Multilayer structure using chemically crosslinked alginic acid

Abstract

There has been required a novel and practicable multilayer structure that has enhanced structure stability and, therefore, is improved in the enlargement of device size, easiness in processing, etc. A structure according to the present invention that comprises a core layer containing a pharmacological ingredient embedded in a chemically crosslinked alginic acid, a cationic polymer layer coating the core layer, and an anionic polymer layer coating the cationic polymer layer.

Description

Multilayer structure using chemically cross-linked alginate

[0001] The present invention relates to a multilayer structure using chemically crosslinked alginate. More specifically, the present invention relates to a structure including a core layer containing chemically crosslinked alginate in which pharmacological components such as cells are embedded, a cationic polymer layer covering the core layer, and an anionic polymer layer such as alginate or chemically crosslinked alginate covering the cationic polymer layer, as well as a method for producing the structure.

[0002] In recent years, techniques for transplanting cells encapsulated in polymer beads or polymer capsules into the body have been investigated. For example, microcapsules using alginate are known as cell delivery devices used for transplantation. Alginate gels instantly in the presence of divalent ions such as calcium ions and barium ions, and is therefore used to encapsulate cells within the gel. Additionally, three-layer alginate capsules of alginate / PLL or PLO / alginate have been reported, in which alginate is coated with cationic polymers such as poly-L-lysine (PLL) or poly-L-ornithine (PLO), which are then further coated with alginate (e.g., Non-Patent Document 1, Patent Document 1).

[0003] Non-Patent Document 2 describes that in three-layered alginate capsules, the outermost layer of alginate decomposes in vivo, exposing PLL, which can trigger an immune response. Non-Patent Document 2 also discloses experimental results comparing the physical properties of alginate capsules coated with PLO instead of PLL with those using PLL. According to this document, alginate capsules coated with PLO have higher physical strength and better permselectivity than those coated with PLL. Non-Patent Document 3 describes that alginate capsules coated with PLO exhibit a lower degree of fibrosis than those coated with PLL.

[0004] Under these circumstances, various alginate capsules with a three-layer structure have been developed using chemically structurally modified materials (e.g., Patent Documents 2 to 6). Chemically crosslinked alginate capsules using the Huisgen reaction (Patent Document 7 and Patent Document 9, published after the application on which the priority of this application is based) and photocrosslinking reaction have been reported (Patent Document 8).

[0005] International Publication No. 2001 / 052871 International Publication No. 2014 / 171842 International Publication No. 2018 / 151186 International Publication No. 98 / 049202 Chinese Publication No. 105078923 International Publication No. 2010 / 139061 International Publication No. 2019 / 240219 International Publication No. 2019 / 168058 International Publication No. 2021 / 125255

[0006] Pharmaceutical Journal, Vol. 125, No. 8, pp. 601-615, 2005; Biomaterials, Vol. 26, No. 34, pp. 6846-52, 2005; American Diabetes Association 60th Scientific Sessions, 2000, Abstract, No. 448-P

[0007] Conventional multilayer structures using alginate as a core layer have been limited in their applications to microcapsules, etc., due to their structural stability. There has been a demand for a new, practical multilayer structure that allows for the upsizing of devices and simplifies processing due to improved structural stability.

[0008] As a result of extensive research, the present inventors have discovered the following (1) to (7). Based on these findings, they have completed the present invention. (1) A structure (hereinafter referred to as the structure of the present invention) in which a core layer prepared using chemically crosslinked alginic acid is coated with a cationic polymer layer and further coated with an anionic polymer layer such as alginic acid or chemically crosslinked alginic acid exhibits higher physical strength than a similar structure using alginic acid as a core layer (hereinafter referred to as the conventional structure) even under conditions in which intermolecular crosslinking of alginic acid by divalent metal ions is not performed. (2) When the structure of the present invention is prepared in the shape of, for example, beads, it can be produced in a larger size than conventional structures. (3) Because the structure of the present invention has high stability, it is possible to produce large, freely shaped structures such as flat plates and fibers, and these structures can maintain structural stability for a long period of time. (4) The structure of the present invention in which cells are encapsulated in the core layer can actually be produced, and cells can survive in the structure of the present invention for a long period of time. (5) Therefore, the structure of the present invention can be used for a wide range of applications, such as storing or culturing cells placed in its stable core layer. (6) When cells derived from pancreatic beta cells were encapsulated in the core layer, insulin was secreted for a long period of time while the structure was maintained, making it possible to use the structure for transplanting pancreatic islets and cell transplantation devices. (7) Since the structure of the present invention uses chemically modified alginic acid or alginic acid as the layer coating the cationic polymer, it is suitable for use in vivo, and since it possesses the structural characteristics described above in (1) to (6), it can be applied not only to cell transplantation but also to various other in vivo applications. Note that chemically crosslinked alginic acid obtained by forming crosslinks between the chemically modified alginic acid derivatives represented by formula (HB-I) or formula (HB-II) described below has not been known until now.

[0009] Exemplary aspects of the present invention are as follows [1] to [13-9].

[0010] [1] A structure comprising a core layer containing a pharmacological ingredient embedded in chemically crosslinked alginate, a cationic polymer layer coating the core layer, and an anionic polymer coating the cationic polymer layer.

[0011] [2] The structure according to [1], wherein the chemically crosslinked alginic acid is obtained by forming a crosslink between a chemically modified alginic acid derivative represented by formula (H-I) and a chemically modified alginic acid derivative represented by formula (H-II): [Chemical modified alginic acid derivative represented by formula (H-I)] The structure according to [1], wherein the chemically crosslinked alginic acid is obtained by forming a crosslink between the two alginic acid derivatives using a chemically modified alginic acid derivative represented by formula (H-I): [In formula (HI), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L １ - is a divalent linker bonding to a cyclic alkyne group (Akn); [Chemical modified alginic acid derivative represented by formula (H-II)] A chemically modified alginic acid derivative represented by the following formula (H-II): [In formula (H-II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L ２ - is a divalent linker that bonds to the azide group.

[0012] [2-1] The structure according to [1] or [2], wherein the chemically crosslinked alginic acid is obtained by forming a crosslink between a chemically modified alginic acid derivative represented by formula (H-I) and a chemically modified alginic acid derivative represented by formula (H-II): [Chemical modified alginic acid derivative represented by formula (H-I)] The structure according to [1] or [2], wherein the chemically crosslinked alginic acid is obtained by forming a crosslink between the chemically modified alginic acid derivative represented by formula (H-I): [In formula (HI), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L １ - is the table below: Akn represents a linker selected from the group consisting of the partial structural formulas shown in the following table: [in each formula, the right side of the dashed line is not included]]; [a chemically modified alginic acid derivative represented by formula (H-II)] The following formula (H-II): (In formula (H-II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L ２ - is the table below: A chemically modified alginic acid derivative represented by the partial structural formula (1) (wherein each formula does not include the area outside the dashed lines at both ends)

[0013] [2-2] Akn-L in the formula (HI) １ -The combinations are as shown in the table below: A chemically modified alginic acid derivative selected from the group consisting of: １ Each symbol shown in - has the following partial structural formula: [In each formula, the areas outside the dashed lines at both ends are not included]), and -L in the formula (H-II). ２ - is of the following formula: The structure described in [2-1] above is produced using a chemically crosslinked alginic acid obtained by forming a crosslink between two alginic acid derivatives using a chemically modified alginic acid derivative selected from the partial structural formulas described in the following [each formula does not include the parts outside the dashed lines at both ends]:

[0014] [2-2b] Akn in formula (HI) is (AK-1), and -L １ [2-2c] The structure according to the above [2-2], which is produced using a chemically modified alginic acid derivative in which - is (LN-4-X), (LN-9-X), (L1-1-X), (L1-2-X), (L1-3-X1), (L1-3-X2), (L1-4-X1), (L1-4-X2), (L1-5-X), (L1-6-X), (L1-8-X1) or (L1-8-X2). １[2-2d] The structure according to the above [2-2], which is produced using a chemically modified alginic acid derivative in which - is (LN-3-X1), (LN-3-X2) or (L1-7-X). １ The structure according to [2-2] above, which is produced using a chemically modified alginic acid derivative in which - is (LN-4-X), (LN-9-X), (L1-1-X), (L1-2-X), (L1-3-X1), (L1-3-X2), (L1-4-X1), (L1-4-X2), (L1-5-X), (L1-6-X), (L1-8-X1) or (L1-8-X2).

[0015] [2-3] The structure according to [2-2] above, which is produced using a chemically modified alginic acid derivative in which Akn in the formula (HI) is (AK-1) or (AK-3).

[0016] [2-4] Akn in the formula (HI) is (AK-1), and -L １ - is (LN-3) or (LN-9), or Akn is (AK-3), and -L １ A chemically modified alginic acid derivative in which - is (LN-3), and -L in the formula (H-II) ２ The structure according to the above [2-1], which is produced using a chemically modified alginic acid derivative in which -L is (LK-2), (LK-4), (LK-5) or (LK-7). Particularly preferably, -L ２ The above structure wherein - is (LK-2).

[0017] [2-4b] Akn in formula (HI) is (AK-1), and -L １ - is (LN-9) or Akn is (AK-3), and -L １ A chemically modified alginic acid derivative in which - is (LN-3), and -L in the formula (H-II) ２ The structure according to the above [2-4], which is produced using a chemically modified alginic acid derivative in which -L is (LK-2), (LK-4), (LK-5) or (LK-7). Particularly preferably, -L ２ The above structure wherein - is (LK-2).

[0018] [2-5] Akn in the formula (HI) is (AK-1), and -L １ - is (LN-3-X1) or (LN-9-X) in the table in [2-2], or Akn is (AK-3), and -L １ A chemically modified alginic acid derivative in which - is (LN-3-X1), and -L in the formula (H-II) ２ The structure according to the above [2-2], which is produced using a chemically modified alginic acid derivative in which -L is (LK-2-X), (LK-4-X), (LK-5-X1) or (LK-7-X1). Particularly preferred is -L ２ The above structure wherein - is (LK-2-X).

[0019] [2-6] The chemically modified alginic acid derivative of formula (HI) is represented by the following formula (A01): The chemically modified alginic acid derivative of formula (H-II) is represented by the following formula (N01): The structure described in [2-1].

[0020] [2-7] The chemically modified alginic acid derivative of formula (H-I) is formula (A01) in [2-6], and the chemically modified alginic acid derivative of formula (H-II) is formula (N02) below. The structure described in [2-1].

[0021] [2-8] The chemically modified alginic acid derivative of formula (H-I) is formula (A01) in [2-6], and the chemically modified alginic acid derivative of formula (H-II) is formula (N03) below. The structure described in [2-1].

[0022] [2-9] The chemically modified alginic acid derivative of formula (H-I) is formula (A01) in [2-6], and the chemically modified alginic acid derivative of formula (H-II) is formula (N04) below. The structure described in [2-1].

[0023] [2-10] The chemically modified alginic acid derivative of formula (HI) is represented by the following formula (A02): The chemically modified alginic acid derivative of formula (H-II) is represented by the following formula (N01): The structure described in [2-1].

[0024] [2-11] The chemically modified alginic acid derivative of formula (H-I) is formula (A02) in [2-10], and the chemically modified alginic acid derivative of formula (H-II) is formula (N02) below. The structure described in [2-1].

[0025] [2-12] The chemically modified alginic acid derivative of formula (H-I) is formula (A02) in [2-10], and the chemically modified alginic acid derivative of formula (H-II) is formula (N03) below. The structure described in [2-1].

[0026] [2-13] The chemically modified alginic acid derivative of formula (H-I) is formula (A02) in [2-10], and the chemically modified alginic acid derivative of formula (H-II) is formula (N04) below. The structure described in [2-1].

[0027] [2-14] In formula (HI), -L １ - is - (CH ２ ) ｎ１ - (where n1=1 to 50, and -CH ２ - may be replaced by 1 to 10 groups selected from -CO-, -CONH-, -NHCO-, -O-CONH-, -NHCO-O-, -O- and -NH-, or a benzene ring, ２ The hydrogen atom of - is C １－3 C substituted with alkyl or phenyl groups １－3 Akn is an 8-membered cyclic alkyne group (which may be further fused with one or two benzene rings, cyclopropane rings, or 1,2,3-triazole rings, and the fused rings may have -L １ -, and -CH in the 8-membered cyclic alkyne group ２ - may be an 8-membered ring group substituted by 1 or 2 groups selected from -C(=O)-, -CONH-, and -NH-, and -CH ２ The hydrogen atom of - is C １－３ an alkyl group, a fluorine atom, a hydroxyl group, or C １－３and wherein in formula (H-II), -L ２ -ga- (CH ２ ) ｎ２ - (where n2=1 to 50, and -CH ２ - may be replaced by 1 to 10 groups selected from -CONH-, -NHCO-, -O- and -NH-, or a benzene ring or a pyridine ring, and the -CH ２ The hydrogen atom of - is C １－3 The structure according to [2] above, wherein the alkyl group is a substituted or unsubstituted alkyl group.

[0028] [2-15] In formula (HI), -L １ - is - (CH ２ ) ｎ１ - (where n1=1 to 15 (e.g., 2 to 15), and -CH ２ - may be replaced by 1 to 5 groups selected from -CO-, -CONH-, -NHCO-, -O-CONH-, -NHCO-O-, -O- and -NH-, or a benzene ring, ２ The hydrogen atom of - is C １－3 C substituted with alkyl or phenyl groups １－3 Akn is an 8-membered cyclic alkyne group (which may be further fused with 1 or 2 benzene rings, and the 8-membered cyclic alkyne group may be fused with -CH ２ In the formula (H-II), -L may be an 8-membered ring group in which - is replaced by -NH-. ２ -ga- (CH ２ ) ｎ２ - (where n2=1 to 15 (e.g., 2 to 15), and -CH ２ The structure according to [2] above, wherein - may be replaced by 1 to 5 groups selected from -CONH-, -NHCO-, -O- and -NH-, or a benzene ring.

[0029] [3-1] The structure according to [1], [2] or [2-1], wherein the chemically crosslinked alginic acid is obtained by forming a crosslink between a chemically modified alginic acid derivative represented by formula (HB-I) and a chemically modified alginic acid derivative represented by formula (HB-II), using the chemically modified alginic acid derivative represented by formula (HB-I): [Chemical modified alginic acid derivative represented by formula (HB-I)] The structure according to the following formula (HB-I): [In formula (HB-I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L １ - is -L described in [2-1] １ A chemically modified alginic acid derivative represented by the following formula (HB-II): wherein, in the partial structural formula of -, represents a linker selected from the group consisting of the partial structural formulas described in (LN-3), (LN-9), and (L1-3) to (L1-10) [in each formula, the areas outside the dashed lines at both ends are not included]; Akn represents a cyclic alkyne group selected from the group consisting of the partial structural formulas described in the partial structural formula of Akn described in [2-1] [in each formula, the area to the right of the dashed line is not included]; [A chemically modified alginic acid derivative represented by the formula (HB-II)] [In formula (HB-II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L ２ - is -L described in [2-1] ２ In the partial structural formula of -, - represents a linker selected from the group consisting of (LK-2) and the partial structural formulas of (L2-1) to (L2-9b) [within each formula, the areas outside the dashed lines at both ends are not included]. (However, in the chemically modified alginic acid derivative represented by formula (HB-I), -L １ a derivative in which - is any one linker selected from the group consisting of (LN-3) and (LN-9); and a chemically modified alginic acid derivative represented by formula (HB-II), in which -L ２ (Excluding chemically cross-linked alginic acid obtained by cross-linking using a derivative in which - is the linker of (LK-2)).

[0030] [3-2] In the formula (HB-I), -L １- is the formula in the table below: Akn is selected from the partial structural formulas described in the table of Akn in [2-1] above [excluding the area to the right of the dashed line in each formula]; -L in formula (HB-II) ２ - is the table below: The structure according to [3-1] above, which is selected from the group consisting of partial structural formulas shown in the following formulas (each formula does not include the areas outside the dashed lines at both ends):

[0031] [3-3] In the formula (HB-II), -L ２ - is the table below: The structure according to [3-2] above, which is selected from the group consisting of partial structural formulas shown in the following formulas (each formula does not include the areas outside the dashed lines at both ends):

[0032] [3-4] -L in the formula (HB-II) ２ - but see the table below: The structure according to [3-3] above, which is selected from the group consisting of partial structural formulas shown in the following formulas (each formula does not include the areas outside the dashed lines at both ends):

[0033] [3-5] -L in the formula (HB-II) ２ - but see the table below: The structure according to [3-3], which is selected from the group consisting of partial structural formulas shown in the following formulas (each formula does not include the areas outside the dashed lines at both ends):

[0034] [3-6] -L in the formula (HB-I) １ - but see the table below: The structure according to [3-3] to [3-5], which is selected from the group consisting of partial structural formulas described in the following [each formula does not include the areas outside the dashed lines at both ends]:

[0035] [4] The structure according to [1] or [2], wherein the chemically crosslinked alginic acid is obtained by forming a crosslink between a chemically modified alginic acid derivative represented by formula (HA-I) and a chemically modified alginic acid derivative represented by formula (HA-II): [Chemical modified alginic acid derivative represented by formula (HA-I)] The structure according to [1] or [2], wherein the chemically crosslinked alginic acid is obtained by forming a crosslink between the two alginic acid derivatives using a chemically modified alginic acid derivative represented by formula (HA-I): [In formula (HA-I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L １ - represents the following partial structural formula (each formula does not include the areas outside the dashed lines at both ends): Akn represents a divalent linker selected from the group consisting of: wherein the asterisk represents a chiral center]; [Chemical modified alginic acid derivatives represented by formula (HA-II)] A chemically modified alginic acid derivative represented by the following formula (HA-II): [In formula (HA-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 (each formula does not include the areas outside the dashed lines at both ends): and wherein the alginic acid derivative is a divalent linker selected from the group consisting of:

[0036] [5] The chemically crosslinked alginic acid is such that any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (H-III-L): [In formula (H-III-L), —CONH— and —NHCO— at both ends represent an amide bond via any carboxyl group of alginic acid; １ - is a divalent linker that connects to a cyclic alkyne group (Akn); -L ２ - is a divalent linker that connects the azide group; X is L ２ an azide group bonded to L １is a divalent group having a cyclic group formed by a 3+2 cycloaddition reaction.

[0037] [5-1] The chemically crosslinked alginic acid is a compound in which any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (H-III-L): [In formula (H-III-L), —CONH— and —NHCO— at both ends represent an amide bond via any carboxyl group of alginic acid; １ - is the same as defined in [2-1]; -L ２ - is the same as defined in [2-1]; X is as defined in the following table: (In each formula, the areas outside the dashed lines at both ends are not included.)

[0038] [5-2] The chemically crosslinked alginic acid is represented by the formula (H-III-L), １ -The combinations are as shown in the table below: (wherein -L in the table is selected from) １ - is as defined in the above [2-2], and each symbol shown in -X- is as defined in the partial structural formula shown in the table in [5-1] (in each formula, the areas outside the dashed lines at both ends are not included), -L ２ The structure according to [5-1] above, wherein - is selected from the structural formulas in [2-2] above.

[0039] [5-3] The structure according to [5-2] above, wherein —X— in the formula (H-III-L) is (TZ-2) or (TZ-6).

[0040] [5-4] In the formula (H-III-L), -X- is (TZ-6) and -L １ - is (LN-3) or (LN-9), or -X- is (TZ-2) and -L １ - is (LN-3) and -L ２The structure according to [5-1] above, wherein - is (LK-2), (LK-4), (LK-5) or (LK-7) in the table in [2-2].

[0041] [5-5] In the formula (H-III-L), -X- is (TZ-6), and -L １ - is (LN-3-X1) or (LN-9X) in the table in [2-2], or -X- is (TZ-2) and -L １ - is (LN-3-X1), and -L ２ The structure according to [5-2] above, wherein - is (LK-2-X), (LK-4-X), (LK-5-X1) or (LK-7-X1) in the table in [2-2].

[0042] [5-6] -L in the formula (H-III-L) １ - is selected from the partial structural formulas shown in the table in [2-14] [each formula does not include the areas outside the dashed lines at both ends], -L ２ The structure according to [5] above, wherein - is selected from the partial structural formulas described in the table in [2-14] [within each formula, the areas outside the dashed lines at both ends are not included], and -X- is selected from the partial structural formulas shown in the table in [5-1] [within each formula, the areas outside the dashed lines at both ends are not included].

[0043] [5-7] -L in the formula (H-III-L) １ - is selected from the partial structural formulas shown in the table in [2-15] [each formula does not include the areas outside the dashed lines at both ends], and -L ２ The structure according to [5] above, wherein - is selected from the partial structural formulas described in the table in [2-15] [within each formula, the areas outside the dashed lines at both ends are not included], and -X- is selected from the partial structural formulas shown in the table in [5-1] [within each formula, the areas outside the dashed lines at both ends are not included].

[0044] [6-1] The chemically crosslinked alginic acid is such that any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (HB-III-L): [In formula (HB-III-L), —CONH— and —NHCO— at both ends represent an amide bond via any carboxyl group of alginic acid; １- is the -L listed in the table in [2-1] １ - represents a linker selected from the group consisting of the partial structural formulas (LN-3), (LN-9), and (L1-3) to (L1-10) in the partial structural formula of - [in each formula, the areas outside the dashed lines at both ends are not included]; -L ２ - is the -L listed in the table in [2-1] ２ In the partial structural formula of -, represents a linker selected from the group consisting of the partial structural formulas (LK-2) and (L2-1) to (L2-9b) [in each formula, the areas outside the dashed lines at both ends are not included]; -X- has the same meaning as the partial structural formula shown in the table in [5-1] [in each formula, the areas outside the dashed lines at both ends are not included], with the proviso that -L １ - is (LN-3) or (LN-9), and -L ２ - is (LK-2) excepted].

[0045] [6-2] -L in the formula (HB-III-L) １ - is selected from the partial structural formulas shown in the table in [3-2] [each formula does not include the areas outside the dashed lines at both ends], and -L ２ The structure according to [6-1] above, wherein - is selected from the partial structural formulas described in the table in [3-2] [within each formula, the areas outside the dashed lines at both ends are not included], and -X- is selected from the partial structural formulas shown in the table in [5-1] [within each formula, the areas outside the dashed lines at both ends are not included].

[0046] [6-3] -L in the formula (HB-III-L) ２ [6-4] The structure according to [6-2] above, wherein - is selected from the partial structural formulas shown in the table in [3-3] [each formula does not include the areas outside the dashed lines at both ends]. ２ [6-5] The structure according to [6-3], wherein - is selected from the partial structural formulas shown in the table in [3-4] [each formula does not include the areas outside the dashed lines at both ends]. ２The structure according to [6-3] above, wherein - is selected from the partial structural formulas shown in the table in [3-5] [each formula does not include the areas outside the dashed lines at both ends]. [6-6] In the formula (HB-III-L), -L １ The structure according to [6-3] to [6-5] above, wherein - is selected from the partial structural formulas described in the table in [3-6] [in each formula, the areas outside the dashed lines at both ends are not included].

[0047] [7] The chemically crosslinked alginic acid is a compound in which any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (HA-III-L): [In formula (HA-III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; １ - is the same as defined in the above [4]; -L ２ - is as defined in the above [4]; and X is as defined in the above [5-1]. The structure according to the above [1], wherein the crosslinking is performed via a group represented by the following formula:

[0048] [8-1] The structure according to [1], wherein the chemically crosslinked alginate is obtained by irradiating a chemically modified alginate derivative having a photoreactive group represented by formula (C-I) introduced therein with light. [Chemically modified alginate derivative having a photoreactive group represented by formula (C-I) introduced therein] A chemically modified alginate derivative having a photoreactive group represented by formula (C-I) introduced thereinto at any one or more carboxyl groups of alginate, wherein the photoreactive group represented by formula (C-I) is introduced thereinto. [The chemically ... [In formula (CI), Ar is C ６～１０ Aryl group (C ６～１０ The aryl group includes a hydroxyl group, a cyano group, a nitro group, a halogen atom, C １～６ Alkyl group, halogenated C １～６ Alkyl group, C １～６ Alkoxy groups, and -NR Ａ R Ｂ Group (-NR Ａ R Ｂ In the group, R Ａ and R Ｂ are each independently a hydrogen atom, C １～６ Alkyl group, C ２～７Alkanoyl group, or C １～６ alkylsulfonyl groups (wherein -NH ２ , —NH(C １～６ alkyl group), and —N(C １～６ alkyl group) ２ hydrogen atoms on the ring may be substituted with 1 to 3 groups arbitrarily selected from the group consisting of C ６～１０ The aryl group may include C １～６ Alkyl group and C １～６ Alkoxy group or two C １～６ When alkoxy groups are adjacently substituted, the C １～６ Alkyl group and C １～６ The alkoxy group is bonded between carbon atoms obtained by removing one arbitrary hydrogen atom from the alkyl group of each group, or the two C １～６ A cyclic ether may be formed by bonding between carbon atoms obtained by removing one arbitrary hydrogen atom from each alkyl group of the alkoxy group, or ６～１０ The aryl group may include C １～６ Alkoxy group and -NHR Ｇ Group (-NHR Ｇ R in the group Ｇ is C ２～７ Alkanoyl group or C １～６ When an alkylsulfonyl group is adjacent to the C １～６ A carbon atom obtained by removing one arbitrary hydrogen atom from the alkyl group of an alkoxy group and the —NHR Ｇ The nitrogen atom from which one hydrogen atom has been removed is bonded to form 3-N-(C ２～７ alkanoyl)oxazolidine ring, 3-N-(C １～６ alkylsulfonyl)oxazolidine ring, 4-N-(C ２～７ alkanoyl)morpholine ring, 4-N-(C １～６ alkylsulfonyl)morpholine ring, 4-N-(C ２～７ alkanoyl)-1,4-oxazepane ring, or 4-N-(C １～６p represents an integer of 1 or 2; -X- represents -O-; -A- represents a group represented by formula (AL-1) to (AL-4) [in each formula, the areas outside the dashed lines at both ends are not included]: (In formulas (AL-1) to (AL-4), n represents an integer of 1 to 18; m represents an integer of 1 to 9; j represents an integer of 0 to 9; a methylene group (—CH ２ -) hydrogen atom is 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, carboxy 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 The alkyl group (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 groups), and 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 The methylene group (—CH ２ -) two hydrogen atoms are C１～６ When substituted with an alkyl group, each C １～６ The alkyl group is bonded to the carbon atom where one hydrogen atom has been removed from each alkyl group. ３～８ a cycloalkyl ring; and the —NH— group in formula (AL-3) or formula (AL-4) may form a non-aromatic heterocycle together with the substituent substituted on the adjacent carbon atom) (provided that in formula (C-I), when Ar is a phenyl group, p is 1, -A- is formula (AL-1), and n is 3, the hydrogen atoms on the phenyl group of Ar are substituted with 1 to 3 groups arbitrarily selected from the group of substituents that may be substituted for the hydrogen atoms on the Ar ring).

[0049] [8-2] In the formula (C-I), Ar is a phenyl group (the phenyl group may have 1 to 3 groups arbitrarily selected from a cyano group, a fluorine atom, a trifluoromethyl group, and a methoxy group substituted for hydrogen atoms on the ring, and when two methoxy groups are substituted adjacent to each other on the phenyl group, the two methoxy groups may be bonded to carbon atoms having one hydrogen atom removed from the methyl group of each methoxy group to form a 1,4-dioxane ring) (however, when Ar is a phenyl group, p is 1, -A- is formula (AL-1), and n is 3 in formula (C-I), 1 to 3 groups arbitrarily selected from the group of substituents which may be substituted for hydrogen atoms on the phenyl group are substituted for hydrogen atoms on the phenyl group in the phenyl group); p is an integer of 1 or 2; -X- is -O-; The structure according to [8-1], which contains a chemically crosslinked alginic acid obtained by irradiating a "chemically modified alginic acid derivative having a photoreactive group introduced therein" with light, wherein -A- is formula (AL-1) or (AL-2) in the above aspect [8-1] [each formula does not include the area outside the dashed lines at both ends].

[0050] [8-3] The structure according to [8-2], which contains a chemically crosslinked alginic acid obtained by irradiating a "chemically modified alginic acid derivative having a photoreactive group introduced therein" with light, wherein Ar in formula (C-I) is a phenyl group, a 4-fluorophenyl group, a 4-(trifluoromethyl)phenyl group, a 4-methoxyphenyl group, a 4-cyanophenyl group, or a 2,3-dihydrobenzo[b][1,4]dioxinyl group.

[0051] [8-4] The structure according to [8-2], which contains a chemically crosslinked alginic acid obtained by irradiating a chemically modified alginic acid derivative having a photoreactive group introduced therein selected from the partial structural formulas shown below in the formula (C-I) [excluding the right side of the dashed line in each formula] with light. [8-5] The structure according to any one of [8-1] to [8-4], wherein the light to be irradiated is light selected from ultraviolet light and LED light.

[0052] [9-1] The chemically crosslinked alginic acid is a compound in which any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (C-II-L-1) or formula (C-II-L-2): [In formula (C-II-L-1) or formula (C-II-L-2), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -A-, -X-, and Ar are as defined in any one of items [8-1] to [8-3]]. The structure according to [1] above,

[0053] [9-2] The chemically crosslinked alginic acid is a compound in which any carboxyl group of the first alginic acid and any carboxyl group of the second alginic acid are represented by the following formula (C-II-L-3): [In formula (C-II-L-3), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -A-, -X-, and Ar are the same as defined in any one of the items [8-1] to [8-3].] The structure according to the above [1], which is crosslinked via a group represented by the following formula:

[0054] [10-1] A method for producing a structure comprising a pharmacological component embedded in the chemically crosslinked alginate core layer as shown in [2] above, comprising the following steps (a) to (c): step (a): a step of contacting a solution of a chemically modified alginic acid derivative containing a pharmacological component [a mixture of a derivative represented by formula (H-I) and a derivative represented by formula (H-II)] with a solution containing a divalent metal ion to form a gel; step (b): a step of contacting the gel obtained in step (a) with a solution containing a cationic polymer to coat the gel with the cationic polymer; and step (c): a step of contacting the product obtained in step (b) with a solution containing an anionic polymer to further coat it with an aniline polymer.

[0055] [10-2] A method for producing the structure according to the above [10-1], further comprising the following step (d): step (d): a step of chelating the structure obtained in step (c) with a chelating agent.

[0056] [10-3] The manufacturing method according to [10-1] or [10-2], wherein the structure is one according to any one of [2-1] to [7] above.

[0057] [11-1] A method for producing a structure containing a pharmacological component embedded in chemically crosslinked alginate, comprising the following steps (a) to (c) and performing light irradiation at any stage after step (a): step (a): a step of contacting a solution of a chemically modified alginate derivative containing a pharmacological component with a solution containing a divalent metal ion to form a gel; step (b): a step of contacting the gel obtained in step (a) with a solution containing a cationic polymer to coat the gel with the cationic polymer; step (c): a step of contacting the product obtained in step (b) with a solution containing an anionic polymer to further coat it with an aniline polymer.

[0058] [11-2] A method for producing the structure according to the above [11-1], further comprising the following step (d): step (d): a step of chelating the structure obtained in step (c) with a chelating agent.

[0059] [11-3] The manufacturing method according to [11-1] or [11-2], wherein the structure is one according to any one of [8-1] to [9-2] above.

[0060] [12-1] The structure according to any one of [1] to [9-2] above, wherein the anionic polymer is alginic acid or chemically crosslinked alginic acid. [12-2] The structure according to any one of [1] to [9-2] above, wherein the anionic polymer is alginic acid. [12-3] The structure according to any one of [1] to [9-2] above, wherein the anionic polymer is the same chemically crosslinked alginic acid as that used in the core layer. [12-4] The structure according to any one of [1] to [9-2] above, wherein the anionic polymer is selected from chemically crosslinked alginic acids having the structural formula according to any one of [1] to [9-2] above.

[0061] [12-5] The structure according to any one of [1] to [9-2] or [12-1] to [12-4] above, wherein the pharmacological component embedded in the chemically crosslinked alginate of the core layer is a cell. In one embodiment, the structure is an insulin-secreting cell. In another embodiment, the structure is according to any one of [1] to [9-2] or [12-1] to [12-4] above, wherein the pharmacological component is a cell that produces a biologically active substance. In another embodiment, the structure is according to any one of [1] to [9-2] or [12-1] to [12-4] above, wherein the pharmacological component is a cell that produces a physiologically active substance. In another embodiment, the structure is according to any one of [1] to [9-2] or [12-1] to [12-4] above, wherein the pharmacological component is a cell that produces a physiologically active natural product. [12-6] The structure according to any one of [1] to [9-2] or [12-1] to [12-5] above, wherein the cationic polymer is poly-L-ornithine or poly-L-lysine. [12-7] The structure according to any one of [1] to [9-2] or [12-1] to [12-6] above, wherein the cationic polymer is poly-L-ornithine. [12-8] The structure according to any one of [1] to [9-2] or [12-1] to [12-7] above, wherein the structure has the shape of beads, a sheet, or a fiber. Preferably, the structure according to any one of [1] to [9-2] or [12-1] to [12-7] above, wherein the structure has the shape of beads. In another embodiment, the structure according to any one of [1] to [9-2] or [12-1] to [12-7] above, wherein the structure has the shape of a sheet. In yet another embodiment, the structure according to any one of the above items [1] to [9-2] or [12-1] to [12-7] is in the form of a fiber. [12-9] The structure according to any one of the above items [1] to [9-2] or [12-1] to [12-8] is produced using alginic acid or a chemically modified alginic acid derivative having a weight-average molecular weight of 100,000 Da to 3,000,000 Da as measured by gel filtration chromatography. [12-10] The structure according to any one of the above items [1] to [9-2] or [12-1] to [12-9] is for use in a living body.[12-11] A medical material containing, as an active ingredient, a pharmacological ingredient encapsulated in the structure according to any one of the above items [1] to [9-2] or [12-1] to [12-9]. [12-12] The medical material according to [12-11] is a wound dressing, a postoperative adhesion barrier, a substrate for sustained drug release, a substrate for cell transplantation, a prosthetic material, a pharmaceutical coating material, or a bioink for use in a bioprinter. The prosthetic material is preferably an implant or an artificial organ.

[0062] [13-1] The manufacturing method according to any one of the above [10-1] to [11-3] for producing a structure in which the anionic polymer is alginic acid or chemically crosslinked alginic acid. [13-2] The manufacturing method according to any one of the above [10-1] to [11-3] for producing a structure in which the anionic polymer is alginic acid. [13-3] The manufacturing method according to any one of the above [10-1] to [11-3] for producing a structure in which the anionic polymer is the same chemically crosslinked alginic acid as used in the core layer. [13-4] The manufacturing method according to any one of the above [10-1] to [11-3] for producing a structure in which the anionic polymer is selected from the chemically crosslinked alginic acids according to any one of the above [1] to [9-2].

[0063] [13-5] The method for producing a construct in which the pharmacological component embedded in the chemically crosslinked alginate of the core layer is a cell, as described in any one of [10-1] to [11-3] or [13-1] to [13-4]. In one embodiment, the method for producing a construct in which the cell is an insulin-secreting cell. [13-6] The method for producing a construct in which the cationic polymer is poly-L-ornithine or poly-L-lysine, as described in any one of [10-1] to [11-3] or [13-1] to [13-5]. [13-7] The method for producing a construct in which the cationic polymer is poly-L-ornithine, as described in any one of [10-1] to [11-3] or [13-1] to [13-6]. [13-8] The manufacturing method according to any one of the above items [10-1] to [11-3] or [13-1] to [13-7], for producing a structure in the form of a bead, sheet, or fiber. Preferably, the manufacturing method according to any one of the above items [10-1] to [11-3] or [13-1] to [13-7], for producing a structure in the form of a bead. In another embodiment, the manufacturing method according to any one of the above items [10-1] to [11-3] or [13-1] to [13-7], for producing a structure in the form of a sheet. In yet another embodiment, the manufacturing method according to any one of the above items [10-1] to [11-3] or [13-1] to [13-7], for producing a structure in the form of a fiber. [13-9] The method for producing a structure produced using alginic acid or a chemically modified alginic acid derivative having a weight average molecular weight of 100,000 Da to 3,000,000 Da as measured by gel filtration chromatography, according to any one of [10-1] to [11-3] or [13-1] to [13-8].

[0064] Examples of preferred embodiments of the structure of the present invention include the following: [14-1] The structure according to any one of [1] to [7] above, wherein the pharmacological component is a bioactive substance-producing cell, the cationic polymer is poly-L-ornithine or poly-L-lysine, the anionic polymer is alginic acid or the same chemically crosslinked alginic acid as used in the core layer, and the structure is in the form of beads or a sheet. [14-2] A medical material containing the pharmacological component as an active ingredient, encapsulated in the structure according to any one of [1] to [7] above, wherein the pharmacological component is a bioactive substance-producing cell, the cationic polymer is poly-L-ornithine or poly-L-lysine, and the anionic polymer is alginic acid or the same chemically crosslinked alginic acid as used in the core layer. [14-3] The structure according to any one of [1] to [7] above, wherein the cationic polymer is poly-L-ornithine or poly-L-lysine, and the anionic polymer is alginic acid or the same chemically crosslinked alginic acid as used in the core layer, which is used to be placed in the body to exert the function of the pharmacological component. [14-4] A method for treating a disease in a human or animal, comprising placing in the body the structure according to any one of [1] to [7] above, wherein the pharmacological component is a bioactive substance-producing cell, the cationic polymer is poly-L-ornithine or poly-L-lysine, and the anionic polymer is alginic acid or the same chemically crosslinked alginic acid as that used in the core layer, particularly a method for treating a disease in which the structure is used for wound dressing, postoperative adhesion prevention, sustained drug release, cell transplantation, implantation, prosthetics such as artificial organs, etc.

[0065] The present invention provides a novel structure, which preferably exhibits at least one of the following advantages:

[0066] (1) Compared to similar structures using alginate as the core layer (hereinafter referred to as conventional structures), it exhibits high physical strength even under conditions in which intermolecular cross-linking of alginate by divalent metal ions is not present. (2) It is possible to fabricate structures larger than conventional structures. (3) It is also possible to fabricate large, freely shaped structures such as flat plates and fibers, and these structures can maintain structural stability for long periods of time. (4) When cells are encapsulated in the core layer, they can be kept viable for long periods of time. (5) It can be used for purposes such as storing or culturing cells placed in the core layer. (6) It can also be used for transplanting pancreatic islets and cell transplantation devices. (7) It is biocompatible, making it applicable to a variety of in vivo uses.

[0067] 1 is a diagram showing an evaluation of the stability of chemically cross-linked alginate. 2 is a diagram showing an evaluation of the stability of chemically cross-linked alginate under EDTA. 3 is a diagram showing an evaluation of the stability of chemically cross-linked alginate. 4 is a diagram showing an evaluation of the stability of chemically cross-linked alginate under EDTA. 5 is a diagram showing an evaluation of the stability of chemically cross-linked alginate. 6 is a diagram showing an evaluation of the stability of chemically cross-linked alginate under EDTA. 7 is a diagram showing an evaluation of the permeability of chemically cross-linked alginate. 8 is a diagram showing an evaluation of the permeability of chemically cross-linked alginate. 9 is a diagram showing an evaluation of the biocompatibility of chemically cross-linked alginate. 10 is a diagram showing an evaluation of the stability of a structure according to Example A-6 after chelate treatment (a) and after shaking for one day (b). 11 is a diagram showing an evaluation of the stability of a structure according to Example A-3 after chelate treatment (a) and after shaking for one day (b). 12 is a diagram showing an evaluation of the stability of a structure according to Example A-2 after chelate treatment (a) and after shaking for one day (b). 13 is a diagram showing an evaluation of the stability of a structure according to Example D-1 after shaking for one day. 14 is a diagram showing an evaluation of the stability of a structure according to Example D-2 after shaking for one day. 1 is a diagram showing an evaluation of the stability of the structure according to Example D-3 after shaking for one day. 2 is a diagram showing an evaluation of the stability of the structure according to Example D-4 after shaking for one day. 3 is a diagram showing an evaluation of the stability of the structures according to Examples D(2)-1 and D(2)-2 after shaking for one day.

[0068] Each aspect of the present invention will be described in more detail below. 1. Multilayer structure using chemically crosslinked alginate In this specification, the term "structure" refers to a multilayer structure using chemically crosslinked alginate that can encapsulate a pharmacological ingredient therein. The structure provided herein is preferably a three-layer structure including a core layer containing a pharmacological ingredient, a cationic polymer layer, and an anionic polymer layer. Unless otherwise specified, the term "structure" refers to a structure including a core layer containing a pharmacological ingredient embedded in chemically crosslinked alginate, a cationic polymer layer coating the core layer, and an anionic polymer layer coating the cationic polymer layer.

[0069] In this specification, "subjecting to a chemical crosslinking reaction" or "carrying out a chemical crosslinking reaction" means that a chemically modified alginic acid derivative of the formula (H-I), (HA-I) or (HB-I) and a chemically modified alginic acid derivative of the formula (H-II), (HA-II) or (HB-II) are used to carry out a Huisgen reaction, thereby forming a chemical crosslink (chemical bond) between these chemically modified alginic acid derivatives; or that a chemically modified alginic acid derivative into which a photoreactive group represented by the formula (C-I) has been introduced is irradiated with light to carry out a photocyclization reaction, thereby forming a chemical crosslink (chemical bond) between the chemically modified alginic acid derivatives represented by the formula (C-I).

[0070] The chemically crosslinked alginate constituting the structure of the present invention will be described in detail later. Chemical crosslinks are formed by crosslinking between "chemically modified alginic acid derivatives" in which a reactive group is condensed to any carboxyl group of alginic acid. It is known that sugar polymers such as alginic acid form ionic crosslinks between two carboxyl groups upon contact with divalent metal ions, forming hydrogels. The chemically crosslinked alginate constituting the structure of the present invention has similar properties. Therefore, the chemically crosslinked alginate constituting the structure of the present invention can exist in two states: (i) a state in which no crosslinks (ionic crosslinks) are formed by divalent metal ions (e.g., a state in which bonds with divalent metal ions are removed by the presence of a chelating agent such as EDTA), and (ii) a state in which both chemical crosslinks and ionic crosslinks are formed, as well as a coexistence of these states. The structure of the present invention can be used by combining ionic crosslinks to form a strong gel, or it can maintain a stable structure even in the absence of ionic crosslinks. In this specification, the term "chemically cross-linked alginic acid" includes any state unless otherwise specified.

[0071] 1-1. Core Layer Containing Pharmacological Components Embedded in Chemically Crosslinked Alginate Pharmacological components that can be encapsulated in the core layer include substances with pharmacological or biological activity, i.e., bioactive substances, as well as cells and microorganisms. The pharmacological component encapsulated in the core layer may be at least one type of pharmacological component, but multiple types of pharmacological components may also be encapsulated simultaneously. In the case of multiple types of pharmacological components, the pharmacological components may each have independent activity, or may interact with each other to exhibit activity, or may be a combination of a main component and a supporting component. Also, combinations of multiple types of bioactive substances, multiple types of cells, multiple types of microorganisms, or combinations of one or more types of bioactive substances, cells, or microorganisms are possible. The bioactive substances that can be encapsulated in the core layer are not particularly limited, but are preferably those used as pharmaceuticals, such as small molecule drugs, medium molecule drugs such as peptides and oligonucleotides, biopharmaceuticals such as proteins, and physiologically active substances. As used herein, the term "biologically active substance" includes not only physiologically active substances inherent in living organisms (biologically active natural products), but also modified or altered physiological substances, substances that activate or inhibit physiological activity, and the like. This term encompasses not only naturally occurring substances, but also substances produced by genetic engineering or chemically synthesized, as well as prodrugs thereof. Specific examples include vitamins, coenzymes, hormones, antibiotics, neurotransmitters, cytokines, enzymes, growth factors, antibodies, and other biological factors. More specific examples of biological factors include insulin, dopamine, factor VIII, and factor IX. Cells that can be encapsulated in the core layer are not particularly limited, but are preferably cells capable of releasing biologically active substances (bioactive substance-producing cells). These include natural cells, artificially modified cells, and cell masses consisting of multiple cells. Examples of cells that can be encapsulated in the core layer include, for example, cells for transplantation. Examples of transplantation cells include cells capable of releasing hormones such as insulin, and specific examples include insulin-secreting cells, pancreatic islets, and pancreatic islet cells.In addition, examples of cells capable of releasing other bioactive substances include dopamine-secreting cells, pituitary cells, growth hormone-secreting cells, parathyroid cells, nerve growth factor-secreting cells, blood coagulation factor-secreting cells, hepatocytes, parathyroid cells, erythropoietin-secreting cells, and norepinephrine-secreting cells. Cells that produce bioactive substances (bioactive substance-producing cells) are preferred. In another embodiment, the cells are cells that produce bioactive natural products.

[0072] "Insulin-secreting cells" refer to cells with the ability to secrete insulin, such as beta cells that secrete insulin among cells constituting pancreatic islets. Furthermore, "insulin-secreting cells" may also refer to cells that have acquired insulin secretion function through differentiation, maturation, modification, or the like. For example, "insulin-secreting cells" may include cells with insulin secretion function obtained by differentiating stem cells such as iPS cells, ES cells, or somatic stem cells (e.g., mesenchymal stem cells), cells with insulin secretion function obtained by maturing immature cells or progenitor cells, and cells conferred insulin secretion function through genetic recombination. Here, differentiating or maturing the cells includes culturing the cells, i.e., cells obtained by differentiation or maturation may include cells obtained by culture. "Pancreatic islets" are also known as islets of Langerhans and are cell clusters composed of an average of approximately 2,000 pancreatic islet cells. Pancreatic islets are composed of five types of cells: α cells that secrete glucagon, β cells that secrete insulin, δ cells that secrete somatostatin, ε cells that secrete ghrelin, and PP (pancreatic polypeptide) cells that secrete pancreatic polypeptide.

[0073] As used herein, the term "pancreatic islet cells" refers to cells that contain at least one of the five types of cells that constitute the pancreatic islets, and preferably includes at least β cells. In some embodiments, the pancreatic islet cells may be a mixture containing all of α cells, β cells, δ cells, ε cells, and PP cells, or may be cells contained in pancreatic islets.

[0074] Furthermore, "pancreatic islet cells" may be cells that have become pancreatic islet cells through differentiation, maturation, modification, etc. In this case, "pancreatic islet cells" may include, for example, pancreatic islet cells obtained by differentiating stem cells such as iPS cells, ES cells, and somatic stem cells (e.g., mesenchymal stem cells), as well as pancreatic islet cells obtained by maturing immature cells or progenitor cells.

[0075] When used for transplantation, "insulin-secreting cells" or "pancreatic islets (including pancreatic islet cells)" preferably have sufficient viability and function to recover a pathological condition of a patient when transplanted into the patient. The function of insulin-secreting cells, pancreatic islets, or pancreatic islet cells is, for example, to secrete insulin, and it is preferred that glucose responsiveness is maintained even after transplantation.

[0076] The donor of "insulin-secreting cells," "pancreatic islets," or "pancreatic islet cells" is an animal, preferably a vertebrate, more preferably a mammal, and specific examples include humans, pigs, monkeys, rats, or mice, and even more preferably humans or pigs. In some embodiments, the donor of "insulin-secreting cells," "pancreatic islets," or "pancreatic islet cells" is a pig, in order to alleviate the shortage of donors. "Insulin-secreting cells," "pancreatic islets," or "pancreatic islet cells" may be either pancreatic islets or pancreatic islet cells obtained from a donor animal, or insulin-secreting cells or pancreatic islet cells obtained from donor-derived cells, and may be, for example, insulin-secreting cells or pancreatic islet cells differentiated from human-derived ES cells or iPS cells.

[0077] When the "insulin-secreting cells," "pancreatic islets," or "pancreatic islet cells" are derived from pigs, examples include adult porcine pancreatic islets, or fetal, neonatal, or perinatal porcine pancreatic islets, or insulin-secreting cells or pancreatic islet cells obtained from such pancreatic islets. The pancreatic islets may be appropriately cultured before use, or islets obtained by maturing fetal, neonatal, or perinatal porcine pancreatic islets may also be used.

[0078] Examples of blood coagulation factor-secreting cells include factor VIII-secreting cells and factor IX-secreting cells.

[0079] Other transplant cells are preferably animal cells, more preferably vertebrate-derived cells, even more preferably mammalian-derived cells, and particularly preferably human-derived or porcine-derived cells. The vertebrate-derived cells (particularly human-derived cells) may be stem cells (e.g., pluripotent stem cells (mutilpotent cells) or somatic stem cells), progenitor cells, or mature cells. Examples of pluripotent stem cells include embryonic stem (ES) cells, germline stem (GS) cells, and induced pluripotent stem (iPS) cells. Examples of somatic stem cells include mesenchymal stem cells (MSCs), hematopoietic stem cells, amniotic cells, umbilical cord blood cells, bone marrow-derived cells, cardiac muscle stem cells, adipose-derived stem cells, and neural stem cells. These stem cells may also be differentiated and used.

[0080] Examples of progenitor cells and mature cells that can be used include cells derived from skin, dermis, epidermis, muscle, cardiac muscle, nerve, bone, cartilage, endothelium, brain, epithelium, heart, kidney, liver, spleen, oral cavity, cornea, bone marrow, umbilical cord blood, amniotic membrane, and hair. Examples of human-derived cells that can be used include ES cells, iPS cells, MSCs, chondrocytes, osteoblasts, osteoblast precursor cells, mesenchymal cells, myoblasts, cardiac muscle cells, cardiac muscle blasts, neurons, hepatocytes, fibroblasts, corneal endothelial cells, vascular endothelial cells, corneal epithelial cells, amniotic cells, umbilical cord blood cells, bone marrow-derived cells, and hematopoietic stem cells. Furthermore, the origin of the cells may be either autologous or allogeneic.

[0081] Preferred embodiments of the pharmacological component that can be encapsulated in the core layer include substances that have pharmacological or biological activity in vivo, or cells or microorganisms that produce such substances. In one embodiment, the components are cells that produce bioactive substances, in another embodiment, cells that produce physiologically active substances, and in another embodiment, cells that produce physiologically active natural products. Microorganisms that can be encapsulated in the core layer are not particularly limited, but are preferably microorganisms that can release bioactive substances, such as aerobic bacteria and anaerobic bacteria. The "chemically cross-linked alginate" used in the core layer is described in detail in "5. Chemically Cross-Linked Alginate (Core Layer and Anionic Polymer Layer)." Furthermore, additives other than the pharmacological component can also be added to the core layer of the structure of one embodiment of the present invention. For example, when the pharmacological component is a bioactive substance, it may be effective to use a formulation for controlled sustained release or a carrier-supported component. In the case of cells, it may be effective to use a substance that maintains the viability and function of the cells, or to add a substance that uniformly disperses the pharmacological component in the core layer. The structure of the present invention encompasses both embodiments in which these are contained and embodiments in which they are not contained. Examples of additives other than pharmacological components include polymeric substances other than alginic acid, and specific components include biocompatible polymeric substances such as polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), gelatin, collagen, and hyaluronic acid. Furthermore, for example, when cells or microorganisms are used as pharmacological components, components of culture media or culture solutions, or formulation components commonly used in pharmaceuticals, may also be included.

[0082] 1-2. Cationic Polymer Layer Coating the Core Layer The cationic polymer layer coats the surface of the core layer through electrostatic interaction with the chemically crosslinked alginate of the core layer. The cationic polymer forming the cationic polymer layer is not particularly limited as long as it can form a membrane through electrostatic interaction with the chemically crosslinked alginate, and examples thereof include polymers having a basic functional group such as an amino group or an imino group. Specific examples include polysaccharides such as chitosan and glycol chitosan; polyamino acids such as polylysine (preferably poly-L-lysine (PLL)), polyornithine (preferably poly-L-ornithine (PLO)), polyarginine (preferably poly-L-arginine), and polyhistidine (preferably poly-L-histidine); proteins such as protamine, collagen, and gelatin; and synthetic polymers such as polyethyleneimine, polyvinylamine, polyvinylpyridine, polyvinylimidazole, and polyallylamine. The cationic polymer is preferably chitosan, PLL, or PLO, more preferably PLL or PLO, and even more preferably PLO. Commercially available PLO products can be used, such as poly-L-ornithine hydrochloride and poly-L-ornithine hydrobromide (SIGMA-ALDRICH).

[0083] When PLO is used as the cationic polymer, the weight-average molecular weight of PLO is preferably 500 to 300,000, more preferably 1,000 to 200,000, still more preferably 5,000 to 100,000, and in one embodiment, 10,000 to 50,000. The weight-average molecular weight of PLO can be measured by gel permeation chromatography (GPC).

[0084] 1-3. Anionic Polymer Layer Coating the Cationic Polymer Layer The anionic polymer layer coats the outside of the cationic polymer layer through electrostatic interaction with the cationic polymer. The anionic polymer forming the anionic polymer layer is not particularly limited as long as it can form a membrane through electrostatic interaction with the cationic polymer, and examples include polymers having acidic functional groups such as carboxyl groups or sulfate groups. Specific examples include polysaccharides such as alginic acid, polygalacturonic acid, pectin, pectic acid, carboxymethylcellulose, carrageenan, hyaluronic acid, and chondroitin sulfate, synthetic polymers such as polyacrylic acid and polystyrene sulfonic acid, and chemically modified versions thereof, including chemically crosslinked versions thereof. Furthermore, the anionic polymer may be a polymer that exhibits neutrality overall, as long as it has the property of being able to form a membrane through electrostatic interaction with the cationic polymer. The anionic polymer layer is preferably a layer composed of a biocompatible anionic polymer. Furthermore, it is advisable to select an anionic polymer or a chemically modified version thereof having appropriate properties depending on the intended use and the environment of the site of use. For example, if adhesion is a concern, an anionic polymer with low cell adhesiveness or cell proliferation properties can be selected.

[0085] In some embodiments, the anionic polymer layer is alginic acid or chemically crosslinked alginic acid. The term "alginic acid" used herein refers to alginic acid (e.g., sodium alginate) as described in "2. Alginic acid (regarding raw alginic acid used in synthesizing chemically modified alginic acid derivatives and alginic acid used in the anionic polymer layer)" below. Furthermore, the term "chemically crosslinked alginic acid" refers to chemically crosslinked alginic acid as described in "5. Chemically crosslinked alginic acid (core layer and anionic polymer layer)" below. The anionic polymer layer is preferably alginic acid.

[0086] 1-4. Shape of the Structure, etc. The structures provided herein may have various shapes. The structure may be, for example, beads, sheets, fibers, or the like, or may be a combination of these shapes or a three-dimensionally deformed shape. Furthermore, the structure may be freely formed using a 3D printer or the like. The size of the structure is not particularly limited as long as it can maintain its shape; however, an appropriate size should be selected depending on the intended use and site of use. The diameter of the core layer of the structure is preferably less than the outer diameter of the structure but is at least 50% of the outer diameter. Furthermore, the thickness of the cationic polymer layer in the structure is not particularly limited as long as it can form and maintain a layer; however, an appropriate thickness should be selected depending on the type of cationic polymer, the intended use and site of use of the structure, etc. In one embodiment, the thickness is 0.01 to 1000 μm, in another embodiment, 0.1 to 200 μm, and in yet another embodiment, 1 to 100 μm. For example, when PLO is used as the cationic polymer, the thickness of the cationic polymer layer is 0.1 to 200 μm in one embodiment, and 1 to 100 μm in another embodiment. The thickness of the anionic polymer layer in the structure is not particularly limited as long as it is thick enough to form and maintain a layer, but an appropriate thickness should be selected depending on the type of anionic polymer, the intended use of the structure, the site of use, etc. In one embodiment, it is 0.01 to 1000 μm, in another embodiment, it is 0.1 to 200 μm, and in yet another embodiment, it is 1 to 100 μm. For example, when alginic acid or chemically modified alginic acid is used as the anionic polymer, the thickness of the anionic polymer layer is in one embodiment 0.1 to 200 μm, and in another embodiment, it is 1 to 100 μm.

[0087] When the structure is in the shape of a bead, the shape of the structure is not particularly limited as long as it is spherical, and it may also be called a capsule. The bead shape is not limited to a symmetrical sphere, but may also be an asymmetrical structure or a deformed shape, and includes, for example, an ellipsoid, a prolate spheroid (oblong ellipsoid), a bean shape, or a marble shape. When the structure is in the shape of a bead, its diameter (hereinafter, in the case of a non-spherical structure, this refers to the major axis or maximum diameter) is preferably 0.01 to 20 mm, and more preferably 0.1 to 10 mm. In one embodiment, it is 1 to 8 mm, or 1 to 6 mm. In another embodiment, it is 2 to 6 mm, 3 to 6 mm, or 4 to 6 mm.

[0088] When the structure is in the shape of a bead, the diameter of the core layer is preferably 0.01 to 20 mm, more preferably 0.1 to 10 mm. In one embodiment, it is 1 to 8 mm, or 1 to 6 mm. The diameter of the core layer is preferably less than but not less than 50% of the diameter of the structure.

[0089] When the structure is in the shape of beads, the thickness of the cationic polymer layer is 0.1 to 200 μm in one embodiment, and 1 to 100 μm in another embodiment.

[0090] When the structure is in the shape of beads, the thickness of the anionic polymer layer is 0.1 to 200 μm in one embodiment, and 1 to 100 μm in another embodiment.

[0091] When the structure is in the form of a sheet, the shape of the structure is not particularly limited as long as it is flat. A flat plate means a flat plate having a substantially uniform thickness and a large area, but the shape of the sheet as a structure, i.e., a flat plate, does not need to be uniform in thickness; for example, it may be a gradient structure in which one side is thick and the other is thin. Furthermore, the shape of the sheet may be a shape obtained by deforming a flat structure, such as a mesh shape, a honeycomb shape, a perforated structure, or a rolled cylindrical structure. Examples of the shape of the sheet include polygonal shapes such as a triangle, a rectangle, or a pentagon, and flat shapes such as a circle. In this specification, a three-layer structure in the case where the structure is in the form of a sheet does not simply mean a structure in which a core layer, a cationic polymer layer, and an anionic polymer layer are laminated in this order, but means a structure in which the upper, lower, and side surfaces of the core layer are coated with a cationic polymer layer, and the upper, lower, and side surfaces of the cationic polymer layer are further coated with an anionic polymer layer. However, a structure in the form of a sheet obtained by coating in this way and then cut to a desired shape is also included in the structure.

[0092] When the structure is in the form of a sheet, the thickness thereof is preferably 0.1 to 10 mm, more preferably 0.2 to 5 mm.

[0093] When the structure is in the form of a sheet, the thickness of the core layer is preferably 0.1 to 10 mm, more preferably 0.2 to 5 mm, and is preferably at least 50% less than the thickness of the structure.

[0094] When the structure is in the form of a sheet, the thickness of the cationic polymer layer is 0.1 to 200 μm in one embodiment, and 1 to 100 μm in another embodiment.

[0095] When the structure is in the form of a sheet, the thickness of the anionic polymer layer is 0.1 to 200 μm in one embodiment, and 1 to 100 μm in another embodiment.

[0096] The thickness of the cationic polymer layer and the anionic polymer layer referred to here refers to the thickness of either the upper surface or the lower surface of the sheet-shaped structure.

[0097] Furthermore, it is preferable that the sheet-shaped structure has a substantially constant thickness throughout the entire plate, and the thickness variation in the plate-shaped structure is preferably within ±20%.

[0098] In some embodiments, the sheet-like structure has, for example, a flat plate shape expressed in length x width x thickness, with a length of 1 to 200 mm, a width of 1 to 200 mm, and a thickness of 0.2 to 5 mm. In other embodiments, the sheet-like structure has, for example, an area expressed in length x width of 1 to 40,000 mm. ２ , and a thickness of 0.2 to 5 mm. In these embodiments, the structures can also be circular, square, hexagonal, octagonal, and the like.

[0099] When the structure is in the form of a fiber, the shape of the structure is not particularly limited as long as it is a fibrous, elongated shape. The fiber shape is not limited to a circular cross-section perpendicular to the central axis, but may also be an asymmetric structure or a deformed shape, for example, a polygonal cross-section such as a triangle, a rectangle, or a pentagon, or an elliptical cross-section. A circular cross-sectional shape is preferred. Also included are fibers formed by connecting both ends to form a ring, or multiple such rings connected to form a chain, as well as fibers bundled or connected in the form of a sheet. When the structure is in the form of a fiber, the diameter of the cross-section (hereinafter, in the case of a non-circular structure, the major axis or maximum diameter) is preferably 0.01 to 20 mm, more preferably 0.1 to 10 mm, even more preferably 0.2 to 5 mm, and particularly preferably 0.2 to 2 mm. Furthermore, when the structure is in the form of a fiber, the cross-sectional diameter of the core layer is preferably 0.01 to 20 mm, more preferably 0.1 to 10 mm, even more preferably 0.2 to 5 mm, and particularly preferably 0.2 to 2 mm. The cross-sectional diameter of the core layer is preferably less than the cross-sectional diameter of the structure but is at least 50% of the cross-sectional diameter. Furthermore, when the structure is in the form of a fiber, the thickness of the cationic polymer layer is, in one embodiment, 0.1 to 200 μm, and in another embodiment, 1 to 100 μm. Furthermore, when the structure is in the form of a fiber, the thickness of the anionic polymer layer is, in one embodiment, 0.1 to 200 μm, and in another embodiment, 1 to 100 μm. Furthermore, when the structure is in the form of a fiber, the length is not limited, but is, for example, 10 mm to 50 m in one embodiment, 10 mm to less than 30 cm in another embodiment, and 30 cm to 50 m in yet another embodiment.

[0100] In this specification, a numerical range indicated using "to" indicates a range that includes the numerical values ​​before and after "to" as the minimum and maximum values, respectively.

[0101] 2. Alginic Acid (Regarding Alginic Acid as a Raw Material Used in Synthesizing Chemically Modified Alginic Acid Derivatives and Alginic Acid Used in the Anionic Polymer Layer) In this specification, the term "alginic acid" refers to at least one alginic acid (sometimes referred to as "alginic acids") selected from the group consisting of alginic acid, alginate esters, and salts thereof (e.g., sodium alginate). The alginic acid used may be naturally occurring or synthetic, but naturally occurring alginic acid is preferred. The preferred alginic acids are bioabsorbable polysaccharides extracted from brown algae such as Lessonia, Macrocystis, Laminaria, Ascophyllum, Durvillea, Ecklonia cava, Eisenia bicolor, and Laminaria kelp, and are linear polymers of two uronic acids, D-mannuronic acid (M) and L-guluronic acid (G). 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 bonded.

[0102] Alginic acid is a natural polysaccharide extracted and purified from brown seaweed. It is a polymer formed by the polymerization of D-mannuronic acid (M) and L-guluronic acid (G). The composition ratio (M / G ratio) of alginic acid to L-guluronic acid, i.e., gel strength, varies primarily depending on the type of organism from which the seaweed is derived and is also influenced by the habitat and season of the organism. It ranges widely from a high G type (M / G ratio of approximately 0.2) to a high M type (M / G ratio of approximately 5). The physicochemical properties of alginic acid, as well as the preferred uses, vary depending on the M / G ratio and the arrangement of M and G moieties. The gelling ability of alginic acids and the properties of the resulting gel are affected by the M / G ratio; it is generally known that a higher G ratio results in higher gel strength. The M / G ratio also affects the hardness, brittleness, water absorption, flexibility, and other properties of the gel. Therefore, the alginic acid used in the present invention should have an appropriate M / G ratio and viscosity depending on the final use.

[0103] Industrial methods for producing alginic acid include the acid method and the calcium method, and alginic acid produced by either method can be used in the present invention. After purification, alginic acid whose quantitative value by HPLC falls within the range of 80 to 120% by mass is preferred, more preferably 90 to 110% by mass, and even more preferably 95 to 105% by mass. In the present invention, alginic acid whose quantitative value by HPLC falls within the above range is referred to as high-purity alginic acid. The alginic acid or its salt used in the present invention is preferably high-purity alginic acid. Commercially available products include those sold by Kimica Co., Ltd. under the Kimica Algin series, preferably high-purity food and pharmaceutical grade products. Commercially available products can also be further purified as appropriate. For example, low-endotoxin treatment is preferred. Purification and low-endotoxin treatment methods, such as those described in JP 2007-75425 A, can be used. The terms "alginate ester" and "alginate salt" used herein are not particularly limited, but must not have functional groups that inhibit the reaction with reaction reagents for ionic crosslinking or chemical crosslinking. Preferred examples of alginate esters include propylene glycol alginate.

[0104] As used herein, alginates include, for example, monovalent salts of alginic acid and divalent salts of alginic acid. Monovalent salts of alginic acid are salts prepared by ion-exchanging the hydrogen ion of the D-mannuronic acid or L-guluronic acid carboxylic acid of alginic acid with a monovalent metal ion such as Na or K. Preferred salts include sodium alginate, potassium alginate, ammonium alginate, etc., more preferably sodium alginate or potassium alginate, and particularly preferably sodium alginate. Preferred divalent salts of alginic acid include calcium alginate, magnesium alginate, barium alginate, strontium alginate, etc.

[0105] When an alginate is used as a component of the structure of the present invention, calcium alginate is used in one embodiment, and sodium alginate in another embodiment. When an alginate is used as a raw material for producing the chemically modified alginic acid derivative of the present invention or in the process of producing the structure of the present invention, a "monovalent metal salt of alginic acid" may be preferably used. Specific examples of monovalent metal salts of alginic acid include sodium alginate and potassium alginate, with sodium alginate being particularly preferred.

[0106] In this specification, alginic acid may be expressed as (ALG)-COOH, where alginic acid is (ALG) and any one of the carboxyl groups of alginic acid is -COOH.

[0107] The alginic acid used in the present invention should have an appropriate weight-average molecular weight depending on its end use. For example, it is preferable to use one with a weight-average molecular weight of 10,000 to 10,000,000, more preferably 100,000 to 5,000,000, and even more preferably 150,000 to 3,000,000. In some embodiments, the alginic acid is sodium alginate. Commercially available sodium alginate can be used. For example, sodium alginate A-1, A-2, A-3, B-1, B-2, and B-3 (sold by Mochida Pharmaceutical Co., Ltd.) listed in the table below can be used. The viscosity, weight-average molecular weight, and M / G ratio of a 1 w / w% aqueous solution of each sodium alginate are shown in the table below.

[0108] 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 methods. The measurement methods are not limited to these methods, but the physical property values ​​may differ from those described above depending on the measurement method. One non-limiting preferred embodiment of sodium alginate used in producing the structure of the present invention is "A-2, A-3, B-2, and B-3," and another embodiment is "A-2 and B-2." An embodiment in which the structure is subjected to a chelate treatment is "A-2 and A-3," and yet another embodiment is "A-2."

[0109] [Viscosity measurement of sodium alginate] Measurement was performed using a rotational viscometer (cone-plate rotational viscometer) according to the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). Specific measurement conditions are as follows: The sample solution was prepared using MilliQ water. The measurement equipment used was a cone-plate rotational viscometer (viscosity and viscoelasticity measuring device Rheostress RS600 (Thermo Haake GmbH) sensor: 35 / 1). The rotation speed was 1 rpm when measuring 1 w / w% sodium alginate solution. The reading time was 2 minutes, and the average value from 1 minute to 2 minutes was used. The average value of three measurements was used as the measured value. The measurement temperature was 20°C.

[0110] [Measurement of weight-average molecular weight of sodium alginate] Measurement was carried out using two methods: (1) gel permeation chromatography (GPC) and (2) GPC-MALS. The measurement conditions were as follows:

[0111] [Pretreatment Method] The sample was dissolved in an eluent, and then filtered through a 0.45 μm membrane filter to prepare a measurement solution.

[0112] (1) Gel Permeation Chromatography (GPC) Measurement [Measurement Conditions (Relative Molecular Weight Distribution Measurement)] Column: TSKgel GMPW-XL x 2 + G2500PW-XL (7.8 mm ID x 300 mm x 3) Eluent: 200 mM sodium nitrate aqueous solution Flow rate: 1.0 mL / min Concentration: 0.05% Detector: RI detector Column temperature: 40°C Injection volume: 200 μL Molecular weight standards: standard pullulan, glucose

[0113] (2) GPC-MALS Measurement [Refractive Index Increment (dn / dc) Measurement (Measurement Conditions)] Differential Refractometer: Optilab T-rEX Measurement Wavelength: 658 nm Measurement Temperature: 40°C Solvent: 200 mM aqueous sodium nitrate solution Sample Concentration: 0.5 to 2.5 mg / mL (5 concentrations)

[0114] [Measurement conditions (measurement of absolute molecular weight distribution)] Column: TSKgel GMPW-XL x 2 + G2500PW-XL (7.8 mm I.D. x 300 mm x 3) Eluent: 200 mM aqueous sodium nitrate solution Flow rate: 1.0 mL / min Concentration: 0.05% Detector: RI detector, MALS light scattering detector Column temperature: 40°C Injection volume: 200 μL In this specification, the molecular weights of alginic acid, chemically modified alginic acid derivatives, and chemically crosslinked alginic acid may be expressed in units of Da (Dalton).

[0115] The composition ratio (M / G ratio) of D-mannuronic acid to L-guluronic acid in alginates varies mainly depending on the type of organism from which seaweed is derived, and is also influenced by the location and season of the organism. It ranges widely, from a high M / G ratio of approximately 0.2 (high G type) to a high M / G ratio of approximately 5 (high M type). The gelling ability of alginates and the properties of the resulting gel are affected by the M / G ratio, and it is generally known that a higher G ratio results in higher gel strength. The M / G ratio also affects the hardness, brittleness, water absorption, flexibility, and other properties of the gel. The M / G ratio of the alginates and / or their salts used is typically 0.1 to 4.0, in some embodiments 0.1 to 3.0, in some embodiments 0.1 to 2.0, in some embodiments 0.5 to 1.8, and in some embodiments 0.8 to 1.2. In other embodiments, it is 0.1 to 0.5.

[0116] Alginic acid is a polymeric polysaccharide, and although it is difficult to accurately determine its molecular weight, its weight-average molecular weight is generally in the range of 1,000 to 10,000,000, preferably 10,000 to 8,000,000, and more preferably 20,000 to 3,000,000. It is known that when measuring the molecular weight of naturally occurring polymeric substances, the values ​​can vary depending on the measurement method.

[0117] In this specification, when specifying the molecular weight of a chemically modified alginic acid derivative or alginic acid or its salt, unless otherwise specified, it is the weight-average molecular weight calculated by size exclusion chromatography (SEC). It is desirable to use alginic acid or its salt for use in the present invention with an appropriate molecular weight distribution depending on its final use.

[0118] For example, under the measurement conditions of gel permeation chromatography (GPC) or gel filtration chromatography (collectively referred to as size exclusion chromatography (SEC)) described in the Examples below, the molecular weight is preferably 100,000 to 5,000,000, more preferably 150,000 to 3,000,000. In one embodiment, the molecular weight is in the range of 500,000 to 3,000,000, more preferably 1,000,000 to 2,500,000, and even more preferably 1,000,000 to 2,000,000.

[0119] Furthermore, for example, the absolute weight-average molecular weight can be measured by GPC-MALS (SEC-MALS). The weight-average molecular weight (absolute molecular weight) measured by GPC-MALS is preferably 10,000 or more, more preferably 50,000 or more, and even more preferably 60,000 or more, and is preferably 1,000,000 or less, more preferably 800,000 or less, even more preferably 700,000 or less, and particularly preferably 500,000 or less. The preferred range is 10,000 to 1,000,000, more preferably 500,000 to 800,000, and even more preferably 60,000 to 500,000.

[0120] Typically, when the molecular weight of a high molecular weight polysaccharide is calculated using the above-mentioned SEC or SEC-MALS method, a measurement error of about 10% to about 30% can occur. For example, if the molecular weight is 500,000, the value can vary within a range of about 350,000 to 650,000, and if the molecular weight is 1,000,000, the value can vary within a range of about 700,000 to 1,300,000. In this specification, when the term "about" is used in describing molecular weight measurements, it can include values ​​up to ±10% of the numerical value, and in some embodiments, up to ±20% of the numerical value.

[0121] Generally, naturally occurring polymeric substances do not have a single molecular weight but are aggregates of molecules with various molecular weights, and are therefore measured as a molecular weight distribution with a certain range. A typical measurement method is gel filtration chromatography. Typical information on molecular weight distribution obtained by gel filtration chromatography includes the weight average molecular weight (Mw), number average molecular weight (Mn), and polydispersity ratio (Mw / Mn).

[0122] The weight average molecular weight is determined by taking into account the contribution of large molecular weight polymers to the average molecular weight, and is expressed by the following formula.

[0123] Mw = Σ(WiMi) / W = Σ(HiMi) / Σ(Hi) The number average molecular weight is calculated by dividing the total weight of polymers by the total number of polymers.

[0124] Mn = W / ΣNi = Σ(MiNi) / ΣNi = Σ(Hi) / Σ(Hi / Mi) where W is the total weight of polymers, Wi is the weight of the i-th polymer, Mi is the molecular weight at the i-th elution time, Ni is the number of molecular weights Mi, and Hi is the height at the i-th elution time.

[0125] It is known that when measuring the molecular weight of naturally occurring polymeric substances, the values ​​can vary depending on the measurement method (for example, hyaluronic acid: Chikako YOMOTA et. al. Bull. Natl. Health Sci., Vol. 117, pp. 135-139 (1999), Chikako YOMOTA et. al. Bull. Natl. Inst. Health Sci., Vol. 121, pp. 30-33 (2003)). Regarding the measurement of the molecular weight of alginic acid, there are documents describing a method of calculation from intrinsic viscosity and a method of calculation by SEC-MALLS (Size Exclusion Chromatography with Multiple Angle Laser Light Scattering Detection) (ASTM F2064-00 (2006), published by ASTM International). In the present invention, the weight average molecular weight can be a value calculated by a conventional method such as that shown in the above document, for example, by measuring the molecular weight by size exclusion chromatography (SEC) and using a calibration curve using pullulan as a standard substance.

[0126] In the present invention, the weight-average molecular weight can be the absolute molecular weight measured by a conventional method such as size exclusion chromatography (SEC)-MALS, as described in the above-mentioned literature. The molecular weight of alginic acids can be measured by conventional methods.

[0127] In this specification, when the molecular weight of alginic acid or a salt thereof is specified, unless otherwise specified, it refers to the weight-average molecular weight calculated by gel filtration chromatography. Typical conditions for using gel filtration chromatography to measure molecular weight include, for example, the conditions in the Examples described below. The column can be, for example, a Superose 6 Increase 10 / 300 GL column (GE Healthcare Sciences). The developing solvent can be, for example, 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl. The molecular weight standards can be, for example, blue dextran, thyroglobulin, ferritin, aldolase, conalbumin, ovalbumin, ribonuclease A, and aprotinin.

[0128] The viscosity of the alginic acid used in this specification is not particularly limited, but when measured as a 1 wt% aqueous solution of alginic acids, it is preferably 10 mPa·s to 1000 mPa·s, more preferably 50 mPa·s to 800 mPa·s.

[0129] The viscosity of an aqueous solution of alginic acid can be measured according to a conventional method. For example, the viscosity can be measured using a rotational viscometer such as a coaxial double-cylinder rotational viscometer, a single-cylinder rotational viscometer (Brookfield viscometer), or a cone-and-plate rotational viscometer (cone-and-plate viscometer). Preferably, the viscosity is measured according to the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). More preferably, a cone-and-plate viscometer is used.

[0130] When alginic acids are first extracted from brown algae, they have a large molecular weight and a high viscosity, but the molecular weight decreases and the viscosity decreases during processes such as thermal drying and purification. Alginic acids with different molecular weights can be produced by controlling conditions such as temperature during the production process, selecting the brown algae used as raw materials, and molecular weight fractionation during the production process. Furthermore, alginic acids with the desired molecular weight can be produced by mixing them with other lots of alginic acids with different molecular weights or viscosities.

[0131] The alginate used herein may be, in some embodiments, alginate that has not been treated with low endotoxins, or in other embodiments, alginate that has been treated with low endotoxins. Low endotoxins refers to an endotoxin level that is low enough to not substantially induce inflammation or fever. Low endotoxin alginates are more preferred.

[0132] 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), the purification of β1,3-glucan by Yoshida et al. (see, for example, Japanese Patent Application Laid-Open No. 8-269102), 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), the purification of polysaccharides by James et al. (see, for example, the pamphlet of International Publication No. 93 / 13136), the method of Lewis et al. (see, for example, the specification of U.S. Patent No. 5,589,591), the purification of alginate by Herman Frank et al. (see, for example, Appl Microbiol Biotechnol (1994) 40:638-643), or methods similar thereto. Low-endotoxin treatment can be carried out by known methods such as, but not limited to, washing, filtration through a filter (endotoxin removal filter, charged filter, etc.), ultrafiltration, purification using a column (endotoxin-adsorbing affinity column, gel filtration column, ion exchange resin column, 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), or an appropriate combination of these. These treatment steps may also be appropriately combined with known methods such as centrifugation. It is desirable to select an appropriate method depending on the type of alginate.

[0133] The endotoxin level can be confirmed by known methods, and can be measured, for example, by a method using Limulus Limulus Aqueous Solution (LAL) or a method using Endospecy (registered trademark) ES-24S Set (Seikagaku Corporation).

[0134] The method of endotoxin treatment used is not particularly limited, but as a result, the endotoxin content of alginic acids is preferably 500 endotoxin units (EU) / g or less, more preferably 100 EU / g or less, particularly preferably 50 EU / g or less, and particularly preferably 30 EU / g or less, when endotoxin is measured using Limulus reagent (LAL). In the present invention, "substantially endotoxin-free" means that the endotoxin value measured by the Japanese Pharmacopoeia endotoxin test is within the above-mentioned numerical range. Low-endotoxin treated sodium alginate is available, for example, from Sea Matrix (registered trademark) (Mochida Pharmaceutical Co., Ltd.), PRONOVA ＴＭ It is available commercially as UP LVG (FMC BioPolymer).

[0135] 3. Chemically Modified Alginic Acid Derivatives 3-1. Chemically Modified Alginic Acid Derivatives (Huisgen Type) In some embodiments, the chemically modified alginic acid derivatives herein are those in which a reactive group in the Huisgen reaction or a reactive group complementary to the reactive group is introduced into any one or more carboxyl groups of alginic acid via an amide bond and a divalent linker. More specifically, the chemically modified alginic acid derivatives are represented by the following formula (H-I): [In formula (HI), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L １ - is a divalent linker that bonds to a cyclic alkyne group (Akn)], and a chemically modified alginic acid derivative represented by the following formula (H-II): [In formula (H-II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L ２- is a divalent linker that bonds to the azide group. １ -or-L ２ Any straight-chain group can be used for the reactive group (-(CH ２ ) ｎ -, n = 1 to 30) (-CH ２ - may 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, or a heterocycle (a 5- or 6-membered aromatic heterocycle or a 5- or 6-membered non-aromatic heterocycle such as a pyridine ring, a piperidine ring, or a piperazine ring), and the -CH ２ The hydrogen atom of - is an oxo group (=O), C １－６ Examples of the alkyl group include, but are not limited to, alkyl groups (e.g., methyl groups, ethyl groups, n-propyl groups, isopropyl groups, etc.), halogen atoms (e.g., fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, etc.), hydroxyl groups (—OH), etc., which may be substituted with a plurality of groups (e.g., 1 to 10 groups, or 1 to 5 groups).

[0136] The cyclic alkyne group (Akn) is a 7- to 10-membered saturated hydrocarbon ring group in which one bond constituting the ring is a triple bond, and is preferably an 8-membered cyclic alkyne group. The cyclic alkyne group may further be condensed with one or two ring groups selected from a benzene ring, a 3- to 8-membered hydrocarbon ring, or an aromatic heterocycle such as a pyridine ring. In addition, the —CH ２ - may be replaced by 1 or 2 groups selected from -C(=O)-, -CONH-, -O-, -NH-, and -S-. ２ The hydrogen atom of - is an oxo group (=O), C １－６ The alkyl group may be substituted with 1 or 2 groups selected from the group consisting of alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl, etc.), halogen atoms (e.g., fluorine and chlorine atoms), hydroxyl groups (—OH), etc. However, the substituents are not limited to these.

[0137] In one embodiment of the chemically modified alginic acid derivative used in producing the structure of the above embodiment [2], in the above formula (HI), -L １ - is - (CH ２ ) ｎ１ - (where n1=1 to 50, and -CH ２ - may be replaced by 1 to 10 groups selected from -CO-, -CONH-, -NHCO-, -O-CONH-, -NHCO-O-, -O- and -NH-, or a benzene ring, ２ The hydrogen atom of - is C １－3 C substituted with alkyl or phenyl groups １－3 Akn is an 8-membered cyclic alkyne group (which may be further fused with one or two benzene rings, cyclopropane rings, or 1,2,3-triazole rings, and the fused rings may have -L １ -, and -CH ２ - may be replaced by 1 or 2 groups selected from -C(=O)-, -CONH-, and -NH-, and the -CH ２ The hydrogen atom of - is C １－３ Alkyl group, fluorine atom, hydroxyl group, C １－３ and wherein in the formula (H-II), -L ２ -ga- (CH ２ ) ｎ２ - (where n2=1 to 50, and -CH ２ - may be replaced by 1 to 10 groups selected from -CONH-, -NHCO-, -O- and -NH-, or a benzene ring or a pyridine ring, and the -CH ２ The hydrogen atom of - is C １－3 The compound is a compound in which the formula (I) is a substituted or unsubstituted alkyl group.

[0138] In the above compounds, n1 is preferably 1 to 20, and more preferably 1 to 15 (for example, 2 to 15). １A preferred embodiment of - is -(CH ２ ) ｎ１ -Medium, -CH ２ - is a group in which - may be replaced by 1 to 5, more preferably 1 to 3, groups selected from -CO-, -CONH-, -NHCO-, -O-CONH-, -NHCO-O-, -O-, and -NH-. Akn is preferably a group in which one or two benzene rings may be condensed, and -CH in the 8-membered cyclic alkyne group ２ n2 is an 8-membered cyclic alkyne group in which - may be replaced by -NH-. In a preferred embodiment, n2 is 1 to 20, and in a more preferred embodiment, it is 1 to 15 (for example, 2 to 15). ２ A preferred embodiment of - is -(CH ２ ) ｎ２ -Medium, -CH ２ - is a group which may be substituted by 1 to 5, more preferably 1 to 3, groups selected from -CONH-, -NHCO-, -O- and -NH-.

[0139] A preferred embodiment of the chemically modified alginic acid derivative used in producing the structure of the above embodiment [2] is a compound represented by the formula (HI): １ - is - (CH ２ ) ｎ１ - (where n1=1 to 15 (e.g., 2 to 15), and -CH ２ - may be replaced by 1 to 5 groups selected from -CO-, -CONH-, -NHCO-, -O-CONH-, -NHCO-O-, -O- and -NH-, or a benzene ring, ２ The hydrogen atom of - is C １－3 C substituted with alkyl or phenyl groups １－3 Akn is an 8-membered cyclic alkyne group (which may be further fused with 1 or 2 benzene rings, and the 8-membered cyclic alkyne group may be fused with -CH ２ In the formula (H-II), -L may be an 8-membered ring group in which - is replaced by -NH-. ２ -ga- (CH ２ ) ｎ２- (where n2=1 to 15 (e.g., 2 to 15), and -CH ２ - may be replaced by 1 to 5 groups selected from -CONH-, -NHCO-, -O- and -NH-, or a benzene ring.

[0140] In one embodiment of the chemically modified alginic acid derivative, in the formula (HI), -L １ In one embodiment of the chemically modified alginic acid derivative, in the formula (H-II), -L ２ - is a compound in which the definition of is the same as that in any one of the above-mentioned embodiments [2-1] to [2-5].

[0141] In some embodiments, the compound represented by the following formula (HA-I): [In formula (HA-I), (ALG), -L １ -, Akn are defined as in the above-mentioned embodiment [4]], and a chemically modified alginic acid derivative represented by the following formula (HA-II): [In formula (HA-II), (ALG), -L ２ The definition of "-" is the same as that in the above embodiment [4].

[0142] In some embodiments, the compound represented by the following formula (HB-I): [In formula (HB-I), (ALG), -L １ -, Akn are defined as in any one of the above-mentioned aspects [3-1] to [3-6]], and a chemically modified alginic acid derivative represented by the following formula (HB-II): [In formula (HB-II), (ALG), -L ２ The definition of "-" is the same as that in any one of the above-mentioned embodiments [3-1] to [3-6].

[0143] In the —NH—CO— group of the chemically modified alginic acid derivatives represented by the formulas (HI) to (HB-II), the hydrogen atom of the imino group (—NH—) can be substituted with a methyl group to form an —N(Me)—CO— group.

[0144] In the chemically modified alginic acid derivatives represented by the formulas (HI) to (HB-II), the linker (-L １ -, -L ２ The bonding mode between the alkyl group and the chemically modified alginic acid is an —NH—CO— bond or an —N(Me)—CO— bond; an —NH—CO— bond is preferred.

[0145] The chemically modified alginic acid derivatives represented by formulas (HI) to (HB-II) in this specification can be produced, for example, by the method shown in the following formula (for details, see 4. Method for synthesizing chemically modified alginic acid derivatives).

[0146] The weight-average molecular weight of the chemically modified alginic acid derivatives represented by formulas (HI) to (HB-II) herein is 100,000 to 3,000,000 Da, preferably 300,000 to 2,500,000 Da, and more preferably 500,000 to 2,000,000 Da. The molecular weight of both alginic acid derivatives can be determined by the method described below.

[0147] In the present specification, Akn-L of formula (HI), formula (HA-I) or formula (HB-I) １ The —NH— group does not necessarily bond to all the carboxyl groups of the alginic acid building block, and the N of formula (H-II), formula (HA-II) or formula (HB-II) ３ -L ２ It is not necessary for the --NH-- group to be bonded to all of the carboxyl groups of the alginic acid building block.

[0148] In the present specification, Akn-L of formula (HI), formula (HA-I) or formula (HB-I) １ When the —NH— group is referred to as a reactive group, it is the N group of formula (H-II), formula (HA-II), or formula (HB-II). ３ -L ２ Conversely, the —NH— group in formula (H-II), formula (HA-II) or formula (HB-II) serves as a complementary reactive group. ３ -L ２ When the —NH— group is referred to as a reactive group, it is Akn-L of formula (HI), formula (HA-I) or formula (HB-I). １ The —NH— group serves as a complementary reactive group.

[0149] In this specification, the introduction rate of the reactive group or the complementary reactive group is 0.1% to 30% or 1% to 30%, respectively, preferably 2% to 20%, and more preferably 3% to 10%.

[0150] The introduction rate of the reactive group or complementary reactive group is the number of uronic acid monosaccharide units into which each reactive group has been introduced, expressed as a percentage, among the uronic acid monosaccharide units, which are the repeating units of alginic acids. Unless otherwise specified, in this specification, % used for the introduction rate of the reactive group or complementary reactive group in the chemically modified alginic acid derivatives (formulas (H-I) to (HB-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.

[0151] In the present specification, the cyclic alkyne group (Akn) in formula (HI), formula (HA-I) or formula (HB-I) and the azide group in formula (H-II), formula (HA-II) or formula (HB-II) form a triazole ring by Huisgen reaction, thereby forming a crosslink.

[0152] 3-1-1. Huisgen Reaction The Huisgen reaction (1,3-dipolar cycloaddition reaction) is a condensation reaction between compounds having a terminal azide group and a terminal alkyne group, as shown in the following formula. As a result of the reaction, a disubstituted 1,2,3-triazole ring is obtained in good yield, and this reaction is characterized by the absence of unnecessary by-products. This reaction is thought to produce a 1,4- or 1,5-disubstituted triazole ring, but the use of a copper catalyst makes it possible to obtain the triazole ring regioselectively. In addition, Wittig and Krebs have reported a Huisgen reaction that does not use a copper catalyst. That is, this reaction is a reaction in which a cycloadduct can be obtained by simply mixing cyclooctyne and phenyl azide (in the following formula, R ３ (=phenyl). Because the triple bond of cyclooctyne is highly distorted, the reaction with phenyl azide relieves this distortion, driving the reaction, which proceeds spontaneously, eliminating the need for a catalyst.

[0153] As described above, the Huisgen reaction can be used with azide compounds having substituted primary azides, secondary azides, tertiary azides, aromatic azides, etc., and compounds having terminal or cyclic alkyne groups, which are reactive groups complementary to the azide group. Furthermore, since the Huisgen reaction reacts almost exclusively with azide groups and alkyne groups, it is possible to substitute various functional groups (e.g., ester groups, carboxyl groups, alkenyl groups, hydroxyl groups, amino groups, etc.) in the reaction substrate.

[0154] In some embodiments, a cyclic alkyne group (e.g., a cyclooctyl group) is used as the alkyne group in the Huisgen reaction to form crosslinks between alginic acid molecules via a 1,2,3-triazole ring in a short time, easily, and efficiently without using a copper catalyst to avoid the production of undesired by-products and to avoid the cytotoxicity of copper catalysts.

[0155] In a preferred embodiment of the crosslinking method for chemically modified alginic acid derivatives, the reaction (Huisgen reaction) hardly produces any undesirable by-products, making it possible to incorporate various pharmacological components, such as cells or biologically active substances, into the core layer of the structure of the present invention.

[0156] 3-2. Chemically modified alginic acid derivative (cinnamic acid type) A chemically modified alginic acid derivative into which a photoreactive group has been introduced is one in which some of the carboxyl groups of alginic acid are modified to the following formula (CI) [in which the right side of the dashed line is not included]: The definitions of [Ar, p, -X-, and -A-] are as described below, and are the same as those in, for example, embodiment [8-1]. Preferably, they are substituted with a photoreactive group as described in [8-2] or [8-3], i.e., they have a photoreactive group at a portion of the carboxyl group of alginic acid. A particularly preferred embodiment is the chemically modified alginic acid derivative described in [8-3]. The photoreactive group (sometimes referred to as a "photocrosslinking group") contains a moiety (photoreactive moiety) that undergoes cyclization in a photoreaction. The photoreactive moiety may be any moiety that undergoes a dimerization reaction (cyclization reaction) or polymerization reaction upon photoirradiation. Specific examples include a cinnamic acid moiety, a substituted cinnamic acid moiety, a phenylpenta-2,4-dienoic acid moiety, a substituted phenylpenta-2,4-dienoic acid moiety, or a heterocycle-substituted acrylic acid moiety. The alkene moiety in formula (C-I) is shown as a trans isomer as one form, but any bonding form may be used as long as it allows a dimerization reaction (cyclization reaction) or a polymerization reaction to proceed upon light irradiation. When p = 1, either a trans isomer or a cis isomer may be used, and when p = 2, a bonding form that combines a trans isomer and a cis isomer may be used. Any bonding form that satisfies the above is considered to be included in formula (C-I).

[0157] Among these photoreactive moieties, a moiety having a vinylene group capable of forming a cyclobutane ring by a dimerization reaction is preferred, and for example, a cinnamic acid moiety, a substituted cinnamic acid moiety, a phenylpenta-2,4-dienoic acid moiety, a substituted phenylpenta-2,4-dienoic acid moiety, and a heterocycle-substituted acrylic acid moiety are preferred.

[0158] The photoreactive group may be bonded to both the photoreactive moiety and alginic acid to maintain a certain distance between them. Derivatives in which a spacer is bonded to a photoreactive moiety such as a cinnamic acid moiety, a substituted cinnamic acid moiety, a phenylpenta-2,4-dienoic acid moiety, a substituted phenylpenta-2,4-dienoic acid moiety, or a heterocyclic-substituted acrylic acid moiety are most preferred as photoreactive groups (photocrosslinking groups).

[0159] Here, "a photoreactive group has been introduced" means that any one or more carboxyl groups of alginic acid form an amide bond with the terminal amino group of the linker in the photoreactive group, thereby bonding any one or more carboxyl groups of alginic acid to the photoreactive group via the linker.

[0160] 3-2-1. Photoreactive moiety and linker Here, in the formula (CI) which is a photoreactive group, the partial structural formula (CI-S) [the formula does not include the right side of the dashed line]: is sometimes referred to as a "photoreactive moiety" and is represented by the following formula -A- [wherein the formula does not include the area outside the dashed line]: is sometimes called a "linker."

[0161] In the photoreactive moiety, Ar is C ６～１０ Aryl group (C ６～１０ The aryl group includes a hydroxyl group, a cyano group, a nitro group, a halogen atom, C １～６ Alkyl group, halogenated C １～６ Alkyl group, C １～６ Alkoxy groups, and -NR Ａ R Ｂ Group (-NR Ａ R Ｂ In the 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 groups (wherein -NH ２ , —NH(C １～６ alkyl group), and —N(C １～６ alkyl group) ２ , and -N(C １～６ alkyl group) ２ hydrogen atoms on the ring may be substituted with 1 to 3 groups arbitrarily selected from the group consisting of C ６～１０ The aryl group may include C １～６ Alkyl group and C １～６ Alkoxy group or two C １～６ When alkoxy groups are adjacently substituted, the C １～６ Alkyl group and C １～６The alkoxy group is bonded between carbon atoms obtained by removing one arbitrary hydrogen atom from the alkyl group of each group, or the two C １～６ A cyclic ether may be formed by bonding between carbon atoms obtained by removing one arbitrary hydrogen atom from each alkyl group of the alkoxy group, or ６～１０ The aryl group may include C １～６ Alkoxy group and -NHR Ｇ Group (-NHR Ｇ R in the group Ｇ is C ２～７ Alkanoyl group or C １～６ When an alkylsulfonyl group is adjacent to the C １～６ A carbon atom obtained by removing one arbitrary hydrogen atom from the alkyl group of an alkoxy group and the —NHR Ｇ The nitrogen atom from which one hydrogen atom has been removed is bonded to form 3-N-(C ２～７ alkanoyl)oxazolidine ring, 3-N-(C １～６ alkylsulfonyl)oxazolidine ring, 4-N-(C ２～７ alkanoyl)morpholine ring, 4-N-(C １～６ alkylsulfonyl)morpholine ring, 4-N-(C ２～７ alkanoyl)-1,4-oxazepane ring, or 4-N-(C １～６ alkylsulfonyl)-1,4-oxazepane ring may be formed).

[0162] The linker (-A-) may be any linear group as long as it does not inhibit the photoreaction of the photoreactive moiety. Specifically, the linker (-A-) may be any linear group as shown in the following formulae (AL-1) to (AL-4) (each formula does not include the areas outside the dashed lines at both ends): (In formulas (AL-1) to (AL-4), n represents an integer of 1 to 18; m represents an integer of 1 to 9; j represents an integer of 0 to 9; a methylene group (—CH ２ -) hydrogen atom is 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, carboxy C１～６ 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 The alkyl group (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 groups), and 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 The methylene group (—CH ２ -) two hydrogen atoms are C １～６ When substituted with an alkyl group, each C １～６ C by bonding between carbon atoms where one arbitrary hydrogen atom has been removed from the alkyl group ３～８ (Also, the -NH- group in formula (AL-3) or formula (AL-4) may form a non-aromatic heterocycle together with the substituent substituted on the adjacent carbon atom.) However, in formula (C-I), when Ar is a phenyl group, p is 1, -A- is formula (AL-1), and n is 3, the hydrogen atoms on the phenyl group of Ar are substituted with 1 to 3 groups arbitrarily selected from the group of substituents that may be substituted for the hydrogen atoms on the phenyl group.

[0163] The -X- is -O-, -NH-, or -N(C１－６ The aforementioned p represents an integer of 1 or 2.

[0164] In this specification, unless otherwise specified, examples of a "halogen atom" include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. 1～6 The term "halogenated" in "alkyl group" and the like means having several, preferably 1 to 5, of the above-mentioned "halogen atoms" as substituents.

[0165] In this specification, unless otherwise specified, 1～6 Examples of the "alkyl group" include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, and hexyl.

[0166] In this specification, unless otherwise specified, "halogenated C 1～6 The "C alkyl group" is defined as 1～6 "Alkyl" means a group optionally substituted with several, preferably 1 to 5, halogen atoms, such as, for example, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 1,1,2,2-tetrafluoroethyl, or pentafluoroethyl.

[0167] In this specification, unless otherwise specified, 1～6 The "alkoxy group" is the same as the "C 1～6 "Alkyl" refers to an alkoxy attached to an oxygen atom, and includes groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, or hexyloxy.

[0168] In this specification, unless otherwise specified, ６～１０ Examples of the "aryl group" include phenyl, 1-naphthyl, 2-naphthyl, indanyl, indenyl, and 1,2,3,4-tetrahydronaphthyl groups.

[0169] In this specification, unless otherwise specified, ３～８ The term "cycloalkyl ring" means a saturated cyclic hydrocarbon ring (including monocyclic and polycyclic rings) having 3 to 8 carbon atoms, and examples thereof include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cyclopentane, cyclooctane, and the like.

[0170] In this specification, unless otherwise specified, the term "non-aromatic heterocycle" means a "3- to 14-membered saturated or unsaturated non-aromatic heterocycle".

[0171] In this specification, unless otherwise specified, the term "3- to 14-membered saturated or unsaturated non-aromatic heterocycle" means a 3- to 14-membered saturated or unsaturated heterocycle containing 1 to 4 heteroatoms selected from oxygen atoms, sulfur atoms, and nitrogen atoms.

[0172] Unless otherwise specified, in this specification, examples of "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, and oxazine. Unless otherwise specified, in this specification, a "5- to 6-membered non-aromatic heterocycle" refers to a 5- or 6-membered non-aromatic heterocycle among the above-mentioned "non-aromatic heterocycles", and in one embodiment, it is piperidine or piperazine.

[0173] Unless otherwise specified, in this specification, the terms "heteroaryl" and "aromatic heterocyclic" group refer to a monocyclic, polycyclic, or fused-ring (which may be partially hydrogenated in the case of a polycyclic or fused-ring) 5- to 14-membered, preferably 5- to 8-membered, more preferably 5- to 7-membered, and even more preferably 5- to 6-membered heteroaryl containing 1 to 5, preferably 1 to 3, heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen atoms.

[0174] In this specification, unless otherwise specified, the terms "5- to 6-membered heteroaryl" and "5- to 6-membered aromatic heterocyclic" refer to a 5- to 6-membered heteroaryl containing 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom, and an oxygen atom, and the term "5- to 6-membered heteroaryl" refers to a monovalent group obtained by removing any hydrogen atom from the heteroaryl ring, unless otherwise specified. Examples 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, and 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.

[0175] In this specification, unless otherwise specified, "heteroaryl C １～６ The term "alkyl group" refers to a group in which any hydrogen atom of the "heteroaryl group" is substituted with any of the "C １～６ It means a group substituted with an "alkyl group," and examples thereof include a 2-pyridylmethyl group, a 4-imidazolylmethyl group, and a 3-indolylmethyl group.

[0176] In some embodiments, the photoreactive group (photocrosslinking group moiety) has photodimerization properties, absorbing light (e.g., at a wavelength of about 180 to 650 nm) and dimerizing (e.g., dimerizing into a truxillic acid derivative).

[0177] Furthermore, by introducing a linker, the photocrosslinking reaction proceeds even when the introduction rate of photoreactive groups (photocrosslinking groups) is low. The photocrosslinking reaction allows the chemically modified alginic acid derivative to form a three-dimensional network structure via the photocrosslinking groups. A preferred chemically modified alginic acid derivative has improved stability after photocrosslinking.

[0178] In this specification, chemically modified alginic acid derivatives having a photoreactive group represented by formula (C-I) introduced therein are introduced into alginic acids by substituting a portion of the carboxyl groups of the alginic acids with the photoreactive group, and can be produced, for example, by the method shown in the following formula. In this specification, both "(ALG)" and "(alginic acid)" represent alginic acid, and in the structural formula, the notation of the amide bond (-NHCO-) ​​via any carboxyl group of alginic acid may differ between "(ALG)" and "(alginic acid)," but in both cases, the amide bond is commonly present between the linker and alginic acid. In other words, -NHCO-(ALG) and -NH-(alginic acid) represent the same structure.

[0179] The weight-average molecular weight of the novel chemically modified alginic acid derivative having a photoreactive group represented by formula (CI) introduced therein is 100,000 to 3,000,000 Da, preferably 300,000 to 2,500,000 Da, and more preferably 500,000 to 2,000,000 Da. The molecular weight of the alginic acid derivative can be determined by the same method as for the alginic acids described above.

[0180] In this specification, the photoreactive group represented by formula (CI) does not necessarily have to be bonded to all of the carboxyl groups of the alginic acid structural unit.

[0181] In this specification, the introduction rate of the photoreactive group represented by formula (CI) in the chemically modified alginic acid derivative is preferably 0.5% to 30%, more preferably 0.5% to 20%, and even more preferably 1.0% to 15%.

[0182] The introduction rate of the photoreactive group represented by formula (C-I) is the number of uronic acid monosaccharide units, which are repeating units of alginic acids, into which the photoreactive group represented by formula (C-I) has been introduced, expressed as a percentage. Unless otherwise specified, in this specification, the % used for the introduction rate of the photoreactive group represented by formula (C-I) in a chemically modified alginic acid derivative means mol%. The introduction rate of the photoreactive group represented by formula (C-I) can be determined by the method described in the Examples below.

[0183] 4. Method for synthesizing chemically modified alginic acid derivatives The chemically modified alginic acid derivatives (Huisgen type) represented by formula (HA-I) or formula (HA-II) can be produced by referring to PCT / JP2019 / 023478 (filed June 13, 2019). The chemically modified alginic acid derivatives (cinnamic acid type) represented by formula (C-I) can be produced by referring to PCT / JP2019 / 007655 (filed February 27, 2019).

[0184] The chemically modified alginic acid derivatives (Huisgen type (B)) represented by formula (HB-I) or formula (HB-II) are each ２ N-L １ -Akn (in the formula, L １ and Akn are the same as defined in the above embodiment [3-1]), or ２ N-L ２ -N ３ (In the formula, L ２ is the same as defined in the above embodiment [3-1]), can be produced by a condensation reaction with any carboxyl group of alginic acids using a condensing agent.

[0185] [Method for producing the chemically modified alginic acid derivative of formula (HB-I)] Using a 0.5% by weight to 1% by weight aqueous solution of alginic acid and an amine represented by formula (AM-1), a chemically modified alginic acid derivative of formula (HB-I) can be produced by a method known in the art, for example, by the method described in "Experimental Chemistry Lectures, 5th Edition" 16, Synthesis of Organic Compounds IV, Carboxylic Acids and Derivatives, Esters, pp. 35-70, Acid Amides and Acid Imides, pp. 118-154, Amino Acids and Peptides, pp. 258-283, 2007, Maruzen Co., Ltd., etc., 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) In the presence of a condensing agent selected from tetrahydrofuran, 1,4-dioxane, and the like, or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), and the like, in a mixed solvent of water and a solvent selected from ether solvents such as tetrahydrofuran and 1,4-dioxane, alcohol solvents such as methanol, ethanol, and 2-propanol, and polar solvents such as N,N-dimethylformamide, 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, a condensation reaction can be carried out at a temperature between 0 ° C. and 50 ° C. to produce a chemically modified alginic acid derivative of formula (HB-I).

[0186] [Method for producing chemically modified alginic acid derivatives of formula (HB-II)] A chemically modified alginic acid derivative of formula (HB-II) can be produced by carrying out a reaction using a 0.5 wt% to 1 wt% aqueous solution of alginic acid and an amine represented by formula (AM-2) in accordance with the above-mentioned [Method for producing chemically modified alginic acid derivatives of formula (HB-I)].

[0187] In the method for producing the chemically modified alginic acid derivative of formula (HB-I) or the chemically modified alginic acid derivative of formula (HB-II), the introduction rate of the amine of formula (AM-1) or formula (AM-2) can be adjusted by appropriately selecting and combining the following reaction conditions (i) to (v), taking into consideration the properties of the amine: (i) increasing or decreasing the equivalent of the condensing agent, (ii) increasing or decreasing the reaction temperature, (iii) extending or shortening the reaction time, (iv) adjusting the concentration of alginic acid as the reaction substrate, (v) adding a water-miscible organic solvent to increase the solubility of the amine of formula (AM-1) or formula (AM-2), etc.

[0188] The following describes a more specific method for producing the amines represented by formula (AM-1) or formula (AM-2).

[0189] In addition, among the following manufacturing methods, m1, n1, m2a, n2a, p2a, m2b, n2b, p2b, m3, n3, p3, m4a, n4a, m4b, n4b, m5a, n5a, p5a, q5a, m5b, n5b, p5b, q5b, m6a, n6a, p6a, m6b, n6b, p6b, m7, n7, m8a, n8a, m8b, n8b, m9a, n9a, p9a, m9b, n9b, p9b, m10, n10, p10, x The definitions of 1, x2, x2a, y2a, x2b, y2b, x3, y3, x4, y4, z4, x5a, y5a, x5b, y5b, x6a, y6a, z6a, v6a, x6b, y6b, z6b, v6b, x7a, y7a, z7a, x7b, y7b, z7b, x8a, y8a, z8a, x8b, y8b, z8b, x9a, y9a, z9a, x9b, y9b, and z9b are the same as those in the above embodiment [3-1]; P １ represents a —C(O)O-tertBu group, a —C(O)O-Bn group, a —C(O)CH ３ group, -C(O)CF ３ group, —SO ２ Ph, -SO ２ PhMe group, -SO ２ Ph(NO ２ E is a leaving group such as a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), an -OTs group, an -OMs group, etc.

[0190] In addition, in each of the following production methods, the protecting group P １ The protection and deprotection of the above can be carried out in accordance with a method known in the literature, for example, the deprotection method described in "Protective Groups in Organic Synthesis 4th Edition, 4th Edition, 2007, John Wiley & Sons, Greene et al."

[0191] [Production Method AM-A] Production method for amine represented by formula (AM-1-B1): The compound of formula (SM-B1) and the compound of formula (RG-B1) [the compound of formula (SM-B1) and the compound of formula (RG-B1) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known in the literature] are subjected to a condensation reaction similar to that described above in [Production method of chemically modified alginic acid derivative of formula (HB-I)], followed by the addition of a protecting group P １ By deprotecting the group, an amine represented by formula (AM-1-B1) or a salt thereof can be produced.

[0192] [Production Method AM-B] Production Method for Amines Represented by Formula (AM-1-B2a) and Formula (AM-1-B2b): An amine represented by formula (AM-1-B2a), or a salt thereof, can be produced by reacting a compound of formula (SM-B2a) and a compound of formula (RG-B2a) [the compounds of formula (SM-B2a) and (RG-B2b) are commercially available compounds or compounds that can be produced from commercially available compounds by production methods known in the literature] in the same manner as in [Production Method AM-A]. Similarly, an amine represented by formula (AM-1-B2b), or a salt thereof, can be produced by reacting a compound of formula (SM-B2b) and a compound of formula (RG-B2b) [the compounds of formula (SM-B2b) and (RG-B2b) are commercially available compounds or compounds that can be produced from commercially available compounds by production methods known in the literature] in the same manner as in [Production Method AM-A].

[0193] [Production Method AM-C] Production Method of Amine Represented by Formula (AM-1-B3):

[0194] An amine represented by formula (AM-1-B3) or a salt thereof can be produced by carrying out a reaction according to the above synthesis scheme (the reactions in each step are similar to those described in [Production Method AM-A]) using a compound of formula (SM-B3) [the compound of formula (SM-B3) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known in the literature]. In the above scheme, the compounds of formula (RG-B3), formula (RG-B3-1), and formula (RG-B3-2) are commercially available compounds or compounds that can be produced from a commercially available compound by a production method known in the literature.

[0195] [Production Method AM-D] Production method for amines represented by formula (AM-1-B5a) and formula (AM-1-B5b):

[0196] By carrying out the reactions according to the above synthesis scheme (the reactions in each step are similar to those described in [Production Method AM-A]), it is possible to produce amines represented by formula (AM-1-B5a) and formula (AM-1-B5b), or salts thereof. In the above scheme, the compounds of formula (SM-B5a), formula (RG-B5a), formula (RG-B5a-1), formula (RG-B5a-2), formula (SM-B5b), formula (RG-B5b), formula (RG-B5b-1), and formula (RG-B5b-2) are commercially available compounds or compounds that can be produced from commercially available compounds by production methods known in the literature.

[0197] [Production Method AM-E] Production method for amines represented by formula (AM-1-B6a) and formula (AM-1-B6b):

[0198] By carrying out the reactions according to the above synthesis scheme (the reactions in each step are similar to those described in [Production Method AM-A]), it is possible to produce amines represented by formula (AM-1-B6a) and formula (AM-1-B6b), or salts thereof. In the above scheme, the compounds of formula (SM-B6a), formula (RG-B6a), formula (SM-B6b), and formula (RG-B6b) are commercially available compounds or compounds that can be produced from commercially available compounds by production methods known in the literature.

[0199] [Production Method AM-F] Production Method of Amine Represented by Formula (AM-1-B10):

[0200] By carrying out the reactions according to the above synthesis scheme (the reactions in each step are similar to those described in [Production Method AM-A]), an amine represented by formula (AM-1-B10) or a salt thereof can be produced. In the above scheme, the compounds of formulae (SM-B10), (RG-B10), (RG-B10-1), and (RG-B10-2) are commercially available compounds or compounds that can be produced from commercially available compounds by production methods known in the literature.

[0201] [Production Method AM-G] Production method for amines represented by formula (AM-1-B4a) and formula (AM-1-B4b):

[0202] The compound of formula (SM-B4) and the compound of formula (RG-B4a) [the compound of formula (SM-B4) and the compound of formula (RG-B4a) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known in the literature] are used in accordance with a method known in the literature, for example, the method described in "Journal of the American Chemical Society, 126(46), pp. 15046-15047, 2004" etc., <Step 1a> silver trifluoromethanesulfonate, AgClO ４ In the presence of a reagent such as toluene or dichloromethane, a compound of formula (IM-B4a-1) is reacted in a reaction-inert solvent such as toluene or dichloromethane to obtain a compound of formula (IM-B4a-2). In step 2a, a debromination reaction is then carried out using a base such as sodium hydride or NaOMe to obtain a compound of formula (IM-B4a-2). In step 3a, a protecting group P １ By deprotecting the compound, an amine represented by formula (AM-1-B4a) or a salt thereof can be prepared.

[0203] Similarly, an amine represented by formula (AM-1-B4b) or a salt thereof can be produced by carrying out a reaction according to the above scheme using formula (RG-B4b) instead of formula (RG-B4a).

[0204] [Production Method AM-H] Production method for amines represented by formula (AM-1-B8a) and formula (AM-1-B8b):

[0205] By carrying out the reactions according to the above synthesis scheme (the reactions in each step are similar to those described in [Production Method AM-A]), it is possible to produce amines represented by formula (AM-1-B8a) and formula (AM-1-B8b), or salts thereof. In the above scheme, formula (SM-B8a) or formula (SM-B8b) is a commercially available compound or a compound that can be produced according to the reactions described in [Production Method AM-G], and formula (RG-B8a) or formula (RG-B8b) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known in the literature.

[0206] [Production Method AM-J] Production method for amine represented by formula (AM-1-B7):

[0207] The compound of formula (SM-B7) and the compound of formula (RG-B7) [the compound of formula (SM-B7) and the compound of formula (RG-B7) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known in the literature] are subjected to a condensation reaction similar to that described above in the [Production method of chemically modified alginic acid derivative of formula (HB-I)], followed by the removal of the protecting group P １ can be deprotected to produce an amine represented by formula (AM-1-B7) or a salt thereof.

[0208] [Production Method AM-J-2] Production Method of Amine Represented by Formula (AM-1-B7):

[0209] The compound of formula (SM-B7) and the compound of formula (RG-B7-2) [the compound of formula (SM-B7) and the compound of formula (RG-B7-2) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known in the literature] are subjected to a condensation reaction similar to that described above in the [Production method of chemically modified alginic acid derivative of formula (HB-I)] (<Step 1> and <Step 2>), followed by the addition of a protecting group P １ is deprotected, and then a condensation reaction similar to that in the above <Step 1> is carried out using a compound of formula (RG-B7-3) (a commercially available compound or a compound that can be produced from a commercially available compound by a production method known in the literature), and then the protecting group P １ can be deprotected to produce an amine represented by formula (AM-1-B7) or a salt thereof.

[0210] [Production Method AM-K] Production method for amines represented by formula (AM-1-B9a) and formula (AM-1-B9b):

[0211] By carrying out the reactions according to the above synthesis scheme (the reactions in each step are similar to those described in [Production Method AM-J]), it is possible to produce amines represented by formula (AM-1-B9a) and formula (AM-1-B9b), or salts thereof. In the above scheme, the compounds of formula (SM-B7), formula (RG-B9a), or formula (RG-B9b) are commercially available compounds or compounds that can be produced from commercially available compounds by production methods known in the literature.

[0212] [Production Method AM-L] Production method for amine represented by formula (AM-2-Z1):

[0213] The compound of formula (SM-Z1) [the compound of formula (SM-Z1) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known in the literature] is used to obtain a compound of formula (SM-Z1) in a reaction-inert solvent such as dimethyl sulfoxide, in accordance with a method known in the literature, for example, the method described in "Organometallics, 29(23), pp. 6619-6622; 2010" or the like. ３ After the azide group is introduced by the reaction of １ By deprotecting the group, an amine represented by formula (AM-2-Z1) or a salt thereof can be produced.

[0214] The amine represented by formula (AM-2-Z1) or a salt thereof may be commercially available.

[0215] [Production Method AM-M] Production Method for Amines Represented by Formula (AM-2-Z2a) and Formula (AM-2-Z2b):

[0216] The compound of formula (SM-Z2a) or the compound of formula (SM-Z2b) [the compound of formula (SM-Z2a) and the compound of formula (SM-Z2b) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known in the literature] were used to prepare NaN in the same manner as in [Production Method AM-L]. ３ After the azide group is introduced by the reaction of １An amine represented by formula (AM-2-Z2a) or formula (AM-2-Z2b), or a salt thereof, can be produced by deprotecting the amine. Some of the amines represented by formula (AM-2-Z2a) or formula (AM-2-Z2b), or salts thereof, are commercially available.

[0217] [Production Method AM-N] Production method of amine represented by formula (AM-2-Z3):

[0218] The compound of formula (SM-Z3) and the compound of formula (RG-Z3) [the compound of formula (SM-Z3) and the compound of formula (RG-Z3) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known in the literature] are subjected to a condensation reaction similar to that described above in [Production method of chemically modified alginic acid derivative of formula (HB-I)], followed by the removal of the protecting group P １ By deprotecting the group, an amine represented by formula (AM-2-Z3) or a salt thereof can be produced.

[0219] [Production Method AM-O] Production Method of Amine Represented by Formula (AM-2-Z4):

[0220] The compound of formula (SM-Z4) and the compound of formula (RG-Z4) [the compound of formula (SM-Z4) and the compound of formula (RG-Z4) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known in the literature] are subjected to a condensation reaction similar to that described above in [Production method of chemically modified alginic acid derivative of formula (HB-I)], followed by the addition of a protecting group P １ By deprotecting the group, an amine represented by formula (AM-2-Z4) or a salt thereof can be produced.

[0221] [Production Method AM-P] Production method for amines represented by formula (AM-2-Z5a) and formula (AM-2-Z5b):

[0222] A compound having a side chain introduced therein can be obtained by reacting a compound of formula (SM-Z5a) and a compound of formula (RG-Z5a) [the compound of formula (SM-Z5a) and the compound of formula (RG-Z5a) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known in the literature] in the presence of a base such as sodium hydride or potassium carbonate in a reaction-inert solvent such as tetrahydrofuran, N,N-dimethylformamide, N-methylpyrrolidone, or dimethyl sulfoxide. １ By deprotecting the group, an amine represented by formula (AM-2-Z5a) or a salt thereof can be produced.

[0223] Similarly, an amine represented by formula (AM-2-Z5b) or a salt thereof can be produced by carrying out a reaction in the same manner using a compound of formula (SM-Z5b) and a compound of formula (RG-Z5b) [the compound of formula (SM-Z5b) and the compound of formula (RG-Z5b) are commercially available compounds or compounds that can be produced from commercially available compounds by production methods known in the literature].

[0224] The amines represented by formula (AM-2-Z6a), (AM-2-Z6b), (AM-2-Z7a), (AM-2-Z7b), (AM-2-Z8a), (AM-2-Z8b), (AM-2-Z9a), and (AM-2-Z9b), or salts thereof, can be produced by the production method shown in the following scheme in accordance with the above-mentioned [Production Method AM-A] to [Production Method AM-P].

[0225] [Production Method AM-T] Production method for amines represented by formula (AM-1-T1) and formula (AM-1-T2):

[0226] <Step 1> Using the compound of formula (SM-T) and the compound of formula (RG-T-1) [the compound of formula (SM-T) and the compound of formula (RG-T-1) are commercially available compounds or compounds that can be produced from commercially available compounds by production methods known in the literature], a Grignard reaction is carried out in accordance with a method known in the literature, for example, the method described in WO2004 / 035017, etc., followed by an oxidation reaction, to obtain the compound of formula (IM-T-1).

[0227] <Step 2> After protecting the carbonyl group of the compound of formula (IM-T-1) (for example, an acetal group), bromine is added to the cyclooctene ring, followed by debromination using a base such as tert-BuOK, followed by deprotection of the carbonyl group and the protecting group P １ is deprotected to produce an amine represented by formula (AM-1-T1) or a salt thereof.

[0228] <Step 3> A condensation reaction is carried out using an amine of formula (AM-1-T1) or a salt thereof and a compound of formula (RG-T-2) [the compound of formula (RG-T-2) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known in the literature] to remove a protecting group P １ is deprotected to produce an amine represented by formula (AM-1-T2) or a salt thereof.

[0229] [Production Method AM-U] Production method for amines represented by formula (AM-1-U1) and formula (AM-1-U2):

[0230] In the above [Production Method AM-T], the compound of formula (SM-T) is replaced with the compound of formula (SM-U) [the compound of formula (SM-U) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known in the literature], and the reaction is carried out in accordance with the method described in [Production Method AM-T], thereby producing amines represented by formula (AM-1-U1) and formula (AM-1-U2), or salts thereof.

[0231] The aldehyde used in <Step 1> of the above-mentioned [Production Method AM-T] and [Production Method AM-U] is replaced with an aldehyde of the following formula (RG-T-3) or formula (RG-T-4) [the compounds of formula (RG-T-3) and formula (RG-T-4) are commercially available compounds or compounds that can be produced from commercially available compounds by a production method known in the literature], thereby producing an amine having a corresponding linker, or a salt thereof.

[0232] [Production Method AM-V] Production method for amines represented by formula (AM-1-V1) and formula (AM-1-V2):

[0233] <Step 1> A compound of formula (SM-V) [the compound of formula (SM-V) is a commercially available compound or can be produced by a method known in the literature, for example, the method described in Bioorganic & Medicinal Chemistry, 23(22), pp. 7150-7157, 2015, etc.] is converted into an acid chloride according to a standard method, and then a Grignard reaction is carried out using a compound of formula (RG-V-1) [the compound of formula (RG-V-1) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known in the literature], followed by removing a protecting group P １ is deprotected to produce an amine represented by formula (AM-1-V1) or a salt thereof.

[0234] <Step 2> A condensation reaction is carried out using an amine of formula (AM-1-V1) or a salt thereof and a compound of formula (RG-T-1), to remove a protecting group P １ is deprotected to produce an amine represented by formula (AM-1-V2) or a salt thereof.

[0235] The compound used in Step 1 of the above-mentioned [Production Method AM-V] is replaced with a compound of the following formula (each compound is a commercially available compound or can be produced from a commercially available compound by a production method known in the literature), and the reaction is carried out to produce an amine having a corresponding linker, or a salt thereof.

[0236] [Production Method AM-W] Production method for amines represented by formula (AM-1-W1) and formula (AM-1-W2):

[0237] In the above [Production Method AM-V], the compound of formula (SM-V) is replaced with the compound of formula (SM-W) [the compound of formula (SM-W) is a commercially available compound or a compound that can be produced from a commercially available compound by a production method known in the literature], and the reaction is carried out in accordance with the method described in [Production Method AM-V], thereby producing amines represented by formula (AM-1-W1) and formula (AM-1-W2), or salts thereof.

[0238] The compound used in Step 1 of the above-mentioned [Production Method AM-W] is replaced with a compound of the following formula (each compound is a commercially available compound or can be produced from a commercially available compound by a production method known in the literature), and the reaction is carried out to produce an amine having a corresponding linker, or a salt thereof.

[0239] An amine (Akn-L) having an alkyne group introduced therein is used to produce a chemically modified alginic acid derivative represented by formula (HB-I) or formula (HB-II). １ -NH ２ ) or an amine having an azide group introduced therein (N ３ -L ２ -NH ２ Regarding the above-mentioned amines, the desired amines can be produced by appropriately combining the reactions described in the above-mentioned [Production Method AM-A] to [Production Method AM-P] and methods known in the literature, for example, the methods described in "Experimental Chemistry Lectures, 5th Edition, various volumes, 2007, Maruzen," "Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 3rd Edition (Edited by Richard C. Larock), 2018," and "Strategic Applications of Named Reactions in Organic Synthesis, (Edited by Laszlo Kurti, Barbara Czako), Academic Press, 2005."

[0240] In this specification, the amines represented by formula (AM-1) or formula (AM-2) (including subformulae of each formula) may form pharmaceutically acceptable salts (e.g., acid addition salts). Such salts are not particularly limited as long as they are pharmaceutically acceptable, and examples thereof include salts with inorganic acids, salts with organic acids, and salts with acidic amino acids. Suitable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Suitable examples of salts with organic acids include salts with aliphatic monocarboxylic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, butyric acid, valeric acid, enanthic acid, capric acid, myristic acid, palmitic acid, stearic acid, lactic acid, sorbic acid, and mandelic acid; salts with aliphatic dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, and tartaric acid; salts with aliphatic tricarboxylic acids such as citric acid; salts with aromatic monocarboxylic acids such as benzoic acid and salicylic acid; salts with aromatic dicarboxylic acids such as phthalic acid; salts with organic carboxylic acids such as cinnamic acid, glycolic acid, pyruvic acid, oxylic acid, salicylic acid, and N-acetylcysteine; salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid; and acid addition salts with acidic amino acids such as aspartic acid and glutamic acid. Suitable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, etc. Among these, pharmaceutically acceptable salts are preferred.

[0241] The salts can be obtained in a conventional manner, for example, by mixing the compound of the present invention with a solution containing an appropriate amount of acid or base to form the desired salt, and then separating and filtering the salt or by distilling off the mixed solvent. A general review of salts is available in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use," Stahl & Wermuth (Wiley-VCH, 2002), which provides a detailed description.

[0242] In the present specification, the amine represented by formula (AM-1) or formula (AM-2) (including subformulas of each formula) or a salt thereof can form a solvate with a solvent such as water, ethanol, or glycerol.

[0243] Unless otherwise specified, in this specification, when a variable substituent is substituted on a cyclic group, the variable substituent is not bonded to a specific carbon atom of the cyclic group. For example, the variable substituent Rs in the following formula A means that it can be substituted on any of carbon atoms i, ii, iii, iv, or v in the formula A.

[0244] In the present specification, the linker (-L) in the chemically modified alginic acid derivative represented by formula (HB-I) or formula (HB-II) １ -or-L ２ When an asymmetric carbon atom is present in the compound (-), each of the optical isomers thereof is also included.

[0245] For example, -L in formula (HB-I) １ - is the formula (L1-8a), m8a = 2, n8a = 1, R １ The following formula (L1-8a-M) when =Me (wherein the formula does not include the areas outside the dashed lines): If R １ The following formula (L1-8a-M-S) represents a carbon atom substituted with a benzyl group in the S configuration, and the following formula (L1-8a-M-R) represents a carbon atom substituted with a benzyl group in the R configuration (both formulas do not include the areas outside the dashed lines): This means that a linker represented by

[0246] The linker (-L) in the chemically modified alginic acid derivative represented by formula (HB-I) or formula (HB-II) １ -or-L ２ When an asymmetric carbon is present in Formula (HB-I) or Formula (HB-II) (when the compound is an optically active compound), in the step of synthesizing an amine derivative (AM-1) corresponding to Formula (HB-I) or Formula (HB-II), it is possible to separate the racemate into each optically active compound by a conventional optical resolution means (separation technique). Furthermore, in the step of synthesizing an amine derivative of Formula (AM-1) or Formula (AM-2) corresponding to Formula (HB-I), one of the optical isomers can be selectively synthesized by using asymmetric synthesis, and each optically active compound can be synthesized.

[0247] 5. Chemically Crosslinked Alginate (Core Layer and Anionic Polymer Layer) Among the chemically crosslinked alginates constituting the structure of the present invention, those used in the core layer and the anionic polymer layer include those produced by chemical crosslinking using the chemically modified alginate derivatives described in Section "3. Chemically Modified Alginate Derivatives." Specifically, these are the chemically crosslinked alginates used in the production of the structures shown in the above embodiments [1] to [9-2]. When chemically crosslinked alginate is used in the anionic polymer layer, it may have the same structure as or a different structure from the chemically crosslinked alginate used in the core layer. For example, the chemically crosslinked alginate used in the core layer may be crosslinked by the Huisgen-type reaction used in the production of the structures shown in the above embodiments [2] to [7], and the chemically crosslinked alginate used in the anionic polymer layer may be crosslinked by the photocrosslinked alginate (percutaneous acid type) used in the production of the structures shown in the above embodiments [8-1] to [9-2]. In another embodiment, when the chemically crosslinked alginate used in the core layer and the chemically crosslinked alginate used in the anionic polymer layer are both crosslinked by the Huisgen-type reaction used in producing the structures shown in the above embodiments [2] to [7], chemically crosslinked alginates with different structures can be used for the core layer and the anionic polymer layer, and for example, alginates with different crosslinking reaction rates can be selected for each layer.

[0248] As described in "1. Multilayer Structure Using Chemically Crosslinked Alginate," the chemically crosslinked alginate constituting the structure of the present invention may be in either of the following states: (i) a state in which crosslinking by divalent metal ions (ionic crosslinking) is not formed (e.g., a state in which bonds with divalent metal ions are removed by the presence of a chelating agent such as EDTA), or (ii) a state in which both chemical crosslinking and ionic crosslinking are formed. For example, a state in which both chemical crosslinking and ionic crosslinking are formed has high structural stability, which may be advantageous when implanted in a living body. In this case, the divalent metal ion is not particularly limited, but examples include calcium ions, magnesium ions, barium ions, strontium ions, zinc ions, etc., with calcium ions or barium ions being preferred, and calcium ions being more preferred. Another preferred embodiment is barium ions.

[0249] The solution containing divalent metal ions is not particularly limited, but examples thereof include solutions containing calcium ions (e.g., aqueous solutions such as calcium chloride, calcium carbonate, and calcium gluconate), and solutions containing barium ions (e.g., aqueous solutions such as barium chloride), with calcium chloride being preferred. The divalent metal ion concentration (e.g., calcium ion or barium ion concentration) of the solution containing divalent metal ions is not particularly limited, but is, for example, in the range of 1 mM to 1 M, or in the range of 5 mM to 500 mM, and more preferably 20 mM to 100 mM. In this specification, the divalent metal ion aqueous solution may be simply referred to as a divalent metal ion solution, and unless otherwise specified, the divalent metal ion solution refers to an aqueous solution.

[0250] The solvent used when preparing a solution of a chemically modified alginic acid derivative, or when preparing a solution containing a divalent metal ion, is not particularly limited, and each of them independently includes, for example, tap water, pure water (e.g., distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water (MilliQ water), culture medium (i.e., cell culture medium (or culture solution)), phosphate-buffered saline (PBS), and saline, with ultrapure water being preferred.

[0251] When chemically crosslinked alginate involves ionic crosslinking, the ionic crosslinking reaction is instantaneous and reversible, whereas the chemical crosslinking reaction proceeds slowly under relatively mild conditions and is irreversible. By utilizing this property and appropriately combining chemical crosslinking and ionic crosslinking, the structure of the present invention can be efficiently produced. For example, the ionic crosslinking reaction makes it easy to instantly create bead- or sheet-like structures from a mixed solution of chemically modified alginic acid derivatives. On the other hand, the present invention uses chemical crosslinking (Huisugen reaction) to strengthen the structure (e.g., to achieve long-term stability, etc.). Therefore, after the structure of the present invention is produced via both chemical crosslinking and ionic crosslinking, the divalent metal ions incorporated by the ionic crosslinking of the chemically crosslinked alginate are gradually and reversibly released, and even if only the crosslinks formed by chemical bonds remain, the structure can be used stably and continuously.

[0252] 5-1. Chemically crosslinked alginate (Huisgen type) A certain embodiment of the chemically crosslinked alginate can be obtained by mixing chemically modified alginic acid derivatives of the formula (H-I) and the formula (H-II) and performing a Huisgen reaction. Another embodiment of the chemically crosslinked alginate can be obtained by mixing chemically modified alginic acid derivatives of the formula (HA-I) and the formula (HA-II) and performing a Huisgen reaction. Another embodiment of the chemically crosslinked alginate can be obtained by mixing chemically modified alginic acid derivatives of the formula (HB-I) and the formula (HB-II) and performing a Huisgen reaction.

[0253] In some embodiments, chemically crosslinked alginate forms a three-dimensional network structure through chemical crosslinking (crosslinking by triazole rings formed from alkyne groups and azide groups). A preferred chemically modified alginate derivative improves the stability of the chemically crosslinked alginate after crosslinking. The physical properties of the chemically crosslinked alginate can be adjusted, for example, by adjusting the introduction rate of reactive groups in the chemically modified alginate used as a raw material.

[0254] In some embodiments, the chemically crosslinked alginate has the following formula (H-III-L): [In formula (H-III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -L １ -, -L ２ -, and X are each the same as any combination of the above embodiments [5] to [5-7]].

[0255] In some embodiments, the chemically cross-linked alginate has the following formula (HA-III-L): [In formula (HA-III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -L １ -, -L ２ -, and X is the same as defined in the above embodiment [7]].

[0256] In addition, some embodiments of the chemically crosslinked alginic acid have the following formula (HB-III-L): [In formula (HB-III-L), -CONH- and -NHCO- at both ends represent an amide bond via any carboxyl group of alginic acid; -L １ -, -L ２ -, and X are each the same as any combination of the above embodiments [6-1] to [6-6]].

[0257] In one embodiment, the chemically crosslinked alginates of formula (H-III-L), formula (HA-III-L), and formula (HB-III-L) can be produced by mixing chemically modified alginic acid derivatives of formula (HI) and formula (H-II), formula (HA-I) and formula (HA-II), and formula (HB-I) and formula (HB-II), respectively.

[0258] In some embodiments, when preparing chemically crosslinked alginic acid, the mixing ratio of the chemically modified alginic acid derivative of formula (H-I), formula (HA-I) or formula (HB-I) to the chemically modified alginic acid derivative of formula (H-II), formula (HA-II) or formula (HB-II) is, for example, 1:1.0 to 4.0, or 1:1.0 to 3.0, or 1:1.0 to 2.0, or 1:1.0 to 1.5, or 1:1; preferably 1:1.0 to 3.0.

[0259] In some embodiments, when preparing chemically crosslinked alginic acid, the mixing ratio of the chemically modified alginic acid derivative of formula (H-II), formula (HA-II), or formula (HB-II) to the chemically modified alginic acid derivative of formula (H-I), formula (HA-I), or formula (HB-I) is, for example, 1:1.0 to 4.0, or 1:1.0 to 3.0, or 1:1.0 to 2.0, or 1:1.0 to 1.5, or 1:1, in terms of the weight ratio of the derivative of formula (H-II), formula (HA-II), or formula (HB-II) to the derivative of formula (H-I), formula (HA-I), or formula (HB-I).

[0260] In some embodiments, when preparing chemically crosslinked alginic acid, the mixing ratio of the chemically modified alginic acid derivative of formula (H-I), formula (HA-I) or formula (HB-I) and the chemically modified alginic acid derivative of formula (H-II), formula (HA-II) or formula (HB-II) is, more preferably, the ratio of the introduction rate (mol%) of the reactive group of the chemically modified alginic acid derivative of formula (H-I), formula (HA-I) or formula (HB-I) to the chemically modified alginic acid derivative of formula (H-II), formula (HA-II) or formula (HB-II) is, for example, 1: 1.0 to 4.0, or 1: 1.0 to 3.0, or 1: 1.0 to 2.0, or 1: 1.0 to 1.5, or 1: 1; preferably 1: 1.0 to 3.0.

[0261] In some embodiments, when preparing chemically crosslinked alginic acid, the mixing ratio of the chemically modified alginic acid derivative of formula (H-II), formula (HA-II) or formula (HB-II) and the chemically modified alginic acid derivative of formula (H-I), formula (HA-I) or formula (HB-I) is more preferably the ratio of the introduction rate (mol%) of the reactive group of the chemically modified alginic acid derivative of formula (H-II), formula (HA-II) or formula (HB-II) to the chemically modified alginic acid derivative of formula (H-I), formula (HA-I) or formula (HB-I), for example, 1:1.0 to 4.0, or 1:1.0 to 3.0, or 1:1.0 to 2.0, or 1:1.0 to 1.5, or 1:1.

[0262] In chemically crosslinked alginic acid, it is not necessary for all carboxyl groups in the constituent units of alginic acid to have crosslinks of the above formula (HA-III-L) or formula (HB-III-L). The introduction rate (also referred to as crosslinking rate) of crosslinks represented by the above formula (HA-III-L) or formula (HB-III-L) in chemically crosslinked alginic acid is, for example, in the range of about 0.1 to about 80%, about 0.3 to about 60%, about 0.5 to about 30%, or about 1.0 to about 10%.

[0263] The concentration of the chemically modified alginic acid derivatives represented by Formulas (HI) to (HB-II) in the Huisgen reaction to obtain chemically crosslinked alginic acid is usually about 1 to 500 mg / mL, preferably about 5 to 100 mg / mL. In one embodiment, the concentration is in the range of 5 to 50 mg / mL, and in another embodiment, the concentration is in the range of 5 to 20 mg / mL.

[0264] The reaction temperature for the Huisgen reaction (the temperature at which chemically crosslinked alginic acid is produced) is usually in the range of about 4 to about 60°C, preferably about 15 to about 37°C.

[0265] 5-2. Photocrosslinked Alginic Acid (Cinnamic Acid Type) Some embodiments of the chemically crosslinked alginic acid constituting the structure of the present invention are chemically crosslinked alginic acids (also referred to as photocrosslinked alginic acids) obtained by irradiating a chemically modified alginic acid derivative into which a photoreactive group (photocrosslinking group) has been introduced, such as a chemically modified alginic acid derivative into which a group represented by formula (C-I) has been introduced, with light. In photocrosslinked alginic acids, the chemically modified alginic acid derivative forms a three-dimensional network structure via the photocrosslinking group (crosslinking by a cyclobutane ring formed by photoreaction). Preferred chemically modified alginic acid derivatives improve the stability of the chemically crosslinked alginic acid after crosslinking.

[0266] The light to be irradiated is not particularly limited as long as it acts on the photoreactive group (photocrosslinking group) and causes a reaction such as polymerization or dimerization (preferably a photodimerization reaction (photocyclization reaction)). For example, ultraviolet light, LED light (LED = Light Emitting Diode), visible light, infrared light, electron beam, etc. can be used as the light to be irradiated. Of these irradiated lights, visible light, LED light, or ultraviolet light is preferred, and ultraviolet light is more preferred. The light wavelength of the light source used is preferably in the range of 180 to 650 nm, more preferably in the range of 300 to 410 nm.

[0267] In some embodiments, the specific light intensity and wavelength for causing the photocrosslinking reaction are as follows: the illuminance of HLR100T-2 (manufactured by Sen Special Light Source Co., Ltd.) is 170 mW / cm ２ (50 mm distance from the light source), the main wavelength is 365 nm, and the irradiation time is 10 minutes. At a distance of 100 mm from the light source, the exposure dose is 25 J / cm ２ is.

[0268] In some embodiments, the photocrosslinked alginic acid is obtained by dimerizing the photoreactive portion (alkene portion) of the photoreactive group represented by formula (CI) through a photocyclization reaction, for example, by forming alginic acid derivatives represented by the following formulae (C-II-L-1) to (C-II-L-3): [In formulas (C-II-L-1) to (C-II-L-3), -CONH- and -NHCO- at both ends represent amide bonds via any carboxyl group of alginic acid; -A-, -X-, and Ar are the same as defined in the above embodiments [9-1] and [9-2]].

[0269] In photocrosslinked alginic acid, it is not necessary for all carboxyl groups in the constituent units of alginic acid to be crosslinked as described above. In chemically crosslinked alginic acid, the introduction rate of crosslinks represented by the above formula (also referred to as crosslinking rate) is, for example, in the range of 0.1 to 80%, 0.3 to 60%, 0.5 to 30%, or 1.0 to 10%.

[0270] The concentration of the chemically modified alginic acid derivative to obtain photocrosslinked alginic acid ranges, for example, from 1 to 500 mg / mL, or from 5 to 100 mg / mL.

[0271] The temperature at which the photocrosslinking reaction is carried out is usually room temperature (about 0° C. to about 35° C.).

[0272] The reaction solvent or reaction solution used in the photocrosslinking reaction is not particularly limited, and examples thereof include tap water, pure water (e.g., distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), ultrapure water (MilliQ water), culture medium (i.e., cell culture medium (or culture solution)), phosphate buffered saline (PBS), and physiological saline, with ultrapure water being preferred.

[0273] 6. Manufacturing Method of a Structure (Multilayer Structure Using Chemically Crosslinked Alginate in a Core Layer) The present invention also relates to a manufacturing method of a structure. The present specification provides a manufacturing method of a structure containing a pharmacological ingredient embedded in chemically crosslinked alginate in a core layer, the manufacturing method comprising the following steps (a) to (c): step (a): contacting a solution of a chemically modified alginic acid derivative containing the pharmacological ingredient with a solution containing a divalent metal ion to form a gel; step (b): contacting the gel obtained in step (a) with a solution containing a cationic polymer to coat the gel with the cationic polymer; and step (c): contacting the product obtained in step (b) with a solution containing an anionic polymer to further coat the gel with the anionic polymer.

[0274] In the method for producing a structure, in "Step (a): gelation step by contacting a solution of a chemically modified alginic acid derivative containing a pharmacological component with a solution containing a divalent metal ion," the solution of the chemically modified alginic acid derivative containing the suspended pharmacological component is gelled by contacting it with a divalent metal ion solution. By contacting the solution of the chemically modified alginic acid derivative with a solution containing a divalent metal ion, ionic crosslinking and chemical crosslinking proceed simultaneously, resulting in the production of a gel. Specifically, in step (a), various pharmacological components described above in "1-1. Core layer containing a pharmacological component embedded in chemically crosslinked alginic acid" are first suspended or dissolved in a solution containing the chemically modified alginic acid derivative. Additives other than the pharmacological component may also be added. For example, in the case of biologically active substances, it may be effective to use a formulation for controlled release or a carrier-supported substance. In the case of cells, it may be effective to use a substance that maintains cell viability and function. It may also be possible to add a substance that uniformly disperses the pharmacological component in the core layer. Examples of additives other than pharmacological components include polymeric substances other than alginic acid. Specific examples of biocompatible polymeric substances include polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), gelatin, collagen, and hyaluronic acid. For example, when cells or microorganisms are used as pharmacological components, culture medium or culture solution components or formulation components commonly used in pharmaceuticals may also be included. Examples of chemically modified alginic acid derivatives include the compounds represented by the aforementioned formulas (H-I) and (H-II), the compounds represented by the aforementioned formulas (HA-I) and (HA-II), the compounds represented by the aforementioned formulas (HB-I) and (HB-II), and the compounds represented by the aforementioned formula (C-I). In step (a), for example, a 0.1 to 5 wt % aqueous or saline solution of the chemically modified alginic acid derivative is prepared, and the various pharmacological components are suspended in the solution in the required amounts.

[0275] In step (a), a solution of the chemically modified alginic acid derivative represented by the aforementioned formula (H-I) and a solution of the chemically modified alginic acid derivative represented by the aforementioned formula (H-II) can be used in combination. In some embodiments, a solution of the chemically modified alginic acid derivative represented by the aforementioned formula (HA-I) and a solution of the chemically modified alginic acid derivative represented by the aforementioned formula (HA-II) can be used in combination. In other embodiments, a solution of the chemically modified alginic acid derivative represented by the aforementioned formula (HB-I) and a solution of the chemically modified alginic acid derivative represented by the aforementioned formula (HB-II) can be used in combination. These solutions and solutions incorporating pharmacological ingredients therein are prepared separately without being mixed. When using each of the aforementioned combinations of solutions, the pharmacological ingredient may be incorporated into only one of them, or into both.

[0276] Here, "contact" includes immersing or adding a solution (e.g., a solution of a chemically modified alginic acid derivative) to another solution (e.g., a solution containing a divalent metal ion), spraying or spraying a solution (e.g., a solution of a chemically modified alginic acid derivative) with another solution (e.g., a solution containing a divalent metal ion), etc.

[0277] Examples of solutions containing "divalent metal ions" used in the structure include solutions containing calcium ions, barium ions, zinc ions, strontium ions, etc. Preferably, the solution contains calcium ions or barium ions, more preferably a solution containing calcium ions. Preferably, the solution is an aqueous calcium chloride solution. Another preferred embodiment is an aqueous barium chloride solution.

[0278] A solution containing a divalent metal ion can be obtained, for example, by dissolving a salt of the divalent metal ion in a solvent. Examples of the salt of the divalent metal ion include calcium chloride, barium chloride, and strontium chloride. Examples of the solvent include water and physiological saline.

[0279] The amount of the solution containing the divalent metal ions to be used is preferably adjusted appropriately depending on the amount of the chemically modified alginic acid derivative to be used, its molecular weight, and the like.

[0280] One of the features of the structures of the present invention is that they offer greater flexibility in molding than conventional structures using alginic acid. For example, structures of various shapes can be produced by the following method. The shape of the core layer, which accounts for the majority of the volume, has the greatest impact on the overall shape of the structure. For example, to produce spherical structures, a method is used in which a solution of a chemically modified alginic acid derivative mixed with a pharmacological ingredient is dripped into a divalent metal ion solution such as a calcium chloride aqueous solution. By varying the diameter of the dripping conduit and the dripping speed, it is possible to produce spheres of various sizes.

[0281] One method for producing fibrous structures involves slowly releasing a solution of chemically modified alginic acid derivatives mixed with pharmacological ingredients into a divalent metal ion solution through an introduction tube attached to the tip of a syringe or similar device. The released solution gradually gels, producing thread-like structures. By changing the size of the introduction tube or controlling the release rate using a syringe pump or similar device, it is possible to produce structures of various sizes.

[0282] In addition, the use of a template and semipermeable membrane allows for the production of structures of any shape. For example, if a flat plate is desired, a template is made using a material that is non-reactive and easily processed, such as silicone rubber, and a solution of a chemically modified alginic acid derivative mixed with a pharmacological ingredient is poured into the template. By using a semipermeable membrane in part of the template, a divalent metal ion solution is allowed to penetrate the interior of the template through the semipermeable membrane, and the contents become gel-like. This method allows the production of structures of various shapes by designing the template to the desired shape. Furthermore, the use of a 3D printer also allows for the creation of free shapes. For example, a method is used in which a solution of a chemically modified alginic acid derivative mixed with a pharmacological ingredient is used as a bioink and contacted with a divalent metal ion solution.

[0283] The high degree of freedom in molding of the structure of the present invention is achieved by using chemically crosslinked alginate, which can be easily produced while maintaining high strength of the structure.

[0284] Next, in "step (b): contacting the gel obtained in step (a) with a solution containing a cationic polymer to coat the gel with the cationic polymer," the gel obtained in step (a) is contacted with a solution containing a cationic polymer such as PLL or PLO. Preferably, the gel obtained in step (a) is contacted with a solution containing PLO. As a result, the surface of the molded article obtained in step (a) is coated with the cationic polymer due to the interaction between the chemically crosslinked alginic acid and the cationic polymer. As the cationic polymer, those described above in "1-2. Cationic polymer layer coating the core layer" are preferably used.

[0285] When PLO is used as the cationic polymer, the concentration of the cationic polymer reacted with the chemically crosslinked alginic acid in the core layer is, for example, 0.02 to 0.2 w / w %, or 0.05 to 0.1 w / w %.

[0286] The temperature when reacting the chemically crosslinked alginate gel and the cationic polymer in the core layer is, for example, room temperature, and the reaction time is, for example, 1 minute to 45 minutes, or 1 minute to 30 minutes.

[0287] Next, in "step (c): contacting the product obtained in step (b) with a solution containing an anionic polymer and further coating with an anionic polymer," the product obtained in step (b) is contacted with a solution containing the anionic polymer. Preferably, the product obtained in step (b) is contacted with a solution containing the anionic polymer described in "1-3. Anionic polymer layer coating the cationic polymer layer." More preferably, the product obtained in step (b) is contacted with a chemically modified alginic acid derivative or alginic acid, and even more preferably, alginic acid. The "chemically modified alginic acid derivative" referred to here is the chemically modified alginic acid described in "2. Chemically modified alginic acid derivative" above. Furthermore, "alginic acid" is the alginic acid (e.g., sodium alginate) described in "2. Alginic acid (regarding the raw material alginic acid used in synthesizing chemically modified alginic acid derivatives and the alginic acid used in the anionic polymer layer)" above. As a result, the surface of the product coated with the cationic polymer is further coated with a chemically modified alginic acid derivative or alginic acid. When a chemically modified alginic acid derivative is used as the anionic polymer, it may be the same as or different from the chemically modified alginic acid derivative used in the core layer. The treatment conditions for step (c) are generally the same as those used in step (b). When alginic acid or a chemically modified alginic acid derivative is used as the anionic polymer, the concentration of the anionic polymer during reaction with the cationic polymer layer is, for example, 0.01 to 4 wt %.

[0288] In some embodiments, the method may then include step (d). In step (d): chelating the structure obtained in step (c) with a chelating agent, a chelating agent is added to the structure produced by the operations up to step (c), thereby transferring the ionically crosslinked divalent metal ions toward the chelating agent. Examples of chelating agents include sodium citrate and ethylenediaminetetraacetic acid (EDTA). It is desirable to appropriately adjust the amount of chelating agent used depending on the amount of chemically modified alginic acid derivative used, etc.

[0289] Among the structures of the present invention, those in which the chemically cross-linked alginate is photocross-linked alginate (transdermal acid type) comprise the steps (a) to (c) of the "method for producing a structure comprising a pharmacological component embedded in chemically cross-linked alginate," in which, in step (a), a solution of a chemically modified alginate derivative represented by formula (C-I) is used as the solution of the chemically modified alginate derivative, and the chemically modified alginate derivative is irradiated with light at any stage after step (a) to cause a chemical cross-linking reaction.

[0290] More specifically, for example, a solution of a chemically modified alginic acid derivative into which a photoreactive group represented by the formula (C-I) has been introduced is dropped into a solution containing divalent metal ions to obtain a specific structure in which ionic crosslinking (crosslinking partially formed by divalent metal ions) is formed. Subsequently, the structure is irradiated with light to cause photocrosslinking (chemical crosslinking: cyclobutane ring (truxillic acid derivative)), thereby obtaining a specific structure, photocrosslinked alginic acid. Light irradiation can be performed at any stage after step (a), or can be performed after step (c).

[0291] The light to be irradiated is not particularly limited as long as it acts on the photoreactive group (photocrosslinking group) and causes a reaction such as polymerization or dimerization (preferably a photodimerization reaction (photocyclization reaction)). For example, ultraviolet light, LED light (LED = Light Emitting Diode), visible light, infrared light, electron beam, etc. can be used as the light to be irradiated. Of these irradiated lights, visible light, LED light, or ultraviolet light is preferred, and ultraviolet light is more preferred. The light wavelength of the light source used is preferably in the range of 180 to 650 nm, more preferably in the range of 300 to 410 nm.

[0292] In some embodiments, the specific light intensity and wavelength for causing the photocrosslinking reaction are as follows: the illuminance of HLR100T-2 (manufactured by Sen Special Light Source Co., Ltd.) is 170 mW / cm ２ (50 mm distance from the light source), the main wavelength is 365 nm, and the irradiation time is 10 minutes. At a distance of 100 mm from the light source, the exposure dose is 25 J / cm ２ is.

[0293] 7. Physical Properties of the Structure (Multilayer Structure) and Chemically Crosslinked Alginate of the Present Invention [Method for Confirming the Stability of the Structure] The stability of the structure of the present invention can be confirmed, for example, by the following test method. More specifically, it can be confirmed by the method described in the Examples below. (1) Chelation Treatment: The structure of the present invention is subjected to step (d) described in "6. Method for Producing a Structure (Multilayer Structure Using Chemically Crosslinked Alginate in the Core Layer)," i.e., "a step of chelating the structure obtained in step (c) with a chelating agent," thereby releasing the chemically crosslinked alginate or ionic crosslinks within the alginate. Specifically, when the structure of the present invention is treated with an aqueous solution of sodium citrate or ethylenediaminetetraacetic acid (EDTA), a structure that is able to maintain its shape can be confirmed as a "stable structure." The structure of the present invention can maintain its shape even in the absence of ionic crosslinks due to the chemical crosslinking of the chemically crosslinked alginate in the core layer and the electrostatic interactions between the core layer, cationic polymer layer, and anionic polymer layer. This test can be used to confirm, for example, long-term stability when placed in a living body.

[0294] (2) Shaking test: The chelating structure is suspended in phosphate buffered saline (PBS) and shaken for a certain period of time, and then the physical strength of the suspension is measured by determining whether the structure can be maintained or not. Specific test methods include, for example, the methods described in the Examples below.

[0295] Some of the advantages of the structure of the present invention are due to the fact that the chemically crosslinked alginate that constitutes it has properties that are optimal for the function of the structure of the present invention. For example, the chemically crosslinked alginate used in the present invention has high physical stability (abbreviated as gel stability) while also having appropriate permeability even in gel form to release the pharmacological ingredient from the core layer. This gel permeability can be confirmed by measuring the gel permeability as described below.

[0296] [Method for measuring gel stability] Phosphate-buffered saline is added to chemically crosslinked alginate placed in a container, and the concentration (μg / mL) of alginate leaked into the PBS is measured. The measured alginate concentration is divided by the total alginate concentration obtained by decomposing the chemically crosslinked alginate, and the resulting value expressed as a percentage is taken as the disintegration rate. Specifically, gel stability can be determined by the method described in the Examples below.

[0297] In this specification, the gel collapse rate of the chemically crosslinked alginate is preferably 0% to 90%, more preferably 0% to 70%, and even more preferably 0% to 50%. The stability of the chemically crosslinked alginate is higher when the concentration of alginic acid leaking into the aqueous solution is lower, i.e., the gel collapse rate is lower.

[0298] [Method for measuring gel permeability] Chemically cross-linked alginate encapsulating fluorescein isothiocyanate-dextran is prepared, placed in a container, and physiological saline is added to measure the concentration of dextran leaked into the physiological saline. The measured dextran concentration is divided by the total dextran concentration obtained by decomposing the fluorescein isothiocyanate-dextran-encapsulated chemically cross-linked alginate, and the resulting value, expressed as a percentage, is the gel permeability. Specifically, the gel permeability can be determined by the method described in the Examples below.

[0299] The gel permeability of chemically crosslinked alginate 24 hours after addition of physiological saline is 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. Furthermore, when encapsulating dextran with a molecular weight of 150,000, for example, if the intended use of the chemically crosslinked alginate is for the release or production of proteins or antibodies, the gel permeability is preferably 1% to 100%, more preferably 10% to 100%, and even more preferably 30% to 100%. Furthermore, if the intended use is for an immune barrier, the gel permeability is preferably 0% to 90%, more preferably 0% to 70%, and even more preferably 0% to 50%.

[0300] The permeability of chemically cross-linked alginate is such that the lower the permeability, the lower the permeability of the contents and substances outside the gel, and the higher the permeability, the higher the permeability of the contents and substances outside the gel.

[0301] The permeability of the gel can be adjusted by the molecular weight and concentration of the alginic acid used, the type and introduction rate of crosslinking groups introduced into the alginic acid, the type and concentration of divalent metal ions used for gelation, or a combination of these.

[0302] 8. Biocompatibility of Structures In this specification, biocompatibility refers to the property of not causing interactions between the structure and the living body, local reactions in tissues adjacent to the structure, or systemic reactions, and the possession of such properties is referred to as having biocompatibility. The chemically modified alginic acid derivatives and chemically crosslinked alginic acid constituting the structures of the present invention have been confirmed to have good biocompatibility, similar to alginic acid (for example, see PCT / JP2019 / 023478 and PCT / JP2019 / 007655 for chemically crosslinked alginic acid).

[0303] Furthermore, among the chemically crosslinked alginates, those produced using chemically modified alginic acid derivatives represented by formula (HB-I) and formula (HB-II) can be confirmed in the examples regarding biocompatibility described below.

[0304] 9. Uses of the Structure Preferred uses of the structure include medical materials such as wound dressings, postoperative adhesion barriers, substrates for sustained drug release, substrates for cell transplantation, prosthetic materials (implants, artificial organs, etc.), and pharmaceutical coating materials. Furthermore, the structure can be used as bioink for a bioprinter, and the resulting structure can be used for the above-mentioned purposes. The structure is preferably used in vivo.

[0305] 10. Methods of Using the Structure Methods that can be used to transplant the structure into a living body include incision and placement, injection, endoscopy, and laparoscopy. The transplant site is not particularly limited, and examples include subcutaneous, intraperitoneal, intrahepatic, intramuscular, intraomental, and subrenal capsule sites, but subcutaneous or intraperitoneal transplantation is preferred.

[0306] All documents cited in this specification, as well as publications, patent publications, and other patent documents, are hereby incorporated by reference.

[0307] Furthermore, the objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description in this specification, and those skilled in the art will be able to easily implement the present invention from the description in this specification. The best mode for carrying out the invention and specific examples show preferred embodiments of the present invention and are shown for the purpose of illustration and explanation, and are not intended to limit the present invention thereto. It will be apparent to those skilled in the art that various modifications can be made based on the description in this specification within the spirit and scope of the present invention disclosed in this specification.

[0308] Next, examples and test examples will be given to explain the present invention in more detail, but these examples are merely examples and test examples and do not limit the present invention, and may be modified within the scope of the present invention.

[0309] Nuclear magnetic resonance spectroscopy (NMR) was measured using a JEOL JNM-ECX400 FT-NMR (JEOL). Liquid chromatography-mass spectroscopy (LC-Mass) was measured using the following method. [UPLC] A Waters AQUITY UPLC system and a BEH C18 column (2.1 mm x 50 mm, 1.7 μm) (Waters) were used, with a mobile phase and gradient conditions of acetonitrile:0.05% aqueous trifluoroacetic acid = 5:95 (0 min) - 95:5 (1.0 min) - 95:5 (1.6 min) - 5:95 (2.0 min).

[0310] １ In the H-NMR data, 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 means heavy water. １ In the H-NMR data, hydroxyl group (OH), amino group (NH​２ ), and signals that cannot be confirmed due to broadband such as the proton of the carboxyl group (COOH) are not shown in the data.

[0311] In the LC-Mass data, M is the molecular weight, RT is the retention time, [M+H] ＋ , [M + Na] ＋ means the molecular ion peak.

[0312] In the examples, "room temperature" generally refers to a temperature of about 0° C. to about 35° C. In the examples, the reactive substituent introduction rate (mol %) is １ H-NMR (D ２ 0) to the number of moles of monosaccharide (guluronic acid and mannuronic acid) units constituting alginic acid.

[0313] In the examples, sodium alginate having the physical properties shown in Table 13 was used as the sodium alginate before the reactive group or the complementary reactive group was introduced.

[0314] Table 15 shows the physical properties of the alginic acid derivatives into which reactive groups have been introduced, obtained in (Example 1) to (Example 20) (specifically, the reactive group introduction rate (mol%), molecular weight, and weight average molecular weight (10,000 Da)). Tables 16-1 to 16-5 show the properties of the intermediates in (Example 1) to (Example 20). １ H-NMR and LCM-Mass of the intermediates in (Example 1) to (Example 20) are shown in Table 17.

[0315] Example 1 Synthesis of 3-azidopropylamino group-introduced alginic acid (EX1-A2):

[0316] To a 1 wt% aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (20 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (56 mg), a solution of commercially available 3-azidopropylamine [CAS REGISTRY NO.: 88192-19-2] (1-1, 5.1 mg) in ethanol (2 mL), and 1 molar sodium bicarbonate solution (50 μL) were added. After stirring at 30°C for 3 hours, sodium chloride (0.2 g) and ethanol (40 mL) were added sequentially, 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 resulting solid was dissolved in water and lyophilized to obtain the title compound EX1-A2 (187 mg) as a white solid.

[0317] Example 2 Synthesis of 2-(2-(2-azidoethoxy)ethoxy)ethane-1-amino group-introduced alginic acid (EX2-A2):

[0318] To a 1 wt% aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (10.9 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (55.83 mg) and 1 molar sodium bicarbonate solution (252.17 μL) were added under ice-cooling and stirring. Subsequently, a solution of commercially available 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine [CAS REGISTRY NO.: 166388-57-4] (2-1, 26.36 mg) in ethanol (1 mL) and water (1 mL) was added and stirred at room temperature for 15 hours. After stirring at room temperature, sodium chloride (100 mg) and ethanol (21.8 mL) were added sequentially and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure to obtain the title compound EX2-A2 (99 mg) as a white solid.

[0319] Example 3 Synthesis of 2-amino-N-(3-azidopropyl)acetamide group-introduced alginic acid (EX3-A2):

[0320] <Step 1> Synthesis of tert-butyl (2-((3-azidopropyl)amino)-2-oxoethyl)carbamate (3-2): To a solution of commercially available 3-azidopropylamine [CAS REGISTRY NO.: 88192-19-2] (1-1, 41 μL) and N-(tert-butoxycarbonyl)glycine [CAS REGISTRY NO.: 4530-20-5] (3-1, 100 mg) in ethanol (2 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (197 mg) was added and stirred at room temperature for 18 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting oil was dissolved in methyl tert-butyl ether (10 mL) and washed successively with saturated sodium bicarbonate water, water, and saturated brine. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain the title compound 3-2 (95 mg) as a colorless oil.

[0321] <Step 2> Synthesis of 2-amino-N-(3-azidopropyl)acetamide hydrochloride (3-3):

[0322] To the compound (3-2, 95 mg) obtained in Step 1 of Example 3, 4N hydrogen chloride / 1,4-dioxane (665 μL) was added under ice-water cooling, and the mixture was stirred at room temperature for 1 hour. Diisopropyl ether (2.0 mL) was added to the reaction mixture, and the mixture was concentrated under reduced pressure. The resulting oil was decanted and washed with methyl tert-butyl ether, and then concentrated under reduced pressure to give the title compound 3-3 (62 mg) as a colorless gum.

[0323] <Step 3> Synthesis of 2-amino-N-(3-azidopropyl)acetamide group-introduced alginic acid (EX3-A2):

[0324] To a 1 wt% aqueous solution (20 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (56 mg), a solution of the compound (3-3, 10.6 mg) obtained in Step 2 of Example 3 in ethanol (2 mL), and 1 molar sodium bicarbonate solution (76 μL) were added. After stirring at 30°C for 3 hours, sodium chloride (0.2 g) and ethanol (40 mL) were added sequentially, 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 resulting solid was dissolved in water and lyophilized to obtain the title compound EX3-A2 (207 mg) as a white solid.

[0325] Example 4 Synthesis of 3-amino-N-(3-azidopropyl)propanamide group-introduced alginic acid (EX4-A2):

[0326] <Step 1> Synthesis of tert-butyl (3-((3-azidopropyl)amino)-3-oxopropyl)carbamate (4-2):

[0327] To a solution of commercially available 3-azidopropylamine [CAS REGISTRY NO.: 88192-19-2] (1-1, 38 μL) and N-(tert-butoxycarbonyl)-β-alanine [CAS REGISTRY NO.: 3303-84-2] (4-1, 100 mg) in ethanol (2 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (146 mg) was added and stirred at room temperature for 18 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed sequentially with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the title compound 4-2 (124 mg) as a white wax.

[0328] <Step 2> Synthesis of 3-amino-N-(3-azidopropyl)propanamide hydrochloride (4-3):

[0329] To the compound (4-2, 124 mg) obtained in Step 1 of Example 4, 4N hydrogen chloride / 1,4-dioxane (868 μL) was added under ice-water cooling, and the mixture was stirred at room temperature for 1 hour. Diisopropyl ether (2.6 mL) was added to the reaction mixture, and the mixture was concentrated under reduced pressure. The resulting oil was decanted and washed with methyl tert-butyl ether, and then concentrated under reduced pressure to give the title compound 4-3 (93 mg) as a colorless gum.

[0330] <Step 3> Synthesis of 3-amino-N-(3-azidopropyl)propanamide group-introduced alginic acid (EX4-A2):

[0331] To a 1 wt% aqueous solution (20 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (56 mg), a solution of the compound (4-3, 12.6 mg) obtained in Step 2 of Example 4 in ethanol (2 mL), and 1 molar sodium bicarbonate solution (76 μL) were added. After stirring at 30°C for 3 hours, sodium chloride (0.2 g) and ethanol (40 mL) were added sequentially, 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 resulting solid was dissolved in water and lyophilized to give the title compound EX4-A2 (211 mg) as a white solid.

[0332] Example 5 Synthesis of N-(4-(aminomethyl)benzyl)-2-azidoacetamide group-introduced alginic acid (EX5-A2):

[0333] <Step 1> Synthesis of tert-butyl (4-((2-azidoacetamido)methyl)benzyl)carbamate (5-2):

[0334] A methylene chloride (1.0 mL) solution of azidoacetic acid chloride, which was prepared from commercially available 2-azidoacetic acid [CAS REGISTRY NO.: 18523-48-3] (41 μL) in the same manner as described in Organic Letters (2017), 19(23), 6400-6403, was added to a methylene chloride (1.0 mL) solution of commercially available 1-(N-tert-butyloxycarbonyl-aminomethyl)-4-(aminomethyl)benzene [CAS REGISTRY NO.: 108468-00-4] (5-1, 100 mg) and triethylamine (118 μL) under ice-water cooling, and the mixture was stirred at room temperature for 2.5 hours. Ethyl acetate (20 mL) and water (5 mL) were added to the reaction solution, and after separation, the organic layer was washed successively with water, saturated aqueous sodium bicarbonate, water, and saturated brine. Insoluble matter was removed by filtration, and the filtrate was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The residue was triturated with methyl tert-butyl ether / n-heptane. The resulting solid was collected by filtration to obtain the title compound 5-2 (91 mg) as a light beige solid.

[0335] <Step 2> Synthesis of N-(4-(aminomethyl)benzyl)-2-azidoacetamide hydrochloride (5-3):

[0336] To the compound (5-2, 91 mg) obtained in Step 1 of Example 5, 4N hydrogen chloride / 1,4-dioxane (637 μL) was added under ice-water cooling, followed by the addition of 1,4-dioxane (627 μL), and the mixture was stirred at room temperature for 3.5 hours. Diisopropyl ether (3.8 mL) was added to the reaction mixture, and the mixture was stirred for 10 minutes. The resulting solid was filtered to obtain the title compound 5-3 (62 mg) as a beige solid.

[0337] <Step 3> Synthesis of N-(4-(aminomethyl)benzyl)-2-azidoacetamide group-introduced alginic acid (EX5-A2):

[0338] To a 1 wt% aqueous solution (25 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (70 mg), the compound (5-3, 16.1 mg) obtained in Step 2 of Example 5, and 1 molar sodium bicarbonate solution (95 μL) were added. After stirring at 30°C for 3 hours, sodium chloride (0.25 g) and ethanol (50 mL) were added sequentially, 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 resulting solid was dissolved in water and lyophilized to obtain the title compound EX5-A2 (239 mg) as a white solid.

[0339] Example 6 Synthesis of 2-(4-azidophenoxy)ethane-1-amino group-introduced alginic acid (EX6-A2):

[0340] <Step 1> Synthesis of tert-butyl (2-(4-azidophenoxy)ethyl)carbamate (6-3):

[0341] To a mixture of commercially available 4-azidophenol [CAS REGISTRY NO.: 24541-43-3] (6-1, 0.3 g), commercially available tert-butyl (2-bromoethyl)carbamate [CAS REGISTRY NO.: 39684-80-5] (6-2, 0.6 g), and N-methylpyrrolidone (3 mL) was added potassium carbonate (0.61 g) at room temperature. The reaction mixture was stirred at 80°C for 6 hours and 30 minutes, cooled to room temperature, and then water (10 mL) and methyl tert-butyl ether (20 mL) were added. The resulting suspension was filtered through Celite, and the residue was washed twice with methyl tert-butyl ether (5 mL). The filtrate was separated, and the organic layer was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in methyl tert-butyl ether (20 mL) and washed successively with 1N aqueous sodium hydroxide solution (5 mL) twice, water (5 mL) twice, and saturated brine (5 mL), and then dried over anhydrous sodium sulfate. The organic layer was filtered and concentrated under reduced pressure to give the title compound 6-3 (0.411 g) as a purple oil.

[0342] <Step 2> Synthesis of 2-(4-azidophenoxy)ethan-1-amine hydrochloride (6-4):

[0343] To a mixture of the compound (6-3, 0.41 g) obtained in Step 1 of Example 6 and 1,4-dioxane (2.87 mL), 4N hydrogen chloride / 1,4-dioxane (2.87 mL) was added under stirring on ice, and the mixture was stirred at room temperature for 18 hours. Diisopropyl ether (40 mL) was added to the reaction mixture, and the suspension was stirred at room temperature for 30 minutes. The precipitate was filtered, and the collected solid was dried under reduced pressure to obtain the title compound 6-4 (0.2834 g) as a pale purple solid.

[0344] <Step 3> Synthesis of 2-(4-azidophenoxy)ethane-1-amino group-introduced alginic acid (EX6-A2): To a 1 wt% aqueous solution (29.66 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (91.52 mg) and 1 molar sodium bicarbonate solution (68.63 μL) were added at room temperature. Subsequently, a solution of the compound (6-4, 14.73 mg) obtained in Step 2 of Example 6 in water (1 mL) and ethanol (1 mL) was added at room temperature, and the mixture was stirred at the same temperature for 42 hours. Then, sodium chloride (300 mg) and ethanol (59.3 mL) were added sequentially, 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 resulting solid was dissolved in water and lyophilized to obtain the title compound EX6-A2 (269 mg) as a pink solid.

[0345] Example 7 Synthesis of N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX7-B2)

[0346] <Step 1> Synthesis of tert-butyl (2-(2,2,2-trifluoroacetamido)ethyl)carbamate (7-2):

[0347] Ethyl trifluoroacetate (2.24 mL) was added dropwise to a solution of commercially available tert-butyl (2-aminoethyl)carbamate (7-1, 3.00 g, [CAS REGISTRY NO.: 57260-73-8]) in tetrahydrofuran (12.0 mL). The reaction mixture was stirred at room temperature for 14.5 hours. The reaction mixture was concentrated under reduced pressure, and the residue was triturated with tert-butyl methyl ether (5 mL) and heptane (25 mL). The solid was collected by filtration and washed with heptane to obtain the title compound 7-2 (4.36 g) as a white solid.

[0348] <Step 2> Synthesis of N-(2-aminoethyl)-2,2,2-trifluoroacetamide hydrochloride (7-3):

[0349] Compound 7-2 (0.50 g) obtained in (Example 7) <Step 1> was suspended in 1,4-dioxane (3.0 mL). Under ice-water cooling, 4N hydrogen chloride / 1,4-dioxane (7.0 mL) was added, and the mixture was stirred at room temperature for 3 hours. Diisopropyl ether (30.0 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 50 minutes. The solid was collected by filtration, washed with diisopropyl ether, and then dried under reduced pressure to obtain the title compound 7-3 (0.70 g) as a white solid.

[0350] <Step 3> Synthesis of N-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)-2,2,2-trifluoroacetamide (7-5):

[0351] To a solution of carboxylic acid (7-4, 300 mg) synthesized according to a literature method (Org. Process Res. Dev. (2018) 22:108-110) in ethanol (2 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (1.09 g), compound 7-3 (380 mg) obtained in (Example 7) <Step 2>, and triethylamine (321 μL) were added and stirred at 30 °C for 3 hours. After stirring at 30 °C for an additional 1 hour, triethylamine (229 μL) was added and stirred at the same temperature. After further stirring at room temperature for 15.5 hours, water (10 mL) and ethyl acetate (50 mL) were added, the mixture was separated, and the aqueous layer was extracted with ethyl acetate (10 mL). The organic layer was washed successively with 0.5 N citric acid, water, and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Tert-butyl methyl ether was added to the residue, and insoluble matter was removed by filtration. The filtrate was concentrated and then purified by silica gel column chromatography (10% ethyl acetate / n-heptane to 40% ethyl acetate / n-heptane) to obtain the title compound 7-5 (322 mg) as a white solid.

[0352] <Step 4> Synthesis of N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (7-6):

[0353] To a solution of compound 7-5 (322 mg) obtained in Step 3 of Example 7 in methanol (4.8 mL) was added a solution of potassium carbonate (278 mg) in water (1.6 mL), and the mixture was stirred at room temperature for 7.5 hours. The reaction mixture was concentrated under reduced pressure, and water (3 mL) was added, followed by saturation with sodium chloride. The aqueous layer was extracted with ethyl acetate (30 mL, 10 mL x 3), dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to give the title compound 7-6 (238 mg) as a colorless oil.

[0354] <Step 5> Synthesis of N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX7-B2):

[0355] To a 1 wt% aqueous solution (120 mL) of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (335 mg), an ethanol (12 mL) solution of compound 7-6 (68 mg) obtained in Step 4 of Example 7, and 1 molar sodium bicarbonate solution (303 μL) were sequentially added with stirring at room temperature, and the mixture was stirred at 30°C for 3 hours. Sodium chloride (1.2 g) was added to the reaction solution, followed by ethanol (240 mL), and the mixture was stirred for 1.5 hours. The resulting precipitate was collected by filtration, washed with ethanol (20 mL x 5), and then dried under reduced pressure. The resulting solid was dissolved in water and lyophilized to obtain the title compound EX7-B2 (1.16 g) as a white solid.

[0356] Example 8 Synthesis of N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX8-A2):

[0357] <Step 1> Synthesis of tert-butyl (2-(2-(2,2,2-trifluoroacetamido)ethoxy)ethyl)carbamate (8-2):

[0358] Ethyl trifluoroacetate (0.6 mL) was added dropwise to a solution of tert-butyl (2-(2-aminoethoxy)ethyl)carbamate (8-1, 1.0 g, [CAS REGISTRY NO.: 127828-22-2]) in tetrahydrofuran (4.0 mL). The reaction mixture was stirred at room temperature for 3.5 hours and concentrated under reduced pressure to give the title crude compound 8-2 (1.5 g) as a colorless oil.

[0359] <Step 2> Synthesis of N-(2-(2-aminoethoxy)ethyl)-2,2,2-trifluoroacetamide hydrochloride (8-3):

[0360] To compound 8-2 (1.5 g) obtained in Step 1 of Example 8, 4N hydrogen chloride / 1,4-dioxane solution (10.3 mL) was added under ice-water cooling, and the mixture was stirred at room temperature for 1 hour. Diisopropyl ether (30 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. The solvent was evaporated under reduced pressure, and the residue was azeotroped with diisopropyl ether and then dried under reduced pressure to obtain the title compound 8-3 (1.3 g) as a colorless oil.

[0361] <Step 3> Synthesis of N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)ethyl)-2,2,2-trifluoroacetamide (8-4):

[0362] Carboxylic acid 7-4 (300 mg), synthesized according to a literature method (Org. Process Res. Dev. (2018) 22:108-110), and compound 8-3 (443 mg) obtained in Step 2 of Example 8 were dissolved in acetonitrile (6.0 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.75 g) and N,N-diisopropylethylamine (920 μL) were added, and the mixture was stirred at room temperature for 2.5 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 sequentially with water (10 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50% ethyl acetate / n-heptane to 70% ethyl acetate / n-heptane) to give the title compound 8-4 (469 mg) as a colorless gum.

[0363] <Step 4> Synthesis of N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (8-5):

[0364] To a solution of compound 8-4 (220 mg) obtained in Step 3 of Example 8 in methanol (3.0 mL) was added a solution of potassium carbonate (103 mg) in water (0.99 mL), and the mixture was stirred at room temperature for 4.5 hours. Methanol was evaporated under reduced pressure, and water (2 mL) was added, followed by saturation with sodium chloride. Extraction was performed with ethyl acetate (15 mL, 10 mL × 4), followed by drying over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was dissolved in ethyl acetate (10 mL), and insoluble matter was removed by filtration. The mixture was then concentrated under reduced pressure to give the title crude compound 8-5 (140 mg) as a pale yellow gum.

[0365] <Step 5> Synthesis of N-(2-(2-aminoethoxy)ethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX8-A2):

[0366] To a 1 wt% aqueous solution (40 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (112 mg), a solution of compound 8-5 (30 mg) obtained in Step 4 of Example 8 in ethanol (4.0 mL), and 1 molar sodium bicarbonate solution (101 μL) were sequentially added with stirring at room temperature, and the mixture was stirred at 30°C for 3 hours. Sodium chloride (0.4 g) was added to the reaction solution, followed by ethanol (80 mL), and the mixture was stirred for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting solid was dissolved in water and lyophilized to obtain the title compound EX8-A2 (410 mg) as a white solid.

[0367] (Examples 9a and 9b) Synthesis of N-(2-aminoethyl)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)acetamido group-introduced alginic acid (EX9a-A2, EX9b-B2):

[0368] <Step 1> Synthesis of tert-butyl (2-oxo-2-((2-(2,2,2-trifluoroacetamido)ethyl)amino)ethyl)carbamate (9-1):

[0369] N-(tert-butoxycarbonyl)glycine (91 mg, [CAS REGISTRY NO.: 4530-20-5]) and the compound (7-3, 100 mg) obtained in Example 7, Step 2 were dissolved in acetonitrile (3.0 mL). O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (217 mg) and N,N-diisopropylethylamine (281 μL) were added, and the mixture was stirred at room temperature for 3.5 hours. Ethyl acetate (15 mL) and water (5 mL) were added to the reaction mixture, and after separation, the organic layer was washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (eluent: 40% ethyl acetate / n-heptane→ethyl acetate) to obtain the title compound 9-1 (180 mg) as a light beige amorphous substance.

[0370] <Step 2> Synthesis of N-(2-(2-aminoacetamido)ethyl)-2,2,2-trifluoroacetamide hydrochloride (9-2):

[0371] To the compound (9-1, 180 mg) obtained in Step 1 of Example 9, 4N hydrogen chloride / 1,4-dioxane (1.2 mL) was added under ice-water cooling, and the mixture was stirred at room temperature for 0.8 hours. Diisopropyl ether (3.6 mL) was added to the reaction mixture, and the mixture was stirred for 30 minutes. The resulting solid was filtered to obtain the title compound 9-2 (114 mg) as a white solid.

[0372] <Step 3> Synthesis of N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)acetamido)ethyl)-2,2,2-trifluoroacetamide (9-3):

[0373] To the carboxylic acid (7-4, 80 mg) synthesized according to a literature method (Org. Process Res. Dev. (2018) 22:108-110) and the compound (9-2, 110 mg) obtained in (Example 9) <Step 2>, ethanol (1.6 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (219 mg), and triethylamine (67 μL) were added and stirred at room temperature for 3 hours. Water (3.2 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes. The solid was then filtered and washed with water. Ethyl acetate / ethanol (1 / 1, 10 mL) was added to the resulting solid, and insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure to give the title compound 9-3 (101 mg) as a white solid.

[0374] <Step 4> Synthesis of N-(2-aminoethyl)-2-(2-(cyclooct-2-yn-1-yloxy)acetamide)acetamide (9-4):

[0375] To a solution of the compound (9-3, 60 mg) obtained in Step 3 of Example 9 in methanol (1.8 mL) was added a solution of potassium carbonate (59 mg) in water (0.3 mL), and the mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure, and then water (2 mL) was added and saturated with sodium chloride. Extraction was performed with ethyl acetate (15 mL, 10 mL × 4), and the extract layer was concentrated under reduced pressure. Ethyl acetate (10 mL) and ethanol (1 mL) were added to the residue, and insoluble matter was removed by filtration. The resulting filtrate was concentrated under reduced pressure to give the title compound 9-4 (49 mg) as a colorless gum.

[0376] <Step 5-1> Synthesis of N-(2-aminoethyl)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)acetamido group-introduced alginic acid (EX9a-A2):

[0377] To a 1 wt% aqueous solution (38 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (106 mg), a solution of the compound (9-4, 30.3 mg) obtained in Step 4 of Example 9 in ethanol (3.8 mL), and 1 molar sodium bicarbonate water (96 μL) were added. After stirring at 30°C for 3.2 hours, sodium chloride (0.38 g) and ethanol (76 mL) were added sequentially, 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 resulting solid was dissolved in water and lyophilized to obtain the title compound EX9a-A2 (381 mg) as a white solid.

[0378] <Step 5-2> Synthesis of N-(2-aminoethyl)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)acetamido group-introduced alginic acid (EX9b-B2):

[0379] To a 1 wt% aqueous solution (38 mL) of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (64 mg), the compound (9-4, 18.2 mg) obtained in Step 4 of Example 9, and 1 molar sodium bicarbonate solution (58 μL) were added. After stirring at 30°C for 3.2 hours, sodium chloride (0.38 g) and ethanol (76 mL) were added sequentially, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting solid was dissolved in water and lyophilized to obtain the title compound EX9b-B2 (366 mg) as a white solid.

[0380] Example 10 Synthesis of N-(2-aminoethyl)-3-(2-(cyclooct-2-yn-1-yloxy)acetamido)propanamide group-introduced alginic acid (EX10-A2)

[0381] <Step 1> Synthesis of tert-butyl (3-oxo-3-((2-(2,2,2-trifluoroacetamido)ethyl)amino)propyl)carbamate (10-1):

[0382] Commercially available N-(tert-butoxycarbonyl)-β-alanine (113 mg, [CAS REGISTRY NO.: 3303-84-2]) and the compound (7-3, 110 mg) obtained in Step 2 of Example 7 were dissolved in acetonitrile (3.3 mL). O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (261 mg) and N,N-diisopropylethylamine (319 μL) were added, and the mixture was stirred at room temperature for 3 hours. Ethyl acetate (15 mL) and water (5 mL) were added to the reaction mixture, and after separation, the organic layer was washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and triturated with tert-butyl methyl ether (20 mL). The solid was collected by filtration and dissolved in ethyl acetate (20 mL). The organic layer was washed successively with 1N citric acid, water, and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was triturated with tert-butyl methyl ether (10 mL), and the solid was collected by filtration to obtain the title compound 10-1 (80 mg) as a white solid.

[0383] <Step 2> Synthesis of 3-amino-N-(2-(2,2,2-trifluoroacetamido)ethyl)propanamide hydrochloride (10-2):

[0384] To the compound (10-1, 80 mg) obtained in Step 1 of Example 10, 4N hydrogen chloride / 1,4-dioxane (1.1 mL) was added under ice-water cooling, and the mixture was stirred at room temperature for 2 hours. Diisopropyl ether (3.4 mL) was added to the reaction mixture, and the mixture was stirred for 1.5 hours. The resulting solid was filtered to obtain the title compound 10-2 (61 mg) as a white solid.

[0385] <Step 3> Synthesis of 3-(2-(cyclooct-2-yn-1-yloxy)acetamido)-N-(2-(2,2,2-trifluoroacetamido)ethyl)propanamide (10-3):

[0386] To the carboxylic acid (7-4, 44 mg) synthesized according to a literature method (Org. Process Res. Dev. (2018) 22:108-110) and the compound (10-2, 61 mg) obtained in (Example 10) <Step 2>, ethanol (1.2 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (115 mg), and triethylamine (39 μL) were added and stirred at room temperature for 2 hours. Water (3.7 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (15 mL, 5 mL). The organic layer was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. To the resulting solid, tert-butyl methyl ether (10 mL) was added, triturated, and filtered. The resulting solid was purified by column chromatography (80% ethyl acetate / n-heptane→ethyl acetate→20% methanol / ethyl acetate) to give the title compound 10-3 (60 mg) as a pale yellow solid.

[0387] <Step 4> Synthesis of N-(2-aminoethyl)-3-(2-(cyclooct-2-yn-1-yloxy)acetamido)propanamide (10-4):

[0388] To a solution of the compound (10-3, 60 mg) obtained in Step 3 of Example 10 in methanol (3.0 mL), a solution of potassium carbonate (42 mg) in water (0.3 mL) was added and stirred at room temperature for 3 hours. A solution of potassium carbonate (42 mg) in water (0.3 mL) was then added and stirred at room temperature for 16.5 hours. The reaction mixture was concentrated under reduced pressure, and then saturated brine (2 mL) was added and the mixture was further saturated with sodium chloride. Extraction was performed with ethyl acetate (15 mL, 10 mL × 4), and the extract layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. Ethyl acetate (5 mL) and a few drops of methanol were added to the residue, and insoluble matter was removed by filtration. The resulting filtrate was concentrated under reduced pressure to give the title compound 10-4 (31 mg) as a colorless oil.

[0389] <Step 5> Synthesis of N-(2-aminoethyl)-3-(2-(cyclooct-2-yn-1-yloxy)acetamido)propanamide group-introduced alginic acid (EX10-A2):

[0390] To a 1 wt% aqueous solution (41 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (114 mg), a solution of the compound (10-4, 30.5 mg) obtained in Step 4 of Example 10 in ethanol (4.1 mL), and 1 molar sodium bicarbonate solution (103 μL) were added. After stirring at 30°C for 3 hours, sodium chloride (0.41 g) and ethanol (82 mL) were added sequentially, 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 resulting solid was dissolved in water and lyophilized to obtain the title compound EX10-A2 (406 mg) as a white solid.

[0391] (Examples 11a and 11b) Synthesis of 2-(cyclooct-2-yn-1-yloxy)ethane-1-amino group-introduced alginic acid (EX11a-A2, EX11b-B2):

[0392] <Step 1> Synthesis of (E)-N-(2-((2-bromocyclooct-2-en-1-yl)oxy)ethyl)-2,2,2-trifluoroacetamide (11-3):

[0393] To a mixture of the dibromo form (11-1, 1 g) synthesized according to a published method (Org. Process Res. Dev. (2018) 22:108-110) and the alcohol form (11-2, 5.28 g) synthesized according to a published method (WO 2015 / 140807), dichloromethane (2 mL) was added at room temperature. While maintaining the internal temperature at room temperature, the reaction vessel was wrapped in aluminum foil and protected from light. Subsequently, silver trifluoromethanesulfonate (1.92 g) was added in one portion at room temperature, and the mixture was stirred at the same temperature for 1 hour. After stirring, saturated brine (5 mL) was added under ice cooling, and the precipitated silver salt was removed by filtration through Celite. The residue was washed with methyl tert-butyl ether (10 mL). The filtrate was separated, and the organic layer was washed twice with water (5 mL). The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (n-heptane / ethyl acetate) to obtain a fraction containing compound 11-3 (0.46 g).

[0394] <Step 2> Synthesis of 2-(cyclooct-2-yn-1-yloxy)ethan-1-amine (11-4):

[0395] To a mixture of the fraction containing compound (11-3, 0.46 g) obtained in Step 1 of Example 11 and dimethyl sulfoxide (1.38 mL), 28% sodium methoxide methanol solution (1.82 mL) was added under stirring and water cooling, and the mixture was stirred at room temperature for 16 hours. Water (10 mL) was added to terminate the reaction, and the methanol was concentrated under reduced pressure. The resulting solution was extracted three times with methyl tert-butyl ether (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the title compound 11-4 (0.196 g) as a crude product in the form of a brown oil.

[0396] <Step 3-1> Synthesis of 2-(cyclooct-2-yn-1-yloxy)ethane-1-amino group-introduced alginic acid (EX11a-A2):

[0397] To a 1 wt% aqueous solution (69.2 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (213.55 mg) was added at room temperature. Subsequently, a solution of the compound (11-4, 26.78 mg) obtained in (Example 11) <Step 2> in water (1 mL) and ethanol (1 mL) was added at room temperature, and the mixture was stirred at the same temperature for 24 hours. Then, sodium chloride (700 mg) and ethanol (138.4 mL) were added sequentially, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration, washed with ethanol, and then dried under reduced pressure. The resulting solid was dissolved in water and lyophilized to obtain the title compound EX11a-A2 (661 mg) as a white solid.

[0398] <Step 3-2> Synthesis of 2-(cyclooct-2-yn-1-yloxy)ethane-1-amino group-introduced alginic acid (EX11b-B2):

[0399] Using a 1 wt% aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (70.1 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (216.4 mg), and the compound (11-4, 27.14 mg) obtained in Step 2 of Example 11, the same procedure as in Step 3-1 of Example 11 was carried out to obtain the title compound EX11b-B2 (648 mg) as a white solid.

[0400] Example 12 Synthesis of 2-(2-(cyclooct-2-yn-1-yloxy)ethoxy)ethane-1-amino group-introduced alginic acid (EX12-A2)

[0401] <Step 1> Synthesis of 2,2,2-trifluoro-N-(2-(2-hydroxyethoxy)ethyl)acetamide (12-2):

[0402] To a solution of commercially available 2-(2-aminoethoxy)ethanol [CAS REGISTRY NO.: 929-06-6] (12-1, 2.0 mL) in tetrahydrofuran (8.0 mL), 2,2,2-trifluoroethyl acetate (2.5 mL) was added dropwise over 5 minutes, and the mixture was stirred at room temperature for 20 hours. The reaction mixture was concentrated under reduced pressure, and then ethyl acetate (30 mL) and water (10 mL) were added, followed by separation. The aqueous layer was extracted with ethyl acetate (10 mL), and the combined organic layer was washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain the title compound 12-2 (3.7 g) as a colorless oil.

[0403] <Step 2> Synthesis of (E)-N-(2-(2-((2-bromocyclooct-2-en-1-yl)oxy)ethoxy)ethyl)-2,2,2-trifluoroacetamide (12-3):

[0404] The dibromo compound (11-1, 0.30 g) synthesized according to a literature method (Org. Process Res. Dev. (2018) 22:108-110) was dissolved in methylene chloride (0.54 mL), and the compound (12-2, 1.86 g) obtained in (Example 12) <Step 1> and silver trifluoromethanesulfonate (0.52 g) were added under a light-shielding aluminum foil. After stirring at room temperature for 1.5 hours under a light-shielding condition, saturated aqueous sodium bicarbonate (2.0 mL) and saturated saline (3.0 mL) were sequentially added to the reaction solution under ice-water cooling. The solid was removed by filtration through Celite and washed with tert-butyl methyl ether (10 mL × 3). The filtrate was separated, and the organic layer was washed sequentially with water and saturated saline. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain the title compound 12-3 (424 mg) as a pale brown oil.

[0405] <Step 3> Synthesis of N-(2-(2-(cyclooct-2-yn-1-yloxy)ethoxy)ethyl)-2,2,2-trifluoroacetamide (12-4):

[0406] The compound (12-3, 100 mg) obtained in Step 2 of Example 12 was dissolved in tetrahydrofuran (0.7 mL) and N,N-dimethylformamide (0.7 mL). 60% sodium hydride (21 mg) was added under ice-cold water, and the mixture was stirred at the same temperature for 3 hours. 60% sodium hydride (10 mg) was added, and the mixture was stirred at room temperature for 1 hour. Further 60% sodium hydride (10 mg) was added, and the mixture was stirred at room temperature for 20 hours. Water (3 mL) was added, and the mixture was extracted with ethyl acetate (15 mL, 10 mL). The organic layer was washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (n-heptane → 50% ethyl acetate / n-heptane) to obtain the title compound 12-4 (37 mg) as a colorless oil.

[0407] <Step 4> Synthesis of 2-(2-(cyclooct-2-yn-1-yloxy)ethoxy)ethan-1-amine (12-5):

[0408] To a solution of the compound (12-4, 37 mg) obtained in Step 3 of Example 12 in methanol (555 μL) was added a solution of potassium carbonate (50 mg) in water (185 μL), and the mixture was stirred at room temperature for 17 hours. The reaction mixture was concentrated under reduced pressure, and then water (1 mL) was added and saturated with sodium chloride. The mixture was extracted with ethyl acetate (10 mL × 4), and the extract layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. Ethyl acetate (10 mL) and a few drops of methanol were added to the residue, and insoluble matter was removed by filtration. The resulting filtrate was concentrated under reduced pressure to give the title compound 12-5 (30 mg) as a colorless oil.

[0409] <Step 5> Synthesis of 2-(2-(cyclooct-2-yn-1-yloxy)ethoxy)ethane-1-amino group-introduced alginic acid (EX12-A2):

[0410] To a 1 wt% aqueous solution (52 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (145 mg), a solution of the compound (12-5, 29 mg) obtained in Step 4 of Example 12 in ethanol (5.2 mL), and 1 molar sodium bicarbonate solution (131 μL) were added. After stirring at 30°C for 3.2 hours, sodium chloride (0.52 g) and ethanol (104 mL) were added sequentially, 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 resulting solid was dissolved in water and lyophilized to obtain the title compound EX12-A2 (522 mg) as a white solid.

[0411] Example 13 Synthesis of 3-amino-N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)ethyl)propanamide group-introduced alginic acid (EX13-A2)

[0412] <Step 1> Synthesis of 3-(2,2,2-trifluoroacetamido)propanoic acid (13-2):

[0413] Commercially available β-alanine [CAS REGISTRY NO.: 107-95-9] (13-1, 2.0 g) was dissolved in methanol (40.0 mL), and triethylamine (3.3 mL) was added. Ethyl 2,2,2-trifluoroacetate (3.4 mL) was added dropwise over 5 minutes under water cooling, and the mixture was stirred at room temperature for 20.5 hours. The reaction mixture was concentrated under reduced pressure, water (20 mL) was added, and the pH was adjusted to 4 with 1N hydrochloric acid. Extraction was performed with ethyl acetate (100 mL x 2, 50 mL), and the organic layer was washed with saturated brine. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain the title compound 13-2 (2.9 g) as a white solid.

[0414] <Step 2> Synthesis of tert-butyl (2-(2-(3-(2,2,2-trifluoroacetamido)propanamido)ethoxy)ethyl)carbamate (13-3):

[0415] To a solution of the compound (13-2, 400 mg) obtained in (Example 13) <Step 1>, tert-butyl (2-(2-aminoethoxy)ethyl)carbamate (8-1, 441 mg, [CAS REGISTRY NO.: 127828-22-2]) in ethanol (4.0 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (897 mg) was added and stirred for 3.5 hours. Water (5 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL, 10 mL). The organic layer was then washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (30% ethyl acetate / n-heptane→ethyl acetate) to obtain the title compound 13-3 (451 mg) as a colorless oil.

[0416] <Step 3> Synthesis of N-(2-(2-aminoethoxy)ethyl)-3-(2,2,2-trifluoroacetamido)propanamide hydrochloride (13-4):

[0417] To the compound (13-3, 451 mg) obtained in Step 2 of Example 13, 4N hydrogen chloride / 1,4-dioxane (3.16 mL) was added under ice-water cooling, and the mixture was stirred at room temperature for 3 hours. Diisopropyl ether (6.4 mL) was added to the reaction mixture, and the mixture was concentrated under reduced pressure to give the title compound 13-4 (433 mg) as a colorless gum.

[0418] <Step 4> Synthesis of N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)ethyl-3-(2,2,2-trifluoroacetamido)propanamide (13-5):

[0419] To the carboxylic acid (7-4, 111 mg) synthesized according to a literature method (Org. Process Res. Dev. (2018) 22:108-110) and the compound (13-4, 215 mg) obtained in (Example 13) <Step 3>, ethanol (1.7 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (253 mg), and triethylamine (102 μL) were added and stirred at room temperature for 21 hours. Water (5 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (15 mL). The organic layer was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography (30% ethyl acetate / n-heptane→ethyl acetate→15% methanol / ethyl acetate) to give the title compound 13-5 (35 mg) as a colorless oil.

[0420] <Step 5> Synthesis of 3-amino-N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)ethyl)propanamide (13-6):

[0421] To a solution of the compound (13-5, 35 mg) obtained in Step 4 of Example 13 in methanol (700 μL) was added a solution of potassium carbonate (33 mg) in water (175 μL), and the mixture was stirred at room temperature for 16.5 hours. The reaction mixture was concentrated under reduced pressure, and then water (2 mL) was added and saturated with sodium chloride. Extraction was performed with ethyl acetate (10 mL × 5), and the extract layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. Ethyl acetate (10 mL) and a few drops of methanol were added to the residue, and insoluble matter was removed by filtration. The resulting filtrate was concentrated under reduced pressure to give the title compound 13-6 (24 mg) as a colorless gum.

[0422] <Step 6> Synthesis of 3-amino-N-(2-(2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)ethyl)propanamide group-introduced alginic acid (EX13-A2):

[0423] To a 1 wt% aqueous solution (28 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (78 mg), a solution of the compound (13-6, 24 mg) obtained in Step 5 of Example 13 in ethanol (2.8 mL), and 1 molar sodium bicarbonate water (71 μL) were added. After stirring at 30°C for 3.5 hours, sodium chloride (0.28 g) and ethanol (56 mL) were added sequentially, 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 resulting solid was dissolved in water and lyophilized to give the title compound EX13-A2 (272 mg) as a white solid.

[0424] (Example 14) Synthesis of N-(4-(2-aminoethoxy)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX14a-A2, EX14b-B2):

[0425] <Step 1> Synthesis of N-(2-bromoethyl)-2,2,2-trifluoroacetamide (14-2):

[0426] To a solution of commercially available 2-bromoethylamine hydrobromide [CAS REGISTRY NO.: 2576-47-8] (14-1, 3 g) in methanol (30 mL) was added triethylamine (4.29 mL) under ice-cooling and stirring. Ethyl trifluoroacetate (1.92 mL) was slowly added to this mixture at the same temperature, and the mixture was stirred at room temperature for 42 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and water (10 mL) was added. 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 to obtain the title compound 14-2 (2.457 g) as a pale brown solid.

[0427] <Step 2> Synthesis of tert-butyl (4-(2-(2,2,2-trifluoroacetamido)ethoxy)benzyl)carbamate (14-4):

[0428] To a mixture of commercially available tert-butyl (4-hydroxybenzyl)carbamate [CAS REGISTRY NO.: 149505-94-2] (14-3, 0.36 g), the compound (14-2, 0.46 g) obtained in (Example 14) <Step 1>, potassium iodide (0.35 g), and N-methylpyrrolidone (3.6 mL), potassium carbonate (0.45 g) was added at room temperature, and the mixture was stirred at 140°C for 5 hours. After completion of the reaction, the mixture was cooled to room temperature and diluted with water (10 mL). The mixture was extracted three times with methyl tert-butyl ether (10 mL), and the organic layer was washed twice with 1N aqueous sodium hydroxide solution (5 mL), water (5 mL), and saturated brine (5 mL), and then dried over anhydrous sodium sulfate. The organic layer was filtered and concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by silica gel column chromatography (n-heptane / ethyl acetate) to obtain the title compound 14-4 (0.202 g) as a white amorphous substance.

[0429] <Step 3> Synthesis of N-(2-(4-(aminomethyl)phenoxy)ethyl)-2,2,2-trifluoroacetamide hydrochloride (14-5):

[0430] The compound (14-4, 0.2 g) obtained in (Example 14) <Step 2> was used to carry out the same operation as in (Example 6) <Step 2> to obtain the title compound 14-5 (0.147 g) as a white solid.

[0431] <Step 4> Synthesis of N-(2-(4-((2-(cyclooct-2-yn-1-yloxy)acetamido)methyl)phenoxy)ethyl)-2,2,2-trifluoroacetamide (14-6):

[0432] To a mixture of carboxylic acid (7-4, 50 mg) synthesized according to a literature method (Org. Process Res. Dev. (2018) 22:108-110), the compound (14-5, 81.96 mg) synthesized in (Example 14) <Step 3>, and ethanol, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (137.22 mg) and triethylamine (38.25 μL) were added under ice-cooling and stirring, and the mixture was stirred at room temperature for 1 hour and 30 minutes. After completion of the reaction, water (2 mL) was added, the suspension was stirred, and methyl tert-butyl ether (0.5 mL) was added. The separated aqueous layer was extracted twice with methyl tert-butyl ether (5 mL), washed successively with water (5 mL) and saturated brine (5 mL), and dried over anhydrous sodium sulfate. The dried organic layer was filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (n-heptane / ethyl acetate) to obtain the title compound 14-6 (99 mg) as a white amorphous substance.

[0433] <Step 5> Synthesis of N-(4-(2-aminoethoxy)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (14-7):

[0434] To a mixture of the compound (14-6, 99 mg) obtained in Step 4 of Example 14 and methanol (1485 μL), potassium carbonate (64.17 mg) and water (495 μL) were added under stirring on ice, and the mixture was stirred at room temperature for 15 hours. After completion of the reaction, the methanol was concentrated under reduced pressure, and the resulting aqueous layer was extracted three times with ethyl acetate (5 mL). The organic layer was washed successively with water (5 mL) and saturated brine (5 mL) and dried over anhydrous sodium sulfate. The dried organic layer was filtered and then concentrated under reduced pressure to obtain the title compound 14-7 (68 mg) as a crude product in the form of a yellow oil.

[0435] <Step 6-1> Synthesis of N-(4-(2-aminoethoxy)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX14a-A2):

[0436] Using a 1 wt% aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (49.44 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (152.54 mg), and the compound (14-7, 37.79 mg) obtained in (Example 14) <Step 5>, the same procedure as in (Example 11) <Step 3-1> was carried out to obtain the title compound EX14a-A2 (479 mg) as a white solid.

[0437] <Step 6-2> Synthesis of N-(4-(2-aminoethoxy)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX14b-B2):

[0438] Using a 1 wt% aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (40.08 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (123.66 mg), and the compound (14-7, 30.64 mg) obtained in Step 5 of Example 14, the same procedure as in Step 3-1 of Example 11 was carried out to obtain the title compound EX14b-B2 (356 mg) as a white solid.

[0439] Example 15 Synthesis of 2-amino-N-[3-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-3-oxopropyl]acetamide group-introduced alginic acid (EX15-A2)

[0440] Step 1: Synthesis of (9H-fluoren-9-yl)methyl-N-[3-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-3-oxopropyl]acetamido-2-carbamate group (15-2):

[0441] Commercially available 3-amino-1-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-1-propanone [CAS REGISTRY NO.: 1255942-06-3] (15-1, 50 mg) and N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine [CAS REGISTRY NO.: 29022-11-5] (54 mg) were dissolved in acetonitrile (1.5 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (76 mg) and N,N-diisopropylethylamine (70 μL) were added, and the mixture was stirred at room temperature for 4.5 hours. Ethyl acetate (15 mL) and water (5 mL) were added to the reaction mixture, and the mixture was separated. The organic layer was washed with water and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain the title compound 15-2 (63 mg) as a pale beige amorphous solid.

[0442] <Step 2> Synthesis of 2-amino-N-[3-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-3-oxopropyl]acetamide (15-3):

[0443] A solution of piperidine (56 μL) in N,N-dimethylformamide (315 μL) was added to the compound (15-2, 63 mg) obtained in Step 1 of Example 15, and the mixture was stirred at room temperature for 30 minutes. Ethyl acetate (15 mL) and water (5 mL) were added to the reaction mixture, and after separation, the organic layer was washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. tert-Butyl methyl ether (5 mL) was added to the resulting solid, and the mixture was triturated and filtered to obtain the title compound 15-3 (10 mg) as a pale beige solid. Additional title compound 15-3 (11 mg) was obtained as a pale yellow gum by recovering it from the filtrate.

[0444] <Step 3> Synthesis of 2-amino-N-[3-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-3-oxopropyl]acetamide group-introduced alginic acid (EX15-A2):

[0445] To a 1% by weight aqueous solution (19 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (106 mg), a solution of the compound (15-3, 21 mg) obtained in Step 2 of Example 15 in ethanol (1.9 mL), and 1 molar sodium bicarbonate solution (48 μL) were added. After stirring at 30°C for 3 hours, sodium chloride (0.19 g) and ethanol (38 mL) were added sequentially, 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 resulting solid was dissolved in water and lyophilized to obtain the title compound EX15-A2 (188 mg) as a white solid.

[0446] Example 16 Synthesis of 2-amino-N-(2-(cyclooct-2-yn-1-yloxy)ethyl)acetamide group-introduced alginic acid (EX16-A2)

[0447] <Step 1> Synthesis of (2,2,2-trifluoroacetyl)glycine (16-2):

[0448] Glycine (16-1, 2 g) was suspended in methanol (10 mL) and cooled to 4°C. Ethyl trifluoroacetate (3.5 mL) and triethylamine (3.71 mL) were added at the same temperature, and the mixture was stirred at room temperature for 23 hours. After completion of the reaction, 1N hydrochloric acid (20 mL) was gradually added until the pH reached 2, and the mixture was extracted three times with ethyl acetate (10 mL) and washed sequentially with water (5 mL) and saturated brine (5 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a pale yellow oil. The resulting oil was dissolved in ethyl acetate (20 mL), and n-heptane (10 mL) was added. This solution was concentrated under reduced pressure to obtain the title compound 16-2 (3.22 g) as a white amorphous substance.

[0449] <Step 2> Synthesis of N-(2-((2-(cyclooct-2-yn-1-yloxy)ethyl)amino)-2-oxoethyl)-2,2,2-trifluoroacetamide (16-3):

[0450] To a mixture of compound 11-4 (80 mg) and the compound (16-2, 81.83 mg) obtained in (Example 16) <Step 1>, ethanol (1600 μL) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (239.21 mg) were added under ice-cooling and stirring, and the mixture was stirred at room temperature for 3 hours. Water (2 mL) was added to quench the reaction, and the mixture was extracted three times with methyl tert-butyl ether (5 mL). The organic layer was washed successively with water (5 mL) and saturated brine (5 mL) and dried over anhydrous sodium sulfate. The organic layer was filtered and concentrated under reduced pressure to obtain a crude product. This crude product was triturated with n-heptane (10 mL), filtered, and dried under reduced pressure to obtain the title compound 16-3 (95.1 mg) as a white solid.

[0451] <Step 3> Synthesis of 2-amino-N-(2-(cyclooct-2-yn-1-yloxy)ethyl)acetamide (16-4):

[0452] The compound (16-3, 60 mg) obtained in (Example 16) <Step 2>, methanol (900 μL), potassium carbonate (51.78 mg), and water (300 μL) were used to carry out the same operation as in (Example 14) <Step 5>, and the title compound 16-4 (15 mg) was obtained as a pale yellow oil.

[0453] <Step 4> Synthesis of 2-amino-N-(2-(cyclooct-2-yn-1-yloxy)ethyl)acetamide group-introduced alginic acid (EX16-A2):

[0454] Using a 1 wt% aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (29.66 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (91.52 mg), and the compound (16-4, 15 mg) obtained in (Example 16) <Step 3>, the same procedure as in (Example 11) <Step 3-1> was carried out to obtain the title compound EX16-A2 (279 mg) as a white solid.

[0455] Example 17 Synthesis of (2S)-2-amino-N-(2-(cyclooct-2-yn-1-yloxy)ethyl)-3-phenylpropanamide group-introduced alginic acid (EX17-A2)

[0456] <Step 1> Synthesis of (2,2,2-trifluoroacetyl)-L-phenylalanine (17-2):

[0457] L-Phenylalanine [CAS REGISTRY NO.: 63-91-2] (17-1, 2 g) was dissolved in methanol (10 mL) and cooled to 4°C. Subsequently, ethyl trifluoroacetate (1.59 mL) and triethylamine (1.69 mL) were added at the same temperature, and the mixture was stirred at room temperature for 16 hours. After completion of the reaction, 1N hydrochloric acid (10 mL) was gradually added until the pH reached 1, and the suspension was stirred for 30 minutes. The suspension was filtered, and the collected solid was dried under reduced pressure to obtain the title compound 17-2 (2.53 g) as a white solid.

[0458] <Step 2> Synthesis of (2S)-N-(2-(cyclooct-2-yn-1-yloxy)ethyl)-3-phenyl-2-(2,2,2-trifluoroacetamido)propanamide (17-3):

[0459] To a mixture of compound 11-4 (60 mg) and the compound (17-2, 93.7 mg) obtained in (Example 17) <Step 1>, ethanol (1200 μL) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (179.41 mg) were added under ice-cooling, and the mixture was stirred at room temperature for 3 hours. Water (2 mL) was added to terminate the reaction, and the mixture was extracted three times with methyl tert-butyl ether (5 mL). The organic layer was washed successively with water (5 mL) and saturated brine (5 mL) and dried over anhydrous sodium sulfate. The organic layer was filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (n-heptane / ethyl acetate) to obtain the title compound 17-3 (57 mg) as a white amorphous substance.

[0460] <Step 3> Synthesis of (2S)-2-amino-N-(2-(cyclooct-2-yn-1-yloxy)ethyl)-3-phenylpropanamide (17-4):

[0461] The compound (17-3, 57 mg) obtained in (Example 17) <Step 2>, methanol (855 μL), potassium carbonate (38.39 mg), and water (285 μL) were used to carry out the same operation as in (Example 14) <Step 5>, and the title compound 17-4 (35 mg) was obtained as a pale yellow oil.

[0462] <Step 4> Synthesis of (2S)-2-amino-N-(2-(cyclooct-2-yn-1-yloxy)ethyl)-3-phenylpropanamide group-introduced alginic acid (EX17-A2):

[0463] Using a 1 wt% aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (47.46 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (146.44 mg), and the compound (17-4, 34.53 mg) obtained in (Example 17) <Step 3>, the same procedure as in (Example 11) <Step 3-1> was carried out to obtain the title compound EX17-A2 (383 mg) as a white solid.

[0464] Example 18 Synthesis of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group-introduced alginic acid (EX18-A2)

[0465] <Step 1> Synthesis of methyl 4-(2-((tert-butoxycarbonyl)amino)ethoxy)benzoate (18-2):

[0466] A solution of diethyl azodicarboxylate (40% toluene solution, 1.92 mL) was added to a solution of triphenylphosphine (0.96 g) in tetrahydrofuran (2.59 mL) under ice-cooling and stirring, and the mixture was stirred at room temperature for 20 minutes. To this solution, a solution of commercially available 4-hydroxybenzoic acid (compound 18-1, 0.37 g) and 2-(tert-butoxycarbonyl)ethanolamine (0.39 g) in tetrahydrofuran (1.1 mL) was added under ice-cooling and stirring, and the mixture was stirred at room temperature for 17 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (5% ethyl acetate / n-heptane to 40% ethyl acetate / n-heptane) to give a mixture of compounds 18-1 and 18-2. This mixture was dissolved in methyl tert-butyl ether (20 mL) and washed twice with 1N aqueous sodium hydroxide solution (5 mL) and then with saturated brine (5 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was then distilled off under reduced pressure to obtain the title compound 18-2 (0.45 g) as a pink oily substance.

[0467] <Step 2> Synthesis of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide hydrochloride (compound 18-4):

[0468] Lithium hydroxide monohydrate (0.25 g) was added to a solution of compound 18-2 (0.44 g) obtained in Step 1 of Example 18 in methanol (4.4 mL), and the mixture was stirred at 60°C for 3 hours and 30 minutes. 1N hydrochloric acid (5 mL) was added to the reaction mixture, 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, and the solvent was distilled off under reduced pressure. The residue was dissolved in acetonitrile (4.4 mL), and 3-azidopropan-1-amine (0.15 g) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.57 g) were added. Subsequently, N,N-diisopropylethylamine (0.52 mL) was added under ice-cooling and stirring, and the mixture was stirred at room temperature for 5 hours. Water (10 mL) was added to the reaction mixture, which was then extracted three times with ethyl acetate (15 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (16% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain a fraction containing compound 18-3 (0.71 g).

[0469] To the fraction containing compound 18-3 (0.71 g), 4N hydrogen chloride / 1,4-dioxane (4.9 mL) was added and stirred at room temperature for 20 minutes. Diisopropyl ether was added to the reaction mixture, and the precipitate was filtered to obtain the title compound 18-4 (0.49 g) as a white solid.

[0470] <Step 3> Synthesis of 4-(2-aminoethoxy)-N-(3-azidopropyl)benzamide group-introduced alginic acid (compound EX18-A2):

[0471] A 1 wt% aqueous solution (19.6 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) was added with stirring to ice-cooling, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (50.19 mg), compound 18-4 (54.37 mg) obtained in (Example 18) <Step 2>, and 1 molar sodium bicarbonate water (181.4 μL) were used in the same manner as in (Example 11) <Step 3-1> to obtain the title compound EX18-A2 (198 mg) as a white solid.

[0472] Example 19 Synthesis of 3-amino-1-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-1-propanone group-introduced alginic acid (EX19-A2)

[0473] A 1 wt% aqueous solution (43.6 mL) of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) was added with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (111.7 mg), 1 molar sodium bicarbonate solution (403.5 μL), and commercially available 3-amino-1-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-1-propanone [CAS REGISTRY NO.: 1255942-06-3] (19-1, 83.6 mg) to carry out the same procedure as in (Example 11) <Step 3-1>, to obtain the title compound EX19-A2 (376 mg) as a pale yellow solid.

[0474] Example 20 Synthesis of N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX20-B2, EX20-A2)

[0475] <Step 1> Synthesis of tert-butyl (4-((2,2,2-trifluoroacetamido)methyl)benzyl)carbamate (Compound 20-2):

[0476] To a mixture of tert-butyl (4-(aminomethyl)benzyl)carbamate (20-1, 0.67 g), synthesized with reference to a literature method (Bioorganic & Medicinal Chemistry (2003) 11:4189-4206), triethylamine (0.39 mL), and methanol (6.67 mL), ethyl trifluoroacetate (0.44 mL) was added dropwise under ice-cooling and stirring. The reaction mixture was warmed to room temperature and stirred at the same temperature for 5 hours. The reaction was quenched with water (10 mL) and extracted three times with ethyl acetate (10 mL). The collected organic layer was washed with saturated brine (5 mL) and dried over anhydrous sodium sulfate. The dried organic layer was filtered and concentrated to obtain the crude title compound 20-2 (0.67 g) as a pale yellow amorphous solid.

[0477] <Step 2> Synthesis of N-(4-(aminomethyl)benzyl)-2,2,2-trifluoroacetamide hydrochloride (compound 20-3):

[0478] To a 1,4-dioxane solution (3.5 mL) of compound 20-2 (0.5 g) obtained in (Example 20) <Step 1>, 4N hydrogen chloride / 1,4-dioxane (3.5 mL) was added under stirring in a water-cooled bath, and the mixture was stirred at room temperature for 3 hours. Diisopropyl ether (40 mL) was added to the reaction solution, and the precipitate was filtered to obtain the title compound 20-3 (0.4 g) as a white solid.

[0479] <Step 3> Synthesis of N-(4-((2-(cyclooct-2-yn-1-yloxy)acetamido)methyl)benzyl)-2,2,2-trifluoroacetamide (compound 20-4):

[0480] To a solution of carboxylic acid (7-4, 0.17 g) synthesized according to a literature method (Org. Process Res. Dev. (2018) 22:108-110) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.26 g) in acetonitrile (1.7 mL), compound 20-3 (0.26 g) obtained in (Example 20) <Step 2> and N,N-diisopropylethylamine (0.51 mL) were added dropwise under ice-cooling and stirring, and the mixture was stirred at room temperature for 1 hour and 30 minutes. Water (5 mL) was added to quench the reaction, and the mixture was extracted three times with ethyl acetate (5 mL). The organic layer was washed with saturated brine (3 mL) and then dried over anhydrous sodium sulfate. The dried organic layer was filtered, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (12% ethyl acetate / n-heptane to 100% ethyl acetate) to obtain the title compound 20-4 (0.19 g) as a white amorphous substance.

[0481] <Step 4> Synthesis of N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (Compound 20-5):

[0482] To a mixture of compound 20-4 (0.18 g) obtained in (Example 20) <Step 3> and methanol (1.8 mL), an aqueous solution (0.9 mL) of potassium carbonate (0.13 g) was added dropwise under ice-cooling and stirring, and the mixture was stirred at room temperature for 17 hours and 30 minutes. Methanol was evaporated under reduced pressure, and the mixture was extracted three times with ethyl acetate (5 mL). The organic layer was washed with saturated brine (5 mL) and then dried over anhydrous sodium sulfate. The organic layer was filtered, and the solvent was evaporated under reduced pressure to obtain crude title compound 20-5 (0.13 g) as a pale yellow oil.

[0483] <Step 5> Synthesis of N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX20-B2):

[0484] Using a 1 wt% aqueous solution of sodium alginate (B-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (50.9 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (0.12 g), and an ethanol (3 mL) solution of compound 20-5 (35 mg) obtained in (Example 20) <Step 4>, the same procedure as in (Example 11) <Step 3-1> was carried out to obtain the title compound EX20-B2 (521 mg) as a white solid.

[0485] <Step 5-2> Synthesis of N-(4-(aminomethyl)benzyl)-2-(cyclooct-2-yn-1-yloxy)acetamide group-introduced alginic acid (EX20-A2):

[0486] Using a 2 wt% aqueous solution of sodium alginate (A-2, manufactured by Mochida Pharmaceutical Co., Ltd.) (250 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (279 mg), 1 molar sodium bicarbonate water (252 μL), and an ethanol (25 mL) solution of compound 20-5 (84 mg) obtained in (Example 20) <Step 4>, the same procedure as in (Example 11) <Step 3-1> was carried out to obtain the title compound EX20-A2 (4.57 g) as a white solid.

[0487] (Examples P1 to P7) The alginic acid derivatives of (Example P1) to (Example P7) shown in the table below were produced using the corresponding amino compound (which may be a pharmaceutically acceptable salt thereof or a solvate thereof) and alginic acid in accordance with the method shown in the above examples.

[0488] Physical properties data of chemically modified alginic acid derivatives

[0489] NMR data of intermediate compounds

[0490] LC-Mass data of intermediate compounds

[0491] [Measurement of introduction rate of reactive group or complementary reactive group] The introduction rate of reactive group or complementary reactive group means the number of reactive groups or complementary reactive groups introduced per uronic acid monosaccharide unit, which is a repeating unit of alginic acid, expressed as a percentage. In this example, the introduction rate of reactive group or complementary reactive group (mol %) is １ The amount of alginic acid required to calculate the introduction rate was measured by the carbazole-sulfuric acid method using a calibration curve, and the amount of reactive groups or complementary reactive groups can also be measured by absorbance measurement using a calibration curve.

[0492] [Molecular weight measurement] The alginic acid solid into which the reactive group or complementary reactive group obtained in the examples was introduced was dissolved in 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl to prepare a 0.1% or 0.2% solution, and then passed through a 0.22 μm pore size polyethersulfone filtration filter (Minisart High Flow Filter, Sartorius) to remove insoluble matter, and then used as a gel filtration sample. The spectrum of each sample was measured using a spectrophotometer DU-800 (Beckman-Coulter), and the measurement wavelength in gel filtration for each compound was determined. For compounds without specific absorption wavelengths, a differential refractometer was used.

[0493] 200 μL of the gel filtration sample was loaded onto a Superose 6 Increase 10 / 300 GL column (GE Healthcare Sciences). Gel filtration was performed using an AKTA Explorer 10S chromatograph and 10 mmol / L phosphate buffer (pH 7.4) containing 0.15 mol / L NaCl as the developing solvent at a flow rate of 0.8 mL / min at room temperature. The sample elution profile was generated by monitoring the absorption at wavelengths determined for each compound. The resulting chromatogram was analyzed using Unicorn 5.31 software (GE Healthcare Sciences) to determine the peak range.

[0494] The molecular weight of alginic acid into which a reactive group or a complementary reactive group had been introduced was determined by gel filtration under the same conditions as for alginic acid into which a reactive group or a complementary reactive group had been introduced, using 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 6,500 Da, GE Healthcare Sciences) as standards, and the eluate volume of each component was determined using Unicorn software. The elution volume of each component was plotted on the horizontal axis against the logarithmic value of the molecular weight on the vertical axis, and linear regression was performed to prepare two calibration curves: one from blue dextran to ferritin, and the other from ferritin to aprotinin.

[0495] Using this calibration curve, the molecular weight (Mi) at elution time i of the previously obtained chromatogram was calculated. Next, the absorbance at elution time i was read and designated as Hi. From these data, the weight average molecular weight (Mw) was calculated using the following formula:

[0496]

[0497] [Measurement of gel stability] (Measurement of gel stability (1)): Stability in PBS Each of the alginic acid derivatives (Ex1-A2), (Ex4-A2), (Ex5-A2), and (Ex19-A2) obtained in Examples 1, 4, 5, and 19 was dissolved in water to a concentration of 1.0% to obtain aqueous alginic acid solutions (1-1), (4-1), (5-1), and (19-1), respectively. These were mixed in equal amounts in combinations of (1-1) and (19-1), (4-1) and (19-1), and (5-1) and (19-1), respectively, and placed in a syringe equipped with an 18-gauge needle. The syringe was placed in a syringe pump set to a flow rate of 1 mL / min, and the mixture was added dropwise to a 30 mmol / L calcium chloride solution for 30 seconds and stirred for 5 minutes to obtain an alginic acid gel. This gel was washed once with 10 mL of PBS and allowed to stand in PBS at 37°C for 10 minutes to undergo chemical crosslinking, resulting in a gel of chemically crosslinked alginate. Chemically crosslinked alginate (beads) made with (Ex18-A2) / (Ex19-A2) were also prepared in the same manner. 19.5 mL of PBS was added to the resulting chemically crosslinked alginate, and the mixture was shaken at 37°C. The aqueous solution was collected over time and replenished with the same amount of PBS as the collected amount. After the test was completed, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and the mixture was shaken at 37°C for more than 3 hours to completely disintegrate the gel, and the aqueous solution was collected. The alginic acid concentration in the recovered aqueous solution was measured using the carbazole-sulfuric acid method. The amount of alginic acid dissolved up to each time point was divided by the total amount of alginic acid calculated from the alginic acid concentrations at all time points and the alginic acid concentration after the test was completed. The percentage obtained was used as the disintegration rate, which was an index of gel stability.

[0498] The results shown in Figure 1 were obtained. The chemically crosslinked alginate (beads) did not disintegrate even after 96 hours, confirming the stability of the gel. This suggests that the chemical crosslinking formed by the Huisgen reaction allows the prepared chemically crosslinked alginate to maintain its structure for a long period of time.

[0499] (Gel Stability Measurement (2)): Stability under EDTA The alginate aqueous solutions obtained in (Gel Stability Measurement (1)) were mixed in equal amounts with combinations of (1-1) and (19-1), (4-1) and (19-1), and (5-1) and (19-1), respectively, and placed in a syringe equipped with an 18-gauge needle. This syringe was placed in a syringe pump set to a flow rate of 1 mL / min. The mixture was added dropwise to a 30 mmol / L calcium chloride solution for 30 seconds and stirred for 5 minutes to obtain an alginate gel. Chemically crosslinked alginate (beads) produced by (Ex18-A2) / (Ex19-A2) were also prepared in the same manner. This gel was washed once with 10 mL of saline and allowed to stand in saline at 37°C for 10 minutes to perform chemical crosslinking, yielding chemically crosslinked alginate. To the resulting chemically crosslinked alginate, 19.5 mL of 5 mM ethylenediaminetetraacetic acid dipotassium salt dihydrate (EDTA 2K) / physiological saline was added, and the mixture was shaken at 37 ° C. The aqueous solution was collected over time, and the collected amount was replenished with the same amount of 5 mM EDTA 2K / physiological saline. 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 at 37 ° C. for 3 hours or more to completely disintegrate the gel, and the aqueous solution was collected. The alginic acid concentration in the collected aqueous solution was measured using the carbazole sulfate method, and the amount of eluted alginic acid up to each time point was divided by the total amount of alginic acid calculated from the alginic acid concentration at all time points and the alginic acid concentration after the test was completed. The value expressed as a percentage was used as an index of gel stability.

[0500] The results are shown in Figure 2. The disintegration rate of the chemically cross-linked alginate (beads) after 24 hours was 2% or less. This suggests that the structure of the chemically cross-linked alginate produced by cross-linking via the Huisgen reaction is maintained even in a calcium ion-free solution (below the physiological concentration for the body).

[0501] (Measurement of gel stability (3)): Stability in PBS Each of the alginic acid derivatives obtained in Examples 3, 5, 6, 9, 10, 12 and 18, (Ex3-A2), (Ex5-A2), (Ex6-A2), (Ex9a-A2), (Ex10-A2), (Ex12-A2) and (Ex18-A2), was dissolved in water to a concentration of 1.0% to obtain aqueous alginic acid solutions (3-1), (5-1), (6-1), (9-1), (10-1), (12-1) and (18-1), respectively. These were mixed in equal amounts in combinations of (6-1) and (9-1), (3-1) and (10-1), (5-1) and (10-1), and (18-1) and (12-1), respectively, and placed in a syringe equipped with an 18-gauge needle. The syringe was placed in a syringe pump set to a flow rate of 1 mL / min, and the mixture was added dropwise to a 30 mmol / L calcium chloride solution for 30 seconds and stirred for 5 minutes to obtain an alginate gel. This gel was washed once with 10 mL of PBS and allowed to stand in PBS at 37°C for 10 minutes to undergo chemical crosslinking, yielding chemically crosslinked alginate. Chemically crosslinked alginate gels (beads) made with (Ex18-A2) / (Ex19-A2) were also prepared in the same manner. 19.5 mL of PBS was added to the obtained chemically crosslinked alginate, and the mixture was shaken at 37 ° C. The aqueous solution was collected over time, and the same amount of PBS 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 at 37 ° C. for 3 hours or more to completely disintegrate the gel, and the aqueous solution was collected. The alginic acid concentration in the collected aqueous solution was measured using the carbazole sulfate method, and the amount of alginic acid eluted up to each time point was divided by the total amount of alginic acid calculated from the alginic acid concentration at all time points and the alginic acid concentration after the test was completed. The value expressed as a percentage was used as the disintegration rate, which was used as an index of gel stability.

[0502] The results shown in Figure 3 were obtained. The chemically cross-linked alginate (beads) did not disintegrate even after 96 hours, confirming the stability of the gel. This suggests that the chemical cross-linking formed by the Huisgen reaction allows the prepared chemically cross-linked alginate to maintain its structure for a long period of time.

[0503] (Gel Stability Measurement (4)): Stability under EDTA The alginate aqueous solutions obtained in (Gel Stability Measurement (3)) were mixed in equal amounts with combinations of (6-1) and (9-1), (3-1) and (10-1), (5-1) and (10-1), and (18-1) and (12-1), respectively, and placed in a syringe equipped with an 18-gauge needle. The syringe was placed in a syringe pump set at a flow rate of 1 mL / min. The mixture was added dropwise to a 30 mmol / L calcium chloride solution for 30 seconds and stirred for 5 minutes to obtain an alginate gel. This gel was washed once with 10 mL of saline and allowed to stand in saline at 37°C for 10 minutes to undergo chemical crosslinking, yielding chemically crosslinked alginate. Chemically crosslinked alginate (beads) produced by (Ex18-A2) / (Ex19-A2) were also prepared in the same manner. To the resulting chemically crosslinked alginate, 19.5 mL of 5 mM ethylenediaminetetraacetic acid dipotassium salt dihydrate (EDTA 2K) / physiological saline was added, and the mixture was shaken at 37 ° C. The aqueous solution was collected over time, and the collected amount was replenished with the same amount of 5 mM EDTA 2K / physiological saline. 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 at 37 ° C. for 3 hours or more to completely disintegrate the gel, and the aqueous solution was collected. The alginic acid concentration in the collected aqueous solution was measured using the carbazole sulfate method, and the amount of eluted alginic acid up to each time point was divided by the total amount of alginic acid calculated from the alginic acid concentration at all time points and the alginic acid concentration after the test was completed. The value expressed as a percentage was used as an index of gel stability.

[0504] The results are shown in Figure 4. The disintegration rate of the chemically cross-linked alginate (beads) after 24 hours was 4% or less. This suggests that the structure of the chemically cross-linked alginate produced by cross-linking via the Huisgen reaction is maintained even in a calcium ion-free solution (below the physiological concentration for the body).

[0505] (Measurement of gel stability (5)): Stability in PBS Each of the alginic acid derivatives obtained in Examples 4, 9, 16, 18 and 20, (Ex4-A2), (EX9a-A2), (Ex16-A2), (Ex18-A2) and (Ex20-B2), was dissolved in water to a concentration of 1.0% to obtain aqueous alginic acid solutions (4-1), (9-1), (16-1), (18-1), and (20-1), respectively. These were mixed in equal amounts in combinations of (4-1) and (20-1), (18-1) and (9-1), and (18-1) and (16-1), respectively, and placed in a syringe equipped with an 18-gauge needle. The syringe was placed in a syringe pump set to a flow rate of 1 mL / min. The solution was added dropwise to a calcium chloride solution with a concentration of 30 mmol / L for 30 seconds and stirred for 5 minutes to obtain an alginic acid gel. This gel was washed once with 10 mL of PBS and allowed to stand in PBS at 37°C for 10 minutes to undergo chemical crosslinking, yielding chemically crosslinked alginate. Chemically crosslinked alginate (beads) made with (Ex18-A2) / (Ex19-A2) were also prepared in the same manner. 19.5 mL of PBS was added to the resulting chemically crosslinked alginate, and the mixture was shaken at 37°C. The aqueous solution was collected over time, and the same amount of PBS 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 at 37°C for 3 hours or more to completely disintegrate the gel, and the aqueous solution was collected. The alginic acid concentration in the recovered aqueous solution was measured using the carbazole-sulfuric acid method. The amount of alginic acid dissolved up to each time point was divided by the total amount of alginic acid calculated from the alginic acid concentrations at all time points and the alginic acid concentration after the test was completed. The percentage obtained was used as the disintegration rate, which was an index of gel stability.

[0506] The results are shown in Figure 5. The chemically cross-linked alginate prepared by this method had a disintegration rate of approximately 12% or less even after 96 hours. This suggests that the chemical cross-linking formed by the Huisgen reaction allowed the prepared chemically cross-linked alginate to maintain its structure.

[0507] (Gel Stability Measurement (6)): Stability under EDTA The alginate aqueous solutions obtained in (Gel Stability Measurement (5)) were mixed in equal amounts with combinations of (4-1) and (20-1), (18-1) and (9-1), and (18-1) and (16-1), respectively, and placed in a syringe equipped with an 18-gauge needle. The syringe was placed in a syringe pump set at a flow rate of 1 mL / min. The mixture was added dropwise to a 30 mmol / L calcium chloride solution for 30 seconds and stirred for 5 minutes to obtain an alginate gel. This gel was washed once with 10 mL of saline and allowed to stand in saline at 37°C for 10 minutes to undergo chemical crosslinking, yielding chemically crosslinked alginate. Chemically crosslinked alginate (beads) produced by (Ex18-A2) / (Ex19-A2) were also prepared in the same manner. To the resulting chemically crosslinked alginate, 19.5 mL of 5 mM ethylenediaminetetraacetic acid dipotassium salt dihydrate (EDTA 2K) / physiological saline was added, and the mixture was shaken at 37 ° C. The aqueous solution was collected over time, and the collected amount was replenished with the same amount of 5 mM EDTA 2K / physiological saline. 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 at 37 ° C. for 3 hours or more to completely disintegrate the gel, and the aqueous solution was collected. The alginic acid concentration in the collected aqueous solution was measured using the carbazole sulfate method, and the amount of eluted alginic acid up to each time point was divided by the total amount of alginic acid calculated from the alginic acid concentration at all time points and the alginic acid concentration after the test was completed. The value expressed as a percentage was used as an index of gel stability.

[0508] The results shown in Figure 6 were obtained. The disintegration rate of the chemically cross-linked alginate (beads) after 24 hours was 18% or less. This suggests that the structure of the chemically cross-linked alginate produced by cross-linking via the Huisgen reaction is maintained even in a calcium ion-free solution (below the physiological concentration for the body).

[0509] [Measurement of Gel Permeability] (Measurement of Gel Permeability (1)) Each of the alginic acid derivatives (Ex1-A2), (Ex3-A2), (Ex4-A2), (Ex5-A2), and (Ex18-A2) obtained in Examples 1, 3, 4, 5, and 18 was dissolved in water to a concentration of 2.0% to prepare an alginic acid aqueous solution. To this alginic acid aqueous solution, 2 / 5 volume of fluorescein isothiocyanate-dextran (Sigma-Aldrich, FD150S) with a molecular weight of 150,000, adjusted to 1 mg / mL, and 3 / 5 volume of water were added to obtain 1.0% alginic acid aqueous solutions (1-2), (3-2), (4-2), (5-2), and (18-2) containing 0.2 mg / mL fluorescein isothiocyanate-dextran.

[0510] Furthermore, each of the alginic acid derivatives (Ex10-A2), (Ex12-A2) and (Ex19-A2) obtained in Examples 10, 12 and 19 was dissolved in water to a concentration of 1.0% to obtain aqueous alginic acid solutions (10-1), (12-1) and (19-1), respectively.

[0511] These were mixed in equal amounts in combinations of (1-2) and (19-1), (4-2) and (19-1), (5-2) and (19-1), (3-2) and (10-1), and (18-2) and (12-1), respectively. 40 mL of 30 mmol / L calcium chloride solution was added and stirred for 5 minutes to obtain alginate gels. The gels were washed once with 10 mL of saline and allowed to stand in saline at 37°C for 10 minutes to undergo chemical crosslinking, yielding fluorescein isothiocyanate-dextran-encapsulated chemically crosslinked alginate. Fluorescein isothiocyanate-dextran-encapsulated chemically crosslinked alginate (Ex18-A2) / (Ex19-A2) was also prepared in the same manner. 19.5 mL of saline was added to the obtained chemically crosslinked alginate, and the mixture was shaken at 37°C. The aqueous solution was collected over time, and the same amount of saline was replenished. After the test was completed, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and the mixture 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 fluorometry (excitation light: 485 nm, fluorescence: 535 nm). The amount of dextran up to each time point was divided by the total amount of dextran after the test, and the value expressed as a percentage was used as the transmittance.

[0512] The results are shown in Figure 7. The transmittance after 24 hours was in the range of 25% to 40%.

[0513] (Measurement of Gel Permeability (2)) Each of the alginic acid derivatives (Ex4-A2), (Ex5-A2), (Ex6-A2), and (Ex18-A2) obtained in Examples 4, 5, 6, and 18 was dissolved in water to a concentration of 2.0% to prepare an alginic acid aqueous solution. To this alginic acid aqueous solution, 2 / 5 volume of fluorescein isothiocyanate-dextran (Sigma-Aldrich, FD150S) with a molecular weight of 150,000, adjusted to 1 mg / mL, and 3 / 5 volume of water were added to obtain 1.0% alginic acid aqueous solutions (4-2), (5-2), (6-2), and (18-2) containing 0.2 mg / mL fluorescein isothiocyanate-dextran.

[0514] Furthermore, each of the alginic acid derivatives (EX9a-A2), (Ex10-A2), (Ex16-A2), and (Ex20-B2) obtained in Examples 9, 10, 16, and 20 was dissolved in water to a concentration of 1.0% to obtain aqueous alginic acid solutions (9-1), (10-1), (16-1), and (20-1), respectively.

[0515] These were mixed in equal amounts in combinations of (4-2) and (20-1), (5-2) and (10-1), (6-2) and (9-1), (18-2) and (9-1), and (18-2) and (16-1), respectively. 40 mL of 30 mmol / L calcium chloride solution was added and stirred for 5 minutes to obtain alginate gels. The gels were washed once with 10 mL of saline and allowed to stand in saline at 37°C for 10 minutes to undergo chemical crosslinking, yielding fluorescein isothiocyanate-dextran-encapsulated chemically crosslinked alginate. Fluorescein isothiocyanate-dextran-encapsulated chemically crosslinked alginate (Ex18-A2) / (Ex19-A2) was also prepared in the same manner. 19.5 mL of saline was added to the obtained chemically crosslinked alginate, and the mixture was shaken at 37°C. The aqueous solution was collected over time, and the same amount of saline was replenished. After the test was completed, 10 μL of alginate lyase (Nippon Gene, 319-08261) was added to the test solution, and the mixture 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 fluorometry (excitation light: 485 nm, fluorescence: 535 nm). The amount of dextran up to each time point was divided by the total amount of dextran after the test, and the value expressed as a percentage was used as the transmittance.

[0516] The results are shown in Figure 8. The transmittance after 24 hours was in the range of 25% to 30%.

[0517] [Biocompatibility Evaluation of Chemically Crosslinked Alginate] Each of the chemically modified alginate derivatives (EX4-A2), (EX5-A2), (EX12-A2), (EX16-A2), (EX18-A2), (EX19-A2), and (EX20-B2) obtained in Examples 4, 5, 12, 16, 18, 19, and 20 was dissolved in water to prepare a reactive group-introduced alginate solution (alginate aqueous solution may be referred to simply as alginate solution in this specification, and unless otherwise specified, alginate solution refers to an aqueous solution of the corresponding alginic acid, chemically modified alginic acid derivative, or a mixture thereof). This was then sterilized by filtration using a Minisart High Flow (Sartorius, 16532GUK), and a 1.0% reactive group-introduced alginate / physiological saline solution was prepared. Cell concentration: 5 x 10 ３ HeLa cells were seeded onto a 96-well plate at 1000 cells / well and cultured for one day. 1.0% reactive group-introduced alginate / physiological saline solution was added to the cells at a final concentration of 0.1% in combinations of (EX18-A2) and (EX19-A2), (Ex5-A2) and (Ex19-A2), (Ex4-A2) and (Ex20-B2), (Ex18-A2) and (Ex12-A2), or (Ex16-A2). After one day of culture, ATP activity was assessed as an indicator of cytotoxicity using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571).

[0518] The results shown in Figure 9 were obtained. ATP activity was confirmed in all chemically cross-linked alginates evaluated by the above method, suggesting that chemically cross-linked alginates are not cytotoxic and that chemically cross-linked alginates formed by the Huisgen reaction are biocompatible.

[0519] [Preparation of alginate three-layer structures] The structures of the present invention were molded into the shapes of beads, disks, and fibers using the above-mentioned chemically modified alginate derivatives by the method described below, and their strengths were compared with those of structures using natural alginate. Furthermore, it was confirmed that when cells were actually encapsulated as pharmacological ingredients, the cells survived in the structures and were able to exert their functions.

[0520] (Preparation of Chemically Modified Alginic Acid Derivative Solutions) Aqueous solutions of chemically modified alginic acid derivatives used in each of the examples described below were prepared by the following method. In each example in which chemically crosslinked alginic acid was produced by the Huisgen reaction, aqueous solutions of the chemically modified alginic acid derivatives of Examples 18, 19, and 20 (EX20-A2) were prepared. In addition, in the examples in which chemically crosslinked alginic acid was produced by a photocrosslinking reaction, chemically modified alginic acid derivatives (C-IA and C-IB) represented by the following formulas were prepared and their aqueous solutions were prepared according to the method described in PCT / JP2019 / 007655 (filed February 27, 2019). These solutions are abbreviated as the solution of Example 18, the solution of Example 19, the solution of Example 20, the solution of Example C-IA, and the solution of Example C-IB, respectively. [In the above formula, the nitrogen atom on the left side of the dashed line is bonded to the carboxyl group of alginic acid on the right side of the dashed line (manufactured using A-2, manufactured by Mochida Pharmaceutical Co., Ltd.)]

[0521] <Aqueous Solutions Used in Examples A to C> 1 wt % solutions of the chemically modified alginic acid derivatives produced by the methods of Examples 18 to 20, and the above compounds C-IA and C-IB were each prepared in physiological saline.

[0522] <Preparation of Aqueous Solution Used in Example D> A 3 wt % aqueous solution of each of the chemically modified alginic acid derivatives produced by the methods of Examples 18 to 20 was prepared using MilliQ water.

[0523] <Preparation of aqueous solution used in Example E> For each of the chemically modified alginic acid derivatives produced by the methods of Example 18 and Example 20, a 2.6 wt% solution in physiological saline was prepared, and this was sterilized and filtered using a Minisart Highflow (Sartorius, 0.2 μm, Cat. 16532GUK). The salt concentration of the aqueous solution after sterilization by filtration was adjusted to a 2.4 wt% solution in physiological saline. In the following examples, unless otherwise specified, each solution used in each step was prepared using physiological saline.

[0524] Example A: Preparation of Alginate Three-Layer Structure (Beads) This test verified that when the structure of the present invention is molded into beads, larger sizes can be produced compared to conventional structures using natural alginate. <Step 1a> Preparation of Alginate Beads (1 mm Beads) Alginate beads with a diameter of approximately 1 mm were prepared using the following method. A commercially available encapsulation device (Nihon Buchi, Encapsulator B-390) was used to prepare the beads. First, a magnetic stirrer was placed under the nozzle of the encapsulation device, and a crystallizing dish measuring 95 mm in diameter and 55 mm in height was placed on top of it. 100 mL of 100 mmol / L calcium chloride solution and a stir bar were added, and the mixture was stirred at 300 rpm. Next, a 1 wt% aqueous solution of sodium alginate (AL100, manufactured by Mochida Pharmaceutical Co., Ltd. [corresponding to A-2 in Table 13] (sodium alginate solution, occasionally referred to as AL100 solution; the same applies below)) was placed in a pressure bottle. The pressure of the oil-free air compressor was adjusted to 1.5–2 bar, and the air pressure was then set to approximately 700 mbar using the pressure regulation system of the device's main body. While observing the water stream illuminated by a strobe light, the flow rate and oscillation frequency were adjusted until the continuous water stream broke into regularly spaced droplets 3–5 cm below the nozzle and could be observed over several centimeters. The electrostatic dispersion unit was started at 500 V and the voltage was increased in 100 V increments until the capsule spheres dispersed into a conical shape 3–10 cm past the electrode. Once a symmetrical and stable circular dispersion was obtained, the beads were dropped into the calcium chloride solution and stirred for 30 minutes.

[0525] <Step 1b> Preparation of Alginate Beads (5 mm Beads) Alginate beads with a diameter of approximately 5 mm were prepared using the following method. First, various alginate solutions prepared at 1 wt% were loaded into a syringe connected to a 12-gauge needle. 40 mL of 100 mmol / L calcium chloride solution was placed so that the distance from the tip of the needle to the liquid surface was 8 mm, and the solution was stirred at 400 rpm using a magnetic stirrer. The alginate solution was added dropwise using a syringe pump at a flow rate of 1 mL / min for 30 seconds and stirred for 30 minutes. The alginate solutions used to prepare the alginate beads were as follows: AL100 solution A solution obtained by mixing the solution of Example 18 and the solution of Example 19 in a 1:1 (volume ratio) (hereinafter also referred to as the solution of Example 18 / 19) A solution obtained by mixing the solution of Example 18 and the solution of Example 20 in a 1:1 (volume ratio) (hereinafter also referred to as the solution of Example 18 / 20) A solution of Example C-IA

[0526] <Step 1c> PLO ​​Coating 5 mL of a 0.1 wt% poly-L-ornithine (SIGMA-ALDRICH, P2533, MW: 15,000-30,000) solution (hereinafter, unless otherwise specified, the poly-L-ornithine solution was prepared using 100 mmol / L calcium chloride-containing saline solution) was added to a plastic Petri dish containing the alginate beads obtained in step 1a or step 1b, and the dish was left to stand for 10 minutes at 37°C. After removing the solution, 5 mL of a 0.05 wt% poly-L-ornithine solution was added, the dish was left to stand for 6 minutes at 37°C, and the dish was washed twice with 10 mL of saline.

[0527] <Step 1d> Alginic acid coating 5 mL of a 0.15 wt % alginic acid solution was added to the plastic dish containing the PLO-coated beads obtained in step 1c, and the dish was left to stand at 37°C for 10 minutes, followed by washing twice with 10 mL of physiological saline. The alginic acid solution used was the same as in step 1b.

[0528] <Step 1e> Light irradiation For beads using an alginate solution (solution of Example C-I) that undergoes a crosslinking reaction upon light irradiation in steps 1b and 1d, UV irradiation was carried out for 10 minutes using a UV irradiation device (manufactured by Sen Lights corp., HB100A-1) after step 1d was completed.

[0529] <Step 1f> Chelation Treatment and Stability Evaluation 20 mL of 55 mM sodium citrate solution was added to a plastic Petri dish containing beads obtained through each of the above steps. After confirming that the alginate beads derived from the AL100 solution prepared in step 1a or 1b had dissolved rapidly, the alginate beads prepared in step 1d or 1e were placed in a tube containing 20 mL of phosphate buffer and shaken (water bath shaker (TAITEC, personal II) at 180 rpm) at 37°C for 1 day, and their shapes were observed. The observation results are shown in Table 18 below. The stability of the alginate beads was evaluated visually based on the following criteria. For reference, photographs of Example A-6, rated "3," Example A-3, rated "2," and Example A-2, rated "1," are shown in Figure 10, 11(b), and Figure 12(b), respectively. Stability evaluation index: 3: No collapse / dissolution / deformation of beads is observed. 2: Major collapse / dissolution / deformation is observed in some beads. 1: Clear collapse / dissolution / deformation is observed in beads, and the function of the structure is not maintained.

[0530]

[0531] Among beads prepared using AL100 as the core layer, stable three-layer structures were produced for those with a diameter of approximately 1 mm (Example A-1). However, for those with a diameter of approximately 5 mm, the beads maintained their shape at the time of chelation treatment (FIG. 12(a)), but collapsed after shaking (FIG. 12(b), Example A-2). On the other hand, the structures of the present invention (Examples A-5 to A-8) prepared from Huisgen-type chemically modified alginic acid derivatives all maintained their shape after shaking (FIG. 10(b) shows a photograph of Example A-6 after shaking). When a chemically modified alginic acid derivative was used in the anionic polymer layer (Examples A-3 and A-4), the beads were more stable than A-2, but deformation was observed after shaking (FIG. 11(b) shows a photograph of Example A-3 after shaking), indicating insufficient strength. A similar tendency was also observed when a photocrosslinked chemically modified alginic acid derivative was used (Examples A-9 to A-12). Thus, the structure of the present invention exhibited good stability even with beads larger than those used conventionally.

[0532] Example B: Preparation of an Alginate Three-Layer Structure (20 mm Diameter (2 mm Thick) Disk (Sheet)) The structure of the present invention can be molded into any shape using a mold. This test verified that disk (flat) structures can be produced and that their strength is superior to that of conventional structures using natural alginate. <Step 2a> Preparation of Alginate Disks Alginate disks were prepared using the following method. First, a 5 cm square semipermeable membrane (Repligen, Catalog No. 132670) was immersed in a 100 mmol / L calcium chloride solution and allowed to soak for at least 30 minutes. Next, a 20 mm diameter disk-shaped silicone rubber mold was placed on top of the semipermeable membrane whose surface had been wiped clean with a Kimwipe, and 1.3 mL of alginate solution was poured into it. The semipermeable membrane whose surface had been wiped clean was then placed on top, and the semipermeable membrane and silicone rubber mold were secured in place using clips. The alginate disk was then immersed overnight in a 100 mmol / L calcium chloride solution, and then removed from the mold. The alginate solution used was the same as in step 1b.

[0533] <Step 2b> PLO ​​Coating 10 mL of a 0.1 wt% poly-L-ornithine solution was added to a 60 mm plastic dish containing the alginate disk obtained in step 2a, and the dish was left to stand for 10 minutes at 37° C. After the solution was removed, 10 mL of a 0.05 wt% poly-L-ornithine solution was added, the dish was left to stand for 6 minutes at 37° C., and the dish was washed twice with 20 mL of physiological saline.

[0534] <Step 2c> Alginic acid coating 10 mL of a 0.15 wt % alginic acid solution was added to the plastic dish containing the PLO-coated disk obtained in step 2b, and the dish was left to stand at 37°C for 10 minutes, followed by washing twice with 20 mL of physiological saline. The alginic acid solution used was the same as in step 1b in Examples B-9 to B-11, except that the solution of Example C-IB was used.

[0535] <Step 2d> Light irradiation For disks using an alginic acid solution (solution of Example C-IB) that undergoes a crosslinking reaction upon light irradiation in steps 2a and 2c, UV irradiation was performed for 10 minutes using a UV irradiation device (HB100A-1, manufactured by Sen Lights corp.) after step 2c was completed.

[0536] <Step 2e> Chelate Stability Evaluation: 40 mL of 55 mM sodium citrate solution was added to the plastic dish containing the disks obtained through each of the above steps. Once it was confirmed that the alginate disks derived from the AL100 solution prepared in step 2a had dissolved, the shape of the remaining disks was observed. The observation results are shown in Table 19 below. The stability of the alginate disks was evaluated visually based on the same indicators as for the stability of alginate beads.

[0537]

[0538] As a result, when a Huisgen-type chemically modified alginic acid derivative was used as the core layer (Examples B-4 to B-7), or when a photocrosslinked chemically modified alginic acid derivative was used (Examples B-10 and B-11), a three-layer structure as stable as that of beads could be produced. In contrast, when AL100 was used as the core layer, or when chemically crosslinked alginic acid was used only in the anionic polymer layer (Examples B-1 to B-3, Examples B8 and B9), the shape of the structure was not maintained at the time of chelation treatment. Thus, the structures of the present invention showed remarkable stability even when molded into a disk shape.

[0539] Example C: Preparation of Alginate Three-Layer Structure (50 mm Diameter (2 mm Thick) Disk (Sheet)) This test verified that when a disk-shaped structure was prepared using the same method as in Example B, a larger diameter structure could be prepared. <Step 3a> Preparation of Alginate Disks Alginate disks were prepared using the following method. First, a 5 cm square semipermeable membrane (Repligen, Catalog No. 132670) was immersed in a 100 mmol / L calcium chloride solution and allowed to soak for at least 30 minutes. A 50 mm diameter disk-shaped silicone rubber mold was then placed on top of the semipermeable membrane, the surface of which had been wiped dry with a Kimwipe. 5.65 mL of alginate solution was poured into the mold, and the semipermeable membrane, the surface of which had been wiped dry, was placed on top. The semipermeable membrane and silicone rubber mold were then secured in place using clips. After overnight immersion in a 100 mmol / L calcium chloride solution, the alginate disk was removed from the mold. The alginate solution used was the same as in step 1b.

[0540] <Step 3b> PLO ​​Coating 20 mL of a 0.1 wt% poly-L-ornithine solution was added to a 100 mm plastic dish containing the alginate disk obtained in step 3a, and the dish was left to stand for 10 minutes at 37°C. After the solution was removed, 20 mL of a 0.05 wt% poly-L-ornithine solution was added, the dish was left to stand for 6 minutes at 37°C, and the dish was washed twice with 40 mL of physiological saline.

[0541] <Step 3c> Alginic acid coating: 20 mL of a 0.15 wt% alginic acid solution was added to the plastic dish containing the PLO-coated disk obtained in step 3b, and the dish was left to stand at 37°C for 10 minutes. The alginic acid solutions used were the AL100 solution, the solutions of Examples 18 / 19, and 18 / 20.

[0542] <Step 3d> Chelate Stability Evaluation 200 mL of 55 mM sodium citrate solution was added to the plastic dish containing the disks obtained through each of the above steps. Once it was confirmed that the alginate disks derived from the AL100 solution prepared in step 3a had dissolved, the shape of the remaining disks was observed. The observation results are shown in Table 20 below. The stability of the alginate disks was evaluated visually based on the same indicators as for the stability of alginate beads.

[0543] As a result, when a Huisgen-type chemically modified alginic acid derivative was used for the core layer (Examples C-2 and C-3), a stable three-layer structure could be produced, whereas when AL100 was used for the core layer (Example C-1), the structure could not maintain its shape at the time of chelate treatment. Thus, the structure of the present invention showed remarkable stability even when molded into a disk shape with a diameter of 50 mm.

[0544] Example D: Preparation of Three-Layer Alginate Structure (Fiber) This test demonstrated that the structure of the present invention can be molded into a fiber shape and that its strength i...

Claims

1. A structure comprising a core layer containing a pharmacological ingredient embedded in chemically crosslinked alginate, a cationic polymer layer coating said core layer, and an anionic polymer layer coating said cationic polymer layer.

2. The structure according to claim 1, wherein the chemically crosslinked alginic acid is obtained by forming a crosslink between a chemically modified alginic acid derivative represented by formula (H-I) and a chemically modified alginic acid derivative represented by formula (H-II): [Chemically modified alginic acid derivative represented by formula (HI)] The following formula (H-I): 【Chemistry 178】 [In formula (HI), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L １ - is a divalent linker that bonds to a cyclic alkyne group (Akn); [Chemically modified alginic acid derivative represented by formula (H-II)] The following formula (H-II): 【Chemistry 179】 [In formula (H-II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L ２ - is a divalent linker that bonds to the azide group.

3. In formula (HI), -L １ - is - (CH ２ ) ｎ１ -(wherein n1=1 to 50, and -CH ２ - may be replaced by 1 to 10 groups selected from -CO-, -CONH-, -NHCO-, -O-CONH-, -NHCO-O-, -O- and -NH-, or a benzene ring, ２ The hydrogen atom of - is C １－3 C substituted with an alkyl group or a phenyl group １－3 which may be substituted by an alkyl group; Akn is an 8-membered cyclic alkyne group (wherein the cyclic alkyne group may further have one or two benzene rings, cyclopropane rings, or 1,2,3-triazole rings condensed thereto, and in the condensed ring, -L １ -, and -CH in the 8-membered cyclic alkyne group. ２ - may be an 8-membered ring group substituted with 1 to 2 groups selected from -C(=O)-, -CONH-, and -NH-, and -CH ２ The hydrogen atom of - is C １－３ Alkyl group, fluorine atom, hydroxyl group, or C １－３ alkyloxy groups, In formula (H-II), -L ２ -ga- (CH ２ ) ｎ２ - (wherein n2=1 to 50, and -CH ２ - may be replaced by 1 to 10 groups selected from -CONH-, -NHCO-, -O- and -NH-, or a benzene ring or a pyridine ring, ２ The hydrogen atom of - is C １－3 3. The structure of claim 2, wherein the aryl group is substituted with an alkyl group.

4. In formula (HI), -L １ - is - (CH ２ ) ｎ１ -(wherein n1=2 to 15, and -CH ２ - may be replaced by 1 to 5 groups selected from -CO-, -CONH-, -NHCO-, -O-CONH-, -NHCO-O-, -O- and -NH-, or a benzene ring, ２ The hydrogen atom of - is C １－3 C substituted with an alkyl group or a phenyl group １－3 which may be substituted by an alkyl group; Akn is an 8-membered cyclic alkyne group (wherein the cyclic alkyne group may further have one or two benzene rings condensed thereto, and the —CH ２ - may be replaced by -NH-, In formula (H-II), -L ２ -ga- (CH ２ ) ｎ２ -(wherein n2=2 to 15, and -CH ２ The structure according to claim 3, wherein - may be replaced by 1 to 5 groups selected from -CONH-, -NHCO-, -O- and -NH-, or a benzene ring.

5. The chemically modified alginic acid derivative represented by formula (H-I) is represented by the following formula (A01) or (A02), and the chemically modified alginic acid derivative represented by formula (H-II) is represented by the following formula (N01), (N02), (N03) or (N04). The structure according to claim 4. 【Chemistry 180】 [In each formula, (ALG) represents alginic acid; -NHCO- bonded to (ALG) represents an amide bond via any carboxyl group of alginic acid] 6. The structure according to claim 1, wherein the anionic polymer forming the anionic polymer layer is alginic acid or chemically crosslinked alginic acid.

7. The structure according to any one of claims 1 to 6, wherein the pharmacological component embedded in the chemically crosslinked alginate of the core layer is a cell.

8. The structure according to any one of claims 1 to 6, wherein the pharmacological component embedded in the chemically crosslinked alginate of the core layer is a biologically active substance-producing cell.

9. The structure according to claim 1, wherein the cationic polymer forming the cationic polymer layer is poly-L-ornithine or poly-L-lysine.

10. The structure according to any one of claims 1 to 9, wherein the structure has a shape of a bead, a sheet or a fiber.

11. A structure according to any one of claims 1 to 10 for use in a living body.

12. A medical material comprising, as an active ingredient, a pharmacological ingredient encapsulated in the structure according to any one of claims 1 to 10.

13. 13. The medical material according to claim 12, which is used as a wound dressing material, a postoperative adhesion barrier material, a substrate for sustained drug release, a substrate for cell transplantation, a prosthetic material, a pharmaceutical coating material, or a bioprinter.

14. A method for producing the structure according to any one of claims 1 to 11, comprising the following steps (a) to (c): Step (a): A step of gelling a solution of a chemically modified alginic acid derivative containing a pharmacological ingredient [a mixture of a derivative represented by formula (H-I) and a derivative represented by formula (H-II)] by contacting the solution with a divalent metal ion; Step (b): contacting the gel obtained in step (a) with a solution containing a cationic polymer to coat the gel with the cationic polymer; Step (c): A step of contacting the product obtained in step (b) with a solution containing an anionic polymer to further coat it with the anionic polymer.

15. A method for producing the structure according to claim 14, further comprising the step (d) of: Step (d): A step of subjecting the structure obtained in step (c) to a chelating treatment using a chelating agent.

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