WO2016143335A1 - Fluorescent compound responding to mitochondrial membrane potential - Google Patents

Fluorescent compound responding to mitochondrial membrane potential Download PDF

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WO2016143335A1
WO2016143335A1 PCT/JP2016/001281 JP2016001281W WO2016143335A1 WO 2016143335 A1 WO2016143335 A1 WO 2016143335A1 JP 2016001281 W JP2016001281 W JP 2016001281W WO 2016143335 A1 WO2016143335 A1 WO 2016143335A1
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group
compound
membrane potential
formula
mitochondrial membrane
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PCT/JP2016/001281
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French (fr)
Japanese (ja)
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川俣　純
康孝 鈴木
博紀 守友
祥平 藤木
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大塚電子株式会社
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Priority to KR1020177020226A priority Critical patent/KR20170126861A/en
Priority to AU2016230561A priority patent/AU2016230561A1/en
Publication of WO2016143335A1 publication Critical patent/WO2016143335A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
    • C07D213/38—Radicals substituted by singly-bound nitrogen atoms having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B6/00—Anthracene dyes not provided for above
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B9/00—Esters or ester-salts of leuco compounds of vat dyestuffs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers

Definitions

  • the present invention relates to a novel fluorescent compound, and more particularly, to a fluorescent compound that changes a localized location in response to a mitochondrial membrane potential.
  • the present invention also relates to a fluorescent dye composition containing the compound.
  • the present invention relates to a method for detecting a change in mitochondrial membrane potential and a method for discriminating cell viability using the fluorescent dye composition.
  • Mitochondria are cell organelles that are involved in the control of apoptosis while producing cellular energy, and involved in the life and death of cells.
  • metabolic diseases such as diabetes, cerebral infarction and myocardial infarction, neurodegenerative diseases such as Alzheimer and Parkinson's disease, and cancer are caused by mitochondrial dysfunction. Therefore, it is important to observe changes in mitochondria in order to elucidate the mechanisms of these diseases and develop therapeutic methods.
  • Mitochondrial membrane potential that accompanies energy production means a potential difference between the inside and outside of the mitochondria and represents the vitality of the mitochondria itself. That is, when there is a membrane potential, energy is produced and the mitochondrial vitality is high. On the other hand, when the membrane potential disappears, no energy is produced and the vitality is low.
  • the mitochondrial membrane potential represents the health state of a cell. When there is a membrane potential, it is a normal cell, and when there is no membrane potential, it is regarded as an abnormal cell.
  • mitochondria are stained with a dye that changes luminescence behavior according to the membrane potential, and changes in luminescence behavior are observed. There is a way to do it.
  • conventional dyes that respond to mitochondrial membrane potential. One is a type of dye whose emission intensity changes according to the mitochondrial membrane potential, and the other is a type of dye whose emission color changes.
  • the type of dye whose emission intensity changes changes the emission intensity due to the mitochondrial membrane potential and the emission intensity change due to photobleaching of the dye itself, so whether the change in fluorescence intensity is due to the change in membrane potential, It is difficult to distinguish whether it is due to photobleaching of the dye, and it cannot be said that it is suitable for detecting changes in membrane potential.
  • the excitation light source and the fluorescence detector may be at least according to the emission color. It is necessary to prepare two systems, and it is necessary to adjust the excitation light source and the fluorescence detector in real time, which increases the cost of the apparatus and makes the experimental operation extremely complicated.
  • both types of dyes have a problem that organic solvents harmful to cells must be used in order to stain cells because of low solubility.
  • the property that the compound BP moves from the mitochondria to the nucleus according to the mitochondrial membrane potential does not change the fluorescence intensity, so the change in membrane potential is detected without being affected by the photobleaching of the compound BP itself. make it possible to do.
  • a special excitation light source and a fluorescence detection device are not required, and a change in membrane potential can be detected by a general fluorescence microscope.
  • compound BP has high water solubility, and it enables staining of cells without using an organic solvent harmful to cells. Therefore, cell death due to organic solvents did not occur, and it was possible to observe living cells over 24 hours.
  • the quantum yield ( ⁇ ) which is the ratio of the number of photons absorbed in the compound by absorption (excitation) to the number of photons emitted by fluorescence, is as low as 0.14, and the light emission efficiency is good
  • a compound with high light emission efficiency is required to detect fluorescence with high sensitivity.
  • a specific compound characterized by the relative bias of the charge distribution of the electron cloud of the molecule, that is, the low polarizability, is localized according to the mitochondrial membrane potential. It has been found that it has the property of transferring the place from the mitochondria to the nucleus, and the present invention has been completed.
  • the compound is water-soluble and has high luminous efficiency, so that it is useful as a fluorescent dye for staining cells.
  • a compound represented by the formula (1) [Wherein R 1 represents a C1-C10 alkyl group, Z ⁇ represents a counter anion with respect to a pyridinium cation, and X represents (Wherein the wavy line represents a bonding site to an adjacent pyridine ring, provided that the naphthylene group (i) may have an electron donating group). Represents. ]
  • the naphthylene group (i) may have an electron donating group.
  • a fluorescent dye composition comprising one or more of the compounds according to any one of (1) to (4) above.
  • the compound of the present invention is fluorescent and has the property of transferring the localization location from the mitochondria to the nucleus in accordance with the mitochondrial membrane potential. Presence / absence can be determined from the location of the compound.
  • the compound of the present invention is water-soluble and does not require the use of an organic solvent that is toxic to cells, living cells can be observed.
  • the compound of the present invention does not change the fluorescence emission color during cell observation, it is sufficient to detect a single fluorescence wavelength emitted by irradiation with a single wavelength excitation light. Can easily observe cells.
  • 1 HNMR chart of naphthalene derivative (I) 1 HNMR chart of anthracene derivative (II) 1 HNMR chart of pyrene derivative (III) 1 HNMR chart of compound of formula (2-1) It is a figure showing an ultraviolet-visible absorption spectrum (solid line) and a fluorescence spectrum (dotted line). It is a figure showing the fluorescence-microscope image of Hek293 cell before the membrane potential fall dye
  • the compound of this invention is a compound represented by Formula (1).
  • R 1 represents a C1-C10 alkyl group
  • Z ⁇ represents a counter anion for the pyridinium cation
  • X is (Wherein the wavy line represents a bonding site to an adjacent pyridine ring, provided that the naphthylene group (i) may have an electron donating group). Represents.
  • the condensed polycyclic groups of X preferably (Wherein the wavy line represents a binding site to an adjacent pyridine ring, provided that the naphthylene group (i) may have an electron-donating group). More preferably, (Wherein the wavy line represents a binding site to an adjacent pyridine ring, provided that the naphthylene group (i-1) may have an electron donating group). is there.
  • the alkyl group having 1 to 10 carbon atoms is a linear or branched alkyl group having 1 to 10 carbon atoms which may have a substituent.
  • the electron donating group that may be possessed by the naphthylene group is not particularly limited as long as it is a group having a property of donating electrons by bonding to an aromatic ring.
  • —O ⁇ —OH, —OR, —NH 2 , —NR 2 2 (R 2 has the same definition as R 1 ), amide, —OCOR 3 (R 3 has the same definition as R 1 ), alkyl group (for example, methyl group, ethyl Group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentylo group, isopentyl group, neopentyl group, n-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decyl group), phenyl group, and conjugated
  • the compound represented by Formula (1) can illustrate the compound shown below.
  • the compound represented by the formula (1) of the present invention is a compound that changes the localization location according to the mitochondrial membrane potential, and is a fluorescent compound that emits light when irradiated with excitation light.
  • the compound represented by the formula (1) of the present invention is localized in mitochondria, and mitochondria stops energy production. When the membrane potential disappears, the compound represented by the formula (1) of the present invention is localized in the nucleus.
  • the compound represented by the formula (1) of the present invention is not particularly limited.
  • a halogen compound (2) having a condensed polycycle and an organoboron compound having a pyridine ring ( 3) is reacted in the presence of a palladium catalyst and a base, and is coupled by the Suzuki-Miyaura coupling reaction (Step I), and the nitrogen of the pyridine of compound (4) is alkylated by an N-alkylating agent (R 1 Z).
  • Step II the compound of the formula (1) can be synthesized.
  • X, Z, and R 1 have the same definition as X, Z, and R 1 in the formula (1).
  • Y 1 and Y 2 may be the same or different, and are a chlorine atom or a bromine atom. And represents any halogen atom selected from iodine atoms.
  • P 1 and P 2 may be the same or different and each represents a hydroxyl group, an alkyl group, an alkoxy group, etc.
  • the commercially available halides can be used as the halide (2).
  • the commercially available halides include 1,3-dibromonaphthalene, 1,5-dibromonaphthalene, 2,7-dibromonaphthalene, 2,6-dibromonaphthalene, 2,3-dibromonaphthalene, 2,6-dibromo.
  • Anthracene, 1,5-dibromoanthracene, 1,8-dibromoanthracene, 9,10-dibromoanthracene, 1,6-dibromopyrene, 2,7-dibromopyrene and the like can be mentioned.
  • the halide (2) can be synthesized by an organic synthesis method.
  • organic synthesis method for example, commercially available naphthalene, anthracene, pyrene, tetracene, phenanthrene, benzo [a] phenanthrene, chlorine (Cl 2 ), bromine (Br 2 ), iodine (I 2 ), N-bromosuccinimide (NBS), N-chlorosuccinimide
  • the halide (2) can be synthesized by acting (NCS), N-iodosuccinimide (NIS) or the like.
  • the naphthylene group may have one or more electron donating groups, but the introduction of the electron donating group is a known synthesis. It can be done by the method. Moreover, you may use the commercially available halide which has an electron-donating group and / or an electron withdrawing group in a naphthylene part.
  • a commercially available organic boron compound can be used as the organic boron compound (3).
  • examples of the commercially available organic boron compound include 4-pyridylboronic acid, 4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) pyridine, and the like. .
  • the palladium catalyst is not particularly limited as long as it is a palladium catalyst used in the Suzuki-Miyaura coupling reaction.
  • triphenylphosphine for example, triphenylphosphine, tri-o-tolylphosphine, 1,3-bis (diphenylphosphino) propane, tri-tert-butylphosphine, tris (o-methoxyphenyl) phosphine, dibutyl
  • a phosphorus ligand such as butylphosphonate and an arsenic ligand such as triphenylarsenic may be added as appropriate.
  • the base is not particularly limited as long as it is a base used in the Suzuki-Miyaura coupling reaction, and amines such as trimethylamine, triethylamine, diisopropylethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, pyridine;
  • examples include inorganic bases such as sodium, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, cesium hydroxide.
  • the alkylating agent is not particularly limited as long as it is an N-alkylating agent usually used for alkylating nitrogen.
  • N-alkylating agent usually used for alkylating nitrogen.
  • the reactions in Steps I and II can be performed in a solvent, but the solvent is appropriately selected depending on the reaction temperature, reactants, and the like.
  • the reaction temperature of the reaction in Steps I and II is appropriately selected depending on conditions such as the boiling point of the solvent used.
  • the resulting reaction solution is concentrated as necessary, and the residue may be used as it is in the next reaction.
  • the formula You may use as a compound represented by 1). Specific methods of post-treatment include known purification such as extraction treatment and / or crystallization, recrystallization, chromatography and the like.
  • fluorescent dye composition Since the compound represented by the formula (1) exhibits fluorescence, it can be used as a fluorescent dye.
  • the compound represented by the formula (1) may be used as a fluorescent dye as it is, but if necessary, an additive usually used for preparing a reagent may be blended and used as a fluorescent dye composition.
  • additives such as solubilizers, pH adjusters, buffers, and tonicity agents can be used as additives for using the reagent in a physiological environment. Is possible.
  • These compositions are generally provided as a composition in an appropriate form such as a powdered mixture, a lyophilized product, a granule, a tablet, or a liquid.
  • the fluorescent dye composition of the present invention utilizes the property of changing the localization location according to the mitochondrial membrane potential of the compound of the formula (1) contained in the composition, that is, the presence or absence of mitochondrial membrane potential of cells, Can determine the presence or absence of mitochondrial vitality.
  • mitochondria have vitality means that the mitochondria are producing energy by aerobic respiration, and if there is no vitality, energy production has stopped due to destruction of the outer membrane of the mitochondria, etc. Refers to the state.
  • the presence or absence of mitochondrial vitality is determined by the following mechanism.
  • the fluorescent dye composition of the present invention When the fluorescent dye composition of the present invention is added to a cell in which mitochondria having a membrane potential exist, that is, a cell in which energy is produced, the formula (1) contained in the mitochondria and the fluorescent dye composition in response to the mitochondrial membrane potential. ) Interacts with the compound of formula (1) in the mitochondria.
  • the cell When the cell is irradiated with excitation light having a predetermined wavelength, fluorescence having a predetermined wavelength is emitted from the compound represented by the formula (1) existing in the mitochondria. As described above, it is determined that mitochondria have vitality by detecting fluorescence from mitochondria.
  • the fluorescent dye composition of the present invention when added to a cell in which mitochondria having a depolarized membrane potential exist, that is, a cell in which energy production is stopped, the mitochondrial membrane potential is depolarized.
  • the compound (1) is not localized, and the compound (1) is localized in the cell nucleus.
  • fluorescence having a predetermined wavelength is emitted from the compound represented by the formula (1) present in the nucleus. As described above, it is determined that mitochondria have no vitality by detecting fluorescence from the nucleus.
  • the compound represented by the formula (1) contained in the fluorescent dye composition of the present invention changes the localization location in the cell in response to the change in mitochondrial membrane potential, and the fluorescence of the compound is detected.
  • the presence or absence of mitochondrial vitality can be determined by confirming the location of the mitochondrion.
  • the detection of a change in mitochondrial membrane potential in the present invention is to determine the presence or absence of mitochondrial vitality.
  • the mitochondrial vitality is present, that is, whether the cell is producing energy. Therefore, by observing the cells with the fluorescent dye composition of the present invention, it is possible to determine whether the cells are normal cells or cells having mitochondria depolarized due to apoptosis, metabolic stress, or the like, that is, whether the cells are alive or dead.
  • the excitation light is not particularly limited as long as it can be absorbed by the compound represented by the formula (1), and is light in the ultraviolet region, visible region, and infrared region. Moreover, the detection of the localization location of the compound represented by Formula (1) can be performed with the fluorescence microscope about the light emission state of this compound. Further, the excitation light may cause two-photon absorption in the compound represented by the formula (1), or may be irradiated with light condensed by a lens.
  • the compound represented by the following formula (2) is also used in the method for detecting a change in mitochondrial membrane potential and the method for determining whether a cell is alive or dead. can do.
  • R 1 represents a C1 to C10 alkyl group
  • Z ⁇ represents any of a condensed polycyclic group represented by a counter anion with respect to a pyridinium cation
  • the naphthylene group has an electron donating group. May be.
  • R 1 has the same definition as R 1 in formula (1).
  • the compound represented by the formula (2) can be synthesized by a known synthesis method with reference to PCT / JP2014 / 004948 and the like.
  • Step 2 Compound 1 (0.2 g, 1 mmol) was dissolved in 25 mL of dichloromethane, iodomethane (CH 3 I, 2 mL) was added, and the mixture was stirred at room temperature for 24 hours. The precipitated solid was washed with dichloromethane to obtain naphthalene derivative (I) as a yellow solid.
  • the 1 HNMR data is shown below.
  • a 1 HNMR chart of the naphthalene derivative (I) obtained is shown in FIG.
  • Step 1 A solution of 2,6-dibromoanthracene (0.34 g, 1 mmol), 4-pyridylboronic acid (0.27 g, 2.2 mmol), and cesium carbonate (0.81 g, 2.5 mmol) in dimethylformamide (DMF, 12 mL) To was added palladium catalyst (5.78 mg, 0.005 mmol). After stirring at 110 ° C. for 70 hours, the mixed solution was extracted with diethyl ether, the organic layer was washed with water, magnesium sulfate (anhydrous) was added to the organic layer, dried and concentrated. The crude crystals were recrystallized from methanol to obtain compound 2.
  • DMF dimethylformamide
  • Step 2 Compound 2 (0.18 g, 0.5 mmol) was dissolved in 25 mL of dichloromethane, iodomethane (CH 3 I, 2 mL) was added, and the mixture was stirred at room temperature for 24 hours. The precipitated solid was washed with dichloromethane to obtain an anthracene derivative (II) as a yellow solid.
  • the 1 HNMR data is shown below. Moreover, the 1 HNMR chart of the obtained anthracene derivative (II) is shown in FIG.
  • Step 1 A solution of 1,6-dibromopyrene (0.36 g, 1 mmol), 4-pyridylboronic acid (0.27 g, 2.2 mmol), and cesium carbonate (0.81 g, 2.5 mmol) in dimethylformamide (DMF, 12 mL) To was added palladium catalyst (5.78 mg, 0.005 mmol). After stirring at 120 ° C. for 50 hours, the mixed solution was extracted with diethyl ether, the organic layer was washed with water, magnesium sulfate (anhydrous) was added to the organic layer, dried and concentrated. The crude crystal was recrystallized from methanol to obtain Compound 3.
  • DMF dimethylformamide
  • Step 2 Compound 3 (0.15 g, 0.4 mmol) was dissolved in 15 mL of dichloromethane, iodomethane (CH 3 I, 2 mL) was added, and the mixture was stirred at room temperature for 24 hours. The precipitated solid was washed with dichloromethane to obtain pyrene derivative (III) as a yellow solid.
  • the 1 HNMR data is shown below.
  • FIG. 3 shows a 1 HNMR chart of the obtained pyrene derivative (III).
  • Embodiment 5 Measurement of UV-visible absorption spectrum and fluorescence spectrum
  • the UV-visible absorption spectrum and fluorescence spectrum of the compound synthesized in Examples 1 to 3 and Comparative Example 1 were measured according to the following conditions.
  • the compound of Comparative Example 1 has the following structure.
  • the ultraviolet-visible absorption spectrum was measured using a V-670-UV-VIS-NIR spectrophotometer (Jasco Co.).
  • the fluorescence spectrum was measured using C9920-03G (Hamamatsu Photonics. K. K.).
  • the fluorescence quantum yield was determined by absolute measurement using an integrating sphere. Measurement was performed using a sample adjusted to a concentration of 10 ⁇ 6 mol / L. The measurement results are shown in Table 1 and FIG.
  • the solid line in FIG. 1 represents the ultraviolet-visible absorption spectrum, and the dotted line represents the fluorescence spectrum.
  • the emission quantum yield ( ⁇ ) of the compound of Comparative Example 1 and the ratio between the number of photons absorbed by the compound and the number of photons emitted by fluorescence are represented.
  • the luminescence quantum yields ( ⁇ ) of the naphthalene derivative (I), the anthracene derivative (II), and the pyrene derivative (III) were 0.92 and 0.92, respectively. 0.58 and 0.43. That is, the compound of the present invention is suitable for use as a fluorescent dye because it has high emission efficiency and can detect strong fluorescence.
  • Hek293 cells which are human fetal kidney cells, were used as model cells for cell culture staining. Hek293 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v / v) fetal bovine serum, 1% (v / v) trypsin and streptomycin under conditions of 37 ° C. and 5% CO 2 . .
  • DMEM Dulbecco's Modified Eagle Medium
  • Hek293 cells were passaged on a 35 mm glass base dish to a cell density of 1 ⁇ 10 5 cells / dish. 24 hours after passage, it was confirmed by microscopic observation that the cells were attached to the dish. The medium was removed from the dish, and the cells were washed twice with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the compounds of Comparative Examples 2 to 4 have the following structures and were obtained by known synthesis methods.
  • CCCP carbonyl cyanide-m-chlorophenylhydrazone
  • the intensity and location of the fluorescence of the compounds of Comparative Examples 2 to 4 did not change before and after the decrease in membrane potential.
  • the compound of the present invention is fluorescent, and has a property of moving the localization location from the mitochondria to the nucleus in accordance with the mitochondrial membrane potential, so that a change in the mitochondrial membrane potential can be detected. Further, by using the compound of the present invention as a fluorescent dye, it is possible to observe living cells, and the cells can be easily observed with a general fluorescence microscope. Furthermore, information on the place where the fluorescence is emitted can be obtained by the fluorescence microscope, and the viability of the cell can be easily determined.
  • the fluorescent dye composition of the present invention can be used for studying prevention of diseases caused by mitochondrial dysfunction and research for elucidating the mechanism of disease occurrence, for example, effects of drugs by the administration of treatment candidates, effects on mitochondrial vitality and cell viability Can be used when testing etc.

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Abstract

The present invention addresses the problem of providing a compound that is a pigment usable for staining cells, is soluble in water, shows a high emission efficiency and is capable of translocating from mitochondria to nucleus depending on mitochondrial membrane potential. A compound represented by formula (1) [wherein: R1 represents a C1-C10 alkyl group; Z- represents a counter anion to a pyridinium cation; and X represents any of condensed polycyclic groups represented by the formulae (wherein a wavy line stands for a binding site to an adjacent pyridine ring, provided that a naphthylene group (i) optionally has an electron donating group)], which is soluble in water, shows a high emission efficiency and is capable of translocating from mitochondria to nucleus depending on mitochondrial membrane potential, is usable as a fluorescent pigment for staining cells.

Description

ミトコンドリア膜電位応答性蛍光性化合物Mitochondrial membrane potential responsive fluorescent compound
　本発明は、新規な蛍光性化合物に関し、さらに詳しくは、ミトコンドリア膜電位に応答して局在場所を変化させる蛍光性化合物に関する。また、該化合物を含有する蛍光色素組成物に関する。さらに、上記蛍光色素組成物を用いた、ミトコンドリア膜電位の変化の検出方法や細胞の生死の判別方法に関する。 The present invention relates to a novel fluorescent compound, and more particularly, to a fluorescent compound that changes a localized location in response to a mitochondrial membrane potential. The present invention also relates to a fluorescent dye composition containing the compound. Furthermore, the present invention relates to a method for detecting a change in mitochondrial membrane potential and a method for discriminating cell viability using the fluorescent dye composition.
　ミトコンドリアは、細胞のエネルギーを生産する一方でアポトーシスの制御にも関わり、細胞の生死に関わる細胞小器官である。また、ミトコンドリアの機能障害に起因して、糖尿病、脳梗塞、心筋梗塞等の代謝疾患、アルツハイマーやパーキンソン病等の神経変性疾患、癌が発病することが指摘されている。そのため、これら疾患のメカニズム解明や、治療方法の開発のためにも、ミトコンドリアの変化を観察することは重要である。 Mitochondria are cell organelles that are involved in the control of apoptosis while producing cellular energy, and involved in the life and death of cells. In addition, it has been pointed out that metabolic diseases such as diabetes, cerebral infarction and myocardial infarction, neurodegenerative diseases such as Alzheimer and Parkinson's disease, and cancer are caused by mitochondrial dysfunction. Therefore, it is important to observe changes in mitochondria in order to elucidate the mechanisms of these diseases and develop therapeutic methods.
　エネルギー生産に伴って生じるミトコンドリア膜電位は、ミトコンドリアの内側と外側で電位差を意味し、ミトコンドリア自体の活力を表す。すなわち、膜電位があるときはエネルギーが生産され、ミトコンドリアの活力が高い状態にある。他方、膜電位が消失しているときは、エネルギー生産がされず、活力が低い状態にある。また、ミトコンドリア膜電位は、細胞の健康状態を表し、膜電位があるときは正常細胞で、膜電位がないときは異常細胞とされる。 Mitochondrial membrane potential that accompanies energy production means a potential difference between the inside and outside of the mitochondria and represents the vitality of the mitochondria itself. That is, when there is a membrane potential, energy is produced and the mitochondrial vitality is high. On the other hand, when the membrane potential disappears, no energy is produced and the vitality is low. The mitochondrial membrane potential represents the health state of a cell. When there is a membrane potential, it is a normal cell, and when there is no membrane potential, it is regarded as an abnormal cell.
　ミトコンドリア膜電位の変化を検出したり、該変化による細胞の健康状態を判別したりするためには、膜電位に応じて発光挙動を変化させる色素によりミトコンドリアを染色し、その発光挙動の変化を観測する方法がある。従来のミトコンドリア膜電位に応答する色素には、２種類のタイプがある。一つは、ミトコンドリア膜電位に応じて発光強度が変化する型の色素、もう一つは、発光色が変化する型の色素である。しかし、発光強度が変化する型の色素は、ミトコンドリア膜電位による発光強度の変化と、色素自体の光退色による発光強度の変化が同時に起こるため、蛍光強度の変化が膜電位の変化によるものか、色素の光退色によるものかを区別することが難しく、膜電位の変化の検出には適するとはいえない。 In order to detect changes in mitochondrial membrane potential and to determine cell health due to such changes, mitochondria are stained with a dye that changes luminescence behavior according to the membrane potential, and changes in luminescence behavior are observed. There is a way to do it. There are two types of conventional dyes that respond to mitochondrial membrane potential. One is a type of dye whose emission intensity changes according to the mitochondrial membrane potential, and the other is a type of dye whose emission color changes. However, the type of dye whose emission intensity changes changes the emission intensity due to the mitochondrial membrane potential and the emission intensity change due to photobleaching of the dye itself, so whether the change in fluorescence intensity is due to the change in membrane potential, It is difficult to distinguish whether it is due to photobleaching of the dye, and it cannot be said that it is suitable for detecting changes in membrane potential.
　また、もう一方の発光色が変化する型の色素においても、膜電位の変化に伴う色素の発光色の変化を観測するために、励起光源及び蛍光検出器は発光色に応じてそれぞれ少なくても２系統用意する必要があり、且つリアルタイムで上記励起光源及び蛍光検出器を調整する必要もあり、装置が高コスト化するとともに、実験操作が極めて煩雑となる。さらに、どちらの型の色素も、溶解度の低さから細胞を染色するには、細胞に対して有害な有機溶媒を用いなければならないという問題もある。 In addition, in the other type of dye whose emission color changes, in order to observe the change in the emission color of the dye due to the change in the membrane potential, the excitation light source and the fluorescence detector may be at least according to the emission color. It is necessary to prepare two systems, and it is necessary to adjust the excitation light source and the fluorescence detector in real time, which increases the cost of the apparatus and makes the experimental operation extremely complicated. In addition, both types of dyes have a problem that organic solvents harmful to cells must be used in order to stain cells because of low solubility.
　本出願人は、ミトコンドリア膜電位に応じて、局在場所をミトコンドリアから核に移す性質を有している以下の化合物ＢＰを見いだし、細胞の染色に利用できることを既に報告している（非特許文献１）。 The present applicant has already found that the following compound BP having the property of transferring the localization location from the mitochondria to the nucleus according to the mitochondrial membrane potential can be found and used for staining cells (non-patent document). 1).
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
　ミトコンドリア膜電位に応じて、化合物ＢＰが局在場所をミトコンドリアから核に移す性質は、蛍光強度が変化する訳ではないので、化合物ＢＰ自身の光退色の影響を受けずに膜電位の変化を検出することを可能にする。また、蛍光色の変化もないため、特別な励起光源及び蛍光検出装置を必要とせず、一般的な蛍光顕微鏡による膜電位の変化の検出を可能にした。また、化合物ＢＰは水溶性が高く、細胞にとって有害な有機溶媒を用いなくても細胞の染色を可能にした。そのため、有機溶媒による細胞死が起こらず、２４時間以上にわたる生きた細胞の観察を可能にした。 The property that the compound BP moves from the mitochondria to the nucleus according to the mitochondrial membrane potential does not change the fluorescence intensity, so the change in membrane potential is detected without being affected by the photobleaching of the compound BP itself. Make it possible to do. In addition, since there is no change in the fluorescence color, a special excitation light source and a fluorescence detection device are not required, and a change in membrane potential can be detected by a general fluorescence microscope. In addition, compound BP has high water solubility, and it enables staining of cells without using an organic solvent harmful to cells. Therefore, cell death due to organic solvents did not occur, and it was possible to observe living cells over 24 hours.
　しかし、化合物ＢＰにおける、吸収（励起）によって化合物に吸収された光子数と蛍光によって放出された光子数との比である量子収率（φ）が０．１４と低く、発光の効率がよいものではなく、高感度で蛍光を検出するためにも発光の効率がよい化合物が必要とされていた。 However, in compound BP, the quantum yield (φ), which is the ratio of the number of photons absorbed in the compound by absorption (excitation) to the number of photons emitted by fluorescence, is as low as 0.14, and the light emission efficiency is good However, a compound with high light emission efficiency is required to detect fluorescence with high sensitivity.
　細胞を染色できる色素であって、水溶性を備え、発光の効率が良く、ミトコンドリア膜電位に応じて局在場所をミトコンドリアから核に移す性質がある化合物を提供することを課題とする。 It is an object of the present invention to provide a compound that can stain cells, has water solubility, has high light emission efficiency, and has a property of transferring a localized location from the mitochondria to the nucleus according to the mitochondrial membrane potential.
　前記課題解決のために鋭意研究の結果、分子の電子雲等がもつ電荷分布の相対的な偏り、すなわち、分極率が低いことを特徴とする特定の化合物が、ミトコンドリア膜電位に応じて局在場所をミトコンドリアから核に移す性質を有していることを見いだし、本発明を完成するに至った。また、当該化合物は水溶性を示し、発光効率がよいため、細胞の染色のための蛍光色素として有用である。 As a result of diligent research to solve the above problem, a specific compound characterized by the relative bias of the charge distribution of the electron cloud of the molecule, that is, the low polarizability, is localized according to the mitochondrial membrane potential. It has been found that it has the property of transferring the place from the mitochondria to the nucleus, and the present invention has been completed. In addition, the compound is water-soluble and has high luminous efficiency, so that it is useful as a fluorescent dye for staining cells.
　すなわち、本発明は以下の発明に関する。
（１）式（１）で表される化合物。
Figure JPOXMLDOC01-appb-C000002
 ［式中、Ｒ１はＣ１～Ｃ１０のアルキル基を表し、Ｚ－はピリジニウムカチオンに対するカウンターアニオンを表し、Ｘは、
Figure JPOXMLDOC01-appb-C000003
 （式中、波線は隣接するピリジン環への結合部位を表す。ただし、ナフチレン基（ｉ）は、電子供与性基を有していてもよい。）で表される縮合多環基のいずれかを表す。］
（２）Ｘが以下の式で表される縮合多環基のいずれかであることを特徴とする上記（１）に記載の化合物。
Figure JPOXMLDOC01-appb-C000004
 （式中、波線は隣接するピリジン環への結合部位を表す。ただし、ナフチレン基（ｉ）は、電子供与性基を有していてもよい。）
（３）Ｘが以下の式で表される縮合多環基のいずれかであることを特徴とする上記（１）に記載の化合物。
Figure JPOXMLDOC01-appb-C000005
 （式中、波線は隣接するピリジン環への結合部位を表す。ただし、ナフチレン基（ｉ－１）は、電子供与性基を有していてもよい。）
　（４）カウンターアニオンが、ハロゲン化物イオン、スルホネートであることを特徴とする上記（１）～（３）のいずれかに記載の化合物。
　（５）上記（１）～（４）のいずれかに記載の化合物の１又は２以上を含有することを特徴とする蛍光色素組成物。
　（６）上記（１）～（４）に記載の化合物及び以下の式（２）で表される化合物；
Figure JPOXMLDOC01-appb-C000006
 （式中、Ｒ１はＣ１～Ｃ１０のアルキル基を表し、Ｚ－はピリジニウムカチオンに対するカウンターアニオンを表し、ナフチレン基は電子供与性基を有していてもよい。）
からなる群より選ばれる少なくとも一つを用いることを特徴とする、ミトコンドリア膜電位の変化の検出方法。
　（７）化合物（２）が以下の化合物（２－１）
Figure JPOXMLDOC01-appb-C000007
 であることを特徴とする、上記（６）に記載のミトコンドリア膜電位の変化の検出方法。
　（８）上記（１）～（４）に記載の化合物及び以下の式（２）で表される化合物；
Figure JPOXMLDOC01-appb-C000008
 （式中、Ｒ１はＣ１～Ｃ１０のアルキル基を表し、Ｚ－はピリジニウムカチオンに対するカウンターアニオンを表し、ナフチレン基は電子供与性基を有していてもよい。）
からなる群より選ばれる少なくとも一つを用いることを特徴とする、細胞の生死を判別する方法。
　（９）化合物（２）が以下の化合物（２－１）
Figure JPOXMLDOC01-appb-C000009
 であることを特徴とする、上記（８）に記載の細胞の生死を判別する方法。 That is, the present invention relates to the following inventions.
(1) A compound represented by the formula (1).
Figure JPOXMLDOC01-appb-C000002
 [Wherein R 1 represents a C1-C10 alkyl group, Z − represents a counter anion with respect to a pyridinium cation, and X represents
Figure JPOXMLDOC01-appb-C000003
 (Wherein the wavy line represents a bonding site to an adjacent pyridine ring, provided that the naphthylene group (i) may have an electron donating group). Represents. ]
(2) The compound as described in (1) above, wherein X is any one of a condensed polycyclic group represented by the following formula:
Figure JPOXMLDOC01-appb-C000004
 (In the formula, a wavy line represents a bonding site to an adjacent pyridine ring. However, the naphthylene group (i) may have an electron donating group.)
(3) The compound as described in (1) above, wherein X is any one of a condensed polycyclic group represented by the following formula:
Figure JPOXMLDOC01-appb-C000005
 (In the formula, a wavy line represents a binding site to an adjacent pyridine ring. However, the naphthylene group (i-1) may have an electron-donating group.)
(4) The compound according to any one of (1) to (3) above, wherein the counter anion is a halide ion or sulfonate.
(5) A fluorescent dye composition comprising one or more of the compounds according to any one of (1) to (4) above.
(6) The compound described in the above (1) to (4) and the compound represented by the following formula (2);
Figure JPOXMLDOC01-appb-C000006
 (In the formula, R 1 represents a C1-C10 alkyl group, Z − represents a counter anion with respect to the pyridinium cation, and the naphthylene group may have an electron-donating group.)
A method for detecting a change in mitochondrial membrane potential, comprising using at least one selected from the group consisting of:
(7) Compound (2) is the following compound (2-1)
Figure JPOXMLDOC01-appb-C000007
 The method for detecting a change in mitochondrial membrane potential according to (6) above, wherein
(8) The compound described in the above (1) to (4) and the compound represented by the following formula (2);
Figure JPOXMLDOC01-appb-C000008
 (In the formula, R 1 represents a C1-C10 alkyl group, Z − represents a counter anion with respect to the pyridinium cation, and the naphthylene group may have an electron-donating group.)
A method for discriminating whether cells are alive or dead, wherein at least one selected from the group consisting of:
(9) Compound (2) is the following compound (2-1)
Figure JPOXMLDOC01-appb-C000009
 The method for discriminating whether a cell is alive or dead as described in (8) above,
　本発明の化合物は、蛍光性があり、また、ミトコンドリア膜電位に応じて局在場所をミトコンドリアから核に移す性質を有していることから、ミトコンドリアの膜電位の変化、すなわち、ミトコンドリアの活力の有無を上記化合物の局在場所から判別することができる。また、本発明の化合物は水溶性を示し、細胞に毒性のある有機溶媒を使用する必要がないため、生きた細胞の観察が可能である。また、本発明の化合物は、細胞観察中に蛍光の発光色の変化がないため、単一波長の励起光の照射によって発光する単一波長の蛍光波長を検出すればよく、一般的な蛍光顕微鏡によって簡単に細胞の観察ができる。 The compound of the present invention is fluorescent and has the property of transferring the localization location from the mitochondria to the nucleus in accordance with the mitochondrial membrane potential. Presence / absence can be determined from the location of the compound. In addition, since the compound of the present invention is water-soluble and does not require the use of an organic solvent that is toxic to cells, living cells can be observed. In addition, since the compound of the present invention does not change the fluorescence emission color during cell observation, it is sufficient to detect a single fluorescence wavelength emitted by irradiation with a single wavelength excitation light. Can easily observe cells.
ナフタレン誘導体（Ｉ）の１ＨＮＭＲチャート 1 HNMR chart of naphthalene derivative (I) アントラセン誘導体（II）の１ＨＮＭＲチャート 1 HNMR chart of anthracene derivative (II) ピレン誘導体（III）の１ＨＮＭＲチャート 1 HNMR chart of pyrene derivative (III) 式（２－１）の化合物の１ＨＮＭＲチャート 1 HNMR chart of compound of formula (2-1) 紫外－可視吸収スペクトル（実線）、蛍光スペクトル（点線）を表す図である。It is a figure showing an ultraviolet-visible absorption spectrum (solid line) and a fluorescence spectrum (dotted line). ナフタレン誘導体（Ｉ）により染色された膜電位低下前と膜電位低下後のＨｅｋ２９３細胞の蛍光顕微鏡画像を表す図である。It is a figure showing the fluorescence-microscope image of Hek293 cell before the membrane potential fall dye | stained with the naphthalene derivative (I) and after a membrane potential fall. アントラセン誘導体（II）により染色された膜電位低下前と膜電位低下後のＨｅｋ２９３細胞の蛍光顕微鏡画像を表す図である。It is a figure showing the fluorescence-microscope image of Hek293 cell before the membrane potential fall dye | stained with the anthracene derivative (II) and after a membrane potential fall. ピレン誘導体（III）により染色された膜電位低下前と膜電位低下後のＨｅｋ２９３細胞の蛍光顕微鏡画像を表す図である。It is a figure showing the fluorescence-microscope image of Hek293 cell before and after membrane potential fall dye | stained with the pyrene derivative (III). 式（２－１）の化合物により染色された膜電位低下前と膜電位低下後のＨｅｋ２９３細胞の蛍光顕微鏡画像を表す図である。It is a figure showing the fluorescence-microscope image of the Hek293 cell before and after a membrane potential fall dye | stained with the compound of Formula (2-1). 比較例２の化合物により染色された膜電位低下前と膜電位低下後のＨｅｋ２９３細胞の蛍光顕微鏡画像を表す図である。It is a figure showing the fluorescence-microscope image of Hek293 cell before the membrane potential fall dye | stained with the compound of the comparative example 2, and after a membrane potential fall. 比較例３の化合物により染色された膜電位低下前と膜電位低下後のＨｅｋ２９３細胞の蛍光顕微鏡画像を表す図である。It is a figure showing the fluorescence-microscope image of Hek293 cell before the membrane potential fall dye | stained with the compound of the comparative example 3, and after a membrane potential fall. 比較例４の化合物により染色された膜電位低下前と膜電位低下後のＨｅｋ２９３細胞の蛍光顕微鏡画像を表す図である。It is a figure showing the fluorescence-microscope image of Hek293 cell before and after a membrane potential fall dye | stained with the compound of the comparative example 4.
（化合物）
　本発明の化合物は、式（１）で表される化合物である。 (Compound)
The compound of this invention is a compound represented by Formula (1).
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-C000010
　式中、Ｒ１はＣ１～Ｃ１０のアルキル基を表し、Ｚ－はピリジニウムカチオンに対するカウンターアニオンを表し、Ｘは、
Figure JPOXMLDOC01-appb-C000011
 （式中、波線は隣接するピリジン環への結合部位を表す。ただし、ナフチレン基（ｉ）は、電子供与性基を有していてもよい。）で表される縮合多環基のいずれかを表す。 Wherein R 1 represents a C1-C10 alkyl group, Z − represents a counter anion for the pyridinium cation, and X is
Figure JPOXMLDOC01-appb-C000011
 (Wherein the wavy line represents a bonding site to an adjacent pyridine ring, provided that the naphthylene group (i) may have an electron donating group). Represents.
　上記Ｘの縮合多環基の中でも、好ましくは、
Figure JPOXMLDOC01-appb-C000012
 （式中、波線は隣接するピリジン環への結合部位を表す。ただし、ナフチレン基（ｉ）は、電子供与性基を有していてもよい。）で表される縮合多環基であり、さらに好ましくは、
Figure JPOXMLDOC01-appb-C000013
 （式中、波線は隣接するピリジン環への結合部位を表す。ただし、ナフチレン基（ｉ－１）は、電子供与性基を有していてもよい。）で表される縮合多環基である。 Among the condensed polycyclic groups of X, preferably
Figure JPOXMLDOC01-appb-C000012
 (Wherein the wavy line represents a binding site to an adjacent pyridine ring, provided that the naphthylene group (i) may have an electron-donating group). More preferably,
Figure JPOXMLDOC01-appb-C000013
 (Wherein the wavy line represents a binding site to an adjacent pyridine ring, provided that the naphthylene group (i-1) may have an electron donating group). is there.
　式（１）における炭素数１～１０のアルキル基とは、置換基を有していてもよい炭素数１～１０の直鎖状または分岐状のアルキル基である。例えば、メチル基、エチル基、ｎ－プロピル基、イソプロピル基、ｎ－ブチル基、イソブチル基、ｓｅｃ－ブチル基、ｔｅｒｔ－ブチル基、ｎ－ペンチル基、イソペンチル基、ネオペンチル基、ｎ－へキシル基、ｎ－ヘプチル基、ｎ－オクチル基、ｎ－ノニル基、ｎ－デシル基等が挙げられる。 In the formula (1), the alkyl group having 1 to 10 carbon atoms is a linear or branched alkyl group having 1 to 10 carbon atoms which may have a substituent. For example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, neopentyl group, n-hexyl group N-heptyl group, n-octyl group, n-nonyl group, n-decyl group and the like.
　上記「置換基を有していてもよい」の置換基としては、ハロゲン原子、アルコキシ基、アリール基等が挙げられる。 As the above-mentioned “optionally substituted” substituent, a halogen atom, an alkoxy group, an aryl group and the like can be mentioned.
　ナフチレン基が有していてもよい電子供与性基としては、芳香環に結合して電子を供与する性質を有する基であれば特に制限はなく、例えば、－Ｏ－、－ＯＨ、－ＯＲ、－ＮＨ２、－ＮＲ２ ２（Ｒ２は、Ｒ１と同じ定義である）、アミド、－ＯＣＯＲ３（Ｒ３は、Ｒ１と同じ定義である）、アルキル基（例えば、メチル基、エチル基、ｎ－プロピル基、イソプロピル基、ｎ－ブチル基、イソブチル基、ｓｅｃ－ブチル基、ｔｅｒｔ－ブチル基、ｎ－ペンチルオ基、イソペンチル基、ネオペンチル基、ｎ－へキシル基、ｎ－ヘプチル基、ｎ－オクチル基、ｎ－ノニル基、ｎ－デシル基）、フェニル基および共役アルケニルが挙げられるが、これらに限定されない。 The electron donating group that may be possessed by the naphthylene group is not particularly limited as long as it is a group having a property of donating electrons by bonding to an aromatic ring. For example, —O − , —OH, —OR, —NH 2 , —NR 2 2 (R 2 has the same definition as R 1 ), amide, —OCOR 3 (R 3 has the same definition as R 1 ), alkyl group (for example, methyl group, ethyl Group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentylo group, isopentyl group, neopentyl group, n-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decyl group), phenyl group, and conjugated alkenyl, but are not limited thereto.
　式（１）で表される化合物は、具体的には、以下に示す化合物を例示することができる。
Figure JPOXMLDOC01-appb-C000014
 Specifically, the compound represented by Formula (1) can illustrate the compound shown below.
Figure JPOXMLDOC01-appb-C000014
　本発明の式（１）で表される化合物は、ミトコンドリア膜電位に応じて局在場所を変化させる化合物であって、且つ、励起光を照射すると発光する蛍光性化合物である。上記局在場所に関して、具体的には、ミトコンドリアがエネルギー生産をして膜電位を有するとき、本発明の式（１）で表される化合物はミトコンドリアに局在し、ミトコンドリアがエネルギー生産を停止して膜電位を消失したとき、本発明の式（１）で表される化合物は核に局在する。 The compound represented by the formula (1) of the present invention is a compound that changes the localization location according to the mitochondrial membrane potential, and is a fluorescent compound that emits light when irradiated with excitation light. Regarding the above-mentioned localization location, specifically, when mitochondria produce energy and have a membrane potential, the compound represented by the formula (1) of the present invention is localized in mitochondria, and mitochondria stops energy production. When the membrane potential disappears, the compound represented by the formula (1) of the present invention is localized in the nucleus.
（化合物の合成）
　本発明の式（１）で表される化合物は、特に制限されるものではないが、例えば、以下に示すように、縮合多環を有するハロゲン化合物（２）とピリジン環を有する有機ホウ素化合物（３）とをパラジウム触媒及び塩基の存在下反応させる、鈴木－宮浦カップリング反応（工程Ｉ）によって連結し、Ｎ－アルキル化剤（Ｒ１Ｚ）により化合物（４）のピリジンの窒素をアルキル化すること（工程II）により式（１）の化合物を合成することができる。 (Synthesis of compounds)
The compound represented by the formula (1) of the present invention is not particularly limited. For example, as shown below, a halogen compound (2) having a condensed polycycle and an organoboron compound having a pyridine ring ( 3) is reacted in the presence of a palladium catalyst and a base, and is coupled by the Suzuki-Miyaura coupling reaction (Step I), and the nitrogen of the pyridine of compound (4) is alkylated by an N-alkylating agent (R 1 Z). By doing (Step II), the compound of the formula (1) can be synthesized.
Figure JPOXMLDOC01-appb-C000015
 （式中、Ｘ、Ｚ、Ｒ１は、前記式（１）におけるＸ、Ｚ、Ｒ１と同じ定義である。Ｙ１、Ｙ２は、同一でも異なっていてもよく、塩素原子、臭素原子、ヨウ素原子から選択されるいずれかのハロゲン原子を表す。Ｐ１、Ｐ２は、同一でも異なっていてもよく、水酸基、アルキル基、アルコキシ基等を表す。）
Figure JPOXMLDOC01-appb-C000015
 (In the formula, X, Z, and R 1 have the same definition as X, Z, and R 1 in the formula (1). Y 1 and Y 2 may be the same or different, and are a chlorine atom or a bromine atom. And represents any halogen atom selected from iodine atoms. P 1 and P 2 may be the same or different and each represents a hydroxyl group, an alkyl group, an alkoxy group, etc.)
　上記ハロゲン化物（２）は、市販のハロゲン化物を用いることができる。例えば、上記市販のハロゲン化物としては、１，３－ジブロモナフタレン、１，５－ジブロモナフタレン、２，７－ジブロモナフタレン、２，６－ジブロモナフタレン、２，３－ジブロモナフタレン、２，６－ジブロモアントラセン、１，５－ジブロモアントラセン、１，８－ジブロモアントラセン、９，１０－ジブロモアントラセン、１，６－ジブロモピレン、２，７－ジブロモピレン等を挙げることができる。 Commercially available halides can be used as the halide (2). For example, the commercially available halides include 1,3-dibromonaphthalene, 1,5-dibromonaphthalene, 2,7-dibromonaphthalene, 2,6-dibromonaphthalene, 2,3-dibromonaphthalene, 2,6-dibromo. Anthracene, 1,5-dibromoanthracene, 1,8-dibromoanthracene, 9,10-dibromoanthracene, 1,6-dibromopyrene, 2,7-dibromopyrene and the like can be mentioned.
　また、上記ハロゲン化物（２）は、有機合成手法により合成することができる。例えば、市販のナフタレン、アントラセン、ピレン、テトラセン、フェナントレン、ベンゾ［ａ］フェナントレンに塩素（Ｃｌ２）、臭素（Ｂｒ２）、ヨウ素（Ｉ２）、Ｎ－ブロモスクシンイミド（ＮＢＳ）、Ｎ－クロロスクシンイミド（ＮＣＳ）、Ｎ－ヨードスクシンイミド（ＮＩＳ）等を作用させて、ハロゲン化物（２）を合成することができる。 The halide (2) can be synthesized by an organic synthesis method. For example, commercially available naphthalene, anthracene, pyrene, tetracene, phenanthrene, benzo [a] phenanthrene, chlorine (Cl 2 ), bromine (Br 2 ), iodine (I 2 ), N-bromosuccinimide (NBS), N-chlorosuccinimide The halide (2) can be synthesized by acting (NCS), N-iodosuccinimide (NIS) or the like.
　上記ハロゲン化物（２）の、Ｘがナフチレン基である場合、該ナフチレン基は１つ又は２つ以上の電子供与性基を有していてもよいが、電子供与性基の導入は公知の合成方法によって行うことができる。また、ナフチレン部分に電子供与性基及び／又は電子吸引性基を有する市販のハロゲン化物を用いてもよい。 When X of the halide (2) is a naphthylene group, the naphthylene group may have one or more electron donating groups, but the introduction of the electron donating group is a known synthesis. It can be done by the method. Moreover, you may use the commercially available halide which has an electron-donating group and / or an electron withdrawing group in a naphthylene part.
　上記有機ホウ素化合物（３）は、市販の有機ホウ素化合物を用いることができる。例えば、上記市販の有機ホウ素化合物としては、４－ピリジルボロン酸、４－（４，４，５，５－テトラメチル－１，３，２－ジオキサボロラン－２－イル）ピリジン等を挙げることができる。 A commercially available organic boron compound can be used as the organic boron compound (3). For example, examples of the commercially available organic boron compound include 4-pyridylboronic acid, 4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) pyridine, and the like. .
　前記パラジウム触媒は、鈴木－宮浦カップリング反応に用いられるパラジウム触媒であれば特に限定されず、例えば酢酸パラジウム（II）、ジクロロビス（トリフェニルホスフィン）パラジウム（II）、テトラキス（トリフェニルホスフィン）パラジウム（０）またはトリス（ジベンジリデンアセトン）ジパラジウム（０）、ビス(ベンゾニトリル)パラジウム(II)ジクロリド、１，１′－ビス（ジフェニルホスフィノ）フェロセン－パラジウム（II）ジクロリド－ジクロロメタン錯体、トリス(ジベンジリデンアセトン)ジパラジウム（０）等の公知のパラジウム錯体が挙げられ、好ましくは、ビス(ベンゾニトリル)パラジウム(II)ジクロリドである。効率よく反応が進行するために、例えばトリフェニルホスフィン、トリ－ｏ－トリルホスフィン、１，３－ビス（ジフェニルホスフィノ）プロパン、トリ－ｔｅｒｔ－ブチルホスフィン、トリス（ｏ－メトキフェニル）ホスフィン、ジブチルブチルホスホネート等リン配位子、トリフェニルヒ素等のヒ素配位子等を適宜添加してもよい。 The palladium catalyst is not particularly limited as long as it is a palladium catalyst used in the Suzuki-Miyaura coupling reaction. For example, palladium (II) acetate, dichlorobis (triphenylphosphine) palladium (II), tetrakis (triphenylphosphine) palladium ( 0) or tris (dibenzylideneacetone) dipalladium (0), bis (benzonitrile) palladium (II) dichloride, 1,1'-bis (diphenylphosphino) ferrocene-palladium (II) dichloride-dichloromethane complex, tris ( Known palladium complexes such as dibenzylideneacetone) dipalladium (0) can be mentioned, and bis (benzonitrile) palladium (II) dichloride is preferred. In order for the reaction to proceed efficiently, for example, triphenylphosphine, tri-o-tolylphosphine, 1,3-bis (diphenylphosphino) propane, tri-tert-butylphosphine, tris (o-methoxyphenyl) phosphine, dibutyl A phosphorus ligand such as butylphosphonate and an arsenic ligand such as triphenylarsenic may be added as appropriate.
　前記塩基は、鈴木－宮浦カップリング反応に用いられる塩基であれば特に限定されず、トリメチルアミン、トリエチルアミン、ジイソプロピルエチルアミン、ジシクロヘキシルアミン、エタノールアミン、ジエタノールアミン、トリエタノールアミン、エチレンジアミン、ピリジン等のアミン類；炭酸ナトリウム、炭酸カリウム、炭酸セシウム、水酸化ナトリウム、水酸化カリウム、水酸化セシウム等の無機塩基類が挙げられる。 The base is not particularly limited as long as it is a base used in the Suzuki-Miyaura coupling reaction, and amines such as trimethylamine, triethylamine, diisopropylethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, pyridine; Examples include inorganic bases such as sodium, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, cesium hydroxide.
　前記アルキル化剤は、通常窒素のアルキル化に用いられるＮ－アルキル化剤であれば特に限定されず、例えば、ヨードメタン、ヨードエタン、１－ヨードプロパン、１－ヨードブタン、１－ヨード－３－メチルプロパン、２－ヨードブタン、２－ヨード－２－メチルプロパン、１－ヨードペンタン、１－ヨード－３－メチルブタン、１－ヨードへキサン、１－ヨードヘプタン、１－ヨードオクタン、１－ヨードノナン、１－ヨードデカン、ジメチル硫酸、トリフルオロメタンスルホン酸メチル等が挙げられる。これらのＮ－アルキル化剤は市販品を用いることができる。 The alkylating agent is not particularly limited as long as it is an N-alkylating agent usually used for alkylating nitrogen. For example, iodomethane, iodoethane, 1-iodopropane, 1-iodobutane, 1-iodo-3-methylpropane 2-iodobutane, 2-iodo-2-methylpropane, 1-iodopentane, 1-iodo-3-methylbutane, 1-iodohexane, 1-iodoheptane, 1-iodooctane, 1-iodononane, 1-iododecane Dimethylsulfuric acid, methyl trifluoromethanesulfonate, and the like. Commercially available products can be used for these N-alkylating agents.
　工程Ｉ、IIの反応は、それぞれ溶媒中で行うことができるが、溶媒は反応温度や反応物等によって適宜選択される。また、工程Ｉ、IIの反応の反応温度は、用いる溶媒の沸点等の条件によって適宜選択される。工程Ｉ、IIの反応で溶媒を用いる場合、得られた反応溶液を必要に応じて濃縮した後、残渣をそのまま次の反応に使用してもよく、適宜な後処理を行った後に、式（１）で表される化合物として用いてもよい。後処理の具体的な方法としては、抽出処理及び／又は晶出、再結晶、クロマトグラフィー等の公知の精製が挙げられる。 The reactions in Steps I and II can be performed in a solvent, but the solvent is appropriately selected depending on the reaction temperature, reactants, and the like. In addition, the reaction temperature of the reaction in Steps I and II is appropriately selected depending on conditions such as the boiling point of the solvent used. When a solvent is used in the reactions of Steps I and II, the resulting reaction solution is concentrated as necessary, and the residue may be used as it is in the next reaction. After appropriate post-treatment, the formula ( You may use as a compound represented by 1). Specific methods of post-treatment include known purification such as extraction treatment and / or crystallization, recrystallization, chromatography and the like.
（蛍光色素組成物）
　式（１）で表される化合物は、蛍光性を示すため、蛍光色素として用いることができる。式（１）で表される化合物は、蛍光色素としてそのまま用いてもよいが、必要に応じて、試薬の調製に通常用いられる添加剤を配合して蛍光色素組成物として用いてもよい。例えば、生理的環境で試薬を用いるための添加剤として、溶解補助剤、ｐＨ調節剤、緩衝剤、等張化剤等の添加剤を用いることができ、これらの配合量は当業者に適宜選択可能である。これらの組成物は、一般的には、粉末形態の混合物、凍結乾燥物、顆粒剤、錠剤、液剤等適宜の形態の組成物として提供される。 (Fluorescent dye composition)
Since the compound represented by the formula (1) exhibits fluorescence, it can be used as a fluorescent dye. The compound represented by the formula (1) may be used as a fluorescent dye as it is, but if necessary, an additive usually used for preparing a reagent may be blended and used as a fluorescent dye composition. For example, additives such as solubilizers, pH adjusters, buffers, and tonicity agents can be used as additives for using the reagent in a physiological environment. Is possible. These compositions are generally provided as a composition in an appropriate form such as a powdered mixture, a lyophilized product, a granule, a tablet, or a liquid.
　本発明の蛍光色素組成物は、上記組成物に含まれる式（１）の化合物のミトコンドリア膜電位に応じて局在場所を変化させる性質を利用して、細胞のミトコンドリア膜電位の有無、すなわち、ミトコンドリアの活力の有無を判別できる。ここで、ミトコンドリアが活力を有するとは、ミトコンドリアが好気呼吸によってエネルギー生産している状態を指し、活力が無いとは、ミトコンドリアの外膜が破壊されること等によってエネルギー生産が停止している状態を指す。ミトコンドリアの活力の有無は、以下の機構で判別される。 The fluorescent dye composition of the present invention utilizes the property of changing the localization location according to the mitochondrial membrane potential of the compound of the formula (1) contained in the composition, that is, the presence or absence of mitochondrial membrane potential of cells, Can determine the presence or absence of mitochondrial vitality. Here, mitochondria have vitality means that the mitochondria are producing energy by aerobic respiration, and if there is no vitality, energy production has stopped due to destruction of the outer membrane of the mitochondria, etc. Refers to the state. The presence or absence of mitochondrial vitality is determined by the following mechanism.
　膜電位を有するミトコンドリアが存在する細胞、すなわち、エネルギー生産されている細胞に本発明の蛍光色素組成物を添加すると、ミトコンドリア膜電位に応答して、ミトコンドリアと蛍光色素組成物に含まれる式（１）の化合物とが相互作用して、ミトコンドリアに式（１）の化合物が局在する。細胞に所定の波長の励起光が照射されると、ミトコンドリアに存在する式（１）で表される化合物より所定の波長の蛍光が発せられる。以上のように、ミトコンドリアから蛍光が検出されることによって、ミトコンドリアは活力を有していると判断される。 When the fluorescent dye composition of the present invention is added to a cell in which mitochondria having a membrane potential exist, that is, a cell in which energy is produced, the formula (1) contained in the mitochondria and the fluorescent dye composition in response to the mitochondrial membrane potential. ) Interacts with the compound of formula (1) in the mitochondria. When the cell is irradiated with excitation light having a predetermined wavelength, fluorescence having a predetermined wavelength is emitted from the compound represented by the formula (1) existing in the mitochondria. As described above, it is determined that mitochondria have vitality by detecting fluorescence from mitochondria.
　他方、膜電位が脱分極したミトコンドリアが存在する細胞、すなわち、エネルギー生産が停止した細胞に本発明の蛍光色素組成物を添加すると、ミトコンドリア膜電位は脱分極しているためミトコンドリアに式（１）の化合物は局在せず、上記（１）の化合物は細胞の核に局在するようになる。細胞に所定の波長の励起光を照射すると、核に存在する式（１）で表される化合物より所定の波長の蛍光が発せられる。以上のように、核から蛍光が検出されることによって、ミトコンドリアは活力が無いと判断される。 On the other hand, when the fluorescent dye composition of the present invention is added to a cell in which mitochondria having a depolarized membrane potential exist, that is, a cell in which energy production is stopped, the mitochondrial membrane potential is depolarized. The compound (1) is not localized, and the compound (1) is localized in the cell nucleus. When cells are irradiated with excitation light having a predetermined wavelength, fluorescence having a predetermined wavelength is emitted from the compound represented by the formula (1) present in the nucleus. As described above, it is determined that mitochondria have no vitality by detecting fluorescence from the nucleus.
　このように、本発明の蛍光色素組成物に含まれる式（１）で表される化合物はミトコンドリア膜電位の変化に応答して細胞中で局在場所を変化させ、該化合物の蛍光が検出される場所を確認することによってミトコンドリアの活力の有無を判別できる。本発明におけるミトコンドリア膜電位の変化の検出とは、ミトコンドリアの活力の有無を判別することである。 Thus, the compound represented by the formula (1) contained in the fluorescent dye composition of the present invention changes the localization location in the cell in response to the change in mitochondrial membrane potential, and the fluorescence of the compound is detected. The presence or absence of mitochondrial vitality can be determined by confirming the location of the mitochondrion. The detection of a change in mitochondrial membrane potential in the present invention is to determine the presence or absence of mitochondrial vitality.
　ミトコンドリア膜電位の変化から、ミトコンドリアの活力の有無、すなわち、細胞のエネルギー生産がされているか否かがわかる。したがって、本発明の蛍光色素組成物による細胞の観察によって、正常な細胞か、アポトーシスや代謝ストレス等により脱分極したミトコンドリアを有する細胞かの判別、すなわち、細胞の生死の判別ができる。 From the change in mitochondrial membrane potential, it can be seen whether the mitochondrial vitality is present, that is, whether the cell is producing energy. Therefore, by observing the cells with the fluorescent dye composition of the present invention, it is possible to determine whether the cells are normal cells or cells having mitochondria depolarized due to apoptosis, metabolic stress, or the like, that is, whether the cells are alive or dead.
　上記励起光としては、式（１）で表される化合物が吸収できる波長であれば特に制限は無く、紫外領域、可視領域、赤外領域の光である。また、式（１）で表される化合物の局在場所の検出は、該化合物の発光状態を蛍光顕微鏡によって行うことができる。さらに、励起光は式（１）で表される化合物に二光子吸収を起こさせるものであってもよく、レンズで集光した光を照射してもよい。 The excitation light is not particularly limited as long as it can be absorbed by the compound represented by the formula (1), and is light in the ultraviolet region, visible region, and infrared region. Moreover, the detection of the localization location of the compound represented by Formula (1) can be performed with the fluorescence microscope about the light emission state of this compound. Further, the excitation light may cause two-photon absorption in the compound represented by the formula (1), or may be irradiated with light condensed by a lens.
　さらに、本発明のミトコンドリア膜電位の変化の検出方法や、細胞の生死の判別する方法には、式（１）で表される化合物に加え、以下の式（２）で表される化合物も使用することができる。 Furthermore, in addition to the compound represented by the formula (1), the compound represented by the following formula (2) is also used in the method for detecting a change in mitochondrial membrane potential and the method for determining whether a cell is alive or dead. can do.
Figure JPOXMLDOC01-appb-C000016
 （式中、Ｒ１はＣ１～Ｃ１０のアルキル基を表し、Ｚ－はピリジニウムカチオンに対するカウンターアニオンで表される縮合多環基のいずれかを表し、ナフチレン基は電子供与性基を有していてもよい。）
Figure JPOXMLDOC01-appb-C000016
 (Wherein R 1 represents a C1 to C10 alkyl group, Z − represents any of a condensed polycyclic group represented by a counter anion with respect to a pyridinium cation, and the naphthylene group has an electron donating group. May be.)
　上記Ｒ１は、式（１）におけるＲ１と同じ定義である。 R 1 has the same definition as R 1 in formula (1).
　式（２）で表される化合物の中でも、好ましくは以下の式（２－１）で表される化合物である。
Figure JPOXMLDOC01-appb-C000017
 Among the compounds represented by the formula (2), a compound represented by the following formula (2-1) is preferable.
Figure JPOXMLDOC01-appb-C000017
　式（２）で表される化合物は、ＰＣＴ／ＪＰ２０１４／００４９４８等を参考に、公知の合成方法によって合成できる。 The compound represented by the formula (2) can be synthesized by a known synthesis method with reference to PCT / JP2014 / 004948 and the like.
　以下に、実施例において本発明をより詳細に説明するが、本発明の技術範囲は、これらに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the technical scope of the present invention is not limited to these examples.
実施例１．ナフタレン誘導体（Ｉ）の合成 Example 1. Synthesis of naphthalene derivative (I)
Figure JPOXMLDOC01-appb-C000018
Figure JPOXMLDOC01-appb-C000018
［工程１］
　２，６－ジブロモナフタレン（０．２９ｇ，１ｍｍｏｌ）、４－ピリジルボロン酸（０．２７ｇ，２．２ ｍｍｏｌ）、及び炭酸セシウム（０．８１ｇ，２．５ｍｍｏｌ）のジメチルホルムアミド（ＤＭＦ，１２ｍＬ）溶液にパラジウム触媒（５．８mｇ，０．００５ｍｍｏｌ）を加えた。１１０℃で４０時間撹拌した後、混合溶液をジエチルエーテルで抽出し、有機層を水で洗浄、有機層に硫酸マグネシウム（無水）を加えて乾燥し、濃縮した。粗結晶をメタノールで再結晶し、化合物１を得た。 [Step 1]
Dimethylformamide (DMF, 12 mL) of 2,6-dibromonaphthalene (0.29 g, 1 mmol), 4-pyridylboronic acid (0.27 g, 2.2 mmol), and cesium carbonate (0.81 g, 2.5 mmol) To the solution was added palladium catalyst (5.8 mg, 0.005 mmol). After stirring at 110 ° C. for 40 hours, the mixed solution was extracted with diethyl ether, the organic layer was washed with water, dried over magnesium sulfate (anhydrous), and concentrated. The crude crystal was recrystallized from methanol to obtain Compound 1.
［工程２］
　化合物１（０．２ｇ，１ｍｍｏｌ）をジクロロメタン２５ｍＬに溶解し、ヨードメタン（ＣＨ３Ｉ，２ｍＬ）を加え室温で２４時間撹拌した。析出した固体をジクロロメタンで洗浄し、ナフタレン誘導体（Ｉ）を黄色固体として得た。以下に、１ＨＮＭＲデータを示す。また、得られたナフタレン誘導体（Ｉ）の１ＨＮＭＲチャートを図１に示す。 [Step 2]
Compound 1 (0.2 g, 1 mmol) was dissolved in 25 mL of dichloromethane, iodomethane (CH 3 I, 2 mL) was added, and the mixture was stirred at room temperature for 24 hours. The precipitated solid was washed with dichloromethane to obtain naphthalene derivative (I) as a yellow solid. The 1 HNMR data is shown below. A 1 HNMR chart of the naphthalene derivative (I) obtained is shown in FIG.
1H NMR (400 MHz, DMSO-d6) d (ppm): 9.11 (d, J = 6.8 Hz, 4H), 8.88 (s, 2H), 8.68 (d, J =7.2 Hz, 4H), 8.35 (d, J = 8.8 Hz, 2H), 8.30 (dd, J = 1.6, 8.8 Hz, 2H), 4.37 (s, 6H)． 1 H NMR (400 MHz, DMSO-d 6 ) d (ppm): 9.11 (d, J = 6.8 Hz, 4H), 8.88 (s, 2H), 8.68 (d, J = 7.2 Hz, 4H), 8.35 ( d, J = 8.8 Hz, 2H), 8.30 (dd, J = 1.6, 8.8 Hz, 2H), 4.37 (s, 6H).
実施例２．アントラセン誘導体（II）の合成 Example 2 Synthesis of anthracene derivative (II)
Figure JPOXMLDOC01-appb-C000019
Figure JPOXMLDOC01-appb-C000019
［工程１］
　２，６－ジブロモアントラセン（０．３４ｇ，１ｍｍｏｌ）、４－ピリジルボロン酸（０．２７ｇ，２．２ｍｍｏｌ）、及び炭酸セシウム（０．８１ｇ，２．５ｍｍｏｌ）のジメチルホルムアミド（ＤＭＦ，１２ｍＬ）溶液にパラジウム触媒（５．７８mｇ，０．００５ｍｍｏｌ）を加えた。１１０℃で７０時間撹拌した後、混合溶液をジエチルエーテルで抽出し、有機層を水で洗浄、有機層に硫酸マグネシウム（無水）を加えて乾燥し、濃縮した。粗結晶をメタノールで再結晶し、化合物２を得た。 [Step 1]
A solution of 2,6-dibromoanthracene (0.34 g, 1 mmol), 4-pyridylboronic acid (0.27 g, 2.2 mmol), and cesium carbonate (0.81 g, 2.5 mmol) in dimethylformamide (DMF, 12 mL) To was added palladium catalyst (5.78 mg, 0.005 mmol). After stirring at 110 ° C. for 70 hours, the mixed solution was extracted with diethyl ether, the organic layer was washed with water, magnesium sulfate (anhydrous) was added to the organic layer, dried and concentrated. The crude crystals were recrystallized from methanol to obtain compound 2.
［工程２］
　化合物２（０．１８ｇ，０．５ｍｍｏｌ）をジクロロメタン２５ｍＬに溶解し、ヨードメタン（ＣＨ３Ｉ，２ｍＬ）を加え室温で２４時間撹拌した。析出した固体をジクロロメタンで洗浄し、アントラセン誘導体（II）を黄色固体として得た。以下に、１ＨＮＭＲデータを示す。また、得られたアントラセン誘導体（II）の１ＨＮＭＲチャートを図２に示す。 [Step 2]
Compound 2 (0.18 g, 0.5 mmol) was dissolved in 25 mL of dichloromethane, iodomethane (CH 3 I, 2 mL) was added, and the mixture was stirred at room temperature for 24 hours. The precipitated solid was washed with dichloromethane to obtain an anthracene derivative (II) as a yellow solid. The 1 HNMR data is shown below. Moreover, the 1 HNMR chart of the obtained anthracene derivative (II) is shown in FIG.
1H NMR (400 MHz, DMSO-d6) d (ppm): 9.10 (d, J = 6.8 Hz, 4H), 9.03 (s, 2H), 8.90 (s, 2H), 8.72 (d, J = 6.8 Hz, 4H), 8.46 (d, J = 8.8 Hz, 2H), 8.21 (dd, J = 1.6, 7.2, 2 Hz, 2H), 4.37 (s, 6H)． 1 H NMR (400 MHz, DMSO-d 6 ) d (ppm): 9.10 (d, J = 6.8 Hz, 4H), 9.03 (s, 2H), 8.90 (s, 2H), 8.72 (d, J = 6.8 Hz, 4H), 8.46 (d, J = 8.8 Hz, 2H), 8.21 (dd, J = 1.6, 7.2, 2 Hz, 2H), 4.37 (s, 6H).
実施例３．ピレン誘導体（III）の合成 Example 3 FIG. Synthesis of pyrene derivative (III)
Figure JPOXMLDOC01-appb-C000020
Figure JPOXMLDOC01-appb-C000020
［工程１］
　１，６－ジブロモピレン（０．３６ｇ，１ｍｍｏｌ）、４－ピリジルボロン酸（０．２７ｇ，２．２ｍｍｏｌ）、及び炭酸セシウム（０．８１ｇ，２．５ｍｍｏｌ）のジメチルホルムアミド（ＤＭＦ，１２ｍＬ）溶液にパラジウム触媒（５．７８mｇ，０．００５ｍｍｏｌ）を加えた。１２０℃で５０時間撹拌した後、混合溶液をジエチルエーテルで抽出し、有機層を水で洗浄、有機層に硫酸マグネシウム（無水）を加えて乾燥し、濃縮した。粗結晶をメタノールで再結晶し、化合物３を得た。 [Step 1]
A solution of 1,6-dibromopyrene (0.36 g, 1 mmol), 4-pyridylboronic acid (0.27 g, 2.2 mmol), and cesium carbonate (0.81 g, 2.5 mmol) in dimethylformamide (DMF, 12 mL) To was added palladium catalyst (5.78 mg, 0.005 mmol). After stirring at 120 ° C. for 50 hours, the mixed solution was extracted with diethyl ether, the organic layer was washed with water, magnesium sulfate (anhydrous) was added to the organic layer, dried and concentrated. The crude crystal was recrystallized from methanol to obtain Compound 3.
［工程２］
　化合物３（０．１５ｇ，０．４ｍｍｏｌ）をジクロロメタン１５ｍＬに溶解し、ヨードメタン（ＣＨ３Ｉ，２ｍＬ）を加え室温で２４時間撹拌した。析出した固体をジクロロメタンで洗浄し、ピレン誘導体（III）を黄色固体として得た。以下に、１ＨＮＭＲデータを示す。また、得られたピレン誘導体（III）の１ＨＮＭＲチャートを図３に示す。 [Step 2]
Compound 3 (0.15 g, 0.4 mmol) was dissolved in 15 mL of dichloromethane, iodomethane (CH 3 I, 2 mL) was added, and the mixture was stirred at room temperature for 24 hours. The precipitated solid was washed with dichloromethane to obtain pyrene derivative (III) as a yellow solid. The 1 HNMR data is shown below. In addition, FIG. 3 shows a 1 HNMR chart of the obtained pyrene derivative (III).
1H NMR (400 MHz, DMSO-d6) δ(ppm): 9.18 (d, J = 6.8 Hz, 4H), 8.62 (d, J = 8.0 Hz, 2H), 8.49 (d, J = 6.8 Hz, 4H), 8.46 (d, J = 9.2 Hz, 2H), 8.29 (d, J = 8.0 Hz,2H), 8.26 (d, J = 9.2 Hz, 2H), 4.47 (s, 6H)． 1 H NMR (400 MHz, DMSO-d 6 ) δ (ppm): 9.18 (d, J = 6.8 Hz, 4H), 8.62 (d, J = 8.0 Hz, 2H), 8.49 (d, J = 6.8 Hz, 4H), 8.46 (d, J = 9.2 Hz, 2H), 8.29 (d, J = 8.0 Hz, 2H), 8.26 (d, J = 9.2 Hz, 2H), 4.47 (s, 6H).
実施例４．式（２－１）で表される化合物の合成 Example 4 Synthesis of compound represented by formula (2-1)
Figure JPOXMLDOC01-appb-C000021
Figure JPOXMLDOC01-appb-C000021
　フレイムドライし、アルゴン置換したシュレンク管に、２，６－ジブロモナフタレン（０．２９ｇ，１ｍｍｏｌ）とジクロロビス（トリフェニルホスフィン）パラジウム（II）（Pd(PPh3)2Cl2，０．０７７ｇ，０．１１ｍｍｏｌ）、４－ビニルピリジン（０．２６ｇ，２．５６ｍｍｏｌ）、ベンゼン１ｍＬ、トリエチルアミン１ｍＬを加え、２４時間、１００℃で加熱・撹拌した。エバポレーターで有機溶媒を除き、得られた固体をジクロロメタンに溶解させ、水と分液した。有機相に無水硫酸マグネシウムを加え、乾燥した後、濃縮し、粗結晶を得た。その後、粗結晶をジエチルエーテルで洗い、アセトンによる再結晶でパールイエローの固体を得た。
　得られた上記固体（０．０４５ｇ，０．１３ｍｍｏｌ）をジクロロメタン１０ｍＬに溶かし、ヨウ化メチル２ｍＬを加えた。一晩、常温で撹拌し、析出した黄色の固体をろ過により得た。以下に、１ＨＮＭＲデータを示す。また、得られた式（２－１）で表される化合物の１ＨＮＭＲチャートを図４に示す。 Flame-dried, argon-substituted Schlenk tubes were charged with 2,6-dibromonaphthalene (0.29 g, 1 mmol) and dichlorobis (triphenylphosphine) palladium (II) (Pd (PPh 3 ) 2 Cl 2 , 0.077 g, 0 .11 mmol), 4-vinylpyridine (0.26 g, 2.56 mmol), 1 mL of benzene, and 1 mL of triethylamine were added, and the mixture was heated and stirred at 100 ° C. for 24 hours. The organic solvent was removed with an evaporator, and the resulting solid was dissolved in dichloromethane and separated from water. Anhydrous magnesium sulfate was added to the organic phase, dried and concentrated to obtain crude crystals. Thereafter, the crude crystals were washed with diethyl ether and recrystallized with acetone to obtain a pearl yellow solid.
The obtained solid (0.045 g, 0.13 mmol) was dissolved in 10 mL of dichloromethane, and 2 mL of methyl iodide was added. The mixture was stirred overnight at room temperature, and the precipitated yellow solid was obtained by filtration. The 1 HNMR data is shown below. Further, FIG. 4 shows a 1 HNMR chart of the compound represented by the formula (2-1) obtained.
1H NMR (500 MHz, DMSO-d6) δ(ppm): 8.91 (d, J = 6.5 Hz, 4H), 8.29 (d, J = 6.5 Hz, 4H), 8.25 (s, 4H), 8.21 (d, J = 16.5 Hz, 2H), 8.21 (s, 2H), 8.12 (d, J = 9.0 Hz, 2H), 8.04 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 16.5 Hz, 2H), 4.25 (s, 6H)． 1 H NMR (500 MHz, DMSO-d 6 ) δ (ppm): 8.91 (d, J = 6.5 Hz, 4H), 8.29 (d, J = 6.5 Hz, 4H), 8.25 (s, 4H), 8.21 ( d, J = 16.5 Hz, 2H), 8.21 (s, 2H), 8.12 (d, J = 9.0 Hz, 2H), 8.04 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 16.5 Hz, 2H), 4.25 (s, 6H).
実施例５．紫外－可視吸収スペクトル及び蛍光スペクトルの測定
　実施例１～３により合成された化合物及び比較例１の紫外－可視吸収スペクトル及び蛍光スペクトルを以下の条件にしたがって測定した。比較例１の化合物は、以下の構造である。 Embodiment 5 FIG. Measurement of UV-visible absorption spectrum and fluorescence spectrum The UV-visible absorption spectrum and fluorescence spectrum of the compound synthesized in Examples 1 to 3 and Comparative Example 1 were measured according to the following conditions. The compound of Comparative Example 1 has the following structure.
Figure JPOXMLDOC01-appb-C000022
Figure JPOXMLDOC01-appb-C000022
　紫外－可視吸収スペクトルは、V-670-UV-VIS-NIR spectrophotometer（Jasco Co.）を用いて測定した。蛍光スペクトルは、C9920-03G（Hamamatsu Photonics. K. K.）を用いて測定した。蛍光量子収率は積分球を用いた絶対測定により決定した。濃度が１０－６ｍｏｌ／Ｌとなるように調整した試料を用いて測定した。測定結果を、表１と図１に示す。なお、図１における実線は紫外－可視吸収スペクトルを表し、点線は蛍光スペクトルを表す。 The ultraviolet-visible absorption spectrum was measured using a V-670-UV-VIS-NIR spectrophotometer (Jasco Co.). The fluorescence spectrum was measured using C9920-03G (Hamamatsu Photonics. K. K.). The fluorescence quantum yield was determined by absolute measurement using an integrating sphere. Measurement was performed using a sample adjusted to a concentration of 10 −6 mol / L. The measurement results are shown in Table 1 and FIG. The solid line in FIG. 1 represents the ultraviolet-visible absorption spectrum, and the dotted line represents the fluorescence spectrum.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　上記表１に示すように、比較例１の化合物の発光量子収率（φ）、化合物に吸収された光子数と蛍光によって放出された光子数との比を表し、数値が大きいほど発光の効率がよいことを意味する）が０．１４であったのに対し、ナフタレン誘導体（Ｉ）、アントラセン誘導体（II）、ピレン誘導体（III）の発光量子収率（φ）は、それぞれ、０．９２、０．５８、０．４３であった。すなわち、本発明の化合物は発光の効率が良く、強い蛍光を検出できるため、蛍光色素としての使用に適する。 As shown in Table 1, the emission quantum yield (φ) of the compound of Comparative Example 1 and the ratio between the number of photons absorbed by the compound and the number of photons emitted by fluorescence are represented. Was 0.14, whereas the luminescence quantum yields (φ) of the naphthalene derivative (I), the anthracene derivative (II), and the pyrene derivative (III) were 0.92 and 0.92, respectively. 0.58 and 0.43. That is, the compound of the present invention is suitable for use as a fluorescent dye because it has high emission efficiency and can detect strong fluorescence.
実施例６．細胞の蛍光化実験と観察 Example 6 Cell fluorescence experiments and observations
細胞の培養
　染色のモデル細胞としてヒト胎児腎細胞であるＨｅｋ２９３細胞を使用した。Ｈｅｋ２９３細胞は、１０％（ｖ／ｖ）のウシ胎児血清、１％（ｖ／ｖ）のトリプシン及びストレプトマイシンを含むダルベッコ改変イーグル培地（ＤＭＥＭ）中、３７℃、５％ＣＯ２条件下で培養した。 Hek293 cells, which are human fetal kidney cells, were used as model cells for cell culture staining. Hek293 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v / v) fetal bovine serum, 1% (v / v) trypsin and streptomycin under conditions of 37 ° C. and 5% CO 2 . .
細胞の蛍光化
　顕微鏡観察を行う準備のために、Ｈｅｋ２９３細胞を３５ｍｍガラスベースディッシュに細胞密度１×１０５ｃｅｌｌｓ／ｄｉｓｈとなるように継代した。継代して２４時間後、細胞がディッシュへ付着していることを顕微鏡観察により確認した。ディッシュより培地を除き、リン酸緩衝生理食塩水（ＰＢＳ）を用いて２回細胞を洗浄した。ナフタレン誘導体（I）、アントラセン誘導体（II）、ピレン誘導体（III）、式（２－１）で表される化合物、比較例２～４の化合物の１×１０－３ｍｏｌ　ｄｍ－３のジメチルスルホキシド（ＤＭＳＯ）溶液２μＬを添加したフェノールレッド不含ＤＭＥＭ培地２ｍＬ（ＰＹ最終濃度１μｍｏｌ　ｄｍ－３、最終ＤＭＳＯ濃度０．１％（ｖ／ｖ））をディッシュに入れ、１２時間インキュベートすることにより染色を行った。顕微鏡観察の直前に、色素を含む培地をディッシュから取り除き、ＰＢＳを用いて２回細胞を洗浄し、フェノールレッド不含ＤＭＥＭ培地２ｍＬをディッシュに加えた。なお、比較例２～４の化合物は以下の構造であり、公知の合成方法により得た。 In preparation for performing fluorescence microscopy of the cells, Hek293 cells were passaged on a 35 mm glass base dish to a cell density of 1 × 10 5 cells / dish. 24 hours after passage, it was confirmed by microscopic observation that the cells were attached to the dish. The medium was removed from the dish, and the cells were washed twice with phosphate buffered saline (PBS). 1 × 10 −3 mol dm −3 dimethyl sulfoxide of naphthalene derivative (I), anthracene derivative (II), pyrene derivative (III), compound represented by formula (2-1), and compounds of Comparative Examples 2 to 4 Stain was obtained by adding 2 mL of phenol red-free DMEM medium (PY final concentration 1 μmol dm −3 , final DMSO concentration 0.1% (v / v)) to which 2 μL of (DMSO) solution was added and incubating for 12 hours. went. Immediately before microscopic observation, the medium containing the dye was removed from the dish, the cells were washed twice with PBS, and 2 mL of phenol red-free DMEM medium was added to the dish. The compounds of Comparative Examples 2 to 4 have the following structures and were obtained by known synthesis methods.
Figure JPOXMLDOC01-appb-C000023
Figure JPOXMLDOC01-appb-C000023
蛍光顕微鏡観察
　蛍光顕微鏡は、オプティカルブロック（Hamamatsu Photonics K. K.）を用いて作成した。光源にはフェムト秒チタンサファイヤレーザー（Mira900、Coherent）を用いた。蛍光の検出には光電子増倍管（R928、Hamamatsu Photonics K. K.）を用い、印加電圧１０００Ｖでプリアンプ（５ＭＨｚ）付きソケットを経てＤＣ検出した。サンプルステージにはKZG0620-Gを用い、対物レンズは倍率４０倍、ＮＡ＝１．１５の無限遠補正対物レンズを用いた。観察により得られた画像を図２～７に示す。
　なお、膜電位の調整、すなわち、膜電位の低下への誘導は、脱共役剤であるカルボニルシアニド－ｍ－クロロフェニルヒドラゾン（ＣＣＣＰ）を用いた。ＣＣＣＰは、ミトコンドリアのプロトン透過性を増加させてミトコンドリア膜電位を崩壊させる化合物である。ＣＣＣＰをＤＭＳＯに溶解させ、１０ｍｍｏｌ　ｄｍ－３の溶液を調製した。調製したＣＣＣＰのＤＭＳＯ溶液を、培地に対して０．１％（ｖ／ｖ）となるよう添加した（最終ＣＣＣＰ濃度　１０（ｍｏｌ　ｄｍ－３）。添加して５分後、蛍光顕微鏡により観察した。 Fluorescence microscope observation The fluorescence microscope was prepared using an optical block (Hamamatsu Photonics K. K.). A femtosecond titanium sapphire laser (Mira900, Coherent) was used as the light source. For detection of fluorescence, a photomultiplier tube (R928, Hamamatsu Photonics K. K.) was used, and DC was detected through a socket with a preamplifier (5 MHz) at an applied voltage of 1000 V. The sample stage used was KZG0620-G, and the objective lens was an infinity corrected objective lens with a magnification of 40 times and NA = 1.15. Images obtained by observation are shown in FIGS.
Note that carbonyl cyanide-m-chlorophenylhydrazone (CCCP), which is an uncoupling agent, was used to adjust the membrane potential, that is, to induce reduction in membrane potential. CCCP is a compound that increases mitochondrial proton permeability and disrupts mitochondrial membrane potential. CCCP was dissolved in DMSO to prepare a 10 mmol dm -3 solution. The prepared CCCP in DMSO was added to the medium so as to be 0.1% (v / v) (final CCCP concentration 10 (mol dm −3 ). 5 minutes after addition, it was observed with a fluorescence microscope. .
　図１０～１２に示されるように、膜電位低下の前と後で、比較例２～４の化合物は蛍光の強度や場所は変化していない。一方、図６～９に示されるように、ナフタレン誘導体（Ｉ）、アントラセン誘導体（II）、ピレン誘導体（III）及び式（２－１）で表される化合物は、膜電位の低下前はミトコンドリアで蛍光が検出され、核では蛍光が検出されなかったが、膜電位の低下後は核での蛍光が検出されるようになり、蛍光が検出される場所が変化した。したがって、本発明の化合物は、ミトコンドリア膜電位に応じて局在場所が変化する化合物である。 As shown in FIGS. 10 to 12, the intensity and location of the fluorescence of the compounds of Comparative Examples 2 to 4 did not change before and after the decrease in membrane potential. On the other hand, as shown in FIGS. 6 to 9, the compounds represented by naphthalene derivative (I), anthracene derivative (II), pyrene derivative (III) and formula (2-1) Fluorescence was detected in the nucleus, but no fluorescence was detected in the nucleus, but after the membrane potential was lowered, fluorescence in the nucleus was detected, and the location where fluorescence was detected changed. Therefore, the compound of the present invention is a compound whose localization location changes according to the mitochondrial membrane potential.
　本発明の化合物は、蛍光性があり、また、ミトコンドリア膜電位に応じて局在場所をミトコンドリアから核に移す性質を有していることから、ミトコンドリアの膜電位の変化を検出することができる。また、本発明の化合物を蛍光色素として用いることにより、生きた細胞の観察が可能で一般的な蛍光顕微鏡によって、簡単に細胞の観察ができる。さらに、蛍光顕微鏡によって、蛍光の発せられる場所の情報を得ることができ、簡単に細胞の生死を判別することができる。本発明の蛍光色素組成物は、ミトコンドリアの機能障害に起因する疾患の予防研究や疾患の発生メカニズム解明の研究、例えば、治療候補薬の投与による薬剤の効果、ミトコンドリア活力や細胞の生死への影響等を試験するときに使用できる。 The compound of the present invention is fluorescent, and has a property of moving the localization location from the mitochondria to the nucleus in accordance with the mitochondrial membrane potential, so that a change in the mitochondrial membrane potential can be detected. Further, by using the compound of the present invention as a fluorescent dye, it is possible to observe living cells, and the cells can be easily observed with a general fluorescence microscope. Furthermore, information on the place where the fluorescence is emitted can be obtained by the fluorescence microscope, and the viability of the cell can be easily determined. The fluorescent dye composition of the present invention can be used for studying prevention of diseases caused by mitochondrial dysfunction and research for elucidating the mechanism of disease occurrence, for example, effects of drugs by the administration of treatment candidates, effects on mitochondrial vitality and cell viability Can be used when testing etc.

Claims (9)

  1. 　式（１）で表される化合物。
    Figure JPOXMLDOC01-appb-C000024
     ［式中、Ｒ１はＣ１～Ｃ１０のアルキル基を表し、Ｚ－はピリジニウムカチオンに対するカウンターアニオンを表し、Ｘは、
    Figure JPOXMLDOC01-appb-C000025
     （式中、波線は隣接するピリジン環への結合部位を表す。ただし、ナフチレン基（ｉ）は、電子供与性基を有していてもよい。）で表される縮合多環基のいずれかを表す。］     The compound represented by Formula (1).
    Figure JPOXMLDOC01-appb-C000024
     [Wherein R 1 represents a C1-C10 alkyl group, Z − represents a counter anion with respect to a pyridinium cation, and X represents
    Figure JPOXMLDOC01-appb-C000025
     (Wherein the wavy line represents a bonding site to an adjacent pyridine ring, provided that the naphthylene group (i) may have an electron donating group). Represents. ]
  2. 　Ｘが以下の式で表される縮合多環基のいずれかであることを特徴とする請求項１に記載の化合物。
    Figure JPOXMLDOC01-appb-C000026
     （式中、波線は隣接するピリジン環への結合部位を表す。ただし、ナフチレン基（ｉ）は、電子供与性基を有していてもよい。）     The compound according to claim 1, wherein X is any one of a condensed polycyclic group represented by the following formula.
    Figure JPOXMLDOC01-appb-C000026
     (In the formula, a wavy line represents a bonding site to an adjacent pyridine ring. However, the naphthylene group (i) may have an electron donating group.)
  3. 　Ｘが以下の式で表される縮合多環基のいずれかであることを特徴とする請求項１に記載の化合物。
    Figure JPOXMLDOC01-appb-C000027
     （式中、波線は隣接するピリジン環への結合部位を表す。ただし、ナフチレン基（ｉ－１）は、電子供与性基を有していてもよい。）     The compound according to claim 1, wherein X is any one of a condensed polycyclic group represented by the following formula.
    Figure JPOXMLDOC01-appb-C000027
     (In the formula, a wavy line represents a binding site to an adjacent pyridine ring. However, the naphthylene group (i-1) may have an electron-donating group.)
  4. 　カウンターアニオンが、ハロゲン化物イオン、スルホネートであることを特徴とする請求項１～３のいずれかに記載の化合物。 The compound according to any one of claims 1 to 3, wherein the counter anion is a halide ion or sulfonate.
  5. 　請求項１～４のいずれかに記載の化合物の１又は２以上を含有することを特徴とする蛍光色素組成物。 A fluorescent dye composition comprising one or more of the compounds according to any one of claims 1 to 4.
  6. 　請求項１～４に記載の化合物及び以下の式（２）で表される化合物；
    Figure JPOXMLDOC01-appb-C000028
     （式中、Ｒ１はＣ１～Ｃ１０のアルキル基を表し、Ｚ－はピリジニウムカチオンに対するカウンターアニオンを表し、ナフチレン基は電子供与性基を有していてもよい。）
    からなる群より選ばれる少なくとも一つを用いることを特徴とする、ミトコンドリア膜電位の変化の検出方法。     The compound according to claims 1 to 4 and a compound represented by the following formula (2);
    Figure JPOXMLDOC01-appb-C000028
     (In the formula, R 1 represents a C1-C10 alkyl group, Z − represents a counter anion with respect to the pyridinium cation, and the naphthylene group may have an electron-donating group.)
    A method for detecting a change in mitochondrial membrane potential, comprising using at least one selected from the group consisting of:
  7. 　化合物（２）が以下の化合物（２－１）
    Figure JPOXMLDOC01-appb-C000029
     であることを特徴とする、請求項６に記載のミトコンドリア膜電位の変化の検出方法。     Compound (2) is the following compound (2-1)
    Figure JPOXMLDOC01-appb-C000029
     The method for detecting a change in mitochondrial membrane potential according to claim 6, wherein:
  8. 　請求項１～４に記載の化合物及び以下の式（２）で表される化合物；
    Figure JPOXMLDOC01-appb-C000030
     （式中、Ｒ１はＣ１～Ｃ１０のアルキル基を表し、Ｚ－はピリジニウムカチオンに対するカウンターアニオンを表し、ナフチレン基は電子供与性基を有していてもよい。）
    からなる群より選ばれる少なくとも一つを用いることを特徴とする、細胞の生死を判別する方法。     The compound according to claims 1 to 4 and a compound represented by the following formula (2);
    Figure JPOXMLDOC01-appb-C000030
     (In the formula, R 1 represents a C1-C10 alkyl group, Z − represents a counter anion with respect to the pyridinium cation, and the naphthylene group may have an electron-donating group.)
    A method for discriminating whether cells are alive or dead, wherein at least one selected from the group consisting of:
  9. 　化合物（２）が以下の化合物（２－１）
    Figure JPOXMLDOC01-appb-C000031
     であることを特徴とする、請求項８に記載の細胞の生死を判別する方法。     Compound (2) is the following compound (2-1)
    Figure JPOXMLDOC01-appb-C000031
     The method for discriminating whether a cell is alive or dead according to claim 8, wherein:
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