JP6620466B2 - Fluorescent compounds with properties localized in mitochondria - Google Patents

Description

  The present invention relates to a novel fluorescent compound, and more particularly to a fluorescent compound having the property of being localized in mitochondria. The present invention also relates to a fluorescent dye composition containing the compound for use in visualization of mitochondria.

  Mitochondria are cell organelles that are involved in the regulation of apoptosis while producing cellular energy, and are involved in cell life and death. 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 visualize and observe mitochondria by staining with fluorescent compounds for elucidating the mechanism of these diseases and developing therapeutic methods.

  In order to visualize mitochondria, the mitochondria are stained with a fluorescent compound that changes its behavior according to the membrane potential of the mitochondria, that is, a fluorescent compound having the property of being localized in the mitochondria, and the fluorescence emitted from the compound is observed. There is a way to do it. The applicant of the present invention is a condensed polycyclic group compound having 2 to 4 rings having two N-alkylpyridinylethenyl groups as substituents, specifically, the following BP, FLW, NP, AC, PY, etc. It has already been reported that it has been found to be localized in mitochondrial according to the mitochondrial membrane potential and can be used for visualization (Non-patent Documents 1, 2, and 3). Moreover, since the said compound has a multiphoton fluorescence characteristic, a three-dimensional image can also be obtained by observing the biological tissue etc. which dye | stained with this compound with a multiphoton excitation microscope.

  However, the brightness of microscopic images of cells stained with the above compounds is not sufficient, and it has been desired to obtain brighter images.

H. Moritomo, K. Yamada, Y. Kojima, Y. Suzuki, H. Kinoshita, A. Sasaki, S. Mikuni, M. Kinjo, J. Kawamata, Cell Struct. Funct. 39 (2014) 125. M. Tominaga, S. Mochida, H. Sugihara, K. Satomi, H. Moritomo, A. Fuji, A. Tomoyuki, Y. Suzuki, J. Kawamata, Chem. Lett. Vol. 43, No. 9 (2014) 1490. Y. Niko, H. Moritomo, H. Sugihara, Y. Suzuki, J. Kawamata, G. Konishi, J. Mater. Chem. B, 3 (2015) 184.

  An object of the present invention is to provide a compound having a property of localizing to mitochondria and capable of obtaining a bright microscopic image when cells are stained.

  In the condensed polycyclic group compound having 2 to 4 rings having two N-alkylpyridinylethenyl groups as substituents, the N-alkyl is a C4 to C8 linear or branched alkyl group. Since the ability of the compound to localize to mitochondria is improved and the degree of accumulation of the compound on the outer mitochondrial membrane is increased, a bright microscopic image can be obtained.

That is, the present invention relates to the following.
[1] A compound represented by formula (1).
[Wherein R ¹ represents a C4 to C8 linear or branched alkyl group,
The wavy line represents the geometric isomers E, Z,
X represents the following condensed polycyclic groups (i) to (v)
(In the formula, R ² represents an electron donating group, a is an integer of 0 to 4, b is an integer of 0 to 6, c and e are integers of 0 to 8, and d is 0 to 0. And R ² may be the same or different when a, b, c, d, and e are integers greater than or equal to ^{2. The} wavy line indicates the bonding position to the adjacent carbon atom. It indicates whether, Z ^- represents a counter anion to a pyridinium cation. ]
[2] The compound of [1] above, wherein the condensed polycyclic group is any of the following condensed polycyclic groups (i ′) to (v ′).
(R ² represents an electron donating group, a is an integer of 0 to 4, b is an integer of 0 to 6, c and e are integers of 0 to 8, and d is an integer of 0 to 3. When a, b, c, d, and e are integers of 2 or more, R ² may be the same or different. The wavy line indicates the bonding position with respect to the adjacent carbon atom.)
[3] The compound of [1] or [2] above, wherein the counter anion is a halide ion, sulfonate, or perchlorate ion.
[4] A fluorescent dye composition characterized by using at least one selected from the group consisting of the compounds of any one of [1] to [3].
[5] The fluorescent dye composition according to the above [4] for use in visualization of mitochondria.

  The compound of the present invention has a mitochondrial localization ability, and a bright microscopic image can be obtained when such a compound is used for staining of living tissue or cells. Therefore, it is possible to obtain a microscopic image sufficient for observation even if the intensity of light irradiated to a biological tissue or cell that is the object of observation is weakened, damage to the tissue or cell due to light irradiation, and photobleaching of the compound Can be suppressed. Further, the compound of the present invention also has multiphoton fluorescence characteristics such as two-photon and three-photon. For example, in observation with a two-photon excitation fluorescence microscope, weak intensity excitation light, that is, a length that reaches deep in the living body. Since light of a wavelength can be used, the observation range in the depth direction can be expanded more than before, and the objects that can be observed with a two-photon excitation fluorescence microscope can be expanded.

The fluorescence microscope image of HEK293 cell dye | stained with the compound of Example 4 and tetramethyl rhodamine methyl ester (TMRM), and the image which overlap | superposed the fluorescence microscope image of the compound of Example 4 and the fluorescence microscope image of TMRM are shown. FIG. It is a figure which shows the image which overlap | superposed the fluorescence microscope image of the compound of Example 5, and the HEK293 cell dye | stained by TMRM, and the fluorescence microscope image of the compound of Example 5, and the fluorescence microscope image of TMRM. It is a figure which shows the image which superimposed the fluorescence microscope image of the compound of Example 6, and the HEK293 cell dye | stained by TMRM, and the fluorescence microscope image of the compound of Example 6, and the fluorescence microscope image of TMRM. It is a figure which shows the image which superimposed the fluorescence microscope image of the compound of the comparative example 2, and the HEK293 cell dye | stained by TMRM, and the fluorescence microscope image of the compound of the comparative example 2, and the fluorescence microscope image of TMRM. FIG. 3 shows a fluorescence microscope image of HEK293 cells stained with the compound of Example 2. FIG. 3 shows a fluorescence microscope image of HEK293 cells stained with the compound of Comparative Example 1. It is a figure which shows light emission only by the focus of a laser beam when condensing and irradiating a laser beam to the compound of Example 1. FIG. It is a figure which shows light emission only by the focus of a laser beam when condensing and irradiating a laser beam to the compound of Example 2. FIG. It is a figure which shows light emission only by the focus of a laser beam when condensing and irradiating a laser beam to the compound of Example 3. FIG. It is a figure which shows light emission only by the focus of a laser beam when condensing and irradiating a laser beam to the compound of Example 4. FIG. It is a figure which shows light emission only by the focus of a laser beam when condensing and irradiating a laser beam to the compound of Example 5. FIG. It is a figure which shows light emission only by the focus of a laser beam when condensing and irradiating a laser beam to the compound of Example 6. FIG.

(Compound)
The compound of this invention is a compound represented by Formula (1).

In the formula, R ¹ represents a C4 to C8 linear or branched alkyl group,
The wavy line represents the geometric isomers E, Z,
X represents the following condensed polycyclic groups (i) to (v)

(In the formula, R ² represents an electron donating group, a is an integer of 0 to 4, b is an integer of 0 to 6, c and e are integers of 0 to 8, and d is 0 to 0. And R ² may be the same or different when a, b, c, d, and e are integers greater than or equal to ^{2. The} wavy line indicates the bonding position to the adjacent carbon atom. It indicates whether, Z ^- represents a counter anion to a pyridinium cation.

  Among the condensed polycyclic groups (i) to (v) of X, preferably

(In the formula, the wavy line indicates the bonding position to the adjacent carbon atom.)
A condensed polycyclic group (i ′) to (v ′) represented by:

A condensed polycyclic group (i ′) represented by:

In formula (1), the C4-C8 linear or branched alkyl group in R ¹ is not particularly limited as long as it is a linear or branched alkyl group having 4 to 8 carbon atoms, and 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 and the like. Among the C4-C8 linear or branched alkyl groups in R ¹ , n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, neopentyl group, n-to A C4-C6 linear or branched alkyl group such as a xyl group is preferred.

  The compound represented by the formula (1) of the present invention is a compound having multiphoton absorption characteristics including two-photons. Multiphoton absorption means that when light is considered as a photon, two or more photons are absorbed at the same time, whereby the molecular state is excited and transitions to a high energy level. For example, in the two-photon absorption, there is a nonlinear phenomenon in which the probability of occurrence is proportional to the square of the light intensity. Therefore, since light absorption is observed only when the light intensity is high, when light is collected by a lens, absorption can occur only near the focal point where the light intensity is high, and the position of the focal point relative to the measurement sample. A three-dimensional image can be obtained by scanning.

Here, when R ¹ is a C1-C3 alkyl group, it exhibits multiphoton absorption characteristics and mitochondrial localization is observed, but the localization is not sufficient, and as a result, the fluorescence intensity is at a practical level. It wasn't. The present invention can enhance the ability to localize mitochondria by making R ¹ a C4-C8 linear or branched alkyl group. This seems to be because the ability to localize mitochondria was greatly improved by controlling the hydrophobicity of the cation sites adsorbed on mitochondria. As a result, the degree of accumulation of the compound on the outer mitochondrial membrane increases, so that the fluorescence intensity is amplified and a very bright microscopic image can be obtained. Here, when R ¹ is C9 or more, the ability to localize to mitochondria is reduced, and another part of the cell is stained, which is not suitable. Therefore, it is excellent in strong multiphoton absorption and mitochondrial localization ability by making R ¹ a C4 to C8 linear or branched alkyl group, particularly a C4 to C6 linear or branched alkyl group. A brighter microscope image can be obtained.

In formula (1), the condensed polycyclic group represented by X is a group represented by (i) to (v), and each is a condensed polycyclic group which may be unsubstituted or substituted. In the case of unsubstituted, in the groups represented by (i) to (v), a to e are 0, which is synthetically advantageous. Further, when substituted, the electron donating group R ² is 1 or more, and by substituting the electron donating group, the electron density of the condensed polycyclic group is increased and the intramolecular charge transfer is large. Thus, the wavelength of multiphoton absorption can be shifted to a longer wavelength, and the fluorescence characteristics can be changed. The electron-donating group R ² is not particularly limited as long as it is an organic group having an effect of increasing the electron density of the condensed polycyclic group. For example, a hydroxyl group, a C1-C10 alkyl group, a C1-C10 alkoxy group, an amino group And an alkyl group having an ether bond and an alkoxy group having an ether bond.

  The C1-C10 alkyl group is not particularly limited as long as it is a linear or branched alkyl group having 1 to 10 carbon atoms. For example, a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, Examples thereof include an isobutyl group, a tert-butyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, a nonyl group, an isononyl group, and a decyl group.

  Examples of the C1-C10 alkoxy group include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a sec-butoxy group, a tert-butoxy group, an n-pentyloxy group, n-hexyloxy group, n-heptyloxy group, isoheptyloxy group, tert-heptyloxy group, n-octyloxy group, isooctyloxy group, tert-octyloxy group, 2-ethylhexyloxy group, etc. it can.

Examples of the amino _group, -NH ^_2, -NHR _3, mention may be made of functional groups represented by ^{-NR 3} R 3 ^'. R ³ and R ^{3 ′} are methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-hepsyl, C1-C10 alkyl group such as n-octyl group, nonyl group, isononyl group, decyl group; C3-C6 cycloalkyl group such as cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group; and phenyl group.

The alkyl group having an ether bond and the alkoxy group having an ether bond may be any alkyl group and alkoxy group having one or more ether bonds, such as —CH ₂ OCH ₃ , —OCH ₂ OCH ₃ , — _{CH 2 OCH 2 CH 3, -OCH} 2 OCH 2 CH 3, - (CH 2) 2 OCH 2 CH 3, -O (CH 2) 2 OCH 2 CH 3, - (CH 2) 2 O (CH 2) 2 _{CH 3, -O (CH 2)} 2 O (CH 2) 2 CH 3, - (CH 2) 3 O (CH 2) 2 CH 3, -O (CH 2) 3 O (CH 2) 2 CH 3, _{- (CH 2) 2 O (} CH 2) 2 O (CH 2) 2 CH 3, -O (CH 2) 2 O (CH 2) 2 O (CH 2) may be mentioned 2 CH ₃ and the like.

  The counter anion for the pyridinium cation is not particularly limited as long as it can form a salt with the pyridinium cation. For example, halide ions such as chlorine ion, bromine ion and iodine ion; methanesulfonate, p-toluenesulfonate, Sulfonates such as trifluoromethane sulfonate and trifluoroethane sulfonate; hexafluoroantimonate, hexafluorophosphate, tetrafluoroborate, perchlorate ion, etc., preferably halide ion, sulfonate, perchlorate ion It is.

  Specifically, the compound represented by Formula (1) can illustrate the compound shown below.

(Synthesis of compounds)
Examples of the method for synthesizing the compound represented by the formula (1) of the present invention include a method in which a condensed polycyclic moiety and a pyridine moiety are linked via a double bond. Specifically, an aldehyde represented by the formula (I) and an N-alkyl-4-methylpyridine-1-ium compound represented by the formula (II) are optionally added in the presence of a catalytic amount of a base. The compound of formula (1) can be synthesized by reacting in an appropriate reaction solvent.

(In the formula, R ¹ , X and Z ⁻ represent the same meaning as described above. Wavy lines represent geometric isomers E and Z.)

  As the aldehyde represented by the formula (I), commercially available 4,4′-biphenyldicarboxaldehyde or the like can be used, but it can also be derived from an aryl compound by a known method. For example, a method in which a commercially available aryl halide is lithiated, followed by a formylation reaction, a method in which a commercially available aryl compound such as naphthalene, anthracene, or pyrene is derived by a Friedel-Crafts reaction, a bis (hydroxymethyl) aryl Examples thereof include a method of inducing a compound by an appropriate oxidation reaction, but are not limited thereto.

  N-alkyl-4-methylpyridine-1-ium compound represented by the formula (II) can be obtained from 4-methyliodopyridine from Zhang, Y .; Wang, J .; Ji, P .; Yu, X .; Liu , H .; Liu, X .; Zhao, N .; Huang, B. Org. Biomol. Chem. 2010, 8, 4582-4588, specifically, 4-methylpyridine and Although it can synthesize | combine by making it react with haloalkane, it is not limited to this. Examples of the haloalkane include 1-iodobutane, 1-iodo-2-methylpropane, 1-iodo-1-methylpropane, 2-iodo-2-methylpropane, 1-iodopentane, 1-iodo-3-methylbutane, Commercially available haloalkanes such as iodo-2,2-dimethylpropane, 1-iodohexane, 1-iodoheptane, 1-iodooctane can also be used.

  The amount ratio of the aldehyde to the N-alkyl-4-methylpyridine-1-ium compound is not particularly limited, but the equivalent ratio of N-alkyl-4-methylpyridine-1-ium to aldehyde is 2 It is suitably selected from the range of 0.0 to 4.0, preferably 2.1 to 3.0.

  Examples of the base include trimethylamine, triethylamine, diisopropylethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, pyridine, piperidine and the like. Although the usage-amount of a base is not specifically limited, As an equivalent ratio with respect to the said aldehyde, it is suitably selected from the range of 0.01-1.0.

Moreover, the method of utilizing a Heck reaction can also be mentioned as a method of connecting the condensed polycyclic moiety and the pyridine moiety via a double bond. Specifically, the aryl halide of formula (III) and 4-vinylpyridine are reacted in the presence of a palladium catalyst and a base, if necessary, in an appropriate reaction solvent to obtain a compound of formula (V). Thereafter, the compound of formula (V) is reacted with an N-alkylating agent (R ¹ Z) in an appropriate reaction solvent as necessary to alkylate the nitrogen of pyridine, thereby converting the compound of formula (1). Can be synthesized.

(In the formula, R ¹ , X and Z ⁻ represent the same meaning as described above, Hal represents a halogen atom. Wavy lines represent geometric isomers E and Z.)

  Commercially available products can be used for the aryl halide and 4-vinylpyridine. Moreover, the usage-amount ratio of the said aryl halide and the said 4-vinyl pyridine compound is 2.0-4.0 as an equivalent ratio with respect to the aryl halide of 4-vinyl pyridine, Preferably it is 2.1-3.0. It is appropriately selected from the range.

  The palladium catalyst may be any palladium catalyst that is usually used for the Heck reaction. For example, palladium acetate, palladium chloride, tris (dibenzylideneacetone) dipalladium, bis (dibenzylideneacetone) palladium, tetrakis (triphenylphosphine) palladium. , [1,1′-bis (diphenylphosphino) ferrocene] dichloropalladium, bis (tri-ortho-tolylphosphine) palladium dichloride, bis (triphenylphosphine) palladium dichloride, palladium acetylacetonate, palladium carbon, dichlorobis (acetonitrile ) Palladium, bis (benzonitrile) palladium chloride, (1,3-diisopropylimidazol-2-ylidene) (3-chloropyridyl) palladium dichloride, bis Tri -tert- butylphosphine) palladium, dichlorobis (triphenylphosphine) palladium (II), dichlorobis (tricyclohexylphosphine) palladium and the like. Although the usage-amount of a catalyst is not specifically limited, As an equivalent ratio with respect to an aryl halide, it is suitably selected from the range of 0.01-0.5, Preferably it is 0.05-0.3.

  The base is not particularly limited as long as it is a base usually used for the Heck reaction, and amines such as trimethylamine, triethylamine, diisopropylethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, pyridine; sodium carbonate, carbonic acid Examples include inorganic bases such as potassium, cesium carbonate, sodium hydroxide, potassium hydroxide, cesium hydroxide. Although the usage-amount of a base is not specifically limited, As an equivalent ratio with respect to an aryl halide, it is suitably selected from the range of 2-20, Preferably 3-10.

  The alkylating agent is not particularly limited as long as it is an N-alkylating agent usually used for alkylating nitrogen. For example, 1-iodobutane, 1-iodo-2-methylpropane, 1-iodo-1-methylpropane 2-iodo-2-methylpropane, 1-iodopentane, 1-iodo-3-methylbutane, 1-iodo-2,2-dimethylpropane, 1-iodohexane, 1-iodoheptane, 1-iodooctane, etc. Examples include commercially available haloalkanes. Although the usage-amount of an alkylating agent is not specifically limited, As an equivalent ratio with respect to the compound represented by Formula (V), it is suitably selected from the range of 1-10, Preferably it is 1-5.

  Moreover, the compound represented by the formula (V) can also be induced by using the Horner-Wadsworth-Emmons reaction. Specifically, the phosphoric acid ester compound represented by the formula (VI) and 4-pyridinecarboxaldehyde are reacted in the presence of a base in an appropriate reaction solvent as necessary to convert the compound of the formula (V). Can be synthesized.

(Wherein X represents the same meaning as described above, and R ⁴ represents an ethyl group or a 2,2,2-trifluoroethyl group. Wavy lines represent geometric isomers E and Z.)

  The phosphate compound represented by the formula (VI) is prepared according to the method described in Iwase, Y .; Kamada, K .; Ohta, K .; Kondo, KJ Mater. Chem. 2003, 13, 1575-1581. Examples include, but are not limited to, a method in which a bis (halomethyl) aryl compound is induced by reacting with triethyl phosphite or tris phosphite (2,2,2-trifluoroethyl). The 4-pyridinecarboxaldehyde can use a commercial item. Moreover, although the usage-amount ratio of the said phosphate ester compound and the said 4-pyridine carboxaldehyde is not specifically limited, As an equivalent ratio with respect to the phosphate ester compound of 4-pyridine carboxaldehyde, it is from the range of 2.0-4.0. It is selected appropriately.

  The base is 1,8-diazabicyclo [5.4.0] -7-undecene, sodium hydride, sodium hexamethyldisilazide, potassium hexamethyldisilazide, benzyltrimethylammonium hydroxide, tert-butoxypotassium, etc. Is mentioned. The usage-amount of a base is suitably selected from the range of 2.0-4.0 as an equivalent ratio with respect to an aryl halide.

  Examples of the solvent used in all the above reactions include aromatic hydrocarbon solvents such as benzene and toluene, amide solvents such as acetonitrile, N, N-dimethylacetamide and N, N-dimethylformamide, tetrahydrofuran, and diethyl. Examples include ether solvents such as ether, alcohol solvents such as methanol, ethanol, and isopropanol, and halogen solvents such as dichloromethane, dichloroethane, and chloroform. These reaction solvents may be used alone or in appropriate combination of two or more. Although the usage-amount of a solvent is not specifically limited, The said aldehyde is suitably selected from the range used as the density | concentration of 0.001-1.0 (mol / L).

  In all the above reactions, the temperature during the reaction is usually 0 to 200 ° C., preferably 20 to 130 ° C., and is appropriately selected depending on the boiling point of the solvent or base used. The reaction may be carried out in an air atmosphere, but usually it is preferably carried out in an inert gas atmosphere. Examples of the inert gas include argon, helium, and nitrogen gas. The reaction product may be used as a reaction product after concentrating the reaction solution after completion of the reaction, if necessary, and then precipitating the crystals as they are or after performing an appropriate post-treatment. Specific methods for the post-treatment include known purification such as extraction, crystallization, recrystallization, chromatography and the like.

(Fluorescent dye composition)
The fluorescent dye composition of the present invention is not particularly limited as long as it contains at least one of the compounds represented by the formula (1), and is an additive usually used for the preparation of reagents in addition to the above compounds. , Solubilizers, pH adjusters, buffers, tonicity agents and the like may be included. In order to facilitate staining of cells and living tissues, the composition preferably further contains a solvent, and dimethyl sulfoxide (DMSO) can be suitably used as the solvent. Examples of the form of the fluorescent dye composition include a powder form, a freeze-dried form, a granule form, a tablet form, and a liquid form.

  The fluorescent dye composition of the present invention can be used for visualization of mitochondria. The above-mentioned visualization of mitochondria refers to obtaining the presence of mitochondria in cells as visual information such as a fluorescence microscope image. Specifically, the fluorescent dye of the present invention is present in a cell in which energy is produced, that is, a cell in which mitochondria having a membrane potential in a state of producing energy by aerobic respiration (also referred to as “active mitochondria”) exists. When the composition is added, in response to the mitochondrial membrane potential, the mitochondria and the compound of formula (1) contained in the fluorescent dye composition interact to localize the compound of formula (1) in the mitochondria. When excitation light having a predetermined wavelength is irradiated to the cell, fluorescence having a predetermined wavelength is emitted from the compound represented by the formula (1) localized in the mitochondria, and the fluorescence is detected by a fluorescence microscope or the like. Thus, a fluorescence microscope image of the observation target can be obtained.

  Moreover, the fluorescent dye composition of the present invention can discriminate whether it is a normal cell or a cell having mitochondria depolarized by apoptosis or metabolic stress, that is, cell viability. As described above, when the cell contains active mitochondria, the compound of formula (1) contained in the fluorescent dye composition is localized in the mitochondria and fluorescence is detected, and it is determined as a normal cell.

  On the other hand, the fluorescence of the present invention is present in cells in which energy production is stopped, that is, cells in which mitochondria (also referred to as “inactive mitochondria”) with depolarized membrane potential in a state where energy production by aerobic respiration is stopped exist. When the dye composition is added, the mitochondrial membrane potential is depolarized, so that the compound of formula (1) does not localize in the mitochondria. Spread. When the cell is irradiated with excitation light of a predetermined wavelength, fluorescence of a predetermined wavelength is emitted from the compound represented by the formula (1) existing in other organelles such as the nucleus, or the formula (1 ) The dispersed fluorescence of the compound represented by) reduces the fluorescence intensity detected, so that the luminescence behavior changes from normal cells, so cells with mitochondria depolarized due to apoptosis, metabolic stress, etc. It is judged.

  Detection of fluorescence emitted from the compound represented by the formula (1) or irradiation of excitation light to cells or living tissues can be performed by a fluorescence microscope. The excitation light is not particularly limited as long as the compound represented by the formula (1) can be absorbed, and is light in the ultraviolet region, visible region, and infrared region. Further, in order to obtain a three-dimensional image of a living tissue, the excitation light is preferably one that causes two-photon absorption in the compound represented by the formula (1), specifically, a wavelength region of 600 to 1200 nm. A three-dimensional image can be obtained by condensing the excitation light with a lens or the like, scanning the focal position, and irradiating the light.

  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 thereto.

Example 1 Synthesis of N, N'-dibutyl-4,4 '-(biphenyl-2,1-ethenediyl) dipyridinium diiodide

1-iodobutane (5 mmol) and 4-methylpyridine (5 mmol) were dissolved in ethyl acetate (5 mL). The solution was stirred at 80 ° C. to yield an oil. After removing the solvent, the resulting oil was washed several times with ethyl acetate to give 4-methyl-1-butylpyridinium iodide.
The obtained 4-methyl-1-butylpyridinium iodide (1 mmol) and 4,4′-biphenyldicarboxaldehyde (0.5 mmol) were dissolved in ethanol (5 mL). Two drops of piperidine were added dropwise thereto and stirred at 60 ° C. After heating for 12 hours, the precipitated yellow powder was filtered and washed several times with cold ethanol, and N, N′-dibutyl-4,4 ′-(biphenyl-2,1-ethenediyl) dipyridinium diiodide (0. 15 g) was obtained.

¹ HNMR (400 MHz, DMSO-d ₆ ): δ = 8.97 (d, J = 6 Hz, 4H), 8.27 (d, J = 6.4 Hz, 4H), 8.11 (d, J = 16 Hz) 4H), 7.94 (d, J = 8 Hz, 4H), 7.898 (d, J = 8 Hz, 4H), 7.63 (d, J = 16 Hz, 4H), 4.54 (t, J = 7.2 Hz, 4H), 1.94 (m, 4H), 1.37 (m, 4H), 0.97 (t, J = 7.6 Hz, 6H)

[Example 2] Synthesis of N, N'-di (2-methylpropyl) -4,4 '-(biphenyl-2,1-ethenediyl) dipyridinium diiodide

  The title compound was obtained according to the method described in Example 1, except that 1-iodo-2-methylpropane was used in place of 1-iodobutane.

¹ HNMR (400 MHz, DMSO-d ₆ ): δ = a = 8.95 (d, J = 6 Hz, 4H), 8.32 (d, J = 6.4 Hz, 4H), 8.12 (d, J = 16 Hz, 4H), 7.95 (d, J = 8 Hz, 4H), 7.95 (d, J = 8 Hz, 4H), 7.65 (d, J = 16 Hz, 4H), 4.37 (d , J = 7.2 Hz, 4H), 1.07 (m, 2H), 0.92 (t, J = 7.6 Hz, 12H)

Example 3 Synthesis of N, N'-dipentyl-4,4 '-(biphenyl-2,1-ethenediyl) dipyridinium diiodide

1-iodopentane (5 mmol) and 4-methylpyridine (5 mmol) were dissolved in ethyl acetate (5 mL). The solution was stirred at 80 ° C. for 3 hours to yield an oil. After heating for 24 hours and removing the solvent, the resulting oily substance was washed several times with ethyl acetate to give 4-methyl-1-pentylpyridinium iodide.
The obtained 4-methyl-1-pentylpyridinium iodide (1 mmol) and 4,4′-biphenyldicarboxaldehyde (0.5 mmol) were dissolved in ethanol (5 mL). Two drops of piperidine were added dropwise thereto and stirred at 60 ° C. After heating for 12 hours, the precipitated yellow powder was filtered and washed several times with cold ethanol, and N, N′-dipentyl-4,4 ′-(biphenyl-2,1-ethenediyl) dipyridinium diiodide (0. 10 g) was obtained.

¹ HNMR (400 MHz, DMSO-d ₆ ): δ = 8.98 (d, J = 6 Hz, 4H), 8.28 (d, J = 6.4 Hz, 4H), 8.12 (d, J = 16 Hz) 4H), 7.95 (d, J = 8 Hz, 4H), 7.89 (d, J = 8 Hz, 4H), 7.64 (d, J = 16 Hz, 4H), 4.53 (t, J = 7.2 Hz, 4H), 1.95 (m, 4H), 1.37 (m, 8H), 0.91 (t, J = 7.6 Hz, 6H)

[Example 4] Synthesis of N, N'-di (2-methylbutyl) -4,4 '-(biphenyl-2,1-ethenediyl) dipyridinium diiodide

1-Iodo-3-methylbutane (5 mmol) and 4-methylpyridine (5 mmol) were dissolved in ethyl acetate (5 mL). When the solution was stirred at 80 ° C. for 1 hour, a solid precipitated. After heating for 24 hours, the precipitated solid was filtered and washed several times with ethyl acetate to obtain 4-methyl-1- (2-methylbutyl) pyridinium iodide (0.5 g).
The obtained 4-methyl-1- (2-methylbutyl) pyridinium iodide (1 mmol) and 4,4′-biphenyldicarboxaldehyde (0.5 mmol) were dissolved in ethanol (5 mL). Two drops of piperidine were added dropwise thereto and stirred at 60 ° C. The precipitated yellow powder was filtered and washed several times with cold ethanol, and N, N′-di (2-methylbutyl) -4,4 ′-(biphenyl-2,1-ethenediyl) dipyridinium diiodide (0.13 g )

¹ HNMR (400 MHz, DMSO-d ₆ ): δ = 9.01 (d, J = 6.4 Hz, 4H), 8.28 (d, J = 6.4 Hz, 4H), 8.12 (d, J = 16 Hz, 4H), 7.95 (d, J = 8 Hz, 4H), 7.89 (d, J = 8 Hz, 4H), 7.64 (d, J = 16 Hz, 4H), 4.55 (t , J = 7.2 Hz, 4H), 1.86 (m, 4H), 1.62 (m, 2H), 0.97 (t, J = 6.4 Hz, 6H)

Example 5 Synthesis of N, N'-dihexyl-4,4 '-(biphenyl-2,1-ethenediyl) dipyridinium diiodide

1-iodohexane (5 mmol) and 4-methylpyridine (5 mmol) were dissolved in ethyl acetate (5 mL). The solution was stirred at 80 ° C. for 3 hours to yield an oil. After heating for 24 hours to remove the solvent, the resulting oily substance was washed several times with ethyl acetate to give 4-methyl-1-hexylpyridinium iodide (0.8 g).
The obtained 4-methyl-1-hexylpyridinium iodide (1 mmol) and 4,4′-biphenyldicarboxaldehyde (0.5 mmol) were dissolved in ethanol (5 mL). Two drops of piperidine were added dropwise thereto and stirred at 60 ° C. The precipitated yellow powder was filtered and washed several times with cold ethanol to obtain N, N′-dihexyl-4,4 ′-(biphenyl-2,1-ethenediyl) dipyridinium diiodide (0.15 g).

¹ HNMR (400 MHz, DMSO-d ₆ ): δ = 8.99 (d, J = 6.4 Hz, 4H), 8.28 (d, J = 6.4 Hz, 4H), 8.12 (d, J = 16 Hz, 4H), 7.95 (d, J = 8 Hz, 4H), 7.90 (d, J = 8 Hz, 4H), 7.64 (d, J = 16 Hz, 4H), 4.53 (t , J = 7.2 Hz, 4H), 1.95 (m, 4H), 1.31 (m, 12H), 0.89 (t, J = 6.4 Hz, 6H)

Example 6 Synthesis of N, N′-dioctyl-4,4 ′-(biphenyl-2,1-ethenediyl) dipyridinium diiodide

1-iodooctane (5 mmol) and 4-methylpyridine (5 mmol) were dissolved in ethyl acetate (5 mL). The solution was stirred at 80 ° C. for 3 hours to yield an oil. After heating for 24 hours to remove the solvent, the resulting oily substance was washed several times with ethyl acetate to give 4-methyl-1-octylpyridinium iodide (0.6 g).
The obtained 4-methyl-1-octylpyridinium iodide (1 mmol) and 4,4′-biphenyldicarboxaldehyde (0.5 mmol) were dissolved in ethanol (5 mL). Two drops of piperidine were added dropwise thereto and stirred at 60 ° C. The precipitated yellow powder was filtered and washed several times with cold ethanol to obtain N, N′-dioctyl-4,4 ′-(biphenyl-2,1-ethenediyl) dipyridinium diiodide (0.17 g).

¹ HNMR (400 MHz, DMSO-d ₆ ): δ = 8.99 (d, J = 6.4 Hz, 4H), 8.28 (d, J = 6.4 Hz, 4H), 8.12 (d, J = 16 Hz, 4H), 7.95 (d, J = 8 Hz, 4H), 7.90 (d, J = 8 Hz, 4H), 7.65 (d, J = 16 Hz, 4H), 4.53 (t , J = 7.2 Hz, 4H), 1.94 (m, 4H), 1.30 (m, 20H), 0.80 (t, J = 6.4 Hz, 6H)

[Example 7] Cell fluorescence and observation

[Cell culture]
Hek293 cells, which are human fetal kidney cells, were used as model cells for staining. Hek293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v / v) fetal bovine serum, 1% (v / v) trypsin and streptomycin under conditions of 37 ° C. and 5% CO ₂ . .

[Cell fluorescence]
In preparation for microscopic observation, 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). 2 mL of phenol red-free DMEM medium supplemented with 2 μL of a 1 × 10 ⁻³ mol dm ⁻³ dimethylsulfoxide (DMSO) solution of the compounds of Examples 2, 4, 5, and 6 and Comparative Examples 1 and 2 (PY final) The concentration was 1 μmol dm ⁻³ , the final DMSO concentration was 0.1% (v / v)), and staining was performed by incubating for 12 hours. 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 1 and 2 have the following structures, and the compound (BP) of Comparative Example 1 was obtained by a known synthesis method. The compound of Comparative Example 2 was obtained by synthesis using the same synthesis procedure as in Example 1.

(Comparative Example 1)
(Comparative Example 2)

[Fluorescence microscope observation]
The fluorescence microscope used was Nikon's ECRIPSE50i. Nikon DS-Ri1 was used for acquisition of fluorescence images. As the objective lens, an infinitely corrected objective lens having a magnification of 100 times and NA = 1.2 was used. Nikon B-2A filter cube was used as the fluorescent filter.

The compounds of Examples 4, 5, 6 and Comparative Example 2 were compared with cells stained with tetramethylrhodamine methyl ester (TMRM), which is a mitochondrial selective probe, in order to confirm localization in mitochondria. Each went. Images obtained by observation are shown in FIG. 1 (Example 4), FIG. 2 (Example 5), FIG. 3 (Example 6), and FIG. 4 (Comparative Example 2).
From the above drawings, it was found that the compounds of Examples 4, 5 and 6 were localized in mitochondria in the same manner as when cells were stained with TMRM. On the other hand, it was found that the compound of Comparative Example 2 was not localized in mitochondria.

Comparison of fluorescence intensity between the compound of Example 2 and the compound of Comparative Example 1 was performed using NIS-Elements. In addition, the value representing the intensity of fluorescence of the compound of Example 2 by image analysis with NIS-Elements was 78, and the value representing the intensity of the compound of Comparative Example 1 was 15. From the above, it was found that the compound of Example 2 was about 5 times stronger in fluorescence intensity than the compound of Comparative Example 1.
Moreover, the image of the cell dye | stained with the compound of Example 2 and the compound of the comparative example 1 is shown in FIG. 5, FIG. 6, respectively. As can be seen from the comparison of FIG. 5 and FIG. 6, the compound of Example 2 emits stronger fluorescence.

[Confirmation of two-photon absorption]
When the compounds of Examples 1 to 6 were dissolved in dimethyl sulfoxide and the cells containing the solutions of the compounds of Examples 1 to 6 were irradiated with focused laser light, light emission was observed only at the focal point of the laser light. (See FIGS. 7 to 12). Therefore, the compound of the present invention can be said to be a compound that emits fluorescence when excited by two-photon absorption.

  Since the compound of the present invention has fluorescence and a property localized in mitochondria, a microscopic image in which mitochondria are visualized can be obtained. In addition, when the compound of the present invention is used for staining cells, the intensity of excitation light applied to the tissue or cells to be observed may be weak, damage to living tissue or cells due to light irradiation, and the compound Can be prevented from fading. In addition, the compound of the present invention has multiphoton fluorescence characteristics. For example, in observation with a two-photon excitation fluorescence microscope, it is necessary to use weak intensity excitation light, that is, light having a long wavelength that reaches deep in the living body. Therefore, the observation range in the depth direction can be expanded more than before, and the objects that can be observed with the two-photon excitation fluorescence microscope can be expanded.

Claims (5)

The compound represented by Formula (1).
[Wherein R ¹ represents a C4 to C8 linear or branched alkyl group,
The wavy line represents the geometric isomers E, Z,
X is the following condensed polycyclic group (i) or (iv)
(In the formula, R ² represents a hydroxyl group, an alkyl group, an alkoxy group or an alkoxyalkyl group, a is an integer of 0 to 4, and d is an integer of 0 to 3. a and d are integers of 2 or more. And R ² may be the same or different. The wavy line represents the bonding position to the adjacent carbon atom.), And Z ⁻ represents the counter anion for the pyridinium cation . ] The compound according to claim 1, wherein the condensed polycyclic group is the following condensed polycyclic group (i ') or (iv') .
(R ² represents a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, a is an integer of 0 to 4, and d is an integer of 0 to 3. When a 1 and d are integers of 2 or more, R ² may be the same or different, and the wavy line indicates the bonding position to the adjacent carbon atom .)   The compound according to claim 1 or 2, wherein the counter anion is a halide ion, a sulfonate, or a perchlorate ion.   A fluorescent dye composition comprising at least one selected from the group consisting of the compounds according to claim 1. The fluorescent dye composition according to claim 4 for use in visualization of mitochondria.