Novel crosslinked alginate structure

Abstract

Provided are a novel crosslinked alginic acid, a crosslinked alginic acid structure, etc., by performing a crosslinking reaction using alginic acid derivatives represented by formula (I) and formula (II). As a result, a novel crosslinked alginic acid, crosslinked alginic acid structure, etc., are provided.

Description

Technical Field

[0001] The present invention relates to a novel crosslinked alginic acid, an alginic acid derivative for forming the crosslinked alginic acid, and the like.

Background Art

[0002] Alginic acid is a biodegradable polysaccharide extracted from brown algae such as Lessonia, Macrocystis, Laminaria, Ascophyllum, Darbilla, Kazime, Arame, and Kombu, and is a polymer in which two types of uronic acids, D-mannuronic acid (M) and L-guluronic acid (G), are polymerized linearly. More specifically, it is a block copolymer in which a homopolymer fraction of D-mannuronic acid (MM fraction), a homopolymer fraction of L-guluronic acid (GG fraction), and a fraction in which D-mannuronic acid and L-guluronic acid are randomly arranged (M / G fraction) are arbitrarily combined. Alginic acid is used in a wide range of fields such as food, medicine, cosmetics, fibers, and paper.

[0003] Alkaline metal salts of alginic acid monovalent salts (for example, sodium alginate, etc.) are water-soluble, but alkaline earth metal salts of alginic acid divalent salts (for example, calcium alginate, etc.) are crosslinked by metal ions and gelled (insolubilized). That is, by adding an aqueous solution containing a divalent metal ion (for example, calcium ion, barium ion, etc.) to an aqueous solution of alginic acid, an alginic acid gel (ion-crosslinked alginic acid) having a three-dimensional network structure formed through ionic crosslinking can be obtained. Attempts have been made to modify or mold it into a suitable form for various applications using this property (Patent Documents 1 to 3). On the other hand, it is known that the alginic acid gel easily breaks down into alginic acid by capturing divalent metal ions that form ionic crosslinks in the gel in the presence of a chelating agent such as ethylenediaminetetraacetic acid (EDTA).

[0004] Polysaccharide derivatives that describe maleimide groups and / or thiol groups as reactive groups for forming chemical crosslinks are known (Patent Documents 4 to 10, Non-Patent Documents 1 to 2).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Patent Document 10

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] In the above situation, there has been a demand for a novel crosslinked alginic acid, an alginic acid derivative for forming the crosslinked alginic acid, a crosslinked alginic acid structure, and methods for producing them. [Means for Solving the Problems]

[0008] As a result of intensive studies to solve the above problems, the present inventors have found that when a crosslinked alginic acid and a crosslinked alginic acid structure (beads / dye-containing beads) are formed using an alginic acid derivative having a predetermined crosslinking group (the alginic acid derivatives represented by the following formulas (I) and (II) described later), the beads have high stability and are a gel having substance permeability, and have thus completed the present invention. That is, the present invention is as follows.

[0009] Here, an alginic acid derivative represented by formula (I) shown in the following aspect, an alginic acid derivative represented by formula (II), a novel crosslinked alginic acid obtained by performing a Michael addition reaction using these, and a gel obtained by dropping the alginic acid derivative into a solution containing a divalent metal ion and subjecting the resulting gel to a Michael addition reaction are provided, as well as a method for producing the alginic acid derivative, the crosslinked alginic acid, and the crosslinked alginic acid structure. That is, exemplary aspects can be as follows in [1] to

[27] below.

[0010] [1] The following formula (I): [Chemical formula] [In formula (I), -L 1 -, -NHCO-, and (ALG) are the same as the definitions in the first aspect described later] and an alginic acid derivative represented by the following formula (II): [Chemical formula] [In formula (II), -L 2 -, -NHCO-, and (ALG) are the same as the definitions in the first aspect described later] and a crosslinked alginic acid obtained by subjecting the alginic acid derivative to a crosslinking reaction.

[0011] [2] The following formula (I): [Chemical formula] [In formula (I), -L 1 -, -NHCO-, and (ALG) are the same as defined in the first or second aspect described below] An alginic acid derivative represented by

[0012] [3] The following formula (BR-1): [Chemical formula] [In formula (BR-1), -L 1 - is the same as defined in the third aspect described below] The introduction rate of the reactive group represented by is 0.5% to 30% or 1% to 30%, and the alginic acid derivative represented by formula (I) described in [1] or [2] above.

[0013] [4] The weight average molecular weight measured by the gel filtration chromatography method of the alginic acid derivative is 100,000 Da to 3,000,000 Da, and the alginic acid derivative represented by formula (I) described in [1] or [2] above.

[0014] [5] The following formula (II-P): [Chemical formula] [In formula (II-P), P 1 , -L 2 -, -NHCO-, and (ALG) are the same as defined in the fifth aspect described below] An alginic acid derivative represented by

[0015] [6-1] The following formula (BR-2): [Chemical formula] [In formula (BR-2), -L 2 - is the same as defined in the 6-1 aspect described below] The introduction rate of the reactive group represented by is 1.0% to 30%, and the alginic acid derivative represented by formula (II) described in [1] above.

[0016] [6-2] The following formula (BR-2-P): [Chemical formula] [In formula (BR-2-P), P 1 , -L 2 - is the same as the definition in the 6-2nd aspect described later], and the introduction rate of the reactive group represented by is 1.0% to 30%. The alginic acid derivative represented by the formula (II-P) described in the above [5].

[0017] [7-1] The alginic acid derivative represented by the formula (II) described in the above [1], wherein the weight average molecular weight measured by the gel filtration chromatography method of the alginic acid derivative is 100,000 Da to 3,000,000 Da.

[0018] [7-2] The alginic acid derivative represented by the formula (II-P) described in the above [5], wherein the weight average molecular weight measured by the gel filtration chromatography method of the alginic acid derivative is 100,000 Da to 3,000,000 Da.

[0019] [8] In the crosslinked alginic acid of the above [1], the chemical crosslinking is represented by the following formula (LK-1): [Chemical formula] [In formula (LK-1), the -CONH- and -NHCO- at both ends, -L 1 -, and -L 2 - is the same as the definition in the 8th aspect described later], and the crosslinked alginic acid described in the above [1] has such a structure.

[0020] [8-1] The following formula (CAL-1): [Chemical formula] [In formula (CAL-1), (ALG), -CONH-, -NHCO-, -L 1 -, and -L 2 - is the same as the definition in the above [1]], and the crosslinked alginic acid is represented by this formula.

[0021] [8-2] The following formula (CAL-1) obtained by the crosslinking reaction described in the above [1]: [Chemical formula] [In formula (CAL-1), (ALG), -CONH-, -NHCO-, -L 1 -, and -L 2 - are the same as defined in the above [1]], a crosslinked alginic acid represented by

[0022] [9] A method for producing the crosslinked alginic acid according to the above [1], comprising adding a solution of an alginic acid derivative represented by formula (I) to a solution of an alginic acid derivative represented by formula (II) and performing a crosslinking reaction.

[0023]

[10] A method for producing the crosslinked alginic acid according to the above [1], comprising adding a solution of an alginic acid derivative represented by formula (II) to a solution of an alginic acid derivative represented by formula (I) and performing a crosslinking reaction.

[0024]

[11] The chemical crosslinking formed by performing a Michael addition reaction using an alginic acid derivative represented by formula (I) and an alginic acid derivative represented by formula (II) is the following formula (LK-1): [Chemical formula] [In formula (LK-1), -CONH- and -NHCO- at both ends, -L 1 -, and -L 2 - are the same as defined in the 11th aspect described below], a method for producing the crosslinked alginic acid according to the above [1] having the structure.

[0025]

[12] A crosslinked alginic acid structure comprising an ionic crosslink formed partially by a divalent metal ion as a crosslink and a chemical crosslink formed by a Michael addition reaction, obtained by dropping a solution of an alginic acid derivative represented by formula (I) into a solution containing a divalent metal ion to obtain a gel and performing a crosslinking reaction in a solution of an alginic acid derivative represented by formula (II).

[0026] A crosslinked alginate structure obtained by subjecting a gel obtained by dropping a solution of an alginate derivative represented by the formula (II) into a solution containing a divalent metal ion to a crosslinking reaction in a solution of an alginate derivative represented by the formula (I), the crosslinked alginate structure including an ionic crosslink formed partially by the divalent metal ion as a crosslink and a chemical crosslink formed by a Michael addition reaction.

[0027] 〔14〕A crosslinked alginate structure obtained by dropping a solution of a composition containing an alginate derivative represented by the formula (I) and an alginate derivative represented by the formula (II) into a solution containing a divalent metal ion, the crosslinked alginate structure including an ionic crosslink formed partially by the divalent metal ion as a crosslink and a chemical crosslink formed by a Michael addition reaction.

[0028] 〔15〕The chemical crosslink formed by performing a Michael addition reaction using an alginate derivative represented by the formula (I) and an alginate derivative represented by the formula (II) is represented by the following formula (LK-1):

Chemical formula

[0029] 〔16〕The crosslinked alginate structure according to any one of the above items 〔12〕 to 〔15〕, which is a fibrous structure, fiber, bead, gel, or substantially spherical gel.

[0030] 〔17〕A medical material containing the crosslinked alginate structure according to any one of the above items 〔12〕 to 〔16〕.

[0031] 〔18〕The medical material according to the above item 〔17〕, which is a fibrous structure, fiber, bead, gel, or substantially spherical gel.

[0032] A composition containing an alginic acid derivative represented by formula (I) and an alginic acid derivative represented by formula (II). 〔19-1〕A composition containing an alginic acid derivative represented by formula (I) and an alginic acid derivative represented by formula (II-P). 〔19-2〕A composition containing an alginic acid derivative selected from an alginic acid derivative represented by formula (I), an alginic acid derivative represented by formula (II), or an alginic acid derivative represented by formula (II-P).

[0033] 〔20〕A method for producing a crosslinked alginic acid structure, which includes dropping a solution of a composition containing an alginic acid derivative represented by formula (I) and an alginic acid derivative represented by formula (II) into a solution containing a divalent metal ion.

[0034] 〔21〕A method for producing a crosslinked alginic acid structure, which includes subjecting a gel obtained by dropping a solution of an alginic acid derivative represented by formula (I) into a solution containing a divalent metal ion to a crosslinking reaction in a solution of an alginic acid derivative represented by formula (II), thereby obtaining a crosslinked alginic acid structure including an ionic crosslink partially formed by a divalent metal ion as a crosslink and a chemical crosslink formed by a Michael addition reaction.

[0035] 〔22〕A method for producing a crosslinked alginic acid structure, which includes subjecting a gel obtained by dropping a solution of an alginic acid derivative represented by formula (II) into a solution containing a divalent metal ion to a crosslinking reaction in a solution of an alginic acid derivative represented by formula (I), thereby obtaining a crosslinked alginic acid structure including an ionic crosslink partially formed by a divalent metal ion as a crosslink and a chemical crosslink formed by a Michael addition reaction.

[0036] 〔23〕The chemical crosslink formed by performing a Michael addition reaction using an alginic acid derivative represented by formula (I) and an alginic acid derivative represented by formula (II) is represented by the following formula (LK-1):

Chemical formula

[20] to

[22] .

[0037]

[24] A crosslinked alginic acid structure having a retention property of the content, obtained by chemically crosslinking an alginic acid derivative represented by formula (I) and an alginic acid derivative represented by formula (II) by ionic crosslinking with a divalent metal ion and Michael addition reaction.

[0038]

[25] The crosslinked alginic acid according to the above [1] or [8] having biocompatibility, or the crosslinked alginic acid structure according to any one of the above

[12] to

[16] and

[24] .

[0039] [26a] The alginic acid derivative represented by formula (I) according to the above [2] and the alginic acid derivative represented by formula (II-P) according to the above [5], which have biocompatibility.

[0040]

[27] The following formula (AM-2):

Chemical formula

Advantages of the Invention

[0041] The present invention provides a novel crosslinked alginic acid in which chemical crosslinking is formed, a crosslinked alginic acid structure, a novel alginic acid derivative that can be used for the formation of the crosslinked alginic acid and the crosslinked alginic acid structure, and an intermediate (amino compound) for producing the derivative, etc. Preferably, the crosslinked alginic acid and the alginic acid derivative as a raw material are expected to be safe for living organisms. In addition, since the crosslinking reaction by the alginic acid derivative of the present invention is completed under mild conditions by Michael addition reaction, it can be used safely and easily. Crosslinked alginic acids in some embodiments are those chemically crosslinked by Michael addition reaction. The crosslinked alginic acid of the present invention can be used in combination with chemical crosslinking and other crosslinking methods, for example, crosslinking using divalent metal ions using calcium ions. By adjusting the reaction conditions, its stability is improved compared to non-crosslinked alginic acid (for example, monosodium salt of alginic acid) or non-chemically crosslinked alginic acid (for example, crosslinked alginic acid crosslinked with calcium ions). In addition, in one embodiment, the gel physical properties of the crosslinked product can be adjusted, and the substance permeability can also be adjusted. The present invention has at least one or more of these effects.

Brief Description of the Drawings

[0042]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Mode for Carrying Out the Invention

[0043] [Specific Embodiment] More specifically, the following embodiments [1] to

[24] may be included.

[0044] [1] The first embodiment is as follows. Crosslinked alginic acid obtained by subjecting an alginic acid derivative represented by the following formula (I) and an alginic acid derivative represented by the following formula (II) to a crosslinking reaction.

[0045] [Alginic Acid Derivative Represented by Formula (I)] The following formula (I):

Chemical Formula

Chemical Formula

[0046] [Alginic acid derivative represented by formula (II)] The following formula (II): [Chemical formula] (In formula (II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 2 - represents the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0047] In the present specification, in the linker (-L 1 -, -L 2 -), in the symbol representing an integer in the structural formula, when the same symbol is used multiple times, each may be the same integer or different integers.

[0048] In the present specification, in formulas (I), (II), (II-P) and (CAL-1), for the amide bond (-NH-CO- or -CO-NH-) bonded to (ALG) in the formula, -CO- is a carbonyl group derived from the carboxy of alginic acid.

[0049] Hereinafter, each group in formulas (I) and (II) in the above aspect [1] will be specifically described. In the description of the compound, for example, "C 1~6 " indicates that the number of constituent carbon atoms is from 1 to 6, and unless otherwise specified, represents the total number of carbon atoms of a linear, branched or cyclic group. For a group containing a chain-like group and a cyclic group, it means "the total number of carbon atoms of the chain and the ring".

[0050] In addition, unless otherwise specified, each group in the sub-aspects of aspect [1] shall also have the same definition as each group in aspect [1].

[0051] In this specification, unless otherwise specified, "C 6~10 aryl group" includes, for example, groups such as phenyl, 1-naphthyl, 2-naphthyl, indanyl, indenyl, or 1,2,3,4-tetrahydronaphthyl, etc.

[0052] In this specification, unless otherwise specified, "heterocyclic group" includes, for example, "heteroaryl group" and "non-aromatic heterocyclic group", etc.

[0053] In this specification, unless otherwise specified, the "heteroaryl group" means a monocyclic, polycyclic or condensed cyclic (wherein, in the case of polycyclic or condensed cyclic, it may be partially hydrogenated) 5- to 14-membered, preferably 5- to 8-membered, more preferably 5- to 7-membered heteroaryl ring containing 1 to 5, preferably 1 to 3 heteroatoms selected from the group consisting of a nitrogen atom, a sulfur atom, and an oxygen atom.

[0054] In this specification, unless otherwise specified, examples of the "heteroaryl group" include, for example, "monocyclic heteroaryl group", "condensed heteroaryl group", "partially hydrogenated condensed heteroaryl group", etc.

[0055] In this specification, unless otherwise specified, the "monocyclic heteroaryl group" is a monocyclic one of the above-described heteroaryl rings, and preferably has 5 to 8, more preferably 5 to 6 ring members (a "5- to 6-membered heteroaryl group").

[0056] In this specification, unless otherwise specified, the "5- to 6-membered heteroaryl group" is a 5- to 6-membered heteroaryl ring containing 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom, and the "5- to 6-membered heteroaryl group" means, unless otherwise specified, a monovalent group formed by removing any hydrogen atom from the heteroaryl ring.

[0057] In this specification, unless otherwise specified, examples of the "5- or 6-membered heteroaryl group" include groups such as pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, furazanyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 2H-1,2,3-thiadiazinyl, 4H-1,2,4-thiadiazinyl, 6H-1,3,4-thiadiazinyl, pyridazin-3(2H)-one, pyrimidin-2(1H)-one, pyrazin-2(1H)-one, or pyridin-2(1H)-one.

[0058] In this specification, unless otherwise specified, the "5-membered heteroaryl group" is a 5-membered heteroaryl ring containing 1 to 4 heteroatoms selected from nitrogen atoms, sulfur atoms, and oxygen atoms, and the "5-membered heteroaryl group" means, unless otherwise specified, a monovalent group formed by removing any hydrogen atom from the heteroaryl ring. Examples of such groups include pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, furazanyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, or tetrazolyl.

[0059] In this specification, unless otherwise specified, the "6-membered heteroaryl group" refers to a 6-membered heteroaryl ring containing 1 to 4 heteroatoms selected from nitrogen atoms, sulfur atoms, and oxygen atoms. Unless otherwise specified, the "6-membered heteroaryl group" means a monovalent group formed by removing any hydrogen atom from the heteroaryl ring. Examples include groups such as pyridyl (pyridinyl), pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 2H-1,2,3-thiadiazinyl, 4H-1,2,4-thiadiazinyl, 6H-1,3,4-thiadiazinyl, pyridazin-3(2H)-one, pyrimidin-2(1H)-one, pyrazin-2(1H)-one, or pyridin-2(1H)-one.

[0060] In this specification, unless otherwise specified, in the "5- to 6-membered heteroaryl C 1~6 alkyl group", the "5- to 6-membered heteroaryl group" means a group in which the "C 1~6 alkyl group" is substituted. Examples include groups such as pyrrolylmethyl, furylmethyl, thienylmethyl, imidazolylmethyl, pyrazolylmethyl, oxazolylmethyl, isoxazolylmethyl, thiazolylmethyl, isothiazolylmethyl, 1,2,3-triazolylmethyl, 1,2,4-triazolylmethyl, 1,2,3-oxadiazolylmethyl, 1,2,4-oxadiazolylmethyl, 1,3,4-oxadiazolylmethyl, furazanylmethyl, 1,2,3-thiadiazolylmethyl, 1,2,4-thiadiazolylmethyl, 1,3,4-thiadiazolylmethyl, tetrazolylmethyl, pyridylmethyl, pyridazinylmethyl, pyrimidinylmethyl, pyrazinylmethyl, 1,2,3-triazinylmethyl, 1,2,4-triazinylmethyl, 1,3,5-triazinylmethyl, 2H-1,2,3-thiadiazinylmethyl, 4H-1,2,4-thiadiazinylmethyl, or 6H-1,3,4-thiadiazinylmethyl.

[0061] The term "partially hydrogenated fused heterocyclic aryl group" means a monovalent group formed by removing any hydrogen atom from a partially hydrogenated fused ring in a fused ring formed by the condensation of a "heterocyclic group" and an "aryl group", or a "heterocyclic group" and a "heteroaryl group". The said any hydrogen atom may be removed from a hydrogen atom in any ring part of the "heterocyclic group", "aryl group" and "heteroaryl group" within the fused ring, or a hydrogen atom in the hydrogenated ring part. For example, if quinoline is partially hydrogenated to tetrahydroquinolyl, examples include 5,6,7,8-tetrahydroquinolyl or 1,2,3,4-tetrahydroquinolyl. These groups are exemplified by, for example, -2-yl, -3-yl, -4-yl, -5-yl, -6-yl, -7-yl, -8-yl, etc. for 5,6,7,8-tetrahydroquinolyl, and, for example, -1-yl, -2-yl, -3-yl, -4-yl, -5-yl, -6-yl, -7-yl, -8-yl, etc. for 1,2,3,4-tetrahydroquinolyl, depending on the position where any hydrogen atom is removed.

[0062] As the "partially hydrogenated condensed heterocyclic aryl group", those having 8 to 12 ring members are preferred. That is, as the "partially hydrogenated 8- to 12-membered condensed heterocyclic aryl group", for example, indolinyl, 2,3-dihydrobenzofuranyl, 4,5,6,7-tetrahydro-benzofuranyl, 2,3-dihydrobenzo[d]oxazolyl, 2,3-dihydrobenzo[d]thiazolyl, 4,5,6,7-tetrahydrobenzo[d]oxazolyl, 4,5,6,7-tetrahydrobenzo[d]thiazolyl, 4,5,6,7-tetrahydro-1H-benzo[d]imidazolyl, benzod[1,3]dioxonyl, 2,3-dihydrobenzo[b][1,4]dioxinyl, 2,3-dihydrobenzo[b][1,4]oxathiinyl, 3,4-dihydro-2H-benzo[b][1,4]oxazinyl, 3,4-dihydro-2H-benzo[b][1,4]thiazinyl, 1,2,3,4-tetrahydroquinoxalinyl, 3,4-dihydro-2H-benzo[b][1,4]dioxepin, 2,3,4,5-tetrahydrobenzo[b][1,4]dioxocinyl, N-acetyl-2,3-dihydrobenzo[d]oxazolyl, N-acetyl-3,4-dihydro-2H-benzo[b][1,4]oxazinyl, N-acetyl-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepinyl, N-acetyl-3,4,5,6-tetrahydro-2H-benzo[b][1,4]oxazocinyl, N-methanesulfonyl-2,3-dihydrobenzo[d]oxazolyl, N-methanesulfonyl-3,4-dihydro-2H-benzo[b][1,4]oxazinyl, N-methanesulfonyl-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepinyl, N-methanesulfonyl-3,4,5,6-tetrahydro-2H-benzo[b][1,4]oxazocinyl, etc. may be mentioned.

[0063] In this specification, unless otherwise specified, the "non-aromatic heterocyclic group" means a "3- to 14-membered saturated or unsaturated non-aromatic heterocyclic group".

[0064] In this specification, unless otherwise specified, the "3- to 14-membered saturated or unsaturated non-aromatic heterocyclic group" means a monovalent group formed by removing any hydrogen atom from a 3- to 14-membered saturated or unsaturated heterocyclic ring containing 1 to 4 heteroatoms selected from an oxygen atom, a sulfur atom, and a nitrogen atom.

[0065] In this specification, unless otherwise specified, examples of the "non-aromatic heterocyclic group" include groups such as aziridinyl, azetidinyl, oxiranyl, thiiranyl, oxetanyl, thietanyl, pyrrolidinyl, tetrahydrofuryl, dihydrofuryl, thioranyl, pyrazolinyl, pyrazolidinyl, imidazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl (2-tetrahydro-2H-pyranyl, 3-tetrahydro-2H-pyranyl, 4-tetrahydro-2H-pyranyl (4-tetrahydro-2H-pyran-4-yl group)), tetrahydrothiopyranyl, piperazinyl, dioxanyl, oxazolidinyl, isoxazolinyl, 1,3-oxazolidinyl, isoxazolidinyl, thiazolinyl, isothiazolinyl, 1,3-thiazolidinyl, isothiazolidinyl, oxadiazolinyl, 1,3,4-oxadiazolidinyl, morpholinyl, thiomorpholinyl, quinuclidinyl, azepanyl, diazepinyl, or oxepanyl.

[0066] In this specification, unless otherwise specified, examples of the "halogen atom" include a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, etc. In this specification, unless otherwise specified, "halogenated" in "halogenated C 1~6 alkyl group" etc. means having several, preferably 1 to 5, of the above-mentioned "halogen atoms" as substituents.

[0067] In this specification, unless otherwise specified, examples of the "C 1~6 alkyl group" include groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, or hexyl, etc.

[0068] In this specification, unless otherwise specified, "halogenated C 1~6 alkyl group" means a group in which the above-mentioned "C 1~6 alkyl" is optionally substituted with several, preferably 1 to 5 halogen atoms, and examples thereof include groups such as fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 1,1,2,2-tetrafluoroethyl, or pentafluoroethyl, etc.

[0069] In this specification, unless otherwise specified, "C 1~6 alkoxy group" means an alkoxy in which the above-mentioned "C 1~6 alkyl" is bonded to an oxygen atom, and examples thereof include groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, or hexyloxy, etc.

[0070] In this specification, unless otherwise specified, "-NR a R b group" means a group in which two hydrogen atoms on the nitrogen atom of the "amino group" are substituted with -R a 、 -R b .

[0071] In this specification, unless otherwise specified, R a and R b are each independently a group selected from a hydrogen atom, a C 1~6 alkyl group, a C 2~7 alkanoyl group, or a C 1~6 alkylsulfonyl group, and examples thereof include groups such as amino, N-methylamino, N-ethylamino, N,N-dimethylamino, N-acetylamino, N-methanesulfonylamino, or N-acetyl-N-methylamino, etc.

[0072] In this specification, unless otherwise specified, "C 2~7 alkanoyl group" means a "C 1~6 alkyl group" to which a carbonyl group is bonded, "C 1~6means an "alkylcarbonyl group", and examples thereof include groups such as acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, heptanoyl, cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, cyclopropylmethylcarbonyl, or 2-methylcyclopropylcarbonyl.

[0073] In this specification, unless otherwise specified, "C" 1~6 The "alkylsulfonyl group" means a group in which the "sulfonyl group: -SO" 2 -" is substituted with the above-mentioned "C" 1~6 "alkyl group", and examples thereof include groups such as methylsulfonyl, ethylsulfonyl, propylsulfonyl, or isopropylsulfonyl.

[0074] In this specification, unless otherwise specified, the "cyclic ether" means an ether having a structure in which the carbon of a cyclic hydrocarbon (for example, a cyclic hydrocarbon (C 3~8 cycloalkyl ring) of 3 to 8 carbon atoms among monocyclic or polycyclic saturated hydrocarbon ring groups) is substituted with oxygen, and examples thereof include cyclic ethers such as epoxide, oxetane, tetrahydrofuran, tetrahydropyran, 1,3-dioxolane, 1,3-dioxane, 1,4-dioxane, 1,3-dioxepane, 1,4-dioxepane, 1,4-dioxocane, or 1,5-dioxocane.

[0075] In this specification, unless otherwise specified, the "3-N-(C 2~7 alkanoyl)oxazolidine ring" means a ring in which the hydrogen atom of the NH group of the oxazolidine ring is substituted with the above-mentioned "C" 2~7 "alkanoyl group", and examples thereof include rings such as 3-N-acetyl-oxazolidine ring, 3-N-ethylcarbonyl-oxazolidine ring, etc.

[0076] In this specification, unless otherwise specified, the "4-N-(C 2~7"(alkanoyl)morpholine ring" means a ring in which the hydrogen atom of the NH group of the morpholine ring is replaced by the said "C" 2~7 "(alkanoyl) group", and examples of such rings include 4-N-acetyl-morpholine ring, 4-N-ethylcarbonyl-morpholine ring, etc.

[0077] In this specification, unless otherwise specified, "4-N-(C" 2~7 "(alkanoyl)-1,4-oxazepane ring" means a ring in which the hydrogen atom of the NH group of the 1,4-oxazepane ring is replaced by the said "C" 2~7 "(alkanoyl) group", and examples of such rings include 4-N-acetyl-1,4-oxazepane ring, 4-N-ethylcarbonyl-1,4-oxazepane ring, etc.

[0078] In this specification, unless otherwise specified, "3-N-(C" 1~6 "(alkylsulfonyl)oxazolidine ring" means a ring in which the hydrogen atom of the NH group of the oxazolidine ring is replaced by the said "C" 1~6 "(alkylsulfonyl) group", and examples of such rings include 3-N-methanesulfonyl-oxazolidine ring, 3-N-ethylsulfonyl-oxazolidine ring, etc.

[0079] In this specification, unless otherwise specified, "4-N-(C" 1~6 "(alkylsulfonyl)morpholine ring" means a ring in which the hydrogen atom of the NH group of the morpholine ring is replaced by the said "C" 1~6 "(alkylsulfonyl) group", and examples of such rings include 4-N-methanesulfonyl-morpholine ring, 4-N-ethylsulfonyl-morpholine ring, etc.

[0080] In this specification, unless otherwise specified, "4-N-(C" 2~7 "(alkanoyl)-1,4-oxazepane ring" means a ring in which the hydrogen atom of the NH group of the 1,4-oxazepane ring is replaced by the said "C" 1~6 "(alkylsulfonyl) group", and examples of such rings include 4-N-methanesulfonyl-1,4-oxazepane ring, 4-N-ethylsulfonyl-1,4-oxazepane ring, etc.

[0081] Unless otherwise specified herein, "hydroxy C 1~6 alkyl group" means a group in which any hydrogen atom of the said "C 1~6 alkyl" is optionally substituted by preferably 1 to 5 hydroxyl groups. Examples thereof include groups such as hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, or 2,2-dimethyl-2-hydroxyethyl (=2-hydroxy-2-methylpropyl), etc.

[0082] Unless otherwise specified herein, "thiol C 1~6 alkyl group" means a group in which the said "C 1~6 alkyl" is optionally substituted by several, preferably 1 to 5 thiol groups (-SH groups). Examples thereof include groups such as thiolmethyl, 2-thiolethyl, or 3-thiopropyl, etc.

[0083] Unless otherwise specified herein, "C 1~6 alkylthio C 1~6 alkyl group" means a group in which the hydrogen atom of the thiol group (-SH group) of the said "thiol C 1~6 alkyl group" is substituted by a "C 1~6 alkyl group". Examples thereof include groups such as methylthiomethyl, methylthioethyl, ethylthiomethyl, or ethylthioethyl, etc.

[0084] Unless otherwise specified herein, "-COO(C 1~6 alkyl) group" means a group in which the hydrogen atom of the said "carboxy group" is substituted by the said C 1~6 alkyl group. Examples thereof include groups such as carboxymethyl, carboxyethyl, carboxypropyl, etc.

[0085] Unless otherwise specified herein, "(R a R b N)-C 1~6 alkyl group" means a group in which any hydrogen atom of the said "C 1~6 alkyl" is "-NR a R b"Base" (in this specification, R a and R b each independently represents a group selected from a hydrogen atom, a C 1~6 alkyl group, a C 2~7 alkanoyl group, or a C 1~6 alkylsulfonyl group. For example, groups such as aminomethyl, aminoethyl, aminopropyl, aminobutyl, N-methylaminomethyl, N-acetylaminomethyl, or N-methanesulfonylaminomethyl can be mentioned.) means a group optionally substituted with

[0086] In this specification, unless otherwise specified, "(R a R b N)C(=O)-C 1~6 alkyl group" refers to any hydrogen atom of the above-mentioned "C 1~6 alkyl" being replaced by "(R a R b N)C(=O)-group" (in this specification, R a and R b each independently represents a group selected from a hydrogen atom, a C 1~6 alkyl group, a C 2~7 alkanoyl group, or a C 1~6 alkylsulfonyl group. For example, groups such as aminocarbonylmethyl, aminocarbonylethyl, N-methylaminocarbonylmethyl, N-acetylaminocarbonylethyl, or N-methanesulfonylaminocarbonylethyl can be mentioned.)

[0087] In this specification, unless otherwise specified, "guanidino C 1~6 alkyl group" means a group in which any hydrogen atom of the "C 1~6 alkyl group" is replaced by a "guanidino group (-NH-C(=NH)-NH 2 )". For example, groups such as guanidinomethyl, guanidinoethyl, or guanidinopropyl can be mentioned.)

[0088] In this specification, unless otherwise specified, "C 7~16 aralkyl group" refers to any hydrogen atom of the above-mentioned "C 1~6 alkyl group" being6~10 means a group substituted with an "aryl group", and examples thereof include groups such as benzyl group, phenethyl group, diphenylmethyl group, trityl group, biphenylmethyl group, naphthylmethyl group, indanylmethyl group, or 1,2,3,4-tetrahydronaphthalen-1-ylmethyl group, etc.

[0089] In this specification, unless otherwise specified, "hydroxy C" 6~10 aryl C 1~6 alkyl group" means a group in which the hydrogen atom of the "C" 7~16 aralkyl group" of the "C" 6~10 aryl group" is optionally substituted with several, preferably 1 to 5, hydroxyl groups, and examples thereof include groups such as 2-hydroxybenzyl group, 3-hydroxybenzyl group, or 4-hydroxybenzyl group, etc.

[0090] In this specification, unless otherwise specified, "heteroaryl C" 1~6 alkyl group" means a group in which any hydrogen atom of the "heteroaryl group" is substituted with the "C" 1~6 alkyl group", and examples thereof include groups such as 2-pyridylmethyl group, 4-imidazolylmethyl group, or 3-indolylmethyl group, etc.

[0091] In this specification, unless otherwise specified, "non-aromatic heterocyclic ring" means "a 3- to 14-membered saturated or unsaturated non-aromatic heterocyclic ring".

[0092] In this specification, unless otherwise specified, "a 3- to 14-membered saturated or unsaturated non-aromatic heterocyclic ring" means a 3- to 14-membered saturated or unsaturated heterocyclic ring containing 1 to 4 heteroatoms selected from oxygen atom, sulfur atom and nitrogen atom.

[0093] In this specification, unless otherwise specified, examples of the "non-aromatic heterocycle" include rings such as aziridine, azetidine, pyrrolidine, pyrazolidine, oxazolidine, thiazolidine, isoxazolidine, isothiazolidine, imidazolidine, piperidine, piperazine, morpholine, thiomorpholine, oxazepane, diazepane, thiazepane, oxazocane, diazocane, thiazocane, or oxazine, etc.

[0094] In this specification, unless otherwise specified, "C 3~8 cycloalkyl ring" means a cyclic saturated hydrocarbon ring having 3 to 8 carbon atoms (including monocyclic or polycyclic), and examples thereof include rings such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cyclopentane, or cyclooctane, etc.

[0095] [1-1] In formula (I) of the above aspect [1], -L 1 - is preferably the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0096] More preferably, -L 1 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0097] More preferably, -L 1 - is the following partial structural formula [in each formula, the outside of the broken lines at both ends is not included]:

Chemical formula

[0098] Particularly preferably, -L 1 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0099] Most preferably, -L 1 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0100] [1 - 2] In the formula (II) of the above aspect [1], -L 2 - is preferably the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0101] More preferably, -L2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]: [Chemical formula] (In formula (L2-3), formula (L2-5) and formula (L2-6), the hydrogen atom of the methylene group (-CH 2 -) is an oxo group (=O), a halogen atom, a hydroxyl group, C 1~6 alkyl group, hydroxy C 1~6 alkyl group, thiol C 1~6 alkyl group, C 1~6 alkylthio C 1~6 alkyl group, -COOH group, -COOM group (M is Li, Na, K, or 1 / 2Ca), -COO(C 1~6 alkyl) group, -NR a R b group, (R a R b N)-C 1~6 alkyl group, (R a R b N)C(=O)-C 1~6 alkyl group (in the -NR a R b group, (R a R b N)-C 1~6 alkyl group, or (R a R b N)C(=O)-C 1~6 alkyl group, R a and R b are each independently a group selected from a hydrogen atom, C 1~6 alkyl group, C 2~7 alkanoyl group, or C 1~6 alkylsulfonyl group), guanidino C 1~6 alkyl group, C 7~16 aralkyl group, hydroxy C 6~10 aryl C 1~6 alkyl group, or heteroaryl C 1~6 alkyl group may be replaced by a plurality of (for example, 1 to 10, or 1 to 5) groups; n2 is an integer from 1 to 9; m3 is an integer from 1 to 6; n3 is an integer from 1 to 6; j2 is a linker selected from the group consisting of integers from 0 to 6;

[0102] More preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0103] Particularly preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0104] Most preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0105] [1-2a] In the formula (II) of the above aspect [1], -L 2 - is preferably the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]: [Chemical formula] (In formula (L2-3), formula (L2-4), formula (L2-5), and formula (L2-6), the hydrogen atom of the methylene group (-CH 2 -) is an oxo group (=O), a halogen atom, a hydroxyl group, C 1~6 alkyl group, hydroxy C 1~6 alkyl group, thiol C 1~6 alkyl group, C 1~6 alkylthio C 1~6 alkyl group, -COOH group, -COOM group (M is Li, Na, K, or 1 / 2Ca), -COO(C 1~6 alkyl) group, -NR a R b (R a R b N)-C 1~6 (R a R b N)C(=O)-C 1~6 alkyl group (in the -NR a R b group, (R a R b N)-C 1~6 alkyl group, or (R a R b N)C(=O)-C 1~6 alkyl group, R a and R b are each independently a hydrogen atom, C 1~6 alkyl group, C 2~7 alkanoyl group, or C 1~6 alkylsulfonyl group selected from the group), guanidino C 1~6 alkyl group, C 7~16 aralkyl group, hydroxy C 6~10 aryl C 1~6 alkyl group, or heteroaryl C 1~6 alkyl group may be replaced by a plurality of (for example, 1 to 10, or 1 to 5) groups selected therefrom; In formula (L2-3), formula (L2-4), formula (L2-5), and formula (L2-6), two hydrogen atoms of the same methylene group (-CH 2 -) are C 1~6When replaced by an alkyl group, the alkyl groups may be bonded to each other to form a C 3~8 cycloalkyl ring; The -NH- groups in formula (L2-3), formula (L2-4), formula (L2-5) and formula (L2-6) may form a non-aromatic heterocyclic ring together with the substituents bonded to the adjacent carbon atoms; n2 is an integer from 1 to 18; m3 is an integer from 1 to 10; n3 is an integer from 1 to 10; j2 is a linker selected from the group consisting of integers from 0 to 9);

[0106] More preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0107] More preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the broken lines at both ends is not included]:

Chemical formula

[0108] Particularly preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the broken lines at both ends is not included]: [Chemical formula] is a linker selected from the group consisting of;

[0109] Most preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]: [Chemical formula] is a linker selected from the group consisting of.

[0110] [1-2b] In formula (II) of the above aspect [1], -L 2 - is preferably the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]: [Chemical formula] (In formula (L2-3), formula (L2-4), formula (L2-5) and formula (L2-6), the hydrogen atom of the methylene group (-CH 2 -) is an oxo group (=O), a halogen atom, a hydroxyl group, C 1~6 alkyl group, hydroxy C 1~6 alkyl group, thiol C 1~6 alkyl group, C 1~6 alkylthio C 1~6 alkyl group, -COOH group, -COOM group (M is Li, Na, K, or 1 / 2Ca), -COO(C 1~6 alkyl) group, -NR a R b group, (R a R b N)-C 1~6 alkyl group, (R a R b N)C(=O)-C 1~6 alkyl group (in the above -NR a R b group, (R a R b N)-C 1~6 alkyl group, or (R a R b N)C(=O)-C 1~6 alkyl group, R ina and R b each independently represents a hydrogen atom, C 1~6 alkyl group, C 2~7 alkanoyl group, or C 1~6 alkylsulfonyl group, and a plurality (e.g., 1 to 10, or 1 to 5) of the guanidino C 1~6 alkyl groups, C 7~16 aralkyl groups, hydroxy C 6~10 aryl C 1~6 alkyl groups, or heteroaryl C 1~6 alkyl groups may be replaced; In formula (L2-3), formula (L2-4), formula (L2-5) and formula (L2-6), when two hydrogen atoms of the same methylene group (-CH 2 -) are replaced by C 1~6 alkyl groups, the alkyl groups may be bonded to each other to form a C 3~8 cycloalkyl ring; In formula (L2-3), formula (L2-4), formula (L2-5) and formula (L2-6), the -NH- group may form a non-aromatic heterocyclic ring together with the substituents bonded to the adjacent carbon atoms; n2 is an integer from 1 to 18; m3 is an integer from 1 to 10; n3 is an integer from 1 to 10; j2 is a linker selected from the group consisting of integers from 0 to 9);

[0111] More preferably, -L 2 - is the following partial structural formula [excluding the outside of the dashed lines at both ends in each formula]:

Chemical formula

[0112] More preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0113] Particularly preferably,

Chemical formula

[0114] Most preferably, -L 2 - is a linker selected from the group consisting of the following partial structural formulas [in each formula, the outside of the dashed lines at both ends is not included]:

Chem.

[0115] Even more preferably, -L 2 - is a linker selected from the group consisting of the following partial structural formulas [in each formula, the outside of the dashed lines at both ends is not included]:

Chem.

[0116] [2] A second aspect is as follows. The following formula (I):

Chem.

Chem.

[0117] [2-1] In Formula (I) of the above aspect [2], -L 1 - is preferably the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0118] More preferably, -L 1 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0119] More preferably, -L 1 - is the following partial structural formula [in each formula, the outside of the broken lines at both ends is not included]:

Chemical formula

[0120] Particularly preferably, -L 1 - is the following partial structural formula [in each formula, the outside of the broken lines at both ends is not included]:

Chemical formula

[0121] Most preferably, -L 1 - is the following partial structural formula [in each formula, the outside of the broken lines at both ends is not included]:

Chemical formula

[0122] [3] The third aspect is as follows. Formula (BR-1): [Chemical formula] (In formula (BR-1), -L 1 - is the same as the definition in the above aspect [1] or [2]), the introduction rate of the group represented by is 1% to 30%, and the alginic acid derivative according to [2] above.

[0123] [4] The fourth aspect is as follows. The weight average molecular weight measured by the gel filtration chromatography method of the alginic acid derivative represented by formula (I) is 100,000 Da to 3,000,000 Da, and the alginic acid derivative according to [2] above.

[0124] [5] The fifth aspect is as follows. The following formula (II-P): [Chemical formula] (In formula (II-P), (ALG) represents alginic acid; -NHCO- represents an amide bond through any carboxyl group of alginic acid; P 1 is a hydrogen atom or a protecting group for a thiol group (-SH group); -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]: [Chemical formula] (In formulas (L2-1) to (L2-6), the hydrogen atom of the methylene group (-CH 2 -) is an oxo group (=O), a halogen atom, a hydroxyl group, C 1~6 alkyl group, hydroxy C 1~6 alkyl group, thiol C 1~6 alkyl group, C 1~6 alkylthio C 1~6 alkyl group, -COOH group, -COOM group (M is Li, Na, K, or 1 / 2Ca), -COO(C 1~6 alkyl) group, -NR a R b group, (R a Rb N)-C 1~6 alkyl group, (R a R b N)C(=O)-C 1~6 alkyl group (wherein the -NR a R b group, (R a R b N)-C 1~6 alkyl group, or (R a R b N)C(=O)-C 1~6 in the alkyl group, R a and R b are each independently a hydrogen atom, C 1~6 alkyl group, C 2~7 alkanoyl group, or C 1~6 alkylsulfonyl group (selected from the group consisting of); guanidino C 1~6 alkyl group, C 7~16 aralkyl group, hydroxy C 6~10 aryl C 1~6 alkyl group, or heteroaryl C 1~6 alkyl group (for example, 1 to 10, or 1 to 5) may be replaced by a group selected from; When two hydrogen atoms of the same methylene group (-CH 2 -) in Formula (L2-1) to Formula (L2-6) are replaced by a C 1~6 alkyl group, the alkyl groups may be bonded to each other to form a C 3~8 cycloalkyl ring; The -NH- group in Formula (L2-3) to Formula (L2-6) may form a non-aromatic heterocyclic ring together with a substituent bonded to an adjacent carbon atom; m2 is an integer from 1 to 9; n2 is an integer from 1 to 18; m3 is an integer from 1 to 10; n3 is an integer from 1 to 10; j2 is an integer from 0 to 9) and is a linker selected from the group consisting of). An alginic acid derivative represented by

[0125] [5-1] In Formula (II-P) of the above aspect [5], preferably, -L 2- is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]: [Chemical formula] (In formula (L2-3), formula (L2-5), and formula (L2-6), the hydrogen atom of the methylene group (-CH 2 -) is an oxo group (=O), a halogen atom, a hydroxyl group, C 1~6 alkyl group, hydroxy C 1~6 alkyl group, thiol C 1~6 alkyl group, C 1~6 alkylthio C 1~6 alkyl group, -COOH group, -COOM group (M is Li, Na, K, or 1 / 2Ca), -COO(C 1~6 alkyl) group, -NR a R b group, (R a R b N)-C 1~6 alkyl group, (R a R b N)C(=O)-C 1~6 alkyl group (in the above -NR a R b group, (R a R b N)-C 1~6 alkyl group, or (R a R b N)C(=O)-C 1~6 alkyl group, R a and R b are each independently a group selected from a hydrogen atom, C 1~6 alkyl group, C 2~7 alkanoyl group, or C 1~6 alkylsulfonyl group), guanidino C 1~6 alkyl group, C 7~16 aralkyl group, hydroxy C 6~10 aryl C 1~6 alkyl group, or heteroaryl C 1~6 alkyl group may be replaced by a plurality of (for example, 1 to 10, or 1 to 5) groups selected therefrom; In formula (L2-3), formula (L2-5), and formula (L2-6), two hydrogen atoms of the same methylene group (-CH 2 -) are C1~6 When replaced by an alkyl group, the alkyl groups may be bonded to each other to form a C 3~8 cycloalkyl ring; In formula (L2-3), formula (L2-5) and formula (L2-6), the -NH- group may form a non-aromatic heterocyclic ring together with the substituents bonded to the adjacent carbon atoms; n2 is an integer from 1 to 18; m3 is an integer from 1 to 10; n3 is an integer from 1 to 10; j2 is a linker selected from the group consisting of integers from 0 to 9);

[0126] More preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0127] More preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the broken lines at both ends is not included]: [Chemical formula] (In formula (L2-5-1) and formula (L2-6-1), the R A groups are, independently of each other, a hydrogen atom, a halogen atom, a hydroxyl group, C 1~6 alkyl group, hydroxy C 1~6 alkyl group, -COOH group, -COOM group (M is Li, Na, K, or 1 / 2Ca), -COO(C 1~6 alkyl) group, or C 7~16 aralkyl group, and the like); n2 is an integer from 1 to 5; m3 is an integer from 1 to 3; n3 is an integer from 1 to 4; j2 is a linker selected from the group consisting of integers from 0 to 3);

[0128] Particularly preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the broken lines at both ends is not included]: [Chemical formula] a linker selected from the group consisting of;

[0129] Most preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]: [Chemical formula] a linker selected from the group consisting of.

[0130] [5-1a] In formula (II-P) of the above aspect [5], -L 2 - is preferably the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]: [Chemical formula] (In formula (L2-3), formula (L2-4), formula (L2-5) and formula (L2-6), the hydrogen atom of the methylene group (-CH 2 -) is an oxo group (=O), a halogen atom, a hydroxyl group, C 1~6 alkyl group, hydroxy C 1~6 alkyl group, thiol C 1~6 alkyl group, C 1~6 alkylthio C 1~6 alkyl group, -COOH group, -COOM group (M is Li, Na, K, or 1 / 2Ca), -COO(C 1~6 alkyl) group, -NR a R b group, (R a R b N)-C 1~6 alkyl group, (R a R b N)C(=O)-C 1~6 alkyl group (in the above -NR a R b group, (R a R b N)-C 1~6 alkyl group, or (R a R b N)C(=O)-C 1~6 alkyl group, R a and R bis, independently of each other, a hydrogen atom, C 1~6 alkyl group, C 2~7 alkanoyl group, or C 1~6 alkylsulfonyl group, and may be replaced by a plurality (for example, 1 to 10, or 1 to 5) of groups selected from 1~6 guanidino C 7~16 alkyl group, C 6~10 aralkyl group, hydroxy C 1~6 aryl C 1~6 alkyl group, or heteroaryl C In the formulas (L2-3), (L2-4), (L2-5) and (L2-6), when two hydrogen atoms of the same methylene group (-CH 2 -) are replaced by a C 1~6 alkyl group, the alkyl groups may be bonded to each other to form a C 3~8 cycloalkyl ring; In the formulas (L2-3), (L2-4), (L2-5) and (L2-6), the -NH- group may form a non-aromatic heterocyclic ring together with substituents bonded to adjacent carbon atoms; n2 is an integer of 1 to 18; m3 is an integer of 1 to 10; n3 is an integer of 1 to 10;

[0131] 2 -L

Chemical formula

[0132] More preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0133] Particularly preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0134] Most preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0135] [5-1b] In the formula (II-P) of the above aspect [5], -L 2 - is preferably the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0136] More preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0137] More preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]: [Chemical formula] (In formula (L2-3-1), formula (L2-4-1), formula (L2-5-1) and formula (L2-6-1), R A groups are each independently a hydrogen atom, a halogen atom, a hydroxyl group, C 1~6 alkyl group, hydroxy C 1~6 alkyl group, -COOH group, -COOM group (M is Li, Na, K, or 1 / 2Ca), -COO(C 1~6 alkyl) group, or C 7~16 aralkyl group; ; In formula (L2-3-1), formula (L2-4-1), formula (L2-5-1) and formula (L2-6-1), the -NH- group may form a non-aromatic heterocyclic ring together with a substituent bonded to an adjacent carbon atom; n2 is an integer from 1 to 5; m3 is an integer from 1 to 3; n3 is an integer from 1 to 4; j2 is a linker selected from the group consisting of integers from 0 to 3);

[0138] Particularly preferably, [Chemical formula] (In formula (L2-4-1), formula (L2-6-1), and formula (L2-6-1-a), R A groups are each independently a hydrogen atom, a halogen atom, a hydroxyl group, a C 1~6 alkyl group, a hydroxy C 1~6 alkyl group, a -COOH group, a -COOM group (M is Li, Na, K, or 1 / 2Ca), a -COO(C 1~6 alkyl) group, or a C 7~16 aralkyl group; n2 is an integer from 1 to 5; m3 is an integer from 1 to 3; n3 is an integer from 1 to 4; j2 is an integer from 0 to 3; k is an integer from 1 to 4) and is a linker selected from the group consisting of;

[0139] Most preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0140] Even more preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0141] [5-2] In formula (II-P) of the above aspect [5], preferably, P 1 is a hydrogen atom, an acetyl group, or a benzoyl group. [5-2a] In formula (II-P) of the above aspect [5], P 1 is preferably a hydrogen atom, an acetyl group, or a benzoyl group, and more preferably a hydrogen atom or a benzoyl group.

[0142] [6-1]Aspect 6-1 is as follows. The introduction rate of the reactive group represented by the following formula (BR-2): [Chemical formula] (in formula (BR-2), -L 2 - is the same as the definition in the above aspect [1] or [5]) is 1% to 30%, and the alginic acid derivative represented by formula (II) described in the above aspect [1].

[0143] [6-2]Aspect 6-2 is as follows. The following formula (BR-2-P): [Chemical formula] (in formula (BR-2-P), P 1 , -L 2 - is the same as the definition in the above aspect [1] or [5]) is 1% to 30%, and the alginic acid derivative represented by formula (II-P) described in the above [5].

[0144] [7-1]Aspect 7-1 is as follows. 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 represented by formula (II) described in the above aspect [1].

[0145] [7-2]Aspect 7-2 is as follows. 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 represented by formula (II-P) described in the above aspect [5].

[0146] [8]Aspect 8 is as follows. In the crosslinked alginic acid of the above aspect [1], the chemical crosslinking is represented by the following formula (LK-1): [Chemical formula] [In formula (LK-1), -CONH- and -NHCO- at both ends represent amide bonds via any carboxyl group of alginic acid; -L1 - and -L 2 - has the same structure as defined in the above aspect [1], and is the crosslinked alginic acid described in [1].

[0147] [8-1] The 8-1st aspect is as follows. In the above aspect [8], the preferable, more preferable, further preferable, particularly preferable, or most preferable linker -L 1 - has the same definition as that in the above aspect [2-1].

[0148] [8-2] The 8-2nd aspect is as follows. In the above aspect [8], the preferable, more preferable, further preferable, particularly preferable, or most preferable linker -L 2 - has the same definition as that in the above aspect [5-1].

[0149] [8-3] The 8-3rd aspect is as follows. In the above aspect [8], the preferable, more preferable, further preferable, particularly preferable, or most preferable linker -L 2 - has the same definition as that in the above aspect [5-1a].

[0150] [8-4] The 8-4th aspect is as follows. In the above aspect [8], the preferable, more preferable, further preferable, particularly preferable, most preferable, or even most preferable linker -L 2 - has the same definition as that in the above aspect [5-1b].

[0151] [8a] The 8a-th aspect is as follows. The following formula (CAL-1):

Chemical formula

[0152] [8a-1] The 8a-1 aspect is as follows. The following formula (CAL-1) obtained by the crosslinking reaction described in the above aspect [1]:

Chemical formula

[0153] [8a-2] The 8a-2 aspect is as follows. In the above aspect [8a] or [8a-1], the preferred, more preferred, further preferred, particularly preferred, or most preferred linker -L 1 - is the same as the definition in the above aspect [2-1].

[0154] [8a-3] The 8a-3 aspect is as follows. In the above aspect [8a] or [8a-1], the preferred, more preferred, further preferred, particularly preferred, or most preferred linker -L 2 - is the same as the definition in the above aspect [5-1].

[0155] [8a-4] The 8a-4 aspect is as follows. In the above aspect [8a] or [8a-1], the preferred, more preferred, further preferred, particularly preferred, or most preferred linker -L 2 - is the same as the definition in the above aspect [5-1a].

[0156] [8a-5] Aspect 8a-5 is as follows. In the above aspect [8a] or [8a-1], a preferred, more preferred, further preferred, particularly preferred, most preferred, or even most preferred linker-L 2 - is the same as defined in the above aspect [5-1b].

[0157] [9] Aspect 9 is as follows. A method for producing the crosslinked alginic acid according to the above aspect [1], which includes subjecting a solution of an alginic acid derivative represented by formula (I) to a crosslinking reaction in a solution of an alginic acid derivative represented by formula (II).

[0158]

[10] Aspect 10 is as follows. A method for producing the crosslinked alginic acid according to the above aspect [1], which includes subjecting a solution of an alginic acid derivative represented by formula (II) to a crosslinking reaction in a solution of an alginic acid derivative represented by formula (I).

[0159]

[11] Aspect 11 is as follows. A chemical crosslink formed by performing a Michael addition reaction using an alginic acid derivative represented by formula (I) and an alginic acid derivative represented by formula (II) has the following formula (LK-1):

Chemical formula

[0160] [11-1] Aspect 11-1 is as follows. In the above aspect

[11] , a preferred, more preferred, further preferred, particularly preferred, or most preferred linker-L 1 - is the same as defined in the above aspect [2-1].

[0161] [11-2] Aspect 11-2 is as follows. In the said aspect

[11] , the preferable, more preferable, further preferable, particularly preferable, or most preferable linker-L 2 - is the same as defined in the said aspect [5-1].

[0162] [11-3] Aspect 11-3 is as follows. In the said aspect

[11] , the preferable, more preferable, further preferable, particularly preferable, or most preferable linker-L 2 - is the same as defined in the said aspect [5-1a].

[0163] [11-4] Aspect 11-4 is as follows. In the said aspect

[11] , the preferable, more preferable, further preferable, particularly preferable, most preferable, or even most preferable linker-L 2 - is the same as defined in the said aspect [5-1b].

[0164]

[12] Aspect 12 is as follows. A crosslinked alginate structure obtained by subjecting a gel obtained by dropping a solution of an alginate derivative represented by formula (I) into a solution containing a divalent metal ion to a crosslinking reaction in a solution of an alginate derivative represented by formula (II), the crosslinked structure including an ionic crosslink formed partially by the divalent metal ion as a crosslink and a chemical crosslink formed by a Michael addition reaction.

[0165]

[13] Aspect 13 is as follows. A crosslinked alginate structure obtained by subjecting a gel obtained by dropping a solution of an alginate derivative represented by formula (II) into a solution containing a divalent metal ion to a crosslinking reaction in a solution of an alginate derivative represented by formula (I), the crosslinked structure including an ionic crosslink formed partially by the divalent metal ion as a crosslink and a chemical crosslink formed by a Michael addition reaction.

[0166]

[14] Aspect 14 is as follows. A crosslinked alginate structure obtained by dropping a solution of a composition containing an alginate derivative represented by formula (I) and an alginate derivative represented by formula (II) into a solution containing a divalent metal ion, the crosslinked alginate structure including an ionic crosslink partially formed by the divalent metal ion as a crosslink and a chemical crosslink formed by a Michael addition reaction.

[0167]

[15] Aspect 15 is as follows. A chemical crosslink formed by performing a Michael addition reaction using an alginate derivative represented by formula (I) and an alginate derivative represented by formula (II) has the following formula (LK-1): [Chemical formula] [In formula (LK-1), -CONH- and -NHCO- at both ends represent an amide bond via an arbitrary carboxyl group of alginic acid; -L 1 -, and -L 2 - are the same as defined in the above aspect [1].] The crosslinked alginate structure according to any one of the above

[12] to

[14] .

[0168] [15-1]Aspect 15-1 is as follows. In the above aspect

[15] , the preferable, more preferable, further preferable, particularly preferable, or most preferable linker -L 1 - is the same as defined in the above aspect [2-1].

[0169] [15-2]Aspect 15-2 is as follows. In the above aspect

[15] , the preferable, more preferable, further preferable, particularly preferable, or most preferable linker -L 2 - is the same as defined in the above aspect [5-1].

[0170] [15-3]Aspect 15-3 is as follows. In the above aspect

[15] , the preferable, more preferable, further preferable, particularly preferable, or most preferable linker -L 2 - is the same as defined in the above aspect [5-1a].

[0171] [15-4] Aspect [15-4] is as follows. In the above aspect

[15] , a preferable, more preferable, still more preferable, particularly preferable, most preferable, or even most preferable linker-L 2 - is the same as defined in the above aspect [5-1b].

[0172]

[16] Aspect

[16] is as follows. A crosslinked alginic acid structure according to any one of aspects

[12] to

[15] , which is a fibrous structure, fiber, bead, gel, or substantially spherical gel.

[0173]

[17] Aspect

[17] is as follows. A medical material containing a crosslinked alginic acid structure according to any one of aspects

[12] to

[16] .

[0174]

[18] Aspect

[18] is as follows. A medical material according to aspect

[17] , which is a fibrous structure, fiber, bead, gel, or substantially spherical gel.

[0175]

[19] Aspect

[19] is as follows. A composition containing an alginic acid derivative represented by formula (I) and an alginic acid derivative represented by formula (II).

[0176] [19-1] Aspect [19-1] is as follows. A composition containing an alginic acid derivative represented by formula (I) and an alginic acid derivative represented by formula (II-P).

[0177] [19-2] Aspect [19-2] is as follows. A composition containing any one alginic acid derivative selected from an alginic acid derivative represented by formula (I), an alginic acid derivative represented by formula (II), or an alginic acid derivative represented by formula (II-P).

[0178]

[20] The 20th aspect is as follows. A method for producing a crosslinked alginate structure, comprising dropping a solution of a composition containing an alginate derivative represented by formula (I) and an alginate derivative represented by formula (II) into a solution containing a divalent metal ion.

[0179]

[21] The 21st aspect is as follows. A method for producing a crosslinked alginate structure, comprising subjecting a gel obtained by dropping a solution of an alginate derivative represented by formula (I) into a solution containing a divalent metal ion to a crosslinking reaction in a solution of an alginate derivative represented by formula (II), to obtain a crosslinked alginate structure including an ionic crosslink partially formed by a divalent metal ion as a crosslink and a chemical crosslink formed by a Michael addition reaction.

[0180]

[22] The 22nd aspect is as follows. A method for producing a crosslinked alginate structure, comprising subjecting a gel obtained by dropping a solution of an alginate derivative represented by formula (II) into a solution containing a divalent metal ion to a crosslinking reaction in a solution of an alginate derivative represented by formula (I), to obtain a crosslinked alginate structure including an ionic crosslink partially formed by a divalent metal ion as a crosslink and a chemical crosslink formed by a Michael addition reaction.

[0181]

[23] The 23rd aspect is as follows. The chemical crosslink formed by performing a Michael addition reaction using an alginate derivative represented by formula (I) and an alginate derivative represented by formula (II) has the structure of the following formula (LK-1):

Chemical formula

[20] to

[22] .

[0182] [23-1] Aspect 23-1 is as follows. In the above aspect

[23] , a preferable, more preferable, further preferable, particularly preferable, or most preferable linker-L 1 - is the same as the definition in the above aspect [2-1].

[0183] [23-2] Aspect 23-2 is as follows. In the above aspect

[23] , a preferable, more preferable, further preferable, particularly preferable, or most preferable linker-L 2 - is the same as the definition in the above aspect [5-1].

[0184] [23-3] Aspect 23-3 is as follows. In the above aspect

[23] , a preferable, more preferable, further preferable, particularly preferable, or most preferable linker-L 2 - is the same as the definition in the above aspect [5-1a].

[0185] [23-4] Aspect 23-4 is as follows. In the above aspect

[23] , a preferable, more preferable, further preferable, particularly preferable, most preferable, or even most preferable linker-L 2 - is the same as the definition in the above aspect [5-1b].

[0186]

[24] Aspect 24 is as follows. A crosslinked alginate structure having content retention property, obtained by subjecting an alginate derivative represented by formula (I) and an alginate derivative represented by formula (II) to ionic crosslinking with a divalent metal ion and chemical crosslinking by Michael addition reaction.

[0187]

[25] Aspect 25 is as follows. The crosslinked alginate according to the above aspect [1] or [8] having biocompatibility, or the crosslinked alginate structure according to any one of the above aspects

[12] to

[16] and

[24] .

[0188] [26a]Aspect 26a is as follows. An alginic acid derivative represented by formula (I) according to aspect [2] that is biocompatible, and an alginic acid derivative represented by formula (II-P) according to aspect [5].

[0189]

[27] Aspect 27 is as follows. The following formula (AM-2):

Chemical formula

[0190] [27-1]In formula (AM-2) of aspect

[27] , -L 2 - is preferably the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0191] More preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0192] More preferably, -L 2 - is the following partial structural formula [in each formula, the outside of the broken lines at both ends is not included]:

Chemical formula

[0193] Particularly preferably, [Chemical formula] (In formulas (L2-4-1), (L2-6-1), and (L2-6-1-a), R A groups are each independently a hydrogen atom, a halogen atom, a hydroxyl group, C 1~6 alkyl group, hydroxy C 1~6 alkyl group, -COOH group, -COOM group (M is Li, Na, K, or 1 / 2Ca), or C 7~16 aralkyl group; n2 is an integer from 1 to 5; m3 is an integer from 1 to 3; n3 is an integer from 1 to 4; j2 is an integer from 0 to 3; k is an integer from 1 to 4) and is a linker selected from the group consisting of.

[0194] [27-2] In formula (AM-2) of the above aspect

[27] , P 1is preferably an acetyl group or a benzoyl group, more preferably a benzoyl group.

[0195]

[28] The 28th aspect is as follows. The following formula (AM-2):

Chemical formula

Chemical formula

[0196] [28-1] In the formula (AM-2) of the above aspect

[28] , -L 2 - is preferably the following partial structural formula [in each formula, the outside of the broken lines at both ends is not included]:

Chemical formula

[0197] More preferably, it is the following partial structural formula [in each formula, the outside of the broken lines at both ends is not included]:

Chemical formula

[0198] More preferably, the following partial structural formulas [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

[0199] Particularly preferably, the following partial structural formulas [in each formula, the outside of the dashed lines at both ends is not included]:

Chemical formula

Chemical formula

[0200] [28-2] In formula (AM-2) of the above aspect

[28] , P 1 is preferably a benzoyl group.

[0201]

[29] The 29th aspect is as follows. The following formula

Chemical formula

[0202] [29-1] In the amino compound or a pharmaceutically acceptable salt thereof according to the aspect

[29] , the amino compound is preferably represented by the following formula

Chemical formula

[0203] 1. Alginate In this specification, when referring to alginate, it means at least one alginate selected from the group consisting of alginate, alginate esters, and their salts (for example, sodium alginate) (sometimes referred to as "alginate-like substances"). The alginate used may be of natural origin or synthetic, but is preferably of natural origin. The preferably used alginate-like substances are biocompatible polysaccharides extracted from brown algae such as Lessonia, Macrocystis, Laminaria, Ascophyllum, Darbilla, Kjellmaniella, Arame, and Kombu, and are polymers in which two types of uronic acids, D-mannuronic acid (M) and L-guluronic acid (G), are polymerized linearly. More specifically, it is a block copolymer in which a homopolymer fraction of D-mannuronic acid (MM fraction), a homopolymer fraction of L-guluronic acid (GG fraction), and a fraction in which D-mannuronic acid and L-guluronic acid are randomly arranged (M / G fraction) are arbitrarily combined. In this specification, when referring to the molecular weight of alginate, alginate derivatives, crosslinked alginate, and crosslinked alginate, Da (dalton) may be appended as a unit.

[0204] In this specification, alginate may be represented as (ALG), with one of any carboxyl groups of alginate as -COOH, and denoted as (ALG)-COOH.

[0205] In some embodiments, the alginic acid is sodium alginate. Commercial sodium alginate can be used. For example, alginic acid having the physical property values shown in Table 1 can be used. Here, in Examples 10 to 12 described below, sodium alginate is sodium alginate A-2 described in Table 1 (manufacturer: Motoda Pharmaceutical Co., Ltd.). The viscosity, weight average molecular weight, and M / G ratio of a 1 w / w% aqueous solution of each sodium alginate are shown in Table 1. Also, in Examples 1 to 9 described below, sodium alginate is ALG-2 (vendor: Kimika Co., Ltd.) and has the weight average molecular weight described in <Measurement of Molecular Weight> below.

[0206]

Table 1

[0207] The physical property values of the sodium alginates A-1, A-2, A-3, B-1, B-2, and B-3 were measured by the following various methods. The measurement method is not limited to this method, but the physical property values may differ from the above depending on the measurement method.

[0208] [Viscosity Measurement of Sodium Alginate] It was measured using a rotational viscometer method (cone and plate type rotational viscometer) in accordance with the viscosity measurement method of the Japanese Pharmacopoeia (16th Edition). The specific measurement conditions are as follows. The sample solution was prepared using MilliQ water. The measuring instrument used was a cone and plate type rotational viscometer (viscosity and viscoelasticity measuring device Rheostress RS600 (Thermo Haake GmbH) sensor: 35 / 1). The rotation speed was 1 rpm when measuring the 1 w / w% sodium alginate solution. The reading time was measured for 2 minutes, and the average value from 1 minute to 2 minutes after the start was taken. The average value of three measurements was taken as the measured value. The measurement temperature was 20°C.

[0209] [Measurement of Weight Average Molecular Weight of Sodium Alginate] (1) Gel permeation chromatography (GPC) and (2) two types of measurement methods, GPC-MALS, were used for measurement. The measurement conditions are as follows.

[0210] [Pretreatment method] After adding the eluent to the sample and dissolving it, the solution filtered through a 0.45 μm membrane filter was used as the measurement solution. (1) Gel permeation chromatography (GPC) measurement [Measurement conditions (relative molecular weight distribution measurement)] Column: TSKgel GMPW-XL × 2 + G2500PW-XL (7.8 mm I.D. × 300 mm × 3 columns) Eluent: 200 mM aqueous sodium nitrate solution Flow rate: 1.0 mL / min Concentration: 0.05% Detector: RI detector Column temperature: 40 °C Injection volume: 200 μL Molecular weight standard: Standard pullulan, glucose

[0211] (2) GPC-MALS measurement [Refractive index increment (dn / dc) measurement (measurement conditions)] Differential refractometer: Optilab T-rEX Measurement wavelength: 658 nm Measurement temperature: 40 °C Solvent: 200 mM aqueous sodium nitrate solution Sample concentration: 0.5 - 2.5 mg / mL (5 concentrations)

[0212] [Measurement conditions (absolute molecular weight distribution measurement)] Column: TSKgel GMPW-XL × 2 + G2500PW-XL (7.8 mm I.D. × 300 mm × 3 columns) Eluent: 200 mM aqueous sodium nitrate solution Flow rate: 1.0 mL / min Concentration: 0.05% Detector: RI detector, light scattering detector (MALS) Column temperature: 40 °C Injection volume: 200 μL

[0213] In this specification, when indicating the molecular weight of alginic acid, alginic acid derivatives, crosslinked alginic acid, and crosslinked alginic acid, Da (Dalton) may be appended as the unit.

[0214] The composition ratio (M / G ratio) of D-mannuronic acid and L-guluronic acid in alginic acids varies mainly depending on the type of organism from which they are derived, such as seaweed, and is also affected by the growth location and season of the organism. The M / G ratio ranges widely from a high G type with an M / G ratio of about 0.2 to a high M type with an M / G ratio of about 5. The gelling ability of alginic acids and the properties of the gels formed are affected by the M / G ratio. Generally, it is known that when the G ratio is high, the gel strength is high. The M / G ratio also affects other properties such as the hardness, brittleness, water absorbency, and flexibility of the gel. The M / G ratio of the alginic acids and / or their salts used is usually 0.2 to 4.0, more preferably 0.4 to 3.0, and even more preferably 0.5 to 3.0.

[0215] In this specification, the numerical range indicated using "~" represents a range that includes the numerical values described before and after "~" as the minimum value and the maximum value, respectively.

[0216] In this specification, the "alginic acid ester" and "alginic acid salt" used are not particularly limited, but in order to react with a crosslinking agent, it is necessary that they do not have a functional group that inhibits the crosslinking reaction. Preferred examples of the alginic acid ester include propylene glycol alginate, etc.

[0217] In this specification, examples of the alginic acid salt include monovalent salts of alginic acid and divalent salts of alginic acid. Preferred examples of the monovalent salt of alginic acid include sodium alginate, potassium alginate, ammonium alginate, etc., more preferably sodium alginate or potassium alginate, and particularly preferably sodium alginate. Preferred examples of the divalent salt of alginic acid include calcium alginate, magnesium alginate, barium alginate, strontium alginate, etc.

[0218] Alginic acid is a high molecular polysaccharide, and it is difficult to accurately determine its molecular weight. Generally, the weight average molecular weight is in the range of 1,000 to 10 million, preferably 10,000 to 8 million, more preferably 20,000 to 3 million. In the measurement of the molecular weight of natural product-derived high molecular substances, it is known that the values may vary depending on the measurement method.

[0219] For example, the weight average molecular weight measured by gel permeation chromatography (GPC) or gel filtration chromatography (collectively also referred to as size exclusion chromatography) is preferably 100,000 or more, more preferably 500,000 or more, and also preferably 5 million or less, more preferably 3 million or less. The preferred range is 100,000 to 5 million, more preferably 150,000 to 3 million.

[0220] Also, for example, according to the GPC-MALS method, the absolute weight average molecular weight can be measured. The weight average molecular weight (absolute molecular weight) measured by the GPC-MALS method is preferably 10,000 or more, more preferably 50,000 or more, even more preferably 60,000 or more, and also preferably 1 million or less, more preferably 800,000 or less, even more preferably 700,000 or less, particularly preferably 500,000 or less. The preferred range is 10,000 to 1 million, more preferably 50,000 to 800,000, even more preferably 60,000 to 700,000, particularly preferably 60,000 to 500,000.

[0221] Generally, when calculating the molecular weight of a high molecular polysaccharide by the above methods, a measurement error of 10% to 20% may occur. For example, if it is 400,000, the value may vary in the range of about 320,000 to 480,000; if it is 500,000, the value may vary in the range of 400,000 to 600,000; if it is 1 million, the value may vary in the range of about 800,000 to 1.2 million.

[0222] The molecular weight of alginic acids can be measured according to conventional methods.

[0223] Typical conditions when using gel filtration chromatography for molecular weight measurement are as described in the examples of this specification below. As the column, for example, a Superose6 Increase 10 / 300 GL column (GE Healthcare Sciences) can be used. As the developing solvent, for example, a 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl can be used. As the molecular weight standards, blue dextran, thyroglobulin, ferritin, aldolase, conalbumin, ovalbumin, ribonuclease A, and aprotinin can be used.

[0224] The viscosity of alginic acid used in this specification is not particularly limited, but when the viscosity is measured as an aqueous solution of 1 w / w% alginic acid, preferably, it is 10 mPa·s to 1000 mPa·s, more preferably, 50 mPa·s to 800 mPa·s.

[0225] The viscosity of the aqueous solution of alginic acid can be measured according to a conventional method. For example, it can be measured using a coaxial double cylindrical rotational viscometer, a single cylindrical rotational viscometer (Brookfield type viscometer), a conical - plate rotational viscometer (cone - plate type viscometer), etc. of the rotational viscometer method. Preferably, it is desirable to follow the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). More preferably, a cone - plate type viscometer is used.

[0226] When initially extracted from brown algae, alginic acids have a large molecular weight and a high viscosity, but during processes such as drying and purification by heat, the molecular weight becomes smaller and the viscosity becomes lower. Alginic acids with different molecular weights can be produced by controlling conditions such as the temperature in the manufacturing process, selecting the brown algae as the raw material, and fractionating the molecular weight in the manufacturing process. Furthermore, it is also possible to obtain alginic acids with the desired molecular weight by mixing different lots of alginic acids with different molecular weights or viscosities.

[0227] The alginic acid used herein may be, in some embodiments, alginic acid that has not been treated with low endotoxins, or in some other embodiments, alginic acid that has been treated with low endotoxins. Low endotoxins means that the endotoxin level is low enough to not substantially cause inflammation or fever. More preferably, alginic acids that have been treated with low endotoxins are desirable.

[0228] The endotoxin reduction treatment can be carried out by a known method or a method similar thereto. For example, the purification of sodium hyaluronate can be carried out by the method of Suga et al. (see, for example, Japanese Patent Application Laid-Open No. 9-324001, etc.), the purification of β1,3-glucan by Yoshida et al. (see, for example, Japanese Patent Application Laid-Open No. 8-269102, etc.), the purification of biopolymer salts such as alginate and gellan gum by Williams et al. (see, for example, Japanese Translation of PCT International Publication No. 2002-530440, etc.), the purification of polysaccharides by James et al. (see, for example, the pamphlet of International Publication No. 93 / 13136, etc.), the method of Lewis et al. (see, for example, the specification of U.S. Patent No. 5,589,591, etc.), the purification of alginate by Herman Frank et al. (see, for example, Appl Microbiol Biotechnol (1994) 40:638-643, etc.), etc., or methods similar thereto. The low endotoxin treatment is not limited to these, but may be performed by known methods such as washing, filtration with a filter (endotoxin removal filter, charged filter, etc.), ultrafiltration, purification using a column (endotoxin adsorption affinity column, gel filtration column, column with ion exchange resin, etc.), adsorption to hydrophobic substances, resins or activated carbon, organic solvent treatment (extraction with organic solvent, precipitation / sedimentation by adding organic solvent, etc.), surfactant treatment (see, for example, JP 2005-036036 A, etc.), or by appropriately combining these. These treatment steps may be appropriately combined with known methods such as centrifugation. It is desirable to select an appropriate method according to the type of alginic acid.

[0229] Endotoxin levels can be confirmed by known methods, for example, measured by methods such as the Limulus reagent (LAL) method, the method using Endospecy® ES-24S set (Seikagaku Corporation), and the like.

[0230] The method for treating endotoxin used is not particularly limited, but as a result, the endotoxin content of alginates is preferably 500 endotoxin units (EU) / g or less, more preferably 100 EU / g or less, particularly preferably 50 EU / g or less, and most preferably 30 EU / g or less when endotoxin measurement is performed by the Limulus reagent (LAL). Sodium alginate treated with low endotoxin can be obtained, for example, from commercially available products such as Sea Matrix® (Mochida Pharmaceutical Co., Ltd.), PRONOVA TM UP LVG (FMC BioPolymer).

[0231] 2. Alginate derivatives In the present specification, novel alginate derivatives are provided. In the present specification, as the alginate derivative, a reactive group in a Michael addition reaction or a complementary reactive group of the reactive group is introduced into any one or more carboxyl groups of alginate via an amide bond and a divalent linker. More specifically, the following formula (I): [Chemical formula] [In formula (I), the definitions of (ALG), -CONH-, and -L 1 - are the same as those defined in the above aspect [1] or [2]] and the alginate derivative represented by the following formula (II): [Chemical formula] [In formula (II), the definitions of (ALG), -CONH-, -L 2 - are the same as those defined in the above aspect [1] or [5]] and the alginate derivative represented by the following formula (II):

[0232] The reactive group of the alginic acid derivative represented by formula (I) is an acrylic acid residue, and the reactive group of the alginic acid derivative represented by formula (II) (the complementary reactive group of the reactive group of the alginic acid derivative represented by formula (I)) is a thiol residue. Both reactive groups of the acrylic acid residue and the thiol residue can form a covalent bond more easily than a Michael addition reaction.

[0233] Examples of the acrylic acid residue include residues that can form an adduct by a Michael addition reaction with a thiol residue. Specifically, an acryloyl group, an acrylic group, a maleyl group, a maleimide group, a fumar group, etc. are mentioned. Preferably, it is an acryloyl group or a maleimide group, and more preferably a maleimide group.

[0234] Examples of the thiol residue include residues that can form an adduct by a Michael addition reaction with an acrylic acid residue. Specifically, a benzylthiol group, a thiophenol group, an alkylthiol group (for example, a residue in which a thiol group is substituted with an alkyl group such as a methanethiol residue, an ethanethiol residue, a cysteine residue, etc.), etc. are mentioned. Preferably, it is a benzylthiol group or an alkylthiol group, and more preferably an alkylthiol group. 1~6 The residue substituted with an alkyl group) etc. are mentioned. Preferably, it is a benzylthiol group or an alkylthiol group, and more preferably an alkylthiol group.

[0235] The above divalent linker (-L 1 - or -L 2 -) can use any linear group as long as it does not inhibit the reaction between the reactive group and the complementary reactive group of the reactive group, and keeps the reactive group or the complementary reactive group and alginic acid at a certain distance. Examples of the divalent linker include a linear alkylene group (-(CH 2 ) n -, n = 1 to 30) (in the alkylene group, -CH 2 - can be replaced by a plurality of (for example, 1 to 10, or 1 to 5) groups such as -C(=O)-, -CONH-, -O-, -NH-, -S-, a benzene ring, a heterocyclic ring (a 5- to 6-membered aromatic heterocyclic ring or a 5- to 6-membered non-aromatic heterocyclic ring such as a pyridine ring, a piperidine ring, a piperazine ring, etc.); -CH 2- The hydrogen atom is an oxo group (=O), a halogen atom, a hydroxyl group, C 1~6 alkyl group, hydroxy C 1~6 alkyl group, thiol C 1~6 alkyl group, C 1~6 alkylthio C 1~6 alkyl group, -COOH group, -COOM group (M is Li, Na, K, or 1 / 2Ca), -COO(C 1~6 alkyl) group, -NR a R b group, (R a R b N)-C 1~6 alkyl group, (R a R b N)C(=O)-C 1~6 alkyl group (in the said -NR a R b group, (R a R b N)-C 1~6 alkyl group, or (R a R b N)C(=O)-C 1~6 alkyl group, R a and R b are each independently a group selected from a hydrogen atom, C 1~6 alkyl group, C 2~7 alkanoyl group, or C 1~6 alkylsulfonyl group), guanidino C 1~6 alkyl group, C 7~16 aralkyl group, hydroxy C 6~10 aryl C 1~6 alkyl group, or heteroaryl C 1~6 alkyl group may be replaced by a plurality of (for example, 1 to 10, or 1 to 5) groups selected therefrom, and when two hydrogen atoms of the same -CH 2 - are replaced by C 1~6 alkyl groups, the alkyl groups may be bonded to each other to form a C 3~8 cycloalkyl ring), but are not limited thereto. Specifically, -L 1 - is the divalent linker described in the said aspect [1] or [2], -L 2- For [1] or [5], examples of the divalent linker described in the above aspect can be cited.

[0236] In the present specification, the linker -L in formula (I) 1 - (in formula (L1-1) to formula (L1-4)), when an asymmetric carbon exists in the formula, it means that each optical isomer thereof is also included.

[0237] For example, -L in formula (I) 1 - is the following formula (L1-3-1-a) (where the outside of both dashed lines is not included):

Chemical formula

Chemical formula

[0238] Similarly, for the linker -L in formula (II) or formula (II-P) 2 - (in formula (L2-1) to formula (L2-6)), when an asymmetric carbon exists in the formula, it means that each optical isomer thereof is also included.

[0239] For example, -L in formula (II) or formula (II-P) 2 - is the following formula (L2-6-1-a) (where the outside of both dashed lines is not included):

Chemical formula

Chemical formula

[0240] In formula (I) herein, when an asymmetric carbon exists in linker -L 1 - (when it is an optically active substance), in the step of synthesizing the amine derivative (AM-1) corresponding to formula (I), it can be separated from its racemate into each optically active substance by ordinary optical resolution means (separation methods), and also in the step of synthesizing the amine derivative (AM-1) corresponding to formula (I), by using asymmetric synthesis, one of the optical isomers can be selectively synthesized, and each optically active substance can be synthesized. By using each of the obtained optically active amine derivatives, it becomes possible to synthesize an alginic acid derivative of formula (I) having an asymmetric carbon (optically active). Also, in formula (II) or formula (II-P) herein, when an asymmetric carbon exists in linker -L 2 - as well, in the step of synthesizing the amine derivative (AM-2) corresponding to each of formula (II) or formula (II-P), in the same manner as the above-mentioned method, it is possible to synthesize an alginic acid derivative of formula (II) or formula (II-P) having each optically active substance and an asymmetric carbon (optically active).

[0241] Examples of the separation methods include optical resolution methods such as fractional recrystallization method, diastereomer method, and chiral column method. Each separation method will be described in detail below. Fractional recrystallization method: A method of ionically bonding an optical resolution agent to a racemate to obtain a crystalline diastereomer, then separating the crystalline diastereomer by fractional recrystallization method, and passing through a step of removing the optical resolution agent if desired to obtain an optically pure compound. Examples of the optical resolution agent include (+)-mandelic acid, (-)-mandelic acid, (+)-tartaric acid, (-)-tartaric acid, (+)-1-phenylethylamine, (-)-1-phenylethylamine, cinchonine, (-)-cinchonidine, and brucine, etc. Diastereomer method: A method in which an optical resolving agent is covalently bonded to a racemic mixture to obtain a mixture of diastereomers, and then the mixture is separated into optically pure diastereomers by ordinary separation means (for example, fractional recrystallization, silica gel column chromatography, HPLC, etc.), and then, through a step of removing the optical resolving agent by a chemical reaction (such as a hydrolysis reaction), an optically pure optical isomer is obtained.

[0242] For example, when the compound or intermediate compound in the present specification has a hydroxyl group or an amino group (primary, secondary), ester or amide diastereomers are obtained from each by a condensation reaction with an optically active organic acid (for example, α-methoxy-α-(trifluoromethyl)phenylacetic acid, (-)-menthoxyacetic acid, etc.). Also, when the compound in the present specification has a carboxyl group, amide or ester diastereomers are obtained from each by a condensation reaction with an optically active amine or an optically active alcohol. The diastereomers obtained by the condensation reaction are separated, and each diastereomer is subjected to a hydrolysis reaction with an acid or a base to be converted into an optically pure optical isomer of the original compound.

[0243] Chiral column method: A method of directly resolving a racemate or its salt by subjecting it to chromatography using a chiral column (a column for separating optical isomers). For example, in the case of high performance liquid chromatography (HPLC), a mixture of optical isomers is added to a chiral column (e.g., CHIRAL series manufactured by Daicel Corporation, etc.), and developed using an elution solvent (single solvents such as water, various buffers (e.g., phosphate buffer), and organic solvents (e.g., ethanol, methanol, isopropanol, acetonitrile, trifluoroacetic acid, and diethylamine, etc.), or a mixed solvent thereof), whereby separation of the optical isomers is possible. Also, for example, in the case of gas chromatography, separation of optical isomers is possible using a chiral column (e.g., CP-Chirasil-DeX CB (manufactured by GL Sciences Inc., etc.)). Also, for example, in the case of supercritical fluid chromatography (SFC), a mixture of optical isomers is added to a chiral column (e.g., CHIRAL series manufactured by Daicel Corporation, etc.), and separation of the optical isomers is possible using carbon dioxide and an appropriate organic solvent (e.g., methanol, ethanol, isopropanol, trifluoroacetic acid, and diethylamine, etc.) as the elution solvent.

[0244] Examples of the asymmetric synthesis for selectively synthesizing one of the optical isomers include: (1) an asymmetric synthesis reaction that enantioselectively reacts a racemic compound to lead to an optically active substance; (2) a method of diastereoselective synthesis from naturally occurring optically active compounds (such as sugars and amino acids), etc.

[0245] The alginate derivatives represented by formula (I) and formula (II), which are novel alginate derivatives in this specification, can be produced, for example, by the method of the following formula (for details, refer to the general production method described later).

[0246]

Chemical formula

[0247] The weight average molecular weight of the alginic acid derivative represented by formula (I), formula (II) or formula (II-P) in this specification is from 100,000 Da to 3,000,000 Da, preferably from 300,000 Da to 2,500,000 Da, and more preferably from 500,000 Da to 2,000,000 Da. The molecular weight of the two alginic acid derivatives can be determined by the method described below.

[0248] In this specification, in the alginic acid derivative represented by formula (I), the following formula (BR-1):

Chemical formula

Chemical formula

Chemical formula

[0249] In this specification, when the group of formula (BR-1) in the alginic acid derivative of formula (I) is referred to as a reactive group, the group of formula (BR-2) in the alginic acid derivative of formula (II) becomes a complementary reactive group. Conversely, when the group of formula (BR-2) in the alginic acid derivative of formula (II) is referred to as a reactive group, the group of formula (BR-1) in the alginic acid derivative of formula (I) becomes a complementary reactive group.

[0250] In this specification, the introduction rate of the reactive group or the complementary reactive group is, for example, 0.1% to 30% or 1% to 30% respectively, preferably 2% to 20%, and more preferably 3% to 10%.

[0251] The introduction rate of the reactive group or complementary reactive group is a value expressed as a percentage of the number of uronic acid monosaccharide units, which are repeating units of alginic acids, into which each reactive group has been introduced. In this specification, unless otherwise specified, the % used for the introduction rate of the reactive group or complementary reactive group in the alginic acid derivative (Formula (I) or Formula (II)) means mol%. The introduction rate of each reactive group or complementary reactive group can be determined by the method described in the Examples below.

[0252] The protecting group P for the thiol group in the alginic acid derivative represented by Formula (II-P) 1 can be appropriately selected from protecting groups that can be easily protected and deprotected. For example, protecting groups known from the literature described in "Protective Groups in Organic Synthesis 5th Edition, 2014, John Wiley & Sons, Greene et al." can be appropriately selected. More specifically, as the protecting group P 1 acetyl group, ethylcarbonyl, etc., C 2~6 alkanoyl groups; benzoyl group, naphthylcarbonyl group, etc., C 6~10 arylcarbonyl groups; trityl group (triphenylmethyl group), diphenylmethyl group; methylaminocarbonyl group, ethylaminocarbonyl group, etc., N-C 1~6 alkyl-carbamoyl groups, etc. can be mentioned, but are not limited thereto.

[0253] In this specification, the maleimide group in the group of Formula (BR-1) in the alginic acid derivative in Formula (I) and the thiol (HS-) group in the alginic acid derivative of Formula (II) form a covalent bond by a Michael addition reaction, thereby forming a crosslink.

[0254] 3. Method for synthesizing alginic acid derivative In this specification, the alginic acid derivative represented by Formula (I) is an amine derivative represented by Formula (AM-1) (wherein -L 1- is the same as the definition in the above aspect [1] or [2]) or a salt thereof and can be produced by a condensation reaction with any carboxyl group of alginic acids. Further, the alginic acid derivative represented by the formula (II) is represented by the formula (AM-2) (wherein -L 2 - and P 1 is the same as the definition in the above aspect [1] or [5]), after obtaining an alginic acid derivative represented by the formula (II-P) by a condensation reaction of an amine derivative represented by the formula (AM-2) or a salt thereof with any carboxyl group of alginic acids, the protecting group P 1 group can be produced by deprotection.

Chemical formula

[0255] Specifically, using an aqueous solution of alginic acid at 0.5% to 1% by weight and an amine derivative represented by formula (AM-1) or formula (AM-2), or a salt thereof, according to a method known in the literature, for example, the method described in "Experimental Chemistry Course, 5th Edition, 16, Synthesis of Organic Compounds IV, Carboxylic Acids and Derivatives, Acid Amides and Acid Imides, pp. 118-154, Amino Acids and Peptides, pp. 258-283, 2007, Maruzen", etc., in the presence of a condensing agent such as 1,3-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC·HCl), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent), bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl), 2-chloro-1,3-dimethylimidazolinium hexafluorophosphate (CIP), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), etc., in water, an ether solvent such as 1,4-dioxane, an alcohol solvent such as methanol or ethanol, a polar solvent such as N,N-dimethylformamide, or a mixed solvent thereof (provided that the mixed solvent is a mixed solvent to the extent that alginic acid does not precipitate), in the presence or absence of an inorganic base such as sodium bicarbonate or sodium carbonate or an organic base such as triethylamine or pyridine, reacting at a temperature of 0°C to 50°C, an alginic acid derivative represented by formula (I) or formula (II-P) can be produced. Further, by deprotecting the protecting group P 1 of formula (II-P), an alginic acid derivative represented by formula (II) can be produced.

[0256] P of formula (II-P) 1 When the group is a C 2~6 alkanoyl group such as an acetyl group or a benzoyl group, or a benzoyl group-based protecting group, it can be deprotected by the following method. An aqueous solution (for example, 0.5% to 1% by weight) of the alginic acid derivative of formula (II-P) is added with the following formula (BR-2-P) introduced:

Chemical Formula

[0257] In the method for producing the alginic acid derivative of formula (I) or the alginic acid derivative of formula (II-P), the introduction rate of the amine derivative of formula (AM-1) or formula (AM-2) can be adjusted by appropriately selecting and combining the following reaction conditions (i) to (v) etc. in consideration of the properties of the amine etc. (i) Increase or decrease the equivalent amount of the condensing agent, (ii) Raise or lower the reaction temperature, (iii) Extend or shorten the reaction time, (iv) Adjust the concentration of the alginic acid as the reaction substrate, (v) Add an organic solvent miscible with water to increase the solubility of the amine derivative of formula (AM-1) or formula (AM-2), etc.

[0258] The following shows a method for producing the amine derivative represented by formula (AM-1) or formula (AM-2).

[0259] [Production Method A] [Synthesis Method of the Amine Derivative of Formula (AM-1) and Its Salt] [Chemical Formula]

[0260] [Production Method A] [Step 1] Formula (III) [The compound of formula (III) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known from the literature. In the formula, P 2is a protecting group for an amino group and can be appropriately selected. Using an amine represented by [ ] and maleic acid, according to a method known in the literature, for example, the method described in "Experimental Chemistry Course, 5th Edition, 16, Synthesis of Organic Compounds IV, Carboxylic Acids and Derivatives, Acid Amides and Acid Imides, pages 146 - 154, 2007, Maruzen", etc., in an ether solvent such as tetrahydrofuran, 1,4 - dioxane, a halogen solvent such as methylene chloride, 1,2 - dichloroethane, a polar solvent such as N,N - dimethylformamide, etc., a solvent selected from these, in the presence of a condensing agent selected from 1,3 - dicyclohexylcarbodiimide (DCC), 1 - ethyl - 3 - (3’ - dimethylaminopropyl)carbodiimide hydrochloride (WSC·HCl), benzotriazol - 1 - yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent), bis(2 - oxo - 3 - oxazolidinyl)phosphinic chloride (BOP - Cl), 2 - chloro - 1,3 - dimethylimidazolinium hexafluorophosphate (CIP), 4 - (4,6 - dimethoxy - 1,3,5 - triazin - 2 - yl) - 4 - methylmorpholinium chloride (DMT - MM), etc., in the presence or absence of a base selected from inorganic bases such as sodium hydrogen carbonate, sodium carbonate, potassium carbonate, or organic bases such as triethylamine, N,N - diisopropylethylamine, pyridine, etc., by reacting at a temperature between 0 °C and 50 °C, the compound of formula (IV) can be produced.

[0261] Also, by reacting maleic anhydride and the amine represented by formula (III) in a solvent such as an alcohol solvent like methanol, ethanol, etc., in the presence or absence of a base such as triethylamine, N,N - diisopropylethylamine, pyridine, etc., the compound of formula (IV) can be produced.

[0262] [Production Method A]<Step 2> By using the compound represented by formula (IV) and a base such as sodium acetate, in a solvent selected from ether solvents such as tetrahydrofuran, 1,4 - dioxane, 1,2 - dimethoxyethane, hydrocarbon solvents such as toluene, benzene, xylene, halogen solvents such as 1,2 - dichloroethane, methylene chloride, chloroform, or acetic anhydride, etc., reacting at a temperature from 40 °C to the reflux temperature of the solvent, the compound of formula (VI) can be produced.

[0263] Also, [Production Method A] By using the crude compound of formula (IV) obtained in <Step 1> and a base such as sodium acetate, reacting in acetic anhydride, the compound of formula (VI) can be produced.

[0264] Also, [Production Method A] After reacting the compound or crude compound of formula (IV) obtained in <Step 1> with a suitable condensing agent to induce an active ester form, and then performing a cyclization reaction, the compound of formula (VI) can be produced.

[0265] [Production Method A]<Step 3> By using the compound represented by formula (V) [the compound of formula (V) is a commercially available compound or a compound that can be produced by a production method known from the literature from commercially available compounds] and maleimide, in a solvent selected from ether solvents such as tetrahydrofuran, 1,4 - dioxane, hydrocarbon solvents such as toluene, benzene, xylene, etc., in the presence of a phosphine reagent such as triphenylphosphine and a Mitsunobu reagent such as diethyl azodicarboxylate, diisopropyl azodicarboxylate, reacting at a temperature from - 78 °C to the reflux temperature of the solvent, the compound of formula (VI) can be produced.

[0266] [Production Method A]<Step 4> The compound represented by formula (VI) is deprotected according to the deprotection method of the protecting group of the amino group described in a method known in the literature, for example, "Protective Groups in Organic Synthesis 5th Edition, 2014, John Wiley & Sons, Greene, etc.", and the protecting group P 2 is deprotected according to its type, whereby an amine derivative of formula (AM-1) can be produced.

[0267] The amine derivative of formula (AM-1) can be obtained as a salt if necessary, and examples of the salt include hydrochloride, trifluoroacetate, and the like.

[0268] P in [Production Method A] 2 represents a protecting group of an amine, and for example, a protecting group described in "Protective Groups in Organic Synthesis 5th Edition, 2014, John Wiley & Sons, Greene, etc." can be appropriately selected. P 2 Examples of P 2 include protecting groups such as -C(O)O-tertBu group, -C(O)O-Bn group, -C(O)O-CH 2 -CH=CH 3 group, -C(O)CH 3 group, -C(O)CF 2 group, -SO 2 Ph, -SO 2 PhMe group, -SO 2 (NO

[0269] P 2When it is, for example, a -C(O)O-tertBu group, deprotection can be achieved by using an acid (such as hydrogen chloride (which can be a solution in 1,4-dioxane, cyclopentyl methyl ether, ethyl acetate, etc. containing hydrogen chloride), trifluoroacetic acid, etc.). More specifically, for example, refer to the methods known in the literature such as "Protective Groups in Organic Synthesis 5th Edition", 2014, John Wiley & Sons, Greene et al., and it is possible to select a deprotection method according to the type of protecting group.

[0270] [Production Method B] Synthesis method of the amine derivative of formula (AM-2) and its salt (Reaction Formula B) [Chemical Formula]

[0271] [Production Method B] <Step 1> Using a compound represented by formula (VIII) [The compound of formula (VIII) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known in the literature. In the formula, P 3 is a protecting group for the amino group and can be appropriately selected], according to a method known in the literature, for example, the method described in "Protective Groups in Organic Synthesis 3rd Edition, PROTECTION FOR THE THIOL GROUP, pages 457 - 486, 1999", etc., by introducing the protecting group P 1 , a compound of formula (IX) can be produced. For example, when P 1 is an acetyl group, acetyl chloride can be used; when it is a benzoyl group, benzoyl chloride can be used; when it is a trityl group, triphenylmethyl chloride can be used; when it is an EtNHCO- group, ethyl isocyanate can be used to introduce the protecting group.

[0272] Furthermore, the compound represented by formula (VIII) and carboxylic acid derivatives such as acetic acid and benzoic acid are used, and a compound of formula (IX) can be produced by performing a condensation reaction according to the method of [Production Method A] <Step 1>.

[0273] [Production Method B] <Step 2> The compound represented by formula (XI) [The compound of formula (XI) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known from the literature. In the formula, P 3 is a protecting group for an amino group and can be appropriately selected. X is a halogen atom and can be selected, for example, from a chlorine atom, a bromine atom, and an iodine atom.] and an acylthio derivative such as thiobenzoic acid, thioacetic acid, or potassium thioacetate are used, and the reaction is carried out in a solvent selected from acetonitrile, methylene chloride, N,N-dimethylformamide, etc. in the presence or absence of a base such as potassium carbonate, whereby a compound of formula (IX) can be produced.

[0274] [Production Method B] <Step 3> The compound represented by formula (IX) is deprotected according to the method for deprotecting a protecting group for an amino group described in a method known from the literature, for example, "Protective Groups in Organic Synthesis 5th Edition", 2014, John Wiley & Sons, Greene et al., etc., according to the type of the protecting group P 3 to produce an amine derivative of formula (AM-2).

[0275] The amine derivative of formula (AM-2) can be obtained as a salt if necessary, and examples of the salt include hydrochloride, trifluoroacetate, etc.

[0276] P in [Production Method B] 3represents a protecting group for an amine. For example, a protecting group can be appropriately selected from those described in "Protective Groups in Organic Synthesis 5th Edition, 2014, John Wiley & Sons, Greene et al." etc. P 3 Examples of P 2 -CH=CH 2 -C(O)CH 3 -C(O)CF 3 -SO 2 Ph, -SO 2 PhMe group, -SO 2 Ph(NO 2 ) group, etc. are protecting groups, but are not limited thereto.

[0277] P 3 When P is, for example, a -C(O)O-tertBu group, deprotection can be achieved by using an acid (hydrogen chloride (a solution of hydrogen chloride in 1,4-dioxane, cyclopentyl methyl ether, ethyl acetate, etc. is also acceptable), trifluoroacetic acid, etc.). More specifically, for example, referring to the methods known in the literature such as "Protective Groups in Organic Synthesis 5th Edition, 2014, John Wiley & Sons, Greene et al.", it is possible to select a deprotection method according to the type of protecting group.

[0278] In this specification, the amine derivatives (amino compounds) represented by formula (AM-1) or formula (AM-2) (including the sub-formulas of each formula) may form pharmaceutically acceptable salts (for example, acid addition salts). Such salts are not particularly limited as long as they are pharmaceutically acceptable salts, and examples thereof include salts with inorganic acids, salts with organic acids, and salts with acidic amino acids. Preferable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Preferable examples of salts with organic acids include salts with aliphatic monocarboxylic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, butyric acid, valeric acid, enanthic acid, capric acid, myristic acid, palmitic acid, stearic acid, lactic acid, sorbic acid, mandelic acid, etc., salts with aliphatic dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, tartaric acid, etc., salts with aliphatic tricarboxylic acids such as citric acid, salts with aromatic monocarboxylic acids such as benzoic acid, salicylic acid, etc., salts of aromatic dicarboxylic acids such as phthalic acid, salts with organic carboxylic acids such as cinnamic acid, glycolic acid, pyruvic acid, oxalic acid, salicylic acid, N-acetylcysteine, etc., salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc., and acid addition salts with acidic amino acids such as aspartic acid, glutamic acid, etc. Preferable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, etc. Among these, pharmaceutically acceptable salts are preferred.

[0279] In this specification, when a carboxyl group is substituted on an amine derivative (amino compound) represented by formula (AM-1) or formula (AM-2) (including the sub-formulas of each formula), it may form a pharmaceutically acceptable salt (for example, a base addition salt). Such salts are not particularly limited as long as they are pharmaceutically acceptable, and examples include metal salts, ammonium salts, salts with organic bases, etc. Preferred examples of metal salts include, for example, alkali metal salts such as lithium salt, sodium salt, potassium salt, cesium salt, alkaline earth metal salts such as calcium salt, magnesium salt, barium salt, and aluminum salt. Preferred examples of salts with organic bases include, for example, salts with methylamine, ethylamine, t-butylamine, t-octylamine, diethylamine, trimethylamine, triethylamine, cyclohexylamine, dicyclohexylamine, dibenzylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, morpholine, pyridine, picoline, lysine, arginine, ornithine, ethylenediamine, N-methylglucamine, glucosamine, phenylglycine alkyl ester, guanidine, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, N,N'-dibenzylethylenediamine, etc.

[0280] The salt can be obtained according to a conventional method. For example, after forming the target salt by mixing a solution containing an appropriate amount of acid or base with the compound of the present invention, it can be obtained by fractional filtration or by distilling off the mixed solvent. As a general review of salts, Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Stahl & Wermuth (Wiley-VCH, 2002) has been published, and detailed descriptions are provided in this book.

[0281] In this specification, the amine compound represented by formula (AM-1) or formula (AM-2) (including the sub-formulas of each formula) or its salt can form a solvate with solvents such as water, ethanol, glycerol, etc.

[0282] 4. Michael Addition Reaction The Michael addition reaction means that a carbon anion, an organometallic compound, an amine, an alkoxide, or a thioalkoxide undergoes a 1,4-addition (conjugate addition) reaction with an electron-deficient double bond conjugated with an electron-withdrawing group (such as an acryloyl group, a cinnamic acid group, a maleimide group, etc.) as shown in the following reaction formula to form a covalent bond.

[0283] [Chemical formula] (In the above reaction formula, EWG represents an electron-withdrawing group (such as COOR, CONHR, etc.); Nu - represents a nucleophilic reagent selected from a carbon anion (R-M), R-NH 2 (or R-NH - ), R-OH (or R-O - ), R-SH (or R-S - ), etc.; R represents various substituents such as a C 1~6 alkyl group; R x or R y represents a hydrogen atom, a C 1~6 alkyl group, or other various substituents; M represents a metal such as Li, Na, etc.)

[0284] More specifically, the Michael addition reaction in this specification is a reaction in which a thiol compound undergoes a 1,4-addition (conjugate addition) reaction with a maleimide compound as shown in the following reaction formula to form a covalent bond. [Chemical formula] (In the above reaction formula, R represents various substituents such as a C 1~6 alkyl group)

[0285] In some embodiments, the Michael addition reaction can form covalent cross-links between alginic acid molecules without generating undesirable by-products, easily, and efficiently in a short time.

[0286] In the method for crosslinking an alginic acid derivative of a preferred embodiment, almost no undesirable by-products are formed in the Michael addition reaction. In this case, in the production of a novel form of biocompatible material using alginic acid and in the formation of an alginic acid hydrogel, it becomes possible to incorporate various bioactive molecules, and also to incorporate cell substances in an alginic acid hydrogel for reconstructive surgery or gene therapy.

[0287] 5. Crosslinked Alginate Crosslinked alginate includes (i) that via divalent metal ion bonds, (ii) that via chemical bonds, or (iii) that via both divalent metal ion bonds and chemical bonds. Any crosslinked alginate has the property of being able to form a gel-like to semi-solid, and in some cases sponge-like form.

[0288] The crosslinked alginate via divalent metal ion bonds reacts extremely rapidly and is reversible, whereas the crosslinked alginate via chemical bonds reacts slowly under relatively mild conditions and is irreversible. The physical properties of crosslinked alginate can be adjusted, for example, by changing the concentration of an aqueous solution containing the divalent metal ion used (for example, an aqueous calcium chloride solution), or the introduction rate of the reactive groups introduced into alginate.

[0289] By utilizing the above crosslinking reaction, it becomes possible to create various alginate structures. For example, a specific structure can be instantaneously created from an alginate solution by an ion crosslinking reaction, and it is possible to utilize a crosslinking reaction by chemical bonds for strengthening the structure of the structure (for example, obtaining long-term stability, etc.). Also, for example, in a crosslinked alginate structure via both divalent metal ion bonds and chemical bonds, it is also possible to reversibly release the divalent metal ions incorporated by ion crosslinking to create a structure in which only the crosslinking by chemical bonds remains.

[0290] A certain embodiment of crosslinked alginate can be obtained by mixing the alginic acid derivatives of the above formula (I) and the above formula (II) and performing a Michael addition reaction.

[0291] In this specification, performing a crosslinking reaction means that, by using the alginic acid derivative represented by the formula (I) and the alginic acid derivative represented by the formula (II), a Michael addition reaction is carried out, so that a chemical crosslinking (chemical bond) is formed between the alginic acid derivative represented by the formula (I) and the alginic acid derivative represented by the formula (II), or that a divalent metal ion is allowed to coexist with the alginic acid derivative represented by the formula (I) and the alginic acid derivative represented by the formula (II), so that an ionic crosslinking (ionic bond) is formed between the respective derivatives of the alginic acid derivative represented by the formula (I) and / or the alginic acid derivative represented by the formula (II), or that both the chemical crosslinking by the Michael addition reaction and the ionic crosslinking by the divalent metal ion are formed.

[0292] In this specification, a mixed solution containing the alginic acid derivative of the formula (I) and the alginic acid derivative of the formula (II-P) (wherein, in the formula (II-P), P 1 is not a hydrogen atom), or a solution of a composition containing the alginic acid derivative of the formula (I) and the alginic acid derivative of the formula (II-P) (wherein, in the formula (II-P), P 1 is not a hydrogen atom), a protecting group P of the alginic acid derivative of the formula (II-P) 1 is deprotected by adding a deprotecting agent (for example, when P 1 is an acyl-based protecting group such as an acetyl group or a benzoyl group, a base such as an aqueous sodium hydroxide solution can be mentioned. The deprotecting agent can be appropriately selected according to the protecting group P 1 ), whereby a chemical crosslinking (chemical bond) can be formed.

[0293] Crosslinked alginic acid in a certain aspect forms a three-dimensional network structure through chemical crosslinking (crosslinking by a covalent bond formed from a maleimide group and a thiol group). A preferred alginic acid derivative is one in which the stability of the crosslinked alginic acid after crosslinking is improved.

[0294] In some aspects of crosslinked alginic acid, any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (LK-1): [Chemical formula] [In formula (LK-1), the -CONH- and -NHCO- at both ends represent amide bonds through any carboxyl group of alginic acid; -L 1 -, and -L 2 - are crosslinked alginic acids amide-bonded through the same as defined in the above aspect [1]].

[0295] In some embodiments, when preparing crosslinked alginic acid, the mixing ratio of the alginic acid derivative of formula (I) and the alginic acid derivative of formula (II) is, in terms of the weight ratio of the derivative of formula (I) to the derivative of formula (II), for example, 1 to 1.5:1, preferably 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1.

[0296] In some embodiments, when preparing crosslinked alginic acid, the mixing ratio of the alginic acid derivative of formula (II) and the alginic acid derivative of formula (I) is, in terms of the weight ratio of the derivative of formula (II) to the derivative of formula (I), for example, 1 to 4.0:1, preferably 1.5 to 4.0:1, or 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1.

[0297] In some embodiments, when preparing crosslinked alginic acid, the mixing ratio of the alginic acid derivative of formula (I) and the alginic acid derivative of formula (II) is more preferably in terms of the ratio of the introduction rate (mol%) of the reactive groups of the alginic acid derivative of formula (I) to the alginic acid derivative of formula (II), for example, 1 to 1.5:1, preferably 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1.

[0298] In some embodiments, when preparing crosslinked alginic acid, the mixing ratio of the alginic acid derivative of formula (II) and the alginic acid derivative of formula (I) is more preferably in terms of the ratio of the introduction rate (mol%) of the reactive groups of the alginic acid derivative of formula (II) to the alginic acid derivative of formula (I), for example, 1 to 4.0:1, preferably 1.5 to 4.0:1, or 1.2 to 1.5:1, or 1 to 1.2:1, more preferably 1:1.

[0299] Furthermore, in the above mixing ratio, it is also possible to replace the alginic acid derivative of formula (I) with the alginic acid derivative of formula (II) and the alginic acid derivative of formula (II) with the derivative of formula (I), respectively.

[0300] For the crosslinked alginic acid, it is not necessary that all carboxyl groups of the constituent units of alginic acid have the crosslinking of the above formula (LK-1). The introduction rate (also referred to as the crosslinking rate) of the crosslinking represented by the above formula (LK-1) in the crosslinked alginic acid is, for example, in the range of 0.1 to 80%, 0.3 to 60%, 0.5 to 30%, or 1.0 to 10%.

[0301] The concentration of the alginic acid derivative of formula (I) or formula (II) in the Michael addition reaction for obtaining the crosslinked alginic acid is usually 1 to 500 mg / mL, preferably in the range of 5 to 100 mg / mL.

[0302] The reaction temperature of the Michael addition reaction is usually an external temperature of 4 to 60 °C, preferably in the range of an external temperature of 15 to 40 °C.

[0303] The stirring time for forming the crosslinked alginic acid (hydrogel) is, for example, several seconds to 24 hours, several seconds to 12 hours, several seconds to 30 minutes, or several seconds to 10 minutes.

[0304] The reaction solvent or reaction solution used in the Michael addition reaction is not particularly limited, and examples thereof include tap water, pure water (for example, distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water, cell culture medium, phosphate buffered saline (PBS), and physiological saline, etc., and preferably ultrapure water.

[0305] The crosslinked alginic acid in some embodiments is a crosslinked alginic acid containing a chemical crosslink by a covalent bond formed by a Michael addition reaction as the crosslink and an ionic crosslink partially formed by a divalent metal ion (for example, calcium ion, etc.).

[0306] 6. Composition There is provided a composition comprising the alginic acid derivative represented by the formula (I) and the alginic acid derivative represented by the formula (II). In each formula, (ALG), -NHCO-, -L 1 -, and -L 2 - are as described in the above aspect.

Chemical formula

[0307] In the compositions of some aspects, the weight ratio of the alginic acid derivative of formula (I) to the alginic acid derivative of formula (II) (alginic acid derivative of formula (I): alginic acid derivative of formula (II)) is, for example, 1:1 to 1.5, preferably 1:1.2 to 1.5, or 1:1 to 1.2, more preferably 1:1.

[0308] In the compositions of some aspects, the weight ratio of the alginic acid derivative of formula (II) to the alginic acid derivative of formula (I) (alginic acid derivative of formula (II): alginic acid derivative of formula (I)) is, for example, 1:1 to 1.5, preferably 1:1.2 to 1.5, or 1:1 to 1.2, more preferably 1:1.

[0309] In the compositions of some aspects, the mixing ratio of the alginic acid derivative of formula (I) and the alginic acid derivative of formula (II) is, in terms of the introduction rate (mol%) ratio of the group of formula (BR-1) in the alginic acid derivative of formula (I) to the group of formula (BR-2) in the alginic acid derivative of formula (II), for example, 1:1 to 1.5, preferably 1:1.2 to 1.5, or 1:1 to 1.2, more preferably 1:1.

[0310] In the compositions of some aspects, the mixing ratio of the alginic acid derivative of formula (II) and the alginic acid derivative of formula (I) is, in terms of the introduction rate (mol%) ratio of the group of formula (BR-2) in the alginic acid derivative of formula (II) to the group of formula (BR-1) in the alginic acid derivative of formula (I), for example, 1:1 to 1.5, preferably 1:1.2 to 1.5, or 1:1 to 1.2, more preferably 1:1.

[0311] There is provided a composition comprising the alginic acid derivative represented by the formula (I) and the alginic acid derivative represented by the formula (II-P). In each formula, (ALG), -NHCO-, P 1 , -L 1 -, and -L 2 - are as described in the above aspect.

Chemical formula

[0312] In the compositions of some aspects, the weight ratio of the alginic acid derivative of formula (I) to the alginic acid derivative of formula (II-P) (alginic acid derivative of formula (I): alginic acid derivative of formula (II-P)) is, for example, 1:1 to 1.5, preferably 1:1.2 to 1.5, or 1:1 to 1.2, more preferably 1:1.

[0313] In the compositions of some aspects, the weight ratio of the alginic acid derivative of formula (II-P) to the alginic acid derivative of formula (I) (alginic acid derivative of formula (II-P): alginic acid derivative of formula (I)) is, for example, 1:1 to 1.5, preferably 1:1.2 to 1.5, or 1:1 to 1.2, more preferably 1:1.

[0314] In the compositions of some aspects, the mixing ratio of the alginic acid derivative of formula (I) to the alginic acid derivative of formula (II-P) is based on the introduction rate (mol%) ratio of the group of formula (BR-1) in the alginic acid derivative of formula (I) to the group of formula (BR-2-P) in the alginic acid derivative of formula (II-P), for example, 1:1 to 1.5, preferably 1:1.2 to 1.5, or 1:1 to 1.2, more preferably 1:1.

[0315] In the compositions of some aspects, the mixing ratio of the alginic acid derivative of formula (II-P) to the alginic acid derivative of formula (I) is based on the introduction rate (mol%) ratio of the group of formula (BR-2-P) in the alginic acid derivative of formula (II-P) to the group of formula (BR-1) in the alginic acid derivative of formula (I), for example, 1:1 to 1.5, preferably 1:1.2 to 1.5, or 1:1 to 1.2, more preferably 1:1.

[0316] 7. Crosslinked alginic acid structure The crosslinked alginic acid structure can be obtained by a method including subjecting the alginic acid derivative to a crosslinking reaction. Specific crosslinked alginic acid structures include, for example, fibrous structures, fibers, beads, gels, substantially spherical gels, and the like. Preferred crosslinked alginic acid structures have improved stability. Also, the crosslinked alginic acid structure may have the ability to retain contents inside thereof (content retention property). The crosslinked alginic acid structure can be prepared, for example, by the following methods, but is not limited thereto.

[0317] [Mixing method (1)] A mixed solution of alginic acid derivatives obtained by mixing an alginic acid derivative of formula (I) and an alginic acid derivative of formula (II), or a solution of a composition containing an alginic acid derivative represented by formula (I) and an alginic acid derivative represented by formula (II) is dropped into a solution containing divalent metal ions, whereby a crosslinked alginic acid structure, which is a specific structure in which chemical crosslinking (crosslinking by covalent bonds formed by Michael addition reaction) and ionic crosslinking (crosslinking partially formed by divalent metal ions) are formed, can be obtained.

[0318] [Mixing method (2)] A mixed solution of alginic acid derivatives obtained by mixing an alginic acid derivative of formula (I) and an alginic acid derivative of formula (II-P) (wherein in formula (II-P), P 1 is not a hydrogen atom), or a solution of a composition containing an alginic acid derivative represented by formula (I) and an alginic acid derivative represented by formula (II-P) (wherein in formula (II-P), P 1 is not a hydrogen atom) is added with a deprotecting agent so that the protecting group P 1 of the alginic acid derivative of formula (II-P) is deprotected. Examples of the deprotecting agent include a base such as an aqueous sodium hydroxide solution when P 1 is an acyl-based protecting group such as an acetyl group or a benzoyl group. The deprotecting agent is the protecting group P 1After adding (which can be appropriately selected according to the situation), it is dropped into a solution containing divalent metal ions, whereby a crosslinked alginic acid structure, which is a specific structure in which chemical crosslinking (crosslinking by covalent bonds formed by Michael addition reaction) and ionic crosslinking (crosslinking partially formed by divalent metal ions) are formed, can be formed.

[0319] [Coating method (1)] A solution containing an alginic acid derivative of formula (I) is dropped into a solution containing divalent metal ions to obtain a specific structure that is partially crosslinked. By adding the structure obtained above, such as a gel, to a solution containing an alginic acid derivative of formula (II) described above, and subjecting the surface of the structure to a further crosslinking reaction (Michael addition reaction), a crosslinked alginic acid structure can be obtained. Incidentally, this method can also be carried out by replacing the alginic acid derivative of formula (I) with the alginic acid derivative of formula (II) and the alginic acid derivative of formula (II) with the alginic acid derivative of formula (I) respectively.

[0320] [Coating method (2)] A solution containing an alginic acid derivative of formula (I) is dropped into a solution containing divalent metal ions to obtain a specific structure that is partially crosslinked. The structure obtained above, such as a gel, is added to a solution containing an alginic acid derivative of formula (II-P) (however, in formula (II-P), P 1 is not a hydrogen atom), and further, the protecting group P 1 of the alginic acid derivative represented by the above formula (II-P) 1 is deprotected. By adding a deprotecting agent (for example, when P 1 is an acyl protecting group such as an acetyl group or a benzoyl group, a base such as an aqueous sodium hydroxide solution can be mentioned. The deprotecting agent can be appropriately selected according to the protecting group P 1 ), and by subjecting the surface of the structure to a further crosslinking reaction (Michael addition reaction), a crosslinked alginic acid structure can be formed.

[0321] Also, the alginic acid derivative of formula (II-P) (however, in formula (II-P), P 1A solution containing (not a hydrogen atom) is dropped into a solution containing a divalent metal ion to obtain a specific structure that is partially cross-linked. The structure obtained above, such as a gel, is added to a solution containing the alginic acid derivative of the aforementioned formula (I), and further, the protecting group P of the alginic acid derivative of the formula (II-P) 1 is deprotected by adding a deprotecting agent (for example, when P 1 is an acyl protecting group such as an acetyl group or a benzoyl group, a base such as an aqueous sodium hydroxide solution can be mentioned. The deprotecting agent can be appropriately selected according to the protecting group P 1 . By adding (), a further cross-linking reaction (Michael addition reaction) is carried out on the surface of the structure, etc., to form a cross-linked alginic acid structure.

[0322] The divalent metal ion used in the above method is not particularly limited, and examples thereof include calcium ion, magnesium ion, barium ion, strontium ion, zinc ion, etc., and calcium ion is preferably used.

[0323] The solution containing calcium ion used in the above method is not particularly limited, and examples thereof include aqueous solutions such as calcium chloride aqueous solution, calcium carbonate aqueous solution, calcium gluconate aqueous solution, etc., and calcium chloride aqueous solution is preferably used.

[0324] The calcium ion concentration of the solution containing calcium ion used in the above method is not particularly limited, and examples thereof include 1 mM to 1 M, preferably 5 mM to 500 mM, and more preferably 10 mM to 300 mM.

[0325] The solvent or solution used in the above method is also not particularly limited, and examples thereof include tap water, pure water (for example, distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water, cell culture medium, phosphate buffered saline (PBS), and physiological saline, etc., and ultrapure water is preferably used.

[0326] The physical properties of the alginate gel can be adjusted by physical property values such as hardness, elasticity, resilience, breaking force, stress at break, and the like.

[0327] 8. Biocompatibility of Alginate Derivatives, Crosslinked Alginate, and Crosslinked Alginate Structures In this specification, alginate derivatives, crosslinked alginate, and crosslinked alginate structures have biocompatibility. In this specification, biocompatibility refers to the property of not causing reactions such as the interaction between a biomaterial (here, an alginate derivative represented by formula (I) or formula (II), and a crosslinked alginate or crosslinked alginate structure produced using both alginate derivatives) and a living body, the local reaction of tissues adjacent to the biomaterial, or a systemic reaction, and is said to have biocompatibility.

[0328] Regarding the biocompatibility of alginate derivatives, crosslinked alginate, or crosslinked alginate structures in this specification, it can be confirmed in the examples regarding biocompatibility described later.

[0329] 9. Stability of Crosslinked Alginate Structures The stability of the crosslinked alginate structure can be confirmed, for example, by measuring gel stability and measuring gel permeability.

[0330] [Method for Measuring Gel Stability] Add phosphate buffered saline (PBS) to the crosslinked alginate structure gel placed in a container, and measure the concentration (μg / mL) of alginate leaked into the PBS. The value obtained by dividing the measured alginate concentration by the total alginate concentration obtained by decomposing the crosslinked alginate structure gel and showing it as a percentage is defined as the disintegration rate. Specifically, the gel stability can be determined by the method described in the examples below.

[0331] In this specification, the gel disintegration rate of the crosslinked alginic acid structure is preferably 0% to 90%, more preferably 0% to 70%, and even more preferably 0% to 50%. The stability of the crosslinked alginic acid structure means that the lower the concentration of alginic acid leaking into the aqueous solution, that is, the lower the gel disintegration rate, the higher the stability.

[0332] [Measurement method of gel permeability] A crosslinked alginic acid structure gel encapsulating fluorescein isothiocyanate-dextran is prepared, physiological saline is added to the gel placed in a container, and the concentration of dextran leaked into the physiological saline is measured. The gel permeability is the value shown as a percentage obtained by dividing the measured concentration of dextran by the total dextran concentration obtained by decomposing the fluorescein isothiocyanate-dextran encapsulated crosslinked alginic acid structure gel. Specifically, the gel permeability can be determined by the method described in the examples below.

[0333] The gel permeability of the crosslinked alginic acid 24 hours after adding physiological saline is, for example, preferably 0% to 90%, more preferably 0% to 70%, and even more preferably 0% to 50% when encapsulating dextran with a molecular weight of 2 million. Also, when encapsulating dextran with a molecular weight of 150,000, for example, if the purpose of using the crosslinked alginic acid structure gel is the release / production of proteins or antibodies, it is preferably 1% to 100%, more preferably 10% to 100%, and even more preferably 30% to 100%. Also, if the purpose of use is an immunological barrier, it is preferably 0% to 90%, more preferably 0% to 70%, and even more preferably 0% to 50%.

[0334] The permeability of the crosslinked alginic acid structure means that the lower the permeability rate, the lower the permeability of the content and gel-external substances, and the higher the permeability rate, the higher the permeability of the content and gel-external substances.

[0335] The transmittance of the gel can be adjusted by the molecular weight and concentration of the alginic acid used, the type and introduction rate of the cross-linking group introduced into the alginic acid, the type and concentration of the divalent metal ion used for gelation, or a combination thereof.

[0336] [Method for preparing a cross-linked alginic acid structure gel encapsulating a content] For example, a cross-linked alginic acid structure gel encapsulating fluorescein isothiocyanate-dextran as a content can be prepared by the following method.

[0337] (1) Mix a solution of an alginic acid derivative represented by formula (I) and a fluorescein isothiocyanate-dextran solution. (2) Mix a solution of an alginic acid derivative represented by formula (II) into the mixed solution obtained in (1). (When formula (I) in (1) is changed to formula (II), formula (II) in (2) will be changed to formula (I)) (3) Drop the mixed solution obtained in (2) into a solution containing calcium ions, and the resulting gel forms chemical cross-links and ionic cross-links in the solution, thereby obtaining a cross-linked alginic acid structure gel encapsulating fluorescein isothiocyanate-dextran.

[0338] 10. Uses of alginic acid derivatives and cross-linked alginic acid structures Alginic acid derivatives can be used in place of conventional alginic acid in a wide range of fields such as food, medicine, cosmetics, textiles, and paper-making. Preferred uses of alginic acid derivatives or cross-linked alginic acid structures specifically include medical materials such as wound dressings, postoperative adhesion preventives, drug sustained-release substrates, cell culture substrates, and cell transplantation substrates. Diseases treatable by cell transplantation include diabetes, Parkinson's disease, hemophilia, and the like.

[0339] Examples of the shape of the cross-linked alginic acid structure when used as a medical material include fibrous structures, fibers, beads, gels, substantially spherical gels, etc. It is preferable to use beads, gels or substantially spherical gels, and more preferably substantially spherical gels.

[0340] In addition, all publications cited in this specification, such as prior art documents, published gazettes, patent gazettes and other patent documents, are incorporated herein by reference.

[0341] Also, the objects, features, advantages, and ideas of the present invention are apparent to those skilled in the art from the description in this specification, and those skilled in the art can easily implement the present invention from the description in this specification. The best mode for carrying out the invention and specific examples, etc. show preferred embodiments of the present invention and are shown for illustration or explanation, and do not limit the present invention thereto. It is apparent to those skilled in the art that various modifications can be made based on the description in this specification within the intention and scope of the present invention disclosed in this specification.

Example

[0342] For the measurement of nuclear magnetic resonance spectrum (NMR), JEOL JNM-ECX400 FT-NMR (JEOL Ltd.) was used. Liquid chromatography-mass spectrometry (LC-Mass) was measured by the following method. Using a Waters AQUITY UPLC system and a BEH C18 column (2.1 mm × 50 mm, 1.7 μm) (Waters), a mobile phase and gradient conditions of acetonitrile: 0.05% trifluoroacetic acid aqueous solution = 5:95 (0 min) - 95:5 (1.0 min) - 95:5 (1.6 min) - 5:95 (2.0 min) were used.

[0343] 1 In the 1H-NMR data, in the pattern of NMR signals, s is singlet, d is doublet, t is triplet, q is quartet, m is multiplet, br is broad, J is coupling constant, Hz is hertz, CDCl 3 is deuterated chloroform, DMSO-d 6 is deuterated dimethyl sulfoxide, D 2 O is heavy water, CD 3 OD means deuterated methanol. 1 In the 1H-NMR data, hydroxyl group (OH), amino group (NH2 ) For signals that are broad bands, such as protons of carboxyl groups (COOH), which cannot be confirmed, they are not described in the data. In LC-Mass data, M represents the molecular weight, and [M+H]+ represents the molecular ion peak.

[0344] "Room temperature" in the examples is generally assumed to indicate a temperature of about 0°C to about 35°C.

[0345] "Introduction rate" in the examples is 2 Measured by 1H-NMR in D2O, and described as "mol% (NMR integration ratio)" from the ratio of the proton integration values of the maleimide group or aromatic ring of the reactive substituent to that of alginic acid.

[0346] (Example 1) Synthesis of 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylamino group-introduced alginic acid (AL-EX-1)

[0347]

Chemical formula

[0348] <Step 1> Synthesis of tert-butyl (2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamate (EX1-IM-1)

[0349] Maleic anhydride (600 mg) was suspended in ethanol (6.0 mL), and a solution of tert-butyl (2-aminoethyl) carbamate (1.03 g) and triethylamine (0.90 mL) in ethanol (3.0 mL) was added thereto under ice-cooling. After the reaction mixture was stirred at room temperature for 2 hours, ethanol was distilled off under reduced pressure. The residue was dissolved in acetic anhydride (6.0 mL), sodium acetate (502 mg) was added, and the mixture was stirred at 70 °C for 1.5 hours. Ethyl acetate (25 mL) and water (10 mL) were added, and the layers were separated. The organic layer was washed successively with saturated aqueous sodium bicarbonate (10 mL, 3 times) and saturated brine (10 mL), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (heptane ~ 50% ethyl acetate / heptane). The obtained oily substance was triturated with heptane (20 mL). The solid was collected by filtration, washed with heptane, and dried under reduced pressure to obtain the title compound (1.01 g) as a white solid.

[0350] <Step 2> Synthesis of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (EX1-IM-2)

[0351] (Example 1) To the compound (500 mg) obtained in <Step 1> was added 4N-hydrogen chloride ethyl acetate solution (5.0 mL), and the mixture was stirred at room temperature for 1.5 hours. After adding ethyl acetate (5.0 mL), the precipitate was collected by filtration and washed with ethyl acetate. The obtained hygroscopic solid was suspended in ethyl acetate, ethyl acetate was distilled off under reduced pressure, and then dried under reduced pressure to obtain the title compound (328 mg) as a white solid.

[0352] <Step 3> Synthesis of alginic acid having 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylamino group introduced (AL-EX-1)

[0353] To an aqueous solution of sodium alginate (ALG-2, manufactured by Kimica Corporation, 20 mL) prepared at 1% by weight, the compound (36 mg) obtained in <Step 2> of (Example 1), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (84 mg), and 1 M aqueous sodium bicarbonate (252 μL) were added, and the mixture was stirred at 30 °C for 3 hours. After adding sodium chloride (200 mg), ethanol (40 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound (183 mg) as a white solid.

[0354] (Example 2) Synthesis of 2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethylamino group-introduced alginic acid (AL-EX-2)

[0355] [Chemical formula]

[0356] <Step 1> Synthesis of tert-butyl (2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamate (EX2-IM-1)

[0357] 1H-Pyrrole-2,5-dione (0.7 g), tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate (1.0 g), and triphenylphosphine (1.4 g) were dissolved in tetrahydrofuran (20 mL). Diisopropyl azodicarboxylate (1.9 mol / L toluene solution, 2.8 mL) was added dropwise under ice-salt cooling, and the mixture was stirred under ice-water cooling for 30 minutes. After stirring at room temperature for 1 hour, ethyl acetate (20 mL) and water (10 mL) were added, and the layers were separated. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (heptane - ethyl acetate) and dried under reduced pressure to obtain the title compound (0.5 g) as a pale yellow oil.

[0358] <Engineering 2> Synthesis of 1-(2-(2-aminoethoxy)ethyl)-1H-pyrrole-2,5-dione trifluoroacetate (EX2-IM-2)

[0359] (Example 2) Trifluoroacetic acid (2.3 mL) was added to the compound (0.5 g) obtained in <Engineering 1> under ice-water cooling, and the mixture was stirred at room temperature for 1 hour. Diisopropyl ether (11.3 mL) was added, and after stirring at room temperature for 30 minutes, the precipitated solid was collected by filtration and washed with diisopropyl ether. The obtained hygroscopic solid was suspended in diisopropyl ether, the solvent was distilled off, and then dried under reduced pressure to obtain the title compound (0.3 g) as a pale yellow solid.

[0360] <Engineering 3> Synthesis of alginic acid (AL-EX-2) with a 2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethylamino group introduced

[0361] Using an aqueous solution of sodium alginate (manufactured by Kimika Co., Ltd., ALG-2) prepared to 1% by weight (20 mL) and the compound (60 mg) obtained in <Engineering 2> of (Example 2), the same operation as <Engineering 3> of (Example 1) was performed to obtain the title compound (183 mg) as a white solid.

[0362] (Example 3) Synthesis of alginic acid (AL-EX-3) with a 2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethylamino group introduced

[0363]

Chemical formula

[0364] <Engineering 1> Synthesis of tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (EX3-IM-1)

[0365] A solution of di-tert-butyl dicarbonate (3.0 g) in methylene chloride (37.5 mL) was added dropwise over 4.75 hours to a solution of 2,2'-(ethane-1,2-diylbis(oxy))ethane-1-amine (3.2 g) and triethylamine (11.5 mL) in methylene chloride (30.0 mL) under ice-water cooling, and then stirred at room temperature for 18.5 hours. The reaction solution was concentrated under reduced pressure, methylene chloride (30 mL) was added to the residue, and the insoluble matter was filtered off. The filtrate was washed successively with water (10 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was dried under reduced pressure to obtain the title crude compound (2.7 g) as a colorless oil.

[0366] <Step 2> Synthesis of tert-butyl (2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethyl)carbamate (EX3-IM-2)

[0367] (Example 3) The compound (500 mg) obtained in <Step 1> and maleic anhydride (217 mg) were suspended in ethanol (5.0 mL) and stirred at room temperature for 30 minutes. Ethanol was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (heptane ~ ethyl acetate) to obtain the amide form (423 mg). Acetic anhydride (4.2 mL) was added to the obtained colorless oil and sodium acetate (100 mg), and the mixture was stirred at 40 °C for 1 hour, then at 60 °C for 1 hour, at 80 °C for 1.5 hours, and at 100 °C for 2 hours. Ethyl acetate (25 mL) and water (10 mL) were added to the reaction solution, and liquid separation was performed. The organic layer was washed successively with saturated aqueous sodium bicarbonate (10 mL), water (10 mL), and saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (heptane ~ 80% ethyl acetate / heptane) to obtain the title compound (275 mg) as a colorless oil.

[0368] <Step 3> Synthesis of 1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-pyrrole-2,5-dione trifluoroacetate (EX3-IM-3)

[0369] (Example 3) Trifluoroacetic acid (1.9 mL) was added to the compound (275 mg) obtained in <Step 2> under ice-cooling, and the mixture was stirred at room temperature for 15 minutes. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate to 30% methanol / ethyl acetate) to obtain the title compound (231 mg) as a colorless oil.

[0370] <Step 4> Synthesis of 2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethylamino group-introduced alginic acid (AL-EX-3)

[0371] Using an aqueous solution of sodium alginate (manufactured by Kimika Co., Ltd., ALG-2) prepared to 1% by weight (20 mL) and the compound (69 mg) obtained in <Step 3> of (Example 3), the same operation as <Step 3> of (Example 1) was carried out to obtain the title compound (145 mg) as a white solid.

[0372] (Example 4) Synthesis of 2-amino-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)acetamide group-introduced alginic acid (AL-EX-4)

Chemical formula

[0373] <Step 1> Synthesis of tert-butyl (2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2-oxoethyl)carbamate (EX4-IM-1)

[0374] To a mixture of commercially available 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride [CAS No. 134272-64-3] (92.43 mg) and water (750 μL), 1 M aqueous sodium bicarbonate solution (578.5 μL) was added at room temperature. To this mixture, a solution of commercially available 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl) glycinate [CAS: 3392-07-2] (150 mg) in tetrahydrofuran (1500 μL) was added at room temperature, and the mixture was stirred at the same temperature for 30 minutes. After completion of the reaction, ethyl acetate (10 mL) and water (5 mL) were added and separated. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (25% ethyl acetate / heptane to 100% ethyl acetate, ethyl acetate to 60% methanol / ethyl acetate) to obtain the title compound (74 mg) as a colorless oily compound.

[0375] <Step 2> Synthesis of 2-amino-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)acetamide trifluoroacetate (EX4-IM-2)

[0376] (Example 4) To a mixture of the compound obtained in <Step 1> (0.074 g) and dichloromethane (0.22 mL), trifluoroacetic acid (0.52 mL) was added under ice-cooling with stirring, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the reaction solution was concentrated, and diisopropyl ether (20 mL) was added. Since a gummy compound was formed, the mixture was concentrated under reduced pressure and dried to obtain the title crude compound (0.097 g) as a pale yellow gummy compound.

[0377] <Step 3> Synthesis of alginic acid (AL-EX-4) with 2-amino-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)acetamide group introduced

[0378] To an aqueous solution of sodium alginate (29.7 mL) prepared at 1 wt% (manufactured by Kimika Co., Ltd., ALG-2), 4-(4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (68.6 mg) and 1 molar concentration of sodium bicarbonate solution (68.6 μL) were added at room temperature. Subsequently, a mixture of the compound (21.4 mg) obtained in <Step 2> of (Example 4), water (1 mL) and ethanol (1 mL) was gradually added at the same temperature, and the mixture was stirred at 40 °C for 4 hours. After adding sodium chloride (300 mg), ethanol (59.3 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound (221.3 mg) as a white cottony compound.

[0379] (Example 5) Synthesis of (S)-2-amino-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3-phenylpropanamide group-introduced alginic acid (AL-EX-5) [Chemical formula]

[0380] <Step 1> Synthesis of tert-butyl (S)-(1-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (EX5-IM-1)

[0381] To a mixture of commercially available 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride [CAS: 134272-64-3] (100 mg), commercially available (tert-butoxycarbonyl)-L-phenylalanine [CAS No. 13734-34-4] (150.23 mg) and dichloromethane (1 mL), triethylamine (78.9 μL) was added under ice-cooling with stirring. To this mixture, N,N'-dicyclohexylcarbodiimide (116.8 mg) was added at the same temperature, and the mixture was stirred at room temperature for 30 minutes. After completion of the reaction, the mixture was diluted with ethyl acetate (20 mL), and the suspension was filtered. The crude product was purified by silica gel column chromatography (12% ethyl acetate / heptane to 100% ethyl acetate) to obtain the title compound (108 mg) as a white amorphous solid.

[0382] <Step 2> Synthesis of (S)-2-amino-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3-phenylpropanamide trifluoroacetate (EX5-IM-2)

[0383] (Example 5) To a mixture of the compound obtained in <Step 1> (0.1 g) and dichloromethane (1.3 mL), trifluoroacetic acid (0.7 mL) was added under ice-cooling with stirring, and the mixture was stirred at room temperature for 30 minutes. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and diisopropyl ether (20 mL) was added. The suspension was filtered to obtain the title compound (0.12 g) as a white solid.

[0384] <Step 3> Synthesis of (S)-2-amino-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3-phenylpropanamide group-introduced alginic acid (AL-EX-5)

[0385] An aqueous solution of sodium alginate (manufactured by Kimica Corporation, ALG-2) prepared at 1% by weight (29.7 mL) and the compound obtained in <Step 2> of (Example 5) (27.5 mg) were used, and the same operation as in <Step 3> of (Example 4) was performed to obtain the title compound (264.8 mg) as a white cottony compound.

[0386] (Example 6) Synthesis of (S)-2-(2-aminoacetamido)-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3-phenylpropanamide Group-Introduced Alginate (AL-EX-6) [Chemical formula]

[0387] <Step 1> Synthesis of (tert-butoxycarbonyl)glycyl-L-phenylalanine (EX6-IM-1)

[0388] To a mixture of commercially available L-phenylalanine [CAS: 63-91-2] (0.12 g) and water (1 mL), 1 molar concentration-sodium bicarbonate water (0.73 mL) was added at room temperature. To this mixture, a solution of commercially available 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)glycinate [CAS: 3392-07-2] (0.2 g) in tetrahydrofuran (4 mL) was added at room temperature, and the mixture was stirred at the same temperature. After 1 hour and 30 minutes, 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)glycinate (0.02 g) was further added, and the mixture was stirred at room temperature for 30 minutes. After completion of the reaction, ethyl acetate (10 mL) and 1N-hydrochloric acid (3 mL) were added and separated. The organic layer was washed successively with water (5 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (25% ethyl acetate / heptane to 100% ethyl acetate, ethyl acetate to 20% methanol / ethyl acetate) to obtain the title compound (0.21 g) as a white amorphous solid.

[0389] <Step 2> Synthesis of tert-butyl (S)-(2-((1-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)carbamate (EX6-IM-2)

[0390] To a mixture of commercially available 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride [CAS: 134272-64-3] (114 mg), the compound obtained in (Example 6) <Step 1> (208 mg), and dichloromethane (2080 μL), triethylamine (90 μL) was added under ice-cooling with stirring. To this mixture, N,N'-dicyclohexylcarbodiimide (133.1 mg) was added at the same temperature, and the mixture was stirred at room temperature for 1 hour and 30 minutes. After completion of the reaction, the mixture was diluted with ethyl acetate (20 mL), and the suspension was filtered. The crude product was purified by silica gel column chromatography (25% ethyl acetate / heptane to 100% ethyl acetate, ethyl acetate to 20% methanol / ethyl acetate). The collected fractions were concentrated under reduced pressure and dissolved in tert-butyl methyl ether (20 mL). This solution was washed successively with saturated aqueous sodium bicarbonate (5 mL), water (5 mL) twice, and saturated brine (5 mL), and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure to obtain the title compound (220 mg) as a white amorphous solid.

[0391] <Step 3> Synthesis of (S)-2-(2-aminoacetamido)-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3-phenylpropanamide trifluoroacetate (EX6-IM-3)

[0392] Using the compound obtained in (Example 6) <Step 2> (0.22 g), the same operation as in (Example 5) <Step 2> was performed to obtain the title compound (0.25 g) as a white solid.

[0393] <Step 4> (S)-2-(2-Aminoacetamido)-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3-phenylpropanamide Group-Introduced Alginate (AL-EX-6) Synthesis

[0394] Using an aqueous solution (49.4 mL) of sodium alginate (manufactured by Kimika Co., Ltd., ALG-2) prepared to 1 wt% and the compound (52.4 mg) obtained in <Step 3> of (Example 6), the same operation as <Step 3> of (Example 4) was performed to obtain the title compound (485 mg) as a white cottony compound.

[0395] (Example 7) S-(4-(2-Aminoethyl)carbamoyl)benzyl)ethanethioate Group-Introduced Alginate (AL-EX-7) Synthesis

[0396] [Chemical formula]

[0397] <Step 1>[ Synthesis of tert-butyl (2-(4-(chloromethyl)benzamide)ethyl)carbamate (EX7-IM-1)

[0398] Dissolve 4-(chloromethyl)benzoyl chloride (2.0 g) in tetrahydrofuran (10.0 mL), and dropwise add a solution of tert-butyl (2-aminoethyl)carbamate (1.7 g) and diisopropylethylamine (3.7 mL) in tetrahydrofuran (10.0 mL) under ice-cooling, and stir at room temperature for 1.5 hours. To the reaction solution, add ethyl acetate (30 mL) and water (10 mL), and separate the layers. The organic layer was washed successively with semi-saturated aqueous sodium bicarbonate (10 mL), water (10 mL), and saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was triturated with tert-butyl methyl ether, and the resulting solid was collected by filtration and washed with tert-butyl methyl ether to obtain the title compound (2.9 g) as a white solid.

[0399] <Step 2>[ S-(4-((2-((tert-Butoxycarbonyl)amino)ethyl)carbamoyl)benzyl)ethanethioate (EX7-IM-2) Synthesis

[0400] (Example 7) The compound obtained in <Step 1> (1.20 g) was suspended in acetonitrile (24.0 mL). Potassium thioacetate (0.53 g) was added, and the mixture was stirred at room temperature for 30 minutes. Ethyl acetate (50 mL) and water (20 mL) were added to the reaction solution, and the layers were separated. The organic layer was washed successively with water (20 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was triturated with tert-butyl methyl ether, and the solid was collected by filtration and washed with tert-butyl methyl ether. The obtained solid was dried under reduced pressure at 40 °C to obtain the title compound (1.27 g) as a white solid.

[0401] <Step 3> S-(4-((2-Aminoethyl)carbamoyl)benzyl)ethanethioate Hydrochloride (EX7-IM-3) Synthesis

[0402] (Example 7) To the compound obtained in <Step 2> (0.60 g), 4 N-hydrogen chloride / 1,4-dioxane (4.2 mL) was added under ice-cooling, and the mixture was stirred at room temperature for 30 minutes. 4 N-hydrogen chloride / 1,4-dioxane (2.1 mL) was added, and the mixture was stirred at room temperature for an additional 30 minutes. Diisopropyl ether (12.6 mL) was added to the reaction solution, and the obtained precipitate was collected by filtration, washed with diisopropyl ether, and dried under reduced pressure to obtain the title compound (0.46 g) as a white solid.

[0403] <Step 4> S-(4-((2-Aminoethyl)carbamoyl)benzyl)ethanethioate Group-Introduced Alginate (AL-EX-7) Synthesis

[0404] An aqueous solution of sodium alginate (manufactured by Kimica Corporation, ALG-2) prepared at 1% by weight (20 mL) and the compound (58 mg) obtained in <Step 3> of (Example 7) were used, and the same operations as in <Step 3> of (Example 1) were performed to obtain the title compound (189 mg) as a white solid.

[0405] (Example 7.1) Preparation of 2-(N-(4-(mercaptomethyl)benzamide))ethylamino group-introduced alginic acid (AL-EX-7.1)

[0406] [Chemical formula]

[0407] (Example 7) The compound (160 mg) obtained in <Step 4> was dissolved in water (8.0 mL), 1N aqueous sodium hydroxide solution (112 μL) was added, and the mixture was stirred at 25 °C for 2 hours to prepare a 2% by weight solution of the title compound. When ethanol precipitation treatment was performed, it became gel-like, so the solution as it was was used for the test. A part was treated with ethanol, and the disappearance of the acetyl group was confirmed by NMR.

[0408] (Example 8) Synthesis of S-(4-(3-((3-aminopropyl)amino)-3-oxopropyl)benzyl)ethanethioate group-introduced alginic acid (AL-EX-8)

[0409] [Chemical formula]

[0410] <Step 1> Synthesis of methyl 4-(3-((3-((tert-butoxycarbonyl)amino)propyl)amino)-3-oxopropyl)benzoate (EX8-IM-1)

[0411] 3-(4-(Methoxycarbonyl)phenyl)propanoic acid (1.15 g) and tert-butyl (3-aminopropyl)carbamate (0.96 g) were dissolved in methanol (11.5 mL). 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.14 g) was added, and the mixture was stirred at room temperature for 2 hours and at 40 °C for 1 hour. Ethyl acetate (20 mL) and water (20 mL) were added to the reaction mixture, and the layers were separated. The aqueous layer was extracted with ethyl acetate (10 mL). The combined organic layers were washed successively with semi-saturated aqueous sodium bicarbonate (10 mL), water (10 mL), and saturated brine (5 mL), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (10% ethyl acetate / heptane to ethyl acetate) to obtain the title compound (0.76 g) as a colorless oil.

[0412] <Step 2> Synthesis of tert-butyl (3-(3-(4-(hydroxymethyl)phenyl)propanamido)propyl)carbamate (EX8-IM-2)

[0413] (Example 8) The compound obtained in <Step 1> (560 mg) was dissolved in tetrahydrofuran (11.2 mL). Lithium aluminum hydride (146 mg) was added over 5 minutes, and the mixture was stirred at room temperature for 1 hour. Under ice-cooling, saturated aqueous sodium sulfate solution (50 drops) was added, and the mixture was stirred at the same temperature for 1 hour. The precipitated insoluble material was filtered off and washed with tetrahydrofuran. The filtrate was concentrated under reduced pressure to obtain the title compound (569 mg) as a colorless oil.

[0414] <Step 3> Synthesis of 4-(3-((3-((tert-butoxycarbonyl)amino)propyl)amino)-3-oxopropyl)benzyl 4-methylbenzenesulfonate (EX8-IM-3)

[0415] (Example 8) The compound (400 mg) obtained in <Step 2> was dissolved in tetrahydrofuran (8.0 mL). p-Toluenesulfonyl chloride (272 mg), N,N-dimethyl-4-aminopyridine (15 mg), and triethylamine (0.33 mL) were added, and the mixture was stirred at 70 °C for 6 hours. Ethyl acetate (25 mL) and water (10 mL) were added to the reaction solution, and the layers were separated. The aqueous layer was extracted with ethyl acetate (5 mL). The organic layers were combined, washed successively with semi-saturated aqueous sodium bicarbonate (10 mL), water (10 mL), and saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was triturated with tert-butyl methyl ether / heptane, and the resulting solid was collected by filtration and washed with heptane to obtain the title compound (224 mg) as a pale beige solid.

[0416] <Step 4> S-(4-(3-((3-((tert-Butoxycarbonyl)amino)propyl)amino)-3-oxopropyl)benzyl)ethanethioate (EX8-IM-4) Synthesis

[0417] (Example 8) The compound (224 mg) obtained in <Step 3> was suspended in acetonitrile (4.5 mL). Potassium thioacetate (87 mg) was added, and the mixture was stirred at room temperature for 30 minutes. Ethyl acetate (20 mL) and water (10 mL) were added to the reaction solution, and the layers were separated. The organic layer was washed successively with water (10 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (10% ethyl acetate / heptane to ethyl acetate) to obtain the title compound (189 mg) as a white solid.

[0418] <Step 5> S-(4-(3-((3-Aminopropyl)amino)-3-oxopropyl)benzyl)ethanethioate Hydrochloride (EX8-IM-5) Synthesis

[0419] (Example 8) Using the compound (189 mg) obtained in <Step 4>, the same operations as in <Step 3> of (Example 7) were performed to obtain the title compound (140 mg) as a white solid.

[0420] <Project 6> Synthesis of Alginate with S-(4-(3-((3-aminopropyl)amino)-3-oxopropyl)benzyl)ethanethioate Group Introduced (AL-EX-8)

[0421] Using an aqueous solution (20 mL) of sodium alginate (manufactured by Kimika Co., Ltd., ALG-2) prepared to 1% by weight and the compound obtained in <Project 5> of Example 8 (67 mg), the same operation as in <Project 3> of Example 1 was carried out to obtain the title compound (189 mg) as a white solid.

[0422] (Example 9) Synthesis of Alginate with S-(2-(4-aminobutanamide)ethyl) Benzothioate Group Introduced (AL-EX-9)

[0423] [Chemical formula]

[0424] <Project 1> Synthesis of S-(2-((tert-butoxycarbonyl)amino)ethyl) Benzothioate (EX9-IM-1) 2-Aminoethane-1-thiol hydrochloride (3.0 g) and triethylamine (4.1 mL) were suspended in methylene chloride (20 mL). Under ice-cooling, a solution of di-tert-butyl dicarbonate (6.3 g) in methylene chloride (10 mL) was added, and the mixture was stirred at room temperature for 2 hours. Under ice-cooling, triethylamine (4.4 mL) and benzoyl chloride (3.7 mL) were added, and the mixture was stirred at room temperature for 1 hour. tert-Butyl methyl ether (100 mL) and water (50 mL) were added to the reaction solution, and the layers were separated. The aqueous layer was extracted with tert-butyl methyl ether (50 mL). The combined organic layers were washed successively with water (50 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (heptane to 30% ethyl acetate / heptane) to obtain the title compound (5.2 g) as a colorless oil.

[0425] <Project 2> Synthesis of S-(2-aminoethyl) benzoate hydrochloride (EX9-IM-2) (Example 9) To the compound (1.0 g) obtained in <Project 1>, 4N-hydrogen chloride / 1,4-dioxane (5.0 mL) was added, and the mixture was stirred at room temperature for 50 minutes. 4N-hydrogen chloride / 1,4-dioxane (1.0 mL) was added, and the mixture was stirred at room temperature for 1 hour. Further, 4N-hydrogen chloride / 1,4-dioxane (1.0 mL) was added, and the mixture was stirred at room temperature for 1.5 hours. Diisopropyl ether (14.0 mL) was added to the reaction solution. The precipitated solid was collected by filtration, washed with diisopropyl ether, and dried under reduced pressure to obtain the title compound (0.76 g) as a white solid.

[0426] <Project 3> Synthesis of S-(2-(4-((tert-butoxycarbonyl)amino)butanamido)ethyl) benzoate (EX9-IM-3) 4-((tert-Butoxycarbonyl)amino)butanoic acid (0.50 g) and triethylamine (0.36 mL) were dissolved in tetrahydrofuran (10.0 mL). Under ice-cooling, isobutyl chloroformate (0.34 mL) was added, and the mixture was stirred at the same temperature for 20 minutes. The compound (0.64 g) obtained in <Project 2> of (Example 9) and triethylamine (0.75 mL) were added at the same temperature, and the mixture was stirred at the same temperature for 1.5 hours. Ethyl acetate (20 mL) and water (10 mL) were added to the reaction solution, and the layers were separated. The organic layer was washed successively with semi-saturated aqueous sodium bicarbonate (10 mL), water (10 mL), and saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (heptane~80% ethyl acetate / heptane) to obtain the title compound (0.74 g) as a white solid.

[0427] <Project 4> Synthesis of S-(2-(4-aminobutanamido)ethyl) benzoate hydrochloride (EX9-IM-4) (Example 9) Using the compound (0.74 g) obtained in <Project 3>, the same operation as in <Project 3> of (Example 7) was carried out to obtain the title compound (0.59 g) as a white solid.

[0428] <Project 5> Synthesis of Alginate with S-(2-(4-aminobutanamido)ethyl) benzothioate Group Introduced (AL-EX-9) Using an aqueous solution (20 mL) of sodium alginate (manufactured by Kimika Co., Ltd., ALG-2) prepared to 1 wt% and the compound (61 mg) obtained in <Project 4> of (Example 9), the same operation as <Project 3> of (Example 1) was performed to obtain the title compound (198 mg) as a white solid.

[0429] (Example 9.1) Preparation of Alginate with 4-((2-mercaptoethyl)amino)-4-oxobutylamino Group Introduced (EX-ALG-9.1)

[0430]

Chemical formula

[0431] (Example 9) The compound (25 mg) obtained in <Project 5> was dissolved in water (2.48 mL), 1 N aqueous sodium hydroxide solution (17 μL) was added, and the mixture was stirred at 25 °C for 2 hours to prepare a 1 wt% solution of the title compound.

[0432] (Example 10) Synthesis of Alginate with Methyl S-benzoyl-N-glycyl-L-cysteinate Group Introduced (AL-EX-10)

Chemical formula

[0433] <Project 1> Synthesis of Methyl (tert-butoxycarbonyl)-L-cysteinate (EX-10-IM-1) With reference to the method known in the literature (Chem. Commun. (2012) 48: 7310-7312), 2.3 molar concentration - sodium bicarbonate water (7.5 mL) was added to a mixture of commercially available L-cysteine methyl hydrochloride [CAS: 18598-63-5] (1 g) and tetrahydrofuran (7.5 mL) under ice-cooling and stirring. Subsequently, di-tert-butyl dicarbonate (1.29 mL) was added at the same temperature, and the mixture was stirred at room temperature for 70 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was acidified with 1 N-hydrochloric acid (10 mL). The solution was extracted twice with ethyl acetate (20 mL), and then the organic layer was washed successively with water (10 mL) and saturated brine (10 mL), and dried over anhydrous sodium sulfate. After filtering the organic layer, it was concentrated under reduced pressure to obtain a crude product of the title compound (1.308 g).

[0434] <Step 2> Synthesis of Methyl S-benzoyl-N-(tert-butoxycarbonyl)-L-cysteinate (EX-10-IM-2) With reference to the method known in the literature (Synthesis (2017) 49: 4879-4886), to a mixture of benzoyl chloride (0.39 mL) and methylene chloride (4 mL), a mixture of the compound EX10-IM-1 (0.4 g) obtained in (Example 10) <Step 1>, triethylamine (0.95 mL) and methylene chloride (4 mL) was added at room temperature, and the mixture was stirred at the same temperature for 1 hour and 30 minutes. After completion of the reaction, water (5 mL) and 1 N-hydrochloric acid (5 mL) were added, and the mixture was extracted three times with ethyl acetate (20 mL) and washed with saturated brine (5 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (5% ethyl acetate / heptane to 40% ethyl acetate / heptane) to obtain the title compound (0.499 g) as a colorless oil.

[0435] <Step 3> Synthesis of Methyl S-benzoyl-L-cysteinate Hydrochloride (EX-10-IM-3) (Example 10) To a mixture of the compound EX10-IM-2 (0.499 g) obtained in <Step 2> and 1,4-dioxane (3.49 mL), 4N-hydrogen chloride / 1,4-dioxane (3.49 mL) was added under water-cooled stirring, and the mixture was stirred at room temperature for 8 hours. After completion of the reaction, diisopropyl ether (40 mL) was added, and the precipitate was filtered. The recovered solid was dried under reduced pressure to obtain the title compound (0.376 g) as a white solid.

[0436] <Step 4> Synthesis of methyl S-benzoyl-N-((tert-butoxycarbonyl)glycyl)-L-cysteinate (EX-10-IM-4) (Example 10) To a mixture of the compound EX10-IM-3 (100 mg) obtained in <Step 3> and tetrahydrofuran (2000 μL), commercially available 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)glycinate [CAS: 3392-07-2] (98.7 mg) and 1 M aqueous sodium bicarbonate (362.6 μL) were added under ice-cooled stirring, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the mixture was extracted three times with ethyl acetate (10 mL), and washed successively with water (5 mL) and saturated brine (5 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (25% ethyl acetate / heptane to 100% ethyl acetate, ethyl acetate to 20% methanol / ethyl acetate) to obtain a fraction containing the title compound (124 mg).

[0437] <Step 5> Synthesis of methyl S-benzoyl-N-glycyl-L-cysteinate hydrochloride (EX-10-IM-5) (Example 10) Using the fraction containing the compound EX10-IM-4 (0.11 g) obtained in <Step 4>, the same deprotection operation as in <Step 4> of (Example 9) was performed to obtain the title compound (0.08 g) as a white solid.

[0438] <Step 6> Synthesis of methyl S-benzoyl-N-glycyl-L-cysteinato group-introduced alginic acid (AL-EX-10) To an aqueous solution (19.78 mL) of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd., A-2) prepared to 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (45.76 mg) and 1 molar concentration-sodium bicarbonate aqueous solution (45.8 μL) were added at room temperature. Subsequently, a mixture of the compound EX10-IM-5 (15.23 mg) obtained in <Step 5> of (Example 10), water (1 mL) and ethanol (1 mL) was added dropwise at the same temperature. The reaction mixture was stirred at 40 °C for 4 hours. After adding sodium chloride (200 mg), ethanol (39.6 mL) was added and stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound (181 mg) as a white solid.

[0439] (Example 10.1) Preparation of methyl glycyl-L-cysteinato group-introduced alginic acid (AL-EX-10.1) [Chemical formula]

[0440] (Example 10) The compound (50 mg) obtained in <Step 6> was dissolved in water (2.48 mL), 1N-sodium hydroxide aqueous solution (21.2 μL) was added, and stirred at room temperature for 2 hours to prepare a 2% by weight solution of the title compound.

[0441] (Example 11) Synthesis of N-(L-phenylalanyl)-S-benzoyl--L-cysteinato group-introduced alginic acid (AL-EX-11) [Chemical formula]

[0442] <Step 1> Synthesis of N-((tert-Butoxycarbonyl)-L-phenylalanyl)-S-trityl-L-cysteine (EX11-IM-1) To a mixture of (tert-Butoxycarbonyl)-L-phenylalanine [CAS: 13734-34-4] (200 mg), tetrahydrofuran (4 mL) and N-methylmorpholine (82.9 μL), isobutyl chloroformate (99 μL) was gradually added under ice-cooling and stirring. The mixture was stirred at the same temperature for 30 minutes. Then, commercially available S-trityl-L-cysteine [CAS: 2799-07-7] (274 mg) was added under ice-cooling and stirring, and the mixture was stirred at the same temperature for 30 minutes and at room temperature for 30 minutes. Then, water (5 mL) and 1 N-hydrochloric acid (5 mL) were added to stop the reaction. The reaction solution was extracted three times with ethyl acetate (10 mL), and the organic layer was successively washed with water (10 mL) and saturated brine (10 mL), and dried over anhydrous sodium sulfate. After filtering the organic layer, it was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (25% ethyl acetate / heptane to 100% ethyl acetate, ethyl acetate to 15% methanol / ethyl acetate) to obtain the title compound (397 mg) as a white amorphous solid.

[0443] <Step 2> Synthesis of N-(L-Phenylalanyl)-S-benzoyl-L-cysteine trifluoroacetate (EX11-IM-2) (Example 11) To a mixture of the compound EX11-IM-1 (100 mg) obtained in <Step 1> and methylene chloride (500 μL), trifluoroacetic acid (1500 μL) and triisopropylsilane (35.4 μL) were added under ice-cooling and stirring, and the mixture was stirred at room temperature for 30 minutes. To this mixture, benzoyl chloride (38 μL) was added dropwise under ice-cooling and stirring. After stirring at room temperature for 25 hours, benzoyl chloride (19 μL) was added at the same temperature, and stirring was continued for another 5 hours. After completion of the reaction, diisopropyl ether (20 mL) was added, and the precipitate was filtered. Since the filtration was insufficient, the filtrate was concentrated, diisopropyl ether (20 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The precipitate was filtered and dried under reduced pressure to obtain the title compound (27.5 mg) as a pale yellow solid.

[0444] <Project 3> An aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd., A-2) (19.78 mL) prepared at 1% by weight and the compound EX10-IM-5 (15.23 mg) obtained in <Project 2> of (Example 11) were used, and by performing the same operations as in <Project 6> of (Example 10), the title compound (179 mg) was obtained as a white solid.

[0445] (Example 11.1) Preparation of Sodium L-Phenylalanyl-L-Cysteinato Group-Introduced Alginate (AL-EX-11.1)

Chemical formula

[0446] (Example 11) The compound AL-EX-11 (50 mg) obtained in <Project 3> was dissolved in water (2.48 mL), 1 N aqueous sodium hydroxide solution (18.2 μL) was added, and the mixture was stirred at room temperature for 2 hours to prepare a 2% by weight solution of the title compound.

[0447] (Example 12) Synthesis of S-Benzoyl-N-Glycylglycyl-L-Cysteinato Group-Introduced Alginate (AL-EX-12)

Chemical formula

[0448] <Project 1> Synthesis of N-(tert-Butoxycarbonyl)Glycylglycyl-S-Trityl-L-Cysteine (EX12-IM-1) To a mixture of commercially available (tert-butoxycarbonyl)glycylglycine [CAS: 31972-52-8] (0.3 g) and tetrahydrofuran (6 mL), triethylamine (0.2 mL) and isobutyl chloroformate (0.19 mL) were added under ice-cooling with stirring, and the mixture was stirred at room temperature for 30 minutes. To this mixture, commercially available S-trityl-L-cysteine [CAS: 2799-07-7] (0.52 g) and triethylamine (0.4 mL) were added under ice-cooling with stirring, and the mixture was stirred at room temperature for 20 hours. The reaction was stopped by adding water (5 mL) and 1 N-hydrochloric acid (5 mL), and the mixture was extracted three times with ethyl acetate (10 mL). The organic layer was washed successively with water (5 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (25% ethyl acetate / heptane to 100% ethyl acetate, ethyl acetate to 20% methanol / ethyl acetate) to obtain the title compound (0.581 g) as a white amorphous solid.

[0449] <Step 2> S-Benzoyl-N-glycylglycyl-L-cysteine trifluoroacetate (EX12-IM-2) synthesis (Example 12) The compound EX12-IM-1 (200 mg) obtained in <Step 1> was dissolved in trifluoroacetic acid (4000 μL), and triisopropylsilane (74.8 μL) was added at room temperature. The reaction mixture was stirred at room temperature for 10 minutes, and then benzoyl chloride (80.4 μL) was added at the same temperature. After stirring at room temperature for 17 hours, benzoyl chloride (80.4 μL) was added at the same temperature, and the mixture was stirred for an additional 4 hours. After completion of the reaction, diisopropyl ether (20 mL) was added under ice-cooling with stirring, and the mixture was stirred at room temperature for 3 days. The suspension was filtered, and the recovered solid was dried under reduced pressure to obtain the title compound (140 mg) as a pale yellow solid.

[0450] <Step 3> S-Benzoyl-N-glycylglycyl-L-cysteine group-introduced alginic acid (AL-EX-12) synthesis To an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd., A-2) (49.44 mL) prepared at 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (114.4 mg) and 1 M sodium bicarbonate aqueous solution (114.4 μL) were added at room temperature. Subsequently, a mixture of the compound EX12-IM-2 (51.86 mg) obtained in <Step 2> of (Example 12), water (1 mL) and ethanol (1 mL) was added dropwise at the same temperature. The reaction mixture was stirred at room temperature for 17 hours. After adding sodium chloride (500 mg), ethanol (98.9 mL) was added and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to obtain the title compound (480 mg) as a white solid.

[0451] (Example 12.1) Preparation of sodium glycylglycyl-L-cysteinato group-introduced alginic acid (AL-EX-12.1)

Chemical formula

[0452] (Example 12) The compound AL-EX-12 (50 mg) obtained in <Step 3> was dissolved in water (2.48 mL), 1 N aqueous sodium hydroxide solution (19.4 μL) was added, and the mixture was stirred at room temperature for 2 hours to prepare a 2% by weight solution of the title compound.

[0453] (Example 13) Synthesis of S-benzoyl-N-glycyl-L-cysteinato group-introduced alginic acid (AL-EX-13)

Chemical formula

[0454] <Step 1> Synthesis of N-((tert-butoxycarbonyl)glycyl)-S-trityl-L-cysteine (EX13-IM-1) To a mixture of commercially available S-trityl-L-cysteine [CAS: 2799-07-7] (0.27 g) and water (1 mL) at room temperature, a solution of commercially available 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl) glycinate [CAS: 3392-07-2] (0.2 g) in tetrahydrofuran (2 mL) was added, and the mixture was stirred at the same temperature for 1 hour and 30 minutes. Subsequently, 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl) glycinate (0.04 g) was added, and after stirring for an additional 30 minutes, ethyl acetate (20 mL) and 1 N-hydrochloric acid (5 mL) were added and separated. The organic layer was washed successively with water (5 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product (0.43 g) of the title compound as a white amorphous solid.

[0455] <Step 2> S-Benzoyl-N-glycyl-L-cysteine trifluoroacetate (EX13-IM-2) synthesis (Example 13) To a mixture of the compound EX13-IM-1 (150 mg) obtained in <Step 1> and methylene chloride (750 μL) under ice-cooling and stirring, trifluoroacetic acid (2250 μL) and triisopropylsilane (62.2 μL) were added. The mixture was stirred at room temperature for 30 minutes, and under ice-cooling and stirring, benzoyl chloride (66.9 μL) was added dropwise, and the mixture was stirred at room temperature for 20 hours and 30 minutes. Subsequently, benzoyl chloride (133.8 μL) was added at room temperature, and after stirring at the same temperature for 24 hours, methylene chloride was concentrated under reduced pressure. Diisopropyl ether (20 mL) was added to the residue, the suspension was stirred at room temperature overnight, filtered, and dried under reduced pressure to obtain the title compound (47.7 mg) as a pale yellow solid.

[0456] <Step 3> S-Benzoyl-N-glycyl-L-cysteine group-introduced alginic acid (AL-EX-13) synthesis An aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd., A-2) prepared at 1% by weight (49.44 mL) and the compound EX13-IM-5 (45.33 mg) obtained in <Step 2> of (Example 13) were used, and by performing the same operations as in <Step 3> of (Example 12), the title compound (468 mg) was obtained as a white solid.

[0457] (Example 13.1) Preparation of Sodium Glycyl-L-Cysteinato Group-Introduced Alginate (AL-EX-13.1) [Chemical formula]

[0458] (Example 13) The compound AL-EX-13 (50 mg) obtained in <Step 3> was dissolved in water (2.478 mL), 1N aqueous sodium hydroxide solution (21.8 μL) was added, and the mixture was stirred at room temperature for 2 hours to prepare a 2% by weight solution of the title compound.

[0459] (Example 14) Synthesis of N-(L-Alanyl)-S-benzoyl-L-cysteinato Group-Introduced Alginate (AL-EX-14) [Chemical formula]

[0460] <Step 1> Synthesis of N-((tert-Butoxycarbonyl)L-alanyl)-S-trityl-L-cysteine (EX14-IM-1) (tert-Butoxycarbonyl)-L-alanine [CAS: 15761-38-3] (260.5 mg) was dissolved in tetrahydrofuran (20 mL). To this solution, N-methylmorpholine (151 μL) and isobutyl chloroformate (181 μL) were gradually added under ice-cooling and stirring. The mixture was stirred at the same temperature for 25 minutes. Then, commercially available S-trityl-L-cysteine [CAS: 2799-07-7] (500.3 mg) was added under ice-cooling and stirring, and the mixture was stirred at the same temperature for 1 hour and at room temperature for 1 hour and 40 minutes. After that, water (10 mL) and 1 N-hydrochloric acid (10 mL) were added to stop the reaction. The reaction solution was extracted three times with ethyl acetate (10 mL), and the organic layer was successively washed with water (5 mL) and saturated brine (5 mL), and dried over anhydrous sodium sulfate. After filtering the organic layer, it was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (25% ethyl acetate / heptane to 100% ethyl acetate, ethyl acetate to 15% methanol / ethyl acetate) to obtain the title compound (0.674 g) as a white amorphous solid.

[0461] <Step 2> Synthesis of N-(L-alanyl)-S-benzoyl-L-cysteine trifluoroacetate (EX14-IM-2) (Example 14) The compound EX14-IM-1 (0.674 g) obtained in <Step 1> was dissolved in trifluoroacetic acid (6.1 mL) under ice-cooling. The reaction mixture was stirred at room temperature for 1 hour and 30 minutes. Triisopropylsilane (0.26 mL) was added under ice-cooling, and the mixture was stirred at room temperature for 30 minutes. Subsequently, benzoyl chloride (0.335 mL) was added dropwise under ice-cooling and stirring, and the mixture was stirred at room temperature for 17 hours. Further, benzoyl chloride (0.335 mL) was added under ice-cooling and stirring, and the mixture was stirred at room temperature for 3 hours. Diisopropyl ether (80 mL) was added, and the suspension was stirred at room temperature overnight. After filtration and drying under reduced pressure, the title compound (0.271 g) was obtained as an off-white solid.

[0462] <Step 3> Synthesis of alginic acid with N-(L-alanyl)-S-benzoyl-L-cysteine group introduced (AL-EX-14) To an aqueous solution (39.5 mL) of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd., A-2) prepared at 1% by weight, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (121.8 mg) and 1 M aqueous sodium bicarbonate solution (91 μL) were added under water-cooled stirring. Subsequently, a mixture of the compound EX14-IM-2 (37.5 mg) obtained in <Step 2> of (Example 14), water (1 mL) and ethanol (1 mL) was added dropwise at the same temperature. The reaction mixture was stirred at room temperature for 66 hours. After adding sodium chloride (400 mg), ethanol (79 mL) was added and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained solid was dissolved in water, filtered, and then freeze-dried to obtain the title compound (0.325 g) as a white solid.

[0463] (Example 14.1) Preparation of Sodium L-Alanyl-L-Cysteinate Group-Introduced Alginate (AL-EX-14.1) [Chemical formula]

[0464] (Example 14) The compound AL-EX-14 (100 mg) obtained in <Step 3> was dissolved in water (4957 μL), 1 N aqueous sodium hydroxide solution (42 μL) was added, and the mixture was stirred at room temperature for 2 hours to prepare a 2% by weight solution of the title compound.

[0465] (Example 15) Synthesis of N-(3-Aminopropanoyl)-S-benzoyl--L-cysteinate Group-Introduced Alginate (AL-EX-15) [Chemical formula]

[0466] <Step 1> Synthesis of N-(3-((tert-Butoxycarbonyl)amino)propanoyl)-S-trityl-L-cysteine (EX15-IM-1) Using commercially available N-(tert-butoxycarbonyl)-β-alanine [CAS: 3303-84-2] (260.5 mg), tetrahydrofuran (20 mL), N-methylmorpholine (151 μL), isobutyl chloroformate (181 μL) and commercially available S-trityl-L-cysteine [CAS: 2799-07-7] (500 mg), the same operation as <Step 1> of (Example 14) was carried out to obtain the title compound (0.445 g) as a white amorphous.

[0467] <Step 2> Synthesis of N-(3-aminopropanoyl)-S-benzoyl-L-cysteine trifluoroacetate (EX15-IM-2) (Example 15) The compound EX15-IM-1 (0.445 g) obtained in <Step 1> was dissolved in trifluoroacetic acid (8.9 mL) under ice-cooling. The reaction mixture was stirred at room temperature for 70 minutes. Triisopropylsilane (0.18 mL) was added under ice-cooling and stirred at room temperature for 20 minutes. Subsequently, benzoyl chloride (0.19 mL) was added at room temperature and stirred at room temperature for 17 hours and 50 minutes. Further, benzoyl chloride (0.095 mL) was added at room temperature, stirred at the same temperature for 4 hours, the same amount of benzoyl chloride was added, and stirred at room temperature for 18 hours. Then, benzoyl chloride (0.19 mL) was added at room temperature and stirred at the same temperature for 2 hours and 40 minutes. Diisopropyl ether (50 mL) was added, the suspension was stirred at room temperature for 4 hours, filtered, and dried under reduced pressure to obtain a mixture of EX15-IM-1 and the title compound (0.445 g). A part of this mixture (0.1876 g) was dissolved in trifluoroacetic acid (1.69 mL). Subsequently, benzoyl chloride (0.18 mL) was added at room temperature and stirred at the same temperature for 3 hours. Diisopropyl ether (20 mL) was added, the suspension was stirred at room temperature overnight, filtered, and dried under reduced pressure to obtain the title compound (0.1055 g) as a white solid.

[0468] <Step 3> Synthesis of N-(3-aminopropanoyl)-S-benzoyl-L-cysteine group-introduced alginic acid (AL-EX-15) An aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd., A-2) prepared at 1% by weight (39.5 mL) and the compound EX15-IM-2 (37.5 mg) obtained in <Step 3> of (Example 15) were used, and the same operation as in <Step 3> of (Example 14) was carried out to obtain the title compound (0.348 g) as a white solid.

[0469] (Example 15.1) Preparation of Sodium (3-aminopropanoyl)-L-cysteinate Group-Introduced Alginate (AL-EX-15.1)

Chemical formula

[0470] (Example 15) The compound AL-EX-15 (100 mg) obtained in <Step 3> was dissolved in water (4957 μL), 1N aqueous sodium hydroxide solution (42 μL) was added, and the mixture was stirred at room temperature for 2 hours to prepare a 2% by weight solution of the title compound.

[0471] (Example 16) Synthesis of S-Benzoyl-N-glycyl-L-prolyl-L-cysteinate Group-Introduced Alginate (AL-EX-16)

Chemical formula

[0472] <Step 1> Synthesis of N-(tert-Butoxycarbonyl)glycyl-L-prolyl-S-trityl-L-cysteine (EX16-IM-1) Using commercially available (tert-butoxycarbonyl)glycyl-L-proline [CAS: 14296-92-5] (374.3 mg), tetrahydrofuran (20 mL), N-methylmorpholine (151 μL), isobutyl chloroformate (181 μL) and commercially available S-trityl-L-cysteine [CAS: 2799-07-7] (500 mg), the same operation as in <Step 1> of (Example 14) was carried out to obtain the title compound (0.453 g) as a white amorphous solid.

[0473] <Engineering 2> S-Benzoyl-N-glycyl-L-prolyl-L-cysteine trifluoroacetate (EX16-IM-2) Synthesis (Example 16) Using the compound EX16-IM-1 (0.453 g) obtained in <Engineering 1>, trifluoroacetic acid (4.1 mL), triisopropylsilane (0.15 mL), and benzoyl chloride (0.45 mL), and performing the same operations as in <Engineering 2> of (Example 14), the title compound (0.2251 g) was obtained as an off-white solid.

[0474] <Engineering 3> S-Benzoyl-N-glycyl-L-prolyl-L-cysteine group-introduced alginic acid (AL-EX-16) Synthesis Using an aqueous solution of sodium alginate (manufactured by Motoda Pharmaceutical Co., Ltd., A-2) prepared to 1 wt% (39.5 mL) and the compound EX16-IM-2 (45.1 mg) obtained in <Engineering 3> of (Example 16), and performing the same operations as in <Engineering 3> of (Example 14), the title compound (0.376 g) was obtained as a white solid.

[0475] (Example 16.1) Preparation of sodium glycyl-L-prolyl-L-cysteinate group-introduced alginic acid (AL-EX-16.1)

Chemical formula

[0476] (Example 16) The compound AL-EX-16 (30 mg) obtained in <Engineering 3> was dissolved in water (1484 μL), 1 N aqueous sodium hydroxide solution (16 μL) was added, and the mixture was stirred at room temperature for 2 hours to prepare a 2 wt% solution of the title compound.

[0477]

Table 2

[0478]

Table 3-1

[0479] [Table 4]

[0480] [Measurement of the introduction rate of reactive groups] The introduction rate of the reactive group means a value expressed as a percentage of the number of reactive groups introduced per uronic acid monosaccharide unit, which is a repeating unit of alginic acid. In the examples in this specification, the introduction rate of the reactive group or complementary reactive group (mol%) was calculated based on the integration ratio of 1H-NMR. Also, the amount of alginic acid required for calculating the introduction rate can be measured by the carbazole-sulfuric acid method using a calibration curve, and the amount of the reactive group or complementary reactive group can also be measured by the absorbance measurement method using a calibration curve. 1

[0481] [Measurement of molecular weight] ​​The crosslinking group-introduced alginic acid solid obtained in the example was weighed, ultrapure water was added, and a 1% aqueous solution was prepared. Subsequently, it was diluted to a 10 mmol / L phosphate buffer (pH 7.4) containing 0.2% alginic acid and 0.15 mol / L NaCl in terms of solution composition. This solution was passed through a Minisart High Flow filter (SARTORIUS) made of polyethersulfone with a pore size of 0.45 μm or 0.22 μm to remove insoluble matters, and then 200 μL of this solution was applied to a Superose6 Increase 10 / 300 GL column (GE Healthcare Sciences) for gel filtration. The gel filtration was carried out using an AKTA Explorer 10S as a chromatograph device and a 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl as a developing solvent under the conditions of a flow rate of 0.8 mL / min at room temperature. The chromatogram of each sample was prepared by monitoring the absorption at 220 nm, 240 nm, or 235 nm. As another method, the absorption at 215 nm was monitored. The peak analysis of the obtained chromatogram was performed using Unicorn 5.31 software (GE Healthcare Sciences).

[0482] The molecular weight of the crosslinking group-introduced alginic acid was determined using as standards blue dextran (molecular weight 2,000,000 Da, SIGMA), thyroglobulin (molecular weight 669,000 Da, GE Healthcare Sciences), ferritin (molecular weight 440,000 Da, GE Healthcare Sciences), aldolase (molecular weight 158,000 Da, GE Healthcare Sciences), conalbumin (molecular weight 75,000 Da, GE Healthcare Sciences), ovalbumin (molecular weight 44,000 Da, GE Healthcare Sciences), ribonuclease A (molecular weight 13,700 Da, GE Healthcare Sciences), and aprotinin (molecular weight 6500 Da, GE Healthcare Sciences). A calibration curve was created from the liquid volume and molecular weight at the absorption peak at 280 nm of each component when gel filtration was performed under the same conditions. Two calibration curves were created, one from blue dextran to ferritin and the other from ferritin to aprotinin. Using this calibration curve, the molecular weight (Mi) at the elution time i of the previously obtained chromatogram was calculated. Next, the absorbance at the elution time i was read as Hi, and the weight average molecular weight (Mw) was determined from these data using the following equation.

[0483] [Number]

[0484] The molecular weight of sodium alginate before introduction of the reactive group was determined as follows. That is, each alginic acid was weighed considering the loss on drying, and ultrapure water was added to prepare a 1% aqueous solution. Next, it was diluted to a 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl with an alginic acid concentration of 0.2%. After removing insolubles with a hydrophilic PVDF Mylex GV33 filter (MERCK-Millipore) with a pore size of 0.22 μm, 200 μL was subjected to gel filtration, and gel filtration was performed under the same conditions as for the crosslinking group-introduced alginic acid. Detection was performed using a differential refractometer. As another method, insolubles were removed by passing through a polyethersulfone Minisart High Flow filter (SARTORIUS) with a pore size of 0.45 μm.

[0485] The weight-average molecular weight of sodium alginate before the introduction of the crosslinking group was determined by the same method as the method for calculating the molecular weight of the crosslinking group-introduced alginic acid. However, Hi was calculated from the data of the differential refractometer.

[0486] (Examples 1) to (Example 3), (Examples 7) to (Example 9), the molecular weight of sodium alginate (manufactured by Kimica Corporation, ALG-2) before the introduction of the crosslinking group was eluted broadly from 2.6 million Da to 145,000 Da, and the weight-average molecular weight was calculated to be 1.46 million Da.

[0487] (Examples 4) to (Example 6), the molecular weight of sodium alginate (manufactured by Kimica Corporation, ALG-2) before the introduction of the crosslinking group was eluted broadly from 9,600 Da to 2.51 million Da, and the weight-average molecular weight was calculated to be 1.38 million Da. (Examples 10) to (Example 16.1), the molecular weight of sodium alginate (manufactured by Motida Pharmaceutical Co., Ltd., A-2) before the introduction of the crosslinking group is the molecular weight described in Table 1 above.

[0488] <Measurement of gel stability (1)> (Example 2) The alginic acid derivative (AL-EX-2) produced in the same manner as in <Step 3> was dissolved in water so that the concentration became 0.5% by weight to obtain an aqueous alginic acid solution (2-1). Furthermore, 3 volumes of phosphate buffered saline (PBS) was added to 2% by weight solutions of alginic acid derivatives (AL-EX-7.1) solution, (AL-EX-10.1) solution, (AL-EX-11.1) solution, (AL-EX-12.1) solution and (AL-EX-13.1) solution produced in the same manner as in (Example 7), (Example 10), (Example 11), (Example 12) and (Example 13) to make it 0.5% by weight, and aqueous alginic acid solutions (7-1), (10-1), (11-1), (12-1) and (13-1) were obtained.

[0489] Equal volumes of 250 μL each of the aqueous alginic acid solutions (2-1), (7-1), (10-1), (11-1), (12-1), or (13-1) were mixed, and 40 mL of a calcium chloride solution with a concentration of 30 mmol / L was added. The mixture was stirred for 5 minutes to obtain an alginic acid gel. This gel was washed once with 10 mL of PBS to obtain a chemically crosslinked alginic acid gel. 19.5 mL of PBS was added to this gel, and the mixture was shaken at 37°C. The aqueous solution was collected over time, and the same volume of PBS as the collected amount was replenished. After the test ended, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and the mixture was shaken overnight at 37°C to completely disintegrate the gel, and the aqueous solution was collected. The alginic acid concentration in the collected aqueous solution was measured by the carbazole-sulfuric acid method, and the value obtained by correcting the amount of alginic acid in the aqueous solution at each time point with the amount of alginic acid already recovered was divided by the total amount of alginic acid calculated from the alginic acid concentration at all time points and the alginic acid concentration after the test ended. The value expressed as a percentage was defined as the disintegration rate and used as an index of gel stability.

[0490] The results of Figure 1 were obtained. Since the disintegration rate of the crosslinked alginic acid gel (beads) was 1% or less after 24 hours and 40% or less after 96 hours, the stability of the gel was confirmed. That is, it was suggested that the structure of the fabricated (bead) structure was maintained over a long period due to the formation of chemical crosslinks by the Michael reaction.

[0491] <Measurement of Gel Stability (2)> The aqueous alginate solutions (2-1), (7-1), (10-1), (11-1), (12-1) or (13-1) obtained in <Measurement of gel stability (1)> were mixed in equal amounts of 250 μL each, and 40 mL of a calcium chloride solution with a concentration of 30 mmol / L was added. The mixture was stirred for 5 minutes to obtain an alginate gel. This gel was washed once with 10 mL of physiological saline to obtain a chemically cross-linked alginate gel. To this gel, 19.5 mL of a 5 mM potassium ethylenediaminetetraacetate dihydrate (EDTA·2K) / physiological saline aqueous solution was added, and the mixture was shaken at 37°C. After 24 hours, the aqueous solution was collected, and the same amount of 5 mM EDTA·2K / physiological saline aqueous solution as the collected amount was replenished. After the test was completed, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and the mixture was shaken overnight at 37°C to completely disintegrate the gel, and the aqueous solution was collected. The alginate concentration in the collected aqueous solution was measured by the carbazole-sulfuric acid method, and the value obtained by correcting the amount of alginate in the aqueous solution at each time point with the amount of alginate already collected was divided by the total amount of alginate calculated from the alginate concentration at all time points and the alginate concentration after the test was completed, and the value expressed as a percentage was defined as the disintegration rate, which was used as an index of gel stability.

[0492] The results shown in Fig. 2 were obtained. The cross-linked alginate gel (beads) had a disintegration rate of about 40% even after 24 hours, and the stability of the gel was confirmed. That is, it was suggested that the structure of the (beads) structure prepared by EDTA treatment was maintained over a long period.

[0493] <Measurement of gel stability (3)> (Example 5) An alginic acid derivative (AL-EX-5) produced in the same manner as in <Process 3> was dissolved in water to a concentration of 1.0% by weight to obtain an aqueous alginic acid solution (5-1). Further, an equal amount of PBS was added to a 2% by weight solution of an alginic acid derivative (AL-EX-7.1) produced in the same manner as in (Example 7) and the solutions (AL-EX-10.1), (AL-EX-11.1), (AL-EX-12.1), and (AL-EX-13.1) obtained in (Example 10), (Example 11), (Example 12), and (Example 13) to make the concentration 1.0% by weight, thereby obtaining aqueous alginic acid solutions (7-2), (10-2), (11-2), (12-2), and (13-2).

[0494] Equal amounts of 250 μL each of the aqueous alginic acid solution (5-1) and the aqueous alginic acid solutions (7-2), (10-2), (11-2), (12-2), or (13-2) were mixed, and 40 mL of a calcium chloride solution having a concentration of 30 mmol / L was added, followed by stirring for 5 minutes to obtain an alginic acid gel. This gel was washed once with 10 mL of PBS to obtain a chemically crosslinked alginic acid gel. 19.5 mL of PBS was added to this gel, and the mixture was shaken at 37°C. The aqueous solution was collected over time, and the same amount of PBS as the collected amount was replenished. After the test, 10 μL of alginic acid lyase (Nippon Gene, 319-08261) was added to the test solution, and the mixture was shaken overnight at 37°C to completely disintegrate the gel, and the aqueous solution was collected. The alginic acid concentration in the collected aqueous solution was measured by the carbazole-sulfuric acid method, and the value obtained by correcting the amount of alginic acid in the aqueous solution at each time point with the amount of alginic acid already collected was divided by the total amount of alginic acid calculated from the alginic acid concentration at all time points and the alginic acid concentration after the end of the test, and the value expressed as a percentage was defined as the disintegration rate and used as an index of gel stability.

[0495] The results are shown in Figure 3. The disintegration rate of each of the crosslinked alginic acid gels (beads) after 96 hours was 26% or less. In addition, since the disintegration rate of the crosslinked alginic acid gel prepared from (AL-EX-7.1) and (AL-EX-2) as a control was 28.8% at the 96-hour time point, it was suggested that the stability was equal to or higher than that of the control.

[0496] <Measurement of Gel Stability (4)> 250 μL each of the aqueous alginic acid solutions (5-1) and (7-2), (10-2), (11-2), (12-2), or (13-2) obtained in <Measurement of Gel Stability (3)> were mixed in equal amounts, and 40 mL of a calcium chloride solution with a concentration of 30 mmol / L was added. The mixture was stirred for 5 minutes to obtain an alginic acid gel. This gel was washed once with 10 mL of physiological saline to obtain a chemically cross-linked alginic acid gel. 19.5 mL of a 5 mM EDTA·2K / physiological saline aqueous solution was added to this gel, and it was shaken at 37°C. After 24 hours, the aqueous solution was recovered, and the same amount of 5 mM EDTA·2K / physiological saline aqueous solution as the recovered amount was replenished. After the test ended, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and it was shaken overnight at 37°C to completely disintegrate the gel, and the aqueous solution was recovered. The alginic acid concentration in the recovered aqueous solution was measured by the carbazole-sulfuric acid method, and the value obtained by correcting the amount of alginic acid in the aqueous solution at each time point with the amount of alginic acid already recovered was divided by the total amount of alginic acid calculated from the alginic acid concentration at all time points and the alginic acid concentration after the test ended, and the value expressed as a percentage was defined as the disintegration rate and used as an index of gel stability.

[0497] The results shown in Fig. 4 were obtained. The cross-linked alginic acid gel (beads) had a disintegration rate of about 30% even after 24 hours, and the stability of the gel was confirmed. That is, it was suggested that the structure of the (beads) structure prepared by EDTA treatment was maintained over a long period.

[0498] <Measurement of Gel Stability (5)> (Example 2) The alginic acid derivative (AL-EX-2) produced in the same manner as in <Step 3> was dissolved in water so that the concentration became 1.0% by weight to obtain an aqueous alginic acid solution (2-3). Furthermore, PBS was added in equal amounts to the 2% by weight alginic acid derivative (AL-EX-7.1) solution and (AL-EX-16.1) solution produced in the same manner as in (Example 7) to make it 1.0% by weight, and aqueous alginic acid solutions (7-2) and (16-1) were obtained. 300 μL each of the aqueous alginic acid solution (2-3) and the aqueous alginic acid solution (7-2) or (16-1) were mixed in equal amounts and pipetted three times. 500 μL of this solution was dispensed into 2.5 mL of a calcium chloride solution with a concentration of 55 mmol / L, gently shaken, and then allowed to stand for 5 minutes. Further, 3.5 mL of a calcium chloride solution with the same concentration was added and allowed to stand for 5 minutes. This gel was washed twice with 5 mL of physiological saline to obtain a chemically cross-linked alginic acid gel. 19.5 mL of PBS was added to this gel, shaken at 37°C, and the aqueous solution was collected over time. The same amount of PBS as the collected amount was replenished. After the test, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, shaken overnight at 37°C to completely disintegrate the gel, and the aqueous solution was collected. The alginic acid concentration in the collected aqueous solution was measured by the carbazole-sulfuric acid method, and the value obtained by correcting the amount of alginic acid in the aqueous solution at each time point with the amount of alginic acid already collected was divided by the total amount of alginic acid calculated from the alginic acid concentration at all time points and the alginic acid concentration after the end of the test, and the value expressed as a percentage was defined as the disintegration rate and used as an index of gel stability.

[0499] The results shown in Fig. 8 were obtained. Since the disintegration rate of the cross-linked alginic acid gel (beads) was 15% or less after 24 hours and 20% or less after 96 hours, the stability of the gel was confirmed. That is, it was suggested that the structure of the produced (bead) structure was maintained over a long period due to the formation of chemical cross-links by the Michael reaction.

[0500] <Measurement of Gel Stability (6)> (Example 2) The alginate derivatives (AL-EX-2) and (AL-EX-5) produced in the same manner as in <Step 3> of (Example 2) and <Step 3> of (Example 5) were dissolved in water to a concentration of 1.0 wt% to obtain alginate aqueous solutions (2-1) and (5-1). Further, a 2 wt% alginate derivative (AL-EX-7.1) solution produced in the same manner as in (Example 7) and the (AL-EX-14.1) solution, (AL-EX-15.1) solution, and (AL-EX-16.1) solution obtained in (Example 14), (Example 15), and (Example 16) were added with an equal amount of PBS to make it 1.0 wt% to obtain alginate aqueous solutions (7-2), (14-1), (15-1), and (16-1). Equal amounts of 300 μL each of the alginate aqueous solution (2-1) or (5-1) and the alginate aqueous solutions (7-2), (14-1), (15-1), or (16-1) were mixed and pipetted several times. 500 μL of this solution was dispensed into 2.5 mL of a calcium chloride solution with a concentration of 55 mmol / L, gently shaken, and then allowed to stand for 5 minutes. Further, 3.5 mL of a calcium chloride solution with the same concentration was added and allowed to stand for 5 minutes. This gel was washed twice with 5 mL of physiological saline to obtain a chemically crosslinked alginate gel. 19.5 mL of a 5 mM dipotassium ethylenediaminetetraacetate dihydrate (EDTA·2K) / physiological saline aqueous solution was added to this gel, shaken at 37°C, and after 24 hours, the aqueous solution was recovered, and the same amount of 5 mM EDTA·2K / physiological saline aqueous solution as the recovered amount was replenished. After the test, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, shaken overnight at 37°C to completely disintegrate the gel, and the aqueous solution was recovered. The alginate concentration in the recovered aqueous solution was measured by the carbazole sulfuric acid method, and the value obtained by correcting the amount of alginate in the aqueous solution at each time point with the amount of alginate already recovered was divided by the total amount of alginate calculated from the alginate concentration at all time points and the alginate concentration after the test was expressed as a percentage, and this value was defined as the disintegration rate and used as an index of gel stability.

[0501] The results of Fig. 9 were obtained. The crosslinked alginic acid gel (beads) had a disintegration rate of about 40% even after 24 hours, and the stability of the gel was confirmed. That is, it was suggested that the structure of the (beads) structure prepared by EDTA treatment was maintained over a long period of time.

[0502] <Measurement of gel permeability> (Example 2) The alginic acid derivative (AL-EX-2) produced by the same method as in <Step 3> and the alginic acid derivative (AL-EX-5) produced by the same method as in <Step 3> of (Example 5) were dissolved in water to a concentration of 2.0% to prepare an aqueous alginic acid solution. To this aqueous alginic acid solution, 4 / 5 volume of fluorescein isothiocyanate-dextran with a molecular weight of 150,000 prepared at 1 mg / mL (Sigma-Aldrich, FD150S), and 2.2 volumes of PBS were added to obtain 0.5% aqueous alginic acid solutions (2-2) and (5-2) containing 0.2 mg / mL fluorescein isothiocyanate-dextran. Furthermore, 3 volumes of PBS were added to 2% alginic acid derivative (AL-EX-7.1) solution, (AL-EX-10.1) solution, (AL-EX-11.1) solution, (AL-EX-12.1) solution and (AL-EX-13.1) solution produced by the same method as in (Example 7), (Example 10), (Example 11), (Example 12) and (Example 13) to make it 0.5 wt%, and aqueous alginic acid solutions (7-1), (10-1), (11-1), (12-1) and (13-1) were obtained.

[0503] An aqueous alginic acid solution (2-2) or (5-2) and aqueous alginic acid solutions (7-1), (10-1), (11-1), (12-1) or (13-1) were each mixed in equal amounts of 250 μL, and 40 mL of a calcium chloride solution with a concentration of 30 mmol / L was added. The mixture was stirred for 5 minutes to obtain an alginic acid gel. This gel was washed once with 10 mL of physiological saline to obtain a fluorescein isothiocyanate-dextran encapsulated chemically crosslinked alginic acid gel. 19.5 mL of physiological saline was added to this gel, and it was shaken at 37°C. The aqueous solution was collected over time, and the same amount of PBS as the collected amount was replenished. After the test, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and it was shaken at 37°C for 3 hours or more to completely disintegrate the gel, and the aqueous solution was collected. The dextran concentration in the collected aqueous solution was measured by fluorescence quantification (excitation light: 485 nm, fluorescence: 535 nm), and the value obtained by dividing the amount of dextran at each time point by the amount of dextran after the test end and expressing it as a percentage was defined as the transmittance.

[0504] The results of Fig. 5 were obtained. The transmittance after 3 hours was about 14 to about 20%. Also, the transmittance after 24 hours was about 30 to about 37%.

[0505] Also, the results of Fig. 6 were obtained. The transmittance after 3 hours was about 17 to about 27%. Also, the transmittance after 24 hours was about 31 to about 42%.

[0506] <Measurement of Gel Transmittance (2)> (Example 2) An alginic acid derivative (AL-EX-2) produced by the same method as in <Step 3> was dissolved in water to a concentration of 2.0% by weight to prepare an aqueous alginic acid solution. To this aqueous alginic acid solution (1.0 mL), fluorescein isothiocyanate-dextran with a molecular weight of 150,000 prepared at 1 mg / mL (Sigma-Aldrich, FD150S) (0.4 mL) and PBS (0.6 mL) were added to obtain a 1.0% aqueous alginic acid solution (2-4) containing 0.2 mg / mL fluorescein isothiocyanate-dextran. Furthermore, the same method as in (Example 7) and (Example 16) was used to produce 2% alginate derivative (AL-EX-7.1) solution and (AL-EX-16.1) solution. An equal amount of PBS was added to these solutions to make the concentration 1.0 wt%, and alginic acid aqueous solutions (7-2) and (16-1) were obtained.

[0507] 300 μL each of alginic acid aqueous solution (2-4) and alginic acid aqueous solution (7-2) or (16-1) were mixed in equal amounts and pipetted three times. 500 μL of this solution was dispensed into 2.5 mL of calcium chloride solution with a concentration of 55 mmol / L, gently shaken, and then left to stand for 5 minutes. Further, 3.5 mL of calcium chloride solution with the same concentration was added and left to stand for 5 minutes. This gel was washed twice with 5 mL of physiological saline to obtain a fluorescein isothiocyanate-dextran encapsulated chemically crosslinked alginate gel. 19.5 mL of physiological saline was added to this gel, and it was shaken at 37°C. The aqueous solution was collected over time, and the same amount of PBS as the collected amount was replenished. After the test, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and it was shaken at 37°C for 3 hours or more until all the gel collapsed, and the aqueous solution was collected. The dextran concentration in the collected aqueous solution was measured by fluorescence quantification method (excitation light: 485 nm, fluorescence: 535 nm), and the value obtained by dividing the dextran amount at each time point by the dextran amount after the end of the test and expressing it as a percentage was taken as the transmittance.

[0508] The results shown in Figure 10 were obtained. The transmittance after 3 hours was about 5 to about 10%. Also, the transmittance after 24 hours was about 26 to about 37%.

[0509] [Biocompatibility Evaluation] (Example 2), (Example 4), (Example 5), (Example 6), (Example 7), the alginate derivatives (AL-EX-2), (AL-EX-4), (AL-EX-5), (AL-EX-6), (AL-EX-7.1) obtained, and (Example 10), (Example 11), (Example 12), (Example 13), (AL-EX-10.1), (AL-EX-11.1), (AL-EX-12.1), and (AL-EX-13.1) produced in the same manner as above were prepared into a PBS solution with a concentration of 1.0 wt% to obtain an aqueous alginate solution. This was filter-sterilized using a Mini Sart High Flow (Sartorius, 16532GUK) to obtain 1.0% cross-linking group-introduced alginate PBS solutions (2), (4), (5), (6), (7), (10), (11), (12), and (13). After seeding HeLa cells cultured for 1 day in a 96-well plate to a cell concentration of 5×10^3 cells / well, a 1.0% cross-linking group-introduced alginate PBS solution was added at a final concentration of 0.1% in combinations of (2) or (5), and (7), (10), (11), (12) or (13), and combinations of (4) or (6) and (7). After culturing for 1 day, ATP activity was evaluated as an index of cytotoxicity using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571).

[0510] The results shown in Fig. 7 were obtained. In all of the cross-linked alginate gels, since ATP activity of a comparable level was confirmed even when compared with the controls, PBS and alginate (A-2), it was suggested that the cross-linked alginate gels were not cytotoxic, and it was suggested that the alginate structure in which a chemical cross-link was formed by the Michael reaction had biocompatibility.

[0511] [Biocompatibility Evaluation (2)] (Example 2), (Example 5), and the alginate derivatives (AL-EX-2), (AL-EX-5), (AL-EX-7.1) obtained in (Example 7), and (AL-EX-14.1), (AL-EX-15.1), and (AL-EX-16.1) produced in the same manner as in (Example 14), (Example 15), and (Example 16) were prepared into a PBS solution with a concentration of 1.0% by weight to obtain an aqueous alginate solution. This was filter sterilized using a Mini Sart High Flow (Sartorius, 16532GUK) to obtain 1.0% crosslinking group-introduced alginate PBS solutions (2), (5), (7), (14), (15), and (16). After seeding HeLa cells cultured for 1 day in a 96-well plate to a cell concentration of 5×10^3 cells / well, a 1.0% crosslinking group-introduced alginate PBS solution was added in combinations of (2) or (5), and (7), (14), (15), or (16) to a final concentration of 0.1%. After culturing for 1 day, the ATP activity was evaluated as an index of cytotoxicity using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571).

[0512] The results shown in Fig. 11 were obtained. In all of the crosslinked alginate gels, it was suggested that the crosslinked alginate gels had no cytotoxicity because the same level of ATP activity was confirmed even when compared with the control PBS and alginate (A-2), suggesting that the alginate structure formed by a Michael reaction with chemical crosslinking has biocompatibility.

Claims

1. The following formula (II-P): 【Chemical 126】 (In formula (II-P), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; P １ is a hydrogen atom or a protecting group for a thiol group (-SH group) selected from the group consisting of an acetyl group, an ethylcarbonyl group, a benzoyl group, a naphthylcarbonyl group, a trityl group, a diphenylmethyl group, a methylaminocarbonyl group, and an ethylaminocarbonyl group; -L ２ - is the following partial structural formula [in each formula, the outside of the dashed lines at both ends is not included]: 【Chemical 57】 (In formula (L2-3), formula (L2-4), formula (L2-5) and formula (L2-6), the hydrogen atom of the methylene group (-CH ２ -) is an oxo group (=O), a halogen atom, a hydroxyl group, C １～６ alkyl group, hydroxy C １～６ alkyl group, thiol C １～６ alkyl group, C １～６ alkylthio C １～６ alkyl group, -COOH group, -COOM group (M is Li, Na, K, or 1 / 2 Ca), -COO(C １～６ alkyl) group, -NR ａ R ｂ group, (R ａ R ｂ N)-C １～６ alkyl group, (R ａ R ｂ N)C(=O)-C １～６ alkyl group (in the above -NR ａ R ｂ group, (R ａ R ｂ N)-C １～６ alkyl group, or (R ａ R ｂ N)C(=O)-C １～６ alkyl group, R ａ and R ｂ are each independently a hydrogen atom, C １～６ alkyl group, C ２～７ alkanoyl group, or C １～６ alkylsulfonyl group), guanidino C １～６ alkyl group, C ７～１６ aralkyl group, hydroxy C ６～１０ aryl C １～６ alkyl group, or heteroaryl C １～６ alkyl group, and may be replaced by 1 to 10 groups selected therefrom; In formula (L2-3), formula (L2-4), formula (L2-5), and formula (L2-6), when two hydrogen atoms of the same methylene group (—CH ２ —) are replaced by C １～６ alkyl groups, the alkyl groups may be bonded to each other to form a C ３～８ cycloalkyl ring; In formula (L2-3), formula (L2-4), formula (L2-5) and formula (L2-6), the -NH- group may form a non-aromatic heterocyclic ring together with the substituent bonded to the adjacent carbon atom; n2 is an integer from 1 to 18; m3 is an integer from 1 to 10; n3 is an integer from 1 to 10; j2 is an integer from 0 to 9), and is a linker selected from the group consisting of) an alginic acid derivative represented by).

2. -L ２ - is the following partial structural formula [in each formula, the outside of the broken lines at both ends is not included]: 【Chemical Formula 60】 The alginic acid derivative according to claim 1, which is a linker selected from the group consisting of.

3. P １ The alginic acid derivative according to claim 1 or 2, wherein P is a hydrogen atom, an acetyl group or a benzoyl group.

4. The following formula (BR-2-P): 【Chemical 128】 (In formula (BR-2-P), P １ , -L ２ - is the same as the definition described in any one of claims 1 to 3), and the introduction rate of the reactive group represented by is 1% to 30%, and the alginic acid derivative according to any one of claims 1 to 3.

5. The alginic acid derivative according to any one of claims 1 to 4, wherein the weight average molecular weight measured by the gel filtration chromatography method of the alginic acid derivative is 100,000 Da to 3,000,000 Da.

Images