JP2014132001A - Novel cell penetration type fluorescent dye - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescent dye molecule capable of being applied as a skeleton of a cell introduction type probe corresponding to novel multicolor imaging, and to provide a synthesis method and a purification method.SOLUTION: There is provided a compound having 7-hydroxy quinoline skeleton represented by the formula (I), a derivative thereof or a salt thereof. In the formula (I), Ris H, a carbonyl group and the like, Ris null or Calkyl, alkylphenyl, Ris H, Calkyl, carbonyl, alkylcarbonyl or alkylamide, Xand Xare each independently selected from (C)alkyl, aryl(C)alkyl, (C)cycloalkyl, hetero(C)cycloalkyl, heteroaryl(C)alkyl, aryl and heteroaryl, which is each substituted or unsubstituted, n is 0 to 3 and m is 0 to 2.

Description

The present invention relates to a novel cell-permeable fluorescent dye. In particular, it relates to a fluorescent probe that supports multi-color imaging.

In the prior art, most of the fluorescent dyes are compounds with small Stokes shift, so it was difficult to use them together in multicolor imaging. In addition, there are rare dye skeletons that can control the fluorescent behavior by slightly changing the molecular design. there were.

Since the 7-hydroxyquinoline skeleton has a large Stokes shift, it is suitable for multicolor imaging. In addition, since the fluorescence characteristics vary depending on the structure of the hydroxyl group at the 7-position, examples of using it extracellularly as a substrate for phosphatase activity have been reported. (Patent Document 1)

ES2246134A1

Francisco Caturla et al, New fluorescent probes for testing combinatorial catalysts with phosphodiesterase and esterase activities., Tetrahedron, 2004, Vol. 60, No. 8, p.1903-1911 In Kyung Rhee et al, Determining Acetylcholinesterase Inhibitory Activity in Plant Extracts Using a Fluorimetric Flow Assay., Phytochem. Anal., 2003, Vol. 14, No. 3, p.145-149

Since the 7-hydroxyquinoline skeleton itself does not penetrate the cell membrane, it was difficult to apply it to a cell-introduced fluorescent probe.

Under such circumstances, it is required to develop a fluorescent dye molecule having a new skeleton, to develop a synthesis method and a purification method thereof, and to apply it as a skeleton of a cell introduction type probe.

Accordingly, the present invention includes the following compounds, derivatives thereof or salts thereof (
Provided are fluorescent compositions (for cell introduction), cell labeling methods using these compounds, and methods for producing these compounds.
[1] A compound represented by the following formula (I), a derivative thereof, or a salt thereof:


(Where
R ₁ is a hydrogen atom, a carbonyl group, a C _1-6 alkylcarbonyl group, or an alkyl ester (such as phosphoric acid);
R ₂ is absent or is C _1-6 alkyl, alkylphenyl, alkylcarbonyl, or alkylamide;
R ₃ is a hydrogen atom, C _1-6 alkyl optionally substituted with a substituent, carbonyl, alkylcarbonyl, or alkylamide;
X ₁ and X ₂ are each independently (C _1-3 ) alkyl, aryl (C _1-3 ) alkyl, (C _3-12 ) cycloalkyl, hetero (C _3-12 ) cycloalkyl, heteroaryl ( C _1-3 ) selected from the group consisting of alkyl, aryl and heteroaryl (each substituted or unsubstituted),
n is 0, 1, 2, or 3;
m is 0, 1, or 2.
[2] R ₁ is a hydrogen atom or a methylcarbonyl group,
R ₂ is absent or is methyl;
R ₃ is methyl ethyl formate;
X ₂ is methyl,
n is 0,
The compound according to [1], a derivative thereof, or a salt thereof, wherein m is 1.
[3] A compound represented by the following formula (II), a derivative thereof, or a salt thereof:

(Where
R ₁ is a hydrogen atom, a carbonyl group, a C _1-6 alkylcarbonyl group, or an alkyl ester (such as phosphoric acid);
R ₂ is absent or is C _1-6 alkyl, alkyl-phenyl, alkyl-carbonyl, or alkyl-amide. ).
[4] Ethyl 2- (7-hydroxy-2-methylquinolin-4-yl) acetate (ethy
l 2- (7-hydroxy-2-methylquinolin-4-yl) acetate);
Ethyl 2- (7-acetoxy-2-methylquinolin-4-yl) acetate (ethyl 2-
(7-acetoxy-2-methylquinolin-4-yl) acetate);
7-acetoxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium;
7-hydroxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium;
These derivatives, or salts thereof.
[5] 7-acetoxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium ;
7-hydroxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium;
These derivatives, or salts thereof.
[6] A fluorescent composition comprising the compound according to any one of [1] to [5], a derivative thereof, or a salt thereof.
[7] A fluorescent composition for introduction into a cell comprising a compound represented by the following formula (III), a derivative thereof, or a salt thereof:


(Wherein R ₁ is a hydrogen atom, a carbonyl group, a C _1-6 alkylcarbonyl group, or an alkyl ester (such as phosphoric acid)),
R ₂ is absent or is C _1-6 alkyl, alkylphenyl, alkylcarbonyl, or alkylamide. ).
[8] R ₁ is a hydrogen atom or a methylcarbonyl group,
The fluorescent composition for intracellular introduction according to [7] above, wherein R ₂ is absent or is methyl.
[9] 7-acetoxy-1-methylquinolinium;
7-hydroxy-1-methylquinolinium;
A fluorescent composition for intracellular introduction, comprising one or more compounds selected from the group consisting of these derivatives and salts thereof, derivatives thereof, or salts thereof.
[10] The compound according to any one of the above [1] to [5], a derivative thereof, or a salt thereof, for labeling or imaging of a cell, or for use as a detection probe for intracellular enzyme activity, The fluorescent composition according to any one of [6] to [9].
[11] A method for labeling cells,
A cell, the compound according to any one of the above [1] to [5], a derivative thereof, or a salt thereof, or the fluorescent composition for intracellular introduction according to any one of the above [6] to [9] Mixing in a solution and incubating for a predetermined time.
[12] Ethyl 2- (7-hydroxy-2-methylquinolin-4-yl) acetate comprising mixing m-aminophenol and 3-oxobutyric acid ethyl ester in the presence of BiCl ₃ -(7-hydroxy-2-methylquinolin-4-yl) acetate) synthesis method.

According to the present invention, a novel derivative of 7-hydroxyquinoline (hereinafter sometimes abbreviated as “HQ”) is provided. As an embodiment of the present invention, the present inventors synthesized a novel derivative obtained by chemically modifying the 2,4-position and N of 7-hydroxyquinoline, and protecting / deprotecting the hydroxyl group of this compound with an arbitrary structure. It was found that by changing the chemical structure, a fluorescent probe that can be spontaneously introduced into a cell and control ON / OFF of fluorescence can be designed. This probe has the advantage that the stokes shift is very large, is compatible with multi-color imaging, and has a high fluorescence quantum yield.

Specifically, for example, ethyl 2- (7-hydroxy-2-methylquinolin-4-yl) acetate (may be abbreviated as “meHQ” in the present specification) is provided as a novel derivative. Normally, 7-hydroxyquinoline (HQ) skeleton cannot be introduced into cells as it is, but the membrane properties are improved by acetylating the hydroxyl group of this new derivative to change its properties so that it does not emit fluorescence, and by N-methylation. The dye 7-acetoxy-4- (2-ethoxy-2-oxoethyl) -1,2- which penetrates the cell membrane and emits yellow fluorescence only inside the cell.
Dimethylquinolinium (in this specification, sometimes abbreviated as “mmeHQ-Ac”) is obtained.

According to the present invention, there is provided a fluorescent probe that is spontaneously introduced into a cell and can control ON / OFF of fluorescence.

According to the present invention, it is possible to develop a fluorescent probe corresponding to multi-color imaging by easy design and synthesis.

It is a figure which shows the compound which concerns on one Embodiment of this invention, and its derivative (s). It is a figure which shows an example of the synthesis method of the derivative | guide_body which concerns on one Embodiment of this invention. (A) 7-acetoxy-1-methylquinolinium (mHQ-Ac) and (B) 7-acetoxy-4- (2-ethoxy-2-oxoethyl) -1,2 according to one embodiment of the present invention FIG. 2 is a graph showing absorption spectra and fluorescence spectra before and after hydrolysis of dimethylquinolinium (mmeHQ-Ac). When both mHQ-Ac and mmeHQ-Ac are hydrolyzed, the absorption wavelength shifts by a long wavelength, and what is non-fluorescent emits yellow fluorescence having a maximum wavelength around 500 nm. (A) 7-acetoxy-1-methylquinolinium and (B) 7-acetoxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium according to an embodiment of the present invention It is a figure which shows a fluorescence-microscope observation result when each is added to the cell. (A) 7-acetoxy-1-methylquinolinium and (B) 7-acetoxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium according to an embodiment of the present invention It is a figure which shows the flow cytometry result when each is added to the cell. The figure which shows the flow cytometry result when 7-acetoxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium which concerns on one Embodiment of this invention is added to a cell, and PI dyeing | staining is carried out. It is.

In one embodiment, the present invention provides a compound represented by the following formula (I), a derivative thereof, or a salt thereof:


(Where
R ₁ is a hydrogen atom, a carbonyl group, a C _1-6 alkylcarbonyl group, or an alkyl ester;
R ₂ is absent or is C _1-6 alkyl, alkylphenyl, alkylcarbonyl, or alkylamide;
R ₃ is a hydrogen atom, C _1-6 alkyl optionally substituted with a substituent, carbonyl, alkylcarbonyl, or alkylamide;
X ₁ and X ₂ are each independently C _1-3 alkyl, aryl (C _1-3 ) alkyl, C _3-12 cycloalkyl, hetero (C _3-12 ) cycloalkyl, heteroaryl (C _1-3 ) Selected from the group consisting of alkyl, aryl and heteroaryl (each substituted or unsubstituted);
n is 0, 1, 2, or 3;
m is 0, 1, or 2. Typically, the compound is a fluorescent dye.

Examples of the “C _1-6 alkyl” in the C _1-6 alkylcarbonyl group represented by R ₁ include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the like. Is mentioned.

Examples of the “alkyl” of the alkyl ester represented by R ₁ include C _1-6 alkyl, and examples of the C _1-6 alkyl include the same as those described above. Examples of esters include phosphate esters.

As the _{C 1-6} alkyl represented by R _2, include those for _{R 1} in the same manner as the above examples of _{C 1-6} alkyl.

As alkyl of alkylphenyl, alkylcarbonyl, and alkylamide represented by R ₂ , for example, C _1-6 alkyl can be mentioned, and as C _1-6 alkyl, for example, the same as those described above for R ₁ Things.

“C ₁ in the“ C _1-6 alkyl _optionally substituted with a substituent ”represented by R ₃
Examples of “ _-6 alkyl” include the same as those described above for R ₁ .

Examples of the “substituent” in the “C _1-6 alkyl which may be substituted with a substituent” represented by R ₃ include a carboxylic acid ester, a phosphoric ester, etc. to which a substituent may be added. Is included.

Examples of alkylcarbonyl represented by R ₃ and alkyl of alkylamide include C _1-6 alkyl, and examples of C _1-6 alkyl include the same as those described above for R _1. It is done.

Examples of the substituent that may be added in the “carboxylic acid ester to which a substituent may be added” include a hydrogen atom, C _1-6 alkyl (eg, methyl, ethyl, propyl, etc.), phenyl, and the like. Etc. are included.

Examples of C _1-3 alkyl represented by X ₁ or X ₂ include methyl, ethyl, propyl, isopropyl and the like.

In a preferred embodiment, R ₁ is a hydrogen atom or a methylcarbonyl group, R ₂ is absent or is methyl, R ₃ is methyl ethyl formate, X ₂ is methyl, and n is 0 And m is 1.

According to a further preferred embodiment of the present invention, a compound represented by the following formula (II), a derivative thereof, or a salt thereof:

(Where
R ₁ is a hydrogen atom, a carbonyl group, a C _1-6 alkylcarbonyl group, or an alkyl ester (eg, alkyl phosphate ester);
R ₂ is absent or is C _1-6 alkyl, alkylphenyl, alkylcarbonyl, or alkylamide. ) Is provided.

Preferred embodiments are:
Ethyl 2- (7-hydroxy-2-methylquinolin-4-yl) acetate,
Ethyl 2- (7-acetoxy-2-methylquinolin-4-yl) acetate,
7-acetoxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium,
7-hydroxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium derivatives thereof or salts thereof.

An even more preferred embodiment is
7-acetoxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium,
7-hydroxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium,
These derivatives or their salts.

The most preferred embodiment is 7-acetoxy-4- (2-ethoxy-2-oxoethyl)
-1,2-dimethylquinolinium.

According to still another embodiment of the present invention, there is provided a fluorescent composition comprising the above compound, a derivative thereof, or a salt thereof. In preferred embodiments, these fluorescent compositions are used for labeling or imaging of cells or as detection probes for intracellular enzyme activity.

According to still another embodiment of the present invention, a fluorescent composition for intracellular introduction comprising a compound represented by the following formula (III), a derivative thereof, or a salt thereof:


(Wherein R ₁ is a hydrogen atom, a carbonyl group, a C _1-6 alkylcarbonyl group, or an alkyl ester (eg, alkyl phosphate ester, etc.);
R ₂ is absent or is C _1-6 alkyl, alkylphenyl, alkylcarbonyl, or alkylamide. ) Is provided.

In a preferred embodiment, R ₁ is a hydrogen atom or a methylcarbonyl group and R ₂ is absent or is methyl.

In a more preferred embodiment, one or more compounds selected from the group consisting of 7-acetoxy-1-methylquinolinium, 7-hydroxy-1-methylquinolinium, derivatives thereof, and salts thereof, Provided is a fluorescent composition for intracellular introduction comprising a derivative or a salt thereof. These fluorescent compositions for introduction into cells are preferably used for labeling or imaging of cells or as detection probes for intracellular enzyme activity.

According to another embodiment of the present invention, the method includes a step of mixing a cell and the compound, a derivative thereof or a salt thereof, or the fluorescent composition for introduction into the cell in a solution and incubating for a predetermined time. A cell labeling method is provided.

According to yet another embodiment, ethyl 2- (7-hydroxy-2-methylquinoline-4-) comprising mixing m-aminophenol and 3-oxobutyric acid ethyl ester in the presence of BiCl _3. Yl) acetate (ethyl 2- (7-hydroxy-2-methylquinolin-4-yl) ace
tate) is provided.

In an exemplary embodiment of the present invention, a substituent in which R ₁ of the fluorescent dye represented by formula (I), (II), or (III) is deprotected from a hydrogen atom by a chemical reaction with any enzyme or the like. It is rendered cell permeable by chemical modification (for example, carbonyl group), introduced into cells, and used for imaging of cells.

EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited to these Examples.

As shown in FIG. 2, various derivatives of HQ and meHQ were synthesized.
[Reference Example 1]

Synthesis of quinolin-7-yl acetate (HQ-Ac)


7-hydroxyquinoline 200 mg (1.38 mmol), dichloromethane 60 ml, triethylamine
4 ml was placed in a 50 ml eggplant flask and stirred at room temperature for 30 minutes. After transferring the reaction solution to an ice bath, ace
tylchloride 0.2 ml (2.46 mmol) was added dropwise, and the mixture was stirred as it was for 7.5 hours. After completion of the reaction, water was added and extracted with dichloromethane. The oil layer was washed with water and brine, and the solvent was distilled off. The product was dissolved in ethyl acetate and separated and purified by column chromatography (hexane: ethyl acetate = 1: 1) to obtain 175 mg of the desired product (yield 68%).

¹ H NMR (270 MHz, CDCl ₃ )
8.91 (dd, 4.3, 1.6 Hz, 1H), 8.16-8.13 (m, 1H), 7.83-7.81 (m, 2H), 7.40-7.31 (m, 2H
), 2.38 (s, 3H)
[Reference Example 2]

7-acetoxy-1-methylquinolinium (mHQ-A
c) Synthesis

7HQ-Ac 73 mg (0.39 mmol) in dichloromethane (15 ml) was placed in a 50 ml eggplant flask, methyl iodide (1.5 ml) was added, and the mixture was stirred at 45 ° C. for 24 hours. The reaction was stopped after confirming the formation of a precipitate. The precipitate was filtered with suction, washed with dichloromethane and dried in vacuo to obtain the desired product.

¹ H NMR (270 MHz, CD ₃ OD)
9.38 (d, 5.9 Hz, 1H), 9.21 (d, 8.1Hz, 1H), 8.48 (d, 8.9 Hz, 1H), 8.36 (d, 1.6 Hz, 1H
), 8.06 (dd, 5.9, 8.4 Hz, 1H), 7.89 (dd, 8.9, 1.9 Hz, 1H), 4.65 (s, 3H), 2.43 (s, 3H
)
[Reference Example 3]

7-hydroxy-1-methylquinolinium (mHQ)
Synthesis of

7HQ 110 mg (0.76 mmol) and dichloromethane 30 ml were placed in a 50 ml eggplant flask, methyl iodide 8.5 ml (60 mmol) was added, and the mixture was stirred at 35 ° C. for 43 hours. After confirming the formation of a precipitate, the reaction was stopped and the precipitate was suction filtered to obtain a powder. This was separated and purified by column chromatography (dichloromethane: methanol = 3: 1) to obtain 71 mg of the desired product (yield 33%).

¹ H NMR (270 MHz, CD ₃ OD)
8.54 (d, 6.2 Hz, 1H), 8.47 (d, 7.8 Hz, 1H), 7.85 (d, 9.2 Hz, 1H), 7.22-7.11 (m, 2H),
6.78 (s, 1H), 4.17 (s, 1H)

Ethyl 2- (7-hydroxy-2-methylquinolin-4-yl) acetate (ethyl 2-
Synthesis of (7-hydroxy-2-methylquinolin-4-yl) acetate) (meHQ)

A mixture of m-aminophenol (550 mg, 5 mmol) and BiCl ₃ was added to 3-
The mixture was stirred in oxobutyric acid ethyl ester (79 mg, 0.25 mmol) at 75 ° C. for 1 hour. The reaction mixture was extracted with chloroform and washed with water. The organic layer was collected and dried over MgSO ₄ . After the solvent can be evaporated, the residue is purified by flash chromatography (Si
O ₂ , hexane-AcOEt 1: 1), ethyl 2- (7-hydroxy-2-methylquinolin-4-yl) acetate (meHQ) 194 mg was obtained in a yield of 11%.

¹ H NMR (270 MHz, CDCl ₃ ): δ 7.50 (d, J = 8.9 Hz, 1H), 7.15 (d, J = 2.4 Hz, 1H), 6
.99 (s, 1H), 6.57 (dd, J = 8.9, 2.2 Hz, 1H), 4.25 (q, J = 7.0 Hz, 2H), 3.97 (s,
2H), 2.60 (s, 3H), 1.31 (t, J = 7.0 Hz, 3H); 13C NMR (67.5 MHz, CD3Cl): δ 170.
2, 159.6, 157.7, 148.0, 140.9, 124.2, 120.1, 119.5, 119.0, 108.1, 61.6, 38.5, 28
.3, 14.4. MS calcd for C14H15NO3 245.1; (ESI) m / z found 246.4 (M + H) +. Anal. Calcd
for C14H15NO3: C, 68.56; H, 6.16; N; 5.71.Found: C, 68.48; H, 6.22; N, 5.55.m
p 205.

Ethyl 2- (7-acetoxy-2-methylquinolin-4-yl) acetate (ethyl 2-
Synthesis of (7-acetoxy-2-methylquinolin-4-yl) acetate) (meHQ-Ac)

Triethylamine (0.27 ml, 1.95 mmol) was added to meHQ (200 mg, 0.7
9 mmol) in dichloromethane (200 ml) and stirred for 20 minutes at room temperature.
Subsequently, acetyl chloride (0.11 ml, 1.56 mmol) was added dropwise to this mixture at 0 ° C., and the mixture was stirred for 2 hours. The reaction was stopped by adding water and extracted with dichloromethane. The organic layer was washed with water and brine. After evaporation of the solvent, the residue was purified by flash chromatography (SiO _2, hexane-AcOEt 1: 1) to give the MEHQ-Ac of 207 mg 92
% Yield.

¹ H NMR (270 MHz, CDCl ₃ ): δ; 7.95 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 2.4 Hz, 1H), 7
.29 (dd, J = 8.9, 2.4 Hz, 1H), 7.21 (s, 1H), 4.16 (q, J = 7.0Hz, 2H), 3.99 (s, 2H),
2.71 (s, 3H), 2.36 (s, 3H), 1.23 (t, J = 7.0 Hz, 3H); 13C NMR (67.5 MHz, CD3Cl)
: δ. 169.7, 168.9, 159.4, 151.0, 148.6, 139.8, 124.4, 123.4, 123.1, 121.0, 120.
1, 61.3, 38.4, 25.2, 21.2, 14.1. Anal.Calcd for C16H17NO4: C, 66.89; H, 5.96;
N; 4.88. Found: C, 66.71; H, 6.05; N; 4.76.

7-acetoxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinori
Synthesis of Ni (mmeHQ-Ac)

meHQ-Ac (100 mg, 0.34 mmol) and methyl iodide (0.5 ml, 8.03 m)
mol) was heated at 70-80 ° C. for 1 hour in a sealed tube. The resulting precipitate was collected by filtration and washed thoroughly with cold AcOEt. The crude product is ethanol /
Purification by recrystallization from diethyl ester gave 41 mg mmeHQ-Ac in 28% yield.

¹ H NMR (270 MHz, CDCl ₃ ): δ; 8.28 (d, J = 9.2 Hz, 1H), 8.28 (s, 1H), 7.99 (s, 1H),
7.71 (d, J = 9.2 Hz, 1H), 4.61 (s, 3H), 4.31 (s, 2H), 4.20 (q, J = 7.0Hz, 2H), 3.31 (
s, 3H), 2.45 (s, 3H), 1.28 (t, J = 7.0 Hz, 3H); 13C NMR (CD3OD, 67.5 MHz): δ.168.
3, 167.9, 160.5, 155.5, 150.9, 140.7, 128.2, 127.1, 125.5, 124.9, 111.9, 62.4, 4
2.3, 39.1, 25.5, 21.6, 14.3. Anal.Calcd for C17H20NO4 + I-: C, 47.57; H, 4.70; N
; 3.26. Found: C, 47.43; H, 4.60; N; 3.10

7-Hydroxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinori
Synthesis of Ni (mmeHQ)

meHQ (100 mg, 0.41 mmol) and methyl iodide (0.5 ml, 8.03 mmo)
The mixture with l) was heated in a sealed tube at 60 ° C. in dichloromethane (2 ml) for 19 hours. The purified precipitate was collected by filtration. The crude product was purified by recrystallization from dichloromethane to give 64 g mmeHQ in 40% yield.

¹ H NMR (270 MHz, CD ₃ OD): δ; 8.29 (d, J = 9.2 Hz, 1H), 7.70 (s, 1H), 7.57 (d, J = 2
.2Hz, 1H), 7.48 (dd, J = 9.2, 2.2 Hz, 1H), 4.32 (s, 3H), 4.19 (q, J = 7.0 Hz, 2H),
3.02 (s, 3H), 1.25 (t, J = 7.0 Hz, 3H); 13C NMR (CD3OD, 67.5 MHz): δ. 170.1, 165.3
, 159.6, 152.2, 143.8, 130.1, 124.2, 123.3, 122.0, 102.5, 62.9, 40.0, 30.8, 23.3
Anal.Calcd for C15H18NO3 + I-: C, 46.53; H, 4.69; N; 3.62. Found: C, 46.
22; H, 4.72; N; 3.35

The measurement and measurement of the absorption and fluorescence properties of mHQ-Ac, mmeHQ-Ac, and their hydrolysates were all carried out in air in a 1% DMSO aqueous solution. The following equipment was used for the measurement.
1) A Shimadzu UV-1600 type visible ultraviolet spectrophotometer was used to measure the absorption spectrum.
2) A Hitachi F-4500 fluorometer was used for the measurement of the fluorescence spectrum and the fluorescence excitation spectrum.
3) For the measurement of the fluorescence quantum yield, a Hitachi F-4500 fluorometer was used as the light source, an ethanol solution of anthracene was used as the standard sample, and the fluorescence quantum yield of anthracene (Φ _f = 0.27).
) And the relative method.

(result)
3A and 3B show the absorption / fluorescence characteristics of meHQ-Ac and mmeHQ-Ac measured before and after hydrolysis. As shown in FIG. 3A, when mHQ-Ac was hydrolyzed to mHQ, the peak of the absorption spectrum shifted from 318 nm to 405 nm, and a fluorescence peak appeared near 517 nm.
Further, as shown in FIG. 3B, when mmeHQ-Ac was hydrolyzed to mmeHQ, the absorption spectrum peak shifted from 324 nm to 405 nm, and a fluorescence peak appeared near 503 nm.

Introduction of mHQ-Ac and mmeHQ-Ac into cells (procedure)
MHQ-Ac and mmeHQ-Ac dissolved in DMSO to 1 mM in human epithelial HEp-2 cells cultured at 37 ° C. with 5% CO 2 in MEM medium containing 10% calf serum. The final concentration was 10 μM. Thereafter, continuous photographing was performed using an Olympus IX70 fluorescence microscope under conditions of an excitation filter of 460-490 nm and an absorption filter of 500 nm. After 50 minutes, the cells were washed twice with MEM medium, MEM medium was added again, and fluorescence imaging was performed under the same conditions.

(result)
As shown in the left of FIG. 4, there was not much change inside and outside the cells after the addition of meHQ-Ac, and when the extracellular fluid was washed with a buffer solution after 50 minutes, the inside of the cells was shining.
On the other hand, as shown in the right side of FIG. 4, it was observed that the inside of the cell was brightly shined immediately after the addition of mmeHQ-Ac. The bottom row in FIG. 4 is a photograph showing the localization of the dye with the magnification increased.
While meHQ-Ac was localized in the nuclear membrane, endoplasmic reticulum, nucleolus, etc., mmeHQ-Ac was distributed throughout the cell.

Flow cytometry when meHQ-Ac and mmeHQ-Ac are added to cells (procedure)
MHQ, mHQ-Ac and mmeHQ- dissolved in DMSO at 1 mM in human epithelial HEp-2 cells cultured at 37 ° C. under 5% CO 2 in MEM medium containing 10% calf serum Ac
Was added to a final concentration of 30 μM. Two hours later, the cells were suspended by trypsin treatment and analyzed by a flow cytometer Flicyme (Mitsui Engineering & Shipbuilding).

(result)
As shown in the upper column of FIG. 5, mHQ-Ac stained 60% of the cells, and mmeHQ-Ac stained 75% of the cells.
As shown in the lower column of FIG. 5, staining was not observed in cells to which no dye was added or cells to which other derivatives were added.

Flow cytometry when adding mmeHQ-Ac to cells and staining with PI (Procedure)
In a MEM medium containing 10% calf serum, mmeHQ-Ac dissolved at 1 mM in DMSO was dissolved in human epithelial HEp-2 cells cultured at 37 ° C. under 5% CO 2 at a final concentration of 10 μM.
It added so that it might become. After 12 hours, the cells are suspended by trypsin treatment to give a final concentration of 2
PI was added so that it might become 0 microgram / mL, and it analyzed by the flow cytometer FACSCalibur (BD). As a comparison with dead cells, analysis was also performed in which PI was added after floating cells were fixed in 70% ethanol.

(result)
As shown in FIG. 6, no significant change was observed in the pattern of FSC / SSC as seen in dead cells by mmeHQ-Ac treatment, indicating that the shape of the cells was not changed from that of living cells. . In addition, more than 90% of dead cells were stained with PI, whereas most cells were not stained with PI in mmeHQ-Ac-treated cells. Therefore, mmeHQ-Ac has low cytotoxicity. Indicated. Similar experiments also showed that all HQ and meHQ derivatives were not toxic under the same conditions.

(Summary)
The dye mmeHQ-Ac as a typical embodiment of the present invention is particularly excellent as a fluorescent dye in the following four points.
1) Absorption and fluorescence characteristics In the structure as it is, the absorption band is in ultraviolet light and non-fluorescence, whereas the hydrolyzed structure (7-hydroxy-4- (2-ethoxy-2-oxoethyl) -1, 2-Dimethylquinolinium (may be abbreviated as “mmeHQ” in this specification)) has an absorption band near 400 nm and emits yellow fluorescence. Further, when the fluorescence quantum yield is compared, the meHQ skeleton dye is a brighter dye than the HQ dye and is more suitable for the probe.
2) Fluorescence ON / OFF switching
When cells were brought into contact with a 10 μM solution of mmeHQ-Ac, only the cells were brightly fluorescent immediately after the addition. (In the case of mHQ-Ac, the difference in brightness inside and outside the cells was not clear at this concentration. Almost no fluorescence was observed with other derivatives.)
The intracellular localization of the dye was distributed throughout the cell in mmeHQ-Ac. (In mHQ-Ac, localization was observed in some tissues of cells.)
3) Cell membrane permeability
When a 30 μM solution of mmeHQ-Ac was added to the cells for 2 hours, 75% of the cells were stained. (60% of cells were stained with mHQ-Ac. 30% of cells were stained with mHQ. No other derivatives were stained.)
4) Cytotoxicity
When a 10 μM solution of mmeHQ-Ac was added to the cells for 12 hours, the cells were not PI-stained and the cell shape was similar to that of living cells, indicating that there was no cytotoxicity under these conditions. (All other derivatives were shown to be non-cytotoxic under similar conditions.)

It is useful as a fluorescent dye corresponding to 405 nm laser excitation, a protein label dye, a fluorescent probe for detecting the activity of a specific physiologically active substance, and the like.
The probe of the present invention can be applied as a probe for detecting any intracellular enzyme activity,
In addition, since the Stokes shift is large, it is useful for multicolor imaging.

Claims (12)

A compound represented by the following formula (I), a derivative thereof, or a salt thereof:


(Where
R ₁ is a hydrogen atom, a carbonyl group, a C _1-6 alkylcarbonyl group, or an alkyl ester;
R ₂ is absent or is C _1-6 alkyl, alkylphenyl, alkylcaribonyl, or alkylamide;
R ₃ is a hydrogen atom, C _1-6 alkyl optionally substituted with a substituent, carbonyl, alkylcarbonyl, or alkylamide;
X ₁ and X ₂ are each independently (C _1-3 ) alkyl, aryl (C _1-3 ) alkyl, (C _3-12 ) cycloalkyl, hetero (C _3-12 ) cycloalkyl, heteroaryl ( C _1-3 ) selected from the group consisting of alkyl, aryl and heteroaryl (each substituted or unsubstituted),
n is 0, 1, 2, or 3;
m is 0, 1, or 2. R ₁ is a hydrogen atom or a methylcarbonyl group,
R ₂ is absent or is methyl;
R ₃ is methyl ethyl formate;
X ₂ is methyl,
n is 0,
The compound according to claim 1, a derivative thereof, or a salt thereof, wherein m is 1. A compound represented by the following formula (II), a derivative thereof, or a salt thereof:

(Where
R ₁ is a hydrogen atom, a carbonyl group, a C _1-6 alkylcarbonyl group, or an alkyl ester;
R ₂ is absent or is C _1-6 alkyl, alkylphenyl, alkylcarbonyl, or alkylamide. ). Ethyl 2- (7-hydroxy-2-methylquinolin-4-yl) acetate;
Ethyl 2- (7-acetoxy-2-methylquinolin-4-yl) acetate;
7-acetoxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium;
7-hydroxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium;
These derivatives, or salts thereof. 7-acetoxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium;
7-hydroxy-4- (2-ethoxy-2-oxoethyl) -1,2-dimethylquinolinium;
These derivatives, or salts thereof. The fluorescent composition containing the compound in any one of Claims 1-5, its derivative (s), or those salts. A fluorescent composition for intracellular introduction, comprising a compound represented by the following formula (III), a derivative thereof, or a salt thereof:


(Wherein R ₁ is a hydrogen atom, a carbonyl group, a C _1-6 alkylcarbonyl group, or an alkyl ester;
R ₂ is absent or is C _1-6 alkyl, alkylphenyl, alkylcarbonyl, or alkylamide. ). R ₁ is a hydrogen atom or a methylcarbonyl group,
Or R ₂ is absent or is methyl, intracellular introduction fluorescent composition according to claim 7. 7-acetoxy-1-methylquinolinium;
7-hydroxy-1-methylquinolinium;
A fluorescent composition for intracellular introduction, comprising one or more compounds selected from the group consisting of these derivatives and salts thereof, derivatives thereof, or salts thereof. The compound according to any one of claims 1 to 5, a derivative thereof, or a salt thereof, or a salt according to claims 6 to 9 for labeling or imaging of a cell, or for use as a detection probe for intracellular enzyme activity. The fluorescent composition according to any one of the above. A method of labeling cells,
A cell, the compound according to any one of claims 1 to 5, a derivative thereof, or a salt thereof,
Alternatively, a method comprising mixing the fluorescent composition for intracellular introduction according to any one of claims 6 to 9 in a solution and incubating for a predetermined time. A method for synthesizing ethyl 2- (7-hydroxy-2-methylquinolin-4-yl) acetate comprising mixing m-aminophenol and 3-oxobutyric acid ethyl ester in the presence of BiCl ₃ .