using the compound [a compound that can be produced by a known manufacturing method], the above [formula (I) A condensate is obtained by performing a condensation reaction similar to the method for producing alginic acid derivatives, followed by storage. Mamoru P 1 By deprotecting the compound, an amine compound represented by formula (AM-2-Z10) is obtained, or It is possible to manufacture that salt.

[0316] [Manufacturing Method AM-R] Method for producing the amine represented by formula (AM-1-B10): [ka]

[0317] <Step 1> Compound of formula (SM-R) [The compound of formula (SM-R) is a commercially available compound or commercialized compound. [A compound that can be produced from the compound by a manufacturing method known in the literature] , for example, “Faming Zhuanli Shenqing, 104529898 In accordance with the method described in ", 22 Apr 2015", etc., (i) a base such as pyridine In the presence of H2NOH-HCl in a solvent that does not participate in the reaction, such as ethanol, the oxidizer is reacted. (ii) A mixture is formed, and then phosphorus pentoxide is reacted in P2O5, methanesulfonic acid. Then, by performing a Beckmann transition, an 8-membered ring lactam is formed, followed by (iii) Using a solvent that does not participate in the reaction, such as ethyl ether, and a reducing agent such as BH3 or LiAlH4, By reducing the amide group, the compound represented by formula (IM-R1) can be produced. can.

[0318] <Step 2> [Manufacturing Method AM-R] Compound of formula (IM-R1) obtained by <Step 1> and compounds of formula (RG-R1) [commercial compounds or commercial compounds Using a compound that can be produced by a manufacturing method known from the literature, the above [Formula (I) A A condensation reaction similar to the method for producing luginic acid derivatives is carried out to obtain a condensate, followed by the addition of bromine. Afterward, an alkyne group is formed by carrying out a debromination reaction using tert-BuOK. Next, protective group P 1 Deprotection results in the amineralization represented by formula (AM-1-B10). A mixture or a salt thereof can be produced.

[0319] [Manufacturing Method AM-S] Method for producing the amine represented by formula (AM-2-Z11): [ka]

[0320] Compound of formula (SM-S) and compound of formula (RG-S1) [Compound of formula (SM-S) and formula The compound (RG-S1) is a commercially available compound or can be produced from a commercially available compound by a manufacturing method known in the literature. Using the compound that can be produced, the same method as the above-mentioned method for producing the alginic acid derivative of formula (I) is used. The reaction is carried out, followed by the protecting group P. 1 By removing the protection, the expression (AM-2-Z11) is expressed The compound or its salt can be produced.

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

[0322] <Step 1> Compound of formula (SM-T) and compound of formula (RG-T-1) [Formula (SM-T The compounds of formula (RG-T-1) and the compound of formula (RG-T-1) are commercially available compounds or are known from literature derived from commercially available compounds. This compound can be produced by the manufacturing method, using a method known in the literature, for example, WO2 The Grignard reaction is carried out according to the method described in 004 / 035017, etc., followed by the addition of acid. By carrying out a chemical reaction, the compound of formula (IM-T-1) is obtained.

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

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

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

[0326] In the above [Manufacturing Method AM-T], the compound of formula (SM-T) is replaced with the compound of formula (SM-U). [The compound of formula (SM-U) is a commercially available compound or can be produced from a commercially available compound by a manufacturing method known in the literature.] Replace with "[a compound that can be produced]" and react according to the method described in [Manufacturing Method AM-T]. By performing this procedure, amines represented by formulas (AM-1-U1) and (AM-1-U2) are obtained. Or manufacture the salt thereof.

[0327] The aldehyde used in <Step 1> of the above-mentioned [Manufacturing Method AM-T] and [Manufacturing Method AM-U] is , an aldehyde of the following formula (RG-T-3) or formula (RG-T-4) [Formula (RG-T-3) The compounds of formula (RG-T-4) are commercially available compounds or compounds known from literature derived from commercially available compounds. By replacing it with "[a compound that can be manufactured by the manufacturing method]", the corresponding linker To produce an amine having or a salt thereof. [ka] [Manufacturing method AM-V] Method for producing amines represented by formulas (AM-1-V1) and (AM-1-V2): [ka]

[0328] <Step 1> Compound of formula (SM-V) [The compound of formula (SM-V) is a commercially available compound or a publicly available compound in the literature. Methods of knowledge, for example, Bioorganic & Medicinal Chemistry Manufactured by the method described in y, 23(22), pp. 7150-7157, 2015, etc. Using [kiru], after converting to an acid chloride according to the conventional method, the compound of formula (RG-V-1) [ Compound (RG-V-1) is a commercially available compound or can be produced from a commercially available compound by a method known from the literature. A Grignard reaction is carried out using a compound that can be manufactured, followed by the protecting group P 1 Deprotect This process produces an amine represented by formula (AM-1-V1) or a salt thereof.

[0329] <Step 2> The amine of formula (AM-1-V1) or its salt and the compound of formula (RG-T-1) Using this, a condensation reaction is carried out, and a protecting group P 1 By removing the protection, the equation (AM-1-V2) To produce the amine or salt thereof shown.

[0330] The compound used in <Step 1> of the above [Manufacturing Method AM-V] is the compound of the following formula [Each compound is Replace with "[Can be produced from a commercially available compound or by a manufacturing method known from a commercially available compound]" and react. By performing this procedure, an amine having a corresponding linker, or a salt thereof, is produced. [ka]

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

[0332] In the above [Manufacturing Method AM-V], the compound of formula (SM-V) is replaced with the compound of formula (SM-W). [The compound of formula (SM-W) is a commercially available compound or can be produced from a commercially available compound by a manufacturing method known in the literature.] Replace with "[a compound that can be produced]" and react according to the method described in [Production Method AM-V]. By performing this procedure, amines represented by formulas (AM-1-W1) and (AM-1-W2) are obtained. Or manufacture the salt thereof.

[0333] The compound used in <Step 1> of the above [Manufacturing Method AM-W] is the compound of the following formula [Each compound is Replace with "[Can be produced from a commercially available compound or by a manufacturing method known from a commercially available compound]" and react. By performing this procedure, an amine having a corresponding linker, or a salt thereof, is produced. [ka]

[0334] A is used to produce alginate derivatives represented by formula (I) or formula (II). Amine with a lukine group introduced (Akn-L 1 Amines with an introduced azide group (-NH2) or amide group (N3-L 2 -NH2) refers to the above [Manufacturing Method AM-A] to [Manufacturing Method AM-P] Each reaction described in, for example, the methods known from the literature, such as in "Experimental Chemistry Course, 5th Edition," each volume, 20 In 2007, Maruzen, "Comprehensive Organic Transformations, A Guide to Functional G roup Preparations, 3rd Edition (Edited by Richard C. Larock), 2018”, “Strate gic Applications of Named Reactions in Organic Synthesis, (Edited by Laszlo Kurt Combine the methods described in "i, Barbara Czako), Academic Press, 2005" as appropriate. This allows for the production of the desired amine.

[0335] In this specification, amine compounds represented by formula (AM-1) or formula (AM-2) (each of the formulas) (Including the lower-order formulas) may form pharmaceutically acceptable salts (e.g., acid addition salts). Yes, such salts are not particularly limited as long as they are pharmaceutically acceptable, but for example, Examples include salts with inorganic acids, salts with organic acids, and salts with acidic amino acids. Suitable examples include, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Examples of salts with organic acids include, for example, formic acid, acetic acid, and trifluor. L-acetic acid, propionic acid, butyric acid, valeric acid, enanthic acid, capric acid, myristic acid, palmitic acid With aliphatic monocarboxylic acids such as tinic acid, stearic acid, lactic acid, sorbic acid, and mandelic acid. Salt, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, tartaric acid, and other fats. Salts with aliphatic dicarboxylic acids, salts with aliphatic tricarboxylic acids such as citric acid, benzoic acid, salicylic acid Salts of aromatic monocarboxylic acids such as acids, salts of aromatic dicarboxylic acids such as phthalic acid, cinnamic acid, Organic compounds such as licolic acid, pyruvic acid, oxylic acid, salicylic acid, and N-acetylcysteine. Salts with rubonic acid, 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 Examples include salts with acidic amino acids, such as aspartic acid and gluten. Examples include salts with tamic acid, etc. Of these, pharmaceutically acceptable salts are preferred.

[0336] The salt is prepared according to a conventional method, for example, by a solution containing the compound of the present invention and an appropriate amount of acid or base. After forming the desired salt by mixing, the mixture is separated by filtration, or the mixed solvent is left to stand. It can be obtained by removing it. As a general overview of salt, see Handbook of Pharmaceutical Salts:Properties, Selecti on, and Use, Stahl & Wermuth (Wiley-VCH, 2002 A book on this topic has been published, and it contains detailed information.

[0337] In this specification, amine compounds represented by formula (AM-1) or formula (AM-2) (each of the formulas) (including the lower-order formulas) or its salts form solvates with solvents such as water, ethanol, and glycerol. It can be formed.

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

[0339] In this specification, phosphorus in chemically modified alginate derivatives represented by formula (I) or formula (II) Car (-L 1 - or -L 2 -) If an asymmetric carbon is present, each optical isomer This means that it is also included.

[0340] For example, -L in equation (I) 1- is equation (L1-8a), where m8a=2 and n8a=1 , R 1 The following formula for the case of =Me (L1-8a-M) (do not include the parts outside the dashed lines in the formula): [ka] If R 1 The following formula (L1-8a-MS) is an example where the stereochemistry of the carbon atom substituted by the group is S-isomer. ) and the following formula (L1-8a-MR) is an R-type carbon atom substituted by the benzyl group. (The areas outside the dashed lines in any of the equations are not included): [ka] This means that a linker represented by [this symbol] is included.

[0341] Linker (-L) in chemically modified alginate derivatives represented by formula (I) or formula (II) 1 - or -L 2 -) If an asymmetric carbon is present (i.e., if it is an optically active compound), then formula ( In the step of synthesizing an amine derivative (AM-1) corresponding to formula (I) or formula (II), The racemic mixture can be separated into its individual optically active compounds using conventional optical resolution methods (separation techniques). It is capable of being an amine derivative corresponding to formula (I), such as formula (AM-1) or formula (AM-2) In the process of synthesizing ), asymmetric synthesis can be used to selectively synthesize one of the optical isomers. It is possible to synthesize each optically active compound.

[0342] 9. Applications of alginic acid derivatives and cross-linked alginic acid structures Alginic acid derivatives are used in a wide range of fields, including food, medicine, cosmetics, textiles, and papermaking, compared to conventional alginic acid derivatives. It can be used as a substitute for alginic acid. Alginic acid derivative or photocrosslinked alginic acid structure. Preferred applications for this material include, specifically, wound dressings, postoperative adhesion prevention materials, and drug release substrates. Examples include medical materials such as cell culture substrates and cell transplantation substrates.

[0343] When used as a medical material, cross-linked alginate structures can take the following forms: tubular, fibrous. Examples include shapes, fibers, beads, gels, and roughly spherical gels, and beads, gels, or roughly spherical It is preferable to form a gel, and more preferably a gel that is approximately spherical in shape.

[0344] Furthermore, all documents and publications mentioned herein, regardless of their purpose, are by reference. The entirety of this is incorporated herein.

[0345] Furthermore, the object, features, advantages, and idea of ​​the present invention are described herein in accordance with the terms of the Industrial Convention. This will be obvious to those skilled in the art, and from the description herein, those skilled in the art will be able to easily implement the present invention. The best mode and specific examples for carrying out the invention are described in the Preferred Embodiments section. These are illustrative examples or explanatory materials, and do not represent the present invention in any way. This is not limited to the intent and scope of the present invention as disclosed herein. It will be obvious to those skilled in the art that various modifications can be made based on the details provided. [Examples]

[0346] Next, examples and test examples will be given to further explain the present invention, but these examples These are merely examples and test cases, and do not limit the present invention or exceed its scope. You may make changes as long as they do not deviate from the original concept.

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

[0348] 1 In the H-NMR data, s represents a singlet and d represents a double 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 the H-NMR data, the hydroxyl group (OH) , amino groups (NH2), carboxyl groups (COOH) protons, etc., in broadband Signals that could not be confirmed are not included in the data.

[0349] In LC-Mass data, M is molecular weight, RT is retention time, and [M+H] + [M+Na] + This indicates a molecular ion peak.

[0350] In the examples, "room temperature" typically refers to a temperature between approximately 0°C and approximately 35°C. The rate of introduction of reactive substituents (mol%) in the examples is: 1 Calculated from H-NMR (D2O) Introduction to the number of moles of monosaccharide units (guluronic acid and mannuronic acid) that make up alginic acid This should indicate the percentage of moles of the reactive substituents.

[0351] In the examples, sodium alginate before the introduction of a reactive group or a complementary reactive group For the substance used, sodium alginate exhibiting the physical properties listed in Table 46 was employed.

[0352] Table 48 shows the argyrates with reactive groups introduced, obtained in (Example 1) to (Example 20). Physical properties of nic acid derivatives (specifically, the rate of reactive group introduction (mol%), molecular weight, and weight average) This shows the molecular weight (in tens of thousands of Da). Tables 49-1 to 49-5 show the 1H-NM intermediates in (Example 1) to (Example 20). Table 50 shows the LCM-Mass of the intermediates in (Example 1) to (Example 20) where R is the value. .

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

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

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

[0356] A 1% by weight aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (1 0.9 mL) is mixed with 4-(4,6-dimethoxy-1,3,5-triazine- under ice-cold stirring. 2-Iyl)-4-methylmorpholinium chloride (DMT-MM) (55.83 mg) Then, 1 molar concentration sodium bicarbonate solution (252.17 μL) was added. Subsequently, commercially available 2-(2-( 2-Azidoethoxy)ethoxy)ethane-1-amine [CAS REGISTRY NO Ethanol (1 mL) of [166388-57-4](2-1, 26.36 mg) Add water (1 mL) solution and stir at room temperature for 15 hours, then add sodium chloride (100 The mixture was then mixed at room temperature for 30 minutes, with ethanol (21.8 mL) added sequentially. The precipitate is filtered, washed with ethanol, and dried under reduced pressure to obtain the labeled compound EX2-A2 (99 mg). ) was obtained as a white solid.

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

[0358] <Process 1> tert-butyl(2-(3-azidopropyl)amino)-2-oxoethyl)carb Synthesis of Mate (3-2): [ka] Commercially available 3-azidopropylamine [CAS REGISTRY NO.:88192- 19-2] (1-1, 41 μL), N-(tert-butoxycarbonyl)glycine [ CAS REGISTRY NO.:4530-20-5](3-1, 100 mg) 4-(4,6-dimethoxy-1,3,5-triazine-) in ethanol (2 mL) solution Add 2-yl)-4-methylmorpholinium chloride (DMT-MM) (197 mg). The mixture was stirred at room temperature for 18 hours. Water was added to the reaction mixture, and after extraction with ethyl acetate, the organic layer was diluted with water. The mixture was sequentially washed with saline solution. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. Dissolve the oily substance in methyl tert-butyl ether (10 mL), then add saturated sodium bicarbonate solution and water. The mixture was then washed sequentially with saturated saline solution. The organic layer was dried with anhydrous sodium sulfate, then concentrated under reduced pressure, and the standard solution was applied. Compound 3-2 (95 mg) was obtained as a colorless oily substance.

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

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

[0361] <Process 3> Alginic acid with 2-amino-N-(3-azidopropyl)acetamide group (EX3-A2 ) Synthesis: [ka]

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

[0363] (Example 4) Alginic acid (EX4-A) with 3-amino-N-(3-azidopropyl)propanamide group introduced 2) Synthesis: [ka]

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

[0365] Commercially available 3-azidopropylamine [CAS REGISTRY NO.:88192- 19-2](1-1, 38 μL), N-(tert-butoxycarbonyl)-β-ara Nin [CAS REGISTRY NO.:3303-84-2] (4-1, 100 m) g) In a 2 mL ethanol solution, add 4-(4,6-dimethoxy-1,3,5-tri Din-2-yl)-4-methylmorpholinium chloride (DMT-MM) (146 mg) Add ) and stir at room temperature for 18 hours. Add water to the reaction mixture, extract with ethyl acetate, and then remove the organic layer. The layers were washed sequentially with water and saturated saline solution. The organic layer was dried with anhydrous sodium sulfate and then concentrated under reduced pressure. The indicated compound 4-2 (124 mg) was obtained as a white, waxy substance.

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

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

[0368] <Process 3> Alginic acid (EX4-A) with 3-amino-N-(3-azidopropyl)propanamide group introduced 2) Synthesis: [ka]

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

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

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

[0372] Commercially available 2-azidoacetic acid [CAS REGISTRY NO.: 18523-48-3] (1-1, 41 μL) from Organic Letters (2017), 19 Salts of azidoacetate chloride prepared in the same manner as described in (23), 6400-6403. A 1.0 mL solution of methylene chloride is added to a commercially available 1-(N-tert-butytoxycarbonyl- (Aminomethyl)-4-(aminomethyl)benzene [CAS REGISTRY NO.: [108468-00-4](5-1, 100 mg), triethylamine (118 μL) The mixture was added to a 1.0 mL solution of methylene chloride under ice water cooling and stirred at room temperature for 2.5 hours. Add ethyl acetate (20 mL) and water (5 mL) to the reaction mixture, and after liquid-liquid separation, the organic layer is diluted with water. The material was washed sequentially with saturated sodium bicarbonate solution, water, and saturated saline solution. Insoluble matter was filtered out, and the filtrate was treated with anhydrous sodium sulfate. After drying with a humectant, the solution was concentrated under reduced pressure. The residue was then treated with methyl-tert-butyl ether / n-heptane. Tritulate was performed. The resulting solid was filtered, and 5-2 (91 mg) of the labeled compound was added to a dilute solution. It was obtained as a solid.

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

[0374] (Example 5) The compound obtained in Step 1 (5-2, 91 mg) was subjected to ice water cooling at 4 N. - After adding hydrogen chloride / 1,4-dioxane (637 μL), 1,4-dioxane (6 After adding 27 μL, the mixture was stirred at room temperature for 3.5 hours. Diisopropyl alcohol was added to the reaction mixture. Add 3.8 mL of tereol and stir for 10 minutes. Filter the resulting solid to obtain the labeled compound. 5-3 (62 mg) was obtained as a beige solid.

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

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

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

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

[0379] Commercially available 4-azidophenol [CAS REGISTRY NO.:24541-43] -3](6-1, 0.3 g), commercially available tert-butyl (2-bromoethyl)carb Mate [CAS REGISTRY NO.:39684-80-5] (6-2, 0.6 To a mixture of g) and N-methylpyrrolidone (3 mL), add potassium carbonate (0) at room temperature. 0.61 g) was added. The reaction mixture was stirred at 80°C for 6 hours and 30 minutes, then cooled to room temperature. Water (10 mL) and methyl tert-butyl ether (20 mL) were added. The suspension was filtered through Celite, and the residue was removed using methyl tert-butyl ether (5 mL). The mixture was washed twice. The filtrate was separated, and the organic layer was concentrated under reduced pressure to obtain the crude product. The crude product is dissolved in methyl tert-butyl ether (20 mL) and 1 N hydroxyl solution is added. Sodium chloride solution (5 mL) twice, water (5 mL) twice, saturated saline solution (5 mL) The layers were sequentially washed and dried with anhydrous sodium sulfate. The organic layer was filtered and then concentrated under reduced pressure. This yielded the indicated compound 6-3 (0.411 g) as a purple oily substance.

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

[0381] (Example 6) Compound obtained in Step 1 (6-3, 0.41 g) and 1,4-diode To the xane (2.87 mL) mixture, under water-cooled stirring, 4 N-hydrogen chloride / 1,4-di After adding oxane (2.87 mL), the mixture was stirred at room temperature for 18 hours. Diisopropyl alcohol was added to the reaction mixture. Add 40 mL of ropil ether and stir the suspension at room temperature for 30 minutes. Filter the precipitate. The recovered solid was dried under reduced pressure, and the labeled compound 6-4 (0.2834 g) was obtained as a pale purple solid. It was obtained as such.

[0382] <Process 3> 2-(4-azidophenoxy)ethane-1-amino group-modified alginate (EX6-A2) Synthesis of: [ka] A 1% by weight aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (2 9.66 mL) at room temperature, 4-(4,6-dimethoxy-1,3,5-triazine-2- Iyl-4-methylmorpholinium chloride (DMT-MM) (91.52 mg) and 1 molar concentration sodium bicarbonate solution (68.63 μL) was added. Then, in (Example 6) <Step 2> The obtained compounds (6-4, 14.73 mg) were dissolved in water (1 mL) and ethanol (1 mL). L) Add the solution at room temperature, stir at the same temperature for 42 hours, then add sodium chloride (300 mg). Then, ethanol (59.3 mL) was added sequentially, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was then... The sample was filtered, washed with ethanol, and then dried under reduced pressure. The resulting solid was dissolved in water and then freeze-dried. The compound EX6-A2 (269 mg) was obtained as a pink solid.

[0383] (Example 7) N-(2-aminoethyl)-2-(cycloocto-2-in-1-yloxy)acetami Synthesis of do-group-introduced alginate (EX7-B2): [ka]

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

[0385] Commercially available tert-butyl(2-aminoethyl)carbamate (7-1, 3.00 g, [CAS REGISTRY NO.:57260-73-8] Tetrahydrofuran To 12.0 mL of the solution, 2.24 mL of ethyl trifluoroethyl was added dropwise. The reaction mixture was stirred at room temperature for 14.5 hours. The reaction solution was concentrated under reduced pressure, and tert-brown phosphate was added to the residue. Add methyl ether (5 mL) and heptane (25 mL) and tritulate. After filtering out the solid, wash with heptane to obtain the labeled compound 7-2 (4.36 g) as a white solid. I obtained it by doing so.

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

[0387] (Example 7) Compound 7-2 (0.50 g) obtained in <Step 1> was treated with 1,4-dioxy Suspended in Sun (3.0 mL). Cooled in ice water, 4 N-hydrogen chloride / 1,4-dioxane ( Add 7.0 mL of diisopropyl ether (3) to the reaction mixture and stir at room temperature for 3 hours. Add 0.0 mL of the solution and stir at room temperature for 50 minutes. Filter out the solid and remove the diisopropyl ether. After washing with a squeegee, the compound 7-3 (0.70 g) was dried under reduced pressure to obtain the indicated compound as a white solid.

[0388] <Process 3> N-(2-(2-(cycloocto-2-in-1-yloxy)acetamide)ethyl)- Synthesis of 2,2,2-trifluoroacetamide (7-5): [ka]

[0389] Methods of publicly available information (Org. Process Res. Dev. (2018) 22: Ethanol (2) of carboxylic acids (7-4, 300 mg) synthesized according to 108-110) In a mL solution, add 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4 - Methylmorpholinium chloride (DMT-MM) (1.09 g), (Example 7) Compound 7-3 (380 mg) obtained in step 2, triethylamine (321 μL) In addition, after stirring at 30°C for 3 hours, add triethylamine (229 μL) and stir at the same temperature for 1 hour. Stirring was continued. After stirring at room temperature for 15.5 hours, water (10 mL) and ethyl acetate (50 mL) were added. Add (mL), separate, and extract the aqueous layer with ethyl acetate (10 mL). The organic layer was extracted using 0.5 mL. Wash sequentially with citric acid, water, and saturated saline solution, dry with anhydrous sodium sulfate, and then concentrate under reduced pressure. Then, tert-butyl methyl ether was added to the residue, insoluble matter was filtered off, and the filtrate was concentrated. Ricagel column chromatography (10% ethyl acetate / n-heptane ~ 40% ethyl acetate) Purified with CHILD / n-heptane, the marked compound 7-5 (322 mg) was obtained as a white solid. I got it.

[0390] <Step 4> N-(2-aminoethyl)-2-(cycloocto-2-in-1-yloxy)acetami Combination of Do (7-6): [ka]

[0391] (Example 7) Methanol (4.5) of compound 7-5 (322 mg) obtained in <Step 3>. Add potassium carbonate (278 mg) in water (1.6 mL) to the 8 mL solution, and in the room The mixture was stirred at warm temperature for 7.5 hours. The reaction mixture was concentrated under reduced pressure, water (3 mL) was added, and then sodium chloride was added. Saturated with sulfur. The aqueous layer was extracted with ethyl acetate (30 mL, 10 mL x 3) and anhydrous sulfur. After drying with sodium acid, the compound 7-6 (238 mg) was concentrated under reduced pressure to obtain a colorless oil. It was obtained as such.

[0392] <Process 5> N-(2-aminoethyl)-2-(cycloocto-2-in-1-yloxy)acetami Synthesis of do-group-introduced alginate (EX7-B2): [ka]

[0393] A 1% by weight aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: B-2) (1 Add 4-(4,6-dimethoxy-1,3,5-triazine-2) to 20 mL of water, stirring at room temperature. -yl)-4-methylmorpholinium chloride (DMT-MM) (335 mg), (actual Example 7) Ethanol (12 mL) of compound 7-6 (68 mg) obtained in <Step 4> The solution and 1 molar concentration sodium bicarbonate solution (303 μL) were added sequentially, and the mixture was stirred at 30°C for 3 hours. After adding sodium chloride (1.2 g) to the solution, add ethanol (240 mL). The mixture was stirred for 1.5 hours. The resulting precipitate was filtered and washed with ethanol (20 mL x 5), It was dried under reduced pressure. The resulting solid was dissolved in water and freeze-dried to obtain the compound EX7-B2(1 0.16 g) was obtained as a white solid.

[0394] (Example 8) N-(2-(2-aminoethoxy)ethyl)-2-(cycloocto-2-in-1-i) Synthesis of alginic acid with a roxyacetamide group (EX8-A2): [ka]

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

[0396] tert-butyl(2-aminoethyl)carbamate (8-1, 1.0 g, [CAS REGISTRY NO.:57260-73-8) Tetrahydrofuran (4.0 Trifluoroethyl acetate (0.6 mL) was added dropwise to the (mL) solution. The reaction mixture was then placed in a room. Stir at warm temperature for 3.5 hours, then concentrate under reduced pressure to obtain the labeled crude compound 8-2 (1.5 g) as a colorless oil. I obtained it as a physical object.

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

[0398] (Example 8) Compound 8-2 (1.5 g) obtained in Step 1 was cooled in ice water to 4 N. -Add hydrogen chloride / 1,4-dioxane solution (10.3 mL) and stir at room temperature for 1 hour. Diisopropyl ether (30 mL) was added to the reaction mixture and stirred at room temperature for 30 minutes. The solvent is removed under reduced pressure, followed by azeotropic distillation with diisopropyl ether, and then dried under reduced pressure to obtain the labeled compound. 1.3 g of substance 8-3 was obtained as a colorless oily substance.

[0399] <Process 3> N-(2-(2-(2-(cycloocto-2-in-1-yloxy)acetamide)ethoxy Synthesis of ethyl(8-4)-2,2,2-trifluoroacetamide: [ka]

[0400] Methods of publicly available information (Org. Process Res. Dev. (2018) 22: Carboxylic acid synthesized according to 108-110) (7-4, 300 mg), (Example 8) The compound 8-3 (443 mg) obtained in step 2> was added to acetonitrile (6.0 mL). It dissolved. O-(7-azabenzotriazol-1-yl)-N,N,N',N'-teto Lamethyluronium hexafluorophosphate (0.75 g), N,N-diisopropyl Add ethylamine (920 μL) and stir at room temperature for 2.5 hours. Add ethyl acetate to the reaction mixture. Add 20 mL of oil and 10 mL of water, then separate the layers. For the organic layer, add 10 mL of water and saturate. The samples were sequentially washed with saline solution (5 mL), dried with anhydrous sodium sulfate, and then concentrated under reduced pressure. Residue Silica gel column chromatography (50% ethyl acetate / n-heptane ~ 70% acetic acid) Purified with ethyl / n-heptane, the labeled compound 8-4 (469 mg) was obtained as a colorless gum-like substance. It was obtained as such.

[0401] <Step 4> N-(2-(2-aminoethoxy)ethyl)-2-(cycloocto-2-in-1-yl) Synthesis of xylacetamide (8-5): [ka]

[0402] (Example 8) Methanol (3.220 mg) of compound 8-4 (220 mg) obtained in <Step 3>. Add potassium carbonate (103 mg) in water (0.99 mL) to the 0 mL solution. The mixture was stirred at room temperature for 4.5 hours. Methanol was removed under reduced pressure, and water (2 mL) was added. Saturated with salt. Extracted with ethyl acetate (15 mL, 10 mL x 4) and anhydrous sodium sulfate. After drying with um, the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (10 mL). Then, after filtering out insoluble matter, the crude compound 8-5 (140 mg) was concentrated under reduced pressure. It was obtained as a pale yellow, gum-like substance.

[0403] <Process 5> N-(2-(2-aminoethoxy)ethyl)-2-(cycloocto-2-in-1-yl) Synthesis of xylacetamide-modified alginate (EX8-A2): [ka]

[0404] A 1% by weight aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (4 0 mL) is mixed at room temperature and 4-(4,6-dimethoxy-1,3,5-triazine-2- (Iyl)-4-methylmorpholinium chloride (DMT-MM) (112 mg), (implementation) Example 8) Ethanol (4.0 mL) of compound 8-5 (30 mg) obtained in <Step 4> The solution and 101 μL of 1 molar concentration sodium bicarbonate solution were added sequentially, and the mixture was stirred at 30°C for 3 hours. After adding sodium chloride (0.4 g) to the solution, add ethanol (80 mL), and 3 The mixture was stirred for 0 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure. The solid was dissolved in water and freeze-dried to obtain the labeled compound EX8-A2 (410 mg) as a white solid. It was obtained as such.

[0405] (Examples 9a, 9b) N-(2-aminoethyl)-2-(2-(cycloocto-2-in-1-yloxy)acetate Synthesis of alginic acid (EX9a-A2, EX9b-B2) with acetamide group introduced: [ka]

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

[0407] N-(tert-butoxycarbonyl)glycine (91 mg, [CAS REGIS TRY NO.:4530-20-5), (Example 7) Compound obtained in Step 2 ( O-(7-aza) was dissolved in acetonitrile (3.0 mL). Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexa Fluorophosphate (217 mg), N,N-diisopropylethylamine (281 μg) Add (L) and stir at room temperature for 3.5 hours. Add ethyl acetate (15 mL) and water (5 After adding (mL), the organic layer was separated and washed sequentially with water and saturated saline. The organic layer was then washed with anhydrous sulfuric acid. After drying with sodium, the residue was concentrated under reduced pressure. The residue was then subjected to column chromatography (elution solvent: 40 Purified with % ethyl acetate / n-heptane → ethyl acetate, and labeled compound 9-1 (180 mg) ) was obtained as a light beige amorphous material.

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

[0409] (Example 9) The compound obtained in Step 1 (9-1, 180 mg) was subjected to ice water cooling for 4 hours. After adding constant-hydrogen chloride / 1,4-dioxane (1.2 mL), stir at room temperature for 0.8 hours. The reaction mixture was then mixed with 3.6 mL of diisopropyl ether and stirred for 30 minutes. The obtained solid was filtered to obtain the labeled compound 9-2 (114 mg) as a white solid.

[0410] <Process 3> N-(2-(2-(2-(cycloocto-2-in-1-yloxy)acetamide)acetamide Synthesis of mido(ethyl)-2,2,2-trifluoroacetamide (9-3): [ka]

[0411] Methods of publicly available information (Org. Process Res. Dev. (2018) 22: Carboxylic acid synthesized according to 108-110) (7-4, 80 mg), (Example 9) To the compound obtained in step 2 (9-2, 110 mg), add ethanol (1.6 mL) and 4 -(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholin Add um chloride (DMT-MM) (219 mg) and triethylamine (67 μL). The mixture was stirred at room temperature for 3 hours. Water (3.2 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. Afterward, the solid was filtered and washed with water. To the obtained solid, ethyl acetate / ethanol (1 / 1. Add 10 mL of the filtrate and filter off the insoluble matter. Concentrate the filtrate under reduced pressure to obtain the labeled compound 9-3 ( 101 mg was obtained as a white solid.

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

[0413] (Example 9) Methanol (1.60 mg) of the compound obtained in Step 3 (9-3.60 mg) Add potassium carbonate (59 mg) in water (0.3 mL) to 8 mL of the solution, and at room temperature. The mixture was stirred for 4 hours. After concentrating the reaction mixture under reduced pressure, water (2 mL) was added and saturated with sodium chloride. The mixture was then diluted. Extraction was performed with ethyl acetate (15 mL, 10 mL x 4), and the extracted layer was concentrated under reduced pressure. Ethyl acetate (10 mL) and ethanol (1 mL) were added to the residue, and insoluble matter was filtered off. The filtrate was concentrated under reduced pressure to obtain the indicated compound 9-4 (49 mg) as a colorless gum-like substance. .

[0414] <Process 5-1> N-(2-(aminoethyl)-2-(2-(cycloocto-2-in-1-yloxy) Synthesis of acetamide-modified alginate (EX9a-A2): [ka]

[0415] A 1% by weight aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (3 8 mL) contains 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (106 mg), (Example 9) <Step A solution of the compound obtained in step 4 (9-4, 30.3 mg) in ethanol (3.8 mL), 1 molar concentration sodium bicarbonate solution (96 μL) was added. After stirring at 30°C for 3.2 hours, sodium chloride was added. Add 0.38 g of ethanol and 76 mL of sodium sequentially, and stir at room temperature for 30 minutes. The obtained precipitate was filtered, washed with ethanol, and dried under reduced pressure. The resulting solid was dissolved in water. After thawing, the compound EX9a-A2 (381 mg) was freeze-dried to obtain the indicated compound EX9a-A2 as a white solid.

[0416] <Step 5-2> N-(2-(aminoethyl)-2-(2-(cycloocto-2-in-1-yloxy) Synthesis of acetamide-modified alginate (EX9b-B2): [ka]

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

[0418] (Example 10) N-(2-aminoethyl)-3-(2-(cycloocto-2-in-1-yloxy) Synthesis of alginic acid (EX10-A2) with a cetamide propanamide group: [ka]

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

[0420] Commercially available N-(tert-butoxycarbonyl)-β-alanine (113 mg, CA [S REGISTRY NO.:3303-84-2]), (Example 7) Obtained in <Step 2> The compound (7-3, 110 mg) was dissolved in acetonitrile (3.3 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl Rhonium hexafluorophosphate (261 mg), N,N-diisopropylethylamine Add (319 μL) of ethyl acetate and stir at room temperature for 3 hours. Add 15 mL of ethyl acetate to the reaction mixture. ), water (5 mL) was added, and after separation, the organic layer was washed sequentially with water and saturated saline solution. After drying with anhydrous sodium sulfate, concentrate under reduced pressure and tert-butyl methyl ether (20 Tritulate with (mL). The solid was filtered and dissolved in ethyl acetate (20 mL). The machine bed was sequentially washed with 1 N citric acid, water, and saturated saline solution, and then dried with anhydrous sodium sulfate. After drying, the solution was concentrated under reduced pressure. The residue was tritubulated with tert-butyl methyl ether (10 mL). After fermentation, the solid was filtered off, and the labeled compound 10-1 (80 mg) was obtained as a white solid. .

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

[0422] (Example 10) The compound obtained in Step 1 (10⁻¹, 80 mg) was subjected to ice water cooling for 4 After adding the standard hydrogen chloride / 1,4-dioxane (1.1 mL), stir at room temperature for 2 hours. Diisopropyl ether (3.4 mL) was added to the reaction mixture and stirred for 1.5 hours. The obtained solid was filtered to obtain the labeled compound 10-2 (61 mg) as a white solid.

[0423] <Process 3> 3-(2-(cycloocto-2-in-1-yloxy)acetamide)-N-(2-(2, Synthesis of 2,2-trifluoroacetamide)ethyl)propanamide(10-3): [ka]

[0424] Methods of publicly available information (Org. Process Res. Dev. (2018) 22: Carboxylic acid synthesized according to 108-110) (7-4, 44 mg), (Example 10) To the compound obtained in step 2 (10⁻², 61 mg), add ethanol (1.2 mL), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorphoyl Nium chloride (DMT-MM) (115 mg), triethylamine (39 μL) In addition, the mixture was stirred at room temperature for 2 hours. Water (3.7 mL) was added to the reaction mixture, and ethyl acetate (15 mL) was added. Extraction was performed using (mL, 5 mL). The organic layer was washed sequentially with water and saturated saline solution, and anhydrous sodium sulfate was used. After drying with lium, it was concentrated under reduced pressure. To the resulting solid, tert-butyl methyl ether (1 (0 mL) was added, triturated, and filtered. The resulting solid was then subjected to column chromatography. (80% ethyl acetate / n-heptane → ethyl acetate → 20% methanol / ethyl acetate) The compound was purified to obtain the labeled compound 10-3 (60 mg) as a pale yellow solid.

[0425] <Step 4> N-(2-(aminoethyl)-3-(2-(cycloocto-2-in-1-yloxy) Synthesis of cetamide propanamide (10-4): [ka]

[0426] (Example 10) Methanol of the compound (10-3, 60 mg) obtained in Step 3 Add potassium carbonate (42 mg) in water (0.3 mL) to the 3.0 mL solution. After stirring at room temperature for 3 hours, add a solution of potassium carbonate (42 mg) in water (0.3 mL). In addition, the mixture was stirred at room temperature for 16.5 hours. After concentrating the reaction mixture under reduced pressure, saturated saline solution (2 mL) was added. In addition, it was further saturated with sodium chloride. Ethyl ethyl (15 mL, 10 mL x 4) Extraction was performed, the extract layer was dried with anhydrous sodium sulfate, and then concentrated under reduced pressure. Ethyl acetate (5) was added to the residue. (mL) and a few drops of methanol were added, and insoluble matter was filtered off. The resulting filtrate was concentrated under reduced pressure and then labeled as standard. Compound 10-4 (31 mg) was obtained as a colorless oily substance.

[0427] <Process 5> N-(2-aminoethyl)-3-(2-(cycloocto-2-in-1-yloxy)acetate Synthesis of alginic acid (EX10-A2) with a tamide propanamide group: [ka]

[0428] A 1% by weight aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (4 1 mL) contains 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (114 mg), (Example 10) Dissolve the compound obtained in step 4 (10⁻⁴, 30.5 mg) in ethanol (4.1 mL). Add 103 μL of 1 molar concentration sodium bicarbonate solution to the liquid. Stir at 30°C for 3 hours, then add sodium chloride. Add thorium (0.41 g) and ethanol (82 mL) sequentially, and stir at room temperature for 30 minutes. The precipitate was filtered, washed with ethanol, and dried under reduced pressure. The resulting solid was then dissolved in water. After dissolution, the compound EX10-A2 (406 mg) was freeze-dried to obtain the labeled compound EX10-A2 as a white solid. .

[0429] (Examples 11a, 11b) 2-(cycloocto-2-in-1-yloxy)ethane-1-amino group-modified alginate ( Synthesis of EX11a-A2 and EX11b-B2: [ka]

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

[0431] Methods of publicly available information (Org. Process Res. Dev. (2018) 22: Dibromo compound (11-1, 1 g) synthesized according to 108-110) and by a method known in the literature ( Alcohol compounds synthesized according to International Publication No. 2015 / 140807 (11 To the mixture of -2 and 5.28 g, dichloromethane (2 mL) was added at room temperature. While maintaining the temperature at room temperature, the reaction vessel was wrapped in aluminum foil to protect it from light. Subsequently, at room temperature Add silver trifluoromethanesulfonate (1.92 g) all at once and stir at the same temperature for 1 hour. After stirring, saturated saline solution (5 mL) was added under ice cooling, and the precipitated silver salt was filtered through Celite. Further removal was performed, and the residue was washed with methyl tert-butyl ether (10 mL). The liquid was separated, and the organic layer was washed twice with water (5 mL). Then, it was dried with anhydrous sodium sulfate. The crude product was obtained by drying, filtering, and then concentrating under reduced pressure. This crude product was then processed using silica gel. The compound 11-3 (0.0) was purified by Lamb chromatography (n-heptane / ethyl acetate). A fraction containing 46 g was obtained.

[0432] <Process 2> Synthesis of 2-(cycloocto-2-in-1-yloxy)ethane-1-amine (11-4) : [ka]

[0433] (Example 11) Fraction containing the compound (11-3, 0.46 g) obtained in Step 1 and To a mixture of dimethyl sulfoxide (1.38 mL), add 28% sodium under water-cooled stirring. Add 1.82 mL of ummethoxidemethanol solution and stir at room temperature for 16 hours. (10 mL) was added to stop the reaction, and methanol was concentrated under reduced pressure. The resulting solution was then mixed with methanol. The organic layer was extracted three times with tert-butyl ether (10 mL). The organic layer was then extracted with anhydrous sodium sulfate. After drying with alum and filtering, the compound 11-4 (0.196) is concentrated under reduced pressure. The crude product of g) was obtained as a brown oily substance.

[0434] <Process 3-1> 2-(cycloocto-2-in-1-yloxy)ethane-1-amino group-modified alginate ( Synthesis of EX11a-A2): [ka]

[0435] A 1% by weight aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (6 9.2 mL) at room temperature, 4-(4,6-dimethoxy-1,3,5-triazine-2-I Add 4-methylmorpholinium chloride (DMT-MM) (213.55 mg). Next, (Example 11) the compound obtained in <Step 2> (11-4, 26.78 m Add the water (1 mL) and ethanol (1 mL) solution from g) at room temperature, and leave at the same temperature for 24 hours. After stirring, add sodium chloride (700 mg) and ethanol (138.4 mL) in sequence. In addition, the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and then dried under reduced pressure. The resulting solid was dissolved in water and then freeze-dried to obtain the compound EX11a-A2(661). mg was obtained as a white solid.

[0436] <Process 3-2> 2-(cycloocto-2-in-1-yloxy)ethane-1-amino group-modified alginate ( Synthesis of EX11b-B2): [ka]

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

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

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

[0440] Commercially available 2-(2-aminoethoxy)ethanol [CAS REGISTRY NO.: 929-06-6](12-1, 2.0 mL) Tetrahydrofuran (8.0 mL) 2,2,2-Trifluoroethyl acetate (2.5 mL) was added dropwise to the solution over 5 minutes. The mixture was then stirred at room temperature for 20 hours. After concentrating the reaction mixture under reduced pressure, ethyl acetate (30 mL) and water were added. (10 mL) was added and the solution was separated. The aqueous layer was extracted with ethyl acetate (10 mL) and combined. The organic layer was washed sequentially with water and saturated saline solution. After drying the organic layer with anhydrous sodium sulfate, it was then subjected to reduced pressure. The compound was concentrated to obtain the labeled compound 12-2 (3.7 g) as a colorless oil.

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

[0442] Methods of publicly available information (Org. Process Res. Dev. (2018) 22: The dibromo compound (11-1, 0.30 g) synthesized according to 108-110) was mixed with methylene chloride. Dissolve in (0.54 mL) and, under light-shielding aluminum foil, obtain (Example 12) <Step 1>. Compound (12-2, 1.86 g), silver trifluoromethanesulfonate (0.52 g) ) was added. After stirring at room temperature for 1.5 hours under the protection of light, saturated sodium bicarbonate solution (2. (0 mL), saturated saline (3.0 mL) was added sequentially. The solid was filtered off with Celite, and t The mixture was washed with ert-butyl methyl ether (10 mL x 3). The filtrate was separated, and the organic layer was removed. The layers were washed sequentially with water and saturated saline solution. The organic layer was dried with anhydrous sodium sulfate and then concentrated under reduced pressure. The indicated compound 12-3 (424 mg) was obtained as a light brown oily substance.

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

[0444] (Example 12) The compound obtained in Step 2 (12-3, 100 mg) was tetrahed Dissolve in Drofuran (0.7 mL) and N,N-dimethylformamide (0.7 mL). 60% sodium hydride (21 mg) was added under ice water and stirred at the same temperature for 3 hours. Add 60% sodium hydride (10 mg) and leave at room temperature for 1 hour, then add 60% sodium hydride... Add 10 mg of lium and stir at room temperature for 20 hours. Add 3 mL of water and acetate. Extraction was performed using chill (15 mL, 10 mL), and the organic layer was sequentially washed with water and saturated saline solution. The layer was dried with anhydrous sodium sulfate and then concentrated under reduced pressure. The residue was subjected to column chromatography. n-heptane → Purified with 50% ethyl acetate / n-heptane) to obtain the labeled compound 12-4(3 7 mg was obtained as a colorless oily substance.

[0445] <Step 4> 2-(2-(cycloocto-2-in-1-yloxy)ethoxy)ethane-1-amine( Synthesis of 12-5): [ka]

[0446] (Example 12) Methanol of the compound obtained in Step 3 (12-4, 37 mg) Add potassium carbonate (50 mg) in water (185 μL) to the 555 μL solution. The mixture was stirred at room temperature for 17 hours. After concentrating the reaction mixture under reduced pressure, water (1 mL) was added, and sodium chloride was added. Saturated with um. Extracted with ethyl acetate (10 mL x 4), and the extract layer was treated with anhydrous sodium sulfate. After drying with a hum, the mixture was concentrated under reduced pressure. Ethyl acetate (10 mL) and a few drops of methanol were added to the residue. The insoluble matter was filtered off. The resulting filtrate was concentrated under reduced pressure to obtain the labeled compound 12-5 (30 mg). It was obtained as a colorless oily substance.

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

[0448] A 1% by weight aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (5 2 mL) contains 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (145 mg), (Example 12) A solution of the compound obtained in step 4 (12-5, 29 mg) in ethanol (5.2 mL), 131 μL of 1 molar concentration sodium bicarbonate solution was added. After stirring at 30°C for 3.2 hours, sodium chloride was added. Add thorium (0.52 g) and ethanol (104 mL) sequentially, and stir at room temperature for 30 minutes. Mixed. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure. The resulting solid was then mixed with water. After dissolution, the compound EX12-A2 (522 mg) was freeze-dried to obtain the marked compound EX12-A2 as a white solid. Ta.

[0449] (Example 13) 3-amino-N-(2-(2-(2-(cycloocto-2-in-1-yloxy)acetate Synthesis of alginic acid (EX13-A2) with a mid-ethoxyethyl propanamide group: [ka]

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

[0451] Commercially available β-alanine [CAS REGISTRY NO.: 107-95-9] (13 -1, 2.0 g) dissolved in methanol (40.0 mL), and triethylamine (3. Add 3 mL of 2,2,2-trifluoroacetic acid (3.4 mL) for 5 minutes under water cooling. After adding the solution dropwise, the mixture was stirred at room temperature for 20.5 hours. The reaction mixture was concentrated under reduced pressure, and water (20 mL) was added. The solution was adjusted to pH 4 with 1 N hydrochloric acid. Extraction was performed with ethyl acetate (100 mL x 2, 50 mL). The organic layer was removed and washed with saturated saline solution. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. Then, the indicated compound 13-2 (2.9 g) was obtained as a white solid.

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

[0453] (Example 13) Compound obtained in <Step 1> (13-2, 400 mg), tert- Butyl(2-aminoethyl)carbamate (8-1, 441 mg, [CAS REGI STRY NO.:57260-73-8) in a 4.0 mL ethanol solution, -(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholin 897 mg of um chloride (DMT-MM) was added and the mixture was stirred for 3.5 hours. Add water (5 mL), extract with ethyl acetate (20 mL, 10 mL), then add water to the organic layer. The layers were then washed sequentially with saturated saline solution. The organic layer was dried with anhydrous sodium sulfate, then concentrated under reduced pressure, and the residue was removed. The solution was purified by column chromatography (30% ethyl acetate / n-heptane → ethyl acetate). The indicated compound 13-3 (451 mg) was obtained as a colorless oily substance.

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

[0455] (Example 13) The compound obtained in Step 2 (13-3, 451 mg) was subjected to ice water cooling. Add 4 N-hydrogen chloride / 1,4-dioxane (3.16 mL) and stir at room temperature for 3 hours. The reaction solution was then mixed with diisopropyl ether (6.4 mL), concentrated under reduced pressure, and labeled. Compound 13-4 (433 mg) was obtained as a colorless gum-like substance.

[0456] <Step 4> N-(2-(2-(2-(cycloocto-2-in-1-yloxy)acetamide)ethoxy C) Ethyl-3-(2,2,2-trifluoroacetamide)propanamide (13-5) Synthesis of: [ka]

[0457] Methods of publicly available information (Org. Process Res. Dev. (2018) 22: Carboxylic acids synthesized according to 108-110) (7-4, 111 mg), (Example 13) To the compound obtained in <Step 3> (13-4, 215 mg), add ethanol (1.7 mL) ), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmol Folinium chloride (DMT-MM) (253 mg), triethylamine (102 μg) Add L) and stir at room temperature for 21 hours. Add water (5 mL) to the reaction mixture and ethyl acetate ( Extraction was performed using 15 mL of water. The organic layer was washed sequentially with water and saturated saline solution, and then with anhydrous sodium sulfate. After drying, the mixture was concentrated under reduced pressure. The resulting residue was subjected to column chromatography (30% ethyl acetate). Purified with n-heptane → ethyl acetate → 15% methanol / ethyl acetate, the marked compound is obtained. 13-5 (35 mg) was obtained as a colorless oily substance.

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

[0459] (Example 13) Methanol of the compound obtained in Step 4 (13-5, 35 mg) Add potassium carbonate (33 mg) in water (175 μL) to the 700 μL solution. The mixture was stirred at room temperature for 16.5 hours. After concentrating the reaction mixture under reduced pressure, water (2 mL) was added, and sodium chloride was added. Saturated with thorium. Extracted with ethyl acetate (10 mL x 5), and the extract layer was converted to anhydrous sodium sulfate. After drying with a humic acid filter, the solution was concentrated under reduced pressure. To the residue, ethyl acetate (10 mL) and a few drops of methanol were added. In addition, insoluble matter was filtered off. The resulting filtrate was concentrated under reduced pressure to obtain the labeled compound 13-6 (24 m). g) was obtained as a colorless gum-like substance.

[0460] <Process 6> 3-amino-N-(2-(2-(2-(cycloocto-2-in-1-yloxy)acetate Synthesis of alginic acid (EX13-A2) with a mid-ethoxyethyl propanamide group: [ka]

[0461] A 1% by weight aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (2 8 mL) contains 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (78 mg), (Example 13) <Step Solution of the compound obtained in step 5 (13-6, 24 mg) in ethanol (2.8 mL), 1 Molar concentration - sodium bicarbonate solution (71 μL) was added. After stirring at 30°C for 3.5 hours, sodium chloride Um (0.28 g) and ethanol (56 mL) were added sequentially, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure. The resulting solid was dissolved in water. The compound EX13-A2 (272 mg) was subsequently freeze-dried to obtain the compound EX13-A2 as a white solid.

[0462] (Example 14) N-(4-(2-aminoethoxy)benzyl)-2-(cycloocto-2-in-1-i) Synthesis of alginic acid with roxyacetamide group (EX14a-A2, EX14b-B2): [ka]

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

[0464] Commercially available 2-bromoethylamine hydrobromide [CAS REGISTRY NO.:2 [576-47-8] (14-1, 3 g) in methanol (30 mL) solution, ice-cold Under stirring, triethylamine (4.29 mL) was added. To this mixture, at the same temperature, 1.92 mL of ethyl fluoroethyl acetate was gradually added, and the mixture was stirred at room temperature for 42 hours. After completion, the reaction mixture was concentrated under reduced pressure and water (10 mL) was added. Ethyl acetate (10 mL) Extract three times using ), then wash the organic layer sequentially with water (5 mL) and saturated saline solution (5 mL), and then anhydrous water. After drying with sodium sulfate and filtering, the compound 14-2(2) is concentrated under reduced pressure. 0.457 g) was obtained as a pale brown solid.

[0465] <Process 2> tert-butyl (4-(2-(2,2,2-trifluoroacetamide)ethoxy) Synthesis of (14-4) carbamates: [ka]

[0466] Commercially available tert-butyl (4-hydroxybenzyl)carbamate [CAS REG [ISTRY NO.:149505-94-2](14-3, 0.36 g), (Example) 14) Compound obtained in <Step 1> (14-2, 0.46 g), potassium iodide (0. To a mixture of 35 g of potassium carbonate and N-methylpyrrolidone (3.6 mL), add potassium carbonate at room temperature. Add 0.45 g of um and stir at 140°C for 5 hours. After the reaction is complete, cool to room temperature. Diluted with water (10 mL). Diluted with methyl tert-butyl ether (10 mL). Extract the organic layer twice, then rinse with 1 N sodium hydroxide aqueous solution (5 mL) and water (5 mL). The layers were then washed sequentially with saturated saline solution (5 mL) and dried with anhydrous sodium sulfate. After filtration, the crude product was concentrated under reduced pressure. The obtained crude product was then collected in silica gel. Purified by microchromatography (n-heptane / ethyl acetate), the labeled compound 14-4(0 0.202 g) was obtained as a white amorphous material.

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

[0468] (Example 14) Using the compound (14-4, 0.2 g) obtained in <Step 2>, (Implementation Example 6) By performing the same procedure as in Step 2, the indicated compound 14-5 (0.147 g) ) was obtained as a white solid.

[0469] <Step 4> N-(2-(4-((2-(cycloocto-2-in-1-yloxy)acetamide)meth Synthesis of phenoxyethyl-2,2,2-trifluoroacetamide (14-6): [ka]

[0470] Methods of publicly available information (Org. Process Res. Dev. (2018) 22: Carboxylic acid synthesized according to 108-110) (7-4, 50 mg), (Example 14) The compound synthesized in step 3 (14-5, 81.96 mg) and a mixture of ethanol were used. Then, under ice-cold stirring, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4 -Methylmorpholinium chloride (DMT-MM) (137.22 mg) and triethyl Add 38.25 μL of luamine and stir at room temperature for 1 hour and 30 minutes. After the reaction is complete, add water ( Add 2 mL of methyl tert-butyl ether (0.5 mL of methyl tert-butyl ether), stir the suspension, and add 2 mL of methyl tert-butyl ether. ) was added. The separated aqueous layer was extracted twice with methyl tert-butyl ether (5 mL). Then, wash sequentially with water (5 mL) and saturated saline solution (5 mL), and dry with anhydrous sodium sulfate. The dry organic layer was filtered and concentrated under reduced pressure. The crude product was collected using silica gel column chromatography. Purified by matrix (n-heptane / ethyl acetate), the labeled compound 14-6 (99 m g) was obtained as a white amorphous material.

[0471] <Process 5> N-(4-(2-aminoethoxy)benzyl)-2-(cycloocto-2-in-1-i) Synthesis of Roxyacetamide (14-7): [ka]

[0472] (Example 14) Compound obtained in Step 4 (14-6, 99 mg) and methanol To (1485 μL) of the mixture, potassium carbonate (64.17 mg) and Water (495 μL) was added and the mixture was stirred at room temperature for 15 hours. After the reaction was complete, methanol was removed under reduced pressure. The mixture was concentrated below, and the resulting aqueous layer was extracted three times with ethyl acetate (5 mL). The organic layer was then diluted with water (5 mL). Washed sequentially with L) and saturated saline solution (5 mL), then dried with anhydrous sodium sulfate. After filtering the resulting organic layer, it was concentrated under reduced pressure to obtain the indicated compound 14-7 (68 mg). The crude product was obtained as a yellow, oily substance.

[0473] <Process 6-1> N-(4-(2-aminoethoxy)benzyl)-2-(cycloocto-2-in-1-i) Synthesis of alginic acid (EX14a-A2) with a roxyacetamide group: [ka]

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

[0475] <Step 6-2> N-(4-(2-aminoethoxy)benzyl)-2-(cycloocto-2-in-1-i) Synthesis of alginic acid (EX14b-B2) with roxyacetamide group: [ka]

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

[0477] (Example 15) 2-amino-N-[3-(11,12-didehydrodibenz[b,f]azosin-5(6 Alginic acid (EX15-A2) with H)-yl)-3-oxopropyl]acetamide group Synthesis: [ka]

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

[0479] Commercially available 3-amino-1-(11,12-didehydrodibenz[b,f]azosin-5( 6H)-Il)-1-Propanone [CAS REGISTRY NO.:1255942] -06-3](15-1, 50 mg), N-[(9H-fluoren-9-ylmethoxy) [Carbonyl Glycine] [CAS REGISTRY NO.: 29022-11-5] (54 mg) was dissolved in acetonitrile (1.5 mL). O-(7-azabenzo) Riazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluoro Add phosphate (76 mg) and N,N-diisopropylethylamine (70 μL), The mixture was stirred at room temperature for 4.5 hours. Ethyl acetate (15 mL) and water (5 mL) were added to the reaction mixture. After separation, the organic layer was washed sequentially with water and saturated saline solution. The organic layer was then dried with anhydrous sodium sulfate. After drying, the compound was concentrated under reduced pressure and purified by column chromatography to obtain the labeled compound 15-2(63 (mg) was obtained as a thin beige amorphous material.

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

[0481] (Example 15) The compound obtained in Step 1 (15-2, 63 mg) was treated with piperidine. Add (56 μL) of N,N-dimethylformamide (315 μL) solution and leave at room temperature for 3 The mixture was expanded for 0 minutes. Ethyl acetate (15 mL) and water (5 mL) were added to the reaction solution, and after separation... The organic layer was then washed sequentially with water and saturated saline solution. After drying the organic layer with anhydrous sodium sulfate, the reduction It was concentrated under pressure. To the resulting solid, tert-butyl methyl ether (5 mL) was added, and After lithuration, filter out 15-3 (10 mg) of the labeled compound and obtain a pale beige solid. The compound was obtained by collecting it from the filtrate and adding 15-3 (11 mg) of the labeled compound to form a pale yellow gum. I obtained it as a physical object.

[0482] <Process 3> 2-amino-N-[3-(11,12-didehydrodibenz[b,f]azosin-5(6 Alginic acid (EX15-A2) with H)-yl)-3-oxopropyl]acetamide group Synthesis: [ka]

[0483] A 1% by weight aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (1 9 mL) contains 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4- Methylmorpholinium chloride (DMT-MM) (106 mg), (Example 15) A solution of the compound obtained in step 2 (15-3, 21 mg) in ethanol (1.9 mL), 1 molar concentration sodium bicarbonate solution (48 μL) was added. After stirring at 30°C for 3 hours, sodium chloride was added. 0.19 g of ethanol and 38 mL of cereal were added sequentially, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered, washed with ethanol, and dried under reduced pressure. After dissolving the resulting solid in water, The compound EX15-A2 (188 mg) was obtained as a white solid by freeze-drying.

[0484] (Example 16) 2-amino-N-(2-(cycloocto-2-in-1-yloxy)ethyl)acetamide Synthesis of modified alginate (EX16-A2): [ka]

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

[0486] Suspend glycine (16-1, 2 g) in methanol (10 mL) and cool to 4°C. At the same temperature, ethyl trifluoroethyl acetate (3.5 mL) and triethylamine (3. Add 71 mL of hydrochloric acid (20 mL) and stir at room temperature for 23 hours. After the reaction is complete, add 1 N hydrochloric acid (20 mL) Gradually add ) until the pH reaches 2, extract three times with ethyl acetate (10 mL), and then add water (5 mL). The layers were sequentially washed with L) and saturated saline solution (5 mL). The layer was dried with anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure to obtain a pale yellow oily substance. The obtained oily substance was then mixed with ethyl acetate (20%). Dissolve in (mL) and add n-heptane (10 mL). Concentrate this solution under reduced pressure. By doing so, the indicated compound 16-2 (3.22 g) was obtained as a white amorphous material.

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

[0488] Compound 11-4 (80 mg) and Compound (16) obtained in (Example 16) <Step 1> -2 (81.83 mg) mixture, under ice-cold stirring, ethanol (1600 μL) and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmol Add pholinium chloride (DMT-MM) (239.21 mg) and stir at room temperature for 3 hours. Then, water (2 mL) was added to stop the reaction, and methyl tert-butyl ether (5 mL) was added. Extraction was performed three times with (mL). The organic layer was washed sequentially with water (5 mL) and saturated saline solution (5 mL). The organic layer was dried with anhydrous sodium sulfate. After filtering, the layer was concentrated under reduced pressure to produce the crude product. A substance was obtained. This crude product was triturated with n-heptane (10 mL), filtered, and reduced. By drying under pressure, the indicated compound 16-3 (95.1 mg) was obtained as a white solid.

[0489] <Process 3> 2-amino-N-(2-(cycloocto-2-in-1-yloxy)ethyl)acetamide Synthesis of (16-4): [ka]

[0490] (Example 16) Compound obtained in Step 2 (16-3, 60 mg), methanol ( Using 900 μL of potassium carbonate (51.78 mg) and water (300 μL), Example 14) By performing the same procedure as in Step 5, the labeled compound 16-4 (15 m g) was obtained as a pale yellow oily substance.

[0491] <Step 4> 2-amino-N-(2-(cycloocto-2-in-1-yloxy)ethyl)acetamide Synthesis of modified alginate (EX16-A2): [ka]

[0492] A 1% by weight aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (2 9.66 mL), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (91.52 mg) and (Examples) 16) Using the compound obtained in <Step 3> (16-4, 15 mg), (Example 11) Perform the same procedure as in step 3-1 to obtain the labeled compound EX16-A2 (279 mg) as a white solid. I acquired it physically.

[0493] (Example 17) (2S)-2-amino-N-(2-(cycloocto-2-in-1-yloxy)ethyl) Synthesis of alginic acid with a 3-phenylpropanamide group (EX17-A2): [ka]

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

[0495] L-phenylalanine [CAS REGISTRY NO.: 63-91-2] (17 -1, 2 g) was dissolved in methanol (10 mL) and cooled to 4°C. Subsequently, the same At warm temperature, add trifluoroethyl acetate (1.59 mL) and triethylamine (1.69 mL) Add (mL) and stir at room temperature for 16 hours. After the reaction is complete, add 10 mL of 1 N hydrochloric acid. Gradually add H1 until the suspension is complete, and stir the suspension for 30 minutes. Filter the suspension and collect the solid. By drying under reduced pressure, the indicated compound 17-2 (2.53 g) was obtained as a white solid.

[0496] <Process 2> (2S)-N-(2-(cycloocto-2-in-1-yloxy)ethyl)-3-pheni Synthesis of ru-2-(2,2,2-trifluoroacetamide)propanamide (17-3): [ka]

[0497] Compound 11-4 (60 mg) and Compound (17) obtained in (Example 17) <Step 1> -2, 93.7 mg) mixture, under ice cooling, ethanol (1200 μL) and 4 -(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholin Um chloride (DMT-MM) (179.41 mg) was added, and the mixture was stirred at room temperature for 3 hours. Add water (2 mL) to stop the reaction, then add methyl tert-butyl ether (5 mL). Extraction was performed three times. The organic layer was washed sequentially with water (5 mL) and saturated saline solution (5 mL), and then anhydrous water was used. The mixture was dried over sodium sulfate. After filtering the organic layer, it was concentrated under reduced pressure to obtain the crude product. The product is purified by silica gel column chromatography (n-heptane / ethyl acetate). Thus, the indicated compound 17-3 (57 mg) was obtained as a white amorphous material.

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

[0499] (Example 17) Compound obtained in Step 2 (17-3, 57 mg), methanol ( Using 855 μL), potassium carbonate (38.39 mg), and water (285 μL), Example 14) By performing the same procedure as in Step 5, the labeled compound 17-4 (35 m g) was obtained as a pale yellow oily substance.

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

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

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

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

[0504] Triphenylphosphine (0.96 g) dissolved in tetrahydrofuran (2.59 mL) To the liquid, add diethyl azodicarboxylate (40% toluene solution, 1.92 mL) under ice-cold stirring. The solution was added and stirred at room temperature for 20 minutes. To this solution, commercially available 4-hydro was added under ice-cold stirring. Xybenzoic acid (compound 18-1, 0.37 g) and 2-(tert-butoxycarbonic acid) Add a solution of ethanolamine (0.39 g) in tetrahydrofuran (1.1 mL). The mixture was stirred at room temperature for 17 hours. The reaction solution was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography. To tography (5% ethyl acetate / n-heptane to 40% ethyl acetate / n-heptane) The mixture was purified to obtain a mixture of compound 18-1 and compound 18-2. This mixture was then methyl te Dissolve in rt-butyl ether (20 mL) and dissolve in 1 N sodium hydroxide aqueous solution (5 The organic layer was washed twice with saturated saline solution (5 mL) and then sequentially with saturated saline solution (5 mL). The organic layer was washed with anhydrous sodium sulfate. After drying, the solvent is removed under reduced pressure, and the labeled compound 18-2 (0.45 g) is pink. It was obtained as a colored oily substance.

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

[0506] (Example 18) Methanol of compound 18-2 (0.44 g) obtained in <Step 1> Add lithium hydroxide monohydrate (0.25 g) to 4.4 mL of the solution and incubate at 60°C for 3 hours. Stirred for 30 minutes. Add 1 N hydrochloric acid (5 mL) to the reaction mixture, then add ethyl acetate (10 mL). Extraction was performed three times. The organic layer was washed sequentially with water (5 mL) and saturated saline solution (5 mL), and then anhydrous water was used. The solution was dried with sodium sulfate and the solvent was removed under reduced pressure. The residue was then treated with acetonitrile (4.4 Dissolve in (mL), and add 3-azidopropan-1-amine (0.15 g) and O-(7-A Zabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexyl Safluorophosphate (0.57 g) was added. Subsequently, under ice-cold stirring, N,N-diisopropyl alcohol was added. Add 0.52 mL of propylethylamine and stir at room temperature for 5 hours. Add water to the reaction mixture. Add (10 mL), extract three times with ethyl acetate (15 mL), and extract the organic layer with anhydrous sodium sulfate. The solution was dried with water, and the solvent was removed under reduced pressure. The residue was then subjected to silica gel column chromatography. Purified with (16% ethyl acetate / n-heptane ~ 100% ethyl acetate), compound 1 A fraction containing 8-3 (0.71 g) was obtained.

[0507] For the fraction containing compound 18-3 (0.71 g), 4 N-hydrogen chloride / 1,4-diode Add xane (4.9 mL) and stir at room temperature for 20 minutes. Add diisopropyl alcohol to the reaction mixture. After adding tel, the precipitate was filtered to obtain the marked compound 18-4 (0.49 g) as a white solution. It was obtained as a solid.

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

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

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

[0511] A 1% by weight aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.: A-2) (4 3.6 mL) contains 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)- 4-Methylmorpholinium chloride (DMT-MM) (111.7 mg), 1 molar concentration -Sodium bicarbonate solution (403.5 μL), commercially available 3-amino-1-(11,12-didehydrodimethyl) [b,f] Azosin-5(6H)-yl)-1-propanone [CAS REGIS TRY NO.:1255942-06-3](19-1, 83.6 mg) was used, Example 11) Perform the same procedure as in Step 3-1 to obtain the compound EX19-A2 (376 (mg) was obtained as a pale yellow solid.

[0512] (Example 20) N-(4-(aminomethyl)benzyl)-2-(cycloocto-2-in-1-yloxy) Synthesis of acetamide-modified alginate (EX20-B2): [ka]

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

[0514] Methods known from the literature (Bioorganic & Medicinal Chemistry) tert-butyl(4-( Aminoethyl(benzyl)carbamate (20-1, 0.67 g), triethylamine A mixture of (0.39 mL) and methanol (6.67 mL) was subjected to ice-cold stirring. Ethyl refluoroethyl acetate (0.44 mL) was added dropwise. The reaction mixture was heated to room temperature. The mixture was stirred for 5 hours. The reaction was stopped with water (10 mL), and then extracted three times with ethyl acetate (10 mL). Removed. The collected organic layer was washed with saturated saline solution (5 mL) and dried with anhydrous sodium sulfate. The dried organic layer was filtered and concentrated to obtain the crude compound 20-2 (0.67 g). It was obtained as a pale yellow amorphous material.

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

[0516] (Example 20) Compound 20-2 (0.5 g) obtained in Step 1 is 1,4-dioxy To 3.5 mL of Sun solution, add 4 N-hydrogen chloride / 1,4-dioxane under water cooling and stirring. (3.5 mL) was added and the mixture was stirred at room temperature for 3 hours. Diisopropyl ether (4 After adding 0 mL, the precipitate was filtered to obtain the labeled compound 20-3 (0.4 g). It was obtained as a white solid.

[0517] <Process 3> N-(4-((2-(cycloocto-2-in-1-yloxy)acetamide)methyl) Synthesis of (zyl)-2,2,2-trifluoroacetamide (compound 20-4): [ka]

[0518] Methods of publicly available information (Org. Process Res. Dev. (2018) 22: Carboxylic acid (7-4, 0.17 g) and O-(7-A) synthesized according to 108-110) Zabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexyl A solution of safluorophosphate (0.26 g) in acetonitrile (1.7 mL) is mixed with ice. Under cold stirring, compound 20-3 (0.26 g) obtained in (Example 20) <Step 2> and N Add N-diisopropylethylamine (0.51 mL) dropwise and stir at room temperature for 1 hour and 30 minutes. Mixed. After adding water (5 mL) to stop the reaction, extract three times with ethyl acetate (5 mL). The organic layer was washed with saturated saline solution (3 mL) and then dried with anhydrous sodium sulfate. The dried organic layer was filtered, and the solvent was removed under reduced pressure. The residue was then subjected to silica gel column chromatography. Purified by matrix (12% ethyl acetate / n-heptane to 100% ethyl acetate). The indicated compound 20-4 (0.19 g) was obtained as a white amorphous material.

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

[0520] (Example 20) Compound 20-4 (0.18 g) obtained in Step 3 and methanol To (1.8 mL) of the mixture, add an aqueous solution of potassium carbonate (0.13 g) under ice-cold stirring. (0.9 mL) was added dropwise, and the mixture was stirred at room temperature for 17 hours and 30 minutes. Methanol was removed by distillation under reduced pressure. The mixture was then extracted three times with ethyl acetate (5 mL). The organic layer was washed with saturated saline solution (5 mL). The mixture was dried with anhydrous sodium sulfate. After filtering the organic layer, the solvent was removed under reduced pressure, and the roughened layer was prepared as indicated. The mixture 20-5 (0.13 g) was obtained as a pale yellow oily substance.

[0521] <Process 5> N-(4-(aminoethyl)benzyl)-2-(cycloocto-2-in-1-yloxy C) Synthesis of acetamide-modified alginate (EX20-B2): [ka]

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

[0523] (Examples P1-P7) The alginic acid derivatives shown in the table below (Examples P1) to (Examples P7) were used in the above examples. In accordance with the method shown, the corresponding amino compound (a pharmaceutically acceptable salt thereof, or the same) It is manufactured using alginic acid and (the solvates of these may also be used). [Table 47]

[0524] Physical property data of alginate derivatives [Table 48]

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

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

[0527] [Measurement of the rate of introduction of reactive groups or complementary reactive groups] The rate of introduction of reactive groups or complementary reactive groups is determined by the uronic acid monounit, which is the repeating unit of alginic acid. This refers to the percentage of reactive groups or complementary reactive groups introduced per sugar unit. do. In this embodiment, the rate of introduction of reactive groups or complementary reactive groups (mol%) is: 1 HN The calculation was performed using the integral ratio of MR. Furthermore, the amount of alginate required to calculate the introduction rate was determined using a calibration curve. The amount of reactive groups or complementary reactive groups was measured using the carbazole sulfuric acid method and measured using a calibration curve. It can also be measured using an absorbance measurement method that utilizes [a specific technology / technology].

[0528] [Measurement of molecular weight] The alginate solid to which the reactive group obtained in the example or a complementary reactive group has been introduced is 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 by passing the sample through (company S), it was prepared as a sample for gel filtration. The spectra of each sample were obtained. The measurements were taken using a spectrophotometer DU-800 (Beckman-Coulter) and for each compound. The measurement wavelength in gel filtration was determined. For compounds that do not have a specific absorption wavelength, A differential refractometer was used.

[0529] 200 μL of sample for gel filtration is added to Superose6 Increase 10 / 30 The samples were subjected to a 0 GL column (GE Healthcare Sciences). Gel filtration was performed by chromatography. The apparatus used was an AKTA Explorer 10S, and the developing solvent was 0.15 mol / Using a 10 mmol / L phosphate buffer (pH 7.4) containing L NaCl, the flow rate was measured at room temperature. The procedure was performed under conditions of 0.8 mL / min. The elution profile of each sample was determined. The absorption at the specified wavelength was monitored and the resulting chromatogram was fabricated. The obtained chromatogram was obtained using Unicorn 5. The peak range was determined by analyzing the data using 31 Software (GE Healthcare Sciences).

[0530] The molecular weight of alginic acid to which a reactive group or a complementary reactive group has been introduced is given by Blue Dextra. (molecular weight 2 million Da, SIGMA), thyroglobulin (molecular weight 669,000 Da, GE Healthcare Sciences) Ferritin (molecular weight 440,000 Da, GE Healthcare Sciences Aldolase (molecular weight 158,000 Da, GE Healthcare Sciences), Conal Bumin (molecular weight 75,000 Da, GE Healthcare Sciences), obalbumin (molecular weight 44,000 Da (GE Healthcare Sciences), Ribonuclease A (Molecular weight 13,700) Da (GE Healthcare Sciences) and aprotinin (molecular weight 6500Da, GE Health Using ScareScience Co., Ltd. as the standard, a reactive group or a complementary reactive group was introduced. Gel filtration was performed under the same conditions as for alginate, and the amount of eluate from each component was measured using Unicorn Softwe. This was determined in A. The horizontal axis represents the amount of eluate for each component, and the vertical axis represents the logarithmic value of the molecular weight. We performed linear regression and created a calibration curve. The calibration curve was derived from blue dextran to ferritin. Up to this point, we have created two types, from ferritin to aprotinin.

[0531] Using this calibration curve, the molecular weight (Mi) at elution time i of the previously obtained chromatogram can be calculated. The following data was calculated. Next, the absorbance at elution time i was read and set to Hi. The weight-average molecular weight (Mw) was calculated from the following formula.

[0532]

number

[0533] [Measurement of gel stability] (Measurement of gel stability (1)): Stability in PBS (Ex1-A2), (Ex4-A2), (Ex) obtained in Examples 1, 4, 5 and 19 The alginic acid derivatives 5-A2) and (Ex19-A2) are dissolved in water to a concentration of 1.0%. Dissolve in (1-1), (4-1), (5-1), and (19-1) respectively to obtain alginic acid aqueous solutions. ) was obtained. These were (1-1) and (19-1), (4-1) and (19-1), Mix equal amounts of (5-1) and (19-1) and insert an 18-gauge injection needle into the syringe. Place the solution in the syringe, then attach this syringe to a syringe pump set to a flow rate of 1 mL / min, and the concentration will be... Add to a 30 mmol / L calcium chloride solution dropwise for 30 seconds, then stir for 5 minutes to form alginate. A gel was obtained. This gel was washed once with 10 mL of PBS and then incubated in PBS at 37°C for 10 minutes. After standing, chemical crosslinking was performed to obtain a chemically crosslinked alginate gel. (Ex18-A2) / (Ex The cross-linked alginate gel (beads) prepared in 19-A2) was prepared in the same manner. Add 19.5 mL of PBS to the gel and shake at 37°C, then collect the aqueous solution over time. Then, the same amount of PBS as the recovered amount was replenished. After the test was completed, alginate lyase was added to the test solution. Add 10 μL of Nippon Gene (319-08261) and shake at 37°C for at least 3 hours. The gel was completely broken down, and the aqueous solution was collected. The alginate concentration in the collected aqueous solution was measured. The amount of eluted alginate up to each time point was measured using the bazole sulfuric acid method, and the total alginate concentration at each time point was measured. The value obtained by dividing the total amount of alginate calculated from the alginate concentration at the time and after the end of the test by the total amount of alginate is expressed as a percentage. The expressed value was defined as the collapse rate and used as an indicator of gel stability.

[0534] The results shown in Figure 1 were obtained. The cross-linked alginate gel (beads) remained unchanged even after 96 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 of the formation process, the created (bead) structure maintains its structure over a long period of time. This was suggested. (Ex18-A2) / (Ex19-A2) Bridged Algebra fabricated The nic acid gel (beads) is the control group in this study.

[0535] (Measurement of gel stability (2)): Stability under EDTA The alginic acid aqueous solutions obtained in (Gel Stability Measurement (1)) were (1-1) and (19) respectively. Mix in equal amounts using the following combinations: (-1), (4-1) and (19-1), and (5-1) and (19-1). Then, the solution is placed in a syringe fitted with an 18-gauge needle, and the syringe is set to a flow rate of 1 mL / min. Place it in a syringe pump and immerse it in a 30 mmol / L calcium chloride solution for 30 seconds. The mixture was added dropwise and stirred for 5 minutes to obtain an alginate gel. (Ex18-A2) / (Ex19-A The cross-linked alginate gel (beads) produced in 2) were prepared in the same manner. Wash once with 10 mL of physiological saline, then let stand in physiological saline at 37°C for 10 minutes and place on a chemical rack. Bridge was performed to obtain a chemically crosslinked alginate gel. 19.5 mL of 5 mM ethylene was added to this gel. Add dipotassium diaminetetraacetate dihydrate (EDTA·2K) / physiological saline solution, 37 The aqueous solution was collected over time by shaking at °C, and the same amount as the collected volume was added to 5 mM EDTA·2K. / The physiological saline solution was replenished. After the test was completed, alginate lyase (Nippon Gene) was added to the test solution. Add 10 μL of (319-08261) and shake at 37°C for at least 3 hours to completely break down the gel. The plant was destroyed, and the aqueous solution was recovered. The alginate concentration in the recovered aqueous solution was measured using the carbazole sulfuric acid method. The amount of eluted alginate up to each time point was measured, along with the total alginate concentration at all time points and the end of the test. The disintegration rate is calculated by dividing the amount of alginate (calculated from the subsequent alginate concentration) by the total amount of alginate, and expressing this value as a percentage. This was used as an indicator of gel stability.

[0536] The results are shown in Figure 2. The cross-linked alginate gel (beads) after 24 hours The decay rate was less than 2%. In other words, crosslinking was formed by the Huisgen reaction. The resulting structure is in a solution without calcium ions (physiologically beneficial to living organisms). It was suggested that the structure is maintained even under conditions (below the concentration). (Ex18-A2) / (E The cross-linked alginate gel (beads) prepared in x19-A2) was used as a control in this study. be.

[0537] (Measurement of gel stability (3)): Stability in PBS (Ex3-A2), (Ex 5-A2), (Ex6-A2), (Ex9a-A2), (Ex10-A2), (Ex12-A 2) Dissolve each of the alginic acid derivatives (Ex18-A2) in water to a concentration of 1.0%. The alginic acid solutions (3-1), (5-1), (6-1), (9-1), ( We obtained (10-1), (12-1), and (18-1). These were (6-1) and (9 -1), (3-1) and (10-1), (5-1) and (10-1), (18-1) and (12 Mix equal volumes of the ingredients in (-1), place in a syringe fitted with an 18-gauge needle, and add to this syringe. The syringe tube is placed in a syringe pump set to a flow rate of 1 mL / min, and the concentration is 30 mmol / L. The mixture was added dropwise to a calcium chloride solution for 30 seconds and stirred for 5 minutes to obtain an alginate gel. Wash the strips once with 10 mL of PBS, then let them stand in PBS at 37°C for 10 minutes to allow chemical crosslinking to occur. This process yielded a chemically crosslinked alginate gel. It was prepared using (Ex18-A2) / (Ex19-A2). Cross-linked alginate gel (beads) was prepared in the same manner. 19.5 Add mL of PBS, shake at 37°C, and collect the aqueous solution over time. The same amount as the collected volume is then added. The PBS was replenished. After the test was completed, alginate lyase (Nippon Gene, 3) was added to the test solution. Add 10 μL of (19-08261) and shake at 37°C for at least 3 hours to completely disintegrate the gel. The solution was then collected. The alginic acid concentration in the collected solution was determined by the carbazole sulfuric acid method. The amount of eluted alginate up to each time point was measured, and the total alginate concentration at all time points was recorded, as well as the amount of alginate eluted up to the end of the test. The disintegration rate is calculated by dividing the alginate concentration by the total amount of alginate, expressed as a percentage. This was used as an indicator of gel stability.

[0538] The results shown in Figure 3 were obtained. The cross-linked alginate gel (beads) remained unchanged even after 96 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 of the formation process, the created (bead) structure maintains its structure over a long period of time. This was suggested. Furthermore, the crosslinked A fabricated with (Ex18-A2) / (Ex19-A2) Luginate gel (beads) is the control for this study.

[0539] (Measurement of gel stability (4)): Stability under EDTA The alginic acid aqueous solutions obtained in (Gel Stability Measurement (3)) were divided into (6-1) and (9- 1), (3-1) and (10-1), (5-1) and (10-1), (18-1) and (12- Mix equal volumes of the ingredients in (1), place them in a syringe fitted with an 18-gauge needle, and administer the injection. The cylinder is placed in a syringe pump set to a flow rate of 1 mL / min, and the concentration is 30 mmol / L. The mixture was added dropwise to a calcium chloride solution for 30 seconds and stirred for 5 minutes to obtain an alginate gel. Wash once with 10 mL of physiological saline, then stand in physiological saline at 37°C for 10 minutes for chemical analysis. Crosslinking was performed to obtain a chemically crosslinked alginate gel. (Ex18-A2) / (Ex19-A2) The cross-linked alginate gel (beads) produced was prepared in the same manner as these. 19 0.5 mL of 5 mM ethylenediaminetetraacetate dipotassium salt dihydrate (EDTA·2K) Add physiological saline solution, shake at 37°C, and collect the aqueous solution over time. The same amount as the collected amount is then used. The test solution was supplemented with 5 mM EDTA·2K / physiological saline. After the test, the test solution was added to the alginate solution. Add 10 μL of acid lyase (Nippon Gene, 319-08261) and incubate at 37°C for 3 hours. The gel was completely broken down by shaking, and the aqueous solution was collected. Alginic acid in the collected aqueous solution The concentration was measured using the carbazole sulfuric acid method, and the amount of eluted alginate up to each time point was calculated as the total amount of alginate at each time point. The value obtained by dividing the alginate concentration by the total amount of alginate calculated from the alginate concentration after the end of the test. The value expressed as a percentage was defined as the disintegration rate and used as an indicator of gel stability.

[0540] The results are shown in Figure 4. The cross-linked alginate gel (beads) after 24 hours The decay rate was 4% or less. In other words, crosslinking was formed by the Huisgen reaction. The resulting structure is in a solution without calcium ions (physiologically beneficial to living organisms). The structure was suggested to be maintained even under conditions (below the concentration limit). Ex18-A2 / Ex19 -The cross-linked alginate gel (beads) prepared in A2 is the control for this study.

[0541] (Measurement of gel stability (5)): Stability in PBS (Ex4-A2), (EX9a-A2) obtained in Examples 4, 9, 16, 18 and 20 Alginate derivatives of (Ex16-A2), (Ex18-A2), and (Ex20-B2) Dissolve the body in water to a concentration of 1.0% to make alginic acid solution (4-1), ( We obtained 9-1), (16-1), (18-1), and (20-1). These were divided into (4 The combinations are (-1) and (20-1), (18-1) and (9-1), and (18-1) and (16-1). Combine equal volumes and pour into a syringe fitted with an 18-gauge needle. Then, inflate the syringe to a flow rate of 1 Install the syringe pump set to mL / min and use calcium chloride at a concentration of 30 mmol / L. The solution was added dropwise for 30 seconds and stirred for 5 minutes to obtain an alginate gel. This gel was then transferred to 10 ml. Wash once with PBS, then stand in PBS at 37°C for 10 minutes to perform chemical crosslinking, and then chemical crosslinking A crosslinked alginate gel was obtained. The crosslinked alginate gel was prepared using (Ex18-A2) / (Ex19-A2). Luginate gel (beads) was prepared in the same manner. 19.5 mL of PBS was added to this gel. Add the solution, shake at 37°C, collect the aqueous solution over time, and supplement with the same amount of PBS as the collected amount. Filled. After the test, add alginate lyase (Nippon Gene, 319-082) to the test solution. Add 10 μL of 61) and shake at 37°C for at least 3 hours to completely disintegrate the gel, then prepare the aqueous solution. It was recovered. The alginic acid concentration in the recovered aqueous solution was measured by the carbazole sulfuric acid method, and at each time The amount of alginate eluted up to the point, the alginate concentration at all time points, and the alginate concentration after the end of the test. The disintegration rate is defined as the value obtained by dividing the total amount of alginate calculated from the degree by the percentage, and the gel stability is also defined. This was used as an indicator.

[0542] The results are shown in Figure 5. The cross-linked alginic acid produced by the above method remained unchanged even after 96 hours. The decay rate was less than approximately 12%. In other words, chemical crosslinking was formed by the Huisgen reaction. This suggests that the resulting (bead) structure will maintain its structure. The cross-linked alginate gel (beads) produced using Ex18-A2 / Ex19-A2 is this This is the control group for the experiment.

[0543] (Measurement of gel stability (6)): Stability under EDTA The alginic acid aqueous solutions obtained in (Gel Stability Measurement (5)) were (4-1) and (20 -1), (18-1) and (9-1), and (18-1) and (16-1) are mixed in equal amounts. Mix the contents, place them in a syringe fitted with an 18-gauge needle, and set the syringe to a flow rate of 1 mL / min. Install in a designated syringe pump and add 30 mmol / L of calcium chloride solution. The mixture was added dropwise for 5 seconds and stirred for 5 minutes to obtain an alginate gel. This gel was then mixed with 10 mL of physiological saline. Wash once, then stand in physiological saline at 37°C for 10 minutes to perform chemical crosslinking, and chemical crosslinked aluminum A ginate gel was obtained. Cross-linked algin prepared using (Ex18-A2) / (Ex19-A2). Acid gels (beads) were prepared in the same manner. 19.5 mL of 5 mM ethyl acetate was added to this gel. Add dipotassium lendiaminetetraacetate dihydrate (EDTA·2K) / physiological saline solution, 3 The aqueous solution was collected over time by shaking at 7°C, and the same amount as the collected volume was added to 5 mM EDTA·2. K / physiological saline was added. After the test was completed, alginate lyase (Nippon G) was added to the test solution. Add 10 μL of (319-08261) and shake at 37°C for at least 3 hours to completely dissolve the gel. The substance was disintegrated, and the aqueous solution was recovered. The alginate concentration in the recovered aqueous solution was measured using the carbazole sulfuric acid method. The amount of eluted alginate up to each time point was measured, and the total alginate concentration at all time points and the final test result were determined. The value obtained by dividing the alginate concentration after processing by the total amount of alginate, expressed as a percentage, is then broken down. This was used as a ratio and as an indicator of gel stability.

[0544] The results shown in Figure 6 were obtained. The cross-linked alginate gel (beads) after 24 hours The decay rate was 18% or less. In other words, there was no cross-linking by the Huisgen reaction. The resulting structure is a calcium ion-free solution (a living organism). It was suggested that the structure is maintained even under conditions below the physicochemical concentration. Ex18-A2 / Ex The cross-linked alginate gel (beads) prepared in 19-A2 is the control for this study. .

[0545] [Measurement of gel permeability] (Measurement of gel permeability (1)) (Ex1-A2), (Ex3-A2), ( obtained in Examples 1, 3, 4, 5 and 18) The alginic acid derivatives Ex4-A2), (Ex5-A2), and (Ex18-A2) were each concentrated Prepare an alginic acid aqueous solution by dissolving it in water to a concentration of 2.0%, and then add to this alginic acid aqueous solution Fluorescein isothiocyanate with a molecular weight of 150,000, prepared at 1 mg / mL in 2 / 5 volume. Add todextran (Sigma-Aldrich, FD150S) and 3 / 5 volume of water. 0.2 mg / mL Fluorescein isothiocyanate-dextran containing 1.0% We obtained ginic acid aqueous solutions (1-2), (3-2), (4-2), (5-2), and (18-2).

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

[0547] These are (1-2) and (19-1), (4-2) and (19-1), and (5-2 The combinations are (19-1), (3-2) and (10-1), and (18-2) and (12-1). Mix equal volumes, then add 40 mL of a 30 mmol / L calcium chloride solution. The gel was stirred for 5 minutes to obtain an alginate gel. This gel was washed once with 10 mL of physiological saline. Chemical crosslinking is performed by standing the mixture in physiological saline at 37°C for 10 minutes, and then fluorescein isothiocyanate is used. An anate-dextran-encapsulated chemically cross-linked alginate gel was obtained. (Ex18-A2) / (E Fluorescein isothiocyanate-dextran encapsulation chemistry prepared using x19-A2) A cross-linked alginate gel was prepared in the same manner. 19.5 mL of physiological saline was added to this gel. Add the solution, shake at 37°C, and collect the aqueous solution over time. The same amount of physiological saline solution is then collected as the amount collected. The test solution was supplemented with alginate lyase (Nippon Gene, 319-0). Add 10 μL of 8261) and shake at 37°C for more than 3 hours to completely disintegrate the gel, then add water. The solution was collected. The dextran concentration in the collected aqueous solution was determined by fluorescence quantification (excitation light: 485n The amount of dextran up to each time point was measured by (m, fluorescence: 535nm), and the total amount after the end of the test was measured. The transmittance was calculated by dividing the value by the amount of quistran and expressing it as a percentage.

[0548] The results shown in Figure 7 were obtained. The transmittance after 24 hours was in the range of 25% to 40%. Fluorescein isothiocyanate prepared using (Ex18-A2) / (Ex19-A2) Todextran-encapsulated chemically cross-linked alginate gel served as the control in this study.

[0549] (Measurement of gel permeability (2)) (Ex4-A2), (Ex5-A2), (Ex) obtained in Examples 4, 5, 6 and 18 The alginic acid derivatives 6-A2) and (Ex18-A2) are dissolved in water to a concentration of 2.0%. Prepare an alginic acid aqueous solution by dissolving it in [a solution], and add 1 mg / 2 / 5 volume to this alginic acid aqueous solution. Fluorescein isothiocyanate-dextran (SIG) prepared in mL Add Maardrich (FD150S) and 3 / 5 volume of water, and add 0.2 mg / mL full 1.0% alginic acid aqueous solution containing olecein isothiocyanate-dextran (4-2) They obtained (5-2), (6-2), and (18-2).

[0550] Furthermore, the (EX9a-A2), (Ex10- Each of the alginic acid derivatives A2), (Ex16-A2), and (Ex20-B2) was used at a concentration of 1. Dissolve in water to make 0% to obtain alginic acid aqueous solutions (9-1), (10-1), and (1 They obtained 6-1) and (20-1).

[0551] These are (4-2) and (20-1), (5-2) and (10-1), and (6-2) respectively. ) and (9-1), (18-2) and (9-1), (18-2) and (16-1) combinations Mix in equal volumes, add 40 mL of calcium chloride solution with a concentration of 30 mmol / L, and 5 The mixture was stirred for minutes to obtain an alginate gel. This gel was washed once with 10 mL of physiological saline. Chemical crosslinking was performed by standing the product in physiological saline at 37°C for 10 minutes, resulting in fluorescein isothiocyanate. A chemically cross-linked alginate gel containing nato-dextran was obtained. (Ex18-A2) / (Ex Fluorescein isothiocyanate-dextran encapsulation chemical frame prepared in 19-A2) Bridged alginate gel was prepared in the same manner. 19.5 mL of physiological saline was added to this gel. Add the solution, shake at 37°C, collect the aqueous solution over time, and add the same amount of physiological saline solution as the collected amount. Replenished. After the test was completed, alginate lyase (Nippon Gene, 319-08) was added to the test solution. Add 10 μL of 261) and shake at 37°C for more than 3 hours to completely disintegrate the gel. The liquid was collected. The dextran concentration in the collected aqueous solution was determined by fluorescence quantification (excitation light: 485 nm). The amount of dextran up to each time point was measured by fluorescence (535nm), and the total amount of dextran after the end of the test was measured. The transmittance was defined as the value obtained by dividing the amount by the strand amount and expressing it as a percentage.

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

[0553] [Evaluation of biocompatibility of cross-linked alginate derivatives (gels)] (EX4-A2), obtained in Examples 4, 5, 12, 16, 18, 19 and 20, EX5-A2), (EX12-A2), (EX16-A2), (EX18-A2), (E The alginic acid derivatives X19-A2 and (EX20-B2) are dissolved in water to form reactive groups. This was used as an introduction alginate solution. This was then used with a Minisart High Flow (Sartorius, 16532G) filter. After sterilization by filtration (UK), a 1.0% reactive group-modified alginate / physiological saline solution was prepared. . Cell concentration 5×10 3 Seeds were seeded into a 96-well plate to achieve a cell / well ratio. HeLa cells cultured for 1 day were then treated with 1.0% reactive group-modified alginate / physiological salt. The aqueous solutions were (EX18-A2) and (EX19-A2), (Ex5-A2) and (Ex19-A 2) (Ex4-A2) and (Ex20-B2), (Ex18-A2) and (Ex12-A2) ) or (Ex16-A2) combination is added to achieve a final concentration of 0.1%, and cultured for 1 day. After nourishment, ATP activity was used as an indicator of cytotoxicity in CellTiter-Glo Lumines. cent Cell Viability Assay (Promega, G7571) I evaluated it using that method.

[0554] The results shown in Figure 9 were obtained. In all cross-linked alginate gels evaluated by the above method, AT The confirmation of P activity suggests that the cross-linked alginate gel is non-cytotoxic. Furthermore, alginate structures (beads) with chemical crosslinks formed by the Huisgen reaction are biologically active. It was suggested that it was compatible.

Claims

1. An amide bond and a divalent linker (-L) are attached to any one or more carboxyl groups of alginic acid. ２ The following formula (II) is formed by introducing an azide group via (-): 【Chemistry 1】 [In formula (II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L ２ - is shown in the table below: Table 1-1 Table 1-2 An alginate derivative represented by [a linker selected from the group consisting of the substructure formulas described in [the formulas excluding the areas outside the dashed lines at both ends]].

2. -L ２ - However, see the table below: Table 2 The alginic acid derivative according to claim 1, which is a linker selected from the group consisting of the substructure formulas described in [excluding the area outside the dashed lines at both ends of each formula].

3. N ３ -L ２ -NH ２ Group (-L ２ The alginic acid derivative according to claim 1, wherein the introduction rate of (where - is the same as the definition in claim 1) is 0.1% to 30%.

4. The alginate derivative according to claim 1, wherein the weight-average molecular weight of the alginate derivative, as measured by gel filtration chromatography, is 100,000 Da to 3,000,000 Da.

5. The alginic acid derivative according to claim 1, which is biocompatible.

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