JP2016008179A - 4h-chromone derivative, method of producing the same and cancer cell detecting method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescence probe that detects a cancer cell in coexistence with a normal cell.SOLUTION: The problem is solved by a 4H-chromone derivative represented by general formula (1).

Description

  The present invention relates to 4H-chromone derivatives useful as a fluorescent probe for cancer cells, a method for producing them, and a method for detecting cancer cells using them.

A highly accurate method for detecting cancer cells mixed in normal cells or normal tissues is an important technique in the early detection and treatment of cancer. Various detection methods for cancer cells have been developed so far, and the detection method using a fluorescent probe is one of highly sensitive detection methods. It has been reported that glutathione-S-transferase (hereinafter referred to as GST) is highly expressed in cancer cells. (Non-Patent Document 1)
Examples of cancer cell lines expressing GST include mouse breast cancer-derived FM3A cells (Non-patent Document 3), human lung adenocarcinoma-derived non-small cell lung cancer PC-9 cells (Non-patent Document 4), and human mammary gland. There are SK-BR-3 cells derived from cancer (Non-patent Document 5).

  By detecting fluorescence of GST with high sensitivity, it is possible to detect cancer cells mixed in normal cells or normal tissues with high accuracy. GST is a detoxifying enzyme that detoxifies foreign substances that have entered cells, and catalyzes a reaction that forms glutathione-foreign substance conjugates from glutathione (hereinafter referred to as GSH) and foreign substances. In particular, when the foreign substance is an electrophilic arylsulfonyl compound, it is known that a conjugate is formed by GSH and an aromatic nucleophilic substitution reaction. A method is disclosed in which an arylsulfonyl compound is introduced into a fluorescent dye by using this reaction, and the arylsulfonyl compound is removed by an aromatic nucleophilic substitution reaction with GSH to detect fluorescence of cells. (Patent Document 1, Non-Patent Document 2).

  However, an arylsulfonyl compound may proceed with an aromatic nucleophilic substitution reaction in an enzyme-independent manner with GSH or other intracellular thiol compounds (for example, cysteine) (Non-Patent Document 2), and in the presence of normal cells. The cancer cell detection method has a problem.

WO2012 / 039499 WO2011 / 149032

Molecular Pharmacology, 1996, 50, 149-159. Organic Letters, 2008, 10, pp. 37-40 Journal of Toxicology Pathology, 2012, 25, 225-228 Japan Journal of Cancer Research, 1988, 79, 301-304. Cancer Research, 2001, 61, 5168-5178 Cancer Research, 1988, 48, 527-537 The Journal of Biological Chemistry, 1974, 249, 7130-7139

  An object of the present invention is to provide a fluorescent probe for detecting cancer cells. In particular, it is an object of the present invention to provide a fluorescent probe for detecting cancer cells in the presence of normal cells.

  As a result of intensive studies to solve the above problems, the present inventors have found that cancer cells can be detected with fluorescence in the presence of normal cells when the 4H-chromone derivative of the present invention is used, and the present invention has been completed. .

That is, the present invention
[1] General formula (1)

(In the formula, R ² and R ³ each independently represent an alkyl group having 1 to 3 carbon atoms. R ^4-1 , R ^4-2 , R ^4-3 , R ^4-4 , R ^5-1 , R ^5-2 and R ^5-3 each independently represent a hydrogen atom, a halogen atom or a methoxy group, R ⁶ represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an aralkyl group having 7 or 8 carbon atoms, or a benzoyl group R ⁷ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, R ⁸ , R ⁹ and R ¹⁰ each independently represents a hydrogen atom, a nitro group or a trifluoromethyl group, provided that R ⁸ , At least one of R ⁹ and R ¹⁰ represents a nitro group, n represents 0 or 1, m represents 0 or 1, and k represents an integer of 1 to 12). Relates to derivatives.
The present invention also relates to [2] 4H-chromone derivative according to [1], wherein R ⁸ is a nitro group or trifluoromethyl group, R ⁹ is a nitro group, R ¹⁰ is a hydrogen atom, and k is an integer of 1 to 6. .
In the present invention, [3] R ² and R ³ are a methyl group, R ^4-1 , R ^4-2 , R ^4-3 , R ^4-4 , R ^5-1 , R ^5-2 and R ^5-3. Relates to the 4H-chromone derivative according to [1] or [2], wherein is a hydrogen atom.

  The present invention also provides [4] general formula (2).

(In the formula, R ² and R ³ each independently represent an alkyl group having 1 to 3 carbon atoms. R ^4-1 , R ^4-2 , R ^4-3 , R ^4-4 , R ^5-1 , R ^5-2 and R ^5-3 each independently represent a hydrogen atom, a halogen atom or a methoxy group, R ⁶ represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an aralkyl group having 7 or 8 carbon atoms, or a benzoyl group N represents 0 or 1, m represents 0 or 1, k represents an integer of 1 to 12, and an amine derivative represented by the general formula (3) is present in the presence of a base.

(Wherein R ⁸ , R ⁹ and R ¹⁰ each independently represents a hydrogen atom, a nitro group or a trifluoromethyl group, provided that at least one of R ⁸ , R ⁹ and R ¹⁰ represents a nitro group. X represents a halogen atom) and is reacted with an arylsulfonyl compound represented by the general formula (1a)

^{(Wherein, R 2, R 3, R} 4-1, R 4-2, R 4-3, R 4-4, R 5-1, R 5-2, R 5-3, R 6, R 8 , R ⁹ , R ¹⁰ , n, m, and k represent the same meaning as described above).

  The present invention also provides [5] general formula (1).

(In the formula, R ² and R ³ each independently represent an alkyl group having 1 to 3 carbon atoms. R ^4-1 , R ^4-2 , R ^4-3 , R ^4-4 , R ^5-1 , R ^5-2 and R ^5-3 each independently represent a hydrogen atom, a halogen atom or a methoxy group, R ⁶ represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an aralkyl group having 7 or 8 carbon atoms, or a benzoyl group R ⁷ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, R ⁸ , R ⁹ and R ¹⁰ each independently represents a hydrogen atom, a nitro group or a trifluoromethyl group, provided that R ⁸ , At least one of R ⁹ and R ¹⁰ represents a nitro group, n represents 0 or 1, m represents 0 or 1, and k represents an integer of 1 to 12). The present invention relates to a fluorescent probe comprising a derivative.

  The present invention also provides [6] general formula (1).

(In the formula, R ² and R ³ each independently represent an alkyl group having 1 to 3 carbon atoms. R ^4-1 , R ^4-2 , R ^4-3 , R ^4-4 , R ^5-1 , R ^5-2 and R ^5-3 each independently represent a hydrogen atom, a halogen atom or a methoxy group, R ⁶ represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an aralkyl group having 7 or 8 carbon atoms, or a benzoyl group R ⁷ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, R ⁸ , R ⁹ and R ¹⁰ each independently represents a hydrogen atom, a nitro group or a trifluoromethyl group, provided that R ⁸ , At least one of R ⁹ and R ¹⁰ represents a nitro group, n represents 0 or 1, m represents 0 or 1, and k represents an integer of 1 to 12). Glutathione, characterized by using a derivative The present invention relates to a method for detecting S-transferase.

  The present invention also provides [7] general formula (1)

(In the formula, R ² and R ³ each independently represent an alkyl group having 1 to 3 carbon atoms. R ^4-1 , R ^4-2 , R ^4-3 , R ^4-4 , R ^5-1 , R ^5-2 and R ^5-3 each independently represent a hydrogen atom, a halogen atom or a methoxy group, R ⁶ represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an aralkyl group having 7 or 8 carbon atoms, or a benzoyl group R ⁷ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, R ⁸ , R ⁹ and R ¹⁰ each independently represents a hydrogen atom, a nitro group or a trifluoromethyl group, provided that R ⁸ , At least one of R ⁹ and R ¹⁰ represents a nitro group, n represents 0 or 1, m represents 0 or 1, and k represents an integer of 1 to 12). Method for detecting cancer cells, characterized by using a derivative It is about the law.

  The 4H-chromone derivative of the present invention is useful as a fluorescent probe for detecting cancer cells because the cancer cells are fluorescently stained in the presence of normal cells and the cancer cells exhibit strong luminescence.

FIG. 10 shows the results of Example 9. FIG. 10 shows the results of Example 9. FIG. 10 shows the results of Example 9. FIG. 10 shows the results of Example 9. It is the figure which showed the result of Example 10 and Comparative Examples 1-2. It is the figure which showed the result of Example 11 and Comparative Example 3. It is the figure which showed the result of Example 11 and Comparative Example 3. It is the figure which showed the result of Example 12. It is the figure which showed the result of Example 12. It is a schematic diagram of a cytodiagnosis chip. It is a schematic diagram of a cytodiagnosis chip. It is the figure which showed the result of Example 13. It is the figure which showed the result of Example 14.

The present invention is described in detail below. ^{Herein, R 2, R 3, R} 4-1, R 4-2, R 4-3, R 4-4, R 5-1, R 5-2, R 5-3, R 6, R ⁷ , definitions of R ⁸ , R ⁹ , R ¹⁰ , X and k will be described.
The alkyl group having 1 to 3 carbon atoms represented by R ² and R ³ may be any of a linear, branched or cyclic alkyl group, and is a methyl group, an ethyl group, a propyl group or a cyclopropyl group. And an isopropyl group. In terms of strong fluorescence intensity, methyl groups are preferred as R ² and R ³ .

Examples of the halogen atom represented by R ^4-1 , R ^4-2 , R ^4-3 and R ^4-4 include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom. In terms of strong fluorescence intensity, R ^4-1 , R ^4-2 , R ^4-3 and R ^4-4 are preferably fluorine atoms.

R ^4-1 , R ^4-2 , R ^4-3 and R ^4-4 are preferably hydrogen atoms in terms of strong fluorescence intensity.

R ^5-1, examples of the halogen atom represented by ^{R 5-2} and ^{R 5-3,} can be exemplified fluorine atom, chlorine atom, bromine atom and iodine atom.

As R ^<5-1> , R ^<5-2> and R ^<5-3> , a hydrogen atom is preferable in terms of strong fluorescence intensity.

The alkyl group having 1 to 4 carbon atoms represented by R ⁶ may be any of linear, branched and cyclic alkyl groups, and includes a methyl group, an ethyl group, a propyl group, a butyl group, an isopropyl group, Examples thereof include a cyclopropyl group, an isobutyl group, a sec-butyl group and a tert-butyl group.

Examples of the aralkyl group having 7 or 8 carbon atoms represented by R ⁶ include a benzyl group and a phenethyl group. In view of strong fluorescence intensity, R ⁶ is preferably a hydrogen atom or a benzyl group.

The alkyl group having 1 to 3 carbon atoms represented by R ⁷ may be linear or branched, and examples thereof include a methyl group, an ethyl group, a propyl group, and an isopropyl group. In terms of strong fluorescence intensity, R ⁷ is preferably a hydrogen atom.

As R ⁸ , R ⁹ and R ^10, it is preferable that R ⁸ is a nitro group or a trifluoromethyl group, R ⁹ is a nitro group, and R ¹⁰ is a hydrogen atom in terms of high detection sensitivity.

  Examples of the halogen atom represented by X include an iodine atom, a bromine atom, and a chlorine atom. Among them, a chlorine atom is desirable in terms of a good yield. Further, k is preferably an integer of 1 to 6 in view of high fluorescence intensity.

  Furthermore, preferred examples of the 4H-chromone derivative (1) of the present invention include (E) -2- [4- (dimethylamino) styryl] -7- [2-[[(2,4-dinitrophenyl) sulfonyl]. Amino] ethoxy] -3-hydroxy-4H-chromen-4-one, (E) -3-benzyloxy-2- [4- (dimethylamino) styryl] -7- [2-[[(2,4- Dinitrophenyl) sulfonyl] amino] ethoxy] -4H-chromen-4-one, (E) -2- [4- (dimethylamino) styryl] -8- [2-[[(2,4-dinitrophenyl) sulfonyl ] Amino] ethoxy] -3-hydroxy-4H-benzo [g] chromen-4-one, 2- [4- (dimethylamino) phenyl] -8- [2-[[(2,4-dinitrophenyl) sulfonyl] ] Ami ] Ethoxy] -3-hydroxy-4H-benzo [g] chromen-4-one, 3-benzyloxy-2- [4- (dimethylamino) phenyl] -8- [2-[[(2,4-dinitro Phenyl) sulfonyl] amino] ethoxy] -4H-benzo [g] chromen-4-one, (E) -2- [4- (dimethylamino) styryl] -7- [2-[[(2,4-dinitro Phenyl) sulfonyl] amino] butoxy] -3-hydroxy-4H-chromen-4-one, (E) -3-benzyloxy-2- [4- (dimethylamino) styryl] -7- [2-[[[ 4-nitro-2- (trifluoromethyl) phenyl] sulfonyl] amino] ethoxy] -4H-chromen-4-one, (E) -3-benzyloxy-2- [4- (dimethylamino) styryl] -7 The - [2 [[(2,4-dinitrophenyl) sulphonyl] methylamino] ethoxy] -4H- chromen-4-one, can be mentioned.

  Next, the production method of the present invention will be described in detail. The 4H-chromone derivative (1) of the present invention can be produced by the following scheme.

^{(Wherein, R 2, R 3, R} 4-1, R 4-2, R 4-3, R 4-4, R 5-1, R 5-2, R 5-3, R 6, R 8 , R ⁹ , R ¹⁰ , X, n, m and k are as defined above, R ^6a represents an alkyl group having 1 to 4 carbon atoms, an aralkyl group having 7 or 8 carbon atoms, or a benzoyl group; ^7a represents an alkyl group having 1 to 3 carbon atoms, Y represents a halogen atom, and Z represents a halogen atom.)
The alkyl group having 1 to 4 carbon atoms represented by R ^6a may be any of linear, branched and cyclic alkyl groups, and includes a methyl group, an ethyl group, a propyl group, a butyl group, an isopropyl group, Examples thereof include a cyclopropyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group.

Examples of the aralkyl group having 7 or 8 carbon atoms represented by R ^6a include a benzyl group and a phenethyl group.

The alkyl group having 1 to 3 carbon atoms represented by R ^7a may be linear or branched, and examples thereof include a methyl group, an ethyl group, a propyl group, and an isopropyl group.

  Examples of the halogen atom represented by Y include an iodine atom and a bromine atom.

  Examples of the halogen atom represented by Z include an iodine atom, a bromine atom, and a chlorine atom.

(1) Step-1 Step-1 is a step of producing an enone derivative (6) by reacting the hydroxyacetophenone derivative (4) with an aldehyde derivative (5) in the presence of a base.

  The hydroxyacetophenone derivative (4) which is a raw material of this step can be prepared with reference to a method described in the literature (Bioorganic Medicinal Chemistry, 16, 7358-7370 (2008)).

  The aldehyde derivative (5) which is the raw material of this step is a method described in the literature (Journal of the American Chemical Society, 133, 6642-6649 (2011), Organic Letters, 5, 777-780 (2003), Synthetic Communications, 18). , 1641-1650 (1988)), and can be prepared from the corresponding 4-aminobenzaldehyde or 4-aminohalobenzene.

  It is essential to carry out the reaction of Step-1 in the presence of a base. Examples of the base include alkali metal alkoxides such as sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, potassium t-butoxide, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, sodium carbonate, potassium carbonate And alkali metal carbonates such as cesium carbonate and sodium hydrogen carbonate. Of these, sodium methoxide and sodium hydroxide are preferred as the base from the viewpoint of good yield.

  The reaction in step-1 may be performed in a solvent as long as it does not inhibit the reaction. Specific examples of solvents that can be used in this step include ethers such as tetrahydrofuran, diethyl ether, 1,4-dioxane, and 1,2-dimethoxyethane, hydrocarbons such as hexane, pentane, and cyclohexane, benzene, toluene, Aromatic hydrocarbons such as xylene, halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, alcohols such as acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, methanol, ethanol Water and the like can be exemplified, and two or more of these solvents may be mixed and used. Of these, dimethylformamide and ethanol are preferred because of their good yield.

  There is no particular limitation on the molar ratio of the hydroxyacetophenone derivative (4) and the aldehyde derivative (5), but a range of 1: 1 to 1:10 is preferable, and 1: 1 to 1: 3 is preferable in terms of good yield. Further preferred. The molar ratio of the hydroxyacetophenone derivative (4) and the base is not particularly limited, but is preferably in the range of 1: 1 to 1:10, and more preferably 1: 1 to 1: 3 in terms of good yield.

  The reaction temperature in step-1 can be carried out at a temperature appropriately selected from the range of -78 ° C to 150 ° C. Among them, the range from room temperature to 120 ° C. is preferable in terms of a good yield.

  The enone derivative (6) obtained by the reaction in Step-1 can be purified from the reaction solution after completion of the reaction, if necessary. The purification method is not particularly limited, but the target product may be purified by a general method such as solvent extraction, silica gel column chromatography, thin layer preparative chromatography, preparative liquid chromatography, recrystallization or sublimation. it can.

(2) About Step-2 Step-2 is a step of oxidizing the enone derivative (6) to produce a 3-hydroxy-4H-chromone derivative (7). The oxidation in step-2 is preferably performed in the presence of an oxidizing agent. Examples of the oxidizing agent that can be used include hydrogen peroxide, tert-butyl hydroperoxide, 3-chloroperbenzoic acid, and perbenzoic acid. Hydrogen peroxide is preferred because of its good yield.

  Moreover, a yield can be improved by carrying out in presence of a base. Examples of the base include sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, alkali metal alkoxides such as potassium t-butoxide, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, sodium carbonate, Examples thereof include alkali metal carbonates such as potassium carbonate, cesium carbonate and sodium hydrogen carbonate. Of these, sodium hydroxide and potassium hydroxide are preferred as the base in terms of good yield.

  The reaction of step-2 may be performed in a solvent as long as it does not inhibit the reaction. Specific examples of solvents that can be used in this step include ethers such as tetrahydrofuran, diethyl ether, 1,4-dioxane, and 1,2-dimethoxyethane, hydrocarbons such as hexane, pentane, and cyclohexane, benzene, toluene, Aromatic hydrocarbons such as xylene, halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, alcohols such as acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, methanol, ethanol Water and the like can be exemplified, and two or more of these solvents may be mixed and used. Of these, ethanol is preferred because of its good yield.

  The molar ratio of the enone derivative (6) and the oxidizing agent is not particularly limited, but is preferably in the range of 1: 1 to 1:30, and more preferably 1:10 to 1:15 in terms of good yield. The molar ratio of the enone derivative (6) and the base is not particularly limited, but is preferably in the range of 1: 1 to 1:30, and more preferably 1:10 to 1:15 in terms of good yield.

  The reaction temperature in step-2 can be carried out at a temperature appropriately selected from the range of -78 ° C to 150 ° C. Among them, the range from room temperature to 120 ° C. is preferable in terms of a good yield.

  The 3-hydroxy-4H-chromone derivative (7) obtained by the reaction in Step-2 can be purified from the reaction solution after the reaction, if necessary. The purification method is not particularly limited, but the target product may be purified by a general method such as solvent extraction, silica gel column chromatography, thin layer preparative chromatography, preparative liquid chromatography, recrystallization or sublimation. it can.

(3) About Step-3 Step-3 is a step of producing a Boc-protected amine (8) by reacting the 3-hydroxy-4H-chromone derivative (7) with a halide (9) in the presence of a base.

  A part of the halide (9) which is a raw material for this step is commercially available, but the method described in the literature (Synlett, 2012, 23, 2692-2698, Tetrahedron, 2002, 58, 8689-8893). Page, Organic Letters, 2001, Vol. 3, pages 3727-3728).

  It is essential to carry out the reaction of Step-3 in the presence of a base. Examples of the base include alkali metal hydrides such as sodium hydride and potassium hydride, alkali metal alkoxides such as sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide and potassium t-butoxide, sodium hydroxide, water Examples include alkali metal hydroxides such as potassium oxide, and alkali metal carbonates such as sodium carbonate, potassium carbonate, cesium carbonate, and sodium bicarbonate. Of these, potassium carbonate is preferred as the base because of its good yield.

  The reaction of step-3 may be performed in a solvent as long as it does not inhibit the reaction. Specific examples of solvents that can be used in this step include ethers such as tetrahydrofuran, diethyl ether, 1,4-dioxane, and 1,2-dimethoxyethane, hydrocarbons such as hexane, pentane, and cyclohexane, benzene, toluene, Aromatic hydrocarbons such as xylene, halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, alcohols such as acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, methanol, ethanol Water and the like can be exemplified, and two or more of these solvents may be mixed and used. Of these, dimethylformamide is preferred because of its good yield.

  The molar ratio of the 3-hydroxy-4H-chromone derivative (7) and the halide (9) is not particularly limited, but is preferably in the range of 1: 1 to 1:10, and 1: 1 in terms of good yield. To 1: 3 is more preferred. The molar ratio of the 3-hydroxy-4H-chromone derivative (7) and the base is not particularly limited, but is preferably in the range of 1: 1 to 1:10, and in particular, 1: 3 to 1: 5 in terms of good yield. Is more preferable.

  The reaction temperature in step-3 can be carried out at a temperature appropriately selected from the range of -78 ° C to 150 ° C. Among these, the range from room temperature to 100 ° C. is preferable from the viewpoint of good yield.

  The Boc-protected amine (8) obtained by the reaction in Step-3 can be purified from the reaction solution after completion of the reaction, if necessary. The purification method is not particularly limited, but the target product may be purified by a general method such as solvent extraction, silica gel column chromatography, thin layer preparative chromatography, preparative liquid chromatography, recrystallization or sublimation. it can.

(4) Step-4 The reaction of Step-4 is a step of producing an amine derivative (2) by acid-treating the 3-hydroxy-4H-chromone derivative (7) or the Boc-protected amine (8). .

  Examples of the acid that can be used in the reaction of Step-4 include Bronsted acids such as hydrochloric acid, sulfuric acid, trifluoroacetic acid, tosylic acid, and mesylic acid.

  The reaction of step-5 may be performed in a solvent as long as it does not inhibit the reaction. Specific examples of solvents that can be used in this step include ethers such as tetrahydrofuran, diethyl ether, 1,4-dioxane, and 1,2-dimethoxyethane, hydrocarbons such as hexane, pentane, and cyclohexane, benzene, toluene, Aromatic hydrocarbons such as xylene, halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, alcohols such as acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, methanol, ethanol Water and the like can be exemplified, and two or more of these solvents may be mixed and used.

  The molar ratio of the 3-hydroxy-4H-chromone derivative (7) or Boc-protected amine (8) and the acid is not particularly limited, but is preferably in the range of 100: 1 to 1:10, and in particular, the yield is good. More preferred is 20: 1 to 1: 1.

  The reaction temperature in step 4 can be carried out at a temperature appropriately selected from the range of -78 ° C to 150 ° C. Among these, the range from room temperature to 100 ° C. is preferable from the viewpoint of good yield.

  The amine derivative (2) obtained by the reaction in Step-4 can be purified from the reaction solution after completion of the reaction, if necessary. The purification method is not particularly limited, but the target product may be purified by a general method such as solvent extraction, silica gel column chromatography, thin layer preparative chromatography, preparative liquid chromatography, recrystallization or sublimation. it can.

(5) About Step-5 The reaction of Step-5 is a step of producing the 4H-chromone derivative (1a) by reacting the amine derivative (2) and the arylsulfonyl compound (3) in the presence of a base.

  The arylsulfonyl compound (3), which is a raw material for the production method of the present invention, is partly commercially available, but is prepared with reference to a method described in the literature (Angewandte Chemie International Edition, 2009, Vol. 48, pages 7591-7594). can do.

  It is essential to carry out the reaction of Step-5 in the presence of a base. Examples of the base include tertiary aliphatic amines such as triethylamine, diisopropylethylamine, triisopropylamine and N-methylmorpholine, aromatic amines such as pyridine, picoline and 4-dimethylaminopyridine, sodium carbonate, potassium carbonate, carbonate Examples thereof include alkali metal carbonates such as cesium and sodium hydrogen carbonate. Of these, diisopropylethylamine is preferred as the base because of its good yield.

  The reaction of step-5 may be performed in a solvent as long as it does not inhibit the reaction. Specific examples of solvents that can be used in this step include ethers such as tetrahydrofuran, diethyl ether, 1,4-dioxane, and 1,2-dimethoxyethane, hydrocarbons such as hexane, pentane, and cyclohexane, benzene, toluene, Aromatic hydrocarbons such as xylene, halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, alcohols such as acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, methanol, ethanol Water and the like can be exemplified, and two or more of these solvents may be mixed and used. Of these, dimethylformamide is preferred because of its good yield.

  There is no particular limitation on the molar ratio of the amine derivative (2) and the arylsulfonyl compound (3), but a range of 1: 1 to 1:10 is preferable, and 1: 1 to 1: 3 is particularly preferable in terms of a good yield. Further preferred. The molar ratio of the amine derivative (2) and the base is not particularly limited, but is preferably in the range of 1: 1 to 1:10, and more preferably 1: 1 to 1: 3 in terms of good yield.

  The reaction temperature in Step-5 can be carried out at a temperature appropriately selected from the range of -78 ° C to 150 ° C. Among them, the range from room temperature to 50 ° C. is preferable in terms of a good yield.

  The 4H-chromone derivative (1a) obtained in step-5 can be purified from the reaction solution after completion of the reaction, if necessary. The purification method is not particularly limited, but the target product may be purified by a general method such as solvent extraction, silica gel column chromatography, thin layer preparative chromatography, preparative liquid chromatography, recrystallization or sublimation. it can.

(6) About Step-6 Step-6 is a step of producing 4H-chromone derivative (1b) by reacting 4H-chromone derivative (1a) with alkyl halide (10) in the presence of a base.

  Alkyl halide (10), which is a raw material for this step, is partially commercially available, but should be prepared with reference to a method described in the literature (The Journal of Organic Chemistry, 1990, 55, 2927-2938). Can do.

  It is essential to carry out the reaction of Step-6 in the presence of a base. Examples of the base include alkali metal hydrides such as sodium hydride and potassium hydride, alkali metal alkoxides such as sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide and potassium t-butoxide, sodium hydroxide, water Examples include alkali metal hydroxides such as potassium oxide, and alkali metal carbonates such as sodium carbonate, potassium carbonate, cesium carbonate, and sodium bicarbonate. Of these, potassium carbonate is preferred as the base because of its good yield.

  The reaction in Step-6 may be performed in a solvent as long as it does not inhibit the reaction. Specific examples of solvents that can be used in this step include ethers such as tetrahydrofuran, diethyl ether, 1,4-dioxane, and 1,2-dimethoxyethane, hydrocarbons such as hexane, pentane, and cyclohexane, benzene, toluene, Aromatic hydrocarbons such as xylene, halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, alcohols such as acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, methanol, ethanol Water and the like can be exemplified, and two or more of these solvents may be mixed and used. Of these, dimethylformamide is preferred because of its good yield.

  The molar ratio of the 4H-chromone derivative (1a) to the alkyl halide (10) is not particularly limited, but is preferably in the range of 1: 1 to 1:10, and 1: 1 to 1: 3 is more preferable. The molar ratio between the 4H-chromone derivative (1a) and the base is not particularly limited, but is preferably in the range of 1: 1 to 1:10, and more preferably 1: 3 to 1: 5 in terms of good yield.

  The reaction temperature in Step-6 can be carried out at a temperature suitably selected from the range of -78 ° C to 150 ° C. Among these, the range from room temperature to 100 ° C. is preferable from the viewpoint of good yield.

  The 4H-chromone derivative (1b) obtained by the reaction in Step-6 can be purified from the reaction solution after completion of the reaction, if necessary. The purification method is not particularly limited, but the target product may be purified by a general method such as solvent extraction, silica gel column chromatography, thin layer preparative chromatography, preparative liquid chromatography, recrystallization or sublimation. it can.

  Next, a fluorescent probe comprising the 4H-chromone derivative (1) of the present invention (hereinafter referred to as the fluorescent probe of the present invention) will be described.

  A fluorescent probe is a substance that specifically binds or distributes to a specific protein, cell, or tissue and emits fluorescence, thereby facilitating observation of the specific protein, cell, or tissue. In general, the term “fluorescent probe” refers to a substance that emits fluorescence, but in the present invention, a precursor that does not emit fluorescence itself but becomes fluorescent by a conversion reaction or the like is also included in the fluorescent probe. Shall.

  Although the fluorescent probe of the present invention does not emit fluorescence by itself, it reacts with GSH by the catalytic action of GST expressed in specific cells such as cancer cells and is converted into an amine derivative (11) that emits fluorescence. The

^{(Wherein, R 2, R 3, R} 4-1, R 4-2, R 4-3, R 4-4, R 5-1, R 5-2, R 5-3, R 6, R 7 , N, m and k have the same meaning as described above.)
The amine derivative (11) has a property of emitting extremely strong fluorescence (for example, fluorescence at 600 nm) when irradiated with predetermined excitation light, for example, excitation light of about 400 to 500 nm. Even when the same level of excitation light is irradiated, it hardly emits fluorescence. The upper limit of the excitation light range for the amine derivative (11) is preferably 500 nm or less, particularly preferably 495 nm or less from the viewpoint of avoiding overlap with the fluorescence wavelength, and the lower limit of the excitation light range is a viewpoint of excitation capability. To 400 nm or more is preferable. From the viewpoint of fluorescence observation, the excitation light is preferably light having a single wavelength obtained through an excitation filter that transmits only a desired wavelength, but a light source having a spectrum having a peak at 400 to 495 nm should be used. You can also.

  In the presence of nucleic acids and proteins such as DNA and RNA, the amine derivative (11) interacts with nucleic acids and proteins such as DNA and RNA and emits strong fluorescence. The amine derivative (11) may change the wavelength of fluorescence depending on the nucleic acid or protein such as DNA or RNA to be bound, but usually emits fluorescence of about 500 to 700 nm, preferably about 510 to 650 nm. Is observed.

  GST is usually highly expressed as a cytoplasmic enzyme, particularly in cancer cells. The fluorescent probe that has permeated the cell membrane of the cancer cell and diffused into the cell reacts with GSH by the catalytic action of GST in the cytoplasm, and is converted to an amine derivative (11), and then a nucleic acid present in the cytoplasm, It interacts with proteins and cancer cells emit strong fluorescence.

  Since the fluorescent probe of the present invention does not emit fluorescence per se, but is converted to an amine derivative (11) that reacts with GSH by the catalytic action of GST and emits strong fluorescence, GST is measured by measuring the change in fluorescence intensity. Can be detected.

  Next, a GST detection method using the fluorescent probe of the present invention will be described. The GST detection method using the fluorescent probe of the present invention includes (a) a step of reacting the fluorescent probe with GST in the presence of GSH; and (b) a step of measuring the luminescence intensity in step (a).

  The type of GST and its origin are not particularly limited, but those belonging to π, α, and μ are preferred as subtypes.

  The solvent used in the step of allowing the fluorescent probe of the present invention to react with GST in the presence of GSH is not particularly limited as long as GST activity is not reduced or deactivated, but a buffer is preferably used. Examples of buffers that can be used include HEPES buffer, Tris-HCl buffer, and phosphate buffer. Furthermore, an organic solvent miscible with the buffer may be added. Examples of the organic solvent include dimethyl sulfoxide, N, N-formamide, N, N-dimethylacetamide, methanol, ethanol, ethylene glycol, and glycerin, and two or more of these organic solvents are mixed. It may be used. The proportion of the organic solvent is preferably in the range of 0% to 10% from the viewpoint that GST performs a catalytic reaction stably.

  The temperature of the step of allowing the fluorescent probe of the present invention to react with GST in the presence of GSH is not particularly limited as long as GST activity is not lowered or deactivated, but GST stably performs a catalytic reaction. It is preferably in the range of 5 ° C to 40 ° C, more preferably in the range of 20 ° C to 37 ° C. The order of adding the fluorescent probe of the present invention, GSH, and GST is not particularly limited.

  The step of measuring the emission intensity can be appropriately set by those skilled in the art according to each measurement method, and is not particularly limited, and a conventionally known fluorescence detection device may be used. Preferred examples of the apparatus include a plate reader, a fluorescence spectrometer, and a fluorescence microscope.

  The step of measuring the luminescence intensity may be performed by measuring the step (a) as it is, but it is preferably performed in the presence of a nucleic acid or protein from the viewpoint that the luminescence intensity increases. There are no particular restrictions on the origin of the nucleic acid that can be used, and it may be a polynucleotide extracted from blood, tissue, cells, or the like, or may be a chemically synthesized oligonucleotide. Moreover, there is no restriction | limiting in particular in the origin of the protein which can be used. In view of easy availability, bovine serum albumin and human serum albumin are preferable. The concentration of the nucleic acid or protein to be used is not particularly limited as long as it does not affect the detection, and may be appropriately set in consideration of the fluorescence intensity.

  Next, a method for detecting cancer cells using the fluorescent probe of the present invention will be described.

  The fluorescent probe of the present invention is a probe for detecting cancer cells, and can also be used in cancer detection, determination, or diagnosis methods. The method for detecting, determining, or diagnosing cancer may be a method performed outside the body or a method performed inside the body.

  The fluorescent probe of the present invention can act on a biological sample held in an observation container generally used in a biological imaging technique such as a preparation, a glass bottom dish, a slide glass, and a multiwell plate. The biological sample held in the biological sample fixing device described in 2 can be acted on.

  As the biological sample fixing apparatus as described above, for example, as shown in FIG. 10, a planar insulator in which a spacer is arranged between an upper electrode substrate and a lower electrode substrate and a plurality of micropores are arranged in an array, and Biological sample fixing device having a structure in which a holding portion made of a light shielding member placed between an insulator and a lower electrode substrate is sandwiched between a spacer and a lower electrode substrate (hereinafter sometimes referred to as a “cell diagnostic chip”) Can be used as a container for holding a biological sample. In the example of FIG. 10, an inlet for introducing a biological sample suspension into the holding unit and an outlet for discharging the solution from the holding unit are provided in the spacer, and the biological sample suspension in the holding unit is provided. The liquid can be supplied and discharged quickly. In addition, the light shielding member is disposed so as to cover the entire surface of the lower electrode substrate other than the bottom of the fine hole, and can reduce optical noise around the fine hole. This makes it possible to detect information such as weak fluorescence emitted from a biological sample in the micropore with higher sensitivity. When the fluorescent probe provided by the present invention is used, only cancer cells are fluorescently labeled out of a group of cells in which white blood cells and cancer cells are mixed. And white blood cells can be accurately identified.

  A biological sample suspension such as a cell is introduced into the holding part of the cytodiagnostic chip having the above-described configuration, and the signal generator and the two electrodes are connected to each other by conducting wires, and a predetermined waveform is generated between the two electrodes. By applying an alternating voltage having the signal generator with the signal generator, the electric lines of force concentrate on the micropores, and the biological sample in the suspension receives a force in the direction in which the electric lines of force concentrate, and the micropores in the holding portion Moves in and is easily fixed. The force generated at this time is called dielectrophoretic force.

  The biological sample held in the observation container is brought into contact by adding the fluorescent probe of the present invention or a solution thereof, and after incubation for a predetermined time, the biological sample is irradiated with excitation light by a fluorescence microscope or a microplate reader, The resulting fluorescence can be detected. Cells that emit fluorescence can be determined as cancer cells. In the present invention, from the viewpoint of determining cancer metastasis, for example, the biological sample may be a tissue or blood, and may be a tissue or a cell group obtained from blood. For example, a group of blood cells collected from a patient suffering from metastatic cancer contains a large amount of blood cells and a small number of cancer cells. By using the fluorescent probe of the present invention, Therefore, it becomes possible to detect cancer cells with high accuracy. When cancer cells are present in the blood cells of the subject, it can be determined or diagnosed that the subject suffers from metastatic cancer.

  Alternatively, after incubation, the number of cancer cells and the acquisition of cancer cells can be performed using image acquisition by flow cytometry or fluorescence microscopy, followed by cell sorting using a micropipette. By measuring the number of cancer cells in the blood cells of the subject, it is possible to diagnose the presence of a minute solid cancer that cannot be detected by image diagnosis such as CT. In addition, by measuring the number of cancer cells in the blood cells of a subject at regular intervals, the subject undergoes surgical treatment, local treatment such as radiation therapy, systemic treatment such as chemotherapy, or treatment such as administration of a molecular target drug. It is possible to determine the effect of. In addition, by obtaining cancer cells in the blood cells of a subject, it becomes possible to separately apply an analysis method to the cancer cells, and it is possible to perform property analysis such as gene analysis, expression analysis, and localization analysis.

  Another aspect of the present invention relates to a method for detecting cancer cells or cancer tissues using the fluorescent probe of the present invention. The detection method includes detecting the fluorescence in the cell or tissue by applying the fluorescent probe of the present invention to the cell or tissue and irradiating the applied cell or tissue with predetermined excitation light. For example, such a method is a method for detecting cancer cells in the blood, in which case the cells in the blood may be fixed to an observation container such as a preparation or fixed by the biological sample fixing device described above. It may be a diagnostic chip. On the other hand, such a detection method can be applied to a living tissue and used for a definitive diagnosis of cancer tissue at the time of surgery. As an application method in that case, it may be applied by spraying or applying to cells or tissues, or may be applied by injection or infusion.

  Still another embodiment of the present invention may relate to a surgical / treatment method including a step of determining a cancer excision region using the cancer cell or cancer tissue detection method of the present invention and excising the region.

  The cancer cell detection method of the present invention can be used for determination of metastatic cancer. That is, when fluorescence higher than the fluorescence serving as an index is detected by the method for detecting cancer cells of the present invention, it can be determined that metastatic cancer exists. Here, the determination method refers to a method of specifying without including a determination by a medical worker including a doctor by comparing with an index. For example, a fluorescent cell can be selected as the fluorescence serving as an index. In this case, when a fluorescent cell is detected, it can be determined that the subject has metastatic cancer. In another aspect, the severity of metastatic cancer can be determined by using the fluorescence intensity or the number of cells having fluorescence as an index.

  As used herein, the term “cancerous tissue” means any tissue containing cancer cells. The term “tissue” can be interpreted in the broadest sense, including a part or the whole of an organ, and should not be limitedly interpreted in any way. Since the fluorescent probe of the present invention has an action of detecting GST that is specifically strongly expressed in cancer tissues, among cancer tissues, tissues that highly express GST are preferable. Cancer tissues are described in, for example, Cancer Research, 48, pp. 527-537, 1988 (Non-Patent Document 6). Examples of cancer include head and neck cancer, pharyngeal cancer, stomach cancer, lung cancer, breast cancer, colon cancer, renal cell cancer, liver cancer, gallbladder cancer, pancreatic cancer, endometrial cancer, ovarian cancer, mesothelioma, skin cancer, melanoma, In addition to typical primary cancers such as bone cancer, lymph tissues such as lymph nodes and tissues of metastasized cancer to surrounding tissues may be mentioned, but are not intended to be limited to these cancer types.

  EXAMPLES Next, although an Example and a reference example demonstrate this invention further in detail, this invention is not limited to these.

  Reference example 1

4 '-[2- (tert-Butoxycarbonylamino) ethoxy] -2'-hydroxyacetophenone (248 mg, 0.84 mmol) and (E) -3- [4- (dimethylamino) phenyl] -2-propenal ( 147 mg, 0.84 mmol) was dissolved in N, N-dimethylformamide (1 mL), sodium methoxide (136 mg, 2.5 mmol) was added, and the mixture was stirred at room temperature for 30 minutes and at 80 ° C. for 12 hours. After completion of the reaction, 1M hydrochloric acid was added to the reaction mixture to adjust the liquidity to pH 7, followed by extraction with ethyl acetate (50 mL × 3). The organic layer was washed with water (25 mL) and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography [in order of 14% ethyl acetate / hexane, 50% ethyl acetate / hexane] to give (2E, 4E) -5- [4- (dimethylamino) phenyl] -1 -[2-Hydroxy-4- [2- (tert-butoxycarbonylamino) ethoxy] phenyl] penta-2,4-dien-1-one (167 mg, 44% yield) was obtained as a red solid, and Reference Example Used for 2. ¹ H-NMR (400 MHz, CDCl ₃ ): δ 13.67 (s, 1H), 7.76 (d, J = 8.7 Hz, 1H), 7.71 (dd, J = 11.0, 14.5 Hz) , 1H), 7.41 (d, J = 8.9 Hz, 2H), 7.03 (d, J = 14.5 Hz, 1H), 6.99 (d, J = 15.2 Hz, 1H), 6 .86 (dd, J = 11.0, 15.2 Hz, 1H), 6.69 (d, J = 8.9 Hz, 2H), 6.45 (dd, J = 2.5, 8.7 Hz, 1H) ), 6.43 (d, J = 2.5 Hz, 1H), 4.97 (brs, 1H), 4.06 (t, J = 5.0 Hz, 2H), 3.55 (m, 2H), 3.03 (s, 6H), 1.46 (s, 9H).
Reference example 2

(2E, 4E) -5- [4- (Dimethylamino) phenyl] -1- [2-hydroxy-4- [2- (tert-butoxycarbonylamino) ethoxy] phenyl] penta- obtained in Reference Example 1 2,4-Dien-1-one (167 mg, 0.37 mmol) was suspended in ethanol (2 mL), 4M aqueous sodium hydroxide solution (0.92 mL, 3.7 mmol), 30% aqueous hydrogen peroxide (420 mg, 3.7 mmol) was added and heated to reflux for 2.5 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, 1M hydrochloric acid was added to adjust the liquid to pH 7, and water was added to precipitate a precipitate. The precipitated precipitate was collected by filtration, and the obtained solid was purified by preparative thin layer chromatography (5% methanol / chloroform) to give (E) -7- [2- (tert-butoxycarbonylamino) ethoxy. ] -2- [4- (Dimethylamino) styryl] -3-hydroxy-4H-chromen-4-one (35 mg, 20%) was obtained as a brown solid and used in Reference Example 3. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ9.21 (brs, 1H), 7.94 (d, J = 8.9 Hz, 1H), 7.51 (d, J = 8.9 Hz, 2H) ), 7.40 (d, J = 16.2 Hz, 1H), 7.21 (brs, 1H), 7.09 (m, 1H), 7.07 (d, J = 16.2 Hz, 1H), 6.98 (dd, J = 2.2, 8.9 Hz, 1H), 6.76 (d, J = 8.9 Hz, 2H), 4.10-4.17 (m, 2H), 3.34 -3.99 (m, 2H), 2.98 (s, 6H), 1.39 (s, 9H).
Reference example 3

(E) -7- [2- (tert-butoxycarbonylamino) ethoxy] -2- [4- (dimethylamino) styryl] -3-hydroxy-4H-chromen-4-one obtained in Reference Example 2 ( 35 mg, 75 μmol) was dissolved in dichloromethane (0.8 mL), trifluoroacetic acid (0.2 mL) was added, and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off under reduced pressure, diethyl ether was added to the resulting residue to solidify, and (E) -7- (2-ammonioethoxy) -2- [4- (dimethylamino) styryl] -3-hydroxy was obtained. -4H-chromen-4-one trifluoroacetate (25 mg, 69%) was obtained as a brown solid and used in Example 1. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 9.28 (brs, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.88-8.17 (m, 3H), 7.52 (d, J = 8.9 Hz, 2H), 7.41 (d, J = 16.2 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.09 (d , J = 16.2 Hz, 1H), 7.04 (dd, J = 2.4, 8.8 Hz, 1H), 6.76 (d, J = 8.9 Hz, 2H), 4.33 (t, J = 4.9 Hz, 2H), 3.24-3.35 (m, 2H), 2.99 (s, 6H).
Reference example 4

(E) -2- [4- (Dimethylamino) styryl] -3-hydroxy-7- [2- (tert-butoxycarbonylamino) ethoxy] -4H-chromen-4-one obtained in Reference Example 3 ( 30 mg, 64 μmol) was dissolved in N, N-dimethylformamide (1 mL), potassium carbonate (27 mg, 0.19 mmol) and benzyl bromide (11 μL, 96 μmol) were added, and the mixture was stirred at room temperature for 12 hours. After completion of the reaction, water (20 mL) was added to the reaction solution, followed by extraction with chloroform (20 mL × 3), and the organic layer was dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the resulting residue was purified by preparative thin layer chromatography (5% methanol / chloroform) to give (E) -3-benzyloxy-7- [2- (tert-butoxycarbonyl). Amino) ethoxy] -2- [4- (dimethylamino) styryl] -4H-chromen-4-one (25 mg, 70%) was obtained as an orange solid. The obtained (E) -3-benzyloxy-7- [2- (tert-butoxycarbonylamino) ethoxy] -2- [4- (dimethylamino) styryl] -4H-chromen-4-one (25 mg, 45 μmol) ) Was dissolved in dichloromethane (0.8 mL), trifluoroacetic acid (0.2 mL) was added, and the mixture was stirred at room temperature for 30 minutes. After completion of the reaction, the solvent was distilled off under reduced pressure, diethyl ether was added to the resulting residue to solidify, and (E) -7- (2-ammonioethoxy) -3-benzyloxy-2- [4- ( [Dimethylamino) styryl] -4H-chromen-4-one trifluoroacetate (15 mg, 58%) was obtained as an orange solid and used in Examples 2 and 7. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 8.03 (d, J = 8.9 Hz, 1H), 7.98 (brs, 3H), 7.48-7.53 (m, 2H), 7.30-7.47 (m, 6H), 7.28 (d, J = 2.3 Hz, 1H), 7.08 (dd, J = 2.3, 8.9 Hz, 1H), 6.88 (D, J = 16, 1 Hz, 1H), 6.76 (d, J = 8.9 Hz, 2H), 5.16 (s, 2H), 4.33 (t, J = 5.0 Hz, 2H) , 3.29-3.35 (m, 2H), 3.00 (s, 6H).
Reference Example 5

6 ′-[2- (tert-Butoxycarbonylamino) ethoxy] -3′-hydroxy-2′-acetonaphthone (511 mg, 1.5 mmol) and (E) -3- [4- (dimethylamino) phenyl]- 2-propenal (285 mg, 1.6 mmol) was dissolved in N, N-dimethylformamide (1.5 mL), sodium methoxide (243 mg, 4.5 mmol) was added, and the mixture was stirred at room temperature for 2.5 hours. Next, ethanol (7.5 mL), 4M aqueous sodium hydroxide solution (3.8 mL, 15 mmol) and 30% aqueous hydrogen peroxide (1.7 g, 15 mmol) were added to the reaction mixture, and the mixture was further heated under reflux for 1.5 hours. did. After completion of the reaction, the reaction mixture was cooled to room temperature, 1M hydrochloric acid was added to adjust the liquid to pH 7, and water was added to precipitate a precipitate. After the deposited precipitate was collected by filtration, the obtained solid was purified by silica gel column chromatography (2.5% methanol / chloroform, 5% methanol / chloroform in this order), and (E) -8- [2- (Tert-Butoxycarbonylamino) ethoxy] -2- [4- (dimethylamino) styryl] -3-hydroxy-4H-benzo [g] chromen-4-one (301 mg, 39%) was obtained as a red solid, Used in Reference Example 6. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 9.26 (s, 1H), 8.67 (s, 1H), 8.13 (d, J = 9.2 Hz, 1H), 8.02 ( s, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 16.2 Hz, 1H), 7.42 (d, J = 2.5 Hz, 1H), 7.14 (dd, J = 2.5, 9.2 Hz, 1H), 7.16 (d, J = 16.2 Hz, 1H), 7.10 (t, J = 5.5 Hz, 1H), 6 .77 (d, J = 8.8 Hz, 2H), 4.14 (t, J = 5.7 Hz, 2H), 3.40 (dt, J = 5.5, 5.7 Hz, 2H), 3. 00 (s, 6H), 1.40 (s, 9H).
Reference Example 6

(E) -8- [2- (tert-butoxycarbonylamino) ethoxy] -2- [4- (dimethylamino) styryl] -3-hydroxy-4H-benzo [g] chromene- obtained in Reference Example 5 4-one (300 mg, 0.58 mmol) was dissolved in dichloromethane (4 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature for 1 hr. After completion of the reaction, the solvent was distilled off under reduced pressure, and diethyl ether was added to the resulting residue to solidify, and (E) -8- (2-ammonioethoxy) -2- [4- (dimethylamino) styryl]. -3-Hydroxy-4H-benzo [g] chromen-4-one trifluoroacetate (282 mg, 95%) was obtained as a brown solid and used in Example 3. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 9.30 (brs, 1H), 8.71 (s, 1H), 8.18 (d, J = 9.2 Hz, 1H), 8.02 ( s, 1H), 7.95-8.07 (m, 3H), 7.57 (d, J = 8.9 Hz, 2H), 7.50 (d, J = 16.2 Hz, 1H), 7. 47 (d, J = 2.3 Hz, 1H), 7.25 (dd, J = 2.3, 9.2 Hz, 1H), 7.17 (d, J = 16.2 Hz, 1H), 6.78 (D, J = 8.9 Hz, 2H), 4.35 (t, J = 5.0 Hz, 2H), 3.35 (m, 2H), 3.00 (s, 6H).
Reference Example 7

6 ′-[2- (tert-Butoxycarbonylamino) ethoxy] -3′-hydroxy-2′-acetonaphthone (494 mg, 1.4 mmol) and 4-dimethylaminobenzaldehyde (235 mg, 1.6 mmol) were added to N, N— After dissolving in dimethylformamide (3 mL) and cooling to 0 ° C., potassium tert-butoxide (513 mg, 4.2 mmol) was added and stirred at 0 ° C. for 30 minutes. Water was added to the reaction solution to deposit a precipitate, which was collected by filtration. The resulting solid was purified by flash chromatography (50% ethyl acetate / hexane) to give (E) -3- [4- (dimethylamino) phenyl] -1- (2-hydroxy-7- [2- ( tert-butoxycarbonylamino) ethoxynaphthalen-3-yl] prop-2-en-1-one (648 mg, 97%) was obtained as an orange solid and used in Reference Example 8. ¹ H-NMR (400 MHz, DMSO) −d ₆ ): δ 12.58 (s, 1H), 8.92 (s, 1H), 7.93 (d, J = 15.3 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 7.84 (d, J = 15.3 Hz, 1H), 7.79 (d, J = 8.9 Hz, 2H), 7.17 (d, J = 2.4 Hz, 1H), 7. 16 (s, 1H), 7.07 (t, J = 5.6 Hz, 1H ), 7.02 (dd, J = 2.4, 9.0 Hz, 1H), 6.79 (d, J = 8.9 Hz, 2H), 4.09 (t, J = 5.6 Hz, 2H) 3.37 (q, J = 5.6 Hz, 2H), 3.05 (s, 6H), 1.39 (s, 9H).
Reference Example 8

(E) -3- [4- (Dimethylamino) phenyl] -1- (2-hydroxy-7- [2- (tert-butoxycarbonylamino) ethoxynaphthalen-3-yl] propylene obtained in Reference Example 7 2-en-1-one (86 mg, 0.18 mmol) was suspended in ethanol (1 mL), 30% aqueous hydrogen peroxide (204 mg, 1.8 mmol), 4M aqueous sodium hydroxide solution (0.45 mL, 1 The reaction solution was cooled to room temperature, 1M hydrochloric acid was added to adjust the liquidity to pH 7, and water was added to precipitate a precipitate. The resulting solid was then purified by flash chromatography (2% methanol / chloroform) to yield 8- [2- (tert-butoxycarbonylamino) ethoxy] -2- [4- (di The Chiruamino) phenyl] -3-hydroxy -4H- benzo [g] chromen-4-one (20 mg, 23%) as a yellow solid which was used in Reference Example ^{9. 1 H-NMR (400MHz} , DMSO-d ₆ ): δ 9.06 (s, 1H), 8.69 (s, 1H), 8.18 (d, J = 9.1 Hz, 2H), 8.13 (d, J = 9.1 Hz, 1H) , 8.05 (s, 1H), 7.42 (d, J = 2.5 Hz, 1H), 7.19 (dd, J = 2.5, 9.1 Hz, 1H), 7.08 (m, 1H), 6.88 (d, J = 9.1 Hz, 2H), 4.14 (t, J = 5.7 Hz, 2H), 3.40 (q, J = 5.7 Hz, 2H), 3. 04 (s, 6H), 1.40 (s, 9H).
Reference Example 9

8- [2- (tert-Butoxycarbonylamino) ethoxy] -2- [4- (dimethylamino) phenyl] -3-hydroxy-4H-benzo [g] chromen-4-one obtained in Reference Example 8 ( 20 mg, 41 μmol) was dissolved in dichloromethane (0.8 mL), trifluoroacetic acid (0.2 mL) was added, and the mixture was stirred at room temperature for 30 minutes. After completion of the reaction, the solvent was distilled off under reduced pressure, diethyl ether was added to the resulting residue to solidify, and 8- (2-ammonioethoxy) -2- [4- (dimethylamino) phenyl] -3-hydroxy was obtained. -4H-benzo [g] chromen-4-one trifluoroacetate (14 mg, 68%) was obtained as a yellow solid and used in Example 4. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 8.73 (s, 1H), 8.19 (d, J = 9.3 Hz, 2H), 8.19 (d, J = 9.2 Hz, 1H) ), 8.12 (s, 1H), 7.46 (d, J = 2.4 Hz, 1H), 7.25 (dd, J = 2.4, 9.2 Hz, 1H), 6.88 (d , J = 9.3 Hz, 2H), 4.34 (t, J = 4.8 Hz, 2H), 3.33 (q, J = 4.8 Hz, 2H), 3.04 (s, 6H).
Reference Example 10

8- [2- (tert-Butoxycarbonylamino) ethoxy] -2- [4- (dimethylamino) phenyl] -3-hydroxy-4H-benzo [g] chromen-4-one obtained in Reference Example 8 ( 50 mg, 0.1 mmol) was dissolved in N, N-dimethylformamide (1 mL), potassium carbonate (42 mg, 0.3 mmol) and benzyl bromide (18 μL, 0.15 mmol) were added, and the mixture was stirred at room temperature for 24 hours. After completion of the reaction, water (20 mL) was added to the reaction solution, followed by extraction with chloroform (20 mL × 3), and the organic layer was dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the resulting residue was purified by flash chromatography (chloroform, then 5% ethyl acetate / chloroform) to give 3-benzyloxy-8- [2- (tert-butoxycarbonylamino) ethoxy. ] -2- [4- (Dimethylamino) phenyl] -4H-benzo [g] chromen-4-one (43 mg, 74%) was obtained as a yellow solid and used in Reference Example 11. ¹ H-NMR (400 MHz, CDCl ₃ ): δ 8.68 (s, 1H), 8.15 (d, J = 9.3 Hz, 1H), 8.08 (d, J = 9.3 Hz, 2H), 8.06 (s, 1H), 7.42-7.46 (m, 3H), 7.29-7.40 (m, 3H), 7.22 (dd, J = 2.3, 9.3 Hz , 1H), 7.07-7.13 (m, 1H), 6.84 (d, J = 9.3 Hz, 2H), 5.07 (s, 2H), 4.14 (t, J = 5 .5 Hz, 2H), 3.40 (q, J = 5.5 Hz, 2H), 3.04 (s, 6H), 1.40 (s, 9H).
Reference Example 11

3-Benzyloxy-8- [2- (tert-butoxycarbonylamino) ethoxy] -2- [4- (dimethylamino) phenyl] -4H-benzo [g] chromen-4-one obtained in Reference Example 10 (43 mg, 74 μmol) was dissolved in dichloromethane (0.8 mL), trifluoroacetic acid (0.2 mL) was added, and the mixture was stirred at room temperature for 30 min. After completion of the reaction, the solvent was distilled off under reduced pressure, diethyl ether was added to the resulting residue to solidify, and 8- (2-ammonioethoxy) -3-benzyloxy-2- [4- (dimethylamino) phenyl ] -4H-benzo [g] chromen-4-one trifluoroacetate (19 mg, 51%) was obtained as a yellow solid and used in Example 5. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 8.72 (s, 1H), 8.20 (d, J = 9.1 Hz, 1H), 8.12 (s, 1H), 8.08 ( d, J = 9.1 Hz, 2H), 8.01 (brs, 3H), 7.45-7.51 (m, 2H), 7.45 (s, 1H), 7.30-7.40 ( m, 4H), 7.27 (dd, J = 2.3, 9.1 Hz, 1H), 6.84 (d, J = 9.1 Hz, 2H), 5.08 (s, 2H), 4. 35 (t, J = 4.8 Hz, 2H), 3.35-3.40 (m, 2H), 3.05 (s, 6H).
Reference Example 12

4 ′-[5- (tert-Butoxycarbonylamino) butyloxy] -2′-hydroxyacetophenone (1.29 g, 3.9 mmol) and (E) -3- [4- (dimethylamino) phenyl] -2- Propenal (699 mg, 3.9 mmol) was dissolved in N, N-dimethylformamide (10 mL), potassium tert-butoxide (1.36 g, 11 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, 1M hydrochloric acid was added to the reaction mixture to adjust the liquidity to pH 1, followed by extraction with chloroform (50 mL × 3). The organic layer was washed with 1M hydrochloric acid (25 mL), saturated aqueous sodium hydrogen carbonate solution (25 mL), saturated brine (25 mL) and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was purified by flash chromatography (10% ethyl acetate / hexane then 25% ethyl acetate / hexane) to give (2E, 4E) -1- [4- [5- (tert-butoxycarbonylamino). ) Butyloxy] -2-hydroxyphenyl] -5- [4- (dimethylamino) phenyl] penta-2,4-dien-1-one (500 mg, 28% yield) was obtained as a red solid. ¹ H-NMR (400 MHz, CDCl ₃ ): δ 13.68 (s, 1H), 7.74 (d, J = 8.9 Hz, 1H), 7.70 (dd, J = 11.0, 14.4 Hz) , 1H), 7.41 (d, J = 8.9 Hz, 2H), 7.03 (d, J = 14.4, 1H), 6.99 (d, J = 15.3 Hz, 1H), 6 .86 (dd, J = 11.0, 15.3 Hz, 1H), 6.69 (d, J = 8.9 Hz, 2H), 6.41-6.46 (m, 2H), 4.59 ( brs, 1H), 4.02 (t, J = 6.1 Hz, 2H), 3.15-3.25 (m, 2H), 3.03 (s, 6H), 1.78-1.88 ( m, 2H), 1.63-1.72 (m, 2H), 1.45 (s, 9H).
Reference Example 13

(2E, 4E) -1- [4- [3- (tert-Butoxycarbonylamino) butoxy] -5- [4- (dimethylamino) phenyl] -2-hydroxyphenyl] penta-2,4-diene-1 -On (100 mg, 0.21 mmol) was suspended in ethanol (1 mL), 4M aqueous potassium hydroxide solution (0.53 mL, 2.1 mmol) and 30% aqueous hydrogen peroxide (236 mg, 2.1 mmol) were added. For 1.5 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, 1M hydrochloric acid was added to bring the liquid to pH 1, and water (25 mL) was further added to dilute the reaction, and the mixture was extracted with chloroform (50 mL × 3). The organic layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was purified by preparative thin layer chromatography (5% methanol / chloroform) to obtain (E) -7- [3- (Tert-Butoxycarbonylamino) butoxy] -2- [4- (dimethylamino) styryl] -3-hydroxy-4H-chromen-4-one (49 mg, 47%) was obtained as a brown solid. Using. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ9.21 (s, 1H), 7.94 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H) ), 7.41 (d, J = 16.1 Hz, 1H), 7.16-7.21 (m, 1H), 7.08 (d, J = 16.1 Hz, 1H), 6.99 (dd , J = 2.0, 8.8 Hz, 1H), 6.85-6.92 (m, 1H), 6.76 (d, J = 8.8 Hz, 2H), 4.13 (t, J = 6.0 Hz, 2H), 2.99-3.04 (m, 2H), 2.99 (s, 6H), 1.70-1.82 (m, 2H), 1.50-1.60 ( m, 2H), 1.38 (s, 9H).
Reference Example 14

Example 1

(E) -7- (2-ammonioethoxy) -2- [4- (dimethylamino) styryl] -3-hydroxy-4H-chromen-4-one = trifluoroacetate (20 mg) obtained in Reference Example 3 , 42 μmol) was dissolved in N, N-dimethylformamide (1 mL), diisopropylethylamine (22 μL, 0.13 mmol) and 2,4-dinitrobenzenesulfonyl chloride (17 mg, 62 μmol) were added, and the mixture was stirred at room temperature for 30 minutes. . Water (5 mL) was added to the reaction solution to stop the reaction, and the mixture was extracted with chloroform (3 × 20 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was purified by preparative thin layer chromatography (5% methanol / chloroform) to give (E) -2- [4 -(Dimethylamino) styryl] -7- [2-[[(2,4-dinitrophenyl) sulfonyl] amino] ethoxy] -3-hydroxy-4H-chromen-4-one (compound 3) (3.0 mg, 12%) as a brown solid and used in Examples 9-14 and Comparative Examples 1-3. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 9.21 (s, 1H), 8.84 (d, J = 2.2 Hz, 1H), 8.55 (dd, J = 2.2, 8 .8 Hz, 1 H), 8.27 (d, J = 8.8 Hz, 1 H), 7.95 (m, 1 H), 7.87 (d, J = 8.8 Hz, 1 H), 7.52 (d , J = 8.7 Hz, 2H), 7.38 (d, J = 16.2 Hz, 1H), 7.07 (1H, J = 16.2 Hz, 1H), 6.97 (d, J = 2. 1 Hz, 1 H), 6.77 (dd, J = 2.1, 8.8 Hz, 1 H), 6.76 (d, J = 8.7 Hz, 2 H), 4.13 (t, J = 4.7 Hz) , 2H), 3.43-3.49 (m, 2H), 2.99 (s, 6H).
Example 2

(E) -7- (2-ammonioethoxy) -3-benzyloxy-2- [4- (dimethylamino) styryl] -4H-chromen-4-one (10 mg, 18 μmol) obtained in Reference Example 4 Was dissolved in N, N-dimethylformamide (1 mL), diisopropylethylamine (9.4 μL, 54 μmol) and 2,4-dinitrobenzenesulfonyl chloride (5.6 mg, 21 μmol) were added, and the mixture was stirred at room temperature for 30 minutes. Water (5 mL) was added to the reaction solution to stop the reaction, and the mixture was extracted with chloroform (3 × 20 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was purified by preparative thin layer chromatography (5% methanol / chloroform) to give (E) -3-benzyloxy. 2- [4- (Dimethylamino) styryl] -7- [2-[[(2,4-dinitrophenyl) sulfonyl] amino] ethoxy] -4H-chromen-4-one (Compound 5) (5.6 mg , 45%) was obtained as a dark red solid and used in Examples 8-14 and Comparative Examples 1-3. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 8.83 (brs, 1H), 8.81 (d, J = 2.2 Hz, 1H), 8.53 (dd, J = 2.2, 8 .8 Hz, 1H), 8.26 (d, J = 8.8 Hz, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.48-7.52 (m, 2H), 7 .42 (d, J = 8.9 Hz, 2H), 7.31-7.43 (m, 4H), 6.98 (d, J = 2.3 Hz, 1H), 6.84 (d, J = 16.1 Hz, 1H), 6.82 (dd, J = 2.3, 8.8 Hz, 1H), 6.76 (d, J = 8.9 Hz, 2H), 5.14 (s, 2H), 4.12 (t, J = 4.8 Hz, 2H), 3.40-3.48 (m, 2H), 3.00 (s, 6H).
Example 3

(E) -8- (2-ammonioethoxy) -2- [4- (dimethylamino) styryl] -3-hydroxy-4H-benzo [g] chromen-4-one = trie obtained in Reference Example 6 Fluoroacetate (13 mg, 25 μmol) is dissolved in N, N-dimethylformamide (1 mL), diisopropylethylamine (13 μL, 75 μmol) and 2,4-dinitrobenzenesulfonyl chloride (10 mg, 38 μmol) are added, and the mixture is stirred at room temperature for 30 minutes. Stir. Water (5 mL) was added to the reaction solution to stop the reaction, and the mixture was extracted with chloroform (3 × 20 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was purified by preparative thin layer chromatography (5% methanol / chloroform) to give (E) -2- [4 -(Dimethylamino) styryl] -8- [2-[[(2,4-dinitrophenyl) sulfonyl] amino] ethoxy] -3-hydroxy-4H-benzo [g] chromen-4-one (compound 7) ( 4.0 mg, 25%) was obtained as a dark purple solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 9.27 (s, 1H), 8.88 (brs, 1H), 8.80 (d, J = 2.3 Hz, 1H), 8.64 ( s, 1H), 8.50 (dd, J = 2.3, 8.7 Hz, 1H), 8.29 (d, J = 8.7 Hz, 1H), 8.04 (d, J = 9.3 Hz) , 1H), 7.97 (s, 1H), 7.58 (d, J = 8.9 Hz, 2H), 7.50 (d, J = 16.0 Hz, 1H), 7.27 (d, J = 2.2 Hz, 1 H), 7.16 (d, J = 16.0 Hz, 1 H), 6.96 (dd, J = 2.2, 9.3 Hz, 1 H), 6.78 (d, J = 8.9 Hz, 2H), 4.17 (t, J = 4.8 Hz, 2H), 3.47-3.55 (m, 2H), 3.00 (s, 6H).
Example 4

8- (2-ammonioethoxy) -2- [4- (dimethylamino) phenyl] -3-hydroxy-4H-benzo [g] chromen-4-one trifluoroacetate (10 mg) obtained in Reference Example 9 , 20 μmol) was dissolved in N, N-dimethylformamide (1 mL), diisopropylethylamine (10 μL, 60 μmol) and 2,4-dinitrobenzenesulfonyl chloride (8 mg, 30 μmol) were added, and the mixture was stirred at room temperature for 30 minutes. Water (5 mL) was added to the reaction solution to stop the reaction, and the mixture was extracted with chloroform (3 × 20 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was purified by preparative thin layer chromatography (5% methanol / chloroform) to give 2- [4- (dimethylamino). ) Phenyl] -8- [2-[[(2,4-dinitrophenyl) sulfonyl] amino] ethoxy] -3-hydroxy-4H-benzo [g] chromen-4-one (Compound 9) (1.0 mg, 8%) was obtained as a brown solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 9.10 (s, 1H), 8.77 (m, 2H), 8.65 (s, 1H), 8.44 (m, 1H), 8 .22 (m, 1H), 8.18 (d, J = 9.3 Hz, 2H), 8.07 (d, J = 8.9 Hz, 1H), 8.01 (s, 1H), 7.28 (D, J = 2.3 Hz, 1H), 7.00 (dd, J = 2.3, 8.9 Hz, 1H), 6.89 (d, J = 9.3 Hz, 2H), 4.09− 4.14 (m, 2H), 3.37-3.43 (m, 2H), 3.04 (s, 6H).
Example 5

8- (2-ammonioethoxy) -3-benzyloxy-2- [4- (dimethylamino) phenyl] -4H-benzo [g] chromen-4-one trifluoroacetate obtained in Reference Example 11 ( 10 mg, 17 μmol) was dissolved in N, N-dimethylformamide (1 mL), triethylamine (7.2 μL, 51 μmol) and 2,4-dinitrobenzenesulfonyl chloride (6.7 mg, 25 μmol) were added, and the mixture was stirred at room temperature for 30 minutes. Stir. Water (5 mL) was added to the reaction solution to stop the reaction, and the mixture was extracted with chloroform (3 × 20 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was purified by preparative thin layer chromatography (5% methanol / chloroform) to give 3-benzyloxy-2- [ 4- (Dimethylamino) phenyl] -8- [2-[[(2,4-dinitrophenyl) sulfonyl] amino] ethoxy] -4H-benzo [g] chromen-4-one (Compound 11) (3.0 mg 25%) as a brown solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 8.88 (brs, 1H), 8.81 (d, J = 2.2 Hz, 1H), 8.65 (s, 1H), 8.50 ( dd, J = 2.2, 8.7 Hz, 1H), 8.28 (d, J = 8.7 Hz, 1H), 8.08 (d, J = 9.0 Hz, 2H), 8.06 (d , J = 9.0 Hz, 1H), 8.01 (s, 1H), 7.43-7.48 (m, 2H), 7.31-7.40 (m, 3H), 7.29 (d , J = 2.3 Hz, 1H) 6.99 (dd, J = 2.3, 9.0 Hz, 1H), 6.84 (d, J = 9.0 Hz, 2H), 5.07 (s, 2H) ), 4.17 (t, J = 5.3 Hz, 2H), 3.48-3.53 (m, 2H), 3.05 (s, 6H).
Example 6

(E) -7- (2-Ammonioboxy) -2- [4- (dimethylamino) styryl] -3-hydroxy-4H-chromen-4-one = trifluoroacetate (10 mg) obtained in Reference Example 14 , 20 μmol) was dissolved in N, N-dimethylformamide (1 mL), diisopropylethylamine (10 μL, 60 μmol) and 2,4-dinitrobenzenesulfonyl chloride (6.5 mg, 24 μmol) were added, and the mixture was stirred at room temperature for 30 minutes. . Water (5 mL) was added to the reaction solution to stop the reaction, and the mixture was extracted with chloroform (3 × 20 mL). The organic layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was purified by preparative thin layer chromatography (5% methanol / chloroform) to give (E) -2- [4 -(Dimethylamino) styryl] -7- [2-[[(2,4-dinitrophenyl) sulfonyl] amino] butoxy] -3-hydroxy-4H-chromen-4-one (Compound 13) (4.0 mg, 32%) was obtained as a brown solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 9.22 (s, 1H), 8.90 (d, J = 2.2 Hz, 1H), 8.64 (dd, J = 2.2, 8 .6 Hz, 1H), 8.53-8, 60 (m, 2H), 7.93 (d, J = 9.0 Hz, 1H), 7.52 (d, J = 8.9 Hz, 2H), 7 .41 (d, J = 16.2 Hz, 1H), 7.15 (d, J = 2.2 Hz, 1H), 7.08 (d, J = 16.2 Hz, 1H), 6.95 (dd, J = 2.2, 9.0 Hz, 1H), 6.76 (d, J = 8.9 Hz, 2H), 4.06-4.14 (m, 2H), 3.02-3.09 (m) , 2H), 2.99 (s, 6H), 1.70-1.85 (m, 2H), 1.55-1.68 (m, 2H).
Example 7

(E) -7- (2-ammonioethoxy) -3-benzyloxy-2- [4- (dimethylamino) styryl] -4H-chromen-4-one = trifluoroacetate obtained in Reference Example 4 ( 50 mg, 88 μmol) was dissolved in N, N-dimethylformamide (1 mL), and diisopropylethylamine (46 μL, 0.26 mmol) and 4-nitro-2- (trifluoromethyl) benzenesulfonyl chloride (31 mg, 0.11 mmol) were added. In addition, the mixture was stirred at room temperature for 30 minutes. Water (10 mL) was added to the reaction solution to precipitate a precipitate, and the resulting precipitate was collected by filtration. The resulting solid was purified by flash chromatography (40% ethyl acetate / hexane) to give (E) -3-benzyloxy-2- [4- (dimethylamino) styryl] -7- [2-[[[[ 4-Nitro-2- (trifluoromethyl) phenyl] sulfonyl] amino] ethoxy] -4H-chromen-4-one (Compound 15) (10 mg, 16%) was obtained as a brown solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 8.75 (brs, 1H), 8.61 (dd, J = 2.3, 8.6 Hz, 1H), 8.52 (d, J = 2) .3 Hz, 1H), 8.39 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 8.9 Hz, 1H), 7.47-7.53 (m, 2H), 7 .42 (d, J = 8.9 Hz, 2H), 7.29-7.47 (m, 4H), 6.99 (d, J = 2.3 Hz, 1H), 6.85 (d, J = 16.1 Hz, 1H), 6.79 (dd, J = 2.3, 8.9 Hz, 1H), 6.76 (d, J = 8.9 Hz, 2H), 5.14 (s, 2H), 4.14 (t, J = 4.9 Hz, 2H), 3.48 (t, J = 4.9 Hz, 2H), 3.00 (s, 6H). ¹⁹ F-NMR (376 MHz, DMSO-d ₆ ): δ-56.8 (s, 3F).
Example 8

(E) -3-Benzyloxy-2- [4- (dimethylamino) styryl] -7- [2-[[(2,4-dinitrophenyl) sulfonyl] amino] ethoxy]-obtained by Example 2 4H-chromen-4-one (compound 5) (23 mg, 33 μmol) was dissolved in N, N-dimethylformamide (1 mL), potassium carbonate (92 mg, 0.66 mmol), methyl iodide (20 μL, 0.33 mmol). And stirred at room temperature for 1 hour. Water (10 mL) was added to the reaction solution to precipitate a precipitate, and the resulting precipitate was collected by filtration. The resulting solid was purified by preparative thin layer chromatography (5% methanol / chloroform) to give (E) -3-benzyloxy-2- [4- (dimethylamino) styryl] -7- [2- [ [(2,4-Dinitrophenyl) sulfonyl] methylamino] ethoxy] -4H-chromen-4-one (Compound 16) (15 mg, 65%) was obtained as a brown solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 8.98 (d, J = 2.3 Hz, 1H), 8.53 (dd, J = 2.3, 8.8 Hz, 1H), 8.31 (D, J = 8.8 Hz, 1H), 7.94 (d, J = 8.8 Hz, 1H), 7.48-7.52 (m, 2H), 7.42 (d, J = 9. 0 Hz, 2H), 7.28-7.45 (m, 4H), 7.10 (d, J = 2.3 Hz, 1H), 6.92 (dd, J = 2.3, 8.8 Hz, 1H) ), 6.85 (d, J = 16.2 Hz, 1H), 6.76 (d, J = 9.0 Hz, 2H), 5.15 (s, 2H), 4.33 (t, J = 5) .2 Hz, 2H), 3.74 (t, J = 5.2 Hz, 2H), 3.06 (s, 3H), 3.00 (s, 6H).

Example 9
Under the following conditions, GST was added to the 4H-chromone derivative (1) (compound 3, compound 5, compound 15, compound 16) of the present invention, and the emission intensity was measured. Similarly, the emission intensity when GST was not added was also measured, and the detection of GST was evaluated by comparing with the emission intensity.

(A) Method reaction solution: phosphate buffer solution (pH 7.4) (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na ₂ HPO ₄ , 1.47 mM KH ₂ PO ₄ )
Compound 3 concentration: 15 μM (1% DMSO)
GSH concentration: 1 mM
Concentration of GST P1-1, Human, Recombinat (manufactured by Oxford Biomedical Research): 20 μg / mL
Reaction temperature: 37 ° C
When GST was not added, the same amount of phosphate buffer solution was added to the reaction solution instead of the GST solution.
The reaction solution was taken out at each time, diluted 10-fold with a phosphate buffer solution containing human serum albumin (200 mg / mL), and the luminescence intensity was measured with a plate reader.
Excitation light: 440 nm
Detection light: 600 nm
(B) Results FIG. 1 shows the time course of fluorescence intensity of Compound 3 in the presence and absence of GST. In FIG. 1, the solid black square indicates the fluorescence intensity in the presence of GST, and the dotted black triangle indicates the fluorescence intensity in the absence of GST. 30 minutes after the start of the reaction, the fluorescence intensity in the presence of GST was about 5.3 times stronger than the fluorescence intensity in the absence. This shows that the 4H-chromone derivative of the present invention can detect GST.

  FIG. 2 shows changes with time in fluorescence intensity of Compound 5 in the presence and absence of GST. In FIG. 2, the solid black square represents the fluorescence intensity in the presence of GST, and the dotted black triangle represents the fluorescence intensity in the absence of GST. 30 minutes after the start of the reaction, the fluorescence intensity in the presence of GST was about 4.8 times stronger than the fluorescence intensity in the absence. This shows that the 4H-chromone derivative of the present invention can detect GST.

  FIG. 3 shows changes with time in fluorescence intensity of Compound 15 in the presence and absence of GST. In FIG. 3, the solid black square indicates the fluorescence intensity in the presence of GST, and the dotted black triangle indicates the fluorescence intensity in the absence of GST. 120 minutes after the start of the reaction, the fluorescence intensity in the presence of GST was about 3.5 times stronger than the fluorescence intensity in the absence. This shows that the 4H-chromone derivative of the present invention can detect GST.

  FIG. 4 shows changes with time in fluorescence intensity of Compound 16 in the presence and absence of GST. In FIG. 4, the solid black square indicates the fluorescence intensity in the presence of GST, and the dotted black triangle indicates the fluorescence intensity in the absence of GST. 120 minutes after the start of the reaction, the fluorescence intensity in the presence of GST was about 3.8 times stronger than that in the absence. This shows that the 4H-chromone derivative of the present invention can detect GST.

Example 10
Under the following conditions, the protein extract of cancer cells was added to the 4H-chromone derivative (1) (compound 3, compound 5) of the present invention, and the luminescence intensity was measured. In addition, when a cancer cell protein extract is not added, when a leukocyte cell protein extract is added (Comparative Example 1), and when a cancer cell protein extract and 2,4-dinitrochlorobenzene (CDNB) are added The luminescence intensity of (Comparative Example 2) was also measured, and GST and cancer cell detection ability were evaluated by comparing with the luminescence intensity.

(A) Method In an EMEM medium containing 10% FBS of FM3A cells derived from mouse breast cancer, in a petri dish for suspension culture 90φ (deep type) (manufactured by Sumitomo Bakelite) in an atmosphere of 5% CO ₂ at 37 ° C. For 24 hours or more.

Cells were harvested after they reached subconfluence. The collected cells were centrifuged (200 × g, 25 ° C., 5 minutes), and the supernatant was discarded, followed by washing with PBS (phosphate buffered saline). The cells were further centrifuged (200 × g, 25 ° C., 5 minutes), the supernatant was discarded, and the cells were suspended in 250 μL of PBS to obtain a cell suspension. The number of cells in the cell suspension was 1.45 × 10 ⁶ . Subsequently, the cell suspension was once frozen in an ultra-low temperature freezer (−80 ° C.) and then thawed. Subsequently, the thawed cell suspension was vigorously mixed with a touch mixer for about 10 minutes while cooling on ice to break the cell membrane. Subsequently, centrifugation (10000 × g, 4 ° C., 10 minutes) was performed, and the supernatant was collected to obtain a cancer cell protein extract. The obtained cancer cell protein extract was stored frozen in an ultra-low temperature freezer (−80 ° C.) until use.

  Under the following conditions, the cancer cell protein extract was added to Compound 3 or Compound 5 of the present invention, and the fluorescence intensity was measured. Similarly, the fluorescence intensity in the case where the cancer cell protein extract was not added was also measured, and the detection of GST contained in the cancer cell protein extract was evaluated by comparison with the fluorescence intensity.

Reaction solution buffer solution: PBS (phosphate buffered saline)
Concentration of Compound 3 or Compound 5: 15 μM (1% DMSO)
GSH concentration: 1 mM
Cancer cell protein extract: 1/10 of the reaction solution volume Reaction temperature: 37 ° C
When the cancer cell protein extract was not added, the same amount of PBS was added to the reaction solution instead of the cancer cell protein extract. The above reaction solution was withdrawn at the lapse of 0 minutes, 30 minutes, 60 minutes, and 120 minutes, and kept frozen in an ultra-low temperature freezer (−80 ° C.) until measurement. The thawed reaction solution was diluted 10-fold with PBS containing bovine serum albumin (200 μg / mL), and the fluorescence intensity was measured with a plate reader.
Compound 3
Excitation light: 470 nm
Detection light: 650 nm
Compound 5
Excitation light: 450 nm
Detection light: 579 nm
(B) Results The results are shown in FIG. When a cancer cell protein extract was added, the fluorescence intensity of the solution containing Compound 3 (FIG. 5A, solid black square) and the fluorescence intensity of the solution containing Compound 5 (FIG. 5B, solid black) with the lapse of reaction time. Square) increased. On the other hand, when the cancer cell protein extract was not added (Compound 3: FIG. 5A, dashed black circle, Compound 5: FIG. 5B, dashed black circle), the change in the fluorescence intensity of the solution over the course of the reaction time was slight.

Comparative Example 1
(A) Method 3 mL of healthy human blood collected in an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo) was diluted with an equal amount of physiological saline to obtain a total of 6 mL of diluted blood. 6 mL of the pre-diluted blood was layered on 2 mL of Ficoll-Paque PREMIUM (manufactured by GE Healthcare Japan), and centrifuged (1100 × g, 10 minutes, 25 ° C.) to recover the white blood cell layer. The collected white blood cells are suspended in physiological saline to make a total of 30 mL, centrifuged (300 × g, 5 minutes, 25 ° C.), the supernatant is discarded, and then suspended in physiological saline again to make a total of 30 mL and centrifuged ( 300 × g, 5 minutes, 25 ° C.) and discarding the supernatant, platelet removal and white blood cell washing were simultaneously performed to obtain white blood cells.

The white blood cells were centrifuged, the supernatant was discarded, and the suspension was suspended in PBS (phosphate buffered saline) to obtain 250 μL of white blood cell suspension. The number of cells in the white blood cell suspension was 3.25 × 10 ⁶ . Subsequently, the cell suspension was once frozen in an ultra-low temperature freezer (−80 ° C.) and then thawed to break the cell membrane. Subsequently, the cell suspension in which the cell membrane was disrupted was vigorously mixed with a touch mixer for about 10 minutes while cooling on ice. Subsequently, the mixture was centrifuged (10000 × g, 4 ° C., 10 minutes), and the supernatant was collected to obtain a leukocyte intracellular protein extract. The obtained leukocyte intracellular protein extract was stored frozen in an ultra-low temperature freezer (−80 ° C.) until use.

Under the following conditions, the white blood cell protein extract was added to the compound 3 or 5 of the present invention, and the fluorescence intensity was measured. Similarly, the fluorescence intensity when no leukocyte intracellular protein extract was added was also measured.
Reaction solution buffer solution: PBS (phosphate buffered saline)
Concentration of Compound 3 or Compound 5: 15 μM (1% DMSO)
GSH concentration: 1 mM
Leukocyte intracellular protein extract: 1/10 of the reaction solution volume Reaction temperature: 37 ° C
When the white blood cell protein extract was not added, the same amount of PBS was added to the reaction solution instead of the white blood cell protein extract. The above reaction solution was withdrawn at the lapse of 0 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes, and kept frozen in an ultra-low temperature freezer (−80 ° C.) until measurement. The thawed reaction solution was diluted 10-fold with PBS containing bovine serum albumin (200 μg / mL), and the fluorescence intensity was measured with a plate reader.
Compound 3
Excitation light: 470 nm
Detection light: 650 nm
Compound 5
Excitation light: 450 nm
Detection light: 579 nm
(B) Results As a result, as shown in FIG. 5A (compound 3: dotted black triangle) and FIG. 5B (compound 5: dotted black triangle), in the presence of the leukocyte intracellular protein extract, the solution with the reaction time elapsed The change in the fluorescence intensity was slight.

Comparative Example 2
(A) Method In the following conditions, Compound 3 or Compound 5 of the present invention is a cancer cell protein extract prepared in the same manner as in Example 10 and a GST substrate, which is competitively inhibited against Compound 3 and Compound 5. 2,4-Dinitrochlorobenzene (CDNB) (Non-patent Document 7) was added, and the fluorescence intensity was measured. Similarly, the fluorescence intensity when CDNB was not added was also measured, and the GST enzyme specificity of Compound 3 or Compound 5 was evaluated by comparing with the fluorescence intensity.
Reaction solution buffer solution: PBS (phosphate buffered saline)
Concentration of Compound 3 or Compound 5: 15 μM (1% DMSO)
GSH concentration: 1 mM
Cancer cell protein extract: 1/10 of reaction solution volume CDNB concentration: 4 mM (4% ethanol)
Reaction temperature: 37 ° C
When CDNB was not added, the same amount of ethanol was added instead of the CDNB ethanol solution. The above reaction solution was withdrawn at the lapse of 0 minutes, 30 minutes, 60 minutes, and 120 minutes, and kept frozen in an ultra-low temperature freezer (−80 ° C.) until measurement. The thawed reaction solution was diluted 10-fold with PBS containing bovine serum albumin (200 μg / mL), and the fluorescence intensity was measured with a plate reader.
Compound 3 and Compound 5
Excitation light: 440 nm
Detection light: 580 nm
(B) Results The results are shown in FIGS. 5C and 5D. When CDNB was not added, the fluorescence intensity of the solution containing Compound 3 (FIG. 5C, dotted black triangle) and the fluorescence intensity of the solution containing Compound 5 (FIG. 5D, dotted black triangle) increased with the reaction time. Increased. On the other hand, when CDNB was added (FIGS. 5C and 5D, solid black squares), the fluorescence intensity of the solution did not change with the lapse of the reaction time.

  From the results of Example 10, Comparative Example 1, and Comparative Example 2, it can be seen that the compounds 3 and 5 of the present invention can detect GST contained in the intracellular protein extract. Moreover, it turns out that a cancer cell and a normal cell can be distinguished by comparing fluorescence intensity.

Example 11
Under the following conditions, the 4H-chromone derivative (1) (compound 3, compound 5) of the present invention was brought into contact with cancer cells, and the fluorescence detection ability of cancer cells was evaluated. Similarly, the fluorescence detection ability of white blood cells was evaluated by contacting with white blood cells (Comparative Example 3).

(A) Method SK-BR-3 cells derived from human breast adenocarcinoma in McCoy's 5a medium containing 10% FBS, and PC-9 cells of non-small cell lung cancer cells derived from human lung adenocarcinoma, In D-MEM / Ham's F-12 medium containing 10% FBS, both in a multiwell plate 24F (for adherent cells, manufactured by Sumitomo Bakelite) at 37 ° C. in a 5% CO ₂ atmosphere. Incubated for more than 24 hours.

  After the cell density is such that both SK-BR-3 cells and PC-9 cells cover about half of the bottom surface, the supernatant medium is discarded, and then DMEM / F-12 medium without phenol red is used. After washing, 150 μL of DMEM / F-12 medium containing 1.5 μM Compound 3 or Compound 5 and not containing phenol red was added.

  Fluorescence images of cells were taken at 10 minutes, 30 minutes, and 60 minutes with an EM-CCD camera through an inverted research microscope (IX71, Olympus) under conditions of excitation wavelengths of 470-495 nm and fluorescence wavelengths of 510-550 nm. (Compound 3: upper part of FIG. 6, compound 5: upper part of FIG. 7).

(B) Results As a result, the cytoplasm of both the SK-BR-3 cells and the PC-9 cells was fluorescently labeled. In addition, the fluorescence intensity emitted by the cells increased with time. The fluorescence intensity of PC-9 cells was higher than that of SK-BR-3.

Comparative Example 3
(A) Method 3 mL of healthy human blood collected in an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo) was diluted with an equal amount of physiological saline to obtain a total of 6 mL of diluted blood. 6 mL of the pre-diluted blood was layered on 2 mL of Ficoll-Paque PREMIUM (manufactured by GE Healthcare Japan), and centrifuged (1100 × g, 10 minutes, 25 ° C.) to recover the white blood cell layer. The collected white blood cells are suspended in physiological saline to make a total of 30 mL, centrifuged (300 × g, 5 minutes, 25 ° C.), the supernatant is discarded, and then suspended in physiological saline again to make a total of 30 mL and centrifuged ( 300 × g, 5 minutes, 25 ° C.) and discarding the supernatant, platelet removal and white blood cell washing were simultaneously performed to obtain white blood cells.

The white blood cells are suspended in a DMEM / F-12 medium containing 1.5 μM Compound 3 or Compound 5 and not containing phenol red (cell concentration 2.0 × 10 ⁶ cells / mL), and a multiwell plate 24F ( 150 μL was seeded on adhesive cells (Sumitomo Bakelite).

Fluorescence images of the white blood cells were passed through an inverted research microscope (IX71, manufactured by Olympus) for 10 minutes, 30 minutes, and 60 minutes under an excitation wavelength of 470-495 nm and a fluorescence wavelength of 510-550 nm. Occasionally images were taken (compound 3: bottom of FIG. 6, compound 5: bottom of FIG. 7).
(B) Results As a result, the change in the fluorescence intensity emitted by the white blood cells was slight with time.

  From the results of Example 11 and Comparative Example 3, it can be seen that Compound 3 and Compound 5 of the present invention fluorescently stain cancer cells, and that white blood cells, which are normal cells, have little fluorescence staining.

Example 12
Under the following conditions, the 4H-chromone derivative (1) (compound 3, compound 5) of the present invention was brought into contact with a mixture of cancer cells and white blood cells, and the fluorescence detection ability of cancer cells was evaluated.

(A) Method 3 mL of healthy human blood collected in an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo) was diluted with an equal amount of physiological saline to obtain a total of 6 mL of diluted blood. 6 mL of the pre-diluted blood was layered on 2 mL of Ficoll-Paque PREMIUM (manufactured by GE Healthcare Japan), and centrifuged (1100 × g, 10 minutes, 25 ° C.) to recover the white blood cell layer. The collected white blood cells are suspended in physiological saline to make a total of 30 mL, centrifuged (300 × g, 5 minutes, 25 ° C.), the supernatant is discarded, and then suspended in physiological saline again to make a total of 30 mL and centrifuged ( (300 × g, 5 minutes, 25 ° C.) and discarding the supernatant, platelet removal and white blood cell washing were simultaneously performed. Next, the obtained white blood cells are suspended in PBS (phosphate buffered saline), 10 μg / mL Hoechst 33342 (manufactured by Dojindo Laboratories) is added to stain the cell nucleus, and at the same time, anti-CD-45 antibody (APC Labeling) (Miltenyi Biotech) was added to perform cell surface staining. Next, centrifuge (300 xg, 5 minutes, 25 ° C), discard the supernatant, and wash the white blood cells with PBS (phosphate buffered saline) to remove the anti-CD45 antibody (APC label) that did not bind to the antigen. did. Next, the mixture was centrifuged (300 × g, 5 minutes, 25 ° C.), the supernatant was discarded, and the suspension was suspended in a DMEM / F-12 medium not containing phenol red to obtain a white blood cell suspension.

PC-9 cells of non-small cell lung cancer cells derived from human lung adenocarcinoma were cultured at 37 ° C. in a D-MEM / Ham's F-12 medium containing 10% FBS in a 5% CO ₂ atmosphere. After the cells reached subconfluence, they were washed with PBS (phosphate buffered saline), and the cells were detached using trypsin-EDTA so that the individual cells were separated. The treated cells were centrifuged (200 × g, 25 ° C., 5 minutes), the supernatant was discarded, and then suspended in a DMEM / F-12 medium not containing phenol red to obtain a PC-9 cell suspension. Next, 10 μg / mL Hoechst 33342 (manufactured by Dojindo Laboratories) was added to stain the cell nucleus.

Subsequently, the PC-9 cell suspension was adjusted so that the cell density of PC-9 cells was 1.0 × 10 ⁵ cells / mL and the cell density of white blood cells was 1.0 × 10 ⁶ cells / mL. The white blood cell suspension was mixed to prepare a mixture of cancer cells and white blood cells.

1.5 μM compound 3 or compound 5 was added to the mixture of cancer cells and white blood cells, and 400 μL was seeded on a multiwell plate 24F (adhesive cells, manufactured by Sumitomo Bakelite).
Fluorescence images of the cancer cell and white blood cell mixture were imaged with an EM-CCD camera through an inverted research microscope (IX71, Olympus) at the time of 10 minutes, 30 minutes, and 60 minutes (Compound 3: FIG. 8). Compound 5: FIG. 9). In addition, the fluorescence image derived from the anti-CD45 antibody (APC label) is imaged under the conditions of the excitation wavelength 600-650 nm and the fluorescence wavelength 670-720 nm, and the fluorescence image derived from the Hoechst 33342 has the excitation wavelength 360-370 nm and the fluorescence wavelength 420-460 nm. Images were taken under conditions, and fluorescence images derived from compound 3 and compound 5 were taken under conditions of excitation wavelengths of 470-495 nm and fluorescence wavelengths of 510-550 nm.

(B) Results When imaging is performed under the above conditions, it can be seen that all nucleated cells are fluorescently stained with Hoechst 3342 and white blood cells are fluorescently stained with anti-CD45 (APC label). Therefore, cells that are fluorescently stained with Hoechst 33342 and fluorescently stained with anti-CD45 antibody (APC label) are leukocytes, and cells that are fluorescently stained with Hoechst 33342 and not fluorescently stained with anti-CD45 antibody (APC label) are PC−. It turns out that it is 9 cells. As a result, the fluorescence intensity derived from Compound 3 and Compound 5 emitted from PC-9 cells increased with time. On the other hand, the fluorescence intensity derived from compound 3 and compound 5 emitted by white blood cells is very small, and it is possible to detect cancer cells from a population of white blood cells from the difference in fluorescence intensity between PC-9 cells and white blood cells. It turns out that.

Example 13
Under the following conditions, the 4H-chromone derivative (1) (compound 3, compound 5) of the present invention was brought into contact with a cancer cell immobilized on a cytodiagnosis chip, and the fluorescence detection ability of the cancer cell was evaluated.

(A) Method Non-small cell lung cancer cell PC-9 cells derived from human lung adenocarcinoma in D-MEM / Ham's F-12 medium containing 10% FBS at 37 ° C. in a 5% CO ₂ atmosphere. In culture. After the cells reached subconfluence, they were washed with PBS (phosphate buffered saline), and the cells were detached using trypsin-EDTA so that the individual cells were separated. The treated cells were centrifuged (200 × g, 25 ° C., 5 minutes), the supernatant was discarded, and the cells were suspended in a 300 mM mannitol aqueous solution so that the cell density was 0.5 × 10 ⁵ cells / mL.

  As shown in FIGS. 10 and 11, the container for observing cells is an insulator having a plurality of fine holes with a diameter of 30 μm and a depth of 30 μm arranged in an array with a hole interval of 100 μm, and an insulator and a lower part. A biological sample fixing device (hereinafter, sometimes referred to as a “cell diagnostic chip”) having a structure in which a holding portion made of a light-shielding chromium film placed between electrode substrates is sandwiched between a spacer and a lower electrode substrate Using.

  When the adjusted cell suspension is introduced into the cell diagnostic chip and a rectangular wave AC voltage having a voltage of 20 Vpp and a frequency of 3 MHz is applied between the electrodes as an AC voltage from the signal generator, the dielectrophoretic force acts on the cells for 1 minute. The cells could be fixed one by one in a plurality of micropores arranged in an array in about the time. “Fixed” means the case where cells entered the micropores, and the same definition was used in the following examples.

  A 300 mM mannitol aqueous solution containing 0.01% poly-L-lysine was introduced into a cytodiagnostic chip in which approximately one cell was fixed in one micropore while continuing to apply an AC voltage, and allowed to stand for 2 minutes. Thereafter, the application of the AC voltage is stopped, the solution in the cytodiagnostic chip is sucked and removed, and PBS (phosphate buffered saline) is immediately introduced into the cytodiagnostic chip, and poly-L-lysine in the cytodiagnostic chip is removed. By washing, cells could be electrostatically bound into the micropores. In addition, since the cells are electrostatically bound in the micropores due to the action of poly-L-lysine, they are not detached from the micropores by washing.

  Subsequently, the solution in the cytodiagnostic chip was removed by suction, and PBS (phosphate buffered saline) containing 5 μM compound 3 or compound 5 was introduced. The fluorescence image of the cytodiagnostic chip was measured with an EM-CCD camera through an inverted research microscope (IX71, manufactured by Olympus Corporation) under the conditions of an excitation wavelength of 470-495 nm and a fluorescence wavelength of 510-550 nm. Images were taken when 60 minutes and 120 minutes passed (Compound 3: FIG. 12A, Compound 5: FIG. 12B).

(B) Results As a result, the fluorescence intensity emitted by the cells increased with time. The labeling rate of PC-9 cells was 100% for both Compound 3 and Compound 5. The “labeling rate” means the proportion of cells that fluoresce among the cells present in the field of view of the fluorescent image photograph, and the same definition is used in the following examples.

Example 14
Under the following conditions, the 4H-chromone derivative (1) (compound 3, compound 5) of the present invention is brought into contact with a mixture of cancer cells and white blood cells immobilized on a cytodiagnostic chip, and the fluorescence detection ability of cancer cells is improved. evaluated.

(A) Method 3 mL of healthy human blood collected in an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo) was diluted with an equal amount of physiological saline to obtain a total of 6 mL of diluted blood. 6 mL of the pre-diluted blood was layered on 2 mL of Ficoll-Paque PREMIUM (manufactured by GE Healthcare Japan), and centrifuged (1100 × g, 10 minutes, 25 ° C.) to recover the white blood cell layer. The collected white blood cells are suspended in physiological saline to make a total of 30 mL, centrifuged (300 × g, 5 minutes, 25 ° C.), the supernatant is discarded, and then suspended in physiological saline again to make a total of 30 mL and centrifuged ( (300 × g, 5 minutes, 25 ° C.) and discarding the supernatant, platelet removal and white blood cell washing were simultaneously performed. Next, the obtained white blood cells are suspended in PBS (phosphate buffered saline), 10 μg / mL Hoechst 33342 (manufactured by Dojindo Laboratories) is added to stain the cell nucleus, and at the same time, anti-CD-45 antibody (APC Labeling) (Miltenyi Biotech) was added to perform cell surface staining. Subsequently, the stained white blood cells were suspended in a 300 mM aqueous mannitol solution to obtain a white blood cell suspension.

Subsequently, after PC-9 cells of non-small cell lung cancer cells derived from human lung adenocarcinoma cultured in the same manner as in Example 13 reached subconfluence, they were washed with PBS (phosphate buffered saline), and trypsin-EDTA. Was used to separate the cells so that the individual cells were separated. 10 μg / mL Hoechst 33342 was added to the treated cells, and cell nuclei were stained. The cells stained with cell nuclei were centrifuged (200 × g, 25 ° C., 5 minutes), the supernatant was discarded, and the cells were suspended in 300 mM mannitol aqueous solution so as to have a cell density of 1 × 10 ⁵ cells / mL. A cell suspension was prepared.

Subsequently, the PC-9 cell suspension was adjusted so that the cell density of PC-9 cells was 0.5 × 10 ⁵ cells / mL and the cell density of white blood cells was 3.5 × 10 ⁵ cells / mL. The white blood cell suspension was mixed to prepare a mixture of cancer cells and white blood cells.

When the mixture of cancer cells and white blood cells is introduced into a cytodiagnosis chip and a rectangular wave AC voltage having a voltage of 20 Vpp and a frequency of 3 MHz is applied between the electrodes as an AC voltage, a dielectrophoretic force acts on the cells for about 1 minute. It was possible to fix the white blood cells and PC-9 cells in the plurality of micropores arranged in an array at the above time. The total cell density of the white blood cells and PC-9 cells introduced into the cytodiagnostic chip is equal to the density of the micropores (4.0 × 10 ⁵ cells / mL). One white blood cell or PC-9 cell was fixed.

  While applying an alternating voltage to the cytodiagnosis chip on which the cells are fixed, a 300 mM mannitol aqueous solution containing 0.01% poly-L-lysine is introduced, and after standing for 2 minutes, the application of the alternating voltage is stopped, The solution in the cytodiagnostic chip is aspirated and removed. Immediately, PBS (phosphate buffered saline) is introduced into the cytodiagnostic chip, and the poly-L-lysine in the cytodiagnostic chip is washed, so that the cells enter the micropores. Could be electrostatically coupled. In addition, since leukocytes and PC-9 cells are electrostatically bound in the micropores by the action of poly-L-lysine, they are not detached from the micropores by washing.

  Subsequently, the solution in the cytodiagnostic chip was removed by suction, and PBS (phosphate buffered saline) containing 5 μM compound 3 or compound 5 was introduced. The fluorescence image of the cytodiagnosis chip was imaged with an EM-CCD camera through an inverted research microscope (IX71, Olympus) at 0, 15, 30, 60, 120 minutes (Compound 3: FIG. 13A, Compound 5: FIG. 13B). In addition, the fluorescence image derived from the anti-CD45 antibody (APC label) is imaged under the conditions of the excitation wavelength 600-650 nm and the fluorescence wavelength 670-720 nm, and the fluorescence image derived from the Hoechst 33342 has the excitation wavelength 360-370 nm and the fluorescence wavelength 420-460 nm. Images were taken under conditions, and fluorescence images derived from compound 3 and compound 5 were taken under conditions of excitation wavelengths of 470-495 nm and fluorescence wavelengths of 510-550 nm. When imaging is performed under the above conditions, it can be seen that all nucleated cells are fluorescently stained with Hoechst 3342 and white blood cells are fluorescently stained with anti-CD45 (APC label). Therefore, cells that are fluorescently stained with Hoechst 33342 and fluorescently stained with anti-CD45 antibody (APC label) are leukocytes, and cells that are fluorescently stained with Hoechst 33342 and not fluorescently stained with anti-CD45 antibody (APC label) are PC- It turns out that it is 9 cells.

(B) Result As a result, the fluorescence intensity derived from Compound 3 and Compound 5 emitted from PC-9 cells increased with time. On the other hand, the fluorescence intensity derived from the compound 3 and the compound 5 emitted from the white blood cells is very small, and because of the difference in fluorescence intensity between the PC-9 cells and the white blood cells, the cancer cells from the group of white blood cells fixed on the cytodiagnosis chip. It was found that it was possible to detect specifically.

1: Lower electrode substrate 2: Upper electrode substrate 3: Micro hole 4: Insulator 5: Light shielding member 6: Spacer 7: Inlet 8: Outlet 9: Holding portion 10: Signal generator 11: Conductor 12: Biological sample fixation Equipment (cell diagnostic chip)
13: Electric field line 14: Dielectrophoretic force 15: Biological sample

Claims (8)

General formula (1)

(In the formula, R ² and R ³ each independently represent an alkyl group having 1 to 3 carbon atoms. R ^4-1 , R ^4-2 , R ^4-3 , R ^4-4 , R ^5-1 , R ^5-2 and R ^5-3 each independently represent a hydrogen atom, a halogen atom or a methoxy group, R ⁶ represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an aralkyl group having 7 or 8 carbon atoms, or a benzoyl group R ⁷ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, R ⁸ , R ⁹ and R ¹⁰ each independently represents a hydrogen atom, a nitro group or a trifluoromethyl group, provided that R ⁸ , At least one of R ⁹ and R ¹⁰ represents a nitro group, n represents 0 or 1, m represents 0 or 1, and k represents an integer of 1 to 12). Derivative. The 4H-chromone derivative according to claim 1, wherein R ⁸ is a nitro group or a trifluoromethyl group, R ⁹ is a nitro group, R ¹⁰ is a hydrogen atom, and k is an integer of 1 to 6. R ² and R ³ are methyl groups, R ^4-1 , R ^4-2 , R ^4-3 , R ^4-4 , R ^5-1 , R ^5-2 and R ^5-3 are hydrogen atoms, Item 4H-chromone derivative according to item 1 or 2. General formula (2)

(In the formula, R ² and R ³ each independently represent an alkyl group having 1 to 3 carbon atoms. R ^4-1 , R ^4-2 , R ^4-3 , R ^4-4 , R ^5-1 , R ^5-2 and R ^5-3 each independently represent a hydrogen atom, a halogen atom or a methoxy group, R ⁶ represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an aralkyl group having 7 or 8 carbon atoms, or a benzoyl group N represents 0 or 1, m represents 0 or 1, k represents an integer of 1 to 12, and an amine derivative represented by the general formula (3) is present in the presence of a base.

(Wherein R ⁸ , R ⁹ and R ¹⁰ each independently represents a hydrogen atom, a nitro group or a trifluoromethyl group, provided that at least one of R ⁸ , R ⁹ and R ¹⁰ represents a nitro group. X represents a halogen atom) and is reacted with an arylsulfonyl compound represented by the general formula (1a)

^{(Wherein, R 2, R 3, R} 4-1, R 4-2, R 4-3, R 4-4, R 5-1, R 5-2, R 5-3, R 6, R 8 , R ⁹ , R ¹⁰ , n, m, and k have the same meanings as described above). General formula (1)

(In the formula, R ² and R ³ each independently represent an alkyl group having 1 to 3 carbon atoms. R ^4-1 , R ^4-2 , R ^4-3 , R ^4-4 , R ^5-1 , R ^5-2 and R ^5-3 each independently represent a hydrogen atom, a halogen atom or a methoxy group, R ⁶ represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an aralkyl group having 7 or 8 carbon atoms, or a benzoyl group R ⁷ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, R ⁸ , R ⁹ and R ¹⁰ each independently represents a hydrogen atom, a nitro group or a trifluoromethyl group, provided that R ⁸ , At least one of R ⁹ and R ¹⁰ represents a nitro group, n represents 0 or 1, m represents 0 or 1, and k represents an integer of 1 to 12). A fluorescent probe comprising a derivative. General formula (1)

(In the formula, R ² and R ³ each independently represent an alkyl group having 1 to 3 carbon atoms. R ^4-1 , R ^4-2 , R ^4-3 , R ^4-4 , R ^5-1 , R ^5-2 and R ^5-3 each independently represent a hydrogen atom, a halogen atom or a methoxy group, R ⁶ represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an aralkyl group having 7 or 8 carbon atoms, or a benzoyl group R ⁷ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, R ⁸ , R ⁹ and R ¹⁰ each independently represents a hydrogen atom, a nitro group or a trifluoromethyl group, provided that R ⁸ , At least one of R ⁹ and R ¹⁰ represents a nitro group, n represents 0 or 1, m represents 0 or 1, and k represents an integer of 1 to 12). Glutathione, characterized by using a derivative A method for detecting S-transferase. General formula (1)

(In the formula, R ² and R ³ each independently represent an alkyl group having 1 to 3 carbon atoms. R ^4-1 , R ^4-2 , R ^4-3 , R ^4-4 , R ^5-1 , R ^5-2 and R ^5-3 each independently represent a hydrogen atom, a halogen atom or a methoxy group, R ⁶ represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an aralkyl group having 7 or 8 carbon atoms, or a benzoyl group R ⁷ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, R ⁸ , R ⁹ and R ¹⁰ each independently represents a hydrogen atom, a nitro group or a trifluoromethyl group, provided that R ⁸ , At least one of R ⁹ and R ¹⁰ represents a nitro group, n represents 0 or 1, m represents 0 or 1, and k represents an integer of 1 to 12). Method for detecting cancer cells, characterized by using a derivative Law.   The method for detecting cancer cells according to claim 7, wherein cancer cells mixed in normal cells are detected.

Images