Anticancer agent containing a metal complex and combination agent with radiation in chemoradiotherapy

Abstract

To provide an anticancer agent or a concomitant drug with radiation in chemoradiotherapy containing a gold complex with an amino acid derivative as a ligand, and a novel gold complex that can be used in the anticancer agent or the concomitant drug with radiation in chemoradiotherapy.SOLUTION: An anticancer agent or a concomitant drug with radiation in chemoradiotherapy, containing a gold complex represented by the formula (1): L1-S-Au-(L2)m (1) (where L1 is an amino acid derivative, L2 is a heterocycle having -SH coordinated to Au shown in the formula (1), optionally having a further substituent, m is 0 or 1).SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an anticancer agent containing a metal complex and a combined agent with radiation in chemoradiotherapy. Furthermore, the present invention relates to a novel metal complex that can be used as a combined agent with radiation in anticancer agents and chemoradiotherapy.

Background Art

[0002] Cancer is the most lethal disease among all diseases, and thus various anticancer agents have been developed.

[0003] Conventionally, anticancer agents containing metal complexes as active ingredients have been widely known, and platinum anticancer drugs such as cisplatin have been clinically used in Japan. In particular, cisplatin has been recognized to have strong anticancer activity and is used as a representative anticancer agent, but problems of severe side effects such as nephrotoxicity, nausea, vomiting, and hearing loss have been pointed out. Therefore, there is a need for an anticancer agent that retains strong anticancer activity while reducing side effects. For example, in Patent Document 1, the synthesis of a novel metal complex showing anticancer activity has been studied.

[0004] The present inventors have hitherto studied the production of a non-cyanide gold plating bath containing thiopronine, which is an amino acid derivative, as a complexing agent, for the purpose of applying it to devices that require safety such as medical devices (see, for example, Patent Document 2). Furthermore, the production of gold plating using a thiopronine gold complex to which a mercaptothiodiazole derivative or a mercaptotriazole derivative is bonded has also been studied (see, for example, Non-Patent Document 1).

[0005] In addition, it has also been reported that four gold preparations containing auranofin, which is already approved as a rheumatoid arthritis treatment drug in Japan, inhibit the adhesion between vascular endothelial cells and cancer cells in a dose-dependent manner (see, for example, Non-Patent Document 2).

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Non-Patent Document

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] In a specific cancer cell culture system, the number of adhesions between vascular endothelial cells and the cancer cells can be an index for evaluating the metastatic ability of the cancer cells. However, for the above auranofin, etc., the effective action concentration against cancer cells, with the half-maximal inhibitory concentration (IC 50 ) in the number of adhesions of both cells in the medium as an index, is as high as ranging from over 0.025 mM to over 0.1 mM, and it is expected to be difficult for clinical application (see Non-Patent Document 2, p. 96). In addition, a chemical structure such as auranofin has low flexibility in molecular design, so it is not easy to synthesize gold complexes of related compounds, and there is also a problem that it is difficult to search for designs to obtain the expected physiological activity.

[0009] The above gold preparations such as auranofin have inorganic compounds or organic ligands which are derivatives of glucose or succinic acid. However, it is not known that gold complexes with amino acid derivatives as ligands, such as thiopronine, exhibit an anticancer effect.

[0010] The present invention has been made in view of the above problems, and an object thereof is to provide an anticancer agent containing a gold complex having an amino acid derivative as a ligand and a combined agent with radiation in chemoradiotherapy. Furthermore, an object is to provide a novel gold complex that can be used in an anticancer agent and a combined agent with radiation in chemoradiotherapy.

Means for Solving the Problems

[0011] The present inventors have found that a gold complex having an amino acid derivative as a ligand has an anticancer effect and is effective as a combined agent with radiation in chemoradiotherapy, and have completed the present invention. Specifically, the present invention provides the following.

[0012] [1] An anticancer agent containing a gold complex represented by the following formula (1) or a combined agent with radiation in chemoradiotherapy. L 1 -S-Au-(L 2 ) m (1) (In the formula, L 1 represents an amino acid derivative, L 2 represents a heterocyclic ring having -SH coordinated to Au represented by the above formula (1) and further may have a substituent, and m represents 0 or 1.) [2] The anticancer agent or the combined agent with radiation in chemoradiotherapy according to [1], wherein the above gold complex is water-soluble. [3] The anticancer agent or the combined agent with radiation in chemoradiotherapy according to [1] or [2], wherein the above L 1 contains -C(=O)NR- or -NRC(=O)- (R represents a hydrogen atom or an acetyl group). [4] The anticancer agent or the combined agent with radiation in chemoradiotherapy according to any one of [1] to [3], wherein the above L 1 contains at least one selected from the group consisting of a carboxy group, a sulfonic acid group, an alkyl group, an alkenyl group, an aryl group, an alkoxy group, a heteroaryl group, a halogen atom, an acetyl group, an acetoxy group, an acetoxymethyl group, and a hydroxyalkyl group. [5] The above L 1It is a hydrocarbon group having 4 to 15 carbon atoms in total, which has a carboxy group and -C(=O)NR- or -NRC(=O)- (wherein R represents a hydrogen atom or an acetyl group), and may further have a substituent, and is an anticancer agent or a combined agent with radiation in chemoradiotherapy according to any one of [1] to [4]. [6] The above L 1 It is an amino acid derivative represented by the following formula (5), and is an anticancer agent or a combined agent with radiation in chemoradiotherapy according to any one of [1] to [5]. R 1 -T-R 2 -** (5) (In the formula, R 1 represents an alkyl group having 1 to 5 carbon atoms, T represents *-C(=O)NR-* or *-NRC(=O)-* (wherein R represents a hydrogen atom or an acetyl group, and * represents a bond with R 1 or R 2 ), R 2 represents a linear or branched alkylene group having 2 to 4 carbon atoms, and ** represents a bond with S in the above -S-Au-(L 2 ) m . R 1 and / or R 2 has a carboxy group.) [7] The above L 1 is a residue obtained by removing -SH from thiopronine, N-acetylpenicillamine or N-acetylcysteine respectively, and is an anticancer agent or a combined agent with radiation in chemoradiotherapy according to any one of [1] to [6]. [8] The above L 2 is a partial structure of a β-lactam antibiotic, and is an anticancer agent or a combined agent with radiation in chemoradiotherapy according to any one of [1] to [7]. [9] The above L 2 is a partial structure related to hydrophobicity and / or cell membrane permeability in a β-lactam antibiotic, and is an anticancer agent or a combined agent with radiation in chemoradiotherapy according to any one of [1] to [8].

[10] The above L 2The above heterocyclic ring in [1] to [9] is a 5- to 7-membered monocyclic ring containing 1 to 4 nitrogen atoms, 0 to 1 oxygen atom, and 0 to 1 sulfur atom as ring-constituting atoms, or a condensed ring composed of a total of 9 to 14 atoms including 1 to 4 nitrogen atoms, 0 to 1 oxygen atom, and 0 to 1 sulfur atom as ring-constituting atoms, which is an anticancer agent or an agent for combined use with radiation in chemoradiotherapy.

[11] The above L 2 The above heterocyclic ring in [1] to

[10] is an anticancer agent or an agent for combined use with radiation in chemoradiotherapy, which contains a structure in which electrons of S (sulfur atom) in the above -SH coordinated to Au represented by the above formula (1) can be delocalized.

[12] The above L 2 The above heterocyclic ring in [1] to

[11] is an anticancer agent or an agent for combined use with radiation in chemoradiotherapy, which is a conjugated heterocyclic ring.

[13] The above L 2 The above heterocyclic ring in [1] to

[12] is an anticancer agent or an agent for combined use with radiation in chemoradiotherapy, which has a structure containing a conjugated heterocyclic ring represented by the following formula (r).

Chemical formula

Chemical formula

[14] The above L 2The above complex ring in [1] to

[13] is selected from the group consisting of a tetrazole ring, a thiadiazole ring, a triazole ring, a thiazole ring, a thiazoline ring, a thiazolidine ring, an imidazoline ring, an imidazolidine ring, a triazine ring, a pyrrolidine ring, a pyrazolo-triazole fused ring, an oxadiazine ring, and a dithiazine ring, and is an anticancer agent described in any one of [1] to

[13] or a combined agent with radiation in chemoradiotherapy.

[15] The above L 2 is 5-mercapto-1-methyltetrazole (MM4), 2-amino-5-mercapto-1,3,4-thiadiazole (AMT), 1,2,4-triazole-3-thiol (MTZ), 3-amino-5-mercapto-1,2,4-triazole (AMZ), 2-mercapto-1,3,4-thiadiazole (MT), 2-mercapto-5-methyl-1,3,4-thiadiazole (MMT), 2-mercapto-5-methylthio-1,3,4-thiadiazole (MMTT), 2-imidazolidinethione (2NN), or 2-mercaptothiazoline (2SN), and the S of -SH in the L 2 coordinates to the Au represented by the above formula (1), and is an anticancer agent described in any one of [1] to

[14] or a combined agent with radiation in chemoradiotherapy.

[16] The above Au is a monovalent gold atom, and is an anticancer agent described in any one of [1] to

[15] or a combined agent with radiation in chemoradiotherapy.

[17] It is an anticancer agent for the treatment of leukemia, liver cancer, or lung cancer, and is an anticancer agent described in any one of [1] to

[16] or a combined agent with radiation in chemoradiotherapy.

[18] Further, it contains a solvent containing at least one selected from the group consisting of water and polyvinyl alcohol, and is an anticancer agent described in any one of [1] to

[17] or a combined agent with radiation in chemoradiotherapy.

[19] A gold complex precursor represented by the following formula (2) used for the synthesis of the above gold complex in the anticancer agent described in any one of [1] to

[18] or a combined agent with radiation in chemoradiotherapy. L 1 -S-Au (2) (In the formula, L 1 represents an amino acid derivative.)

[20] A gold complex represented by the following formula (3) (wherein, in the formula, L 1 is *-C(CH 3 )CONHCH 2 COOH (* represents a bond with -S- represented by formula (3)), L 2 is 2-amino-5-mercapto-1,3,4-thiadiazole (AMT), 1,2,4-triazole-3-thiol (MTZ), 3-amino-5-mercapto-1,2,4-triazole (AMZ), 2-mercapto-5-methylthio-1,3,4-thiadiazole (MMTT), 2-mercapto-5-methyl-1,3,4-thiadiazole (MMT), 2-mercapto-1,3,4-thiadiazole (MT), 5-mercapto-1-methyltetrazole (MM4), 2-imidazolidinethione (2NN), or 2-mercaptothiazoline (2SN), and the S of -SH in the L 2 excludes the gold complex in which the S coordinates to the Au represented by formula (3).). L 1 -S-Au-L 2 (3) (In the formula, L 1 represents an amino acid derivative, and L 2 represents a heterocyclic ring having -SH that coordinates to the Au represented by the above formula (3) and may further have a substituent.).

[21] A gold complex represented by the following formula (3) (wherein, in the formula, L 1 is *-C(CH 3 )CONHCH 2 COOH (* represents a bond with -S- represented by formula (3)), L 2 is 2-amino-5-mercapto-1,3,4-thiadiazole (AMT), and the S of -SH in the L 2 excludes the gold complex in which the S coordinates to the Au represented by formula (3).). L 1 -S-Au-L 2 (3) (In the formula, L 1 represents an amino acid derivative, and L 2 represents a heterocyclic ring having -SH that coordinates to the Au represented by the above formula (3) and may further have a substituent.).

[22] The above-mentioned gold complex is the gold complex described in

[20] or

[21] and is water-soluble.

[23] The above-mentioned L 1 is the gold complex described in any one of

[20] to

[22] and contains -C(=O)NR- or -NRC(=O)- (wherein R represents a hydrogen atom or an acetyl group).

[24] The above-mentioned L 1 is the gold complex described in any one of

[20] to

[23] and contains at least one selected from the group consisting of a carboxy group, a sulfonic acid group, an alkyl group, an alkenyl group, an aryl group, an alkoxy group, a heteroaryl group, a halogen atom, an acetyl group, an acetoxy group, an acetoxymethyl group, and a hydroxyalkyl group.

[25] The above-mentioned L 1 is a hydrocarbon group having 4 to 15 carbon atoms in total, which has a carboxy group and -C(=O)NR- or -NRC(=O)- (wherein R represents a hydrogen atom or an acetyl group) and may further have a substituent, and is the gold complex described in any one of

[20] to

[24] .

[26] The above-mentioned L 1 is an amino acid derivative represented by the following formula (5) and is the gold complex described in any one of

[20] to

[25] . R 1 -T-R 2 -** (5) (In the formula, R 1 represents an alkyl group having 1 to 5 carbon atoms, T represents *-C(=O)NR-* or *-NRC(=O)-* (wherein R represents a hydrogen atom or an acetyl group, and * represents a bond with R 1 or R 2 ), R 2 represents a linear or branched alkylene group having 2 to 4 carbon atoms, ** represents a bond with S in the above-mentioned -S-Au-(L 2 ) m in the formula. R 1 and / or R 2 has a carboxy group.)

[27] The above-mentioned L 1 is a residue obtained by removing -SH from thiopronine, N-acetylpenicillamine, or N-acetylcysteine respectively, and is the gold complex described in any one of

[20] to

[26] .

[28] L above 2 The gold complex according to any one of

[20] to

[27] , wherein R is a partial structure of a β-lactam antibiotic.

[29] L above 2 The gold complex according to any one of

[20] to

[28] , wherein the moiety is a partial structure involved in hydrophobicity and / or cell membrane permeability in a β-lactam antibiotic.

[30] The above L 2 The gold complex according to any one of

[20] to

[29] , wherein the heterocycle is a 5- to 7-membered monocycle containing 1 to 4 nitrogen atoms, 0 to 1 oxygen atom, and 0 to 1 sulfur atom as ring-constituting atoms, or a fused ring containing 1 to 4 nitrogen atoms, 0 to 1 oxygen atom, and 0 to 1 sulfur atom as ring-constituting atoms, with a total of 9 to 14 ring-constituting atoms.

[31] L above 2 The gold complex according to any one of

[20] to

[30] , wherein the heterocycle in the above formula (1) includes a structure that allows delocalization of electrons of S (sulfur atom) in the above -SH coordinated to Au.

[32] The above L 2 The gold complex according to any one of

[20] to

[31] , wherein the heterocycle is a conjugated heterocycle.

[33] L above 2 The gold complex according to any one of

[20] to

[32] , wherein the heterocycle is a structure containing a conjugated heterocycle represented by the following formula (r): [ka] (In the formula, Z 1 represents a nitrogen atom, a sulfur atom, an oxygen atom, or a carbon atom; [ka] represents a single bond or a double bond, and ring A 1 is the NCZ represented by the above formula (r) 1 represents a 5- to 7-membered ring containing three atoms consisting of the ring A 1 may be one ring constituting a condensed ring with another 5- to 7-membered ring, 1 Among the constituent atoms, the nitrogen atom and / or the carbon atom may have a substituent.

[34] The above L 2 Among them, the above heterocyclic ring is a gold complex selected from the group consisting of a tetrazole ring, a thiadiazole ring, a triazole ring, a thiazole ring, a triazine ring, a pyrrolidine ring, a pyrazolo-triazole fused ring, an oxadiazine ring, and a dithiazine ring, as described in any one of

[20] to

[33] .

[35] The above L 2 is 5-mercapto-1-methyltetrazole (MM4), and the S of the mercapto group coordinates to the Au represented by the above formula (3), which is a gold complex described in any one of

[20] to

[34] .

[36] The above Au is a monovalent gold atom, which is a gold complex described in any one of

[20] to

[35] .

[37] A method for producing a gold complex described in any one of

[20] to

[36] , the method includes coordinating a heterocyclic compound L 2 to a gold complex precursor represented by the following formula (2), The above heterocyclic compound L 2 is a compound containing a heterocyclic ring having -SH and optionally having a substituent. L 1 -S-Au (2) (In the formula, L 1 represents an amino acid derivative.)

[38] A method for screening a cell growth inhibitor or an anticancer agent, the method includes a step of coordinating a heterocyclic compound L 2 to a gold complex precursor represented by the following formula (2) to obtain a gold complex described in any one of

[20] to

[36] , and a step of evaluating the cell growth inhibitory ability of the above gold complex and includes, The above heterocyclic compound L 2 is a compound containing a heterocyclic ring having -SH and optionally having a substituent. L 1 -S-Au (2) (In the formula, L 1 represents an amino acid derivative.)

Advantages of the Invention

[0013] According to the present invention, it is possible to provide an anticancer agent containing a gold complex having an amino acid derivative as a ligand and a combined agent with radiation in chemoradiotherapy. Furthermore, it is possible to provide a novel gold complex that can be used as an anticancer agent and a combined agent with radiation in chemoradiotherapy.

Brief Description of Drawings

[0014]

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Mode for Carrying Out the Invention

[0015] Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be implemented with appropriate modifications within the scope of the object of the present invention. In addition, in this specification, thiopronine may sometimes be referred to as "TPN".

[0016] In this specification, the number of carbon atoms in the alkyl group is 1 to 6, preferably 1 to 4, and more preferably 1 or 2. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, and an n-hexyl group. Among these, a methyl group and an ethyl group are preferred, and a methyl group is more preferred.

[0017] In this specification, the number of carbon atoms of the cycloalkyl group is 3 to 8, preferably 3 to 7, and more preferably 4 to 6. Specific examples of the cycloalkyl group having 3 to 8 carbon atoms include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group.

[0018] In this specification, the number of carbon atoms of the alkoxy group is 1 to 6, preferably 1 to 4, and more preferably 1 or 2. Specific examples of the alkoxy group having 1 to 6 carbon atoms include a methoxy group, an ethoxy group, an n-propyloxy group, an isopropyloxy group, an n-butyloxy group, an isobutyloxy group, a sec-butyloxy group, a tert-butyloxy group, an n-pentyloxy group, and an n-hexyloxy group. Among these, the methoxy group and the ethoxy group are preferable, and the methoxy group is more preferable.

[0019] In this specification, the number of carbon atoms of the cycloalkyloxy group is 3 to 8, preferably 3 to 7, and more preferably 4 to 6. Specific examples of the cycloalkyloxy group having 3 to 8 carbon atoms include a cyclopropyloxy group, a cyclobutyloxy group, a cyclopentyloxy group, a cyclohexyloxy group, a cycloheptyloxy group, and a cyclooctyloxy group.

[0020] In this specification, the number of carbon atoms of the aryl group is 6 to 20, preferably 6 to 12. Specific examples of the aryl group having 6 to 20 carbon atoms include a phenyl group, an α-naphthyl group, a β-naphthyl group, a biphenyl-4-yl group, a biphenyl-3-yl group, a biphenyl-2-yl group, an anthracen-1-yl group, an anthracen-2-yl group, an anthracen-9-yl group, a phenanthren-1-yl group, a phenanthren-2-yl group, a phenanthren-3-yl group, a phenanthren-4-yl group, and a phenanthren-9-yl group. Among these, the phenyl group, the α-naphthyl group, the β-naphthyl group, the biphenyl-4-yl group, the biphenyl-3-yl group, and the biphenyl-2-yl group are preferable, and the phenyl group is more preferable.

[0021] In this specification, the number of carbon atoms of the aralkyl group is 7 to 20, preferably 7 to 12. Specific examples of the aralkyl group having 7 to 20 carbon atoms include a benzyl group, a phenethyl group, a 3-phenyl-n-propyl group, a 4-phenyl-n-butyl group, an α-naphthylmethyl group, a β-naphthylmethyl group, a 2-(α-naphthyl)ethyl group, and a 2-(β-naphthyl)ethyl group. Among these groups, a benzyl group and a phenethyl group are preferred, and a benzyl group is more preferred.

[0022] In this specification, examples of the halogen atom contained in the alkyl halide group include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. The number of carbon atoms of the alkyl halide group is 1 to 6, preferably 1 to 4, and more preferably 1 or 2. Specific examples of the alkyl halide group having 1 to 6 carbon atoms include a chloromethyl group, a dichloromethyl group, a trichloromethyl group, a fluoromethyl group, a difluoromethyl group, a trifluoromethyl group, a 1,1-difluoroethyl group, a 2,2,2-trifluoroethyl group, and a pentafluoroethyl group.

[0023] In this specification, the number of carbon atoms of the aliphatic acyl group is 2 to 7, preferably 2 to 5, and more preferably 2 or 3. Specific examples of the aliphatic acyl group having 2 to 7 carbon atoms include an acetyl group, a propionyl group, a butanoyl group, a pentanoyl group, a hexanoyl group, and a heptanoyl group. Among these, an acetyl group and a propanoyl group are preferred, and an acetyl group is more preferred.

[0024] In this specification, examples of the halogen atom contained in the halogenated aliphatic acyl group include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. The number of carbon atoms in the halogenated aliphatic acyl group is from 2 to 7, preferably from 2 to 5, and more preferably 1 or 2. Specific examples of the halogenated aliphatic acyl group having 2 to 7 carbon atoms include a chloroacetyl group, a dichloroacetyl group, a trichloroacetyl group, a fluoroacetyl group, a difluoroacetyl group, a trifluoroacetyl group, and a pentafluoropropionyl group.

[0025] In this specification, the number of carbon atoms in the arylcarbonyl group is from 7 to 20, preferably from 7 to 13. Specific examples of the arylcarbonyl group having 7 to 20 carbon atoms include a benzoyl group, an α-naphthoyl group, and a β-naphthoyl group.

[0026] In this specification, the number of carbon atoms in the carboxyalkyl group is from 2 to 7, preferably from 2 to 5, and more preferably 2 or 3. Specific examples of the carboxyalkyl group having 2 to 7 carbon atoms include a carboxymethyl group, a 2-carboxyethyl group, a 3-carboxy-n-propyl group, a 4-carboxy-n-butyl group, a 5-carboxy-n-pentyl group, and a 6-carboxy-n-hexyl group. Among these, the carboxymethyl group is preferred.

[0027] Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Among these, a fluorine atom, a chlorine atom, and a bromine atom are preferred, and a chlorine atom and a bromine atom are more preferred.

[0028] In this specification, the number of carbon atoms in the alkylthio group is from 1 to 6, preferably from 1 to 4, more preferably 1 or 2. Specific examples of the alkylthio group having 1 to 6 carbon atoms include a methylthio group, an ethylthio group, an n-propylthio group, an isopropylthio group, an n-butylthio group, an isobutylthio group, a sec-butylthio group, a tert-butylthio group, an n-pentylthio group, and an n-hexylthio group. Among these, the methylthio group and the ethylthio group are preferable, and the methylthio group is more preferable.

[0029] Specific examples of the alkyl group contained in the monoalkylamino group containing an alkyl group having 1 to 6 carbon atoms and the dialkylamino group containing an alkyl group having 1 to 6 carbon atoms are the same as the specific examples of the above alkyl group. As the monoalkylamino group containing an alkyl group having 1 to 6 carbon atoms, an ethylamino group and a methylamino group are preferable, and the methylamino group is more preferable. As the dialkylamino group containing an alkyl group having 1 to 6 carbon atoms, a diethylamino group and a dimethylamino group are preferable, and the dimethylamino group is more preferable.

[0030] <Anticancer agent and combined agent with radiation in chemoradiotherapy> The anticancer agent of the present invention and the combined agent with radiation in chemoradiotherapy contain a gold complex represented by the following formula (1). L 1 -S-Au-(L 2 ) m ···(1) (In the formula, L 1 represents an amino acid derivative, L 2 represents a heterocyclic ring having -SH coordinated to Au represented by the above formula (1) and may further have a substituent, and m represents 0 or 1.)

[0031] When m is 0 in the above formula (1), it can be represented by the following formula (2). L 1 -S-Au ···(2) (In the formula, L 1 is the same as in formula (1).) When m is 1 in the above formula (1), it can be represented by the following formula (3). L 1 -S-Au-L 2 ···(3) (In the formula, L 1 and L 2 are the same as those in formula (1).)

[0032] The gold complex represented by the above formula (3) can be produced by a method including coordinating a heterocyclic compound L 2 to the gold complex represented by the above formula (2). Since the gold complex represented by the above formula (2) can be said to be a precursor of the gold complex represented by the above formula (3), it may be referred to as a "gold complex precursor" in this specification. The above heterocyclic compound L 2 is a compound containing a heterocyclic ring having -SH and optionally having a substituent. The heterocyclic compound L 2 is such that the -SH coordinates to the Au represented by the above formula (1) to formula (3), thereby constituting L 2 in the above formula (1) to formula (3). This will be described later.

[0033] <L 1 -S-Au-(L 2 ) m > The anticancer agent and the agent for combined use with radiation in chemoradiotherapy include the gold complex represented by the above formula (1), that is, a gold complex in which an amino acid derivative is bonded to a gold atom (Au).

[0034] It is said that gold (Au) in a reduced state of zero valence (Au(0)) or monovalent (Au(I)) binds to the SH groups possessed by various in vivo proteins, thereby modifying the functions of those proteins. Typical proteins targeted by gold include NF-κB, which is one of the transcription factors, and thioredoxin reductase. These proteins are overexpressed in cancer tissues with high metastatic ability (high malignancy), and the gold complex plays a role as a carrier for efficiently reaching gold atoms to such tissues.

[0035] The gold atom may be zero-valent or monovalent, and is preferably monovalent. A gold complex in which Au in the above formula (1) is a zero-valent gold and a gold complex in which Au is a monovalent gold may coexist. In the above formula (1), L 1 represents an amino acid derivative. Note that L 1 and Au are bonded via a sulfur atom (-S-) in the thiol group of the amino acid-derived compound L 1 -SH.

[0036] <L 1 > L 1 is an amino acid derivative. In this specification, the "amino acid derivative" refers to a compound in which a part of the molecular structure of an amino acid is changed. In this embodiment, the amino acid derivative as L 1 in the above formula (1) is specifically a residue obtained by removing -SH from the amino acid-derived compound "L 1 -SH" (wherein L 1 is the same as in formula (1)).

[0037] L 1 The amino acid derivative or the amino acid-derived compound L 1 -SH used for is not particularly limited. For example, derivatives obtained from known amino acids as raw materials can be used. Also, derivatives obtained from newly synthesized novel amino acids as raw materials can be used.

[0038] Examples of known amino acids include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine, etc.

[0039] L 1 preferably contains -C(=O)NR- or -NRC(=O)- (wherein R represents a hydrogen atom or an acetyl group). L 1It is more preferable that it has a structure represented by the following formula (5). R 1 -T-R 2 -** (5) (In the formula, R 1 represents an alkyl group having 1 to 5 carbon atoms, T represents *-C(=O)NR-* or *-NRC(=O)-* (in the formula, R represents a hydrogen atom or an acetyl group, and * represents a bond with R 1 or R 2 ). R 2 represents a linear or branched alkylene group having 2 to 4 carbon atoms, and ** represents a bond with S in -S-Au-(L 2 ) m represented by the above formula (1). R 1 and / or R 2 has a carboxy group.)

[0040] Also, an amino acid is generally a compound having at least one amino group (-NH 2 ) and at least one carboxy group (-COOH). As the amino acid-derived compound L 1 -SH, a compound in which the amino group of the amino acid is changed to an amide bond (-C(=O)N- or -NC(=O)-) is preferable. As the amino acid derivative as L 1 , a residue obtained by removing -SH from a compound in which the amino group of the amino acid is changed to an amide bond (the same as above) is preferable, and it more preferably contains a carboxy group and -C(=O)NR- or -NRC(=O)- (R represents a hydrogen atom or an acetyl group).

[0041] Also, although an α-amino acid contains -N-C-C(=O)- in its molecular structure from its definition, as the amino acid-derived compound L 1 -SH, a compound in which the amino group of the α-amino acid is changed to an amide bond (-C(=O)N- or -NC(=O)-) is more preferable. As the amino acid derivative as L 1 , a residue obtained by removing -SH from a compound in which the amino group of the α-amino acid is changed to an amide bond (-C(=O)N- or -NC(=O)-) is preferable. Therefore, the amino acid-derived compound L1 -SH or L 1 As for the amino acid derivative as, it is more preferable to contain -C(=O)N-C-C(=O)-, and it is further preferable to contain -C(=O)NR-C-C(=O)- (R represents a hydrogen atom or an acetyl group) or -C(=O)N-C-COOH, and it is even more preferable to contain -C(=O)NR-C-COOH (R represents a hydrogen atom or an acetyl group).

[0042] L 1 L also preferably contains at least one selected from the group consisting of a carboxy group, a sulfonic acid group, an alkyl group, an alkenyl group, an aryl group, an alkoxy group, a heteroaryl group, a halogen atom, an acetyl group, an acetoxy group, an acetoxymethyl group, and a hydroxyalkyl group. Among these, the acetyl group, the acetoxy group, the acetoxymethyl group, and the hydroxyalkyl group may be, for example, those obtained by substituting the carboxy group possessed by the amino acid derivative with these groups.

[0043] L 1 The carboxy group (-COOH) that may be contained in L may be substituted with, as described above, an acetyl group (-C(=O)CH 3 ), an acetoxy group (-OC(=O)CH 3 ), an acetoxymethyl group (-CH 2 OC(=O)CH 3 ), and a hydroxyalkyl group (-C n H 2n OH (n is 1 to 10)), etc. Note that the alkyl group in the above hydroxyalkyl group preferably has a branched structure such as a dimethylated form (-C(CH 3 )) 2 -), an ethylmethylated form (-C(CH 3 )(C 2 H 5 ))-), a diethylated form (-C(C 2 H 5 )) 2 -).

[0044] Also, L 1The carboxy group that may be included may be esterified with pivaloyloxymethyl (POM), 5-methyl-2-oxo-1,3-dioxol-4-ylmethyl (DMDO), or the like. The carboxy group esterified with POM is represented by the following formula (s1). The carboxy group esterified with DMDO is represented by the following formula (s2).

Chemical formula

Chemical formula

[0045] L 1 may further have a substituent and is preferably a hydrocarbon group having a total of 4 to 15 carbon atoms. Examples of the hydrocarbon group include an alkyl group, etc., and it may be a linear or branched alkyl group.

[0046] L 1 The hydrogen atom bonded to a carbon atom other than the carboxy group in may be substituted with the following substituents or atoms. (i) Each substituent of alkyl, alkenyl, aryl, alkoxy, heteroaryl (ii) Each halogen element of fluorine, chlorine, bromine, iodine

[0047] L 1 is also 1-mercaptoethane-1-amide (-HNC(=O)-C(CH 3)-SH), or a structure containing 1-mercaptoethane-2-amide, that is, N-amidated 1-mercaptoethane-2-amine (RHN-C-C-SH; wherein R represents a derivative of an acid group such as a carboxy group or a sulfonic acid group) is preferred. As the derivative of the acid group represented by R above, a structure containing -C(=O)- is preferred, and an acetyl group is more preferred. As the structure containing 1-mercaptoethane-1-amide, for example, thiopronine represented by the following formula is preferred, and as the structure containing 1-mercaptoethane-2-amide, for example, N-acetylpenicillamine and N-acetylcysteine (D, L, DL) represented by the following formula are preferred. [Chemical formula]

[0048] From the viewpoints such as being able to chelate zero-valent or monovalent gold atoms with appropriate strength, being easy to explore the synthesis of related compounds and the design for obtaining the expected physiological activity, and having few concerns about side effects, L 1 The amino acid derivative or amino acid-derived compound L used 1 -SH is preferably thiopronine, N-acetylpenicillamine or N-acetylcysteine (D, L, DL), and more preferably thiopronine. In this case, L 1 is a residue obtained by removing the SH group from thiopronine, N-acetylpenicillamine or N-acetylcysteine, respectively. For example, while thiopronine has been evaluated to have a stable metal chelating action in the field of surface engineering, it is also an approved pharmaceutical as an antidote with few side effects, and its safety is ensured.

[0049] <L 1 -S-Au and Its Synthesis Method> The gold complex (L 1 -S-Au) represented by the above formula (2) can be produced by reacting a gold raw material with the above L 1 -SH. For the production, any conditions and methods employed in the production of gold complexes can be used. As the gold raw material, trivalent gold (Au 3+) or a monovalent gold (Au 1+ )-containing compound or the like can be mentioned, and it is preferable to use an acid or a salt containing trivalent gold (Au 3+ ) or monovalent gold (Au 1+ ).

[0050] As the trivalent gold, for example, a compound represented by Au(III)-anion or cation-Au(III)-anion can be used. Examples of the above anion include halide ions such as Cl - , Br - , I - ; OH - ; anions of organic acids such as acetic acid (CH 3 COO - ) and the like. Examples of the above cation include H + ; NH 4 + ; monovalent alkali metal ions such as Li + , Na + , K + , Rb + , Cs + ; divalent alkaline earth metal ions such as Mg 2+ , Ca 2+ and the like.

[0051] Examples of the compound represented by the above Au(III)-anion include gold trihalide, gold hydroxide (Au(OH) 3 ), gold acetate ((CH 3 COO) 3 Au). Examples of gold trihalide include gold trichloride (AuCl 3 ), gold tribromide (AuBr 3 ), gold triiodide (AuI 3 ) and the like.

[0052] Examples of the compound represented by the above cation-Au(III)-anion include tetrahalogenoauric acid and tetrahalogenoaurate. Examples of tetrahalogenoauric acid include tetraauric acid (HAuCl 4 ) and tetraauric acid (HAuBr 4) etc. Examples of tetrahalogen aurates include tetrachloroaurates and tetrabromoaurates. Examples of tetrachloroaurates include sodium tetrachloroaurate (NaAuCl 4 ) and potassium tetrachloroaurate (KAuCl 4 ). Examples of tetrabromoaurates include sodium tetrabromoaurate (NaAuBr 4 ) and potassium tetrabromoaurate (KAuBr 4 ).

[0053] In addition, a trivalent gold-containing complex of an Au(III)-containing amine complex can also be used as a gold raw material. The Au(III)-containing amine complex is represented as R 3 N:Au 3+ (anion) 3- (where R represents a hydrogen atom, an alkyl group, an aryl group, or a cyclic ring portion (such as pyridine, pyrrole, etc., the same applies below)). Here, R 3 N includes, for example, NH 3 , trimethylamine, pyridine, etc. Examples of Au(III)-containing amine complexes include ammonium-chloroauric acid complex (H 3 N[AuCl 3 ), trimethylamine-chloroauric acid complex ((CH 3 ) 3 N[AuCl 3 ), pyridine-chloroauric acid complex (Trichloro(pyridine)gold(III); chemical formula C 5 H 5 N[AuCl 3 ), etc.

[0054] As the monovalent gold, for example, a compound represented by Au(I)-anion can be used.

[0055] Examples of the compound represented by the above Au(I)-anion include sodium gold(I) sulfite (Gold(I) trisodium disulphite; chemical formula Na 3 [Au(SO 3 ) 2) Potassium gold sulfite or gold halide can be mentioned. Examples of gold halide include gold chloride (AuCl), gold bromide (AuBr), gold iodide (AuI), etc.

[0056] In addition, a monovalent gold-containing complex such as an Au(I)-containing phosphine complex can also be used as the gold raw material. The Au(I)-containing phosphine complex is R 3 PAu 1+ (Anion) 1- (R represents a hydrogen atom, an alkyl group, or an aryl group.) It is shown as, and here R 3 Examples of P include trimethylphosphine, triethylphosphine, triisopropylphosphine, triphenylphosphine, etc. Examples of the Au(I)-containing phosphine complex include trimethylphosphine-gold chloride complex ((CH 3 ) 3 P[AuCl]), triisopropylphosphine-gold chloride complex (Chloro(triisopropylphosphine)gold; chemical formula (i-Pr) 3 P[AuCl]), etc.

[0057] Gold cyanide, the above-mentioned L 1 or L 2 Thiolate gold complexes other than the compounds that can be used as may not be preferably used as the gold raw material in some cases.

[0058] When using the above trivalent gold as the gold raw material, the scheme shown by the following reaction formula is obtained. Au 3+ +3L 1 -SH → L 1 -S-Au(I) polymer + L 1 -S-S-L 1

[0059] In addition, when using the above monovalent gold as the gold raw material, the scheme shown by the following reaction formula is obtained. Au 1+ +1L 1 -SH → L 1 -S-Au(I) polymer

[0060] L 1 The synthesis method of -S-Au will be described. L 1 -S-Au is easily synthesized with reference to the method described in Non-Patent Document 1. First, gold is dissolved in aqua regia at 100 °C. After the gold is dissolved, the solution is continuously concentrated at 100 °C, and the replenishment of the evaporated volume with hydrochloric acid is repeated to reduce nitric acid, and finally the excess hydrochloric acid is also reduced in the concentrated state to synthesize chloroauric acid (tetrachloroauric acid). Next, chloroauric acid reacts with L 1 the raw material L 1 -SH aqueous solution. The reaction temperature is preferably 10 to 40 °C, more preferably 15 to 35 °C, and even more preferably 20 to 30 °C. The molar ratio of L 1 in the -SH aqueous solution to the gold in chloroauric acid is preferably 1 to 5, more preferably 2 to 4, even more preferably 3 to 3.5, and most preferably 3.2. As an example of a specific method, L 1 is reacted with a trivalent gold raw material in a molar amount of 3 times or more (preferably 3.2 times) that of gold, and then, if necessary, the pH of the reaction solution is adjusted to 2 or less, and L 1 -S-Au polymer is precipitated, purified, and isolated. 1 -S-Au polymer is precipitated, purified, and isolated.

[0061] As an example, L 1 The production method of a gold complex (hereinafter referred to as "TPN-Au") using thiopronin as L will be described in detail. TPN-Au is represented by the following formula (2-1) and is easily synthesized with reference to the method described in Non-Patent Document 1. The solution obtained by dissolving the above-mentioned gold in aqua regia is reacted with hydrochloric acid to synthesize chloroauric acid (tetrachloroauric acid). Next, chloroauric acid and L 1React the aqueous solution of thiopronine, which serves as the raw material, at room temperature. The molar ratio of thiopronine in the aqueous solution of thiopronine to gold in chloroauric acid is preferably 1 to 5, more preferably 2 to 4, even more preferably 3 to 3.5, and most preferably 3.2. Finally, pure TPN-Au can be obtained by removing the remaining hydrochloric acid, nitric acid, thiopronine disulfide, and excess thiopronine through purification.

Chemical formula

[0062] Also, when synthesizing TPN-Au by reacting thiopronine with sodium gold sulfite, the molar ratio of thiopronine to gold in sodium gold sulfite is preferably 0.1 to 5, more preferably 0.5 to 2, even more preferably 1 to 1.5, and most preferably 1.2.

[0063] L 1 As such, when using other amino acid derivatives other than thiopronine, it can be synthesized in the same manner. Also, it can be easily synthesized by referring to the method described in Patent Document 2.

[0064] The synthesized TPN-Au forms a tetramer represented by the following formula (4).

Chemical formula

[0065] The synthesized gold complex represented by the above formula (2) has an anticancer effect.

[0066] <L 1 -S-Au-L 2 > In the production of pharmaceutical molecules, for the purpose of improving the selectivity and affinity for the target molecule and controlling the conformation, substitution of heterocyclic compounds is carried out. From such a perspective, the anticancer agent is a heterocyclic compound L further attached to the gold atom of the gold complex represented by the above formula (2).2 It preferably contains a gold complex to which 2 is bonded.

[0067] In the following formula (3), L 1 represents an amino acid derivative, and L 2 represents a heterocyclic compound having -SH that coordinates to Au in the formula and may further have a substituent. L 1 -S-Au-L 2 ···(3)

[0068] The gold complex represented by the above formula (3) has a structure in which an amino acid derivative is bonded via -S- as one ligand and a heterocyclic compound is bonded or coordinated via -SH as the other ligand to zero-valent gold or monovalent gold. Such a gold complex is presumed to have an optimal strength for chelating gold atoms and to have different pharmacokinetics inside and outside the cell membrane compared to gold preparations such as auranofin.

[0069] Also in this case, L 1 is not particularly limited, but due to the reasons described above, thiopronine, N-acetylpenicillamine, and N-acetylcysteine (D, L, DL) are preferable. For example, when L 1 is thiopronine, the gold complex is represented by the following formula (3b).

Chemical formula

[0070] The gold complex represented by the above formula (3) has an anticancer effect and is also useful as a combined agent with radiation in chemoradiotherapy.

[0071] <L 2 > L 2 is not particularly limited as long as it contains a heterocycle having -SH (mercapto group). L 2 may be a conjugated heterocycle or a non-conjugated heterocycle, and a conjugated heterocycle is preferable. L 2As the above-mentioned heterocyclic ring, a 5- to 7-membered monocyclic ring containing 1 to 4 nitrogen atoms, 0 to 1 oxygen atom, and 0 to 1 sulfur atom as ring-constituting atoms, or a condensed ring containing a total of 9 to 14 ring-constituting atoms including 1 to 4 nitrogen atoms, 0 to 1 oxygen atom, and 0 to 1 sulfur atom, is preferably a nitrogen-containing heterocyclic compound.

[0072] L 2 The above-mentioned heterocyclic ring in is preferably a structure in which the electrons of S (sulfur atom) in the -SH coordinated to Au represented by the above formula (1) can be delocalized, more preferably a structure in which the electron density of the lone pair of electrons of the S (sulfur atom) can be dispersed in the above-mentioned heterocyclic ring, and still more preferably a structure in which the -SH is connected to a heterocyclic ring capable of attracting the electrons or the electron density of the lone pair of electrons of the S (sulfur atom). It is still more preferably a structure in which tautomerism as represented by the following formula of N=C-SH and HN-C=S is possible. The delocalization of the electrons of the S (sulfur atom) may be delocalized over the entire heterocyclic ring, but it does not have to be delocalized over the entire heterocyclic ring.

Chemical formula

[0073] L 2 The above-mentioned heterocyclic ring in is more preferably a structure containing a conjugated heterocyclic ring represented by the following formula (r).

Chemical formula

Chemical formula

[0074] As the conjugated heterocyclic ring represented by the above formula (r), S represented by the formula (r) is at least N and / or Z which are the carbon atom to which S is bonded and the ring-constituting atoms adjacent to the carbon atom 1 together with a structure that constitutes a conjugated system or in which the electrons of S can be delocalized. The delocalization of the conjugated system or the electrons of S is represented by ring A represented by the following formula 2 as shown, ring A 1 is more preferably carried out throughout the whole.

Chemical formula

Chemical formula

[0075] The above ring A 1 and ring A 2Examples include the tetrazole ring in 5-mercapto-1-methyltetrazole (hereinafter also referred to as "MM4"), 2-amino-5-mercapto-1,3,4-thiadiazole (hereinafter also referred to as "AMT"), 2-mercapto-1,3,4-thiadiazole (hereinafter also referred to as "MT"), 2-mercapto-5-methyl-1,3,4-thiadiazole (hereinafter also referred to as "MMT"), 2-mercapto-5-methylthio-1,3,4-thiadiazole (hereinafter also referred to as "MMTT"), the thiadiazole ring in 1,2,4-triazole-3-thiol (hereinafter also referred to as "MTZ"), 3-amino-5-mercapto-1,2,4-triazole (hereinafter also referred to as "AMZ"), the triazole ring in 2-imidazolidinethione (ethylene thiourea; hereinafter also referred to as "2NN"), the thiazolidine ring in 2-mercaptothiazoline (hereinafter also referred to as "2SN"), and in the chemical structure of the compound represented by formula (6) described below, a 5- to 7-membered monocyclic ring in which -SH is directly bonded, and a 5- to 7-membered monocyclic ring included in the side chain substituent forming the partial structure of the β-lactam antibiotic described below, etc. The monocyclic ring may be, for example, one of the rings constituting the condensed ring, like the compound having a condensed ring in the compound represented by formula (6).

[0076] L 2 Preferable examples of the nitrogen-containing heterocyclic compound in L include a tetrazole ring, a thiadiazole ring, a triazole ring, a thiazole ring, a thiazoline ring, a thiazolidine ring, a triazine ring, a pyridine ring, a pyridazine ring, a pyrazine ring, a pyrimidine ring, a tetrazine ring, a pyrrole ring, a pyrazole ring, an imidazole ring, an imidazoline ring, an imidazolidine ring, an oxazole ring, an isoxazole ring, an isothiazole ring, an oxadiazole ring, a pyrrolidine ring, an oxadiazine ring, a dithiazine ring, a piperidine ring, and a pyrazolo-triazole condensed ring, etc.

[0077] L 2As particularly preferred specific examples of the heterocyclic ring, a tetrazole ring, a thiadiazole ring, a triazole ring, a thiazole ring, and a triazine ring represented by the following formula are preferable. In the present specification, isomers having different positions among the ring-constituting atoms of the heteroatoms consisting of N, O, and S in the chemical structural formula exemplifying the heterocyclic ring may also be used.

Chem.

[0078] L 2 As a preferable specific example of, a compound represented by the following formula (6) is also included.

Chem.

[0079] L 2 As a specific example of the heterocyclic ring of, a heterocyclic ring in a thiocarbonyl compound (thiocarbonyl heterocyclic ring compound) in which a carbon atom, which is a ring-constituting atom of the heterocyclic ring, also constitutes a thiocarbonyl group (S═C—) is also included. For example, in a thiocarbonyl heterocyclic ring compound such as 2NN and 2SN shown on the right side of each of the following formulas, the thiocarbonyl group coordinates to Au represented by the above formula (3) to form a gold complex as shown on the left side of each of the following formulas (L 1 -S-Au-2NN, L 1 -S-Au-2SN), the heterocyclic ring (the imidazolidine ring in 2NN, the thiazolidine ring in 2SN) is included as the heterocyclic ring of L 2 As the heterocyclic ring of. In the following formula, L 1 -S-Au- is a part of the structure represented by the above formula (3).

Chem.

[0080] L 2 As a preferable specific example of, a heterocyclic ring compound having -SH shown on the right side of the following formula is also included, and the compound can have tautomerism as represented by the following formula of N═C—SH and HN—C═S as described above. In the following formula, R is, L 2Represents a substituent that the complex ring in may have.

Chemical formula

[0081] L 2 As a preferred specific example of, a compound represented by the following formula (r1) is also included. X in the following formula (r1) 1 and X 2 One of them is SH and the other is H. Also, in the following formula (r1), R represents a substituent that the complex ring in L 2 may have.

Chemical formula

[0082] L 2 In addition to -SH, may further have a substituent. The substituent is not particularly limited, and examples include an alkyl group, a cycloalkyl group, an alkoxy group, a cycloalkyloxy group, an aryl group, an aralkyl group, a halogenated alkyl group, an aliphatic acyl group, a halogenated aliphatic acyl group, an arylcarbonyl group, a carboxyalkyl group, a halogen atom, a hydroxyl group, a mercapto group, an alkylthio group, an amino group, a monoalkylamino group containing an alkyl group, a dialkylamino group containing an alkyl group, a nitro group, a cyano group, etc.

[0083] L 2 In addition to -SH, may have a plurality of substituents. When the number of substituents is plural, the plural substituents may be the same or different. Also, when these substituents include an aliphatic hydrocarbon ring, an aromatic hydrocarbon ring, etc., these rings may further have the same substituents as the substituents that L 2 may have.

[0084] L 2 As a specific example of the substituent that may have, in the case of being derived from an antibiotic described later, for example, a substituent represented by the following formula can be mentioned. Note that * represents a bond to the complex ring.

Chemical formula

[0085] In addition, among the side-chain substituents that form the partial structure of the antibiotic used in pharmaceuticals that have been approved, are in pre-clinical development, or are in clinical development, the heterocyclic ring or the structure containing a heterocyclic ring has a known correlation between action and structure, and since it is easy to predict safety and production costs, L 2 can preferably be used. Specifically, a compound having such a heterocyclic ring or a structure containing a heterocyclic ring and having -SH can preferably be used as L 2 When -SH already bonded to the above heterocyclic ring is present in the above antibiotic, a compound composed of the heterocyclic ring or the structure containing a heterocyclic ring and the -SH can be used as L 2 When a divalent sulfur atom (-S-) bonded to the above heterocyclic ring is present in the above antibiotic, a compound composed of -SH formed by converting the -S- and the heterocyclic ring or the structure containing a heterocyclic ring can be used as L 2 When -SH or -S bonded to the above heterocyclic ring does not exist in the above antibiotic, a compound obtained by adding an -SH group to the heterocyclic ring or extending the side-chain carbon chain as a substituent of the heterocyclic ring to add an -SH group can be used as L 2 can be used.

[0086] Examples of the heterocyclic ring contained in the chemical structure of the antibiotic that has been approved, is in pre-clinical development, or is in clinical development include, from the viewpoint of having many compounds having a heterocyclic ring, new quinolone antibiotics, β-lactam antibiotics, etc., preferably the heterocyclic ring contained in β-lactam antibiotics. In this case, L 2 is preferably a side-chain substituent that forms the partial structure of the β-lactam antibiotic, specifically a heterocyclic ring or a structure containing a heterocyclic ring, and in the β-lactam antibiotic, it is preferably a partial structure related to hydrophobicity and / or cell membrane permeability, more preferably a partial structure related to hydrophobicity and cell membrane permeability, and still more preferably a partial structure related to the improvement of cell membrane permeability due to an increase in hydrophobicity.

[0087] Examples of β-lactam antibiotics include ceftriaxone, cefapirin, cephalothin, cefepime, cefpirome, cefbuperazone, cefoperazone, latamoxef, cefazolin, cefatrizine, flomoxef, panipenem, meropenem, biapenem, doripenem, and the like.

[0088] For example, the triazine ring contained in ceftriaxone shown in the following formula can be preferably used as L 2

Chemical formula

[0089] The pyridine ring contained in cefapirin shown in the following formula can be preferably used as L by substituting the -OH group with an -SH group. 2

Chemical formula

[0090] The thiophene ring contained in cephalothin, the pyrrolidine ring contained in cefepime, the condensation of the thiazole ring and the imino group contained in cefpirome, and the piperazine rings contained in cefbuperazone and cefoperazone shown in the following formula can be preferably used as L by adding an -SH group or extending the side-chain carbon chain to add an -SH group. 2

Chemical formula

[0091] The pyrrolidine rings contained in panipenem, meropenem, and doripenem, and the piarazolo-triazole condensed ring contained in biapenem shown in the following formula can be preferably used as L. 2

Chemical formula

[0092] ​​​​As a preferred specific example of the above partial structure, 5-mercapto-1-methyltetrazole (MM4) represented by the following formula can be mentioned. MM4 is used in the 3-position side chain of the hetero six-membered ring of a plurality of cephem antibiotics (such as cefoperazone and latamoxef), and is also preferable in terms of safety. The methyl group of MM4 may further be another alkyl group with an extended carbon chain length.

Chemical formula

[0093] Moreover, as a preferred specific example of the above partial structure, 1,2,4-triazole-3-thiol (MTZ), 3-mercapto-4-methyl-4H-1,2,4-triazole, 3-amino-5-mercapto-1,2,4-triazole (AMZ), 2-amino-5-mercapto-1,3,4-thiadiazole (AMT), 2-mercapto-1,3,4-thiadiazole (MT), 2-mercapto-5-methyl-1,3,4-thiadiazole (MMT), 2-mercapto-5-methylthio-1,3,4-thiadiazole (MMTT) shown in the following formula can be mentioned.

Chemical formula

[0094] Also, for example, heterocyclic compounds such as 2-mercapto-4-methyl-5-carboxymethylthiazole, 5-mercapto-1-dimethylaminoethyltetrazole, 3-mercapto-2-methyl-5-oxotriazine, which are frequently used as side chains in approved pharmaceuticals, can also be preferably used as L 2 as well.

[0095] The inventors of the present invention used thiopronin as L 1 and MM4 as L 2 and bonded them to a gold atom to synthesize a new gold complex. The new gold complex is hereinafter also referred to as "TPN-Au-MM4". TPN-Au-MM4 is represented by the following formula (3-1).

Chemical formula

[0096] Also, as an example, when thiopronine is L 1 and AMT is L 2 and the compound bonded to a gold atom (hereinafter also referred to as "TPN-Au-AMT") is represented by the following formula (3-2). Also, when thiopronine is L 1 and MTZ is L 2 and the compound bonded to a gold atom (hereinafter also referred to as "TPN-Au-MTZ") is represented by the following formula (3-3). [Chemical formula] [Chemical formula]

[0097] Also, as an example, when thiopronine is L 1 and MMT is L 2 and the compound bonded to a gold atom (hereinafter also referred to as "TPN-Au-MMT") is represented by the following formula (3-4), and when thiopronine is L 1 and MMTT is L 2 and the compound bonded to a gold atom (hereinafter also referred to as "TPN-Au-MMTT") is represented by the following formula (3-5), and when thiopronine is L 1 and MT is L 2 and the compound bonded to a gold atom (hereinafter also referred to as "TPN-Au-MT") is represented by the following formula (3-6), and when thiopronine is L 1 and AMZ is L 2 and the compound bonded to a gold atom (hereinafter also referred to as "TPN-Au-AMZ") is represented by the following formula (3-7), and when thiopronine is L 1 and 2NN is L 2 and the compound bonded to a gold atom (hereinafter also referred to as "TPN-Au-2NN") is represented by the following formula (3-8), and when thiopronine is L 1 and 2SN is L 2 and the compound bonded to a gold atom (hereinafter also referred to as "TPN-Au-2SN") is represented by the following formula (3-9). [Chemical formula]

[0098] <L 1 -S-Au-L 2 Synthesis method of The gold complex represented by the above formula (3) can be produced using any conditions and methods employed in the production of gold complexes. As an example, L 1 The gold complex in the case where is thiopropionine will be described in detail.

[0099] The TPN-Au tetramer represented by the above formula (4) and L 2 The raw materials are mixed and reacted in a solution. The solvent is not particularly limited and may be water, or an organic solvent such as dimethyl sulfoxide (DMSO), sulfonate, N,N-dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), propylene carbonate, etc. These solvents may be used alone or in combination of multiple types. As the mixed solvent of multiple types, a mixed solution of water and an organic solvent (mixed solvent), such as a mixed solution of water and DMSO, is preferable. Since the solubility of many conventional thiolate gold compounds in aqueous solutions is low, it is preferable to devise the pH of the solution, the type and composition of the solvent, etc. For example, in order to increase the solubility, it is preferable to mix hydrolyzed saccharides (such as mannitol), corn sugar, polyvinyl alcohol (PVA), etc. The mixing of these substances into the aqueous solution is 2 preferably applied when using MM4, AMT, MMTT, MTZ, AMZ, MT, MMT, 2NN, 2SN, etc. as L. Also, in order to increase the solubility and stabilize the monomer in the solution without precipitation, it is also preferable to mix ethanol into the aqueous solution. In that case, the content of ethanol with respect to the total volume of water and ethanol is preferably in the range of 0.5 to 50% by volume, more preferably in the range of 1 to 30% by volume, and even more preferably 5 to 20% by volume. The mixing of ethanol into such an aqueous solution is 2 preferably applied when using MM4, etc. as L.

[0100] TPN-Au tetramer and L 2 The reaction solution of the TPN-Au tetramer and the raw material of L may contain a pH adjuster such as potassium hydroxide, potassium carbonate, sodium hydroxide, etc. if necessary. For the reaction of TPN-Au and the raw material of L 2 it is preferable to use a carbonate such as potassium carbonate. When using a carbonate, the mercapto group (-SH) in the raw material of L 2 may be less likely to be oxidized or can prevent oxidation during synthesis. Industrially, oxidation of the mercapto group can also be prevented by placing it under an inert atmosphere such as oxygen-free. The pH of the solution, particularly a solution containing water, is preferably in the range of 3 to 14, and more preferably in the range of 4 to 14, so that the synthesized gold complex (TPN-Au-L 2 monomer) is in a monomeric and soluble state.

[0101] When the TPN-Au tetramer and the raw material of L 2 are mixed in a solution, the two react naturally to produce the TPN-Au-L 2 monomer. Note that if the pH is less than 3, it can be returned to the TPN-Au tetramer. A series of reactions including the synthesis of the above-mentioned TPN-Au (including the TPN-Au tetramer in this paragraph) actually involves, for example, dissolving gold or a gold raw material in aqua regia and reacting it with thiopropionine, which is the raw material of L 1 to synthesize TPN-Au. Without isolating and drying the obtained TPN-Au from the solution, the supernatant of the generated pure TPN-Au precipitate is discarded and replaced several times, for example, about three times, and then the solution is neutralized with potassium hydroxide or the like to prepare a solution of a high-concentration salt of TPN-Au of about 0.5 mol / L (for example, a potassium salt of TPN-Au, etc.). Then, the TPN-Au salt solution and the raw material of L 2 are reacted to synthesize a solution of the TPN-Au-L 2 monomer. In this embodiment, in this way, the TPN-Au-L 2 monomer can be obtained economically by a simple method.

[0102] As an example, TPN-Au-MM4 is synthesized from TPN-Au tetramer and MM4 according to the scheme shown in the following reaction formula. [Chemical Formula]

[0103] TPN-Au-L 2 The monomer can be isolated by the method described below. First, TPN-Au-L in solution 2 The potassium salt of the monomer is synthesized. For the synthesis of the potassium salt of the above TPN-Au-L 2 Potassium salts such as potassium carbonate can be used. For the synthesis method of the potassium salt of TPN-Au-L 2 For example, a method of mixing an aqueous solution containing a raw material of L 2 and a potassium salt such as potassium carbonate with TPN-Au or an aqueous solution of TPN-Au can be mentioned. The lower limit of the pH of the solution is preferably 2 or more, more preferably 3 or more, and even more preferably 5 or more. Also, the upper limit of the pH of the solution is preferably 14 or less, more preferably 12 or less, and even more preferably 8 or less. As a pH adjuster, for example, a base (alkali), etc., specifically, a metal carbonate or carbonate such as an alkali metal carbonate or an alkaline earth metal carbonate; a hydroxide or metal hydroxide such as an alkali metal or an alkaline earth metal; ammonia (NH 3 )), other amines, alkaline substances, etc. can be used. Examples of the above carbonates include potassium carbonate and sodium carbonate, and examples of the above metal hydroxides include potassium hydroxide and sodium hydroxide. Also, for example, when a carbonate is used, carbonic acid can be removed by heating the synthesized metal salt such as the potassium salt of TPN-Au-L 2 monomer. The heating temperature is preferably 70 to 90 °C, more preferably 75 to 85 °C, and even more preferably 80 °C.

[0104] Next, TPN-Au-L 2A solution (such as an aqueous solution) containing the potassium salt of the monomer is subjected to rotary evaporation, reduced pressure, etc. to remove water and other solvents, thereby obtaining a white powder. Note that the white powder usually exhibits strong water absorption. The white powder thus obtained is redissolved in a heavy solvent (DMSO-d6 / D 2 O 1:4, etc.) and the NMR spectrum is measured. When compared with a known spectrum, it was confirmed that no structural change occurred even when dried and isolated. Taking TPN-Au-MM4 as an example, the L 1 -S-Au-L 2 The synthesis method will be described in detail in the above and in the examples. However, even when other compounds are used as L 1 and L 2 they can be synthesized by a similar method.

[0105] <Composition> A composition containing the gold complex represented by the above formulas (1) to (3) is useful as an anticancer agent, a combined agent with radiation in chemoradiotherapy, an in vitro diagnostic agent, a research reagent, a reagent for cancer stem cell analysis research, etc., which will be described later. The composition may contain other components in addition to the gold complex as long as the intended action is not impaired. Examples of other components include solvents, pH adjusters, solubilizers, excipients, bases, emulsifiers, stabilizers, etc.

[0106] The gold complex synthesized by the above method exhibits water solubility. Therefore, the gold complex represented by the above formula (3) can be prepared as a solution in a solvent containing water. The content of the solvent in the solution of the gold complex represented by the above formula (3) may be 0 mass% or more and 100 mass% or less, preferably 1 mass% or more and 70 mass% or less, and more preferably 5 mass% or more and 50 mass% or less. Examples of the solvent include the above-mentioned TPN-Au tetramer and L 2It may also be contained in the reaction solution with the raw materials. Specifically, it may contain water, dimethyl sulfoxide (DMSO), sulfonate, N,N-dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), propylene carbonate, polyvinyl alcohol (PVA), ethanol and other organic solvents. It is preferably to contain at least one selected from the group consisting of water and PVA, and more preferably to contain water and PVA. When the solution of the gold complex represented by the above formula (3) contains PVA, the content of PVA in the solution may be, for example, 0.1% by mass or more and 20% by mass or less, preferably 1% by mass or more and 10% by mass or less, and more preferably 1% by mass or more and 3% by mass or less.

[0107] The solution of the gold complex represented by the above formula (3) may further contain hydrolyzed saccharides (such as mannitol) and dissolution accelerators such as corn sugar to increase solubility. It may also contain polymers such as polyvinylpyrrolidone (PVP), hydroxyethyl cellulose (HEC), polyethylene glycol (PEG), polypropylene glycol (PPG), and polymerized saccharides. The content of such polymers in the solution of the gold complex represented by the above formula (3) may be, for example, more than 0% by mass and 70% by mass (up to solubility) or less, preferably 0.5% by mass or more and 50% by mass or less, and more preferably 1% by mass or more and 20% by mass or less. Examples of the pH adjuster include pH adjusters that can also be used in the above synthesis method, such as potassium hydroxide, potassium carbonate, sodium hydroxide, etc. When the above composition is a solution, especially a solution containing water, the pH of the solution is preferably in the range of 3 to 14, and more preferably in the range of 4 to 14. The content of the gold complex represented by the above formula (3) in the solution of the gold complex may be, for example, more than 0% by mass and 70% by mass or less (up to the solubility), preferably 0.5% by mass or more and 50% by mass or less, more preferably 1% by mass or more and 20% by mass or less. Also, for example, it may be more than 0 and 0.5 mol / L or less (or the solubility upper limit), preferably 0.001 mol / L or more and 0.3 mol / L or less, more preferably 0.01 mol / L or more and 0.25 mol / L or less, and even more preferably 0.05 mol / L or more and 0.2 mol / L or less.

[0108] <Anticancer agent> Since the gold complex represented by the above formula (1) has anticancer activity, it is useful as an anticancer agent. The gold complex contained in the anticancer agent is not particularly limited as long as it is the gold complex represented by the above formula (1). However, from the viewpoint of having higher anticancer activity, the gold complex represented by the above formula (3) is preferred. More preferably, it contains at least one selected from the group consisting of TPN-Au-MM4, TPN-Au-MTZ, TPN-Au-AMT, TPN-Au-MMT, TPN-Au-MMTT, TPN-Au-MT, TPN-Au-AMZ, TPN-Au-2NN, and TPN-Au-2SN. Even more preferably, it contains at least one selected from the group consisting of TPN-Au-MM4, TPN-Au-MTZ, TPN-Au-AMT, TPN-Au-MMT, TPN-Au-MMTT, TPN-Au-MT, and TPN-Au-AMZ. Even more preferably, it contains at least one selected from the group consisting of TPN-Au-MM4, TPN-Au-MMT, and TPN-Au-MMTT. Particularly preferably, it contains TPN-Au-MM4.

[0109] The above-mentioned gold preparation auranofin having anticancer activity is lipophilic. On the other hand, the gold complex represented by the above formula (1) is water-soluble. Due to such a difference in properties, it is speculated that the gold complex represented by the above formula (1) shows intracellular kinetics different from that of auranofin. Also, the fact that it is water-soluble and has a high solubility in water is also suitable as a component of a pharmaceutical agent.

[0110] The cancer targeted by the anticancer agent is not particularly limited, and examples include leukemia, lung cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, colorectal cancer, osteosarcoma, malignant lymphoma, and the like. Among these, from the viewpoint of being able to exhibit a higher cancer cell growth inhibitory effect, leukemia, liver cancer, and lung cancer can be preferably treated.

[0111] The anticancer agent can be produced by any method adopted in the pharmaceutical field or a method with appropriate improvements.

[0112] The anticancer agent may further contain other components as necessary. The other components are not particularly limited as long as they are pharmaceutically or physiologically acceptable, and examples include bases, carriers, excipients, binders, disintegrants, lubricants, and coloring agents. Further, other nutritional components (such as carbohydrates), salts (such as NaCl), and pH adjusters (such as edible acids) may be included.

[0113] Examples of the carrier and excipient include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, and crystalline cellulose. Examples of the binder include starch, gelatin, syrup, tragacanth gum, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, and carboxymethyl cellulose. Examples of the disintegrant include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, sodium alginate, sodium carboxymethyl cellulose, and calcium carboxymethyl cellulose. Examples of the lubricant include magnesium stearate, hydrogenated vegetable oil, talc, and macrogol. The coloring agent may be any coloring agent that is permitted to be added to pharmaceuticals.

[0114] The anticancer agent can be formulated into any dosage form by mixing, dissolving, granulating, tableting, emulsifying, encapsulating, freeze-drying, etc. together with a carrier.

[0115] The anticancer agent can be administered orally or parenterally. The formulation is not particularly limited, and examples include oral formulations and injection formulations. Suitable oral formulations include a liquid preparation in which an effective amount of the gold complex is dissolved in a diluent such as water, physiological saline, and orange juice; a capsule, sachet, or tablet containing an effective amount of the gold complex as a solid or granule; a suspension in which the gold complex is dispersed in a suitable dispersion medium; and an emulsion in which a solution in which an effective amount of the gold complex is dissolved is dispersed and emulsified in a suitable dispersion medium, etc.

[0116] As the above injection formulation, aqueous and non-aqueous isotonic sterile injection solutions are suitable, and the injection may contain antioxidants, buffers, bacteriostatic agents, and tonicity agents, etc. It is also preferable to use aqueous and non-aqueous sterile suspension preparations, and such suspension preparations may contain suspending agents, solubilizing agents, thickening agents, stabilizing agents, and preservatives, etc. The injection formulation can be enclosed in a container such as an ampoule or vial in unit doses or multiple doses per container. Also, the gold complex and a pharmaceutically acceptable carrier can be lyophilized and stored, and dissolved or suspended in a sterile suitable vehicle immediately before use.

[0117] The anticancer agent can be used in a treatment method (i.e., chemoradiotherapy) administered in combination with radiotherapy.

[0118] The application target of the anticancer agent is not particularly limited, but it can exhibit an anticancer effect when administered to mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, or humans), especially humans.

[0119] The dosage, frequency of administration, dosing interval, etc. of the anticancer agent are not limited because they depend on the condition of the administration subject, administration route, dosage form, etc. For example, it can be administered in the range of 0.0001 mg / kg·day to 100 mg / kg·day. For example, the daily dose per 1 kg of body weight of patients with TPN-Au-MMTT, TPN-Au-MMT, TPN-Au-MTZ, TPN-Au-MM4, and TPN-Au-AMT is, for example, for the reduction of human liver cancer and / or human lung cancer, 1 mg / kg·day to 10 mg / kg·day is preferred, 0.1 mg / kg·day to 1 mg / kg·day is more preferred, and for the suppression of metastasis of human liver cancer and / or human lung cancer, 0.1 mg / kg·day to 1 mg / kg·day is preferred, and 0.01 mg / kg·day to 0.1 mg / kg·day is more preferred.

[0120] <Combined agent and combined therapy with radiation in chemoradiotherapy> The gold complex represented by the above formula (1) is useful as a combined agent with radiation in chemoradiotherapy. The gold complex used as a combined agent with radiation in chemoradiotherapy is not particularly limited as long as it is the gold complex represented by the above formula (1), but from the viewpoint of achieving a higher therapeutic effect, the gold complex represented by the above formula (3) is preferred, and TPN-Au-MM4 is more preferred. Conventionally, in chemoradiotherapy, anticancer agents such as cisplatin (CDDP), fluorouracil (5-FU), and mitomycin C (MMC) have sometimes been used in combination. However, these drugs originally have the potential to exhibit strong toxicity to the human body, such as kidney damage caused by cisplatin and bone marrow suppression caused by fluorouracil or mitomycin C. When such drugs are used in combination with radiation in chemoradiotherapy, the frequency of occurrence of adverse events may increase. In contrast, in this embodiment, by using the highly safe gold complex represented by the above formula (1) in place of some or all of these conventional drugs in chemoradiotherapy, a highly safe chemoradiotherapy with no or reduced side effects becomes possible.

[0121] By using the complex as an adjunct to radiation in chemoradiotherapy, it is possible to suppress the growth of cancer cells additively with radiation, and in relation to the cancer cell growth inhibitory effect of radiation, it can function as an adjuvant. As a result, the cell-killing effect of radiation on cancer cells can be increased, and the radiation dose required clinically can also be reduced. It is also expected that reducing the total radiation dose will contribute to preventing radiation damage.

[0122] Here, the biological effects of ionizing radiation are considered separately as direct action and indirect action. The direct action here is the action in which radiation itself directly damages DNA by ionization or excitation, and the indirect action is the action in which radical molecules and reactive oxygen species (ROS), which are products thereof, generated as a result of ionization or excitation occurring in water molecules, which are the main constituent molecules of living organisms, indirectly damage DNA. In particular, the indirect action is said to account for most of the biological effects in low-LET radiation such as X-rays, γ-rays, and β-rays (here, LET means linear energy transfer and is an index representing the quality of radiation. The unit is keV / μm). In order to fully exert this indirect effect, it is necessary for a high oxygen concentration to be present in the tissue during radiation exposure. This is due to the property that oxygen molecules themselves have a high electron affinity and thus continuously produce highly reactive radical molecules by taking in electrons (the so-called oxygen effect). However, one of the reasons why many cases where sufficient effects cannot be obtained in cancer radiotherapy is the low oxygenation (so-called hypoxia) in the center of cancer tissue. Therefore, the above-mentioned oxygen effect cannot be expected, and the cell-killing effect due to DNA damage of cancer cells also remains low.

[0123] As described below, by adding TPN-Au-MM4 to the THP1 cell line (human acute monocytic leukemia cell line), a transient and rapid increase in intracellular reactive oxygen species (ROS) levels was confirmed (see Fig. 23). This suggests that for cancer tissues composed of cell types sensitive to the drug, by administering the drug before radiation irradiation, DNA damage by reactive oxygen species (ROS) can be imparted in advance. TPN-Au-MM4 was confirmed to enhance radiation-induced cell death in the THP1 cell line (see Fig. 21B). This is presumably because DNA damage to cancer cells by reactive oxygen species (ROS) occurred in advance due to the addition of TPN-Au-MM4, and radiation irradiation was performed before DNA repair, resulting in a synergistic enhancement of the effect of radiation irradiation. Therefore, the gold complex can be applied as a co-agent with radiation in chemoradiotherapy that can enhance the effect of radiotherapy.

[0124] The co-agent with radiation in chemoradiotherapy can be produced by any method adopted in the field of pharmaceuticals or a method with appropriate modifications. Also, other substances, administration methods, etc. that may be included in the co-agent with radiation in chemoradiotherapy can use the substances and methods, etc. described above for anticancer drugs.

[0125] Moreover, the gold complex represented by the above formula (1) is useful for use in combination therapy with radiotherapy. The gold complex used in combination therapy with radiotherapy is not particularly limited as long as it is the gold complex represented by the above formula (1), but from the viewpoint of achieving a higher therapeutic effect, the gold complex represented by the above formula (3) is preferred, and it is more preferred to contain TPN-Au-MM4.

[0126] Any method adopted in the field of cancer treatment can be used for the combination therapy with radiotherapy.

[0127] <In Vitro Diagnostic Agents and Research Reagents> The in vitro diagnostic agents and research reagents are not particularly limited as long as they contain the gold complex represented by the above formula (1), but from the viewpoints described below, it is preferably those containing the gold complex represented by the above formula (3), and more preferably those containing TPN-Au-MM4.

[0128] In the anti-cancer activity evaluation test of TPN-Au-MM4 described below, when TPN-Au-MM4 was added to the H1299 cell line (human lung cancer cell line) and HepG2 cell line (human liver cancer cell line) so that the final concentration was 1 μM, a significant cell growth inhibitory effect was shown. On the other hand, when TPN-Au-MM4 was added to the A549 cell line (human lung cancer cell line) so that the final concentration was the same, no significant cell growth inhibitory effect was shown (see Fig. 19).

[0129] Here, it is known that the A549 cell line overexpresses the oxidative stress-responsive transcription factor Nrf2. The inventors considered that the difference in the cell growth inhibitory effect of TPN-Au-MM4 due to the above-described difference in cell lines might be caused by Nrf2. Therefore, an anti-cancer activity evaluation test of TPN-Au-MM4 was performed using the A549 cell line in which the expression of Nrf2 was suppressed by siRNA (small interfering RNA). As a result, in the cells into which siRNA against Nrf2 was introduced and TPN-Au-MM4 was added, the cell survival rate decreased compared to the cells into which control siRNA was introduced and TPN-Au-MM4 was added (see Fig. 22). That is, it was confirmed that in the cells in which the expression of Nrf2 was suppressed, the cell growth inhibitory effect by TPN-Au-MM4 became greater. From these results, it was suggested that the cell growth inhibitory effect by TPN-Au-MM4 is related to the expression of Nrf2.

[0130] It was also shown that the amount of intracellular reactive oxygen species in the THP1 cell line temporarily increased by the addition of TPN-Au-MM4 (see Fig. 23).

[0131] From these results, it is presumed that the cell growth inhibitory effect of TPN-Au-MM4 is related to the oxidative stress response mechanism and the redox control mechanism. Therefore, the gold complex can be used as an in vitro diagnostic agent useful for the diagnosis of diseases related to the oxidative stress response mechanism and the redox control mechanism, and as a research reagent for the study of these mechanisms.

[0132] Also, as shown in Fig. 19, compared with the H1299 cell line and the HepG2 cell line, the IC 50 value of TPN-Au-MM4 in the A549 cell line is predicted to be clearly higher. As described above, this is presumably due to the fact that the A549 cell line used in the colony assay overexpresses Nrf2, which is one of the transcription factors. Here, it is known that cancer cells overexpressing Nrf2 tend to be resistant to the growth inhibitory effect against radiation and the addition of anticancer drugs. From these facts, it is also possible to simply screen the suitability of a patient for radiation and anticancer drug treatments by separating and culturing cancer cells from a specimen obtained from the patient, adding a gold complex thereto, and performing a colony assay. Therefore, the gold complex can also be used as an in vitro diagnostic agent for application to such screening.

[0133] The in vitro diagnostic agent and the research reagent can be produced by any method adopted in the field of diagnostic agents and research reagents or a method with appropriate improvements.

[0134] In addition, the form and usage method, etc. of the in vitro diagnostic agent and the research reagent can adopt the form and method, etc. used in conventional diagnostic agents and research reagents.

[0135] The application subjects of the in vitro diagnostic agents and research reagents are not particularly limited, and examples thereof include the above-mentioned animals, animal cells, etc. The types of cells are also not particularly limited, and examples thereof include blood cells, germ cells, fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatic cells, keratinocytes, muscle cells, epidermal cells, endocrine cells, stem cells such as ES cells, iPS cells, hematopoietic stem cells, tissue stem cells, etc., progenitor cells, differentiated cells, cancer cells, etc.

[0136] <Reagents for cancer stem cell analysis and research> The reagent for cancer stem cell analysis and research is not particularly limited as long as it contains a gold complex represented by the above formula (1). However, from the viewpoint described below, it is preferable that it contains a gold complex represented by the above formula (3), and it is even more preferable that it contains TPN-Au-MM4.

[0137] As described above, when TPN-Au-MM4 was added to the H1299 cell line and the HepG2 cell line at a final concentration of 1 μM, a significant cell proliferation inhibitory effect was observed, whereas when it was added to the A549 cell line at the same final concentration, no significant cell proliferation inhibitory effect was observed (see FIG. 19). Here, it is known that the H1299 cell line and the HepG2 cell line show a cancer stem cell-like proliferation pattern, whereas the A549 cell line does not show a cancer stem cell-like proliferation pattern. These findings suggest that TPN-Au-MM4 may suppress the proliferation of cancer cells by acting specifically on cancer stem cells.

[0138] Stem cells, including cancer stem cells, are known to exist in a place called the "microenvironment (niche)," which functions as a shield to protect against cytotoxic drugs and substances. However, the microenvironment (niche), including detailed signaling pathways, is largely unknown. Gold complexes can be used as research reagents to help elucidate the microenvironment (niche) surrounding cancer stem cells at the molecular level.

[0139] The reagent for cancer stem cell analysis research can be produced by any method adopted in the field of research reagents or a method with appropriate improvements.

[0140] Regarding the form, usage method, application targets, etc. of the reagent for cancer stem cell analysis research, the forms and methods described above for research reagents can be adopted, and the above-mentioned animals, animal cells, etc. can be used as application targets.

[0141] <Screening method> The gold complex represented by the above formula (3) can be used for drug repositioning. Specifically, for L in formula (3) 2 as the heterocyclic ring-containing structure, which is a partial structure of the above-mentioned β-lactam antibiotics, is used as a candidate compound to prepare the gold complex represented by formula (3), and its anticancer activity is evaluated, so it is expected to efficiently develop new drugs. This embodiment can be said to be a variant method of drug repositioning in that the partial structure of an existing drug is combined in an organically stable form (i.e., the gold complex represented by formula (3) is prepared) to find or exhibit new drug effects. β-Lactam antibiotics that have been approved, are in preclinical development, or are in clinical development are generally compounds with high safety such as no side effects or reduced side effects, and it is expected that the safety of the gold complex represented by formula (3) obtained by using the partial structure of the compound is also high. Specifically, the following screening method can be used.

[0142] Compounds suitable for L contained in a cell growth inhibitor or an anticancer agent can be selected by a method including the following steps (i) and (ii). 2 (i): A step of coordinating a candidate compound of L to the gold complex precursor represented by the above formula (2) to obtain the gold complex represented by the above formula (3) 2 (ii): A step of evaluating the cell growth inhibitory ability of the gold complex obtained in (i)

[0143] In addition, the screening of a cell growth inhibitor or an anticancer agent can be carried out by a method including the following steps (i) and (ii). (i): Coordinating a candidate compound of L 2 to the gold complex precursor represented by the above formula (2) to obtain a gold complex represented by the above formula (3). (ii): Evaluating the cell growth inhibitory ability of the gold complex obtained in (i).

Examples

[0144] <Synthesis of Gold Complex> [Synthesis Example 1: Synthesis of TPN-Au] Gold (20.0 g, 102 mmol) was added to a solution of 65% by mass nitric acid (25 mL) and 35% by mass hydrochloric acid (75 mL). The temperature was gradually raised from 20°C to 100°C, and the mixture was stirred at 100°C until all the gold was dissolved. After the gold was dissolved, the solution was distilled until it reached a volume of 25 mL. Then, an additional 35% by mass hydrochloric acid (25 mL) was added, and the solution was distilled again until it reached a volume of 25 mL. The addition of 35% by mass hydrochloric acid (25 mL) and the distillation until it reached a volume of 25 mL were repeated once more to obtain a solution of chloroauric acid (HAuCl 4 ).

[0145] The chloroauric acid solution was poured into 1 L of an aqueous solution of thiopropionine (55 g, 336.6 mmol) with rapid stirring, and the reaction mixture was stirred at 20°C for 6 hours. TPN-Au was formed as a white precipitate. This mixture (suspension) was transferred to a centrifuge tube and centrifuged at 3000 rpm for 10 minutes, and the supernatant was removed by decantation. Further, the white precipitate was resuspended in distilled water, centrifuged again at 3000 rpm for 10 minutes, and the supernatant was removed by decantation. The steps of resuspension in distilled water, centrifugation, and removal of the supernatant by decantation were repeated three times to remove residual hydrochloric acid, nitric acid, thiopropionine disulfide, and excess thiopropionine from TPN-Au. By quantitatively drying the white precipitate, pure TPN-Au (36.6 g, 102 mmol) was obtained. TPN-Au produced 20.0 mg of pure gold upon thermal decomposition of 36.6 mg.

[0146] K2 CO 3 neutralized D with 2 The quantitative formation of TPN-Au in 1 H-NMR and 13 C-NMR spectra was confirmed by analysis. The results are shown in Figures 4 and 5.

[0147] When compared with the NMR spectra of thiopronine shown in Figures 1 to 3, the 1 H-NMR signals and 13 C-NMR signals around the sulfur atom (SH group) and the absorption at 340 nm in the NMR spectrum and ultraviolet-visible spectrum broadened, indicating that the synthesized TPN-Au is a TPN-Au polymer characterized as a cyclic tetramer. The formation of the TPN-Au tetramer was confirmed by observing the deprotonated molecular ion in the Matrix assisted laser desorption / ionization (MALDI) mass negative ion spectrum and the molecular ion containing 1 to 4 potassium atoms in the positive ion spectrum.

[0148] [Synthesis Example 2: Synthesis of TPN-Au-MM4] TPN-Au (9.0 g, 25 mmol) was dissolved in an aqueous solution containing potassium hydroxide (1.4 g, 25 mmol) to a volume of 50 mL. Next, the TPN-Au aqueous solution was added to an aqueous solution containing 200 mL of 5-mercapto-1-methyltetrazole (MM4; 3.0 g, 26 mmol) and potassium carbonate (3.6 g, 13 mmol) to a total volume of 250 mL. By mixing both aqueous solutions, an initial reaction occurred instantaneously at room temperature, and by stirring at 80 °C for 3 hours, TPN-Au-MM4 was quantitatively produced.

[0149] K 2 CO 3 neutralized D with 2 The quantitative formation of TPN-Au-MM4 in 1 H-NMR and 13It was confirmed by analyzing the 13C-NMR spectrum. The results are shown in FIGS. 6 and 7. As a feature, the broadened 1 1H-NMR signal and 13 13C-NMR signal of the TPN-Au tetramer became sharp, and it was confirmed that the 1 1H-NMR signal and 13 13C-NMR signal of MM4 were slightly broadened.

[0150] [Synthesis Example 3: Synthesis of TPN-Au-AMT] TPN-Au (9.0 g, 25 mmol) was dissolved in an aqueous solution containing potassium hydroxide (1.4 g, 25 mmol) to a volume of 50 mL. Next, the TPN-Au aqueous solution was added to an aqueous solution containing 200 mL of 2-amino-5-mercapto-1,3,4-thiadiazole (AMT; 3.5 g, 26 mmol) and potassium carbonate (1.8 g, 13 mmol) to a total volume of 250 mL. The mixed solution was stirred at 60° C. until AMT dissolved. TPN-Au and AMT immediately reacted to quantitatively produce TPN-Au-AMT.

[0151] K 2 CO 3 neutralized D in 2 O, the quantitative formation of TPN-Au-AMT was confirmed by analyzing the 1 1H-NMR and 13 13C-NMR spectra. The results are shown in FIGS. 8 and 9. The formation of the TPN-Au-AMT monomer was confirmed by observing the deprotonated molecular ion in the MALDI mass negative ion spectrum and the molecular ion containing 1 to 4 potassium atoms in the positive ion spectrum. MS (MALDI Neg.) m / z found 490.9886, calcd for C 7 H 10 N 4 O 3 S 3 Au 490.9580 (M-H) - . From this result, it was confirmed that the TPN-Au tetramer did not exist and that TPN-Au-AMT was formed.

[0152] As a feature, the 1 H-NMR signal and 13 C-NMR signal of the broadened TPN-Au tetramer became sharp, and it was confirmed that the 1 H-NMR signal and 13 C-NMR signal of AMT were slightly broadened.

[0153] [Synthesis Example 4: Synthesis of TPN-Au-MTZ] TPN-Au (9.0 g, 25 mmol) was dissolved in an aqueous solution containing potassium hydroxide (1.4 g, 25 mmol) to a volume of 50 mL. Next, the TPN-Au aqueous solution was added to an aqueous solution containing 200 mL of 1,2,4-triazole-3-thiol (MTZ; 2.6 g, 26 mmol) and potassium carbonate (1.8 g, 13 mmol) to a total volume of 250 mL. The mixed solution was stirred at 60 °C until MTZ dissolved. TPN-Au and MTZ reacted immediately, and TPN-Au-MTZ was quantitatively produced.

[0154] K 2 CO 3 neutralized D in 2 O, the quantitative formation of TPN-Au-MTZ was confirmed by 1 H-NMR and 13 C-NMR spectra analysis. The results are shown in FIGS. 10 and 11. As a feature, the 1 H-NMR signal and 13 C-NMR signal of the broadened TPN-Au tetramer became sharp, and it was confirmed that the 1 H-NMR signal and 13 C-NMR signal of MTZ were slightly broadened.

[0155] [Synthesis Example 5: Synthesis of PVA Solution of TPN-Au-MM4] An aqueous solution of TPN-Au potassium salt with a gold concentration of 0.5 mol / L (5 mL, [Au] 2.5 mmol) was added to a 2% PVA aqueous solution (20 mL) containing 5-mercapto-1-methyltetrazole (MM4; 0.30 g, 2.6 mmol) and potassium carbonate (0.18 g, 1.3 mmol). Stirring was carried out at 80 °C for 1 hour, and carbon dioxide gas was removed from the solution. TPN-Au and 5-mercapto-1-methyltetrazole immediately reacted to quantitatively produce TPN-Au-MM4 in volume. It was adjusted to a volume of 25 mL to obtain a solution containing the dipotassium salt of TPN-Au-MM4 with a gold concentration of 0.1 mol / L.

[0156] [Synthesis Example 6: Synthesis of PVA solution of TPN-Au-AMT] An aqueous solution of TPN-Au potassium salt with a gold concentration of 0.5 mol / L (5 mL, [Au] 2.5 mmol) was added to a 2% PVA aqueous solution (20 mL) containing 2-amino-5-mercapto-1,3,4-thiadiazole (AMT; 0.35 g, 2.6 mmol) and potassium carbonate (0.18 g, 1.3 mmol). Stirring was carried out at 80 °C for 1 hour, and carbon dioxide gas was removed from the solution. TPN-Au and 2-amino-5-mercapto-1,3,4-thiadiazole immediately reacted to quantitatively produce TPN-Au-AMT in volume. It was adjusted to a volume of 25 mL to obtain a solution containing the dipotassium salt of TPN-Au-AMT with a gold concentration of 0.1 mol / L.

[0157] [Synthesis Example 7: Synthesis of PVA solution of TPN-Au-MTZ] An aqueous solution of TPN-Au potassium salt with a gold concentration of 0.5 mol / L (5 mL, [Au] 2.5 mmol) was added to a 2% PVA aqueous solution (20 mL) containing 2-mercapto-1,3,4-triazole (MTZ; 0.26 g, 2.6 mmol) and potassium carbonate (0.18 g, 1.3 mmol). Stirring was carried out at 80 °C for 1 hour, and carbon dioxide gas was removed from the solution. TPN-Au and 2-mercapto-1,3,4-triazole immediately reacted to quantitatively produce TPN-Au-MTZ in volume. It was adjusted to a volume of 25 mL to obtain a solution containing the dipotassium salt of TPN-Au-MTZ with a gold concentration of 0.1 mol / L.

[0158] [Synthesis Example 8: Synthesis of PVA Solution of TPN-Au-MMT] An aqueous solution of TPN-Au potassium salt with a gold concentration of 0.5 mol / L (5 mL, [Au] 2.5 mmol) was added to a 2% PVA aqueous solution (20 mL) containing 2-mercapto-5-methyl-1,3,4-thiadiazole (MMT; 0.34 g, 2.6 mmol) and potassium carbonate (0.18 g, 1.3 mmol). Stirring was carried out at 80 °C for 1 hour, and carbon dioxide gas was removed from the solution. TPN-Au and 2-mercapto-5-methyl-1,3,4-thiadiazole immediately reacted to quantitatively produce TPN-Au-MMT. The volume was adjusted to 25 mL to obtain a solution containing the dipotassium salt of TPN-Au-MMT with a gold concentration of 0.1 mol / L.

[0159] [Synthesis Example 9: Synthesis of PVA Solution of TPN-Au-MMTT] An aqueous solution of TPN-Au potassium salt with a gold concentration of 0.5 mol / L (5 mL, [Au] 2.5 mmol) was added to a 2% PVA aqueous solution (20 mL) containing 2-mercapto-5-methylthio-1,3,4-thiadiazole (MMTT; 0.42 g, 2.6 mmol) and potassium carbonate (0.18 g, 1.3 mmol). Stirring was carried out at 80 °C for 1 hour, and carbon dioxide gas was removed from the solution. TPN-Au and 2-mercapto-5-methylthio-1,3,4-thiadiazole immediately reacted to quantitatively produce TPN-Au-MMTT. The volume was adjusted to 25 mL to obtain a solution containing the dipotassium salt of TPN-Au-MMTT with a gold concentration of 0.1 mol / L.

[0160] [Synthesis Example 10: Synthesis of PVA Solution of TPN-Au-2NN] An aqueous solution of TPN-Au potassium salt with a gold concentration of 0.5 mol / L (5 mL, [Au] 2.5 mmol) was added to a 2% PVA aqueous solution (20 mL) containing 2-imidazolidinethione (ethylene thiourea; 2NN; 0.27 g, 2.6 mmol) and potassium carbonate (0.18 g, 1.3 mmol). Stirring was carried out at 80 °C for 1 hour, and carbon dioxide gas was removed from the solution. TPN-Au and 2-imidazolidinethione immediately reacted to quantitatively produce TPN-Au-2NN in volume. It was adjusted to a volume of 25 mL to obtain a solution containing the dipotassium salt of TPN-Au-2NN with a gold concentration of 0.1 mol / L.

[0161] [Synthesis Example 11: Synthesis of PVA solution of TPN-Au-2SN] An aqueous solution of TPN-Au potassium salt with a gold concentration of 0.5 mol / L (5 mL, [Au] 2.5 mmol) was added to a 2% PVA aqueous solution (20 mL) containing 2-mercapto-thiazoline (2SN; 0.31 g, 2.6 mmol) and potassium carbonate (0.18 g, 1.3 mmol). Stirring was carried out at 80 °C for 1 hour, and carbon dioxide gas was removed from the solution. TPN-Au and 2-mercapto-thiazoline immediately reacted to quantitatively produce TPN-Au-2SN in volume. It was adjusted to a volume of 25 mL to obtain a solution containing the dipotassium salt of TPN-Au-2SN with a gold concentration of 0.1 mol / L.

[0162] [Synthesis Example 12: Synthesis of PVA solution of TPN-Au-MT] An aqueous solution of TPN-Au potassium salt with a gold concentration of 0.5 mol / L (5 mL, [Au] 2.5 mmol) was added to a 2% PVA aqueous solution (20 mL) containing 2-mercapto-1,3,4-thiadiazole (MT; 0.30 g, 2.6 mmol) and potassium carbonate (0.18 g, 1.3 mmol). Stirring was carried out at 80 °C for 1 hour, and carbon dioxide gas was removed from the solution. TPN-Au and 2-mercapto-1,3,4-thiadiazole immediately reacted to quantitatively produce TPN-Au-MT in volume. It was adjusted to a volume of 25 mL to obtain a solution containing the dipotassium salt of TPN-Au-MT with a gold concentration of 0.1 mol / L.

[0163] [Synthesis Example 13: Synthesis of PVA Solution of TPN-Au-AMZ] An aqueous solution of TPN-Au potassium salt with a gold concentration of 0.5 mol / L (5 mL, [Au] 2.5 mmol) was added to a 2% PVA and 20% ethanol aqueous solution (20 mL) containing 3-amino-5-mercapto-1,2,4-triazole (AMZ; 0.30 g, 2.6 mmol). Stirring was carried out at 80 °C for 1 hour, and carbon dioxide gas was removed from the solution. TPN-Au and 3-amino-5-mercapto-1,2,4-triazole immediately reacted to quantitatively produce TPN-Au-AMZ. The volume was adjusted to 25 mL to obtain a solution containing the potassium salt of TPN-Au-AMZ with a gold concentration of 0.1 mol / L.

[0164] <Evaluation Test> Using the HepG2 cell line (human hepatocellular carcinoma cell line), A549 cell line (human lung cancer cell line), H1299 cell line (human lung cancer cell line), and THP1 cell line (human acute monocytic leukemia cell line), each evaluation test described below was performed. The HepG2 cell line was obtained from the Human Science Research Resources Bank (currently the JCRB Bioresource Bank), the A549 cell line and THP1 cell line were obtained from the RIKEN Cell Bank, and the H1299 cell line was obtained from the American Type Culture Collection. Each cell was cultured in a culture medium supplemented with 10% fetal bovine serum (manufactured by Japan Bioserum) and 1% penicillin / streptomycin (manufactured by Wako) under a 5% CO 2 atmosphere. The culture medium used was low-glucose DMEM medium (manufactured by Sigma-Aldrich) for the HepG2 cell line and A549 cell line, and RPMI1640 medium (manufactured by Gibco) for the H1299 cell line and THP1 cell line.

[0165] [Evaluation 1: Anticancer Activity Evaluation Test of Gold Complex (1)] Using the HepG2 cell line, the anti-cancer activity of the gold complex was evaluated by the colony assay method. The anti-cancer activity evaluation was performed by confirming the colony survival rate of the cells. The colony survival rate is one of the general characteristic values indicating cell proliferation ability, and a decrease in the colony survival rate indicates a decrease in cell proliferation ability. The test solutions were prepared by dissolving each of the gold complexes TPN-Au, TPN-Au-MM4, TPN-Au-MTZ, and TPN-Au-AMT synthesized by the above method in distilled water to a predetermined concentration. After the dissolved test solutions were filtered through a syringe filter (pore size 0.45 μm), they were used for subsequent evaluations.

[0166] Before adding the test solution, the HepG2 cell line was seeded in a culture vessel at a concentration of 300 cells / 2 mL to 1000 cells / 2 mL and cultured at 37 °C in a 5% CO 2 atmosphere for 6 hours. Then, each test solution containing any of the gold complexes TPN-Au, TPN-Au-MM4, TPN-Au-MTZ, and TPN-Au-AMT was added to the medium so that the final concentration of the gold complex was 1 μM, 5 μM, or 10 μM. A control without the addition of the gold complex was used. After culturing at 37 °C in a 5% CO 2 atmosphere for 10 to 14 days, the colony survival rate was evaluated. The colony survival rate was measured with a cell population consisting of 50 or more cells as one colony and determined as a relative value to the control. The same experiment was performed 3 times to obtain the average value and the standard deviation value. The results are shown in Figure 12.

[0167] As shown in Figure 12A, when each test solution containing any of the above gold complexes was added to the medium of the HepG2 cell line, a concentration-dependent decrease in the colony survival rate was shown. Also, for each gold complex, when added to a concentration of 5 μM, the colony survival rate was below 50%.

[0168] Also, 2 even when TPN-Au without L 2 coordination was added, an anti-cancer cell growth inhibitory effect was shown. From this, it was confirmed that TPN-Au has anti-cancer activity regardless of the presence or absence of L

[0169] Figure 12B shows a comparison of the colony survival rates of the HepG2 cell line when each gold complex was added to a final concentration of 5 μM, indicating that the colony survival rate was lowest when TPN-Au-MM4 was added. From this, it was confirmed that among the gold complexes tested, TPN-Au-MM4 has the highest anticancer activity. In addition, a significant difference in colony survival rate was found between TPN-Au without a coordinated heterocyclic ring and compounds in which the heterocyclic rings of MM4 and AMT were coordinated to TPN-Au, respectively. These results provide useful data for molecular design in the development of new gold complexes as drugs.

[0170] [Evaluation 2: Anticancer Activity Evaluation Test of Gold Complexes (2)] Using the human hepatoma cell line HepG2, the anticancer activity of gold complexes was evaluated by counting viable cells using the trypan blue dye exclusion method. As the HepG2 cell line, the same one as used in Evaluation 1 was prepared by culturing, except that 10% fetal bovine serum manufactured by Sigma-Aldrich was used as the fetal bovine serum added to the culture medium. As test solutions, PVA solutions (3) to (9) of the following gold complexes synthesized by the above method and the following (1) and (2) as controls were used. The following Au is Au(I), and the PVA concentration is v / v%.

[0171] [Test Compounds] (1) Control (1.6% PVA) (2) RSG2 (1.6% PVA) (3) RSG2-2NN (1.6% PVA) (4) RSG2-2SN (1.6% PVA) (5) RSG2-MMTT (1.6% PVA) (6) RSG2-MMT (1.6% PVA) (7) RSG2-AMT (1.6% PVA) (8) RSG2-MTZ (1.6% PVA) (9) RSG2-MM4 (1.6% PVA)

[0172] Before adding the test solution, the HepG2 cell line was adjusted to 5×104 Seeded in a 12-well plate (BD Falcon) at a concentration of cells per well, and cultured at 37 °C in a 5% CO 2 atmosphere for 6 hours. Then, each test solution of the above PVA solutions (3) to (9) was added to the medium so that the final concentration of the gold complex became 100 μM, and each test solution of the above (1) and (2) in the same amount as the test solution was added to the medium. Note that those without the addition of the test solution were used as controls ("nontreatment" in Fig. 13). After culturing at 37 °C in a 5% CO 2 atmosphere for 72 hours, viable cells were counted by the trypan blue dye exclusion method. The same experiment was independently conducted 3 times, and the average value and standard deviation value were determined. The results are shown in Fig. 13.

[0173] As shown in Fig. 13, when each test solution containing any of the above gold complexes was added to the medium of the HepG2 cell line, an inhibitory effect on cancer cell growth was shown. Also, 2 even when TPN-Au without the coordination of L was added, an inhibitory effect on cancer cell growth was shown. From this, it was confirmed that TPN-Au has anti-cancer activity against the HepG2 cell line regardless of the presence or absence of the coordination of L. 2

[0174] The cancer cell growth inhibitory effect was particularly high when TPN-Au-MMT, TPN-Au-MMTT, TPN-Au-MTZ, TPN-Au-MM4, TPN-Au-AMT, and TPN-Au-2NN were added. Among them, the inhibitory effect was particularly high when TPN-Au-MMT, TPN-Au-MMTT, TPN-Au-MTZ, TPN-Au-MM4, and TPN-Au-AMT were added. In particular, it was shown that the inhibitory effect was high when TPN-Au-MMT, TPN-Au-MMTT, and TPN-Au-MTZ were added. From this, it was confirmed that among the gold complexes tested, these gold complexes had the highest anticancer activity. In addition, a significant difference in the cancer cell growth inhibitory effect was found between TPN-Au without a coordinated heterocyclic ring and compounds in which heterocyclic rings such as MMT, MMTT, MTZ, MM4, and AMT were coordinated to TPN-Au, respectively. These results, similar to the results of Evaluation 1, provide useful data for molecular design in the creation of new gold complexes for drug development.

[0175] [Evaluation 3: Anticancer Activity Evaluation Test of Gold Complexes (3)] For TPN-Au-MMTT, TPN-Au-MMT, TPN-Au-AMT, TPN-Au-MTZ, and TPN-Au-MM4, which showed a high cancer cell growth inhibitory effect in Evaluation 2, the above-mentioned PVA solutions (5) to (9) were added to the medium so that the final concentration of the gold complex was the concentration shown in Fig. 14. Evaluation was carried out in the same manner as in Evaluation 2, except that the viable cells were counted not only 72 hours after the addition but also 24 hours and 48 hours after the addition. The results are shown in Fig. 14.

[0176] As shown in Fig. 14, a decrease in the number of viable cells was observed for TPN-Au-MMTT at a concentration of 25 μM or higher, for TPN-Au-MMT and TPN-Au-MTZ at a concentration of 50 μM or higher, and for TPN-Au-AMT and TPN-Au-MM4 at a concentration of 100 μM.

[0177] [Evaluation 4: Anticancer Activity Evaluation Test of Gold Complexes (4)] For TPN-Au-MMTT, TPN-Au-MMT, and TPN-Au-MTZ, which showed high cancer cell growth inhibitory effects in Evaluation 2, each of the test solutions of the above PVA solutions (5), (6), and (8) was added to the medium so that the final concentration of the gold complex was 50 μM. Also, the same amount of the PVA control of the above (1) as the test solution was added to the medium. Evaluation was performed in the same manner as in Evaluation 2, except that cell death analysis by Annexin V-FITC / PI staining similar to Evaluation 9 described below was performed instead of the trypan blue dye exclusion method. The results are shown in Fig. 15B.

[0178] As shown in Fig. 15B, the proportion of cell death after 72 hours of culture when TPN-Au-MMTT, TPN-Au-MMT, and TPN-Au-MTZ were added at final concentrations of 50 μM each increased by about 4 to 10 times compared to the control. Specifically, it increased by about 5 times in the case of TPN-Au-MTZ, about 8 times in the case of TPN-Au-MMT, and about 9 times in the case of TPN-Au-MMTT. From these results, it was confirmed that these gold complexes exhibit an effect of inducing cell death against HepG2.

[0179] Among them, for TPN-Au-MMTT, which showed a high cell death induction effect, evaluation was performed in the same manner as in the above Evaluation 4, except that the test solution of the above PVA solution (5) was added to the medium so that the final concentration of the gold complex was 25 μM. The results are shown in Fig. 15A. As shown in Fig. 15A, even when TPN-Au-MMTT was added at a final concentration of 25 μM, the proportion of cell death after 72 hours of culture after the addition increased by about 5 times compared to the control. From this result, it was confirmed that TPN-Au-MMTT exhibits a high cell death induction effect against HepG2.

[0180] [Evaluation 5: Anticancer Activity Evaluation Test of Gold Complex (5)] Using human lung cancer cells H1299, the anti-cancer activity of the gold complex was evaluated by counting viable cells using the trypan blue dye exclusion method. As the H1299 cell line, the one prepared by culturing in the same manner as the H1299 cell line used in Evaluation 1 was used, except that fetal bovine serum (FBS) added to the culture medium was from Sigma-Aldrich. The test solution was the same as in Evaluation 2. Evaluation was performed in the same manner as in Evaluation 2, except that the H1299 cell line was used instead of the HepG2 cell line. The results are shown in Fig. 16B.

[0181] As shown in Fig. 16B, when the test solution containing any of the above gold complexes was added to the medium of the H1299 cell line, an inhibitory effect on cancer cell growth was shown. Also, L 2 Even when TPN-Au without L coordination was added, an inhibitory effect on cancer cell growth was shown. From this, it was confirmed that TPN has anti-cancer activity against the H1299 cell line regardless of the presence or absence of L coordination. 2

[0182] Among them, the inhibitory effect on cancer cell growth was particularly high when TPN-Au-MMT, TPN-Au-MMTT, and TPN-Au-MTZ were added. In particular, it was shown that the inhibitory effect was very high when TPN-Au-MMT and TPN-Au-MMTT were added. However, it was also shown that the inhibitory effect was high even when TPN-Au-MM4, TPN-Au-AMT, TPN-Au-2SN, and TPN-Au-2NN were added. From this, it was confirmed that all the gold complexes tested had the highest anti-cancer activity.

[0183] Among them, for TPN-Au-MMTT and TPN-Au-MMT, which had a high cell death induction effect, evaluation was performed in the same manner as in Evaluation 5 above, except that the test solutions of the above PVA solutions (5) and (6) were added to the medium so that the final concentration of the gold complex was 10 μM. The results are shown in Fig. 16A. As shown in Fig. 16A, even when TPN-Au-MMTT and TPN-Au-MMT were added to a final concentration of 10 μM, it was confirmed that they exhibited an inhibitory effect on cancer cell growth after culturing for 72 hours.

[0184] [Evaluation 6: Anticancer Activity Evaluation Test of Gold Complexes (6)] Regarding TPN-Au-MMTT and TPN-Au-MMT, which showed a high inhibitory effect on cancer cell growth in Evaluation 5, each of the test solutions of the above PVA solutions (5) and (6) was added to the medium so that the final concentration of the gold complex was 25 μM, 50 μM, or 100 μM, and the evaluation was performed in the same manner as in Evaluation 5 except that live cells were counted not only 72 hours after the addition but also 24 hours and 48 hours after the addition. 2 TPN-Au without coordinated L was also evaluated in the same manner using the above PVA solution (2). The results are shown in Fig. 17.

[0185] As shown in Fig. 17, for both TPN-Au-MMTT and TPN-Au-MMT, a decrease in the number of live cells was observed at a concentration of 25 μM or higher, especially at a concentration of 50 μM or higher. Since the decrease in the number of live cells was similar between 50 μM and 100 μM for TPN-Au-MMTT and TPN-Au-MMT, it was suggested that they were saturated in terms of the inhibitory effect on cancer cell growth at 50 μM. For TPN-Au, a decrease in the number of live cells was observed at a concentration of 100 μM, but the degree was lower than that of TPN-Au-MMTT and TPN-Au-MMT at the same concentration. Also, for TPN-Au, the degree of decrease in the number of live cells was small at concentrations of 25 μM and 50 μM.

[0186] [Evaluation 7: Anticancer Activity Evaluation Test of Gold Complexes (7)] Regarding TPN-Au-MMTT and TPN-Au-MMT, which showed a high cancer cell growth inhibitory effect in Evaluation 5, the above-mentioned test solutions of PVA solutions (5) and (6) were added to the medium so that the final concentration of the gold complex was 25 μM and 50 μM, respectively. Also, the same amount of the above-mentioned PVA control of (1) and the PVA solution of TPN-Au of (2) as the test solution were added to the medium. Except for performing cell death analysis by Annexin V-FITC / PI staining similar to Evaluation 9 described below instead of the trypan blue dye exclusion method, the evaluation was performed in the same manner as Evaluation 5. The results are shown in Fig. 18.

[0187] As shown in Fig. 18, the proportion of cell death after 72-hour culture when TPN-Au-MMTT and TPN-Au-MMT were added at a final concentration of 25 μM each was higher than that of the control, and the proportion of cell death when the final concentration was 50 μM was even higher, increasing by about 6 to 8 times compared to the control. It was also confirmed that the difference in effect from TPN-Au was large. From these results, it was confirmed that these gold complexes exhibit an effect of inducing cell death against H1299.

[0188] [Evaluation 8: Anticancer Activity Evaluation Test of TPN-Au-MM4 against Solid Cancer] The anticancer activity of TPN-Au-MM4 was evaluated using the A549 cell line, H1299 cell line, and HepG2 cell line. The anticancer activity evaluation was performed by confirming the colony survival rate of the cells in the same manner as in Evaluation 1. The test solution was prepared in the same manner as in Evaluation 1 using TPN-Au-MM4.

[0189] Before adding the test solution, each cell line was seeded in a culture vessel at a concentration of 100 cells / 2 mL to 1200 cells / 2 mL and cultured at 37 °C in a 5% CO 2 atmosphere for 6 hours. Then, the test solution was added to the medium so that the final concentration of the gold complex was 1 μM, 5 μM, and 10 μM. Note that the one without the gold complex added was used as the control. After culturing at 37 °C in a 5% CO 2 atmosphere for 7 to 15 days, the colony survival rate was evaluated in the same manner as in Evaluation 1. The same experiment was performed 3 times, and the average value and standard deviation value were obtained. The results are shown in Fig. 19.

[0190] As shown in FIGS. 19B and C, the colony survival rate when added to a final concentration of 1 μM was approximately 19% in the H1299 cell line and approximately 35% in the HepG2 cell line, and both of these were significant values. From the above results, it is predicted that the IC 50 values by the colony assay method in both cell lines are both below 1 μM. Note that the concentration of 1 μM is lower than the concentration in the IC 50 of platinum complexes (cisplatin and its derivatives) widely used as anticancer agents. From this, it was confirmed that TPN-Au-MM4 has anticancer activity equivalent to or higher than that of conventional platinum anticancer agents.

[0191] Also, in terms of quantitatively evaluating the survival rate and growth rate of cultured cells, it can be said that the MTS method is in principle common with the colony assay method. Below, the IC 50 values evaluated by the MTS method for the effects of the administration of auranofin and cisplatin are shown (see Elizabeth et al., Cancers, 2014, 6, 2243 - 2258).

[0192]

Table 1

[0193] The IC 50 value in this evaluation and the IC 50 value by the above administration of auranofin and cisplatin are compared. Despite the difference in the test cell types, it can be said that the cell growth inhibitory effect of TPN-Au-MM4 is equivalent to or exceeds that compared with auranofin.

[0194] On the other hand, as shown in Fig. 19A, when the A549 cell line was used, the colony survival rate was approximately 86% when added to achieve a final concentration of 1 μM, and no significant cell growth inhibitory effect was observed. Even when the A549 cell line was used, a significant cell growth inhibitory effect was confirmed when added at a final concentration of 5 μM or 10 μM.

[0195] [Evaluation 9: Anticancer Activity Evaluation Test of TPN-Au-MM4 against Blood Cancer] The anticancer activity of the gold complex was evaluated using the THP1 cell line. The anticancer activity evaluation was performed by measuring the cell count and cell death analysis. The test solution was prepared in the same manner as in Evaluation 1 using TPN-Au-MM4.

[0196] Before adding the test solution, the THP1 cell line was seeded in a culture vessel at a concentration of 10×10 5 / mL and cultured at 37°C in a 5% CO 2 atmosphere for 3 to 6 hours. Then, the test solution was added to the medium so that the final concentrations were 25 μM and 50 μM. A sample without the addition of the gold complex was used as a control. Culturing was carried out at 37°C in a 5% CO 2 atmosphere, and the cell count was measured at 24 hours, 48 hours, and 72 hours after the start of culturing. Also, after culturing for 72 hours, the cells were collected and cell death analysis was performed.

[0197] Cell death analysis was performed by FITC-Annexin V / PI (propidium iodide) staining. The cells were collected, washed twice with PBS(-), centrifuged (1200 rpm, 5 min, room temperature), and suspended in 100 μL of Annexin V Binding Buffer. 4.5 μL each of FITC-Annexin V (90 μg / mL) and PI (1 mg / mL) were added to the cell suspension and reacted at room temperature in the dark for 15 minutes. After the reaction, 320 μL of Annexin V Binding Buffer was added, and the cell death population was analyzed using a flow cytometer (Cytomics FC500 (Beckman-Coulter)). The same experiment was performed three times, and the average and standard deviation were calculated. The results are shown in Figure 20. The Annexin V-positive cell death population on the vertical axis in FIG. 20C is the sum of the Annexin V-positive, PI-negative population (early apoptotic cell population) and the Annexin V-positive, PI-positive population (late apoptotic cell / necrotic cell population).

[0198] As shown in Figures 20A and 20B, the number of proliferating cells after 72 hours of culture when TPN-Au-MM4 was added to a final concentration of 25 μM or 50 μM was reduced by about 37% or about 70%, respectively, compared to the control. In addition, as shown in Figure 20C, the rate of cell death when TPN-Au-MM4 was added to a final concentration of 25 μM was increased by about 2 to 3 times compared to the control. These results confirmed that gold complexes have a cell proliferation inhibitory effect not only on solid cancers but also on blood cancers such as leukemia.

[0199] From the results of evaluations 8 and 9, it was confirmed that the TPN-Au-MM4 concentration that produces a cell proliferation inhibitory effect differs for each cell type. This suggests that differences in the expression of antioxidant stress response genes for each cell type affect the action and effect of TPN-Au-MM4.

[0200] [Evaluation 10: Anticancer activity evaluation test when TPN-Au-MM4 is added and irradiated] Using the THP1 cell line, in addition to the addition of TPN-Au-MM4, the anti-cancer activity was evaluated when irradiated with radiation (X-rays). The anti-cancer activity evaluation was performed by measuring the cell count and cell death analysis. The test solution was prepared in the same manner as in Evaluation 1 using TPN-Au-MM4.

[0201] Before adding the test solution, the THP1 cell line was seeded in a culture vessel at a concentration of 10×10 5 / mL and cultured at 37°C in a 5% CO 2 atmosphere for 3 to 6 hours. Then, the test solution was added to the medium so that the final concentrations were 10 μM and 25 μM. Note that the one without the gold complex added was used as a control. After culturing at 37°C in a 5% CO 2 atmosphere for 1 hour, a predetermined amount of radiation was irradiated. The radiation irradiation was performed using an X-ray generator (model: MBR-1520R-3, manufactured by Hitachi). The irradiation conditions were set with a tube voltage of 150 kVp, a tube current of 20 mA, an irradiation distance of 45 cm, a filter of 0.5 mm Al / 0.3 mm Cu, and a dose rate of ~1.00 Gy / min. After that, after culturing for 72 hours, the cells were collected and the cell count and cell death analysis were performed. The cell count was measured according to the conventional method, and the cell death analysis was performed in the same manner as in Evaluation 9. The same experiment was performed 3 times, and the average value and standard deviation value were obtained. The results are shown in Figure 21.

[0202] As shown in Figure 21A, when TPN-Au-MM4 was added at a final concentration of 25 μM and irradiated with 2 Gy of X-rays, a decrease in the cell count of about 37% was shown compared to the case where the same amount of X-rays was irradiated to the control. Similarly, when TPN-Au-MM4 was added at a final concentration of 25 μM and irradiated with 4 Gy of X-rays, a decrease in the cell count of about 49% was shown compared to the case where the same amount of X-rays was irradiated to the control.

[0203] Also, as shown in Fig. 21B, when TPN-Au-MM4 was added to a final concentration of 25 μM and irradiated with 2 Gy of X-rays, an approximately 2.6-fold increase in cell death was shown compared to when the same amount of X-rays was irradiated to the control. Similarly, when TPN-Au-MM4 was added to a final concentration of 25 μM and irradiated with 4 Gy of X-rays, an approximately 1.32-fold increase in cell death was shown compared to when the same amount of X-rays was irradiated to the control.

[0204] All of these results were significant, and it was confirmed that TPN-Au-MM4 increases the killing effect of cancer cells by radiation.

[0205] [Evaluation 11: Anticancer Activity Evaluation Test against Nrf2 Expression Suppression Strain] Using the RNA interference method with siRNA (small interfering RNA), the anticancer activity of TPN-Au-MM4 was evaluated when the expression of Nrf2 was suppressed. The A549 cell line with suppressed Nrf2 expression was used for the evaluation. The anticancer activity was evaluated by confirming the colony survival rate of the cells. The test solution was prepared in the same manner as in Evaluation 1 using TPN-Au-MM4.

[0206] For siRNA, Silencer Select Pre-designed siRNA (manufactured by Ambion) was used. The ID: s9491 of this product was designated as siNrf2#1, and the ID: s9492 was designated as siNrf2#2. Also, for the control siRNA (siCont), Silencer Select Negative No.1 Control siRNA (cat. No. AM4611, manufactured by Ambion) was used.

[0207] Transfection of siRNA was performed using RNAiMAX (manufactured by Invitrogen). A549 cells were seeded at 3.0×10 4The culture vessels were seeded so as to obtain cells / well, and after overnight culture, transfection was performed according to the protocol attached to RNAiMAX. The cells transfected for 48 hours were collected and used for subsequent experiments. The concentration of the siRNA used was 10 nM.

[0208] The cells were washed twice with PBS(-) and then dissolved in 1× Laemmli Sample Buffer (manufactured by Bio-Rad Laboratories) containing 2.5% 2-mercaptoethanol. The sample that had been frozen and thawed once was boiled for 10 minutes and subjected to SDS-PAGE electrophoresis. SDS-PAGE was performed using 4-20% Mini-PROTEAN TGX Precast Gels (manufactured by Bio-Rad Laboratories). After completion, transfer to polyvinylidene difluoride membranes (PVDF membranes) was performed using the Trans-Blot Turbo Transfer System and Trans-Blot Turbo Transfer Pack (both manufactured by Bio-Rad Laboratories). The transferred PVDF membrane was blocked in TBST buffer (10 mM HCl, pH 7.5, 100 mM NaCl, 0.1% Tween-20) containing 5% low-fat skim milk at room temperature for 1 hour. The blocked membrane was reacted with the primary antibody diluted with TBST buffer containing 5% low-fat skim milk or Can Get Signal Immunoreaction Enhancer Solution1 (manufactured by TOYOBO) at 4°C overnight. Subsequently, it was reacted with the secondary antibody diluted with TBST buffer containing 5% low-fat skim milk or Can Get Signal Immunoreaction Enhancer Solution2 (manufactured by TOYOBO) at room temperature for 1 hour. Chemiluminescence detection was performed using Claity Western ECL Substrate (manufactured by Bio-Rad Laboratories). As the primary antibodies, an anti-human Nrf2 antibody (H-300, sc-13032, manufactured by SANTA CRUZ) and an anti-β-Actin antibody (#4967, manufactured by Cell Signaling Technology) were used. As the secondary antibody, an HRP-labeled anti-rabbit IgG antibody (#7074, manufactured by Cell Signaling Technology) was used.

[0209] As shown in Fig. 22A, it was confirmed that the expression of Nrf2 was suppressed in the A549 cell line transfected with siRNA of siNrf2#1 or siNrf2#2.

[0210] Before adding the test solution, the A549 cell line with suppressed Nrf2 expression was seeded at a concentration of 100 cells / 2 mL to 600 cells / 2 mL and cultured at 37 °C in a 5% CO 2 atmosphere for 6 hours. Then, the test solution was added to the medium so that the final concentration was 5 μM or 10 μM. After culturing for 7 to 12 days, the colony survival rate was evaluated in the same manner as in Evaluation 1. The same experiment was conducted 3 times, and the average value and standard deviation value were determined. The results are shown in Fig. 22B. In Fig. 22B, the cell group without adding the gold complex is shown as Control, and the cell group transfected with the above control siRNA is shown as siControl.

[0211] As shown in Fig. 22B, in the cells with suppressed Nrf2 expression by the introduction of siNrf2#1 or siNrf2#2, the decrease in the colony survival rate due to the addition of TPN-Au-MM4 was significantly increased compared to the cells transfected with the control siRNA.

[0212] [Evaluation 12: Intracellular reactive oxygen species evaluation test] Using the THP1 cell line, the amount of intracellular reactive oxygen species was evaluated when TPN-Au-MM4 was added. The test solution was prepared in the same manner as in Evaluation 1 using TPN-Au-MM4.

[0213] Before adding the test solution, the THP1 cell line was adjusted to 10×10 5They were seeded in a culture vessel at a concentration of / mL and cultured for 3 to 6 hours. Then, the test solution was added to the medium so that the final concentration became 25 μM or 50 μM. Note that the one without the addition of the gold complex was used as a control. After the addition of the test solution, after culturing for 1 to 24 hours, the reactive oxygen species detection fluorescent probe CM-H2DCFDA (manufactured by Thermo Fisher Scientific) was added to the culture medium so that the final concentration became 10 μM, and the reaction was carried out at 37 °C in a 5% CO 2 atmosphere for 30 minutes. Then, it was washed twice with PBS(-), suspended in 450 μL of RPMI1640 medium, and the amount of intracellular reactive oxygen species was evaluated using a flow cytometer (Cytomics FC500, manufactured by Beckman-Coulter). The same experiment was conducted 4 times, and the average value and the standard deviation value were obtained. The results are shown in Fig. 23. Note that the solid-line histogram in Fig. 23A shows the test solution addition group, and the dashed-line histogram shows the control group. Also, the numbers described in Fig. 23A are the relative values of the fluorescence intensity of the test solution addition group with respect to the fluorescence intensity of the control group.

[0214] As shown in Figs. 23A and 23B, the amount of reactive oxygen species at the time point 1 hour after the addition of the test solution was larger than that of the control group. On the other hand, as shown in Fig. 23A, the amount of reactive oxygen species at the time points 3 hours and 24 hours after the addition was equivalent to that of the control group. From this, it was confirmed that the increase in the amount of reactive oxygen species occurs temporarily following the addition of TPN-Au-MM4. The above results suggest the use of TPN-Au-MM4 as a combined agent with radiation in chemoradiotherapy.

Claims

1. An anticancer agent containing a gold complex represented by the following formula (1) or an agent for combined use with radiation in chemoradiotherapy. L １ -S-Au-L ２ (1) (In the formula, L １ is a residue obtained by removing -SH from thioproline, L １ The carboxy group (-COOH) contained in ３ may be substituted by an acetyl group (-C(=O)CH ３ ), an acetoxy group (-OC(=O)CH ２ ), an acetoxymethyl group (-CH ３ OC(=O)CH ｎ H ２ｎ OH (n is 1 to 10)), and may be esterified by pivaloyloxymethyl (POM) or 5-methyl-2-oxo-1,3-dioxol-4-ylmethyl (DMD0). L １ The hydrogen atom bonded to a carbon atom other than the carboxy group in L ２ represents a heterocyclic ring having -SH coordinated to Au represented by the formula (1) and may further have a substituent. The ２ heterocyclic ring in L is selected from the group consisting of a tetrazole ring, a thiadiazole ring, and a triazole ring, or Said L ２ is 2-imidazolidinethione (2NN) or 2-mercaptobenzothiazole (2SN), the Au is a monovalent gold atom.)

2. The anticancer agent or the agent for combined use with radiation in chemoradiotherapy according to Claim 1, wherein the gold complex is water-soluble.

3. Said L ２ is 5-mercapto-1-methyltetrazole (MM4), 2-amino-5-mercapto-1,3,4-thiadiazole (AMT), 1,2,4-triazole-3-thiol (MTZ), 3-amino-5-mercapto-1,2,4-triazole (AMZ), 2-mercapto-1,3,4-thiadiazole (MT), 2-mercapto-5-methyl-1,3,4-thiadiazole (MMT), 2-mercapto-5-methylthio-1,3,4-thiadiazole (MMTT), 2-imidazolidinethione (2NN), or 2-mercaptothiazoline (2SN), and the S of the -SH in the L ２ is coordinated to the Au represented by the formula (1), the anticancer agent according to claim 1 or 2, or a combined agent with radiation in chemoradiotherapy.

4. The anticancer agent or the agent for combined use with radiation in chemoradiotherapy according to any one of Claims 1 to 3, which is an anticancer agent for the treatment of leukemia, liver cancer, or lung cancer.

5. Furthermore, the anticancer agent or the agent for combined use with radiation in chemoradiotherapy according to any one of Claims 1 to 4, which contains a solvent containing at least one selected from the group consisting of water and polyvinyl alcohol.

6. Use of a gold complex represented by the following formula (3) for producing the anticancer agent containing the gold complex or the agent for combined use with radiation in chemoradiotherapy. L １ -S-Au-L ２ (3) (In the formula, L １ is a residue formed by removing -SH from thiopronine, L １ The carboxy group (-COOH) contained in ３ may be substituted by an acetyl group (-C(=O)CH ３ ), an acetoxy group (-OC(=O)CH ２ ), an acetoxymethyl group (-CH ３ OC(=O)CH ｎ H ２ｎ OH (n is 1 to 10)), or may be esterified by pivaloyloxymethyl (POM) or 5-methyl-2-oxo-1,3-dioxol-4-ylmethyl (DMDO). L １ The hydrogen atom bonded to a carbon atom other than the carboxy group in L may be substituted with an alkyl group, an aryl group, an alkoxy group, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, L ２ represents a heterocyclic ring having -SH coordinated to Au represented by the formula (3) and may further have a substituent, and the heterocyclic ring in the L ２ is selected from the group consisting of a tetrazole ring, a thiadiazole ring, and a triazole ring. the Au is a monovalent gold atom.)

7. Use of a gold complex represented by the following formula (3) for producing the anticancer agent containing the gold complex or the agent for combined use with radiation in chemoradiotherapy. L １ -S-Au-L ２ (3) (In the formula, L １ is a residue obtained by removing -SH from thiopronine, L １ The carboxy group (-COOH) contained in ３ may be substituted with an acetyl group (-C(=O)CH ３ ), an acetoxy group (-OC(=O)CH ２ ), an acetoxymethyl group (-CH ３ OC(=O)CH ｎ H ２ｎ OH (n is 1 to 10)), or may be esterified with pivaloyloxymethyl (POM) or 5-methyl-2-oxo-1,3-dioxol-4-ylmethyl (DMD0). L １ The hydrogen atom bonded to a carbon atom other than the carboxy group in L may be substituted by an alkyl group, an aryl group, an alkoxy group, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, L ２ represents a heterocyclic ring having -SH coordinated to Au represented by the formula (3) and may further have a substituent, and the heterocyclic ring in the L ２ is selected from the group consisting of a tetrazole ring, a thiadiazole ring, and a triazole ring, or Said L ２ is 2-imidazolidinethione (2NN) or 2-mercaptobenzothiazole (2SN), the Au is a monovalent gold atom.)

8. The use according to Claim 6 or 7, wherein the gold complex is water-soluble.

9. Said L ２ is 5-mercapto-1-methyltetrazole (MM4), 2-mercapto-5-methylthio-1,3,4-thiadiazole (MMTT), or 2-mercapto-5-methyl-1,3,4-thiadiazole (MMT), and the S of the mercapto group coordinates to the Au represented by the formula (3). The use according to any one of claims 6 to 8.

10. A method for producing an anticancer agent containing a gold complex represented by the following formula (3) or an agent for combined use with radiation in chemoradiotherapy, The method includes coordinating a heterocyclic compound L to a gold complex precursor represented by the following formula (2). ２ A method. L １ -S-Au(2) (In the formula, L １ is a residue formed by removing -SH from thiopronine, L １ The carboxy group (-COOH) contained in ３ may be substituted with an acetyl group (-C(=O)CH ３ ), an acetoxy group (-OC(=O)CH ２ ), an acetoxymethyl group (-CH ３ OC(=O)CH ｎ ), or a hydroxyalkyl group (-C ２ｎ H OH (n is 1 to 10)), and may be esterified with pivaloyloxymethyl (POM) or 5-methyl-2-oxo-1,3-dioxol-4-ylmethyl (DMD0). L １ The hydrogen atom bonded to a carbon atom other than the carboxy group in L may be substituted by an alkyl group, an aryl group, an alkoxy group, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, The complex ring compound L2 is a compound containing a heterocyclic ring having -SH and optionally having a substituent, and the L ２ The heterocyclic ring in is selected from the group consisting of a tetrazole ring, a thiadiazole ring, and a triazole ring, or Said L ２ is 2-imidazolidinethione (2NN) or 2-mercaptothiazoline (2SN), the Au is a monovalent gold atom.) L １ -S-Au-L ２ (3) (wherein, L １ , L ２ , and Au are as described above.)

11. A method for screening an anticancer agent containing a gold complex represented by the following formula (3) or an agent for combined use with radiation in chemoradiotherapy, The method includes a step of coordinating a heterocyclic compound L to a gold complex precursor represented by the following formula (2) to obtain the gold complex, and ２ a step of which includes a step of evaluating the cell growth inhibitory ability of the gold complex The method. L １ -S-Au(2) (In the formula, L １ is a residue obtained by removing -SH from thiopronine, L １ The carboxy group (-COOH) contained in ３ may be substituted by an acetyl group (-C(=O)CH ３ ), an acetoxy group (-OC(=O)CH ２ ), an acetoxymethyl group (-CH ３ OC(=O)CH ｎ H ２ｎ OH (n is 1 to 10)), or may be esterified by pivaloyloxymethyl (POM) or 5-methyl-2-oxo-1,3-dioxol-4-ylmethyl (DMD0). L １ The hydrogen atom bonded to a carbon atom other than the carboxy group in L may be substituted by an alkyl group, an aryl group, an alkoxy group, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, The complex ring compound L2 is a compound containing a heterocyclic ring having -SH and optionally having a substituent, and the L ２ in the heterocyclic ring is selected from the group consisting of a tetrazole ring, a thiadiazole ring, and a triazole ring, or Said L ２ is 2-imidazolidinethione (2NN) or 2-mercaptothiazoline (2SN), the Au is a monovalent gold atom.) L １ -S-Au-L ２ (3) (wherein L １ , L ２ , and Au are as described above.)

12. A gold complex represented by the following formula (3). L １ -S-Au-L ２ (3) (In the formula, L １ is a residue formed by removing -SH from thioproline, L ２ represents a heterocyclic ring having -SH coordinated to Au represented by the formula (3) and may further have a substituent. Said L ２ is 5-mercapto-1-methyltetrazole (MM4), 2-mercapto-5-methyl-1,3,4-thiadiazole (MMT), 2-imidazolidinethione (2NN), or 2-mercaptobenzothiazoline (2SN), and the S of the -SH in the L ２ coordinates to the Au represented by the formula (3), the Au is a monovalent gold atom.)

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