Novel polyrotaxane

Abstract

To provide a novel polyrotaxane. [Solution] A polyrotaxane or salt thereof comprising an axial molecule having an axial portion and two stopper portions bonded to both ends of the axial portion, and at least one cyclic molecule penetrated by the axial molecule, wherein the cyclic molecule has at least one hydroxyl group where the hydrogen atom is represented by the following formula (I) [wherein L represents a divalent organic group, R 1 and R 2 Each of these independently represents a monovalent organic group. A polyrotaxane or a salt thereof is a cyclodextrin that is substituted with a group represented by ] and which may have at least one other hydroxyl group further modified or substituted. TIFF2026131209000029.tif22149

Description

[Technical Field]

[0001] This disclosure relates to novel polyrotaxanes. Furthermore, this disclosure relates to a method for producing novel polyrotaxanes, and to a coating agent for cell culture substrates containing the novel polyrotaxanes. [Background technology]

[0002] Polyrotaxanes are supramolecules comprising at least one cyclic molecule and an axial molecule that penetrates it. The axial molecule has bulky structures (stopper regions) at both ends of a linear polymer, such as a polymer. Because the cyclic molecule cannot penetrate the stopper regions, the state in which the axial molecule penetrates the cyclic molecule is maintained.

[0003] Polyrotaxanes are expected to have various applications due to their characteristic that cyclic molecules can move while penetrating the axial molecule. Regarding polyrotaxanes and their applications, for example, Patent Document 1 describes a polyrotaxane in which at least one hydroxyl group is C 1-6 A coating agent containing an alkyl group-modified polyrotaxane has been disclosed. Patent Document 2 discloses a polyrotaxane having an amino group and a coating agent containing the same. Patent Document 3 discloses a polyrotaxane having a hydroxyl group or a sulfo group and a coating agent containing the same. Patent Documents 1 to 3 also disclose the relationship between surface modification of culture substrates by these polyrotaxane-containing coating agents and cell differentiation. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2022 / 102663 [Patent Document 2] International Publication No. 2022 / 113940 [Patent Document 3] International Publication No. 2022 / 113944 [Overview of the project] [Problems that the invention aims to solve]

[0005] Polyrotaxanes are expected to have diverse applications due to their unique properties, and therefore, there is a need for further expansion of their variations.

[0006] This disclosure aims to provide novel polyrotaxanes. Furthermore, this disclosure aims to provide a method for producing novel polyrotaxanes and a coating agent for cell culture substrates containing the novel polyrotaxanes. [Means for solving the problem]

[0007] The inventors have developed a novel polyrotaxane in which the hydroxyl group of cyclodextrin is modified via an organic group with a group containing a phosphorus atom, as a cyclic molecule.

[0008] This disclosure relates, for example, to the following: [1] An axis molecule having an axis portion and two stopper portions connected to both ends of the axis portion, At least one cyclic molecule penetrated by the aforementioned axial molecule, A polyrotaxane or a salt thereof comprising, The aforementioned cyclic molecule has a hydrogen atom in at least one hydroxyl group that is in the following formula (I): [ka] [In the formula, L represents a divalent organic group, R 1 and R 2 Each of these independently represents a monovalent organic group. A polyrotaxane or a salt thereof, which is a cyclodextrin substituted with a group represented by and which may have at least one other hydroxyl group further modified or substituted. [2] The polyrotaxane or salt thereof according to [1], wherein L in formula (I) represents an alkylene having 2 to 10 carbon atoms. [3] R in formula (I) above 1 and R2 The polyrotaxane or its salt according to [1] or [2], wherein each independently represents a hydrogen atom or an alkyl group. [4] R in the formula (I) 1 and R 2 The polyrotaxane or its salt according to any one of [1] to [3], wherein each represents a hydrogen atom or each represents an alkyl group having 1 to 5 carbon atoms. [5] R in the formula (I) 1 and R 2 The polyrotaxane or its salt according to any one of [1] to [4], wherein each represents a hydrogen atom. [6] The polyrotaxane or its salt according to any one of [1] to [5], wherein the cyclodextrin is α-cyclodextrin or β-cyclodextrin. [7] The polyrotaxane or its salt according to any one of [1] to [6], wherein the average number of hydroxyl groups substituted with the group represented by the formula (I) per molecule of the cyclic molecule is 2.5 or more. [8] The polyrotaxane or its salt according to any one of [1] to [7], wherein the axial site contains polyethylene glycol or a copolymer containing polyethylene glycol and polypropylene glycol. [9] The stopper site is represented by the following formula (II):

Chemical formula

[10] A method for producing polyrotaxane A or a salt thereof, comprising the step of contacting polyrotaxane B or a salt thereof with a base in an aqueous medium to convert it into polyrotaxane A or a salt thereof, The aforementioned polyrotaxane A is An axial molecule having an axial portion and two stopper portions connected to both ends of the axial portion, At least one cyclic molecule penetrated by the aforementioned axial molecule, Equipped with, The aforementioned cyclic molecule has a hydrogen atom in at least one hydroxyl group that is in the following formula (I): [ka] [In the formula, L represents a divalent organic group, R 1 and R 2 Each of these independently represents a hydrogen atom. A cyclodextrin which is substituted with a group represented by and which may have at least one other hydroxyl group further modified or substituted, It is a polyrotaxane, The polyrotaxane B is the same as the polyrotaxane A, in which the R in formula (I) 1 and R 2 However, the manufacturing method is for a polyrotaxane in which each hydrogen atom represents an alkyl group.

[11] The manufacturing method according to

[10] , wherein L in formula (I) is an alkylene having 2 to 10 carbon atoms.

[12] The manufacturing method according to

[10] or

[11] , wherein L in formula (I) is an alkylene having 3 or more carbon atoms and 5 or fewer carbon atoms.

[13] The polyrotaxane B is the same as the polyrotaxane A, in which the R in formula (I) 1 and R 2 The manufacturing method according to any one of

[10] to

[12] , wherein the polyrotaxane is characterized by each atom having an alkyl group with 1 to 5 carbon atoms instead of a hydrogen atom.

[14] The method for producing a product according to any one of

[10] to

[13] , wherein the cyclodextrin is α-cyclodextrin or β-cyclodextrin.

[15] The manufacturing method according to any one of

[10] to

[14] , wherein the aqueous medium is water or a mixed solvent of water and dimethyl sulfoxide.

[16] The manufacturing method according to any one of

[10] to

[15] , wherein the polyrotaxane B or a salt thereof is brought into contact with the base at a temperature of 35°C to 80°C.

[17] Polyrotaxane C or a salt thereof is dissolved in an organic solvent in the following formula (III): [ka] [In the formula, L, R 1 and R 2 This is similar to that in polyrotaxane B, where X represents a monovalent leaving group. The process further includes contacting a compound represented by and a base to convert it into polyrotaxane B or a salt thereof, The method for producing polyrotaxane C according to any one of

[10] to

[16] , wherein the polyrotaxane C is a polyrotaxane in which at least one hydrogen atom in the hydroxyl group is not substituted with a group represented by formula (I) in the polyrotaxane A.

[18] The manufacturing method according to

[17] , wherein X in formula (III) is Cl, Br, or I.

[19] The method for producing the organic solvent according to

[17] or

[18] , wherein the organic solvent is dimethyl sulfoxide. The manufacturing method according to claim 10, which is a method for producing a polyrotaxane or a salt thereof as described in any one of

[20] [1] to [9]. A coating agent for cell culture substrates, comprising a polyrotaxane or a salt thereof as described in any one of

[21] [1] to [9].

[22] The coating agent described in

[21] for promoting osteoblast differentiation of mesenchymal stem cells.

[23] A cell culture method for promoting osteoblast differentiation of mesenchymal stem cells, A method comprising culturing mesenchymal stem cells on the surface of a substrate coated with a composition containing a polyrotaxane or a salt thereof as described in any one of [1] to [9]. A cell culture substrate comprising a substrate coated with a composition containing a polyrotaxane or a salt thereof as described in any one of

[24] [1] to [9]. A composition containing a polyrotaxane or a salt thereof as described in any one of

[25] [1] to [9].

[26] The composition according to

[25] , which is a pharmaceutical composition.

[27] The composition according to

[25] or

[26] , which is a pharmaceutical composition for treating and / or preventing diseases caused by the accumulation of cholesterol in cells.

[28] A pharmaceutical composition for treating and / or preventing Niemann-Pick disease type C, metabolic disorder-related steatohepatitis, Alzheimer's disease, focal segmental glomerulosclerosis, age-related macular degeneration, arteriosclerosis, or diabetic kidney disease, according to any one of

[25] to

[27] .

[29] The composition according to any one of

[25] to

[28] , wherein the stopper portion comprises trityl which may have substituents. [Effects of the Invention]

[0009] This disclosure provides novel polyrotaxanes. The inventors have succeeded in developing a novel polyrotaxane, which has not been previously reported, in which the hydroxyl group of a cyclodextrin is modified via an organic group with a group containing a phosphorus atom as a cyclic molecule.

[0010] This disclosure provides a method for producing a newly developed polyrotaxane. According to one aspect of this disclosure, the production method allows for the production of the target polyrotaxane in high yield, even when using raw materials in which the supramolecular structure of the polyrotaxane has already been formed.

[0011] This disclosure provides a coating agent containing a newly developed polyrotaxane. The inventors have found that using a culture substrate whose surface has been treated with the coating agent according to this disclosure can efficiently induce osteoblast differentiation of cells. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows the results of gel permeation chromatography (GPC) measurements performed on PO3Et2-PRX obtained in Example 1, the raw material unmodified polyrotaxane, and the raw material α-CD. [Figure 2] This figure shows the results of 1H nuclear magnetic resonance (NMR) measurements performed on PO3Et2-PRX obtained in Example 1 and the unmodified polyrotaxane used as the raw material. [Figure 3] This figure shows the results of 31P NMR measurements performed on PO3Et2-PRX obtained in Example 1, the unmodified polyrotaxane used as a starting material, and the compound obtained in Example 12. [Figure 4] This figure shows the results of 1H NMR measurements performed on PO3Et2-PRX obtained in Example 1, the compound obtained in Comparative Example 1, and the compound obtained in Comparative Example 6. [Figure 5] This figure shows the results of 1H NMR measurements performed on the compounds obtained in Examples 10, 11, 12, and 15. [Figure 6] This figure shows the results of 1H NMR measurements performed on PO3Et2-PRX-VB, PO3Na2-PRX-VB, and the raw material PRX-VB obtained in Example 18. [Figure 7] This figure shows the results of 1H NMR measurements performed on PO3Et2-PRX-b-PBnMA, PO3Na2-PRX-b-PBnMA, and the unmodified PRX-b-PBnMA used as a starting material, obtained in Example 19. [Figure 8] This figure shows the results of 1H NMR measurements performed on PO3Et2-βCD-PRX, PO3Na2-βCD-PRX, and the unmodified βCD-PRX used as a starting material, obtained in Example 20. [Modes for carrying out the invention]

[0013] The following describes the forms for implementing this disclosure, but this disclosure should not be construed as being limited to the following embodiments.

[0014] <Definition> C X Alkyl, C X Alkenil, C X Alkylene (where X is an integer greater than or equal to 1) refers to an alkyl, alkenyl, or alkylene molecule with X carbon atoms. C X-Y Alkyl, C X-Y Alkenil, C X-Y Alkylene (where X and Y are integers greater than or equal to 1) refers to an alkyl, alkenyl, or alkylene compound with X to Y carbon atoms.

[0015] Alkyl refers to a group formed by removing one hydrogen atom from an alkane. Alkyl groups may be linear or cyclic, and in one embodiment may be linear. Furthermore, linear alkyl groups may be linear or branched, and in one embodiment may be linear. For example, alkyl groups are C 1-10 Alkyl, C 1-6 Alkyl, C 1-4 Alkyl, C 1-3 Alkyl, C 1-2 The C1 alkyl group may be alkyl or C1 alkyl. The C1 alkyl group is methyl. The C2 alkyl group is ethyl. The C3 alkyl group is n-propyl, isopropyl, or cyclopropyl, and in one embodiment may be n-propyl or isopropyl. The C4 alkyl group may be, for example, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, or cyclopropylmethyl, and in one embodiment may be n-butyl, isobutyl, sec-butyl, or tert-butyl.

[0016] An alkenyl is a group in an alkane in which one of the carbon-carbon single bonds is replaced with a carbon-carbon double bond, and one hydrogen atom is removed from each carbon atom forming the double bond. Alkenyls may be linear or cyclic, and in one embodiment they may be linear. Furthermore, linear alkenyls may be linear or branched, and in one embodiment they may be linear. For example, an alkenyl is C 2-10 Alkenil, C 2-6 Alkenil, C 2-4 Alkenyl or C 2-3 It may be an alkenyl. A C2 alkenyl is vinyl. A C3 alkenyl may be, for example, an allyl.

[0017] Alkylene refers to a group formed by removing two hydrogen atoms from an alkane. Alkylenes may be linear or cyclic, and in one embodiment they may be linear. Furthermore, linear alkylenes may be linear or branched, and in one preferred embodiment they may be linear. Alkylenes include, for example, C 1-10 Alkylene, C 1-6 Alkylene, C 1-4 Alkylene, C 1-3 Alkylene, C 1-2 It may be alkylene or C1 alkylene. X-Y Alkylenes are, for example, the C mentioned above. X-Y The group may also be an alkyl group from which one more hydrogen atom has been removed. In one embodiment, the alkylene is a linear C 2-5 It may be an alkylene, and in another embodiment, the alkylene may be ethylene or propylene.

[0018] In this disclosure, a “may have substituents” group may have one, two, three, four, or five or more substituents. In this disclosure, substituents in a “may have substituents” group may be, for example, alkyl, alkenyl, alkoxy, phenyl, benzyl, amino, azide, carboxy, alkylcarbonyl, alkoxycarbonyl, nitro, hydroxy, halogeno, mercapto, alkylthio, sulfo, phosphate, or phosphoryl. An alkoxy is a group consisting of a monoalkylated oxygen atom. An amino may be a primary, secondary, tertiary, or quaternary amino, and in one embodiment may be a primary amino. A halogeno is, for example, fluoro(F), chloro(Cl), bromo(Br), or iodo(I). An alkylthio is a group consisting of a monoalkylated sulfur atom. In a preferred embodiment, a “may have substituents” group may be unsubstituted.

[0019] For the purposes of this disclosure, molar equivalent refers to the equivalent relationship between the amounts of substance (moles) of multiple substances. For example, "adding 10 molar equivalents of substance B to 1 molecule of substance A" or "adding 10 molar equivalents of substance B to 1 molar equivalent of substance A" means adding substance B in an amount (number of moles) that is 10 times that of substance A.

[0020] <Polyrotaxane> A first embodiment of this disclosure relates to a polyrotaxane comprising an axial molecule having an axial portion and two stopper portions bonded to both ends of the axial portion, and at least one cyclic molecule penetrated by the axial molecule. Hereafter, this embodiment will also be referred to as the polyrotaxane of the first embodiment. In this disclosure, a polyrotaxane means a supramolecular molecule, such as a rotaxane or a derivative thereof, in which there may be two or more cyclic molecules penetrated by the axial molecule. In the polyrotaxane of the first embodiment, since the cyclic molecule cannot penetrate the stopper portion, a supramolecular structure in which the axial molecule penetrates the cyclic molecule is maintained.

[0021] The polyrotaxane of this embodiment may be in the form of the supramolecular itself (free form) or in the form of a salt. The salt of the polyrotaxane may be, for example, a pharmaceutically acceptable salt or a pharmaceutically acceptable salt. The salt of the polyrotaxane may be formed by, for example, a cyclic molecule, by an axial molecule, or by both a cyclic molecule and an axial molecule forming salts. An example of a polyrotaxane salt is R, which will be described later. 1 and / or R 2 In cases where the cyclic molecule exhibits a hydrogen atom, it may form a salt with a monovalent cation instead of the hydrogen atom. The countercation in the polyrotaxane salt of this embodiment is not particularly limited, but for example, a sodium ion (Na) + ), potassium ions (K + ), calcium ions (Ca 2+ ), magnesium ions (Mg 2+ ), aluminum ions (Al 3+ ), ammonium ion (NH4 + ) or lithium ions (Li + ) may be. The counteranion in the polyrotaxane salt of this embodiment is not particularly limited, but for example, chloride ions (Cl - ), sulfate ions (SO4 2- ), nitrate ion (NO3 - ), carbonate ions (CO3 2- ), bicarbonate ions (HCO3) - ), acetate ion (CH3COO - ), fluoride ions (F - ), bromide ions (Br - ), iodide ion (I - ), phosphate ion (PO4 3- ), hydrogen phosphate ion (HPO4 2- ) or hydroxide ion (OH - ) is acceptable.

[0022] In this disclosure, unless otherwise specified, polyrotaxanes or salts thereof may also be in the form of solvates thereof. The solvates are not particularly limited, but may include, for example, hydrates or dimethyl sulfoxides.

[0023] In the polyrotaxane of this embodiment, the number of cyclic molecules penetrated by the axial molecule may be, for example, 2 or more, 5 or more, 10 or more, 20 or more, 40 or more, 50 or more, 60 or more, or 70 or more, and may also be 300 or less, 200 or less, 150 or less, 120 or less, 100 or less, or 90 or less, and these upper and lower limits can be freely combined. Furthermore, when the number of cyclic molecules penetrated by the axial molecule is 2 or more, the structures of each cyclic molecule in the polyrotaxane of this embodiment may be identical or different from each other.

[0024] The polyrotaxane of this embodiment can be obtained, for example, as the final product or synthetic intermediate of the manufacturing method of the second embodiment described later.

[0025] <Cyclic molecule> The cyclic molecule of the polyrotaxane in this embodiment is a cyclodextrin in which at least one hydrogen atom in one hydroxyl group is substituted with a group represented by formula (I) described later (hereinafter also referred to as the formula (I) group), and at least one other hydroxyl group may be further modified or substituted.

[0026] Cyclodextrins are monocyclic oligosaccharides formed by the α-1,4-glucosidic linkage of multiple D-glucose molecules. The number of D-glucose monomers forming the cyclodextrin according to this disclosure may be, for example, 5 or more, 6 or more, 10 or less, 9 or less, 8 or less, or 7 or less, and these upper and lower limits can be freely combined. A cyclodextrin consisting of 6 D-glucose monomers is known as α-cyclodextrin (hereinafter also referred to as "α-CD"). A cyclodextrin consisting of 7 D-glucose monomers is known as β-cyclodextrin (hereinafter also referred to as "β-CD"). A cyclodextrin consisting of 8 D-glucose monomers is known as γ-cyclodextrin (hereinafter also referred to as "γ-CD"). The cyclodextrin relating to this disclosure may be α-CD, β-CD, or γ-CD in one embodiment, α-CD or β-CD in one preferred embodiment, or α-CD in another preferred embodiment.

[0027] In the cyclic molecule according to this embodiment, at least one hydrogen atom in a hydroxyl group is substituted with a group of formula (I). The group of formula (I) is represented by the following formula (I). The substitution of a hydrogen atom in a hydroxyl group with a group of formula (I) means that of the two bonds of the oxygen atom contained in the hydroxyl group, one forms a single bond with the base carbon atom located on the cyclodextrin skeleton side, and the other forms a single bond with L in the group of formula (I) instead of the hydrogen atom. [ka]

[0028] In formula (I), L represents a divalent organic group. Examples of organic groups include alkylene groups having 1 to 10 carbon atoms, alkenylene groups having 1 to 20 carbon atoms, and alkylylene groups having 1 to 20 carbon atoms. These groups may have oxo groups at any position, or they may be connected via oxy or imino groups, or combinations thereof. For example, combining a carbon atom with an oxo group with an oxy group forms an ester bond, and combining a carbon atom with an oxo group with an imino group forms an amide bond. Alkylene groups having multiple oxy groups are also called polyoxyalkylene groups, and include, for example, polyoxyethylene groups having 1 to 10 carbon atoms and polyoxypropylene groups having 1 to 10 carbon atoms. Specific examples of organic groups include alkylene groups such as methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonanylene, and decylene; alkenylene groups such as propynylene, butenylene, pentenylene, hexenylene, heptenylene, octenylene, nonenylene, and decenylene; and alkylylene groups such as propargyl, butynylene, pentynylene, hexynylene, heptynylene, octynylene, nonylene, and decynylene. For organic groups having an oxo, oxy, or imino group, it is preferable that they form an ester bond, a carbonate ester bond, or a urethane bond with an oxygen atom derived from the hydroxyl group of cyclodextrin. Examples of organic groups having an oxo group, an oxy group, or an imino group include carbonylalkylene groups such as carbonylmethylene group (-C(=O)CH2-); alkylene carbonyl groups such as methylenecarbonyl group (-CH2C(=O)-); alkylene carbonylamino groups such as methylenecarbonylamino group (-CH2C(=O)NH-); carbonylaminoalkylene groups such as carbonylaminomethylene group (-C(=O)NHCH2-) and carbonylaminoethylene group (-C(=O)NHCH2CH2-); carbonylaminoalkylene oxyalkylene groups such as carbonylaminoethyleneoxyethylene group (-C(=O)NHCH2CH2OCH2CH2-); and carbonylaminoethylene poly(oxyethylene) group (-C(=O)NHCH2CH2(OCH2CH2)). tExamples include carbonylaminoethylene poly(oxyalkylene) groups such as (-) (where t is between 2 and 1000 in the formula) and oxyalkylene groups such as the oxypropylene group (-OCH2CH2CH2-).

[0029] In a preferred embodiment, L is, for example, C 2-10 It can be alkylene. 2-10 Being an alkylene allows for easier synthesis and handling of polyrotaxanes while maintaining the desired functionality. For similar reasons, in one preferred embodiment, L may be, for example, C 2-6 Alkylene, C 2-4 Alkylene or C 2-3 It may be an alkylene. Also, for similar reasons, L in one preferred embodiment may be, for example, n-ethylene, n-propylene, n-butylene, n-pentylene or n-hexylene, and may be n-ethylene, n-propylene or n-butylene, or n-ethylene or n-propylene.

[0030] In formula (I), R 1 and R 2 Each independently represents a monovalent organic group. The monovalent organic group may be, for example, a hydrogen atom, or an alkyl, alkenyl, benzyl, or phenyl group which may have substituents. In one embodiment, the monovalent organic group may be a hydrogen atom, an alkyl, an alkenyl, or a benzyl group. In another embodiment, the monovalent organic group may be a hydrogen atom, or an alkyl group which may have substituents. In a preferred embodiment, the monovalent organic group may be a hydrogen atom or an alkyl group.

[0031] A preferred direct route, R 1 and R 2 Each of these may independently represent a hydrogen atom or an alkyl group. In this disclosure, R 1 and R 2 The alkyl in this context is, for example, C 1-10 Alkyl, C 1-5 Alkyl, C 1-3 Alkyl or C 2-3 Alkyl is also acceptable. In a more preferred embodiment, R 1 and R 2 each represent a hydrogen atom or each may represent an alkyl of C 1-5 . In this case, it can be said that the polyrotaxane in which R 1 and R 2 each represent an alkyl of C 1-5 is a compound in which the phosphoryl group in the polyrotaxane in which R 1 and R 2 each represent a hydrogen atom is protected with an alkyl. In the most preferred embodiment, R 1 and R 2 may represent a hydrogen atom. The group of formula (I) in this case is a group represented by the following formula (IV). In formula (IV), L is the same as that described in formula (I). [Chemical formula]

[0032] In the polyrotaxane according to the present disclosure, when R 1 and / or R 2 represents a hydrogen atom, the cyclic molecule may form a salt with a monovalent cation instead of the hydrogen atom. That is, in these embodiments, when R 1 and / or R 2 represents a hydrogen atom, it includes the case where the hydrogen atom is ionized and a monovalent cation is added instead (that is, R 1 and / or R 2 is a monovalent cation). The monovalent cation in this case may be, for example, a sodium ion or a potassium ion.

[0033] Cyclodextrin has hydroxyl groups equal to the number of primary hydroxyls and twice the number of secondary hydroxyls as the number of D-glucose monomers, but the hydroxyl groups substituted with the (I) group may be primary hydroxyls and / or secondary hydroxyls. In one embodiment, all primary hydroxyls may be substituted and then secondary hydroxyls may also be substituted, while in another embodiment, only primary hydroxyls may be substituted, for example, only primary hydroxyls may be substituted.

[0034] In the cyclic molecule according to this embodiment, the average number of hydroxyl groups per cyclic molecule in which hydrogen atoms are substituted with a group of formula (I) may be, for example, 0.05 or more, 0.09 or more, 0.5 or more, 1.0 or more, 2.0 or more, 2.5 or more, 3.0 or more, 3.5 or more, 4.0 or more, 5.0 or more, 6.0 or more, 7.0 or more, 8.0 or more, or 10.0 or more, and may also be 20.0 or less, 15.0 or less, 12.0 or less, 9.0 or less, 8.0 or less, 7.0 or less, or 6.0 or less, and these upper and lower limits can be freely combined. The polyrotaxane of this embodiment can also be produced by introducing a group of formula (I) after the polyrotaxane skeleton has been formed, as will be described later in the production method of the second embodiment. Therefore, the polyrotaxane of this embodiment may be a polyrotaxane with a large number of substitutions in the cyclic molecule, which would be difficult to achieve by methods that first introduce substituents to the cyclic molecule and then form a supramolecule. For example, the average number of hydroxyl groups substituted with the (I) group per hydrogen atom per cyclic molecule may be 2.5 or more, 6.0 or more, or 7.0 or more. Furthermore, if the upper limit of the average number of hydroxyl groups is less than or equal to the number of D-glucose monomers that form cyclodextrin, it is possible to obtain a polyrotaxane with substitutions where all or most of the relatively reactive primary hydroxyls are present, making it easy to produce in high yield. The average number of hydroxyl groups substituted with the (I) group per hydrogen atom per cyclic molecule can be determined, for example, using nuclear magnetic resonance spectroscopy, and more specifically, 1 This can be determined based on the ratio of the peak area of ​​the proton at position 1 of the glucose monomer in the cyclic molecule to the peak area of ​​the proton in the (I) group in 1H-NMR.

[0035] In the polyrotaxane of this embodiment, the total number of hydroxyl groups substituted with formula (I) groups may be, for example, 5.0 or more, 10.0 or more, 30.0 or more, 60.0 or more, 100 or more, 200 or more, 300 or more, or 400 or more, and may also be 2000 or less, 1500 or less, 1200 or less, 1000 or less, 800 or less, 600 or less, or 500 or less, and these upper and lower limits can be freely combined.

[0036] The cyclic molecule of the polyrotaxane of this embodiment may have at least one other hydroxyl group that is not substituted with the group of formula (I) further modified or substituted. In this case, the number of such further modified or substituted hydroxyl groups (hereinafter also referred to as "additional modified / substituted hydroxyl groups") may be, on average per cyclic molecule, 0.05 or more, 0.09 or more, 0.5 or more, 1.0 or more, 2.0 or more, 2.5 or more, 3.0 or more, 3.5 or more, 4.0 or more, 5.0 or more, 6.0 or more, 7.0 or more, 8.0 or more, or 10.0 or more, and may also be 20.0 or less, 15.0 or less, 12.0 or less, 9.0 or less, 8.0 or less, 7.0 or less, or 6.0 or less, and these upper and lower limits can be freely combined. The average number of additional modification / substituted hydroxyl groups per cyclic molecule can be determined, for example, using nuclear magnetic resonance spectroscopy, and more specifically, 1 This can be determined based on the ratio of the peak area of ​​the proton at position 1 of the glucose monomer in the cyclic molecule to the peak area of ​​the proton in the additionally modified / substituted hydroxyl group in 1H-NMR.

[0037] Modification of a hydroxyl group in an additionally modified / substituted hydroxyl group means that a hydrogen atom in the hydroxyl group is replaced by a substituent. Such substituents may be alkyl, alkenyl, phenyl, benzyl, or alkylcarbonyl, and in one embodiment, such substituents may be alkyl or alkenyl.

[0038] Substitution of a hydroxyl group in an additionally modified / substituted hydroxyl group means that the hydroxyl group is replaced by another substituent (i.e., the substituent is attached to the base carbon to which the hydroxyl group was attached in a cyclic molecule). Such substituents may be, for example, alkoxy, amino, azide, carbonyl, halogeno, mercapto, alkylthio, sulfo, phosphate, or phosphoryl, and in one embodiment may be an azide or a primary amino.

[0039] <Axis molecule> The axial molecule has an axial portion and two stopper portions attached to both ends of the axial portion. In polyrotaxanes, the cyclic molecule cannot penetrate the stopper portions, so the state in which the axial molecule penetrates the cyclic molecule is maintained.

[0040] <Axis part> The axial portion is a linear portion that serves as an axis through which the cyclic molecule is held movably. The axial portion consists of a linear polymer that serves as the axis (hereinafter also referred to as the "axial polymer"), and has bonding hands at both ends that bind to the stopper portion. The position of these bonding hands is not particularly limited as long as it is at the end of the axial polymer, but the bonding hand may be formed by removing one hydrogen atom from a functional group at the end of the axial polymer. For example, the bonding hand may be formed by removing one hydrogen atom from an alkyl or hydroxyl group at the end of the axial polymer.

[0041] The axial polymer may have a cyclic structure, but in one embodiment it may not have a cyclic structure. In other words, the axial polymer may be chain-like in one embodiment. The chain-like axial polymer may be linear or branched. The length of the side chains in a branched axial polymer may be, for example, less than or equal to the length of a C5 alkyl group, less than or equal to the length of a C3 alkyl group, less than or equal to the length of a C2 alkyl group, or less than or equal to the length of a methyl group. For example, the axial polymer may be linear or branched, with the length of the side chains being less than or equal to the length of a C3 alkyl group. The side chains in a branched axial polymer may be, for example, C 1-5 Alkyl, C 1-3 Alkyl, C 1-2The polymer may be alkyl or methyl. For example, the axial polymer may be linear or branched, with methyl side chains.

[0042] As the axial polymer, any axial polymer can be used if it is used as the axial part of rotaxanes such as polyrotaxanes. The axial polymer may include, for example, polyalkylene glycol, vinyl polymer, polysiloxane, polysilane, polyamine, polyester, polyamide, polyurethane, polyurea, polyimine, polysulfide, polyphosphate, or a composite thereof, and may be any of these. In one embodiment, it may also include polyalkylene glycol, or it may be polyalkylene glycol. Polyalkylene glycols are, for example, C 2-5 It may be a polymer of at least one monomer selected from the group consisting of alkylene oxides, preferably at least one polymer selected from the group consisting of ethylene oxide, propylene oxide, triethylene oxide and tetrahydrofuran, more preferably at least one polymer selected from the group consisting of ethylene oxide and propylene oxide, even more preferably polyethylene glycol, polypropylene glycol or copolymers containing the same, and even more preferably polyethylene glycol or copolymers containing polyethylene glycol and polypropylene glycol. In these cases, the polymer consisting of two or more monomers is a copolymer. In these cases, the copolymer is preferably a block copolymer, and more preferably a block copolymer having one type of polymer at both ends (i.e., having a polymer B-polymer A-polymer B structure). The vinyl polymer may be a polymer of at least one monomer selected from the group consisting of vinyl alcohol, acrylamide, N-vinyl-2-pyrrolidone, ethylene, propylene, methyl vinyl ether, isobutylene, isoprene, and butadiene, and may be polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polyethylene, polypropylene, polymethyl vinyl ether, polyisobutylene, polyisoprene, or polybutadiene. In these cases, a polymer consisting of two or more monomers is a copolymer. Polysiloxanes are, for example, polydimethylsiloxane. Polysilanes include, for example, polydimethylsilane. Polyamines include, for example, polyethyleneimine, polyaniline, or poly-ε-lysine. Polyester is, for example, polylactic acid.

[0043] Among the above, the axial polymer is preferably polyethylene glycol, or a copolymer containing polyethylene glycol and polypropylene glycol; more preferably a block copolymer containing polyethylene glycol, or polyethylene glycol and polypropylene glycol; more preferably a block copolymer containing polyethylene glycol, or polyethylene glycol and polypropylene glycol; and more preferably a block copolymer having polyethylene glycol at both ends of polyethylene glycol or polypropylene glycol (i.e., having a polyethylene glycol-polypropylene glycol-polyethylene glycol structure).

[0044] When the axial polymer is a polymer, the polymerization number of monomers may be, for example, 50 or more, 100 or more, or 300 or more, and may also be 1000 or less, 700 or less, or 500 or less.

[0045] The number-average molecular weight [g / mol] of the axial polymer may be, for example, 1000 or more, 3000 or more, 5000 or more, 10000 or more, or 15000 or more, and may also be 100000 or less, 50000 or less, or 30000 or less, and these upper and lower limits can be freely combined.

[0046] For example, if the axial polymer is polyethylene glycol, the number-average molecular weight [g / mol] of the axial polymer may be, for example, 1000 or more, 3000 or more, 5000 or more, 10000 or more, or 15000 or more, and may also be 100000 or less, 60000 or less, 40000 or less, or 25000 or less, and these upper and lower limits can be freely combined.

[0047] For example, if the axial polymer is a copolymer containing polyethylene glycol and polypropylene glycol, the number-average molecular weight [g / mol] of the axial polymer may be, for example, 1000 or more, 3000 or more, or 5000 or more, and may also be 100000 or less, 40000 or less, 20000 or less, or 10000 or less, and these upper and lower limits can be freely combined.

[0048] <Stopper part> The stopper portion only needs to be one that the cyclic molecule cannot penetrate, for example, one that prevents the cyclic molecule from penetrating due to steric hindrance. The stopper portion has a bulky group (hereinafter also referred to as a "blocking group") that prevents the cyclic molecule from penetrating, and may consist of blocking groups. The blocking group may be, for example, a monovalent group.

[0049] The choke-off group may be one that can be used as a choke-off group for rotaxanes such as polyrotaxanes. The choke-off group may be, for example, a group represented by the following formula (II), optionally substituted adamantyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted diphenylmethyl, optionally substituted trityl, optionally substituted polycyclic aromatic hydrocarbon group, or a group in which one hydrogen atom of methyl is substituted with one of these. In one embodiment, the choke-off group may be a group represented by the following formula (II), optionally substituted adamantyl, optionally substituted para-vinylphenyl on the aromatic ring, or optionally substituted trityl. In one preferred embodiment, the choke-off group may be a group represented by the following formula (II). The group represented by the following formula (II) is suitable for using polyrotaxane as a component of a coating agent. Furthermore, since trityl is eliminated in an acidic environment, it is suitable when using polyrotaxane as a prodrug that releases a cyclic molecule in an acidic environment. [ka]

[0050] R a This represents a hydrogen atom or a methyl atom, and in one embodiment, it may represent a methyl atom. R b represents a benzyl, alkyl, alkenyl, or phenyl which may have substituents, and in one embodiment, benzyl, C 3-12 Alkyl, C 3-12 The material may represent an alkenyl or phenyl compound, and in a preferred embodiment, it may represent a benzyl compound. R c This includes hydrogen atoms, methyl, mercapto, and -SR. s -SC(=S)-R s Or -SC(=S)-SR s This shows that R s benzyl, alkyl (e.g., C 3-12 Alkyl), alkenyl (e.g., C 3-12 R represents an alkenyl or phenyl. In one embodiment, R c is -SC(=S)-R sThis can be shown, and here R s It is benzyl, C 3-12 Alkyl, C 3-12 Represents alkenyl or phenyl. m represents an integer between 1 and 1000, and in one embodiment, it may represent an integer between 10 and 800 or between 100 and 600.

[0051] The stopper portion may have a linker between the sealing group and the axial portion, in addition to the sealing group. The structure of the linker is not particularly limited as long as it can form a bond with the sealing group and the axial portion. For example, if the sealing group is a monovalent group, the linker may be a divalent organic group, and in one embodiment, it may be a divalent organic group consisting of a carbon atom, a hydrogen atom, a nitrogen atom, an oxygen atom and a sulfur atom, or it may be a divalent organic group consisting of a carbon atom, a hydrogen atom, a nitrogen atom and an oxygen atom.

[0052] <Example of stopper part> An example of a stopper portion relating to the polyrotaxane of this embodiment is the following structure. In the formula, R b , R c And m are defined the same as in equation (II) above. [ka]

[0053] <Example of an axial molecule> Examples of axial molecules relating to the polyrotaxane of this embodiment include the structures shown in the following formulas (A1) to (A4). [ka] In formula (A1), n ​​is a positive even number, and the number-average molecular weight of the axial polymer may be, for example, 500 or more, 2000 or more, or 10000 or more, and may also be 200000 or less, 100000 or less, or 40000 or less, and as an example, it may be 20000. [ka] In formula (A2), n is a positive even number, and the number-average molecular weight of the axial polymer may be, for example, 500 or more, 2000 or more, or 10000 or more, and may also be 200000 or less, 100000 or less, or 40000 or less, and as an example, it may be 20000. [ka] In equation (A3), R a , R b , R c And m are as described for the group represented by formula (II). In one embodiment, the axial molecule represented by formula (A3) may be the axial molecule represented by the following formula (A3a). In formula (A3a), m is as described for the group represented by formula (II). [ka] In formulas (A3) and (A3a), n is a positive even number, and the number-average molecular weight of the axial polymer may be, for example, 500 or more, 2000 or more, or 10000 or more, and may be 200000 or less, 100000 or less, or 40000 or less, and for example, it may be 20000. [ka] In formula (A4), m and n are each positive integers, and the two n may be different from each other. In formula (A4), m may be, for example, 10 or more, 30 or more, or 50 or more, and may be 200 or less, 100 or less, or 70 or less. In formula (A4), n may be, for example, 10 or more, 20 or more, or 30 or more, and may be 300 or less, 100 or less, or 50 or less. In formula (A4), the axial polymer may, for example, have a number-average molecular weight of the polypropylene glycol portion that is less than the sum of the number-average molecular weights of the two polyethylene glycol portions. Examples of the axial polymer in formula (A4) include the Pluronic® series. The axial polymer in formula (A4) may be, for example, Pluronic® P105.

[0054] <Uses of polyrotaxane> The polyrotaxane according to this embodiment can be used, for example, as described later in a third embodiment, as a component of a coating agent for cell culture substrates. The coating agent containing the polyrotaxane according to this embodiment is suitable for promoting osteoblast differentiation of mesenchymal stem cells. For this application, a polyrotaxane is preferred in which the cyclodextrin in the cyclic molecule is α-CD and the axial molecule has a group represented by formula (II) above as a chokeholding group.

[0055] The polyrotaxane according to this embodiment can also be used as a prodrug containing a cyclic molecule as an active ingredient, for example. Cyclodextrins are known to encapsulate sterols and fatty acids, and the pharmaceutical applications of cyclodextrins using their encapsulation effect are being studied. Among them, β-CD and γ-CD have the ability to encapsulate cholesterol and are therefore expected to be active ingredients in the treatment of cholesterol-related diseases. In particular, intracellular cholesterol accumulation is known to be involved in various diseases, such as Niemann-Pick disease type C, metabolic disorders-associated steatohepatitis (MASH), Alzheimer's disease (AD), focal segmental glomerulosclerosis (FSGS), age-related macular degeneration (AMD), arteriosclerosis, and diabetic nephropathy (DKD). Therefore, by employing a structure that detaches in the therapeutic target environment (e.g., the intracellular environment or a disease-specific environment) as a stopper site, for example, it can become a prodrug that releases a cyclic molecule in the therapeutic target environment to exert its therapeutic effect, and may be suitable as an active ingredient in a therapeutic agent for diseases caused by high cholesterol levels or a therapeutic agent that exerts its therapeutic effect by promoting cholesterol excretion. For this application, a suitable polyrotaxane is one in which the cyclodextrin relating to the cyclic molecule is β-CD or γ-CD, preferably β-CD, and the axial molecule has trityl, which may have substituents as a chokeholding group, preferably trityl as a chokeholding group. Although we do not wish to be bound by any theory, such a polyrotaxane functions as a prodrug that, after being taken up by cells via endocytosis or the like, releases the cyclic molecule, which is the pharmacoactive component, within the cell by detaching the chokeholding group in an acidic intracellular environment such as an endosome.

[0056] <Method for producing polyrotaxane> A second embodiment of this disclosure relates to a method for producing a polyrotaxane. Hereafter, the polyrotaxane that is the final product produced by the production method of this embodiment will be referred to as "polyrotaxane A". The produced polyrotaxane A may be in the form of the supramolecular itself (free form) or in the form of a salt. The detailed form of the salt of polyrotaxane A may be the same as that of the polyrotaxane in the first embodiment.

[0057] Polyrotaxane A comprises an axial molecule having an axial portion and two stopper portions attached to both ends thereof, and at least one cyclic molecule penetrated by the axial molecule. The axial molecule of polyrotaxane A is the same as that described for the polyrotaxane of the first embodiment. Furthermore, the matters described for the polyrotaxane of the first embodiment regarding the cyclic molecule are also the same for polyrotaxane A, except for the structure of the cyclic molecule.

[0058] The cyclic molecule relating to polyrotaxane A is a cyclodextrin in which at least one hydrogen atom in one hydroxyl group is substituted with a group represented by the following formula (I), and at least one other hydroxyl group may be further modified or substituted, wherein L in the formula is the same as described for the polyrotaxane of one embodiment, and R in the formula 1 and R 2 Each of these independently represents a hydrogen atom. [ka] In other words, the cyclic molecule relating to polyrotaxane A is a cyclodextrin in which at least one hydrogen atom in one hydroxyl group is substituted with a group represented by the following formula (IV), and at least one other hydroxyl group may be further modified or substituted, wherein L in the formula is the same as described for the polyrotaxane of one embodiment. [ka] To put it another way, the cyclic molecule relating to polyrotaxane A is the R group represented by formula (I) in the polyrotaxane of the first embodiment. 1 and R 2 Each of these is a cyclic molecule representing a hydrogen atom. Furthermore, in the above, the cyclic molecule is R 1 and R 2 Instead of the hydrogen atom in the compound, a monovalent cation may be formed to create a salt.

[0059] In polyrotaxane A, L is C 3-5 It is preferably an alkylene, and more preferably a C3 alkylene. When L is one of these, the efficiency of introducing the phosphoryl group tends to be higher in the manufacturing method of this embodiment.

[0060] <Conversion from polyrotaxane B to polyrotaxane A> The manufacturing method of this embodiment includes a step of converting polyrotaxane B or a salt thereof into the above-mentioned polyrotaxane A or a salt thereof. Polyrotaxane B is polyrotaxane A in which R in formula (I) 1 and R 2 However, it is a polyrotaxane in which each hydrogen atom is represented by an alkyl group. In other words, the cyclic molecule relating to polyrotaxane B is the R of the group represented by formula (I) in the polyrotaxane of the first embodiment. 1 and R 2 These are cyclic molecules each exhibiting alkyl groups. In these, the alkyl group is, for example, C 1-5It may be alkyl, and in one embodiment, it may be ethyl. Polyrotaxane B may be in the form of the supramolecular itself (free form) or in the form of a salt. The detailed form of the salt of polyrotaxane B may be the same as that of the polyrotaxane of the first embodiment.

[0061] In this process, polyrotaxane B or a salt thereof is converted to polyrotaxane A or a salt thereof by contacting it with a base in an aqueous medium. Although we do not wish to be bound by any theory, in this process, the alkyl protection of the phosphoryl of polyrotaxane B is deprotected by base hydrolysis.

[0062] The aqueous medium in this step may be any medium that deprotects the alkyl protection of the phosphoryl, and may be, for example, water or a mixed solvent of water and one or more organic solvents, or water or a mixed solvent of water and one or more hydrophilic solvents. In this disclosure, a hydrophilic solvent means an organic solvent that can be mixed with water in any proportion. Such hydrophilic solvents may be, for example, dimethyl sulfoxide (DMSO), tetrahydrofuran, methanol, ethanol, or isopropanol. The aqueous medium in this step may preferably be water or a mixed solvent of water and DMSO, and more preferably a solvent with a water:DMSO ratio of 100:0 to 10:90.

[0063] The base used in this step may be any base that deprotects the alkyl protection of the phosphoryl, and may be an organic or inorganic base. From the viewpoint of solubility in an aqueous medium, the base used in this step may be a salt containing hydroxide ions or carbonate ions, and in one embodiment it may be a metal hydroxide, and in a detailed example it may be sodium hydroxide.

[0064] The concentration of the base in this process can be any amount that deprotects the alkyl protection at the phosphoryl, such as 0.01 mol / L to 10.0 mol / L, 0.1 mol / L to 5.0 mol / L, 0.3 mol / L to 2.0 mol / L, or 0.5 mol / L to 1.0 mol / L.

[0065] The reaction temperature in this step should be such that the alkyl protection of the phosphoryl is deprotected, but it is preferably 35°C to 80°C, 40°C to 70°C, or 45°C to 60°C. When the reaction temperature in this step is within the above range, the deprotection efficiency of the alkyl protection tends to be high, and even in deprotection reactions lasting several days or less, the deprotection reaction proceeds with a yield of 80% or more or 90% or more.

[0066] The reaction temperature in this step should be such that the alkyl protection of the phosphoryl is deprotected, and may be, for example, 10 minutes to 30 days, 1 hour to 168 hours, or 6 hours to 96 hours.

[0067] Polyrotaxane B is a polyrotaxane comprising a cyclodextrin as a cyclic molecule, for example, in which at least one hydroxyl group may be modified or substituted, R 1 and R 2 It can be prepared by introducing groups represented by formula (I) that each represent an alkyl group, or by forming a supramolecular with the cyclic and axial molecules of polyrotaxane B according to a conventional method. Of these, the former is preferred from the viewpoint of ease of synthesis.

[0068] <Conversion from polyrotaxane C to polyrotaxane B> The manufacturing method of this embodiment may further include a step of converting polyrotaxane C or a salt thereof to polyrotaxane B or a salt thereof before the above step. Polyrotaxane C is a polyrotaxane in which, in polyrotaxane A, the hydrogen atom in at least one hydroxyl group is not substituted with the group represented by formula (I). In other words, polyrotaxane C is a polyrotaxane in which, in the polyrotaxane of the first embodiment, the group represented by formula (I) is substituted with a hydrogen atom. Polyrotaxane C may be in the form of the supramolecular itself (free form) or in the form of a salt. The detailed form of the salt of polyrotaxane C may be the same as that of the polyrotaxane of the first embodiment.

[0069] In this process, polyrotaxane C or a salt thereof is converted to polyrotaxane B or a salt thereof by contacting it with a compound represented by formula (III) below and a base in an organic solvent. Although we do not wish to be bound by any theory, in this process, the hydrogen atoms in the hydroxyl groups of the cyclic molecules in polyrotaxane C are replaced by a Williamson ether synthesis reaction, thereby converting it to polyrotaxane B. [ka]

[0070] In equation (III), L, R 1 and R 2 This is the same as in polyrotaxane B. X is a monovalent leaving group and can be any leaving group commonly used in Williamson ether synthesis. X may be, for example, a halogen, tosyloxy (TsO-), trifluoromethanesulfonyloxy (TfO-), or mesyloxy (MsO-), and in one embodiment may be Cl, Br, or I. The compound represented by formula (III) may be obtained from a distributor, or it may be prepared by conventional methods using a compound in which X in formula (III) is hydroxyl as a starting material.

[0071] The organic solvent used in this step can be any solvent that alkylates the hydroxyl groups of the cyclic molecules in polyrotaxane C, such as dimethyl sulfoxide (DMSO), acetonitrile, tetrahydrofuran, diethyl ether, or N,N-dimethylformamide (DMF). Since polyrotaxane is readily soluble in these organic solvents, the hydroxyl groups of the cyclic molecules in polyrotaxane C are readily alkylated. In a preferred embodiment, the organic solvent used in this step may be DMSO.

[0072] The base used in this step can be any base that alkylates the hydroxyl group of the cyclic molecule in polyrotaxane C, and may be an organic or inorganic base. From the viewpoint of increasing the number of hydroxyl group substitutions in polyrotaxane B, the base used in this step may be, for example, a salt containing hydroxide ions or carbonate ions, and in one embodiment it may be a metal hydroxide, and in a detailed example it may be sodium hydroxide.

[0073] The amount of base used in this process is sufficient to alkylate the hydroxyl groups of the cyclic molecules in polyrotaxane C. For example, the amount of base may be 5 to 500 molar equivalents, 10 to 200 molar equivalents, 15 to 100 molar equivalents, or 25 to 60 molar equivalents per molar equivalent of polyrotaxane C.

[0074] The reaction temperature in this process should be such that the hydroxyl groups of the cyclic molecules in polyrotaxane C are alkylated. For example, it may be between 10°C and 80°C, between 10°C and 50°C, or between 10°C and 30°C, and as an example, it may be room temperature or 23°C.

[0075] The reaction temperature in this step should be such that the hydroxyl groups of the cyclic molecules in polyrotaxane C are alkylated, for example, between 10 minutes and 168 hours, between 20 minutes and 24 hours, between 1 hour and 12 hours, or between 2 hours and 6 hours. If the reaction temperature in this step is below the above upper limit, the yield of polyrotaxane B in this step tends to be higher.

[0076] Polyrotaxane C can be prepared according to general methods for producing polyrotaxanes that contain cyclodextrins as cyclic molecules, for example, according to the methods described in Patent Documents 1-3 or the literature (Macromolecules 2005, 38, 7524-7527, Journal of Applied Polymer Science 2021, 138, e49706, Polymer Chemistry 2014, 5, 4511-4520, Science and Technology of Advanced Materials 2016, 17, 361-374, etc.). Therefore, if the production method of this embodiment includes this step, phosphoryl can be introduced later into polyrotaxane prepared according to conventional methods, making synthesis simple and structural development easy.

[0077] <Coating agent> A third embodiment of this disclosure is a coating agent for cell culture substrates containing the polyrotaxane or a salt thereof of the first embodiment. The coating agent of this embodiment can also be described as a composition for coating cell culture substrates.

[0078] The coating agent of this embodiment may contain a solvent and optional additives in addition to the polyrotaxane or its salt described above. Examples of solvents include dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), N,N-dimethylformamide (DMF), methanol, 2-propanol, chloroform, and methylene chloride. Examples of additives include antioxidants.

[0079] The polyrotaxane or salt content in the coating agent of the third embodiment may be 0.0005 to 5% by mass, preferably 0.01 to 1% by mass, and more preferably 0.02 to 0.5% by mass, based on the mass of the coating agent.

[0080] The polyrotaxane or salt content in the coating agent of the third embodiment may be 0.01 to 100 mg / mL, preferably 0.03 to 30 mg / mL, and more preferably 0.1 to 10 mg / mL, based on the volume of the coating agent.

[0081] The coating agent of this embodiment is used to coat the surface of a substrate that can be used as a culture substrate. The substrate that can be used as a culture substrate may be one that is well known to those skilled in the art. Examples of substrate materials include glass, polystyrene, polypropylene, polyethylene, polyolefin, polycarbonate, and acrylic block copolymer (BCF).

[0082] The coating agent of this embodiment may be intended for coating a substrate by, for example, casting, spin coating, gravure coating, die coating, knife coating, bar coating, blade coating, or roll coating, and in a preferred embodiment, it may be intended for coating by casting. Thus, in another embodiment, it can be said that the cell culture substrate includes a substrate coated with the coating agent or composition of this embodiment.

[0083] The coating agent of this embodiment is applied to the substrate at a concentration of 0.05 to 500 μg / cm². 2 It may be intended for coating, and from the viewpoint of increasing the efficiency of differentiation induction, 0.5-100 μg / cm³ 2 , 1.0~30 μg / cm³ 2 , 1.5~10 μg / cm³ 2 Or 2.0-4.0 μg / cm³ 2 And it can also be something that is meant to be coated.

[0084] In one embodiment, the coating agent of this embodiment may be used to promote osteoblast differentiation of mesenchymal stem cells. Osteoblast differentiation of mesenchymal stem cells refers to the differentiation of mesenchymal stem cells into osteoblasts. In osteoblast differentiation of mesenchymal stem cells, bone tubers are formed as the differentiation progresses (also called calcification). The inventors have found that when mesenchymal stem cells are cultured on a cell culture substrate containing a substrate coated with the coating agent of this embodiment, the mesenchymal stem cells are more likely to differentiate into osteoblasts.

[0085] The differentiation of mesenchymal stem cells into osteoblasts can be evaluated based on the staining of bone nodules by cell staining with Alizarin Red S. Furthermore, the differentiation of mesenchymal stem cells into osteoblasts can also be evaluated based on an increase in the expression of molecular markers of osteoblast differentiation within the cells. Examples of differentiation markers for osteoblast differentiation include RUNX2, alkaline phosphatase, osteocalcin, osteopontin, bone sialoprotein, and type I collagen. In this disclosure, for example, mesenchymal stem cells cultured on a substrate coated with the coating agent of this embodiment may be evaluated as having promoted osteoblast differentiation if they are more easily stained by Alizarin Red S and / or if they express higher levels of molecular markers of osteoblast differentiation compared to cells cultured on a substrate not coated with the coating agent of this embodiment.

[0086] <Cell culture method> A fourth embodiment of the present invention is a cell culture method for promoting osteoblast differentiation of mesenchymal stem cells, comprising culturing mesenchymal stem cells on the surface of a substrate coated with a composition containing the polyrotaxane or a salt thereof of the first embodiment. In this embodiment, the "composition containing the polyrotaxane or a salt thereof of the first embodiment" and the coating method thereof are the same as those described in the coating agent of the third embodiment. The promotion of osteoblast differentiation of mesenchymal stem cells can be evaluated in the same way as described in the coating agent of the third embodiment.

[0087] In the cell culture method according to this embodiment, a substrate coated with a composition containing the polyrotaxane or a salt thereof from the first embodiment is used as the culture substrate. Cell culture is carried out by adhering cells to the surface of the coated substrate.

[0088] The culture substrate is a substrate coated with a polyrotaxane or a salt thereof according to the first embodiment. The substrate may be one that is well known to those skilled in the art. Examples of substrate materials include glass, polystyrene, polypropylene, polyethylene, polyolefin, polycarbonate, and acrylic block copolymer (BCF). The substrate may also be a commercially available glass substrate or plastic substrate.

[0089] In the cell culture method according to this embodiment, a culture medium is added to a coated surface on a substrate so that the cells are submerged, and the cells to be cultured are seeded and cultured. The culture medium may be replaced with a new medium as needed. Alternatively, a step may be added to grow the seeded cells by adding a growth medium before differentiation. In this case, after growing until a sufficient number of cells are obtained, the growth medium is replaced with a differentiation medium. The growth medium and differentiation medium can be any medium known to those skilled in the art. For example, as a medium for osteoblast differentiation of mesenchymal stem cells, Mesenchymal Stem Cell Osteogenic Differentiation Medium (C-28013) from PromoCell GmbH (Heidelberg, Germany) can be used. The cell culture environment can be arbitrarily set under conditions known to those skilled in the art.

[0090] <Composition> A fifth embodiment of this disclosure relates to a composition containing the polyrotaxane or a salt thereof of the first embodiment. As described above, the composition of the fifth embodiment contains a polyrotaxane or a salt thereof in which a structure (e.g., trityl which may have substituents) is used as a stopper site to detach in the environment of the therapeutic target (e.g., the intracellular environment or a disease-specific environment), thereby becoming a functional supramolecular (e.g., a prodrug) that releases a cyclic molecule in the environment of the therapeutic target and exerts a pharmacokinetic effect.

[0091] In one embodiment, the composition of this embodiment may contain the polyrotaxane or a salt thereof of the first embodiment as an active ingredient. The composition of this embodiment may be a composition for an application achieved, for example, by encapsulating a substance, and may be a composition for an application achieved by encapsulating cholesterol. The composition of this embodiment may be a composition for an application achieved, for example, by promoting the excretion of cholesterol from cells.

[0092] The composition of this embodiment may contain the polyrotaxane or salt thereof of the first embodiment in amounts of, for example, 0.01% or more, 0.1% or more, 1.0% or more, 3.0% or more, 10% or more, 40% or more, 70% or more, 90% or more, 95% or more, or 99% or more, relative to the total amount of the composition, and may also contain less than 100% by mass, 97% or less, 92% or less, 80% or less, 50% or less, 30% or less, 15% or less, 8.0% or less, or 2.0% or less, and these upper and lower limits can be freely combined. Furthermore, the form of the composition of this embodiment is not particularly limited and may be, for example, in the form of a solid, semi-solid, or liquid.

[0093] The composition of this embodiment may be, for example, a pharmaceutical composition, a cosmetic composition, or a food composition, and may be a pharmaceutical composition or a cosmetic composition, or it may be a pharmaceutical composition.

[0094] The pharmaceutical composition may be, for example, a pharmaceutical composition for treating and / or preventing diseases caused by the accumulation of cholesterol in cells. The pharmaceutical composition may be, for example, a pharmaceutical composition for treating and / or preventing Niemann-Pick disease type C, metabolic disorders-associated steatohepatitis (MASH), Alzheimer's disease (AD), focal segmental glomerulosclerosis (FSGS), age-related macular degeneration (AMD), arteriosclerosis, or diabetic nephropathy (DKD).

[0095] The composition of this embodiment may be, for example, a composition to be administered, applied, or ingested orally or parenterally. The target to which the composition of this embodiment is administered, applied, or ingested may be, for example, a human or a non-human animal, a human or a non-human mammal, or a human. If the composition of this embodiment is administered, applied, or ingested in a human, the amount administered, applied, or ingested may be, for example, 0.1 μg to 1 g or 10 μg to 10 mg per day or per dose as polyrotaxane or its salt of the first embodiment, and this amount may be, for example, a value based on an adult male weighing 60 kg.

[0096] The composition of this embodiment may contain, in addition to the polyrotaxane or salt thereof of the first embodiment as the active ingredient, additives commonly used in the field of its application. The additives may be at least one selected from the group consisting of, for example, excipients, solvents, buffers, stabilizers, antioxidants, binders, disintegrants, fillers, emulsifiers, flavorings, colorants, sweeteners, thickeners, pH adjusters, and preservatives. [Examples]

[0097] The present disclosure will be described in more detail below using examples, but the present disclosure should not be construed as being limited to the following examples.

[0098] <Example 1: Diethyl propylphosphonate modification reaction of polyrotaxane> Polyrotaxanes were synthesized using polyethylene glycol (Sigma-Aldrich, number-average molecular weight 20,000, sometimes referred to as "PEG") as the axial polymer, α-cyclodextrin (Tokyo Chemical Industries, sometimes referred to as "α-CD") as the cyclic molecule, and 1-adamantylamine (Fujifilm Wako Pure Chemical Industries) as the chelating group, according to the literature (Macromolecules 2005, 38, 7524-7527). The number and penetration rate of α-CD in the polyrotaxane were 84.2% and 37.1%, respectively, and the number-average molecular weight was 102,200. This polyrotaxane was used in the following reactions. This polyrotaxane corresponds to "Unmodified PRX" in Figure 1, and "Example 1 Raw Material: PRX" in Figures 2 and 3.

[0099] 500 mg (4.89 μmol, α-CD moles in polyrotaxane: 412 μmol) of the aforementioned polyrotaxane was weighed into a glass vial, and 20 mL of dimethyl sulfoxide (Fujifilm Wako Pure Chemical Industries, hereinafter sometimes referred to as "DMSO") was added and dissolved. 445 mg (11.1 mmol, 27 molar equivalents per α-CD molecule in polyrotaxane) of powdered sodium hydroxide (Fujifilm Wako Pure Chemical Industries) was weighed in a mortar and added to the polyrotaxane solution, and stirred for 5 minutes. 478 μL (2.47 mmol, 6 molar equivalents per α-CD molecule in polyrotaxane) of diethyl (3-bromopropyl)phosphonate (Tokyo Chemical Industries) was weighed and added to the polyrotaxane solution, and stirred at 23°C for 4 hours. After the reaction, the solution was sealed in a dialysis membrane (spectral pore, molecular weight cutoff 6000-8000) and dialyzed against pure water for 3 days to remove unreacted substances. Subsequently, freeze-drying was performed to obtain 471 mg of polyrotaxane in which the hydroxyl groups were substituted with diethyl propylphosphonate. Hereafter, "polyrotaxane in which the hydroxyl groups were substituted with diethyl propylphosphonate" may be collectively referred to as "PO3Et2-PRX". [ka]

[0100] <Example 2> In Example 1, the reaction was carried out in the same manner except that 10 molar equivalents of (3-bromopropyl)phosphonate diethyl were added relative to the α-CD1 molecules in the polyrotaxane to obtain PO3Et2-PRX.

[0101] <Example 3> In Example 1, the reaction was carried out in the same manner except that 20 molar equivalents of (3-bromopropyl)phosphonate diethyl were added relative to the α-CD1 molecules in the polyrotaxane to obtain PO3Et2-PRX.

[0102] <Example 4> In Example 1, the reaction was carried out in the same manner except that 54 molar equivalents of sodium hydroxide were added relative to the α-CD1 molecules in the polyrotaxane to obtain PO3Et2-PRX.

[0103] <Example 5> In Example 1, the reaction was carried out in the same manner except that the reaction time was 24 hours, and PO3Et2-PRX was obtained.

[0104] <Example 6> In Example 4, the reaction was carried out in the same manner except that the reaction time was 24 hours, and PO3Et2-PRX was obtained.

[0105] <Example 7> In Example 6, the reaction was carried out in the same manner except that 40 molar equivalents of (3-bromopropyl)phosphonate diethyl were added to the α-CD1 molecules in the polyrotaxane, to obtain PO3Et2-PRX.

[0106] <Example 8> In Example 1, the reaction was carried out in the same manner as in Example 1, except that 6 molar equivalents of diethyl 2-bromoethylphosphonate (Tokyo Chemical Industry Co., Ltd.) were added to the α-CD1 molecules in the polyrotaxane, 54 molar equivalents of sodium hydroxide were added to the α-CD1 molecules in the polyrotaxane, and 20 molar equivalents of diethyl 2-bromoethylphosphonate were added to the α-CD1 molecules in the polyrotaxane, to obtain PO3Et2-PRX.

[0107] <Example 9> In Example 8, the reaction was carried out in the same manner except that the reaction time was set to 24 hours, and PO3Et2-PRX was obtained.

[0108] <Evaluation of PO3Et2-PRX> Gel permeation chromatography (GPC) measurement was performed on the PO3Et2-PRX obtained in Example 1. The same measurement was also carried out on the unmodified polyrotaxane as the raw material for comparison. The results are shown in Fig. 1. The unmodified polyrotaxane as the raw material showed a peak at an elution volume of 13.2 minutes, while in Example 1, a single-peak peak was confirmed at an elution volume of 12.5 minutes, suggesting an increase in molecular weight. In addition, the peak derived from α-CD of the raw material was observed at an elution time of 17.2 minutes, but in Example 1, no peak derived from α-CD was observed, suggesting that no decomposition of polyrotaxane occurred during the reaction. GPC measurement was also performed on the PO3Et2-PRX obtained in Examples 2-9, and the same results were obtained.

[0109] The PO3Et2-PRX obtained in Example 1 was dissolved in deuterated DMSO (FUJIFILM Wako Pure Chemical Corporation) at a concentration of 10 mg / mL, 1 1H nuclear magnetic resonance (NMR) measurement was performed. The same measurement was also carried out on the unmodified polyrotaxane as the raw material for comparison. The results are shown in Fig. 2. In the PO3Et2-PRX obtained in Example 1, peaks derived from the ethyl groups of the phosphonate ester were newly observed at 1.24 and 3.99 ppm, confirming that diethyl propylphosphonate was chemically modified. Based on the integral area ratio of the proton at the α-CD 1-position (4.79 ppm) in polyrotaxane and the peak derived from the ethylene group of the phosphonate ester (3.99 ppm), the modification number of diethyl propylphosphonate with respect to polyrotaxane was 110, which was 1.3 when converted to the modification of 1 molecule of α-CD in polyrotaxane. Also, for the PO3Et2-PRX obtained in Examples 2-9 11H NMR measurements were performed, and the number of modifications was calculated. The results are shown in Table 1. Based on Table 1, the number of propylphosphonic acid diethyl modifications changed depending on the amount of 2-bromoethylphosphonic acid diethyl, the amount of sodium hydroxide, and the reaction time. Since there are 18 hydroxyl groups in α-CD, it was suggested that the number of propylphosphonic acid diethyl modifications can be adjusted over a wide range by appropriately setting these amounts. Furthermore, it was suggested that not only propylphosphonic acid diethyl but also ethylphosphonic acid diethyl can be modified in a similar manner, indicating that phosphonic acid esters of various structures can be introduced.

[0110] Using a double tube containing a solution of PO3Et2-PRX obtained in Example 1 dissolved in deuterated DMSO at a concentration of 10 mg / mL, and an aqueous solution of dipotassium hydrogen phosphate, 31 1P NMR measurements were performed. For comparison, the same measurements were also performed on the unmodified polyrotaxane used as the raw material. The results are shown in Figure 3. In the PO3Et2-PRX obtained in Example 1, a new peak originating from phosphorus in the phosphonic acid ester was observed at 32.4 ppm, confirming that diethyl propylphosphonate had been chemically modified. The same was also observed for the PO3Et2-PRX obtained in Examples 2-9. 31 P NMR measurements were performed, and similar results were obtained.

[0111] [Table 1]

[0112] <Comparative Example 1: Deprotection reaction of phosphonate esters using a silylating agent> 50 mg (411 nmol, phosphonate ester moles: 45 μmol) of PO3Et2-PRX obtained in Example 1 was weighed into a glass vial and dissolved in 2 mL of DMSO. 58.3 μL (450 μmol, 10 molar equivalents per molecule of phosphonate ester in PO3Et2-PRX) of bromotrimethylsilane (Tokyo Chemical Industries) was added to the PO3Et2-PRX solution and stirred at 23°C for 24 hours. After the reaction, the solution was sealed in a dialysis membrane (molecular weight cutoff 6000-8000) and unreacted products were removed by dialysis with pure water for 3 days. Subsequently, freeze-drying was performed to obtain a powdered product. [ka]

[0113] <Comparative Example 2> In Comparative Example 1, the reaction was carried out in the same manner except that 138 molar equivalents of bromotrimethylsilane were added to one molecule of phosphonic acid ester in PO3Et2-PRX. The product was an insoluble brown solid, suggesting that a side reaction was occurring due to the excess amount of bromotrimethylsilane.

[0114] <Evaluation of deprotection reactions using silylaters> The compound obtained in Comparative Example 1 was dissolved in deuterated DMSO at a concentration of 10 mg / mL. 1 1H NMR measurements were performed. The results are shown in Figure 4. In addition, the reaction conditions for Comparative Examples 1 and 2 were compared. 1 Table 2 shows the deprotection efficiency determined from the integration ratio of 1H NMR. In Comparative Example 1, the peak area of ​​the ethylene group derived from the phosphonic acid ester decreased only slightly from before the reaction, suggesting that the deprotection reaction did not proceed.

[0115] <Comparative Example 3: Deprotection reaction of phosphonic acid esters by acid hydrolysis> 50 mg (411 nmol, phosphonic acid ester molars: 45 μmol) of PO3Et2-PRX obtained in Example 1 was weighed into a glass vial and dissolved in 1.67 mL of dimethyl sulfoxide. 334 μL of 6N hydrochloric acid (Fujifilm Wako Pure Chemical Industries) was added to the PO3Et2-PRX solution and stirred at 23°C for 24 hours. After the reaction, the solution was sealed in a dialysis membrane (molecular weight cutoff 6000-8000) and unreacted products were removed by dialysis with pure water for 3 days. Subsequently, freeze-drying was performed to obtain a powdered product.

[0116] <Comparative Example 4> In Comparative Example 3, the reaction was carried out in the same manner except that the reaction temperature was 50°C, and the product was obtained.

[0117] <Comparative Example 5> In Comparative Example 3, the reaction was carried out in the same manner except that 835 μL of 6N hydrochloric acid was added, and the product was obtained.

[0118] <Comparative Example 6> In Comparative Example 5, the reaction was carried out in the same manner except that the reaction temperature was 50°C, and the product was obtained.

[0119] The compounds obtained in Comparative Examples 3-6 were dissolved in deuterated DMSO at a concentration of 10 mg / mL. 1 1H NMR measurements were performed. As a representative result, the compounds obtained in Comparative Example 6 were measured. 1 The results of the 1H NMR measurement are shown in Figure 4. Furthermore, the reaction conditions for Comparative Examples 3-6 and 1 Table 2 shows the deprotection efficiency determined from the integration ratio of 1H NMR. In the products of Comparative Examples 3-6, the peak area of ​​the ethylene group derived from the phosphonic acid ester decreased only partially from before the reaction, suggesting that the deprotection reaction did not proceed.

[0120] <Example 10: Deprotection reaction of phosphonic acid esters by alkaline hydrolysis> 50 mg (411 nmol, phosphonate ester molars: 45 μmol) of PO3Et2-PRX obtained in Example 1 was weighed into a glass vial, and 1.67 mL of pure water and 185 μL of 5N sodium hydroxide aqueous solution (Fujifilm Wako Pure Chemical Industries) were added to dissolve the PO3Et2-PRX. After stirring at 23°C for 24 hours, the solution was sealed in a dialysis membrane (Spectrumpore, molecular weight cutoff 6000-8000) and dialyzed against pure water for 3 days to remove unreacted substances. Subsequently, freeze-drying was performed to obtain a powdered product. <Example 11> The reaction was carried out in the same manner as in Example 10, except that the reaction time was 72 hours, to obtain the product. <Example 12> In Example 10, the reaction was carried out in the same manner except that the reaction temperature was 50°C, and the product was obtained. <Example 13> In Example 10, the reaction was carried out in the same manner as described above, except that the reaction time was 48 hours and the reaction temperature was 50°C, and the product was obtained. <Example 14> In Example 10, the reaction was carried out in the same manner as described above, except that 294 μL of 5N sodium hydroxide aqueous solution was added, and the product was obtained. <Example 15> In Example 10, the reaction was carried out in the same manner as described above, except that 417 μL of 5N sodium hydroxide aqueous solution was added, and the product was obtained. <Example 16> In Example 15, the reaction was carried out in the same manner as described above, except that the reaction temperature was set to 50°C, and the product was obtained. <Example 17> In Example 12, the reaction was carried out in the same manner as in Example 6, except that 50 mg of PO3Et2-PRX obtained in Example 6 was used as the PO3Et2-PRX, and the product was obtained.

[0121] Using a double tube containing a solution of the compound obtained in Example 12 dissolved in deuterated DMSO at a concentration of 10 mg / mL, and an aqueous solution of dipotassium hydrogen phosphate, 31The results of the P NMR measurement are shown in Figure 3. The compounds obtained in Examples 10-17 were dissolved in deuterated DMSO at a concentration of 10 mg / mL. 1 1H NMR measurements were performed. Representative results were obtained for the compounds obtained in Examples 10, 11, 12, and 15. 1 The results of the 1H NMR measurement are shown in Figure 5. Furthermore, the reaction conditions for Examples 10-17 and 1 Table 2 shows the deprotection efficiency determined from the integration ratio of 1H NMR. In Example 10, the peak area of ​​the ethylene group derived from the phosphonic acid ester decreased by 50.3% from before the reaction. In Example 11, the deprotection efficiency was 77.1%, and deprotection progressed further by extending the reaction time. In Example 12, the deprotection efficiency was 97.5%, indicating that increasing the reaction temperature is effective in improving deprotection efficiency. In Example 15, the reaction was carried out at 23°C with an increased alkali concentration, but the deprotection efficiency at 23°C was approximately 75.1%. From the results in Table 2, it was found that reaction conditions of 50°C with a sodium hydroxide concentration of 0.5M or higher are suitable for quantitative deprotection. Furthermore, based on Example 17, it was suggested that diethyl ethylphosphonate can also be quantitatively deprotected by alkaline hydrolysis, and that this method can be applied to phosphonic acid esters of various structures. In Table 2, the crude yield is calculated based on the mass of the deprotected product, using the mass of the deprotected product as the reference, based on the mass of the product obtained by freeze-drying after being subjected to each deprotection condition (i.e., the total mass of the deprotected product and the protected product).

[0122] [Table 2]

[0123] Using a double tube containing a solution of PO3Na2-PRX obtained in Example 12 dissolved in deuterated DMSO at a concentration of 10 mg / mL, and an aqueous solution of dipotassium hydrogen phosphate, 31 P NMR measurements were performed. The results are shown in Figure 3. Example 1 31Compared with the 1P NMR spectrum, the phosphorus-derived peak shifted to 21.8 ppm, suggesting that diethyl propylphosphonate was deprotected and sodium phosphonate was produced. Furthermore, the products obtained in Examples 13, 16, and 17 were also analyzed. 31 P NMR measurements were performed, and similar results were obtained.

[0124] <Example 18: Diethyl propylphosphonate modification reaction of polyrotaxane having vinylbenzyl groups at both ends of the axial polymer> Polyrotaxanes (hereinafter sometimes referred to as "PRX-VB") having vinylbenzyl groups at both ends of the axial polymer were synthesized using polyethylene glycol (manufactured by Sigma-Aldrich, number average molecular weight 20,000, hereinafter sometimes referred to as "PEG") as the axial polymer, α-cyclodextrin (manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter sometimes referred to as "α-CD") as the cyclic molecule, and vinylbenzoic acid (Tokyo Chemical Industry Co., Ltd.) as the chelating group, according to the literature (Journal of Applied Polymer Science 2021, 138, e49706). The number and penetration rate of α-CD in the polyrotaxane were 75.9 and 33.4%, respectively, and the polyrotaxane with a number average molecular weight of 94,600 was used in the following reaction.

[0125] 200 mg (2.1 μmol, 160 μmol of α-CD in polyrotaxane) of PRX-VB was weighed into a glass vial and dissolved in 8 mL of DMSO. 173 mg (4.3 mmol, 27 molar equivalents per α-CD molecule in polyrotaxane) of powdered sodium hydroxide was weighed in a mortar and added to the polyrotaxane solution and stirred for 5 minutes. 620 μL (3.2 mmol, 20 molar equivalents per α-CD molecule in polyrotaxane) of diethyl (3-bromopropyl)phosphonate was added to the polyrotaxane solution and stirred at 23°C for 4 hours. After the reaction, the solution was sealed in a dialysis membrane (molecular weight cutoff 6000-8000) and unreacted substances were removed by dialysis with pure water for 3 days. Subsequently, freeze-drying was performed to obtain 160 mg of PRX-VB (hereinafter sometimes referred to as "PO3Et2-PRX-VB") in which the hydroxyl groups were replaced with diethyl propylphosphonate (yield 75.2%). [ka]

[0126] 70 mg (693 nmol, phosphonate ester molar count: 68.3 μmol) of the aforementioned PO3Et2-PRX-VB was weighed into a glass vial, and 2.33 mL of pure water and 259 μL of 5N sodium hydroxide aqueous solution were added to dissolve the PO3Et2-PRX-VB. After stirring at 50°C for 24 hours, the solution was sealed in a dialysis membrane (molecular weight cutoff 6000-8000) and dialyzed against pure water for 3 days to remove unreacted substances. Subsequently, freeze-drying was performed to obtain 52 mg of powder of PRX-VB modified with sodium propylphosphonate salt (hereinafter sometimes referred to as "PO3Na2-PRX-VB") (yield 74.0%).

[0127] The PO3Et2-PRX-VB and PO3Na2-PRX-VB obtained above were dissolved in deuterated DMSO at a concentration of 10 mg / mL. 11H NMR measurements were performed. For comparison, the same measurements were also performed on the unmodified PRX-VB, the starting material. The results are shown in Figure 6. In PO3Et2-PRX-VB, new peaks originating from the ethyl group of the phosphonate were observed at 1.23 and 3.99 ppm, confirming that diethyl propylphosphonate had been chemically modified. Based on the integral area ratio of the proton at the α-CD1 position in the polyrotaxane (4.80 ppm) and the peak originating from the ethylene group of the phosphonate (3.99 ppm), the number of modifications by diethyl propylphosphonate to the polyrotaxane was 35.7, which translates to 0.47 modifications to the α-CD1 molecule in the polyrotaxane. Furthermore, in PO3Na2-PRX-VB, the peak originating from the ethyl group in the phosphonate disappeared, confirming that the ethyl group was deprotected to obtain PO3Na2-PRX-VB.

[0128] <Example 19: Diethyl propylphosphonate modification reaction of a polyrotaxane block copolymer with polybenzyl methacrylate at both ends of the axial polymer> Polyrotaxane block copolymer (PRX-b-PBnMA) was synthesized using polyethylene glycol (Sigma-Aldrich, number average molecular weight 20,000, sometimes referred to as "PEG") as the axial polymer and α-cyclodextrin (Tokyo Chemical Industries, sometimes referred to as "α-CD") as the cyclic molecule, with polybenzyl methacrylate attached to both ends of the axial polymer according to the literature (Polymer Chemistry 2014, 5, 4511-4520). The penetration number and penetration rate of α-CD in the polyrotaxane block copolymer were 84.1 and 37.1%, respectively, and the degree of polymerization of polybenzyl methacrylate was 466 per end, with a number average molecular weight of 266,000. This PRX-b-PBnMA was used in the following reaction.

[0129] 150 mg (563 nmol, α-CD moles in polyrotaxane: 47.4 μmol) of the aforementioned PRX-b-PBnMA was weighed into a glass vial and dissolved in 6 mL of DMSO. 102 mg (2.6 mmol, 54 molar equivalents per α-CD molecule in polyrotaxane) of powdered sodium hydroxide was weighed in a mortar and added to the polyrotaxane solution and stirred for 5 minutes. 137 μL (711 μmol, 15 molar equivalents per α-CD molecule in polyrotaxane) of diethyl (3-bromopropyl)phosphonate was added to the polyrotaxane solution and stirred at 23°C for 4 hours. After the reaction, the solution was sealed in a dialysis membrane (molecular weight cutoff 6000-8000) and unreacted substances were removed by dialysis with pure water for 3 days. Subsequently, freeze-drying was performed to obtain 142 mg of PRX-b-PBnMA (hereinafter sometimes referred to as "PO3Et2-PRX-b-PBnMA") in which the hydroxyl groups were substituted with diethyl propylphosphonate (yield 80.6%). [ka]

[0130] 100 mg (320 nmol, phosphonate ester moles: 83 μmol) of the aforementioned PO3Et2-PRX-b-PBnMA was weighed into a glass vial, and 3.33 mL of DMSO was added to dissolve the PO3Et2-PRX-b-PBnMA. 370 μL of 5N sodium hydroxide aqueous solution was added, and the mixture was stirred at 50°C for 24 hours. After the reaction was complete, the solution was sealed in a dialysis membrane (molecular weight cutoff 6000-8000), and unreacted substances were removed by dialysis against pure water for 3 days. Subsequently, freeze-drying was performed to obtain 75 mg of powder of PRX-VB modified with sodium propylphosphonate salt (hereinafter sometimes referred to as "PO3Na2-PRX-b-PBnMA") (yield 75.6%).

[0131] The PO3Et2-PRX-b-PBnMA and PO3Na2-PRX-b-PBnMA obtained above were dissolved in deuterated DMSO at a concentration of 10 mg / mL. 11H NMR measurements were performed. For comparison, the same measurements were also performed on the raw material, unmodified PRX-b-PBnMA. The results are shown in Figure 7. In PO3Et2-PRX-b-PBnMA, new peaks originating from the ethyl group of the phosphonic acid ester were observed at 1.24 and 3.99 ppm, confirming that diethyl propylphosphonate had been chemically modified. Based on the integral area ratio of the proton at the α-CD1 position in the polyrotaxane (4.79 ppm) and the peak originating from the ethylene group of the phosphonic acid ester (3.99 ppm), the number of modifications by diethyl propylphosphonate to the polyrotaxane was 258, which translates to 3.1 modifications to the α-CD1 molecule in the polyrotaxane. Furthermore, in PO3Na2-PRX-b-PBnMA, the peak originating from the ethyl group in the phosphonic acid ester disappeared, confirming that the ethyl group was deprotected to obtain PO3Na2-PRX-b-PBnMA.

[0132] <Example 20: Diethyl propylphosphonate modification reaction of polyrotaxane containing β-cyclodextrin> Using Pluronic P105 (BASF, number-average molecular weight 6,600) as the axial polymer, β-cyclodextrin (Tokyo Chemical Industries, hereinafter sometimes referred to as "β-CD") as the cyclic molecule, and N-triphenylmethylglycine (Tokyo Chemical Industries) as the chelating group, a polyrotaxane containing β-cyclodextrin (βCD-PRX) was synthesized according to the literature (Science and Technology of Advanced Materials 2016, 17, 361-374). The number and penetration rate of β-CD in the polyrotaxane were 12.8 and 46.7%, respectively, and the number-average molecular weight was 21,700. This polyrotaxane was used in the following reactions.

[0133] 300 mg of βCD-PRX (13.8 μmol, 177 μmol of β-CD in polyrotaxane) was weighed into a glass vial and dissolved in 12 mL of DMSO. 191 mg of powdered sodium hydroxide (4.8 mmol, 27 molar equivalents per β-CD molecule in polyrotaxane) was weighed in a mortar and added to the polyrotaxane solution, and stirred for 5 minutes. 684 μL of diethyl (3-bromopropyl)phosphonate (3.5 mmol, 20 molar equivalents per β-CD molecule in polyrotaxane) was added to the polyrotaxane solution, and stirred at 23°C for 4 hours. After the reaction, the solution was sealed in a dialysis membrane (molecular weight cutoff 6000-8000) and unreacted substances were removed by dialysis with pure water for 3 days. Subsequently, freeze-drying was performed to obtain 238 mg of βCD-PRX (hereinafter sometimes referred to as "PO3Et2-βCD-PRX") in which the hydroxyl groups were substituted with diethyl propylphosphonate (yield 73.5%). [ka]

[0134] 40 mg (1.69 μmol, phosphonate ester molars: 16 μmol) of the aforementioned PO3Et2-βCD-PRX was weighed into a glass vial, and 1.33 mL of pure water and 1480 μL of 5N sodium hydroxide aqueous solution were added to dissolve the PO3Et2-PRX. After stirring at 50°C for 24 hours, the solution was sealed in a dialysis membrane (molecular weight cutoff 6000-8000) and dialyzed against pure water for 3 days to remove unreacted substances. Subsequently, freeze-drying was performed to obtain 31.5 mg of βCD-PRX modified with sodium propylphosphonate salt (hereinafter sometimes referred to as "PO3Na2-βCD-PRX") (yield 79.2%).

[0135] The PO3Et2-βCD-PRX and PO3Na2-βCD-PRX obtained above were dissolved in deuterated DMSO at a concentration of 10 mg / mL. 11H NMR measurements were performed. For comparison, the same measurements were also performed on the raw material, unmodified βCD-PRX. The results are shown in Figure 8. In PO3Et2-βCD-PRX, new peaks originating from the ethyl group of the phosphonic acid ester were observed at 1.24 and 3.99 ppm, confirming that diethyl propylphosphonate had been chemically modified. Based on the integral area ratio of the proton at the β-CD1 position in the polyrotaxane (4.84 ppm) and the peak originating from the ethylene group of the phosphonic acid ester (3.99 ppm), the number of modifications by diethyl propylphosphonate to the polyrotaxane was 9.5, which translates to 0.74 modifications to the α-CD1 molecule in the polyrotaxane. Furthermore, in PO3Na2-βCD-PRX, the peak originating from the ethyl group in the phosphonic acid ester disappeared, confirming that the ethyl group was deprotected to obtain PO3Na2-βCD-PRX.

[0136] <Example 21: Preparation of coated culture substrate 1> The solid sample of propylphosphonic acid-modified PRX-b-PBnMA prepared in Example 19 was dissolved in DMSO to a concentration of 1 mg / mL. This solution was passed through a PTFE membrane filter (Advantec Toyo, model 13JP050AN) to prepare a coating solution. The fixation amount was 5.3 μg / cm³. 2 The coating solution was dropped into the center of each well of a 24-well cell culture plate (Thermo Fisher Scientific, model 142475) in the manner described. The plate, with the lid removed, was placed in a dry oven (Tokyo Rikakikai, model VOS-310C) and dried at 60°C under atmospheric pressure. Afterwards, it was washed twice with phosphate buffer solution (hereinafter sometimes referred to as "PBS") (Fujifilm Wako Pure Chemical Industries).

[0137] <Example 22: Preparation of coated culture substrate 2> Example 21 and the amount of propylphosphonic acid-modified PRX-b-PBnMA fixed at 2.6 μg / cm³ 2 The 24-well cell culture plates were coated using the same method, except that the coating solution was added dropwise to achieve the desired result.

[0138] <Comparative Example 7: Preparation of Coated Culture Substrate 3> The 24-well cell culture plates were used in the experiment without any coating treatment.

[0139] <Comparative Example 8: Preparation of Coated Culture Substrate 4> Using unmodified PRX-b-PBnMA, the raw material for Example 19, a coating solution was prepared in the same manner as in Example 21, with a fixation amount of 2.6 μg / cm³. 2 The coating solution was added dropwise to achieve the desired result. Drying and washing were performed in the same manner as in Example 21, and the sample was used for testing.

[0140] <Example 23: Static Contact Angle Measurement> The static contact angles of the plate surfaces prepared in Examples 21 and 22, and Comparative Examples 7 and 8 were measured. 1 μL of pure water was dropped onto the plate surface, and the contact angle was measured using a contact angle meter (Eiko Seiki, OCA20) by the droplet method. Three measurements were taken for each plate, and the average value was taken as the plate's contact angle. Three plates were measured in the same manner, and the average value ± standard deviation was calculated. The results are shown in Table 3. It was found that the contact angle did not change significantly with each PRX-b-PBnMA coating.

[0141] <Example 24: X-ray photoelectron spectroscopy measurement> X-ray photoelectron spectroscopy (hereinafter sometimes referred to as "XPS") measurements were performed on the plate surfaces prepared in Examples 21 and 22, and Comparative Examples 7 and 8, using a K-α (Thermo Fisher Scientific) analyzer. The resulting percentage of phosphorus atoms is shown in Table 3. Coating the propylphosphonic acid-modified PRX-b-PBnMA plates of Examples 21 and 22 increased the percentage of phosphorus atoms, confirming that the coating was effective.

[0142] <Example 25: Cell adhesion test> Cell adhesion was evaluated using human bone marrow-derived mesenchymal stem cells (hereinafter sometimes referred to as "hMSCs") (Lonza, model number PT-2501) on plates prepared in Examples 21 and 22, and Comparative Examples 7 and 8. 2.5 × 10⁶ hMSCs were placed in each well. 4 cells / cm 2 Cells were seeded at a density and cultured for 5 days at 37°C in a humid atmosphere containing 5% CO2 using the growth medium MSCGM BulletKit (Lonza, model PT-3001) until the cell density reached confluence. The growth medium was then replaced with Mesenchymal Stem Cell Osteogenic Differentiation Medium (PromoCell, model C-28013), and the cells were cultured for 14 days. The medium was replaced with fresh medium every 3-4 days. After 14 days of differentiation induction, the formation of calcified nodules in the cells was evaluated by staining with Alizarin Red S. After washing the cells twice with PBS, they were fixed by treatment with 4% paraformaldehyde solution (Fujifilm Wako Pure Chemical Industries) at 23°C for 10 minutes. The cells were then washed twice with ultrapure water and stained with Alizarin Red S solution at 23°C for 10 minutes. After removing the staining solution, the cells were washed four times with ultrapure water and observed under a microscope (Keyence, BZ-X700). The stained area was analyzed using the analysis software included with the microscope. Three points were calculated for each well prepared under the same conditions, and the mean ± standard deviation was determined. The results are shown in Table 3. The improvement in osteoblast differentiation efficiency was confirmed by coating with propylphosphonic acid-modified PRX-b-PBnMA from Examples 21 and 22.

[0143] [Table 3]

Claims

1. An axial molecule having an axial portion and two stopper portions connected to both ends of the axial portion, At least one cyclic molecule penetrated by the aforementioned axial molecule, A polyrotaxane or a salt thereof comprising, The aforementioned cyclic molecule has a hydrogen atom in at least one hydroxyl group that is in the following formula (I): 【Chemistry 1】 [In the formula, L represents a divalent organic group, R １ and R ２ Each of these independently represents a monovalent organic group. A polyrotaxane or a salt thereof, which is a cyclodextrin substituted with a group represented by and which may have at least one other hydroxyl group further modified or substituted.

2. The polyrotaxane or salt thereof according to claim 1, wherein L in formula (I) represents an alkylene having 2 to 10 carbon atoms.

3. R in formula (I) １ and R ２ The polyrotaxane or salt thereof according to claim 1, wherein each independently represents a hydrogen atom or an alkyl group.

4. R in formula (I) １ and R ２ The polyrotaxane or salt thereof according to claim 1, wherein each represents either a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.

5. R in formula (I) １ and R ２ The polyrotaxane or salt thereof according to claim 1, wherein each represents a hydrogen atom.

6. The polyrotaxane or salt thereof according to claim 1, wherein the cyclodextrin is α-cyclodextrin or β-cyclodextrin.

7. The polyrotaxane or salt thereof according to claim 1, wherein the average number of hydroxyl groups per molecule in which hydrogen atoms are substituted with the group represented by formula (I) is 2.5 or more.

8. The polyrotaxane or salt thereof according to claim 1, wherein the shaft portion comprises polyethylene glycol, or a copolymer comprising polyethylene glycol and polypropylene glycol.

9. The stopper portion is defined by the following formula (II): 【Chemistry 2】 [In the formula, R ａ represents a hydrogen atom or a methyl group, R ｂ This represents a benzyl, alkyl, alkenyl, or phenyl compound, which may have substituents. R ｃ This is a hydrogen atom, methyl, mercapto, -S-R ｓ , -S-C(=S)-R ｓ or -S-C(=S)-S-R ｓ Show, Here R ｓ This represents benzyl, alkyl, alkenyl, or phenyl. m represents an integer between 1 and 1000. A polyrotaxane or salt thereof according to claim 1, comprising a group represented by [the group].

10. A method for producing polyrotaxane A or a salt thereof, comprising the step of contacting polyrotaxane B or a salt thereof with a base in an aqueous medium to convert it into polyrotaxane A or a salt thereof, The aforementioned polyrotaxane A is An axial molecule having an axial portion and two stopper portions connected to both ends of the axial portion, At least one cyclic molecule penetrated by the aforementioned axial molecule, Equipped with, The aforementioned cyclic molecule has a hydrogen atom in at least one hydroxyl group that is in the following formula (I): 【Transformation 3】 [In the formula, L represents a divalent organic group, R １ and R ２ Each of these independently represents a hydrogen atom. A cyclodextrin which is substituted with a group represented by and which may have at least one other hydroxyl group further modified or substituted, It is a polyrotaxane, The polyrotaxane B is the same as the polyrotaxane A, in which the R in formula (I) １ and R ２ However, the manufacturing method is for a polyrotaxane in which each hydrogen atom represents an alkyl group.

11. Polyrotaxane C or a salt thereof is dissolved in an organic solvent in the following formula (III): 【Chemistry 4】 [In the formula, L, R １ and R ２ This is similar to that in polyrotaxane B, where X represents a monovalent leaving group. The process further includes contacting a compound represented by and a base to convert it into polyrotaxane B or a salt thereof, The polyrotaxane C is a polyrotaxane in which, in the polyrotaxane A, the hydrogen atom in at least one hydroxyl group is not substituted with the group represented by formula (I). The manufacturing method according to claim 10.

12. A coating agent for cell culture substrates, comprising a polyrotaxane or a salt thereof as described in any one of claims 1 to 9.

13. A coating agent according to claim 12 for promoting osteoblast differentiation of mesenchymal stem cells.

14. A cell culture method for promoting osteoblast differentiation of mesenchymal stem cells, A method comprising culturing mesenchymal stem cells on the surface of a substrate coated with a composition containing a polyrotaxane or a salt thereof according to any one of claims 1 to 9.

15. A cell culture substrate comprising a substrate coated with a composition containing a polyrotaxane or a salt thereof as described in any one of claims 1 to 9.

16. A composition containing a polyrotaxane or a salt thereof as described in any one of claims 1 to 9.

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