Protein folding agent, compound, protein folding method, pharmaceutical composition, and agent for suppressing cytotoxicity by transition metal

Abstract

Provided is a protein folding agent comprising at least one compound selected from the group consisting of (A)-(B) below: (A) cyclic polyamines that comprise a disulfide bond or a thiol group in a quantity of one or greater and that are capable of binding to a transition metal ion; (B) compounds in which two or more of the cyclic polyamines (A) are bonded by a disulfide bond between the thiol groups; (C) complexes of the cyclic polyamines (A) or the compounds (B) and a transition metal ion; (D) salts of the cyclic polyamines (A) or the compounds (B); and (E) solvates of the cyclic polyamines (A) or the compounds (B).

Description

Protein folding agent, compound, protein folding method, pharmaceutical composition, and inhibitor of transition metal-induced cytotoxicity

[0001] The present invention relates to a protein folding agent, a compound, a protein folding method, a pharmaceutical composition, and an inhibitor of transition metal-induced cytotoxicity. This application claims priority to Japanese Patent Application No. 2023-140709 filed on August 31, 2023, and Japanese Patent Application No. 2024-082219 filed on May 20, 2024, the contents of which are incorporated herein by reference.

[0002] Protein folding is a reaction that restores a denatured protein to its native, active structure. Denatured proteins tend to aggregate irreversibly, so protein folding efficiency is usually low. Compounds that promote protein folding are important additives for efficiently obtaining active proteins, and are therefore important in the production of industrially important proteins, such as pharmaceutical proteins. Denatured proteins are also causative agents of folding diseases, such as neurodegenerative disorders. Therefore, compounds that promote folding are also important as drug candidates for the prevention and / or treatment of folding diseases.

[0003] Proteins can be broadly divided into two types: those with intramolecular disulfide bonds and those without. In vivo, folding occurs via different mechanisms. The folding of proteins without disulfide bonds is promoted by molecular chaperones, such as GroEL. The folding of proteins with disulfide bonds is promoted by oxidoreductases, such as protein disulfide isomerase (PDI), which form and exchange disulfide bonds. Proteins without disulfide bonds are often found intracellularly, i.e., in the cytoplasm. On the other hand, many membrane proteins and proteins secreted extracellularly contain disulfide bonds. Many proteins useful as pharmaceuticals, such as antibodies and insulin, also contain disulfide bonds.

[0004] In general, proteins irreversibly aggregate in the presence of transition metal ions such as nickel and copper ions (Non-Patent Document 1). This property not only limits the conditions for protein synthesis, but is also thought to be a cause of protein-degenerative diseases (Non-Patent Documents 2-6).

[0005] Thiol compounds that promote redox reactions have been reported as compounds that promote the folding of proteins having disulfide bonds, such as oxidized glutathione (GSSH), reduced glutathione (GSH), and nitrogen-containing thiol compounds (Patent Document 1).

[0006] Japanese Patent Application Laid-Open No. 2022-135954

[0007] R. John et al., Probing the Role of Cu(II) Ions on Protein Aggregation Using Two Model Proteins. ACS Omega 2021, 6, 35559-35571.AC Kim et al., Metal Ion Effects on Aβ and Tau Aggregation. International Journal of Molecular Sciences 2018, 19(1), 128.A. Prakash et al., Brain biometals and Alzheimer's disease - boon or bane? International Journal of Neuroscience 2017, 127, 99.PB Gabriel et al. Aggregation of biologically important peptides and proteins: inhibition or acceleration depending on protein and metal ion concentrations. RSC Advances 2020, 10, 215.M. Bisaglia, and L. Bubacco, Copper Ions and Parkinson's Disease: Why Is Homeostasis So Relevant? Biomolecules 2020, 10, 195.M. Tamas et al. Heavy Metals and Metalloids As a Cause for Protein Misfolding and Aggregation. Biomolecules 2014, 4, 252.

[0008] Among conventional folding agents such as glutathione (GSH / GSSH), none are known to be capable of promoting protein folding in the presence of transition metal ions.

[0009] Therefore, an objective of the present invention is to provide a protein-folding agent that can promote protein folding in the presence of transition metal ions, a compound that can be used for the protein-folding agent, a protein folding method that uses the protein-folding agent, a pharmaceutical composition containing the protein-folding agent, and an agent for suppressing cytotoxicity caused by transition metals.

[0010] The present invention includes the following aspects. [1] A protein-folding agent comprising at least one compound selected from the group consisting of (A) and (B) below: (A) a cyclic polyamine containing one or more thiol groups or disulfide bonds and capable of binding to a transition metal ion; (B) a compound in which two or more cyclic polyamines of (A) are bonded together via disulfide bonds between the thiol groups; (C) a complex of the cyclic polyamine of (A) or the compound of (B) with a transition metal ion; (D) a salt of the cyclic polyamine of (A) or the compound of (B); and (E) a solvate of the cyclic polyamine of (A) or the compound of (B). [2] The protein-folding agent according to [1], wherein the cyclic polyamine of (A) is a compound represented by the following general formula (A):

[0011] [In the formula, Rx １ ~Rx ３ each independently represents a hydrogen atom or a monovalent organic group. １ ~Rx ３ At least one of Rx is a monovalent organic group containing one or more thiol groups. １ ~Rx ３ When Y contains two or more thiol groups in total, the thiol groups may be bonded to each other to form a disulfide bond. １ ~Y ３ each independently represents an alkylene group having 1 to 5 carbon atoms; m0 represents an integer of 1 to 3; when m0 is an integer of 2 or more, there are 2 or more Rx ３ may be the same or different, and two or more Y ３ may be the same as or different from each other.

[0012] [3] The protein-folding agent according to [2], wherein the cyclic polyamine (A) is a compound represented by the following general formula (A-1) or (A-2):

[0013] [In the formula, Rx ２ and Rx ３ each independently represents a hydrogen atom or a monovalent organic group. １ ~Y ３ each independently represents an alkylene group having 1 to 5 carbon atoms. １ and Lx ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom; m0 represents an integer of 1 to 3; when m0 is an integer of 2 or more, there are 2 or more Rx ３ may be the same or different, and two or more Y ３ may be the same as or different from each other.

[0014] [4] The protein-folding agent according to [3], wherein the cyclic polyamine (A) is a compound represented by the following general formula (A-1-1), and the compound (B) is a compound represented by the following general formula (B-1-1):

[0015] [In the formula, Y １ , Y ２ , Y １１ , Y １２ , Y ２１ , and Y ２２ each independently represents an alkylene group having 1 to 5 carbon atoms; R, R １ , and R ２ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; L, L １ , and L ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.]

[0016] [5] The protein-folding agent according to [4], which comprises at least one compound selected from the group consisting of the compound (B), a metal complex of the compound (B), a salt of the compound (B), and a solvate of the compound (B). [6] The protein-folding agent according to [4], wherein the compound (B) is a compound represented by the following formula (B-1-1-1):

[0017]

[0018] [7] The protein-folding agent according to [4], wherein the cyclic polyamine (A) is a compound represented by the following formula (A-1-1-1), and the compound (B) is a compound represented by the following formula (B-1-1-1):

[0019]

[0020] [8] The protein-folding agent according to [3], wherein the cyclic polyamine (A) is a compound represented by the following general formula (A-1-2), and the compound (B) is a compound represented by the following general formula (B-1-2):

[0021] [In the formula, Y １ , Y ２ , Y ３ , Y １１ , Y １２ , Y １３ , Y ２１ , Y ２２ , and Y ２３ each independently represents an alkylene group having 1 to 5 carbon atoms; R, R １ , and R ２ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; L, L １ , and L ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.

[0022] [9] The protein-folding agent according to [3], wherein the cyclic polyamine (A) is a compound represented by the following formula (A-1-2-1), and the compound (B) is a compound represented by the following formula (B-1-2-1):

[0023]

[0024]

[10] The protein-folding agent according to any one of [1] to [9], comprising at least one compound selected from the group consisting of the cyclic polyamine (A), a metal complex thereof, a salt thereof, and a solvate thereof, and at least one compound selected from the group consisting of the compound (B), a metal complex thereof, a salt thereof, and a solvate thereof.

[11] The protein-folding agent according to [3], wherein the cyclic polyamine (A) is a compound represented by the following formula (A-2-1):

[0025] [In the formula, Y １ ~Y ３ each independently represents an alkylene group having 1 to 5 carbon atoms; R represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; L １ , and L ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.]

[0026]

[12] The protein-folding agent according to

[11] , wherein the cyclic polyamine (A) is a compound represented by the following formula (A-2-1-1):

[0027]

[0028]

[13] The protein folding agent according to any one of [1] to

[12] , which is used for folding a protein in the presence of a transition metal ion.

[14] The protein folding agent according to

[13] , which is used for folding a protein containing a transition metal ion.

[15] The protein folding agent according to

[13] or

[14] , wherein the transition metal ion is at least one selected from the group consisting of copper ions, nickel ions, iron ions, manganese ions, zinc ions, and molybdenum ions.

[16] A compound represented by the following general formula (B-1), (A-1-2), or (A-2):

[0029] [In general formula (B-1), Rx１２ , Rx １３ , Rx ２２ and Rx ２３ each independently represents a hydrogen atom or a monovalent organic group. １１ , Y １２ , Y １３ , Y ２１ , Y ２２ , and Y ２３ each independently represents an alkylene group having 1 to 5 carbon atoms. １ and Lx ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom. m01 and m02 each independently represent an integer of 1 to 3. When m01 is an integer of 2 or more, two or more Rx １３ may be the same or different, and two or more Y １３ When m02 is an integer of 2 or more, two or more Rx ２３ may be the same or different, and two or more Y ２３ In general formula (A-1-2), Y may be the same as or different from each other. １ ~Y ３ each independently represents an alkylene group having 1 to 5 carbon atoms; each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; and L represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom. ３ represents a hydrogen atom or a monovalent organic group. １ ~Y ３ each independently represents an alkylene group having 1 to 5 carbon atoms. １ and Lx ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom; m0 represents an integer of 1 to 3; when m0 is an integer of 2 or more, two or more Rx ３ may be the same or different, and two or more Y ３ may be the same as or different from each other.

[0030]

[17] The compound according to

[16] , which is a compound represented by the following formula (B-1-1-1), (A-1-2-1), (B-1-2-1), or (A-2-1-1):

[0031]

[0032]

[0033]

[18] A method for folding a protein, comprising a step of incubating a protein in the presence of the protein-folding agent according to any one of [1] to

[12] .

[19] The method for folding a protein according to

[18] , wherein the protein is an unfolded protein or a misfolded protein.

[20] The method for folding a protein according to

[18] or

[19] , wherein the incubation is carried out in the presence of a transition metal ion.

[21] The method for folding a protein according to

[20] , wherein the folded protein after the incubation step contains the transition metal ion.

[22] The method for folding a protein according to

[20] , wherein the transition metal ion is at least one selected from the group consisting of copper ions, nickel ions, iron ions, manganese ions, zinc ions, and molybdenum ions.

[23] A pharmaceutical composition comprising the protein-folding agent according to any one of [1] to

[12] and a pharmaceutically acceptable carrier.

[24] The pharmaceutical composition according to

[23] , which is used for treating or preventing a disease caused by protein aggregation.

[25] The pharmaceutical composition according to

[24] , wherein the disease caused by protein aggregation is a folding disease.

[26] The pharmaceutical composition according to

[25] , wherein the folding disease is at least one selected from the group consisting of amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies, spinocerebellar degeneration, frontotemporal dementia, Pick's disease, Creutzfeldt-Jakob disease, familial amyloidotic polyneuropathy, and type 2 diabetes.

[27] An inhibitor of transition metal-induced cytotoxicity, comprising at least one compound selected from the group consisting of (A) and (B) below: (A) a cyclic polyamine containing one or more thiol groups or disulfide bonds and capable of binding to a transition metal ion; (B) a compound in which two or more cyclic polyamines of (A) are bonded together via disulfide bonds between the thiol groups; (C) a complex of the cyclic polyamine of (A) or the compound of (B) with a transition metal ion; (D) a salt of the cyclic polyamine of (A) or the compound of (B); and (E) a solvate of the cyclic polyamine of (A) or the compound of (B).

[0034] According to the present invention, there are provided a protein-folding agent capable of promoting protein folding in the presence of transition metal ions, a compound usable for the protein-folding agent, a protein folding method using the protein-folding agent, and a pharmaceutical composition containing the protein-folding agent.

[0035] The structure of Compound 5, which is an intermediate of Compounds (A-1-1-1) and (B-1-1-1) synthesized in Experimental Example 1, was confirmed. １ The results of H NMR measurement are shown. The results of MALDI-TOF mass spectrometry measurement are shown, which confirmed the structure of Compound 5, which is an intermediate of Compounds (A-1-1-1) and (B-1-1-1), synthesized in Experimental Example 1. The results of H NMR measurement are shown. The results of MALDI-TOF mass spectrometry measurement are shown, which confirmed the structure of Compound 5, which is an intermediate of Compounds (A-1-1-1) and (B-1-1-1), synthesized in Experimental Example 1. １ The results of H NMR measurement are shown. The results of MALDI-TOF mass spectrometry measurement are shown, which confirmed the structure of the compound (B-1-1-1) synthesized in Experimental Example 1. The results of MALDI-TOF mass spectrometry measurement are shown. The results of H NMR measurement are shown. The results of MALDI-TOF mass spectrometry ... １ 1 shows the results of H NMR measurement. 1 shows the results of MALDI-TOF mass spectrometry measurement, which confirmed the structure of compound (A-1-1-1) synthesized in Experimental Example 1. 1 shows the results of a folding test of reduced modified bovine pancreatic trypsin inhibitor (BPTI) using oxidized glutathione (GSSG) and reduced glutathione (GSH) in Experimental Example 1. 1 shows the results of a folding test of reduced modified BPTI using compound (A-1-1-1) and compound (B-1-1-1) in Experimental Example 1. 1 shows the results of a folding test of reduced modified BPTI using compound (A-1-1-1) and compound (B-1-1-1) in Experimental Example 1. 1 shows the results of a folding test of reduced modified BPTI using compound (A-1-1-1) and compound (B-1-1-1) using nickel (II) ions (Ni ２＋ 1 shows the results of a folding test of reduced and modified BPTI in the presence of copper (II) ions (Cu) using compound (A-1-1-1) and compound (B-1-1-1) in Experimental Example 1. ２＋1 shows the results of a folding test of reduced modified BPTI in the presence of zinc (II) ions (Zn ) using GSSG and GSH, or compounds (A-1-1-1) and (B-1-1-1) in Experimental Example 1. ２＋ 1 shows the results of a folding test of reduced and modified BPTI in the presence of copper (II) ions (Cu ２＋ The results of a test evaluating the inhibitory effect of compound (B-1-1-1) on the cytotoxicity of copper (II) ions (Cu ２＋ The structure of Compound 8, which is an intermediate of Compounds (A-1-2-1) and (B-1-2-1), synthesized in Experimental Example 2, was confirmed. １ The results of H NMR measurement are shown. The results of MALDI-TOF mass spectrometry measurement are shown, which confirmed the structure of Compound 8, which is an intermediate of Compounds (A-1-2-1) and (B-1-2-1) synthesized in Experimental Example 2. The results of H NMR measurement are shown. The results of MALDI-TOF mass spectrometry measurement are shown, which confirmed the structure of Compound 8, which is an intermediate of Compounds (A-1-2-1) and (B-1-2-1) synthesized in Experimental Example 2. １ The results of H NMR measurement are shown. The results of MALDI-TOF mass spectrometry measurement are shown, which confirmed the structure of the compound (B-1-2-1) synthesized in Experimental Example 2. The results of MALDI-TOF mass spectrometry measurement are shown. The results of H NMR measurement are shown. The results of MALDI-TOF mass spectrometry ... １ 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. ２＋1 shows the results of a folding test of reduced modified BPTI in the presence of zinc (II) ions (Zn ２＋ 1 shows the results of a folding test of reduced and modified BPTI in the presence of zinc (II) ions (Zn ２＋ The results of the folding test of reduced and modified BPTI in the presence of BPTI were shown. The structure of the compound (B-2-1-1) synthesized in Experimental Example 3 was confirmed. １ 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. 1H NMR measurement results are shown. ２＋ 1 shows the results of a folding test of reduced and modified BPTI in the presence of nickel (II) ions (Ni ２＋ 1 shows the results of a folding test of reduced modified BPTI in the presence of a catalytic amount of compound (B-2-1-1) in Experimental Example 3. 1 shows the results of a folding test of reduced modified BPTI in the presence of a catalytic amount of compound (B-2-1-1) in Experimental Example 3. ２＋ 1 shows the results of a folding test of reduced and modified BPTI in the presence of a catalytic amount of compound (B-2-1-1) in Experimental Example 3. ２＋ 1 shows the results of a folding test of reduced modified BPTI in the presence of copper (II) ions (Cu) using GSSG and GSH, or compounds (A-1-1-1) and (B-1-1-1) in Experimental Example 4. ２＋ 1 shows the results of a folding test of reduced, denatured RNAase A in the presence of ATP.

[0036] A numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0037] The term "comprise" means that it may contain components other than the target component. The term "consist of" means that it does not contain components other than the target component. The term "consist essentially of" means that it does not contain components other than the target component in a manner that would exert a special function (such as a manner that would completely lose the effects of the invention). In this specification, when "comprise" is used, it includes both "consist of" and "consist essentially of" embodiments.

[0038] [Protein Folding Agent] A first aspect of the present disclosure is a protein folding agent. The protein folding agent according to this aspect comprises at least one compound selected from the group consisting of the following (A) and (B): (A) a cyclic polyamine containing one or more thiol groups or disulfide bonds and capable of binding to a transition metal ion (hereinafter also referred to as "compound (A)"); (B) a compound in which two or more cyclic polyamines of (A) are bonded together via disulfide bonds between the thiol groups (hereinafter also referred to as "compound (B)"); (C) a complex of the cyclic polyamine of (A) or the compound of (B) with a transition metal ion (hereinafter also referred to as "compound (C)"); (D) a salt of the cyclic polyamine of (A) or the compound of (B) (hereinafter also referred to as "compound (D)"); and (E) a solvate of the cyclic polyamine of (A) or the compound of (B) (hereinafter also referred to as "compound (E)").

[0039] <Compound (A)> In this specification, the term "cyclic polyamine" refers to a compound containing an aliphatic heterocycle containing two or more nitrogen atoms in the ring structure. The cyclic polyamine preferably contains two or more amino groups selected from the group consisting of secondary amino groups and tertiary amino groups in the ring structure. The number of thiol groups or disulfide bonds contained in compound (A) is, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and even more preferably 1.

[0040] Compound (A) can be bonded to a transition metal ion through a coordinate bond. Transition metals are elements that exist between Groups 3 and 12 of the periodic table. Examples of transition metal ions include, but are not limited to, copper ions, nickel ions, iron ions, manganese ions, zinc ions, and molybdenum ions.

[0041] Examples of the compound (A) include compounds represented by the following general formula (A).

[0042] [In the formula, Rx １ ~Rx ３ each independently represents a hydrogen atom or a monovalent organic group. １ ~Rx ３ At least one of Rx is a monovalent organic group containing one or more thiol groups. １ ~Rx ３ When Y contains two or more thiol groups in total, the thiol groups may be bonded to each other to form a disulfide bond. １ ~Y ３ each independently represents an alkylene group having 1 to 5 carbon atoms; m0 represents an integer of 1 to 3; when m0 is an integer of 2 or more, there are 2 or more Rx ３ may be the same or different, and two or more Y ３ may be the same as or different from each other.

[0043] In the formula (A), Rx １ ~Rx ３The monovalent organic group in Rx may be a monovalent hydrocarbon group which may have a substituent. The monovalent hydrocarbon group may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group. １ ~Rx ３ The monovalent organic group in the formula (I) is an organic group having 1 to 20 carbon atoms, preferably 1 to 16 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms.

[0044] Rx １ ~Rx ３ The aliphatic hydrocarbon group in the formula (I) may have 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms, even more preferably 1 or 2 carbon atoms, and particularly preferably 1 carbon atom. The aliphatic hydrocarbon group may be saturated or unsaturated, but is preferably saturated. The aliphatic hydrocarbon group may be linear, branched, or may contain a ring structure. The aliphatic hydrocarbon group is preferably linear or branched, and more preferably linear. The aliphatic hydrocarbon group is preferably a linear or branched alkyl group. The linear alkyl group may have 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms, even more preferably 1 or 2 carbon atoms, and particularly preferably 1 carbon atom. The branched alkyl group may have 3 to 10 carbon atoms, preferably 3 to 6 carbon atoms, more preferably 3 or 4 carbon atoms, and even more preferably 3 carbon atoms.

[0045] Rx １ ~Rx ３ The aromatic hydrocarbon group in Rx is a hydrocarbon group containing one or more aromatic rings. １ ~Rx ３The aromatic hydrocarbon group in the formula (I) may have 1 to 20 carbon atoms, preferably 1 to 16 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms. The aromatic ring contained in the aromatic hydrocarbon group may be a monocyclic or polycyclic ring, but a monocyclic ring is preferred. The aromatic ring may be an aromatic hydrocarbon ring or an aromatic heterocyclic ring. Examples of the aromatic hydrocarbon ring include a benzene ring and a naphthalene ring. Examples of the aromatic heterocyclic ring include a nitrogen-containing aromatic heterocyclic ring such as pyrrole, a sulfur-containing aromatic heterocyclic ring such as thiophene, and an oxygen-containing aromatic heterocyclic ring such as furan. Rx １ ~Rx ３ Examples of the aromatic hydrocarbon group in the formula (I) include a group in which one hydrogen atom has been removed from an aromatic ring, a group in which one carbon atom of an aromatic ring has been substituted with an alkylene group, and a group in which an aromatic ring is interposed midway through an alkylene group.

[0046] Rx １ ~Rx ３ The monovalent hydrocarbon group in may have a substituent. Examples of the substituent include a thiol group, a hydroxy group, an amino group, a halogen atom, an alkoxy group, and an acyl group. The substituent may be a divalent group that substitutes a methylene group. Examples of such a substituent include a carbonyl group, an ether bond (—O—), an ester bond (—COO—), and an amide bond (—NHCO—).

[0047] Rx １ ~Rx ３ At least one of the thiol group-containing groups is a monovalent organic group containing a thiol group (hereinafter also referred to as a "thiol group-containing group"). The number of thiol group-containing groups may be, for example, 1 to 5, preferably 1 to 3, and more preferably 1 or 2. When there are two or more thiol group-containing groups, two thiol group-containing groups may be bonded to each other to form a disulfide bond. The number of thiol groups contained in one thiol group-containing group is preferably 1 to 5, more preferably 1 to 3, even more preferably 1 or 2, and even more preferably 1.

[0048] In the formula (A), Y １ ~Y ３The alkylene group having 1 to 5 carbon atoms in the formula (I) may be linear or branched, but is preferably linear. The alkylene group more preferably has 1 to 4 carbon atoms, and even more preferably has 2 or 3 carbon atoms.

[0049] In the formula (A), m0 is preferably 1 or 2.

[0050] Examples of the compound (A) include a compound represented by the following general formula (A-1) (hereinafter also referred to as "compound (A-1)") and a compound represented by the following general formula (A-2) (hereinafter also referred to as "compound (A-2)").

[0051] [In the formula, Rx ２ and Rx ３ each independently represents a hydrogen atom or a monovalent organic group. １ ~Y ３ each independently represents an alkylene group having 1 to 5 carbon atoms. １ and Lx ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom; m0 represents an integer of 1 to 3; when m0 is an integer of 2 or more, there are 2 or more Rx ３ may be the same or different, and two or more Y ３ may be the same as or different from each other.

[0052] In the formulas (A-1) and (A-2), Rx ２ , Rx ３ , Y １ ~Y ３ , and m0 represents Rx in the formula (A). ２ , Rx ３ , Y １ ~Y ３ , and m0, respectively.

[0053] In the formulas (A-1) and (A-2), Lx １ and Lx ２ The alkylene group having 1 to 10 carbon atoms in the formula (Lx) may be linear or branched, but is preferably linear. １ and Lx ２The alkylene group in the formula (I) preferably has 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms, even more preferably 1 to 4 carbon atoms, still more preferably 1 to 3 carbon atoms, and particularly preferably 2 carbon atoms. １ and Lx ２ The alkylene group in may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom. １ and Lx ２ Examples of the group containing at least one selected from the group consisting of an oxygen atom and a nitrogen atom that the alkylene group may contain include an ether bond (—O—), —NR Ｌ A group represented by - (R Ｌ is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms), an ester bond (—COO—), and an amide bond (—CONH—). Ｌ is preferably a hydrogen atom, a methyl group, or an ethyl group, more preferably a hydrogen atom or a methyl group. １ and Lx ２ represents a methylene group (-CH ２ -) is -O-, -NR Ｌ It may be a group substituted with at least one selected from the group consisting of -, -COO- and -CONH-.

[0054] Lx １ and Lx ２ Specific examples of the alkylene group in the above formula include a methylene group, an ethylene group, an n-propylene group, an isopropylene group, an n-butylene group, an isobutylene group, a tert-butylene group, an n-pentylene group, an isopentylene group, a neopentylene group, a hexylene group, a heptylene group, an octylene group, a nonylene group, and a decylene group. ２ -) is a group containing at least one selected from the group consisting of an oxygen atom and a nitrogen atom (for example, -O-, -NR Ｌ -, -COO-, and / or -CONH-). １ and Lx ２may be a polyoxyalkylene group. Specific examples of the polyoxyalkylene group include a polyoxyethylene group and a polyoxypropylene group. １ and Lx ２ As the alkylene group, an alkylene group having 1 to 6 carbon atoms or a polyoxyalkylene group having 1 to 6 carbon atoms is preferred, an alkylene group having 1 to 6 carbon atoms, a polyoxyethylene group having 1 to 6 carbon atoms, or a polyoxypropylene group having 1 to 6 carbon atoms is more preferred, an ethylene group or a propylene group is even more preferred, and an ethylene group is particularly preferred.

[0055] Examples of the compound (A-1) include a compound represented by the following general formula (A-1-1) (hereinafter also referred to as "compound (A-1-1)") and a compound represented by the following general formula (A-1-2) (hereinafter also referred to as "compound (A-1-2)").

[0056] [In the formula, Y １ and Y ２ each independently represents an alkylene group having 1 to 5 carbon atoms; each R independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; and L represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.

[0057] [In the formula, Y １ ~Y ３ each independently represents an alkylene group having 1 to 5 carbon atoms; each R independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; and L represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.

[0058] In the formula (A-1-1), Y １ and Y ２ represents Y in the formula (A-1). １ and Y ２ are the same as Y. １ is preferably a linear alkylene group having 2 or 3 carbon atoms, more preferably a propylene group. １may be the same or different, but are preferably the same. ２ is preferably a linear alkylene group having 2 or 3 carbon atoms, more preferably an ethylene group. ２ may be the same or different, but are preferably the same. １ ~Y ３ represents Y in the formula (A-1). １ ~Y ３ are the same as Y. １ ~Y ３ Y is preferably a linear alkylene group having 2 or 3 carbon atoms, more preferably an ethylene group. １ ~Y ３ may be the same or different, but are preferably the same.

[0059] In the formulas (A-1-1) and (A-1-2), the alkyl group having 1 to 3 carbon atoms for R may be linear or branched, but is preferably linear. The linear alkyl group preferably has 1 or 2 carbon atoms, and more preferably has 1 carbon atom. When the alkyl group for R has 3 carbon atoms, it may be a branched alkyl group. Examples of the alkyl group for R include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group, and a methyl group or an ethyl group is preferred, and a methyl group is more preferred. In the formulas (A-1-1) and (A-1-2), the three Rs may be the same or different, but are preferably the same. R is preferably a hydrogen atom.

[0060] In the formulas (A-1-1) and (A-1-2), L represents Lx in (A-1). １ L is preferably an alkylene group having 1 to 6 carbon atoms or a polyoxyalkylene group having 1 to 6 carbon atoms, more preferably an alkylene group having 1 to 6 carbon atoms, a polyoxyethylene group having 1 to 6 carbon atoms, or a polyoxypropylene group having 1 to 6 carbon atoms, still more preferably an ethylene group or a propylene group, and particularly preferably an ethylene group.

[0061] Examples of the compound (A-1-1) include a compound represented by the following general formula (A-1-1a) (hereinafter, also referred to as "compound (A-1-1a)").

[0062] [In the formula, Z １ and Z ２ each independently represents a single bond or a methylene group; each R independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; and each L independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.

[0063] In the formula (A-1-1a), two Z １ may be the same or different, but are preferably the same. １ In the formula (A-1-1a), two Z ２ may be the same or different, but are preferably the same. ２ is preferably a single bond.

[0064] In the formula (A-1-1a), the alkyl group for R preferably has 1 or 2 carbon atoms, more preferably 1 carbon atom. When the alkyl group for R has 3 carbon atoms, it may be a branched alkyl group. Examples of the alkyl group for R include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group, with a methyl group or an ethyl group being preferred, and a methyl group being more preferred. In the formula (A-1-1a), the three R may be the same or different, but are preferably the same. R is preferably a hydrogen atom.

[0065] In the formula (A-1-1a), the alkylene group for L may be linear or branched, but is preferably linear. The alkylene group for L preferably has 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms, even more preferably 1 to 4 carbon atoms, still more preferably 1 to 3 carbon atoms, and particularly preferably 2 carbon atoms. The alkylene group for L may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom. Examples of groups containing at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom that the alkylene group for L may contain include an ether bond (—O—), —NR Ｌ A group represented by - (R Ｌ is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms), an ester bond (—COO—), and an amide bond (—CONH—). Ｌ is preferably a hydrogen atom, a methyl group, or an ethyl group, and more preferably a hydrogen atom or a methyl group. L is a methylene group (—CH ２ -) is -O-, -NR Ｌ It may be a group substituted with at least one selected from the group consisting of -, -COO- and -CONH-.

[0066] Specific examples of the alkylene group for L include a methylene group, an ethylene group, an n-propylene group, an isopropylene group, an n-butylene group, an isobutylene group, a tert-butylene group, an n-pentylene group, an isopentylene group, a neopentylene group, a hexylene group, a heptylene group, an octylene group, a nonylene group, and a decylene group. ２ A part of the group (for example, —O—, —NR Ｌ-, -COO-, and / or -CONH-). L may be a polyoxyalkylene group. Specific examples of the polyoxyalkylene group include a polyoxyethylene group and a polyoxypropylene group. Among these, L is preferably an alkylene group having 1 to 6 carbon atoms or a polyoxyalkylene group having 1 to 6 carbon atoms, more preferably an alkylene group having 1 to 6 carbon atoms, a polyoxyethylene group having 1 to 6 carbon atoms, or a polyoxypropylene group having 1 to 6 carbon atoms, still more preferably an ethylene group or a propylene group, and particularly preferably an ethylene group.

[0067] The compound (A-1-1a) is preferably a compound represented by the following formula (A-1-1b) (hereinafter also referred to as "compound (A-1-1b)").

[0068] [In the formula, each R independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; and L represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.]

[0069] R in the formula (A-1-1b) is the same as R in the formula (A-1-1). R is preferably a hydrogen atom or a methyl group, more preferably a hydrogen atom.

[0070] L in the formula (A-1-1b) is the same as L in the formula (A-1-1). L is preferably an alkylene group having 1 to 6 carbon atoms or a polyoxyalkylene group having 1 to 6 carbon atoms, more preferably an alkylene group having 1 to 6 carbon atoms, a polyoxyethylene group having 1 to 6 carbon atoms, or a polyoxypropylene group having 1 to 6 carbon atoms, still more preferably an ethylene group or a propylene group, and particularly preferably an ethylene group.

[0071] A specific example of the compound (A-1-1b) is a compound represented by the following formula (A-1-1-1) (hereinafter also referred to as "compound (A-1-1-1)").

[0072]

[0073] Specific examples of the compound (A-1-2) include a compound represented by the following formula (A-1-2-1) (hereinafter also referred to as "compound (A-1-2-1)").

[0074]

[0075] Examples of the compound (A-2) include a compound represented by the following general formula (A-2-1) (hereinafter also referred to as "compound (A-2-1)").

[0076] [In the formula, Y １ ~Y ３ each independently represents an alkylene group having 1 to 5 carbon atoms; R represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; L １ , and L ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.]

[0077] In the formula (A-2-1), Y １ ~Y ３ and R represents Y in the formula (A-1-2). １ ~Y ３ , and R, respectively.

[0078] In the formula (A-2-1), L １ and L ２ As for Lx in (A-1), １ The same as above can be mentioned. １ and L ２ As L, an alkylene group having 1 to 6 carbon atoms or a polyoxyalkylene group having 1 to 6 carbon atoms is preferred, an alkylene group having 1 to 6 carbon atoms, a polyoxyethylene group having 1 to 6 carbon atoms, or a polyoxypropylene group having 1 to 6 carbon atoms is more preferred, an ethylene group or a propylene group is even more preferred, and an ethylene group is particularly preferred. １ and L ２ may be the same or different, but are preferably the same.

[0079] Specific examples of the compound (A-2-1) include a compound represented by the following formula (A-2-1-1) (hereinafter also referred to as "compound (A-2-1-1)").

[0080]

[0081] <Compound (B)> Compound (B) is a compound in which two or more molecules of compound (A) are bonded together via disulfide bonds between thiol groups. Compound (B) is, for example, a compound in which two molecules of compound (A) are bonded together via a disulfide bond (-S-S-). The two or more molecules of compound (A) bonded together via a disulfide bond may be the same compound or different compounds, but are preferably the same compound.

[0082] The compound (B) may be a compound in which two molecules of the compound (A-1) are bonded by a disulfide bond. The two molecules of the compound (A-1) bonded by a disulfide bond may be the same compound or different compounds, but are preferably the same compound.

[0083] Examples of the compound (B) include a compound represented by the following general formula (B-1) (hereinafter also referred to as "compound (B-1)"). The compound (B-1) is a compound in which two molecules of the compound (A-1) are bonded via a disulfide bond.

[0084] [In the formula, Rx １２ , Rx １３ , Rx ２２ and Rx ２３ each independently represents a hydrogen atom or a monovalent organic group. １ ~Y ３ each independently represents an alkylene group having 1 to 5 carbon atoms. １ and Lx ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom. m01 and m02 each independently represent an integer of 1 to 3. When m01 is an integer of 2 or more, two or more Rx １３ may be the same or different, and two or more Y １３When m02 is an integer of 2 or more, two or more Rx ２３ may be the same or different, and two or more Y ２３ may be the same as or different from each other.

[0085] In the formula (B-1), Y １１ , Y １２ , Y １３ , Y ２１ , Y ２２ , and Y ２３ The explanation for Y in formula (A-1) is as follows: １ , Y ２ , Y ３ It is the same as Y １１ , Y １２ , and Y １３ may be the same or different. ２１ , Y ２２ , and Y ２３ may be the same or different. １１ and Y ２１ may be the same or different, but are preferably the same. １２ and Y ２２ may be the same or different, but are preferably the same. １３ and Y ２３ may be the same or different, but are preferably the same. １１ , Y １２ , Y １３ , Y ２１ , Y ２２ , and Y ２３ is preferably an ethylene group or a propylene group.

[0086] Rx in the formula (B-1) １２ , Rx １３ , Rx ２２ and Rx ２３ The explanation for Rx in formula (A-1) is as follows: ２ and Rx ３ It is the same as Rx １２ and Rx １３ are preferably the same as each other, and Rx ２２ and Rx ２３ are preferably the same as each other, and Rx１２ , Rx １３ , Rx ２２ and Rx ２３ It is more preferable that Rx １２ , Rx １３ , Rx ２２ and Rx ２３ It is more preferable that all of are hydrogen atoms or all of are methyl groups.

[0087] Lx in the formula (B-1) １ and Lx ２ The explanation for Lx in the formula (A-1) is １ It is the same as Lx １ and Lx ２ may be the same or different, but are preferably the same.

[0088] The explanation for m01 and m02 in the formula (B-1) is the same as that for m0 in the formula (A-1). m01 and m02 may be the same or different from each other, but are preferably the same. m01 and m02 are preferably 1 or 2.

[0089] Examples of the compound (B-1) include a compound represented by the following general formula (B-1-1) (hereinafter also referred to as "compound (B-1-1)") and a compound represented by the following general formula (B-1-2) (hereinafter also referred to as "compound (B-1-2)"). The compound (B-1-1) is a compound in which two molecules of the compound (A-1-1) are bonded together via a disulfide bond. The compound (B-1-2) is a compound in which two molecules of the compound (A-1-2) are bonded together via a disulfide bond.

[0090] [In the formula, Y １１ , Y １２ , Y ２１ , and Y ２２ each independently represents an alkylene group having 1 to 5 carbon atoms; R １ , and R ２ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; L １ , and L ２each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.]

[0091] [In the formula, Y １１ , Y １２ , Y １３ , Y ２１ , Y ２２ , and Y ２３ each independently represents an alkylene group having 1 to 5 carbon atoms; R １ , and R ２ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; L １ , and L ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.]

[0092] In the formula (B-1-1), Y １１ , Y １２ , Y ２１ , and Y ２２ The explanation for Y in the formula (A-1-1) is as follows: １ , and Y ２ It is the same as two Y １１ may be the same or different, but are preferably the same. １２ may be the same or different, but are preferably the same. ２１ may be the same or different, but are preferably the same. ２２ may be the same or different, but are preferably the same. １１ and Y ２１ may be the same or different, but are preferably the same. １２ and Y ２２ may be the same or different, but are preferably the same. １１ and two Y ２１ It is preferable that all two Y １２ and two Y ２２ It is preferable that all of Y are the same. １１ and Y２１ Y is preferably a linear alkylene group having 2 or 3 carbon atoms, and more preferably a propylene group. １２ and Y ２２ As the alkyl group, a linear alkylene group having 2 or 3 carbon atoms is preferred, and an ethylene group is more preferred.

[0093] In the formula (B-1-2), Y １１ , Y １２ , Y １３ , Y ２１ , Y ２２ , and Y ２３ The explanation for Y in formula (A-1-2) is as follows: １ ~Y ３ It is the same as Y １１ , Y １２ , and Y １３ may be the same or different, but are preferably the same. ２１ , Y ２２ , and Y ２３ may be the same or different, but are preferably the same. １１ and Y ２１ may be the same or different, but are preferably the same. １２ and Y ２２ may be the same or different, but are preferably the same. １３ and Y ２３ may be the same or different, but are preferably the same. １１ , Y １２ , Y １３ , Y ２１ , Y ２２ , and Y ２３ are preferably all the same. １１ , Y １２ , Y １３ , Y ２１ , Y ２２ , and Y ２３ is preferably an ethylene group or a propylene group, more preferably an ethylene group.

[0094] R in the formulas (B-1-1) and (B-1-2) １ and R ２The explanation for the three R in the formula (B-1-1) is the same as that for the R in the formula (A-1-1) and (A-1-2). １ and the three R's ２ may be the same or different, but are preferably the same. １ are preferably the same as each other, and the three R ２ are preferably the same as each other, and the three R １ and the three R's ２ It is more preferable that all three R １ and the three R's ２ It is more preferable that all of R in formula (B-1-2) are hydrogen atoms or all of R are methyl groups. １ and R ２ The same is true for .

[0095] L in the formulas (B-1-1) and (B-1-2) １ and L ２ The explanation for L in the formula (B-1-1) is the same as that for L in the formula (A-1-1) and (A-1-2). １ and L ２ may be the same or different, but are preferably the same. １ and L ２ may be the same or different, but are preferably the same.

[0096] Examples of the compound (B-1-1) include a compound represented by the following general formula (B-1-1a) (hereinafter also referred to as "compound (B-1-1a)"). Compound (B-1-1a) is a compound in which two molecules of compound (A-1-1a) are bonded via a disulfide bond.

[0097] [In the formula, Z １１ , Z １２ , Z ２１ , and Z ２２ each independently represents a single bond or a methylene group; R １ , and R ２ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; L １ , and L２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.

[0098] The two compounds (A-1-1a) linked by a disulfide bond may be the same or different, but are preferably the same.

[0099] Z in the general formula (B-1-1a) １１ and Z ２１ The explanation for Z in general formula (A-1-1a) １ It is the same as two Z １１ and two Zs ２１ may be the same or different, but are preferably the same. １１ are preferably the same as each other, and two Z ２１ are preferably the same as each other, and two Z １１ and two Zs ２１ It is more preferable that all are the same, and two Z １１ and two Zs ２１ It is more preferable that all of are methylene groups.

[0100] Z in the general formula (B-1-1a) １２ and Z ２２ The explanation for Z in general formula (A-1-1a) ２ It is the same as two Z １２ and two Zs ２２ may be the same or different, but are preferably the same. １２ are preferably the same as each other, and two Z ２２ are preferably the same as each other, and two Z １２ and two Zs ２２ It is more preferable that all are the same, and two Z １２ and two Zs ２２ It is more preferable that all of are single bonds.

[0101] R in the general formula (B-1-1a) １ and R ２ The explanation for the three R's is the same as that for R in general formula (A-1-1a).１ and the three R's ２ may be the same or different, but are preferably the same. １ are preferably the same as each other, and the three R ２ are preferably the same as each other, and the three R １ and the three R's ２ It is more preferable that all three R １ and the three R's ２ More preferably, all of are hydrogen atoms or methyl groups.

[0102] L in the general formula (B-1-1a) １ and L ２ The explanation for is the same as for L in general formula (A-1-1a). １ and L ２ may be the same or different, but are preferably the same.

[0103] The compound (B-1-1a) is preferably a compound represented by the following general formula (B-1-1b) (hereinafter also referred to as "compound (B-1-1b)"). Compound (B-1-1b) is a compound in which two molecules of compound (A-1-1b) are bonded via a disulfide bond.

[0104] [In the formula, R １ and R ２ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; L １ and L ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.

[0105] R in the formula (B-1-1b) １ and R ２ represents R in the formula (B-1-1a). １ and R ２ are the same as １ and R ２ is preferably a hydrogen atom or a methyl group, more preferably a hydrogen atom.

[0106] L in the formula (B-1-1b)１ and L ２ represents L in the formula (B-1-1a). １ and L ２ It is the same as L １ and L ２ is preferably an alkylene group having 1 to 6 carbon atoms or a polyoxyalkylene group having 1 to 6 carbon atoms, more preferably an alkylene group having 1 to 6 carbon atoms, a polyoxyethylene group having 1 to 6 carbon atoms, or a polyoxypropylene group having 1 to 6 carbon atoms, still more preferably an ethylene group or a propylene group, and particularly preferably an ethylene group.

[0107] A specific example of compound (B-1-1b) is a compound represented by the following formula (B-1-1-1) (hereinafter also referred to as "compound (B-1-1-1)"). Compound (B-1-1-1) is a compound in which two molecules of compound (A-1-1-1) are bonded via a disulfide bond.

[0108]

[0109] A specific example of the compound (B-1-2) is a compound represented by the following formula (B-1-2-1) (hereinafter also referred to as "compound (B-1-2-1)"). Compound (B-1-2-1) is a compound in which two molecules of compound (A-1-2-1) are bonded via a disulfide bond.

[0110]

[0111] <Compound (C)> Compound (C) is a complex of compound (A) or compound (B) with a transition metal ion. Examples of the transition metal ion include ions of elements that exist between Group 3 and Group 12 of the periodic table. Examples of the transition metal ion include copper ions (e.g., Cu ＩＩ ions, Cu Ｉ ions), nickel ions (e.g., Ni ＩＩ ions), iron ions (e.g., Fe ＩＩ ions, Fe ＩＩＩ ions), manganese ions (e.g., Mn ＩＩ ions, Mn ＩＩＩ ions), zinc ions (e.g., Zn ＩＩions), and molybdenum ions (e.g., Mo ＩＶ Ion, Mo ＶＩ ions), but are not limited to these.

[0112] Examples of the complex of compound (A) and a transition metal ion include a complex formed by a transition metal ion being coordinated to the polyamine ring of compound (A) and a complex formed by a transition metal ion being coordinated to two polyamine rings of compound (B).

[0113] <Compound (D)> Compound (D) is a salt of compound (A) or compound (B). The salt is not particularly limited and may be a salt with an acid or a salt with a base. Examples of salts with acids include salts with inorganic acids such as hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, and sulfurous acid; and salts with organic acids such as formic acid, acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, malic acid, mandelic acid, methanesulfonic acid, p-toluenesulfonic acid, and trifluoroacetic acid. Examples of salts with bases include salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as magnesium and calcium; and ammonium salts.

[0114] <Compound (E)> Compound (E) is a solvate of compound (A) or compound (B). Examples of the solvate include hydrates and solvates with organic solvents such as alcohols (e.g., methanol, ethanol, propanol, isopropanol), acetone, tetrahydrofuran, dioxane, DMF, and DMSO.

[0115] A protein folding agent refers to a drug that has the effect of promoting protein folding. "Promoting protein folding" means that when a folding reaction of a reduced-denatured protein is carried out in the presence of the protein folding agent, a greater amount of native structure protein (folded protein) is produced compared to when the protein folding agent is not present. For example, when a reduced-denatured protein (e.g., 30 μM) and the protein folding agent are dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) and incubated at 30°C for 1 hour, the rate of formation of native structure protein is 5% or more, preferably 10% or more. The protein folding agent of this embodiment can be used for folding proteins in which the native structure protein has one or more disulfide bonds.

[0116] The protein-folding agent of this embodiment preferably contains at least one compound selected from the group consisting of compound (B), a metal complex of compound (B), a salt of compound (B), and a solvate of compound (B) (hereinafter referred to as "compound (B) or the like"), and preferably contains compound (B). The protein-folding agent of this embodiment preferably contains, in addition to compound (B) or the like, at least one compound selected from the group consisting of compound (A), a metal complex of compound (A), a salt of compound (A), and a solvate of compound (A) (hereinafter referred to as "compound (A) or the like"), and preferably contains compound (A) and compound (B). When the protein-folding agent of this embodiment contains compound (A) or the like and compound (B) or the like, the ratio of compound (A) or the like to compound (B) or the like is not particularly limited. The molar ratio of the compound (A) etc. to the compound (B) etc. is, for example, 1:10 to 20:1, preferably 1:5 to 15:1, more preferably 1:3 to 10:1, even more preferably 1:2 to 10:1, still more preferably 1:1 to 8:1, and particularly preferably 2:1 to 7:1. When the compound (A) is the compound (A-1-2) and the compound (B) is the compound (B-1-2), the molar ratio of the compound (A) etc. to the compound (B) etc. is, for example, 1:10 to 30:1, preferably 1:5 to 20:1, more preferably 1:3 to 20:1, even more preferably 1:2 to 15:1, still more preferably 1:1 to 15:1, and particularly preferably 2:1 to 10:1.

[0117] The protein-folding agent of the present embodiment may contain at least one compound selected from the group consisting of compound (A-2), a metal complex of compound (A-2), a salt of compound (A-2), and a solvate of compound (A-2) (hereinafter referred to as "compound (A-2) or the like"), and preferably contains compound (A-2).

[0118] (Optional Components) The protein folding agent of this embodiment may contain optional components in addition to compound (A) etc. and / or compound (B) etc. Examples of optional components include oxidizing agents and / or reducing agents other than compound (A) etc. and compound (B) etc. Examples of reducing agents include compounds having a thiol group. Examples of reducing agents include reduced glutathione (GSH), 1,4-dithiothreitol, (±)-trans-1,2-bis(2-mercaptoacetamido)cyclohexane (BMC) (KJ Woycechowsky et al., Chem. Biol., 1999, 6, 871-879), aromatic thiols (DJ Madar et al., J. Biotech., 2009, 142, 214-219), cyclic selenoxides (K. Arai, K et al., Chem. Eur. J., 2011, 17, 481-485), peptides having a Cys-XX-Cys structure (X is any amino acid, Cys is cysteine) (WJ Lees et al., Curr. Opin. Chem. Biol., 2008, 12, 740-745), selenoglutathione (J. Beld et al., Biochemistry. 2007 May 8;46(18):5382-90), selenol-containing peptides (S. Tsukagoshi et al., Chem. Asian J. 2020 September 1;15(17):2646-52), and thiol compounds described in JP 2022-135954 A. Examples of the oxidizing agent include compounds having a disulfide bond. Examples of the oxidizing agent include oxidized compounds of the above-mentioned reducing agents.

[0119] The protein folding agent preferably contains a reducing agent, and more preferably contains both a reducing agent and an oxidizing agent. When the protein folding agent contains at least one compound selected from the group consisting of compound (A-1), a metal complex of compound (A-1), a salt of compound (A-1), and a solvate of compound (A-1) (hereinafter referred to as "compound (A-1) or the like"), it preferably contains compound (A-2) or the like, compound (B) or the like, or an oxidizing agent other than compound (B) or the like, as the oxidizing agent. When the protein folding agent contains compound (A-2) or the like, or compound (B) or the like, it may contain a reducing agent other than compound (A-1) or the like or compound (A-1) or the like as the reducing agent.

[0120] The protein folding agent of this embodiment can suppress protein aggregation and promote protein folding in the presence of transition metal ions. The protein folding agent of this embodiment can promote protein folding more in the presence of transition metal ions than in the absence of transition metal ions. Therefore, the protein folding agent of this embodiment can be suitably used for protein folding in the presence of transition metal ions. Examples of the transition metal include ions of elements present in Groups 3 to 12 of the periodic table. Examples of transition metal ions include copper ions (e.g., Cu ＩＩ ions, Cu Ｉ ions), nickel ions (e.g., Ni ＩＩ ions), iron ions (e.g., Fe ＩＩ ions, Fe ＩＩＩ ions), manganese ions (e.g., Mn ＩＩ ions, Mn ＩＩＩ ions), zinc ions (e.g., Zn ＩＩ ions), and molybdenum ions (e.g., Mo ＩＶ Ion, Mo ＶＩ Examples of the transition metal ions include, but are not limited to, copper ions (e.g., Cu ＩＩ ions, Cu Ｉ ions), nickel ions (e.g., Ni ＩＩ ions), and zinc ions (e.g., ZnＩＩ At least one selected from the group consisting of cations is preferred.

[0121] Some proteins contain metal ions within their molecules (metalloproteins). For example, some metal ion-containing enzymes contain a metal ion in their active center. When such metalloproteins are chemically or biologically synthesized and folded into their native structure, the folding reaction must be carried out in the presence of metal ions. However, in the presence of transition metal ions, there is a risk of protein aggregation, so the reaction conditions must be strictly controlled.

[0122] The protein-folding agent of this embodiment can suppress protein aggregation and promote protein folding into its native structure in the presence of transition metal ions, and therefore can be suitably used for folding proteins containing transition metal ions.

[0123] Examples of transition metal ions contained in proteins include, but are not limited to, copper ions, nickel ions, iron ions, manganese ions, zinc ions, and molybdenum ions. Examples of proteins containing copper ions include cytochrome c oxidase. Examples of proteins containing nickel ions include urease. Examples of proteins containing iron ions include catalase, cytochrome (heme), nitrogenase, and hydrogenase. Examples of proteins containing manganese ions include arginase. Examples of proteins containing zinc ions include alcohol dehydrogenase, carbonic anhydrase, and DNA polymerase. Examples of proteins containing molybdenum include nitrate reductase.

[0124] The protein folding agent of this embodiment can be used for folding unfolded proteins or refolding misfolded proteins. An unfolded protein is a protein that is not folded. In an unfolded protein, disulfide bonds formed in a protein in its native structure are reduced to thiol groups. A protein that has been reduced and denatured by a reducing agent is an unfolded protein. A misfolded protein is a protein in which one or more disulfide bonds are formed in a structure other than the thermodynamically most stable native structure. Unfolded proteins and misfolded proteins cannot exhibit the activity of proteins in their native structure. By applying the protein folding agent of this embodiment to such unfolded or misfolded proteins, they can be folded into the thermodynamically most stable native structure.

[0125] [Compound] The second aspect of the present disclosure is the above-mentioned compound (B-1), compound (A-1-2), or compound (A-2). The explanation of compound (B-1), compound (A-1-2), or compound (A-2-1) is as described above. As described above, these compounds can be used as protein-folding agents according to the first aspect.

[0126] <Method for producing compound (B-1)> Compound (B-1) can be produced by combining known methods. For example, １２ , Rx １３ , Rx ２２ and Rx ２３ The compound (B-1) in which is a hydrogen atom can be synthesized by the following synthesis scheme. In the following synthesis scheme, TsCl represents paratoluenesulfonyl chloride, Boc ２ O represents di-tert-butyl dicarbonate, Ts represents a paratoluenesulfonyl group, and Boc represents a tert-butoxycarbonyl group.

[0127]

[0128] [In the formula, Y １１ , Y １２ , Y １３ , Y ２１ , Y ２２ , and Y ２３ each independently represents an alkylene group having 1 to 5 carbon atoms. １ and Lx ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom. m01 and m02 each independently represent an integer of 1 to 3. When m01 is an integer of 2 or more, two or more Y １３ When m02 is an integer of 2 or more, two or more Y ２３ may be the same as or different from each other.

[0129] The reaction of compound a with TsCl to obtain compound b can be carried out in the presence of a base such as triethylamine. Examples of the reaction solvent that can be used include dichloromethane. The reaction temperature can be 0 to 30°C. The reaction time can be 20 to 30 hours. For example, the method described in P. Jain et al. (Langmuir 2019, 35, 1864) can be referenced.

[0130] Compound c1 and Boc ２ O to give compound d1, and the reaction of compound c2 with Boc ２ The reaction with O to obtain compound d2 can be carried out in the presence of a base such as triethylamine. Dichloromethane, for example, can be used as the reaction solvent. The reaction temperature can be −15 to 30° C. The reaction time can be 20 to 30 hours. For example, the method described in Boitrel et al. (Tetrahedron Lett. 1995, 36, 4995.) can be referenced.

[0131] The reaction of compound d1, compound d2, and compound b to obtain compound e1 can be carried out in the presence of a base such as potassium carbonate. Examples of the reaction solvent that can be used include dehydrated acetonitrile. The reaction temperature can be 40 to 80°C. The reaction time can be 10 to 40 hours. The reaction can be carried out under a nitrogen atmosphere.

[0132] The reaction of obtaining compound (B-1) (a compound represented by general formula (B-1')) by removing Boc from compound e1 can be carried out under acidic conditions using concentrated hydrochloric acid or the like. An ethanol solution can be used as the reaction solvent. The reaction temperature can be 10 to 40°C. The reaction time can be 1 to 10 hours. The reaction can be carried out in an air atmosphere.

[0133] For example, Rx １２ , Rx １３ , Rx ２２ and Rx ２３ is a methyl group, and Lx １ and Lx ２ Compound (B-1), in which is an alkylene group having 2 to 10 carbon atoms (which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom), can be synthesized by the following synthetic scheme (JA Halfen et al., Chem. Commun. 2003, 2894-2895.). In the synthetic scheme, TsCl represents paratoluenesulfonyl chloride, EtOTFA represents ethyltrifluoroacetate, Ts represents a paratoluenesulfonyl group, and TFA represents a trifluoroacetyl group.

[0134]

[0135] [In the formula, Y １１ , Y １２ , Y １３ , Y ２１ , Y ２２ , and Y ２３ each independently represents an alkylene group having 1 to 5 carbon atoms. １ ' and. Lx ２m01 represents an alkylene group having 1 to 9 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom. m01 and m02 each independently represent an integer of 1 to 3. When m01 is an integer of 2 or more, two or more Y １３ When m02 is an integer of 2 or more, two or more Y ２３ may be the same as or different from each other.

[0136] The reaction of compound c1 with EtOTFA to obtain compound f1 can be carried out in the presence of a base such as triethylamine. Examples of the reaction solvent that can be used include methanol. The reaction temperature can be 0 to 40°C. The reaction time can be 10 to 30 hours.

[0137] The reaction of compound f1 with TsCl to obtain compound g1 can be carried out in the presence of a base such as triethylamine. Dichloromethane, for example, can be used as the reaction solvent. The reaction can be carried out by refluxing the reaction solution for 10 to 30 hours.

[0138] The reaction of obtaining compound h1 by hydrolysis of compound g1 can be carried out in the presence of a base such as potassium hydroxide. As a reaction solvent, for example, methanol can be used. The reaction can be carried out by refluxing the reaction solution for 1 to 20 hours.

[0139] The methylation of compound h1 to give compound i1 can be carried out using formic acid and formaldehyde by refluxing the reaction mixture for 1 to 20 hours.

[0140] The reaction of obtaining compound j1 by detosylation of compound i1 can be carried out in the presence of a strong acid such as hydrogen bromide. The reaction can be carried out using, for example, phenol as a reaction solvent. The reaction temperature can be 60 to 120°C. The reaction time can be 10 to 40 hours.

[0141] The reaction of compound j1 with compound k1 to obtain compound l1 can be carried out using a reaction solvent such as benzene. The reaction temperature can be 50 to 150°C. The reaction time can be 10 to 40 hours.

[0142] Compound 12 can be obtained in the same manner as above.

[0143] The reaction for obtaining compound (B-1) (a compound represented by general formula (B-1")) by the oxidation reaction of compound l1 and compound l2 can be carried out using an oxidizing agent such as oxidized DTT. Water can be used as the reaction solvent. The reaction temperature can be 0 to 40°C. The reaction time can be 1 to 30 hours.

[0144] After the completion of each reaction, each compound in the reaction solution may be isolated and purified. For isolation and purification, a conventionally known method can be used, for example, concentration, solvent extraction, distillation, crystallization, recrystallization, chromatography, etc., which can be used alone or in combination of two or more of these. The structure of the compound obtained by each reaction is as follows: １ This can be confirmed by general organic analysis methods such as H-nuclear magnetic resonance (NMR) spectroscopy and MALDI-TOF mass spectrometry.

[0145] (Method for producing compound (B-1-1a)) As an example of compound (B-1), an example of a method for producing compound (B-1-1a) is shown below. Compound (B-1-1a) can be produced by combining known methods. For example, １ and R ２ The compound (B-1-1a) in which is a hydrogen atom can be synthesized by the following synthesis scheme. In the following synthesis scheme, TsCl represents paratoluenesulfonyl chloride, Boc ２ O represents di-tert-butyl dicarbonate, Ts represents a paratoluenesulfonyl group, and Boc represents a tert-butoxycarbonyl group.

[0146] [In the formula, Z １ and Z ２ each independently represents a single bond or a methylene group; L １ and L ２each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.

[0147] The reaction of compound a with TsCl to obtain compound b can be carried out in the presence of a base such as triethylamine. Examples of the reaction solvent that can be used include dichloromethane. The reaction temperature can be 0 to 30°C. The reaction time can be 20 to 30 hours. For example, the method described in P. Jain et al. (Langmuir 2019, 35, 1864) can be referenced.

[0148] Compound c and Boc ２ The reaction with O to obtain compound d can be carried out in the presence of a base such as triethylamine. Dichloromethane, for example, can be used as the reaction solvent. The reaction temperature can be −15 to 30° C. The reaction time can be 20 to 30 hours. For example, the method described in Boitrel et al. (Tetrahedron Lett. 1995, 36, 4995.) can be referenced.

[0149] The reaction of compound d with compound (b) to obtain compound e can be carried out in the presence of a base such as potassium carbonate. For example, dehydrated acetonitrile can be used as the reaction solvent. The reaction temperature can be 40 to 80°C. The reaction time can be 10 to 40 hours. The reaction can be carried out under a nitrogen atmosphere.

[0150] The reaction of obtaining compound (B) (a compound represented by general formula (B-1-1a')) by removing Boc from compound e can be carried out under acidic conditions using concentrated hydrochloric acid or the like. An ethanol solution can be used as the reaction solvent. The reaction temperature can be 10 to 40°C. The reaction time can be 1 to 10 hours. The reaction can be carried out in an air atmosphere.

[0151] For example, R １ and R ２ is a methyl group, and L １ and L ２is an alkylene group having 2 to 10 carbon atoms (which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom), can be synthesized by the following synthetic scheme (JA Halfen et al., Chem. Commun. 2003, 2894-2895.). In the synthetic scheme, TsCl represents paratoluenesulfonyl chloride, EtOTFA represents ethyltrifluoroacetate, Ts represents a paratoluenesulfonyl group, and TFA represents a trifluoroacetyl group.

[0152] [In the formula, Z １ and Z ２ each independently represents a single bond or a methylene group; L １ ' represents an alkylene group having 1 to 9 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.

[0153] The reaction of compound c with EtOTFA to obtain compound f can be carried out in the presence of a base such as triethylamine. Examples of the reaction solvent that can be used include methanol. The reaction temperature can be 0 to 40°C. The reaction time can be 10 to 30 hours.

[0154] The reaction of compound f with TsCl to obtain compound g can be carried out in the presence of a base such as triethylamine. Dichloromethane, for example, can be used as the reaction solvent. The reaction can be carried out by refluxing the reaction solution for 10 to 30 hours.

[0155] The reaction of obtaining compound h by hydrolysis of compound g can be carried out in the presence of a base such as potassium hydroxide. As the reaction solvent, for example, methanol can be used. The reaction can be carried out by refluxing the reaction solution for 1 to 20 hours.

[0156] The methylation of compound h to give compound i can be carried out using formic acid and formaldehyde by refluxing the reaction mixture for 1 to 20 hours.

[0157] The reaction of detosylation of compound i to obtain compound j can be carried out in the presence of a strong acid such as hydrogen bromide. The reaction can be carried out using, for example, phenol as a reaction solvent. The reaction temperature can be 60 to 120°C. The reaction time can be 10 to 40 hours.

[0158] The reaction of compound j with compound k to obtain compound l can be carried out using a reaction solvent such as benzene. The reaction temperature can be 50 to 150°C. The reaction time can be 10 to 40 hours.

[0159] The reaction for obtaining compound (B) (a compound represented by general formula (B-1-1a″)) by oxidation of compound 1 can be carried out using an oxidizing agent such as oxidized DTT. Water can be used as the reaction solvent. The reaction temperature can be 0 to 40° C. The reaction time can be 1 to 30 hours.

[0160] After the completion of each reaction, each compound in the reaction solution may be isolated and purified. For isolation and purification, a conventionally known method can be used, for example, concentration, solvent extraction, distillation, crystallization, recrystallization, chromatography, etc., which can be used alone or in combination of two or more of these. The structure of the compound obtained by each reaction is as follows: １ This can be confirmed by general organic analysis methods such as H-nuclear magnetic resonance (NMR) spectroscopy and MALDI-TOF mass spectrometry.

[0161] The compound (B-1-1) and the compound (B-1-2) can also be produced in a similar manner.

[0162] <Method for producing compound (A-1-2)> Compound (A-1-2) can be obtained by carrying out a reduction reaction of compound (B-1-2). The reduction reaction of compound (B-1-2) can be carried out under acidic conditions, for example, using concentrated hydrochloric acid or the like. An ethanol solution can be used as the reaction solvent. The reaction temperature can be 10 to 40°C. The reaction time can be 1 to 10 hours. The reaction can be carried out under an air atmosphere. The reducing agent can be, for example, dithiothreitol (DTT).

[0163] <Method for producing compound (A-2)> Compound (A-2) can be produced by combining known methods. Compound (A-2) can be synthesized, for example, by the following synthesis scheme. In the following synthesis scheme, NaOH aq represents an aqueous sodium hydroxide solution.

[0164] [In the formula, Rx ３ represents a hydrogen atom or a monovalent organic group. １ ~Y ３ each independently represents an alkylene group having 1 to 5 carbon atoms. １ and Lx ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom; m0 represents an integer of 1 to 3; when m0 is an integer of 2 or more, there are 2 or more Rx ３ may be the same or different, and two or more Y ３ may be the same as or different from each other.

[0165] The reaction for obtaining compound n1 from compound m1 can be carried out in the presence of a base such as sodium hydroxide. The reaction temperature can be 0 to 30°C. The reaction time can be 20 to 30 hours. For example, the methods described in P. Jain et al. (Langmuir 2019, 35, 1864) and CA Barta et al. (Inorg Chem. 2008, 47, 2280) can be referenced.

[0166] The reaction of compound n1 with compound b to obtain compound (A-2) can be carried out in the presence of a base such as potassium carbonate. Examples of the reaction solvent that can be used include dehydrated acetonitrile. The reaction temperature can be 40 to 80°C. The reaction time can be 10 to 40 hours. The reaction can be carried out under a nitrogen atmosphere.

[0167] After the completion of each reaction, each compound in the reaction solution may be isolated and purified. For isolation and purification, a conventionally known method can be used, for example, concentration, solvent extraction, distillation, crystallization, recrystallization, chromatography, etc., which can be used alone or in combination of two or more of these. The structure of the compound obtained by each reaction is as follows: １ This can be confirmed by general organic analysis methods such as H-nuclear magnetic resonance (NMR) spectroscopy and MALDI-TOF mass spectrometry.

[0168] [Protein Folding Method] A third aspect of the present disclosure is a protein folding method, which comprises the step of incubating a protein in the presence of the protein folding agent according to the first aspect.

[0169] <Protein Incubation Step; Protein Folding Step> Protein incubation can be carried out by dissolving the protein and the protein folding agent according to the first aspect (e.g., compound (A) etc. and compound (B) etc.) in an appropriate buffer to prepare a folding reaction solution, and then incubating the solution. This incubation allows the protein folding reaction to occur.

[0170] The buffer solution is not particularly limited as long as it allows the protein folding reaction to proceed, and solutions commonly used in the field of biochemistry can be used without particular limitation. Examples of buffer solutions include amine-based buffer solutions such as Tris-HCl buffer, Tris buffer, MES buffer, and Tricine buffer; phosphate buffer; and Good's buffers. The pH of the buffer solution may be 4 to 10. The pH of the buffer solution is preferably 5 to 9, more preferably 7 to 9, and even more preferably 7 to 8. A preferred buffer solution is a Tris-HCl buffer solution containing added sodium chloride. A specific example of the buffer solution is a buffer solution containing 50 mM trishydroxymethylaminomethane and 30 mM sodium chloride, adjusted to pH 7.5 with hydrochloric acid.

[0171] The concentration of the protein folding agent in the folding reaction solution is not particularly limited. When the protein folding agent contains compound (A) or the like, the concentration of compound (A) or the like is, for example, 1 μM to 1000 mM, preferably 10 μM to 100 mM, more preferably 300 μM to 50 mM, even more preferably 500 μM to 10 mM, and particularly preferably 800 μM to 5 mM. When the protein folding agent contains compound (B) or the like, the concentration of compound (B) or the like is, for example, 1 μM to 1000 mM, preferably 10 μM to 100 mM, more preferably 50 μM to 1 mM, even more preferably 100 to 500 μM, and particularly preferably 100 to 300 μM. When the protein folding agent comprises compound (A) or the like and compound (B) or the like, the molar ratio of compound (A) or the like to compound (B) or the like is, for example, 1:10 to 20:1, preferably 1:5 to 15:1, more preferably 1:3 to 10:1, even more preferably 1:2 to 10:1, even more preferably 1:1 to 8:1, and particularly preferably 2:1 to 7:1. When compound (A) is compound (A-1-2) and compound (B) is compound (B-1-2), the molar ratio of compound (A) or the like to compound (B) or the like is, for example, 1:10 to 30:1, preferably 1:5 to 20:1, more preferably 1:3 to 20:1, even more preferably 1:2 to 15:1, even more preferably 1:1 to 15:1, and particularly preferably 2:1 to 10:1.

[0172] The protein used for incubation may be a protein having one or more disulfide bonds. The number of disulfide bonds in the protein is not particularly limited, as long as it is one or more. Examples of the number of disulfide bonds in the protein include 1 to 20, 1 to 15, 1 to 10, or 1 to 5. The molecular weight of the protein is not particularly limited, but may be, for example, 1,000 to 10,000,000, with 1,000 to 250,000 being preferred. An unfolded protein or a misfolded protein may also be used. For example, a protein that has been reduced and denatured with a reducing agent may also be used.

[0173] The protein-folding agent according to the first aspect promotes protein folding, particularly in the presence of transition metal ions. Therefore, the protein is preferably a protein that contains a transition metal ion in its native structure. Specific examples of transition metal ions and proteins containing transition metal ions include those described above.

[0174] The protein concentration in the folding reaction solution is not particularly limited and can be set appropriately depending on the type of protein. For example, the protein concentration in the folding reaction solution is 0.1 to 1000 μM, preferably 0.5 to 500 μM, more preferably 1 to 300 μM, and even more preferably 5 to 100 μM.

[0175] The folding reaction is preferably carried out in the presence of transition metal ions. By carrying out the folding reaction in the presence of transition metal ions, a protein containing a transition metal can be obtained. The folding reaction solution preferably contains transition metal ions. The transition metal ions can be added to the folding reaction solution, for example, in the form of a transition metal salt. Examples of transition metal salts include, but are not limited to, hydrochlorides, sulfates, nitrates, carbonates, and phosphates. Specific examples of copper ion salts include, for example, copper(II) chloride (CuCl ２ ), copper(II) sulfate (CuSO ４ ) and the like. Specific examples of nickel ion salts include nickel (II) chloride (NiCl ２ ), nickel(II) sulfate (NiSO ４ Specific examples of iron ion salts include iron(II) chloride (FeCl ２ ), iron(II) sulfate (FeSO ４ ) and the like. Specific examples of salts of manganese ions include manganese(II) chloride (MnCl ２ ), manganese(II) sulfate (MnSO ４ ) and the like. Specific examples of zinc ion salts include zinc chloride (II) (ZnCl ２ ), zinc sulfate (II) (ZnSO ４) and the like. Specific examples of salts of molybdenum ions include molybdenum(V) chloride (Mo ２ Cl １０ ) etc.

[0176] The concentration of the transition metal ion in the folding reaction solution is not particularly limited, but may be, for example, 10 to 1000 μM, preferably 10 to 500 μM, and more preferably 20 to 300 μM. When a protein contains a transition metal ion in its molecule, the molar concentration is preferably higher than the molar concentration of the protein, and may be, for example, 1.2 to 20 times higher than the molar concentration of the protein.

[0177] When the folding reaction is carried out in the presence of a transition metal ion, the concentration of compound (A) etc. and compound (B) etc. in the folding reaction solution may be a catalytic amount. For example, the concentration of compound (A) etc. and compound (B) etc. may be 0.01 to 20 μM, preferably 0.1 μM to 10 μM, more preferably 0.5 μM to 10 μM, and even more preferably 1 to 10 μM.

[0178] The incubation temperature is not particularly limited as long as it allows the protein folding reaction to proceed. The incubation temperature can be appropriately selected depending on the type of protein. Examples of incubation temperatures include 10 to 50°C, preferably 20 to 40°C, and more preferably 25 to 35°C. The incubation time can be appropriately selected depending on the type of protein. Examples of incubation times include 1 to 300 minutes, preferably 10 to 200 minutes, and more preferably 30 to 100 minutes. The folding state can be observed over time, and the incubation can be terminated when the most stable native structure is achieved. To observe the folding state, a thiol group modifying agent such as 4-Acetamido-4'-Maleimidylstilbene-2,2'-Disulfonic Acid (AMS) can be used.

[0179] <Optional Steps> The method of this embodiment may include optional steps in addition to the incubation step. Examples of optional steps include a step of unfolding the protein and a step of isolating the folded protein.

[0180] (Protein Unfolding Step) The method of this embodiment may include a step of unfolding the protein prior to the incubation step (protein folding step). The protein unfolding step can be carried out by incubating the protein in the presence of a reducing agent. Examples of reducing agents include reduced glutathione (GSH), β-mercaptoethanol, and 1,4-dithiothreitol. Protein unfolding may also be carried out using a protein denaturant (e.g., guanidine hydrochloride, urea, etc.). The protein unfolding reaction may be carried out by preparing a protein unfolding reaction solution by dissolving the protein, reducing agent, and protein denaturant in a medium such as water or a buffer solution, and then incubating the solution. The protein concentration in the unfolding reaction solution may be, for example, 0.1 to 100 mg / mL. The reducing agent concentration in the unfolding reaction solution may be, for example, 0.1 to 1000 mM. The concentration of the oxidizing agent is preferably 1 to 500 mM, more preferably 10 to 300 mM, and even more preferably 50 to 200 mM. The concentration of the denaturing agent in the unfolding reaction solution is, for example, 0.1 to 100 M. The concentration of the denaturing agent is preferably 0.5 to 50 M, more preferably 1 to 30 M, and even more preferably 1 to 20 M. The pH of the unfolding reaction solution is, for example, 5 to 10, preferably 6 to 9.5, more preferably 7 to 9, and even more preferably 8 to 9.

[0181] Buffers that can be used in the unfolding reaction solution include those similar to those mentioned above. Specific examples of buffers include 0.1 M Tris-HCl buffer.

[0182] The incubation temperature is not particularly limited as long as it allows the protein unfolding reaction to proceed. The incubation temperature can be appropriately selected depending on the type of protein. Examples of incubation temperatures include 10 to 60°C, preferably 20 to 55°C, and more preferably 20 to 50°C. The incubation time can be appropriately selected depending on the type of protein. Examples of incubation times include 1 to 600 minutes, preferably 10 to 500 minutes, more preferably 30 to 400 minutes, and even more preferably 60 to 300 minutes. The unfolding state can be observed over time, and the incubation can be terminated when all disulfide bonds have been reduced.

[0183] After incubation, the unfolding reaction solution may be subjected to dialysis or the like to remove the denaturant, reducing agent, and the like.

[0184] (Protein Isolation Step) The method of this embodiment may include a step of isolating the folded protein after the incubation step (protein folding step). Any known protein isolation method can be used for protein isolation without any particular limitation. Examples of protein isolation methods include salting out, dialysis, column chromatography, etc.

[0185] [Pharmaceutical Composition] A fourth aspect of the present disclosure is a pharmaceutical composition. The pharmaceutical composition according to this aspect comprises the protein folding agent according to the first aspect and a pharmaceutically acceptable carrier.

[0186] The pharmaceutical composition according to this aspect can be used to treat or prevent diseases caused by protein aggregation. It is known that transition metal ions such as copper ions and nickel ions can cause protein aggregation in vivo, leading to the risk of disease. The pharmaceutical composition according to this aspect contains the protein folding agent according to the first aspect as an active ingredient. The protein folding agent inhibits protein aggregation in the presence of transition metals and promotes protein folding into native structural forms. Therefore, it is possible to prevent the onset of diseases caused by protein aggregation, inhibit the progression of the diseases, and / or ameliorate the symptoms caused by the diseases.

[0187] Diseases caused by protein aggregation include folding diseases, which are diseases caused by the aggregation and / or deposition of misfolded proteins. Examples of folding diseases include, but are not limited to, neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies, spinocerebellar degeneration, frontotemporal dementia, Pick's disease, Creutzfeldt-Jakob disease, and familial amyloidotic polyneuropathy; and type 2 diabetes.

[0188] The subject to which the pharmaceutical composition is administered is not particularly limited, and can be used for animals that develop diseases caused by protein aggregation. The subject to which the pharmaceutical composition is administered is preferably a mammal, and may be a human or a non-human mammal. Examples of non-human mammals include, but are not limited to, primates (monkeys, chimpanzees, gorillas, etc.), rodents (mice, hamsters, rats, etc.), rabbits, dogs, cats, cows, goats, sheep, horses, etc.

[0189] The term "pharmaceutically acceptable carrier" refers to a carrier that does not inhibit the physiological activity of an active ingredient and is not substantially toxic to a recipient. "Not substantially toxic" means that the ingredient is not toxic to a recipient at a commonly used dose. In the pharmaceutical composition of this embodiment, the pharmaceutically acceptable carrier is a carrier that does not inhibit the protein folding-promoting activity of the protein-folding agent according to the first aspect and is not substantially toxic to a recipient. Pharmaceutically acceptable carriers include any known pharmaceutically acceptable ingredient that is typically considered an inactive ingredient. Pharmaceutically acceptable carriers include, but are not limited to, solvents, diluents, vehicles, excipients, glidants, binders, granulating agents, dispersing agents, suspending agents, wetting agents, lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, etc. Pharmaceutically acceptable carriers may be used singly or in combination.

[0190] The pharmaceutical composition may contain other ingredients in addition to the above-mentioned ingredients. The other ingredients are not particularly limited, and those commonly used in the pharmaceutical field can be used without particular limitation. Examples of other ingredients include pharmaceutical additives other than those mentioned above. Examples of pharmaceutical additives include, but are not limited to, preservatives (e.g., antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, colorants, etc. The pharmaceutical composition may contain an active ingredient other than the protein folding agent according to the first aspect. Examples of active ingredients include, but are not limited to, antiviral agents, antibiotics, anti-inflammatory agents, antipyretics, analgesics, etc.

[0191] The dosage form of the pharmaceutical composition is not particularly limited and can be any dosage form commonly used for pharmaceutical preparations. The pharmaceutical composition of this embodiment may be an oral or parenteral formulation, with parenteral formulations being preferred. Oral formulations include, for example, tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drops, and emulsions. Parenteral formulations include, for example, injections, suppositories, nasal drops, enteral preparations, and inhalants. Pharmaceutical compositions in these dosage forms can be formulated according to standard methods (e.g., methods described in the Japanese Pharmacopoeia).

[0192] The administration route of the pharmaceutical composition of this embodiment is not particularly limited, and can be oral or parenteral, although parenteral administration is preferred. Examples of parenteral administration routes include intravenous administration, intranasal administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, and enteral administration.

[0193] The pharmaceutical composition can be administered in a therapeutically effective amount of the protein-folding agent according to the first aspect. The term "therapeutically effective amount" refers to the amount of a drug effective for treating or preventing a target disease. For example, the therapeutically effective amount of the protein-folding agent can be an amount effective for refolding an aggregated protein. The therapeutically effective amount can be determined appropriately based on the patient's symptoms, body weight, age, and sex, as well as the dosage form and administration method of the pharmaceutical composition. For example, the pharmaceutical composition can be administered in a single dose of 0.01 to 1,000 mg of the antiviral agent per kg of the body weight of the subject. The dosage may be 0.05 to 500 mg / kg, 0.1 to 300 mg / kg, 0.2 to 200 mg / kg, or 0.3 to 100 mg / kg.

[0194] The pharmaceutical composition may contain a therapeutically effective amount of the protein-folding agent per unit dosage form. For example, the content of the protein-folding agent in the pharmaceutical composition may be 0.01 to 90% by mass, 0.05 to 80% by mass, or 0.1 to 60% by mass.

[0195] The pharmaceutical composition may be administered in a single dose or multiple doses. In the case of multiple doses, the administration interval may be appropriately determined depending on the symptoms, body weight, age, sex, etc. of the patient, as well as the dosage form of the pharmaceutical composition and the administration method, etc. The administration interval may be, for example, every few hours, 2 to 3 times a day, once a day, once every 2 to 3 days, once a week, once a month, once every few months, etc.

[0196] [Inhibitor of Transition Metal Cytotoxicity] A fourth aspect of the present disclosure is an inhibitor of transition metal cytotoxicity. The inhibitor comprises at least one compound selected from the group consisting of the following (A) to (E): (A) a cyclic polyamine (compound (A)) containing one or more thiol groups or disulfide bonds and capable of binding to a transition metal ion; (B) a compound (compound (B)) in which two or more cyclic polyamines of (A) are bonded together via disulfide bonds between the thiol groups; (C) a complex of the cyclic polyamine of (A) or the compound of (B) with a transition metal ion (compound (C)); (D) a salt of the cyclic polyamine of (A) or the compound of (B) (compound (D)); and (E) a solvate of the cyclic polyamine of (A) or the compound of (B) (compound (E)).

[0197] The above (A) to (D) are the same as (A) to (D) in the first embodiment. The compounds (A) to (D) are the same as the compounds (A) to (D) described above.

[0198] Transition metal ions may be cytotoxic. Compounds (A) to (E) can suppress cytotoxicity caused by transition metal ions. Therefore, they can be used as inhibitors of cytotoxicity caused by transition metal ions. The cytotoxicity inhibitor preferably contains at least one selected from the group consisting of compound (A), compound (B), compound (D), and compound (E). The cytotoxicity inhibitor preferably contains at least one selected from the group consisting of compound (B) or compound (A-2), a salt of compound (B) or compound (A-2), and a solvate of compound (B) or compound (A-2). The cytotoxicity inhibitor may further contain at least one selected from the group consisting of compound (A-1), a salt of compound (A-1), and a solvate of compound (A-1).

[0199] Examples of the transition metal ion include the same as those described above. Specific examples of the transition metal ion include copper ions (e.g., Cu ＩＩ ions, Cu Ｉ ions), nickel ions (e.g., Ni ＩＩ ions), iron ions (e.g., Fe ＩＩ ions, Fe ＩＩＩ ions), manganese ions (e.g., Mn ＩＩ ions, Mn ＩＩＩ ions), zinc ions (e.g., Zn ＩＩ ions), and molybdenum ions (e.g., Mo ＩＶ Ion, Mo ＶＩ ions), but are not limited to these.

[0200] Compound (A) and compound (B) stably bind to transition metal ions to form complexes with the transition metal ions, which is presumably capable of suppressing the cytotoxicity caused by the transition metal ions.

[0201] The present invention will be described below with reference to experimental examples, but the present invention is not limited to the following experimental examples.

[0202] Experimental Example 1 Synthesis Example of Compound (A-1-1-1) and Compound (B-1-1-1) Compound (A-1-1-1) and compound (B-1-1-1) were synthesized according to the following synthesis scheme: The percentages in the synthesis scheme represent the yields in each reaction.

[0203]

[0204] (Synthesis of Compound 2 and Compound 4) Compound 2 and Compound 4 were synthesized according to previous reports (P. Jain et al., Langmuir 2019, 35, 1864.; B. Boitrel et al., Tetrahedron Lett. 1995, 36, 4995.). In the above reaction scheme, TsCl represents paratoluenesulfonyl chloride, Et ３ N is triethylamine, Boc ２ O represents di-tert-butyl dicarbonate, Ts represents a paratoluenesulfonyl group, and Boc represents a tert-butoxycarbonyl group. Compound 1 is 2-hydroxyethyl disulfide, and compound 3 is 1,4,8,11-tetraazacyclotetradecane (cyclam).

[0205] (Synthesis of Compound 5) Compound 2 (139 mg, 0.300 mmol) was dissolved in 1.5 mL of dehydrated acetonitrile (MeCN) and mixed with potassium carbonate (103 mg, 0.747 mmol, 2.5 eq). Compound 4 (315 mg, 0.629 mmol, 2.1 eq) dissolved in 1.5 mL of dehydrated acetonitrile was added, and the mixture was stirred at 60°C under a nitrogen atmosphere. 44 hours after the start of the reaction, compound 4 (30 mg, 0.060 mmol, 0.2 eq) was added again, and the mixture was stirred for 24 hours. The resulting suspension was filtered by suction, and the filtrate was dried under reduced pressure and purified by silica gel chromatography to obtain compound 5 (214 mg, 0.192 mmol, 64%). The resulting compound was １ H NMR measurement (400MHz, CDCl ３ , 323K; FIG. 1) and was identified by MALDI-TOF mass spectrometry (FIG. 2).

[0206] (Synthesis of Compound (B-1-1-1)) Compound 5 (60 mg, 0.054 mmol) was dissolved in ethanol (H２ 280 μL of concentrated hydrochloric acid was added to 280 μL of 1H 2 O / EtOH under ice-cooling, and the mixture was stirred for 10 minutes and then at room temperature for 3 hours. The resulting white suspension was ice-cooled and suction filtered to obtain a white solid. This was further purified by reverse-phase HPLC to obtain compound (B-1-1-1) (12 mg, 0.023 mmol, 43%). The obtained compound was １ H NMR measurement (400MHz, D ２ 0,323K; FIG. 3) and MALDI-TOF mass spectrometry (FIG. 4).

[0207] (Synthesis of Compound (A-1-1-1)) 700 μL of concentrated hydrochloric acid was added to an ethanol solution (700 μL) of Compound 5 (150 mg, 0.134 mmol), and the mixture was stirred at room temperature for 15 hours. The resulting white suspension was ice-cooled and suction filtered to obtain a white solid. Subsequently, 1,4-dithiothreitol (DTT) (19 mg, 0.12 mmol, 0.9 eq) and 2 mL of ultrapure water were added, and the mixture was stirred at room temperature for 15 hours. Purification by reverse-phase HPLC yielded Compound (A-1-1-1) (11 mg, 16% from Compound 5). The resulting compound was １ H NMR measurement (400MHz, D ２ 0,323K; FIG. 5) and MALDI-TOF mass spectrometry (FIG. 6).

[0208] (Reagents) 2-Hydroxyethyl disulfide was purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Paratoluenesulfonyl chloride was purchased from Kishida Chemical Co., Ltd. (Tokyo, Japan). Dichloromethane, dehydrated acetonitrile, potassium carbonate, and di-tert-butyl dicarbonate were purchased from Kanto Chemical Co., Ltd. (Tokyo, Japan). 1,4,8,11-tetraazacyclotetradecane (cyclam) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Triethylamine and hydrochloric acid were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 1,4-Dithiothreitol was purchased from Nacalai Tesque, Inc. (Kyoto, Japan).

[0209] (Apparatus) NMR measurements were performed using a JNM-ECX400 instrument manufactured by JEOL Ltd. MALDI-TOF mass spectrometry was performed using an autoflex speed instrument manufactured by Bruker Corporation. HPLC purification was performed at a flow rate of 18.9 mL / min using a PU-4086-Binary pump system and UV-4075 detector manufactured by JASCO Corporation, a YMC-Actus Triart C18 column (250 x 20.0 mm) manufactured by YMC Corporation, and a CHF122SC fraction collector manufactured by Advantec Toyo Co., Ltd.

[0210] <Protein Folding Test> Bovine Pancreatic Trypsin Inhibitor (BPTI), which has three disulfide bonds in the molecule, was used as a model protein for analyzing the folding process.

[0211] (Quantitative determination of compound (A-1-1-1) concentration) A solution of compound (A-1-1-1) dissolved in 10 mM aqueous hydrochloric acid was mixed with 10 mM Ellman's reagent (5,5'-dithio-bis-(2-nitrobenzoic acid)) in 50 mM Tris-HCl buffer (pH 7.5, 0.3 M NaCl), and UV absorption measurement was carried out at 30° C. The concentration of the compound (A-1-1-1) solution was determined from the absorbance at 412 nm.

[0212] (Preparation of reduced and modified BPTI) BPTI (10 mg) was dissolved in 0.1 M Tris-HCl (pH 8.0, 1.0 mL) containing 30 mM 1,4-dithiothreitol and 8 M urea, and the solution was incubated at 50°C for 3 hours and then purified by reversed-phase HPLC. HPLC purification was performed using a PU-4180 pump and UV-4075 detector manufactured by JASCO Corporation (Tokyo, Japan) and an InertSustain C18 column (4.6 mm x 250 mm) manufactured by GL Sciences Inc. (Tokyo, Japan) at a flow rate of 1.0 mL min. －１ The collected fractions were freeze-dried and the resulting powder was stored at -30°C.

[0213] (Reagents) BPTI was purchased from Pro-Spec-Tany TechnoGene Ltd. (Rehovot, Israel). 1,4-Dithiothreitol and urea were purchased from Nacalai Tesque, Inc. (Kyoto, Japan).

[0214] (Apparatus) For analysis of the folding process by reversed-phase HPLC, a Primeide HPLC apparatus manufactured by Hitachi High-Tech Corporation (Tokyo, Japan) and a TSKgel Protein C4-300 column (φ4.6×150 mm) manufactured by Tosoh Corporation (Tokyo, Japan) were used.

[0215] (Protein folding test using glutathione) Reduced and modified BPTI (30 μM) was dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) in the presence of 200 μM oxidized glutathione (GSSG) and 1.0 mM reduced glutathione (GSH), and incubated at 30°C. Aliquots of the reaction solution (200 μL) were taken at 0, 1, 5, 10, 30, and 60 minutes after the start of the reaction, mixed with an equivalent amount of 1 M hydrochloric acid, and analyzed by reverse-phase HPLC. Flow rate: 1.0 mL min －１ The measurement was performed at a wavelength of 229 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; content of solvent A: 95% at 0 minutes, 80% at 15 minutes, and 65% at 45 minutes).

[0216] The results are shown in Figure 7. It was confirmed that the reduced denatured BPTI was folded over time to form the native structural form of BPTI.

[0217] (Protein folding test using compound (A-1-11) and compound (B-1-1-1)) Reduced and modified BPTI (30 μM) was dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) in the presence of 200 μM compound (B-1-1-1) and 1.0 mM compound (A-1-1-1), and incubated at 30°C. Aliquots of the reaction solution (200 μL) were taken at 0, 1, 5, 10, 30, and 60 minutes after the start of the reaction, mixed with an equivalent amount of 1 M hydrochloric acid, and analyzed by reverse-phase HPLC. Flow rate: 1.0 mL min －１ The measurement was performed at a wavelength of 229 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; content of solvent A: 95% at 0 minutes, 80% at 15 minutes, and 65% at 45 minutes).

[0218] The results are shown in Figure 8. It was confirmed that the reduced denatured BPTI was folded over time to form the native structure of BPTI.

[0219] (Protein folding test using compound (A-1-1-1) and compound (B-1-1-1) in the presence of transition metal ions) In the presence of 200 μM compound (B-1-1-1) and 1.0 mM compound (A-1-1-1), 120 μM metal salt (NiCl ２ or CuCl ２ ) and reduced modified BPTI (30 μM) were dissolved and incubated at 30°C. One hour after the start of the reaction, the reaction solution (200 μL) was mixed with an equivalent amount of 1 M hydrochloric acid and analyzed by reversed-phase HPLC. The flow rate was 1.0 mL min －１ The measurement was performed at a wavelength of 229 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; content of solvent A: 95% at 0 minutes, 80% at 15 minutes, and 65% at 45 minutes).

[0220] Nickel (II) ions (Ni ２＋The results of the folding test in the presence of nickel(II) ions are shown in Figure 9. It was confirmed that the higher the concentration of nickel(II) ions, the greater the proportion of native BPTI formed, and folding was promoted.

[0221] Copper (II) ions (Cu ２＋ The results of the folding test in the presence of copper(II) ions are shown in Figure 10. It was confirmed that the higher the concentration of copper(II) ions, the greater the proportion of native structure-formed BPTI, and folding was promoted. These results confirmed that compound (A-1-1-1) and compound (B-1-1-1) promote the folding of reduced, denatured proteins in the presence of transition metal ions.

[0222] (Protein folding test under various conditions) Reduced modified BPTI (30 μM) and each of the following compounds were dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) to prepare reaction solutions (1) to (5): (1) GSSG (200 μM), GSH (1.0 mM), and CuCl ２ (120μM) (2) GSSG (200μM), GSH (1.0mM), cyclam (1.4mM), and CuCl ２ (120 μM) (3) GSSG (200 μM) and GSH (1.0 mM) (4) Compound (A-1-1-1) (1.0 mM), compound (B-1-1-1) (200 μM), and CuCl ２ (120 μM) (5) Compound (A-1-1-1) (1.0 mM) and compound (B-1-1-1) (200 μM)

[0223] Each of the reaction solutions (1) to (5) above was incubated at 30°C. One hour after the start of the reaction, the reaction solution (200 μL) was mixed with an equivalent amount of 1 M hydrochloric acid and analyzed by reverse-phase HPLC. The flow rate was 1.0 mL min －１ The measurement was performed at a wavelength of 229 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; content of solvent A: 95% at 0 minutes, 80% at 15 minutes, and 65% at 45 minutes).

[0224] The results are shown in Figure 11. When glutathione (GSH / GSSH) was used, folding did not proceed in the presence of copper ions, and native structure-type BPTI was hardly formed (reaction solution (1)). When cyclam was added to glutathione (GSH / GSSH), folding did not proceed in the presence of copper ions, and native structure-type BPTI was hardly formed (reaction solution (2)). On the other hand, when compound (A-1-1-1) and compound (B-1-1-1) were used, folding was more promoted in the presence of copper ions than in the absence of copper ions (reaction solutions (3) and (4)). These results confirmed that compound (A-1-1-1) and compound (B-1-1-1) promote the folding of reduced, denatured proteins in the presence of transition metal ions.

[0225] (Zn using compound (A-1-1-1) and compound (B-1-1-1) ＩＩ Protein folding test in the presence of ions) Protein folding test was performed in the presence of 200 μM oxidized glutathione (GSSG) and 1.0 mM reduced glutathione (GSH), or in the presence of 200 μM compound (B-1-1-1) and 1.0 mM compound (A-1-1-1), with ZnSO in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5). ４ and reduced modified BPTI (30 μM) were dissolved and incubated at 30° C. One hour after the start of the reaction, the reaction solution (200 μL) was mixed with an equivalent amount of 1 M hydrochloric acid and analyzed by reversed-phase HPLC. The flow rate was 1.0 mL min －１ The measurement was performed at a wavelength of 229 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; content of solvent A: 95% at 0 minutes, 80% at 15 minutes, and 65% at 45 minutes).

[0226] The results are shown in Figure 12. When GSSG and GSH were used, Zn ＩＩ In the presence of Zn ions, the rate of formation of native structure BPTI was less than 1%. This result indicates that the protein folding promoting effect of GSSG and GSH is ＩＩOn the other hand, when the compound (A-1-1-1) and the compound (B-1-1-1) were used, the activity was significantly inhibited by Zn ions. ＩＩ In the presence of Zn ions, ＩＩ The rate of formation of native structure-type BPTI was improved compared to that in the absence of Zn ions. This result indicates that the protein folding promoting effect of Compound (A-1-1-1) and Compound (B-1-1-1) is enhanced by the addition of Zn ions. ＩＩ This indicates that the reaction is promoted by ions.

[0227] <Cytotoxicity Evaluation Test> (Cytotoxicity Test of Compound (A-1-1-1) and Compound (B-1-1-1)) HeLa cells were seeded in a 96-well plate at 5,000 cells / well and incubated at 37°C and 5% CO ２ The cells were cultured under the same conditions for 24 hours. Subsequently, a fixed amount of a mixed solution (10:1) of compound (A-1-1-1) and compound (B-1-1-1) was added. After 24 hours, a Cell Counting Kit-8 (Dojindo Laboratories) solution was added. After 2 hours, the absorbance at 450 nm was measured to estimate the cell viability.

[0228] The results are shown in Figure 13. A 10:1 mixture of compounds (A-1-1-1) and (B-1-1-1) did not significantly affect the viability of HeLa cells within the tested concentration range. This result confirmed that the cytotoxicity of compounds (A-1-1-1) and (B-1-1-1) was limited.

[0229] (Cu ＩＩ Inhibitory effect of compound (A-1-1-1) and compound (B-1-1-1) on cytotoxicity of ions) HeLa cells were seeded in a 96-well plate at 5,000 cells / well and incubated at 37°C, 5% CO ２ The cells were cultured for 24 hours under the conditions of CuCl ２ Add an aqueous solution containing 100 μM Cu ＩＩ After 4 hours, a certain amount of a mixed solution (10:1) of compound (A-1-1-1) and compound (B-1-1-1), or a mixed solution (10:1) of oxidized glutathione (GSSG) and reduced glutathione (GSH) was added. ２24 hours after the addition, Cell Counting Kit-8 solution was added, and 2 hours later, the absorbance at 450 nm was measured to estimate the cell viability.

[0230] The results are shown in Figure 14. ＩＩ The addition of ions reduced the cell viability of HeLa cells to about 30%. The addition of GSH and GSSG had almost no effect on improving the cell viability of HeLa cells. On the other hand, the addition of compound (A-1-1-1) and compound (B-1-1-1) significantly improved the viability of HeLa cells. From these results, it can be seen that the mixture of compound (A-1-1-1) and compound (B-1-1-1) ＩＩ It was confirmed that it has the effect of reducing ion-induced cytotoxicity.

[0231] (Cu ＩＩ Inhibitory effect of compound (B-1-1-1) on cytotoxicity of ions) HeLa cells were seeded in a 96-well plate at 5,000 cells / well and incubated at 37°C, 5% CO ２ The cells were cultured for 24 hours under the conditions of CuCl ２ Add an aqueous solution containing 100 μM Cu ＩＩ After 4 hours, a certain amount of compound (B-1-1-1) solution or oxidized glutathione (GSSG) solution was added. ２ 24 hours after the addition, Cell Counting Kit-8 solution was added. After 2 hours, the absorbance at 450 nm was measured to estimate the cell viability.

[0232] The results are shown in Figure 15. ＩＩ The addition of ions reduced the cell viability of HeLa cells to about 20%. The addition of GSSG had almost no effect on improving the cell viability of HeLa cells. On the other hand, the addition of compound (B-1-1-1) significantly improved the viability of HeLa cells. From these results, it can be seen that compound (B-1-1-1) has a significant effect on the cell viability of HeLa cells. ＩＩ It was confirmed that it has the effect of reducing ion-induced cytotoxicity.

[0233] Experimental Example 2 Synthesis Example of Compound (A-1-2-1) and Compound (B-1-2-1) Compound (A-1-2-1) and compound (B-1-2-1) were synthesized according to the following synthesis scheme: The percentages in the synthesis scheme represent the yields in each reaction.

[0234]

[0235] (Synthesis of Compounds 2 and 7) Compounds 2 and 7 were synthesized according to previous reports (P. Jain et al., Langmuir 2019, 35, 1864; M. Starck et al., Bioconjugate Chem. 2020, 31, 229). In the above reaction scheme, TsCl represents p-toluenesulfonyl chloride, Et ３ N is triethylamine, Boc ２ O represents di-tert-butyl dicarbonate, Ts represents a paratoluenesulfonyl group, and Boc represents a tert-butoxycarbonyl group. Compound 1 is 2-hydroxyethyl disulfide, and compound 6 is 1,4,7-triazacyclononane trihydrochloride (TACN·3HCl).

[0236] (Synthesis of Compound 8) Compound 8 (245 mg, 0.745 mmol) was dissolved in 3.5 mL of dehydrated acetonitrile and mixed with potassium carbonate (103 mg, 0.747 mmol, 2.5 eq). Compound 2 (117 mg, 0.849 mmol, 2.5 eq) was added, and the mixture was stirred at 60°C under a nitrogen atmosphere for 39 hours. The resulting suspension was filtered by suction, and the filtrate was dried under reduced pressure and purified by silica gel chromatography to obtain compound 8 (215 mg, 0.276 mmol, 181%). The obtained compound 8 was １ The compound was identified by 1 H NMR measurement (FIG. 16) and MALDI-TOF mass spectrometry measurement (FIG. 17).

[0237] (Synthesis of Compound (B-1-2-1)) To an ethanol solution (320 μL) of Compound 8 (44 mg, 0.057 mmol), 320 μL of concentrated hydrochloric acid was added under ice cooling, and the mixture was stirred for 10 minutes and then at room temperature for 3 hours. The resulting white suspension was ice cooled and suction filtered to obtain a white solid. This was further purified by reverse phase HPLC to obtain Compound (B-1-2-1) (4.5 mg, 0.012 mmol, 21%). The obtained Compound (B-1-2-1) was １ The compound was identified by 1 H NMR measurement (FIG. 18) and MALDI-TOF mass spectrometry measurement (FIG. 19).

[0238] (Synthesis of Compound (A-1-2-1)) 320 μL of concentrated hydrochloric acid was added to an ethanol solution (320 μL) of Compound 8 (44 mg, 0.057 mmol), and the mixture was stirred for 10 minutes and then at room temperature for 3 hours. The resulting white suspension was ice-cooled and suction filtered to obtain a white solid. Subsequently, 1,4-dithiothreitol (41 mg, 0.0.26 mmol, 10 eq) and 1.1 mL of ultrapure water were added, and the mixture was stirred at room temperature for 45 hours. Purification by reverse-phase HPLC yielded Compound (A-1-2-1) (3.6 mg, 0.0192 mmol, 17% from Compound 8). The resulting Compound (A-1-2-1) was １ The compound was identified by 1 H NMR measurement (FIG. 20) and MALDI-TOF mass spectrometry measurement (FIG. 21).

[0239] <Protein Folding Test> BPTI, which has three disulfide bonds in the molecule, was used as a model protein for analyzing the folding process.

[0240] (Quantitative determination of compound (A-1-2-1) concentration) A solution of compound (A-1-2-1) dissolved in 10 mM aqueous hydrochloric acid was mixed with 10 mM Ellman's reagent (5,5'-dithio-bis-(2-nitrobenzoic acid)) in 50 mM Tris-HCl buffer (pH 7.5, 0.3 M NaCl), and UV absorption measurement was carried out at 30° C. The concentration of the compound (A-1-2-1) solution was determined from the absorbance at 412 nm.

[0241] (Preparation of reduced modified BPTI) Reduced modified BPTI was prepared in the same manner as in Experimental Example 1.

[0242] (Protein folding test using compound (A-1-2-1) and compound (B-1-2-1)) Reduced modified BPTI (30 μM) was dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) in the presence of 90 μM compound (B-1-2-1) and 900 μM compound (A-1-2-1), or in the presence of 90 μM oxidized glutathione (GSH) and 900 μM reduced glutathione (GSSG), and incubated at 30°C. One hour after the start of the reaction, the reaction solution (200 μL) was mixed with an equivalent amount of 1 M hydrochloric acid and analyzed by reverse-phase HPLC. Flow rate: 1.0 mL min －１ The measurement was performed at a wavelength of 229 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; content of solvent A: 95% at 0 minutes, 80% at 15 minutes, and 65% at 45 minutes).

[0243] The results are shown in Figure 22. Compound (A-1-2-1) and compound (B-1-2-1) formed a higher proportion of native structure-type BPTI than GSH and GSSG. These results confirmed that compound (A-1-2-1) and compound (B-1-2-1) have a stronger effect on promoting protein folding than GSH and GSSG.

[0244] (Zn using compound (B-1-2-1) ＩＩ Protein folding test in the presence of ions) In the presence of 90 μM of compound (B-1-2-1), 90 μM of Zn was added to Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5). ＩＩ ions (ZnSO ４ ) and reduced modified BPTI (30 μM) were dissolved and incubated at 30°C. One hour after the start of the reaction, the reaction solution (200 μL) was mixed with an equivalent amount of 1 M hydrochloric acid and analyzed by reversed-phase HPLC. The flow rate was 1.0 mL min －１The measurement was performed at a wavelength of 229 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; content of solvent A: 95% at 0 minutes, 80% at 15 minutes, and 65% at 45 minutes).

[0245] The results are shown in Figure 23. In the presence of compound (B-1-2-1), Zn ＩＩ The higher the ion concentration, the greater the proportion of native structure BPTI formed. ＩＩ It was confirmed that the higher the ion concentration, the greater the effect of compound (B-1-2-1) in promoting protein folding. ＩＩ Since the native structure of BPTI was formed even at an ion concentration of 0 μM, compound (B-1-2-1) was ＩＩ It was confirmed that the protein folding promoting effect was achieved even in the absence of ions.

[0246] (Zn using compound (A-1-2-1) and compound (B-1-2-1) ＩＩ Protein folding test in the presence of ions) In the presence of 90 μM compound (B-1-2-1) and 900 μM compound (A-1-2-1), or in the presence of 90 μM oxidized glutathione (GSH) and 900 μM reduced glutathione (GSSG), 180 μM Zn was added to Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5). ＩＩ ions (ZnSO ４ ) and reduced modified BPTI (30 μM) were dissolved and incubated at 30°C. One hour after the start of the reaction, the reaction solution (200 μL) was mixed with an equivalent amount of 1 M hydrochloric acid and analyzed by reversed-phase HPLC. The flow rate was 1.0 mL min －１ The measurement was performed at a wavelength of 229 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; content of solvent A: 95% at 0 minutes, 80% at 15 minutes, and 65% at 45 minutes).

[0247] The results are shown in Figure 24. ＩＩIn the presence of ions, when GSH and GSSG were used, the protein aggregated and folding hardly proceeded. On the other hand, when Compound (A-1-2-1) and Compound (B-1-2-1) were used, Zn ＩＩ Even in the presence of Zn ions, ＩＩ From this result, it is clear that compounds (A-1-2-1) and (B-1-2-1) have the same structure as BPTI in the absence of Zn. ＩＩ It was confirmed that the compound has the effect of promoting protein folding both in the presence and absence of ions.

[0248] (Zn using compound (B-1-2-1) ＩＩ Protein folding test in the presence of ions) In the presence of 90 μM of compound (B-1-2-1) or 900 μM of reduced glutathione (GSSG), 90 μM of Zn was added to Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5). ＩＩ ions (ZnSO ４ ) and reduced modified BPTI (30 μM) were dissolved and incubated at 30°C. One hour after the start of the reaction, the reaction solution (200 μL) was mixed with an equivalent amount of 1 M hydrochloric acid and analyzed by reversed-phase HPLC. The flow rate was 1.0 mL min －１ The measurement was performed at a wavelength of 229 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; content of solvent A: 95% at 0 minutes, 80% at 15 minutes, and 65% at 45 minutes).

[0249] The results are shown in Figure 25. ＩＩ In the presence of ions, when GSSG was used, the protein aggregated and folding hardly proceeded. On the other hand, when compound (B-1-2-1) was used, Zn ＩＩ In the presence of Zn ions, ＩＩ The formation rate of native structure BPTI was higher than that in the absence of Zn ions. ＩＩ It was confirmed that the protein folding was more effectively promoted in the presence of ions.

[0250] Experimental Example 3 Synthesis Example of Compound (B-2-1-1) Compound (B-2-1-1) was synthesized according to the following synthesis scheme: The percentages in the synthesis scheme represent the yields in each reaction.

[0251]

[0252] (Synthesis of Compounds 2 and 9) Compounds 2 and 9 were synthesized according to previous reports (P. Jain et al., Langmuir 2019, 35, 1864.; CA Barta et al., Inorg Chem. 2008, 47, 2280.). In the above reaction scheme, MeCN represents acetonitrile.

[0253] (Synthesis of Compound (B-2-1-1)) Compound 9 (14 mg, 0.11 mmol) was dissolved in 4 mL of dehydrated acetonitrile and mixed with potassium carbonate (38 mg, 0.28 mmol, 2.5 eq). Compound 2 (51 mg, 0.11 mmol, 1.0 eq) was added, and the mixture was stirred at 60°C under a nitrogen atmosphere for 24 hours. The resulting suspension was filtered by suction, and the filtrate was dried under reduced pressure to obtain a yellow solid. This was further purified by reverse phase HPLC to obtain compound (B-2-1-1) (1.3 mg, 0.0052 mmol, 5%). The obtained compound was １ The compound was identified by 1 H NMR measurement (FIG. 26) and MALDI-TOF mass spectrometry measurement (FIG. 27).

[0254] <Protein Folding Test> BPTI, which has three disulfide bonds in the molecule, was used as a model protein for analyzing the folding process.

[0255] (Preparation of reduced modified BPTI) Reduced modified BPTI was prepared in the same manner as in Experimental Example 1.

[0256] (Protein folding test using compound (B-2-1-1)) Reduced and modified BPTI (30 μM) was dissolved in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) in the presence of 90 μM compound (B-2-1-1) or 90 μM oxidized glutathione (GSSG), and incubated at 30°C. One hour after the start of the reaction, the reaction solution (200 μL) was mixed with an equivalent amount of 1 M hydrochloric acid and analyzed by reverse-phase HPLC. Flow rate: 1.0 mL min －１ The measurement was performed at a wavelength of 229 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; content of solvent A: 95% at 0 minutes, 80% at 15 minutes, and 65% at 45 minutes).

[0257] The results are shown in Figure 28. Compound (B-2-1-1) exhibited the same level of protein folding promoting effect as GSSG.

[0258] (Protein folding test using compound (B-2-1-1) in the presence of transition metal ions) In the presence of 90 μM compound (B-2-1-1), 90 μM Cu was added to Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5). ＩＩ ions (CuCl ２ ) or 90 μM Ni ＩＩ ions (NiCl ２ ) and reduced modified BPTI (30 μM) were dissolved and incubated at 30°C. One hour after the start of the reaction, the reaction solution (200 μL) was mixed with an equivalent amount of 1 M hydrochloric acid and analyzed by reversed-phase HPLC. The flow rate was 1.0 mL min －１ The measurement was performed at a wavelength of 229 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; content of solvent A: 95% at 0 minutes, 80% at 15 minutes, and 65% at 45 minutes).

[0259] Cu ＩＩ The results in the presence of Cu ions are shown in Figure 29. In the presence of compound (B-2-1-1), ＩＩThe higher the ion concentration, the greater the proportion of native structure BPTI formed. ＩＩ It was confirmed that the higher the ion concentration, the greater the effect of compound (B-2-1-1) in promoting protein folding.

[0260] Ni ＩＩ The results in the presence of ions are shown in Figure 30. In the presence of compound (B-2-1-1), ＩＩ The higher the ion concentration, the greater the proportion of native structure BPTI formed. ＩＩ It was confirmed that the higher the ion concentration, the greater the effect of compound (B-2-1-1) in promoting protein folding.

[0261] (Ni using a catalytic amount of compound (B-2-1-1) ＩＩ Protein folding test in the presence of ions) In the presence of 6 μM of compound (B-2-1-1), NiCl was added to Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5). ２ Reduced and modified BPTI (30 μM) (0 μM or 1 μM) was dissolved and incubated at 30°C. After a certain time from the start of the reaction, the reaction solution (200 μL) was mixed with an equivalent amount of 1 M hydrochloric acid and analyzed by reverse-phase HPLC. Flow rate: 1.0 mL min －１ The measurement was performed at a wavelength of 229 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; content of solvent A: 95% at 0 minutes, 80% at 15 minutes, and 65% at 45 minutes).

[0262] Ni ＩＩ The results in the absence of ions are shown in Figure 31. It was confirmed that even a catalytic amount of compound (B-2-1-1) had the effect of promoting protein folding.

[0263] Ni ＩＩ The results in the presence of Ni ions are shown in Figure 32. ＩＩ In the presence of ions, Ni ＩＩ Compared to the case in the absence of ions (FIG. 31), the protein folding promoting effect of compound (B-2-1-1) was significantly improved.

[0264] (Cu using a catalytic amount of compound (B-2-1-1) ＩＩ Protein folding test in the presence of CuCl in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5) in the presence of 30 μM of compound (B-2-1-1). ２ (5 μM) and reduced modified BPTI (30 μM) were dissolved and incubated at 30°C. After a certain time from the start of the reaction, the reaction solution (200 μL) was mixed with an equivalent amount of 1 M hydrochloric acid and analyzed by reversed-phase HPLC. Flow rate: 1.0 mL min －１ The measurement was performed at a wavelength of 229 nm, and a linear gradient was applied to the elution solvent ratio during measurement (solvent A: water containing 0.05% TFA, solvent B: acetonitrile containing 0.05% TFA; content of solvent A: 95% at 0 minutes, 80% at 15 minutes, and 65% at 45 minutes).

[0265] The results are shown in Figure 33. ＩＩ In the presence of Cu ions, ＩＩ Compared to the case in the absence of ions (FIG. 31), the protein folding promoting effect of compound (B-2-1-1) was significantly improved.

[0266] Since the native structure of BPTI contains three disulfide bonds, three equivalents of an oxidizing agent, i.e., compound (B-2-1-1), are required for folding of BPTI. ＩＩ Or Cu ＩＩ In the presence of transition metal ions, the addition of a catalytic amount of compound (B-2-1-1) promoted folding far exceeding the theoretical yield of an equivalent reaction. Therefore, it is speculated that the complex of compound (B-2-1-1) with transition metal ions catalytically promoted the oxidative folding of BPTI.

[0267] [Experimental Example 4] (Cu using compounds (A-1-1-1) and (B-1-1-1)) ＩＩFolding test of reduced-denatured RNase A in the presence of ions) RNase A (16 mg) was dissolved in 0.2 mM Tris-HCl (pH 8.7, 0.5 mL) containing 6.0 M guanidine hydrochloride and 100 mM DTT, and the solution was incubated at 25°C for 2 hours. The solution was then dialyzed against 10 mM hydrochloric acid for 2 hours to remove guanidine hydrochloride and DTT. This dialysis procedure was repeated three times, yielding a reduced-denatured RNase A solution. The reduced-denatured RNase A solution after dialysis was stored at -30°C until use in a folding experiment.

[0268] In the presence of 32 μM oxidized glutathione (GSSG) and 320 μM reduced glutathione (GSH), or 320 μM compound (B-1-1-1) and 32 μM compound (A-1-1-1), 64 μM CuCl was added to Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5). ２ and reduced denatured RNase A (8 μM) were dissolved and incubated at 30°C. After a certain time from the start of the reaction, the reaction solution was mixed with a substrate oligonucleotide fluorescently labeled at both ends, and the enzymatic activity of RNase A was quantified by measuring the change in fluorescence intensity associated with cleavage of the substrate. The substrate oligonucleotide used was 5'-FAM-dA rU dA dA-TAMRA-3' (dA: deoxyadenosine, rU: uridine) labeled with 6-carboxyfluorescein (FAM) and 5-carboxytetramethylrhodamine (TAMRA). Fluorescence intensity was measured using a spectrofluorometer (RF-6000, Shimadzu Corporation).

[0269] The results are shown in Figure 34. When GSSG / GSH (oxidized glutathione and reduced glutathione), which are existing protein folding promoters, were used, Cu ＩＩ The addition of Cu ions significantly reduced the recovery rate of RNase A enzyme activity. ＩＩ It was confirmed that the oxidative folding of RNase A to its native structure was inhibited in the presence of Cu ions.ＩＩ Regardless of the presence or absence of ions, the recovery rate of RNase A enzyme activity was significantly higher than when GSH / GSSG was used. These results confirmed that compounds (A-1-1-1) / (B-1-1-1) promote the oxidative folding of RNase A even in the presence of transition metal ions, and that their folding efficiency exceeds that of existing folding agents.

[0270] According to the present invention, there are provided a protein-folding agent capable of promoting protein folding in the presence of transition metal ions, a compound usable for the protein-folding agent, a protein folding method using the protein-folding agent, a pharmaceutical composition containing the protein-folding agent, and an agent for suppressing cytotoxicity caused by transition metals.

[0271] Although the preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments. Addition, omission, substitution, and other modifications of the configuration are possible within the scope of the spirit of the present invention. The present invention is not limited by the above description, but is limited only by the scope of the appended claims.

Claims

1. A protein folding agent comprising at least one compound selected from the group consisting of (A) and (B) below: (A) a cyclic polyamine containing one or more thiol groups or disulfide bonds and capable of binding to a transition metal ion; (B) a compound in which two or more of the cyclic polyamines of (A) are bonded together via disulfide bonds between the thiol groups; (C) a complex of the cyclic polyamine of (A) or the compound of (B) with a transition metal ion; (D) a salt of the cyclic polyamine of (A) or the compound of (B); and (E) a solvate of the cyclic polyamine of (A) or the compound of (B).

2. The protein-folding agent according to claim 1, wherein the cyclic polyamine (A) is a compound represented by the following general formula (A): [In the formula, Rx １ ~Rx ３ Each independently represents a hydrogen atom or a monovalent organic group. １ ~Rx ３ At least one of Rx is a monovalent organic group containing one or more thiol groups. １ ~Rx ３ When Y contains a total of two or more thiol groups, the thiol groups may be bonded to each other to form a disulfide bond. １ ~Y ３ Each independently represents an alkylene group having 1 to 5 carbon atoms. m0 represents an integer of 1 to 3. When m0 is an integer of 2 or more, Rx ３ may be the same or different, and two or more Y ３ may be the same or different from each other.

3. The protein-folding agent according to claim 2, wherein the cyclic polyamine (A) is a compound represented by the following general formula (A-1) or (A-2): [In the formula, Rx ２ and Rx ３ Each of Y independently represents a hydrogen atom or a monovalent organic group. １ ~Y ３ Each of Lx independently represents an alkylene group having 1 to 5 carbon atoms. １ and Lx ２ Each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom. m0 represents an integer of 1 to 3. When m0 is an integer of 2 or more, Rx ３ may be the same or different, and two or more Y ３ may be the same or different from each other.

4. The protein-folding agent according to claim 3, wherein the cyclic polyamine (A) is a compound represented by the following general formula (A-1-1), and the compound (B) is a compound represented by the following general formula (B-1-1): [In the formula, Y １ , Y ２ , Y １１ , Y １２ , Y ２１ , and Y ２２ each independently represents an alkylene group having 1 to 5 carbon atoms; R and R １ , and R ２ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; L, L １ , and L ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.] 5. The protein-folding agent according to claim 4, comprising at least one compound selected from the group consisting of the compound (B), a metal complex of the compound (B), a salt of the compound (B), and a solvate of the compound (B).

6. The protein-folding agent according to claim 4, wherein the compound (B) is a compound represented by the following formula (B-1-1-1):

7. The protein-folding agent according to claim 4, wherein the cyclic polyamine (A) is a compound represented by the following formula (A-1-1-1), and the compound (B) is a compound represented by the following formula (B-1-1-1):

8. The protein-folding agent according to claim 3, wherein the cyclic polyamine (A) is a compound represented by the following general formula (A-1-2), and the compound (B) is a compound represented by the following general formula (B-1-2): [In the formula, Y １ , Y ２ , Y ３ , Y １１ , Y １２ , Y １３ , Y ２１ , Y ２２ , and Y ２３ each independently represents an alkylene group having 1 to 5 carbon atoms; R and R １ , and R ２ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; L, L １ , and L ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.] 9. The protein-folding agent according to claim 3, wherein the cyclic polyamine (A) is a compound represented by the following formula (A-1-2-1), and the compound (B) is a compound represented by the following formula (B-1-2-1):

10. The protein-folding agent of claim 1, comprising: at least one compound selected from the group consisting of the cyclic polyamine (A), a metal complex of the cyclic polyamine (A), a salt of the cyclic polyamine (A), and a solvate of the cyclic polyamine (A); and at least one compound selected from the group consisting of the compound (B), a metal complex of the compound (B), a salt of the compound (B), and a solvate of the compound (B).

11. The protein-folding agent according to claim 3, wherein the cyclic polyamine (A) is a compound represented by the following formula (A-2-1): [In the formula, Y １ ~Y ３ each independently represents an alkylene group having 1 to 5 carbon atoms; R represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; L １ , and L ２ each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom.] 12. The protein-folding agent according to claim 11, wherein the cyclic polyamine (A) is a compound represented by the following formula (A-2-1-1):

13. The protein-folding agent according to any one of claims 1 to 12, which is used for folding a protein in the presence of a transition metal ion.

14. The protein-folding agent of claim 13, which is used for folding a protein containing a transition metal ion.

15. The protein-folding agent of claim 13, wherein the transition metal ion is at least one selected from the group consisting of copper ions, nickel ions, iron ions, manganese ions, zinc ions, and molybdenum ions.

16. A compound represented by the following general formula (B-1), (A-1-2), or (A-2): [In general formula (B-1), Rx １２ , Rx １３ , Rx ２２ and Rx ２３ Each of Y independently represents a hydrogen atom or a monovalent organic group. １１ , Y １２ , Y １３ , Y ２１ , Y ２２ , and Y ２３ Each of Lx independently represents an alkylene group having 1 to 5 carbon atoms. １ and Lx ２ Each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom. Each of m01 and m02 independently represents an integer of 1 to 3. When m01 is an integer of 2 or more, Rx １３ may be the same or different, and two or more Y １３ may be the same or different from each other. When m02 is an integer of 2 or more, Rx ２３ may be the same or different, and two or more Y ２３ In the general formula (A-1-2), Y １ ~Y ３ Each of R independently represents an alkylene group having 1 to 5 carbon atoms; each of R independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; and L represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom. ３ represents a hydrogen atom or a monovalent organic group. １ ~Y ３ Each of Lx independently represents an alkylene group having 1 to 5 carbon atoms. １ and Lx ２ Each independently represents an alkylene group having 1 to 10 carbon atoms which may contain at least one atom selected from the group consisting of an oxygen atom and a nitrogen atom. m0 represents an integer of 1 to 3. When m0 is an integer of 2 or more, Rx ３ may be the same or different, and two or more Y ３ may be the same or different from each other.

17. The compound according to claim 16, which is represented by the following formula (B-1-1-1), (A-1-2-1), (B-1-2-1), or (A-2-1-1).

18. A method for folding a protein, comprising the step of incubating the protein in the presence of a protein folding agent according to any one of claims 1 to 12.

19. The method for folding a protein according to claim 18, wherein the protein is an unfolded protein or a misfolded protein.

20. The protein folding method according to claim 18, wherein the incubation is carried out in the presence of a transition metal ion.

21. The method of claim 20, wherein the folded protein after the incubating step comprises the transition metal ion.

22. The protein folding method according to claim 20, wherein the transition metal ion is at least one selected from the group consisting of copper ions, nickel ions, iron ions, manganese ions, zinc ions, and molybdenum ions.

23. A pharmaceutical composition comprising the protein folding agent according to any one of claims 1 to 12 and a pharma- ceutically acceptable carrier.

24. The pharmaceutical composition according to claim 23, which is used for treating or preventing a disease caused by protein aggregation.

25. The pharmaceutical composition according to claim 24, wherein the disease caused by protein aggregation is a protein folding disease.

26. The pharmaceutical composition according to claim 25, wherein the folding disease is at least one selected from the group consisting of amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies, spinocerebellar degeneration, frontotemporal dementia, Pick's disease, Creutzfeldt-Jakob disease, familial amyloidotic polyneuropathy, and type 2 diabetes.

27. An inhibitor of transition metal-induced cytotoxicity, comprising at least one compound selected from the group consisting of (A) and (B) below: (A) a cyclic polyamine containing one or more thiol groups or disulfide bonds and capable of binding to a transition metal ion; (B) a compound in which two or more cyclic polyamines of (A) are bonded together via disulfide bonds between the thiol groups; (C) a complex of the cyclic polyamine of (A) or the compound of (B) with a transition metal ion; (D) a salt of the cyclic polyamine of (A) or the compound of (B); and (E) a solvate of the cyclic polyamine of (A) or the compound of (B).

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