Compounds, compositions, fluorescent dyes, kits, and methods for detecting cells, tissues, or organs.

Abstract

To provide a novel fluorescent dye and kit for readily detecting a compound and a composition or a cell capable of suitably using in a fluorescent dye etc. as well as, a new method for readily detecting a cell etc., and etc.SOLUTION: By applying a fluorescent dye containing a compound and a composition that can be used for a red fluorescent dye based on a specific pyrene structure to stain a cell, a tissue, or an organ, a detection method capable of readily detecting a cell, a tissue, or an organ is provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to fluorescent dyes used for cell detection, compounds and compositions that can be used therefor, kits, and methods for detecting cells, tissues, or organs. [Background technology]

[0002] Multiphoton fluorescence imaging is one of the advanced fluorescence imaging techniques that involves introducing a fluorescent dye into a living organism, exciting and causing it to emit light (multiphotons) by laser irradiation, and then capturing the emission point to create an image. Compared to methods such as nuclear magnetic resonance imaging (MRI), it can observe tissues and organs in living organisms with high spatiotemporal resolution, and is being widely used, mainly in basic life sciences (see, for example, Patent Document 1).

[0003] However, this multiphoton fluorescence imaging has limitations, such as the shallow depth of observation possible within living organisms, and further technological development is needed. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2019 / 093400 [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention aims to provide compounds and compositions that can be suitably used as fluorescent dyes and the like.

[0006] Furthermore, the present invention aims to provide novel fluorescent dyes and kits for the simple detection of cells, tissues, or organs.

[0007] Furthermore, the present invention aims to provide a novel method for easily detecting cells, tissues, or organs.

Means for Solving the Problem

[0008] The inventors of the present invention have found that a fluorescent dye agent containing a compound, a composition, etc. that can be used for a red fluorescent dye based on a specific pyrene structure, etc. can be applied to staining of cells, tissues, or organs (hereinafter also referred to as "cells, etc."), etc., and cells, etc. can be easily detected, and thus completed the present invention.

[0009] That is, the present invention provides the following compounds.

[0010] [1] A compound represented by the following formula (1). [Chemical Formula] (In formula (1), R a and R b are each independently a pyridinium ring, an indolenium ring, or a benzoindolenium ring substituted with R 1 and R 2 on the N atom. R 1 and R 2 are each independently a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, or halogen. R m1 and R m2 are each independently a hydrogen atom, a methyl group, or an ethyl group. R n1 and R n2 are each independently a hydrogen atom, a methyl group, or an ethyl group. m and n are each independently an integer of 2 to 4. R 5 and R 6Each of these is independently a hydrophilic substituent containing one or more substituted or unsubstituted C1-C12 alkyl groups, C2-C12 alkenyl groups, C2-C12 alkynyl groups, C5-C12 aryl groups, C5-C12 heteroaryl groups, halogen atoms, or atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, or halogens. p and q are independent integers between 0 and 4. X S― This is an anion with an S-valence. s is either 1 or 2. t is either 1 or 2. However, when s is 1, t is 2, and when s is 2, t is 1.

[0011] [2] The compound described in [1], represented by the following formula (2). [ka] (In formula (2), R 1 and R 2 Each of these is independently a hydrophilic substituent containing one or more atoms selected from the group consisting of a substituted or unsubstituted C1-C12 alkyl group, a C2-C12 alkenyl group, a C2-C12 alkynyl group, or boron, nitrogen, oxygen, phosphorus, sulfur, or halogen. R 3 and R 4 Each of these is independently a hydrophilic substituent containing one or more atoms selected from the group consisting of a substituted or unsubstituted C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or boron, nitrogen, oxygen, phosphorus, sulfur, or halogen. a and b are independent integers between 0 and 4. R m1 and R m2 Each of these is independently a hydrogen atom, a methyl group, or an ethyl group. R n1 and R n2 Each of these is independently a hydrogen atom, a methyl group, or an ethyl group. m and n are independent integers between 2 and 4. R 5 and R 6 Each of these is independently a hydrophilic substituent containing one or more substituted or unsubstituted C1-C12 alkyl groups, C2-C12 alkenyl groups, C2-C12 alkynyl groups, C5-C12 aryl groups, C5-C12 heteroaryl groups, halogen atoms, or atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, or halogens. p and q are independent integers between 0 and 4. X S― This is an anion with an S-valence. s is either 1 or 2. t is either 1 or 2. However, when s is 1, t is 2, and when s is 2, t is 1.

[0012] [3] The compound according to [1] or [2], wherein m and n are each independently integers between 2 and 3.

[0013] [4] The above R 1 and R 2 The compound according to any one of [1] to [3], wherein each is independently a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decyl group, an n-dodecyl group, or a 2-ethylhexyl group.

[0014] [5] The above R 3 and R 4 The compound according to any one of [1] to [4], wherein each is independently a methyl group, an ethyl group, an n-pyropyr group, an n-butyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decyl group, an n-dodecyl group, or a 2-ethylhexyl group, a hydroxyl group, or an amino group.

[0015] [6] The above X S― Each of them is independent, Cl - , I - , Br - , OH - , monovalent organoboron anions, p-toluenesulfonate anions, methanesulfonate anions, trifluoromethanesulfonate anions, or (COO - )2, the compound described in any one of [1] to [5].

[0016] [7] The above R 5 and R 6 The compound according to any one of [1] to [6], wherein each is independently a methyl group, an ethyl group, an n-pyropyr group, an n-butyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decyl group, an n-dodecyl group, or a 2-ethylhexyl group, a hydroxyl group, or an amino group.

[0017] [8] The compound described in any one of [1] to [7], wherein the absorption maximum wavelength is 400 to 700 nm in 20 mM phosphate buffer (pH 7.4) at 25°C.

[0018] [9] The compound is one of any one of [1] to [8], having a molecular weight of 500 to 3000.

[0019] Furthermore, the present invention provides the following compositions.

[0020]

[10] A composition comprising a compound described in any one of [1] to [9] and a surfactant.

[0021]

[11] The composition according to

[10] , which is an emulsion or micelles.

[0022] Furthermore, the present invention provides the following fluorescent dyes.

[0023]

[12] A fluorescent dye comprising any one of [1] to [9], or the composition described in

[10] or

[11] .

[0024]

[13] A fluorescent dye according to

[12] for morphological detection of cells, tissues, or organs.

[0025]

[14] A fluorescent dye according to

[12] , for staining or visualizing biological samples.

[0026]

[15] A fluorescent dye for fluorescence imaging, as described in any one of

[12] to

[14] .

[0027] Furthermore, the present invention provides the following kit.

[0028]

[16] A kit containing any one of the fluorescent dyes described in

[12] to

[15] .

[0029] Furthermore, the present invention provides a method for detecting the following cells, tissues, or organs.

[0030]

[17] A method for detecting cells, tissues, or organs, comprising the step (1) of staining cells, tissues, or organs with a fluorescent dye described in any one of

[12] to

[15] .

[0031]

[18] The detection method according to

[17] , further comprising step (2) of evaluating the target using the measured fluorescence spectrum after step (1) above.

[0032]

[19] The detection method according to

[17] , further comprising step (3) of evaluating the target using fluorescence imaging after step (1) above.

[0033]

[20] The above fluorescence imaging is multiphoton excitation fluorescence imaging, as described in

[19] . [Effects of the Invention]

[0034] The compounds and compositions of the present invention can be suitably used as fluorescent dyes and the like.

[0035] Furthermore, by using the fluorescent dye and kit of the present invention, cells and other organisms can be stained simply and quickly.

[0036] Furthermore, by using the cell detection method of the present invention, cells and the like can be detected easily. [Brief explanation of the drawing]

[0037] [Figure 1] This is a chart showing the 1H NMR spectrum measurement of compound a in the example. [Figure 2] This is a chart showing the 1H NMR spectrum measurement of compound b in the example. [Figure 3] This is a chart showing the 1H NMR spectrum measurements of compound c in the examples. [Figure 4] This is a chart showing the 1H NMR spectrum measurement of compound A in the example. [Figure 5] This chart shows the one-photon absorption spectrum (dotted line) and fluorescence spectrum (solid line) measurements of compound A in organic solvents (ethanol, dimethyl sulfoxide) in the examples. [Figure 6] This chart shows the one-photon absorption spectrum (dotted line) and fluorescence spectrum (solid line) measurements of composition A in organic solvents (ethanol, dimethyl sulfoxide) in the examples. [Figure 7] 3D stacked images of cerebral blood vessels of living mice obtained by two-photon excitation fluorescence imaging using composition A in the examples. [Modes for carrying out the invention]

[0038] The embodiments of the present invention will be described in detail below, but the present invention is not limited to these embodiments.

[0039] [Compound] The compound of the present invention is a compound represented by the following formula (1) (hereinafter also referred to as "compound (1)"). [ka] (In formula (1), R a and R b Each of them independently has R on the N atom. 1 and R 2 These are pyridinium rings, indorene rings, or benzoindorene rings, respectively, that are substituted with . R 1 and R 2 Each of these is independently a hydrophilic substituent containing one or more atoms selected from the group consisting of a substituted or unsubstituted C1-C12 alkyl group, a C2-C12 alkenyl group, a C2-C12 alkynyl group, or boron, nitrogen, oxygen, phosphorus, sulfur, or halogen. R m1 and R m2 Each of these is independently a hydrogen atom, a methyl group, or an ethyl group. R n1 and R n2 Each of these is independently a hydrogen atom, a methyl group, or an ethyl group. m and n are independent integers between 2 and 4. R 5 and R 6 Each of these is independently a hydrophilic substituent containing one or more substituted or unsubstituted C1-C12 alkyl groups, C2-C12 alkenyl groups, C2-C12 alkynyl groups, C5-C12 aryl groups, C5-C12 heteroaryl groups, halogen atoms, or atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, or halogens. p and q are independent integers between 0 and 4. X S― This is an anion with an S-valence. s is either 1 or 2. t is either 1 or 2. However, when s is 1, t is 2, and when s is 2, t is 1.

[0040] The above-mentioned compounds may be compounds that exhibit single-photon excitation luminescence or multi-photon excitation luminescence, and can be suitably used, for example, as fluorescent dyes.

[0041] The above-mentioned compounds may also include derivatives such as substituted products in which part or all of the structure is substituted, or solvates of hydrates, etc., as appropriate.

[0042] In the above equation (1), R a and R b Each of them independently has R on the N atom. 1 and R 2 These are pyridinium rings, indorene rings, or benzoindorene rings, respectively, that are substituted with .

[0043] In the above equation (1), R a and R b The pyridinium ring, indorhenium ring, or benzoindorhenium ring can bond to a carbon-carbon double bond site at any position other than the N atom.

[0044] In the above equation (1), R a and R b The pyridinium ring, indorene ring, or benzoindorene ring may each be independently substituted.

[0045] R a and R b Examples of substituents on the above ring include unsubstituted C1-C6 alkyl groups, C2-C6 alkenyl groups, C2-C6 alkynyl groups, or hydrophilic substituents containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, or halogens. The number of substituents on the above ring is not limited.

[0046] In the above equation (1), R m1 and R m2Each of these is independently a hydrogen atom, a methyl group, or an ethyl group.

[0047] In addition, in the above equation (1), R m1 and R m2 The wavy line represents an arbitrary bond configuration in relation to a directly bonded double bond. m1 and R m2 Each of these can be in any coupling configuration, such as cis or trans.

[0048] Furthermore, in equation (1) above, R n1 and R n2 Each of these is independently a hydrogen atom, a methyl group, or an ethyl group.

[0049] In addition, in the above equation (1), R n1 and R n2 The wavy line represents an arbitrary bond configuration in relation to a directly bonded double bond. n1 and R n2 Each of these can be in any coupling configuration, such as cis or trans.

[0050] In the above equation (1), R 1 and R 2 Each of these is independently a hydrophilic substituent containing one or more atoms selected from the group consisting of a substituted or unsubstituted C1-C12 alkyl group, a C2-C12 alkenyl group, a C2-C12 alkynyl group, or boron, nitrogen, oxygen, phosphorus, sulfur, or halogen.

[0051] The above R 1 and R 2 Examples of C1-C12 alkyl groups represented by include linear or branched alkyl groups, such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, iso-butyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, n-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decanyl group, n-undecanyl group, and n-dodecanyl group.

[0052] The above R 1 and R 2 Examples of alkenyl groups having 2 to 12 carbon atoms, represented by , include linear or branched alkenyl groups, such as vinyl groups and allyl groups.

[0053] The above R 1 and R 2 Examples of alkynyl groups having 2 to 12 carbon atoms, represented by this formula, include linear or branched alkenyl groups, such as ethynyl groups and propargyl groups.

[0054] Also, the above R 1 and R 2 Examples of substituents such as alkyl groups represented by include methyl groups, ethyl groups, n-propyl groups, isopropyl groups, carboxyl groups, hydroxyl groups, sulfo groups, amide groups, halogen atoms, or salts thereof. When multiple substituents are present, they may be substituted individually or in combination of two or more.

[0055] The above R 1 and R 2 The hydrophilic substituent represented by is preferably a group containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, or halogen, from the viewpoint of improving water solubility and promoting dispersion into the cell membrane and intracellularly. In particular, from the viewpoint of suppressing reactions with biomolecules, the hydrophilic substituent preferably contains one or more atoms selected from the group consisting of tertiary amino groups, quaternary ammonium groups, and carbonyl groups (excluding aldehyde groups).

[0056] In the above equation (1), R 1 and R 2 Each of these groups can be independently, for example, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decyl group, an n-dodecyl group, or a 2-ethylhexyl group.

[0057] In the above formula (1), m and n are each independently an integer of 2 to 4. It is preferable that m and n are each independently an integer of 2 to 3.

[0058] In the above formula (1), R 5 and R 6 are each independently a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, an aryl group having 5 to 12 carbon atoms, a heteroaryl group having 5 to 12 carbon atoms, a halogen atom, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, or halogen.

[0059] The above R 5 and R 6 Examples of the alkyl group having 1 to 12 carbon atoms represented by include linear or branched alkyl groups, such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, iso-butyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, n-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decanyl group, n-undecanyl group, n-dodecanyl group, etc.

[0060] The above R 5 and R 6 Examples of the alkenyl group having 2 to 12 carbon atoms represented by include linear or branched alkenyl groups, such as vinyl group, allyl group, etc.

[0061] The above R 5 and R 6 Examples of the alkynyl group having 2 to 12 carbon atoms represented by include linear or branched alkenyl groups, such as ethynyl group, propargyl group, etc.

[0062] The above R 5 and R 6Examples of the aryl group having 5 to 12 carbon atoms represented by include aromatic hydrocarbon groups having 5 to 12 carbon atoms, such as a phenyl group, a tolyl group, a xylyl group, a naphthyl group, a methylnaphthyl group, an anthracyl group, or an indenyl group.

[0063] The above R 5 and R 6 Examples of the heteroaryl group having 5 to 12 carbon atoms represented by include aromatic hydrocarbon groups having 5 to 12 carbon atoms, and those in which one or more carbon atoms in the aromatic ring are substituted with atoms such as a nitrogen atom, an oxygen atom, or a sulfur atom. For example, a pyrrolyl group, a pyridinyl group, an imidazolyl group, a thienyl group, a furanyl group, a pyrazolyl group, an oxazolyl group, a thiazolyl group, etc. can be mentioned.

[0064] The above R 5 and R 6 Examples of the halogen atom represented by include, for example, a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0065] Also, as substituents such as the alkyl group represented by the above R 5 and R 6 each independently, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a carboxyl group, a hydroxyl group, a sulfo group, an amide group, a halogen atom, or a group which is a salt thereof can be mentioned. When there are a plurality of substituents, these may be substituted alone or may be substituted in combination of two or more kinds.

[0066] The above R 5 and R 6 The hydrophilic substituents represented by can be appropriately used in the same manner as those described in the column of the hydrophilic substituents represented by the above R 1 and R 2 In the above formula (1), R

[0067] and R 5 and R 6Each of these can independently be, for example, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decyl group, an n-dodecyl group, or a 2-ethylhexyl group, a hydroxyl group, or an amino group.

[0068] In equation (1) above, p and q are independent integers between 0 and 4. If the sum of p and q is 2 or more, there exists an R 5 and R 6 These may be the same, or they may be a combination of two or more types.

[0069] In the above equation (1), X S― This is an anion with an S-valence.

[0070] Furthermore, in equation (1) above, s is either 1 or 2. Also, in equation (1) above, etc., t is either 1 or 2. However, when s is 1, t is 2, and when s is 2, t is 1.

[0071] The above X S― When s is 1, t is 2. In this case, the above X S― Each of these can be independent, for example, Cl - , I - , Br - , OH - Examples include monovalent organoboron anions, p-toluenesulfonate anions (tosylate anions), methanesulfonate anions (mesylate anions), trifluoromethanesulfonate anions (triflate anions), and tetrakis(pentafluorophenyl)borate anions. Among these, bulky anions such as tetrakis(pentafluorophenyl)borate anions are more preferable because they can suppress intersubstance interactions, reduce crystallinity, and improve lipophilicity.

[0072] The above X S― When s is 2, t is 1. In this case, the above X S― These are independent of each other, for example, (COO -)It is possible to award 2nd place.

[0073] The above compound (1) is preferably, for example, a compound represented by the following formula (2) (hereinafter also referred to as "compound (2)"). [ka] (In formula (2), R 1 and R 2 Each of these is independently a hydrophilic substituent containing one or more atoms selected from the group consisting of a substituted or unsubstituted C1-C12 alkyl group, a C2-C12 alkenyl group, a C2-C12 alkynyl group, or boron, nitrogen, oxygen, phosphorus, sulfur, or halogen. R 3 and R 4 Each of these is independently a hydrophilic substituent containing one or more atoms selected from the group consisting of a substituted or unsubstituted C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or boron, nitrogen, oxygen, phosphorus, sulfur, or halogen. a and b are independent integers between 0 and 4. m and n are independent integers between 2 and 4. R 5 and R 6 Each of these is independently a hydrophilic substituent containing one or more substituted or unsubstituted C1-C12 alkyl groups, C2-C12 alkenyl groups, C2-C12 alkynyl groups, C5-C12 aryl groups, C5-C12 heteroaryl groups, halogen atoms, or atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, or halogens. p and q are independent integers between 0 and 4. X S― This is an anion with an S-valence. s is either 1 or 2. t is either 1 or 2. However, when s is 1, t is 2, and when s is 2, t is 1.

[0074] The above-mentioned compounds may also include derivatives such as substituted products in which part or all of the structure is substituted, or solvates of hydrates, etc., as appropriate.

[0075] In the above equation (2), 1 and R 2 This is the same as in the case of equation (1) above.

[0076] In equation (2) above, R 3 and R 4 Each of these is independently a hydrophilic substituent containing one or more atoms selected from the group consisting of a substituted or unsubstituted C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or boron, nitrogen, oxygen, phosphorus, sulfur, or halogen.

[0077] The above R 3 and R 4 Examples of C1-C6 alkyl groups represented by include linear or branched alkyl groups, such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, iso-butyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, and n-hexyl group.

[0078] The above R 3 and R 4 Examples of alkenyl groups having 2 to 12 carbon atoms, represented by , include linear or branched alkenyl groups, such as vinyl groups and allyl groups.

[0079] The above R 3 and R 4 Examples of alkynyl groups having 2 to 12 carbon atoms, represented by this formula, include linear or branched alkenyl groups, such as ethynyl groups and propargyl groups.

[0080] Also, the above R 3 and R 4Examples of substituents such as alkyl groups represented by can be independently methyl groups, ethyl groups, n-propyl groups, isopropyl groups, carboxyl groups, hydroxyl groups, sulfo groups, amide groups, halogen atoms, or salts thereof. When multiple substituents are present, they may be substituted individually or in combination of two or more.

[0081] The above R 3 and R 4 The hydrophilic substituent represented by the above R is, for example, 1 and R 2 The hydrophilic substituents listed in the column can be used in the same manner as appropriate.

[0082] In equation (2) above, R 3 and R 4 Each of these can independently be, for example, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decyl group, an n-dodecyl group, or a 2-ethylhexyl group, a hydroxyl group, or an amino group.

[0083] In equation (2) above, a and b are independent integers between 0 and 4. If the sum of a and b is 2 or more, there exists a R 3 and R 4 These may be the same, or they may be a combination of two or more types.

[0084] In equation (2) above, R m1 and R m2 This is the same as in the case of equation (1) above.

[0085] In equation (2) above, R n1 and R n2 This is the same as in the case of equation (1) above.

[0086] In equation (2) above, m and n are the same as in equation (1) above.

[0087] In equation (2) above, R 5and R 6 This is the same as in the case of equation (1) above.

[0088] In equation (2) above, p and q are the same as in equation (1) above.

[0089] In equation (2) above, X S― s and t are the same as in the case of equation (1) above.

[0090] Examples of the above compound (2) include the compound represented by the following formula (3). [ka]

[0091] In equation (3) above, R 1 , R 2 , R 3 , R 4 a, b, R m1 , R m2 , R n1 , R n2 , m, n, R 5 , R 6 , p, q, X S― s and t are the same as in the case of equation (2) above.

[0092] Furthermore, in compound (3) above, the bonding mode of the vinyl group may be independently either cis or trans, or any other form of bonding.

[0093] Furthermore, the absorption maximum wavelength of the above compound is, for example, between 400 and 700 nm in 20 mM phosphate buffer (pH 7.4) at 25°C. The absorption maximum wavelength of the above compound may be within the range of any two values ​​selected from the group consisting of 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 660 nm, 660 nm, 670 nm, 680 nm, and 690 nm.

[0094] Furthermore, the wavelength of the two-photon absorption maximum of the above compound is preferably between 600 and 1200 nm in 20 mM phosphate buffer (pH 7.4) at 25°C. More preferably, it is a wavelength longer than 900 nm. Such compounds are suitable for use in two-photon microscopy because they can be excited by light of wavelengths that are not easily absorbed by biomolecules in tissues.

[0095] Furthermore, the emission maximum wavelength of the above compound is, for example, between 650 and 800 nm in 20 mM phosphate buffer (pH 7.4) at 25°C. The emission maximum wavelength of the above compound may be within the range of any two values ​​selected from the group consisting of 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, 760 nm, 770 nm, 780 nm, and 790 nm.

[0096] Furthermore, the fluorescence quantum yield of the above compound is, for example, between 0.1 and 1.0. The emission maximum wavelength of the fluorescence quantum yield of the above compound may be within the range of any two values ​​selected from the group consisting of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9. The fluorescence quantum yield is assumed to have been measured using an absolute PL quantum yield measuring device.

[0097] Furthermore, the molecular weight of the above compound is, for example, between 500 and 3000. The molecular weight of the above compound can be, for example, within the range of any two numbers selected from the group consisting of 100, 150, 300, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, and 2900.

[0098] Furthermore, in the present invention, the above-mentioned compounds also include derivatives produced by chemical changes in small parts within the molecule, and include simple structural substitutions, adducts, hydrates, etc., as well as what are called analogs.

[0099] [Method for producing compounds] The compounds of the present invention can be produced using known methods as appropriate.

[0100] For example, compound A can be synthesized as shown in the following scheme. [ka]

[0101] The synthesis of compound A can be carried out, for example, by the reaction scheme described above. First, both bromo groups of 1,6-dibromopyrene are replaced with 2-ethylhexyl groups (compound a), and then the pyrene ring is dibrominated (compound b). Next, the bromo group of compound b is converted to an ethanealdehyde group (compound c), and then it is reacted with 1-(2-ethylhexyl)-4-methylpyridinium iodide to convert it to compound A.

[0102] Furthermore, in the production of the above compound, an anion exchange treatment of the above compound may be performed.

[0103] [Composition] The composition of the present invention comprises the above compound and a surfactant.

[0104] Furthermore, the above composition may be in the form of a liquid, paste, semi-solid, solid, etc., but is preferably an emulsion or micelle (hereinafter also referred to as "emulsion, etc.").

[0105] The above emulsion, for example, consists of nano-sized particles with an oil core and a surfactant core, and may possess excellent encapsulation capabilities for lipid-soluble substances as well as high biocompatibility.

[0106] The above-mentioned micelles are, for example, nano-sized particles made of low-molecular-weight or high-molecular-weight surfactants, and when the inner core consists of the hydrophobic portion of the surfactant, they may have excellent encapsulation ability for lipid-soluble substances and high biocompatibility.

[0107] The emulsions mentioned above include, for example, those called nanoemulsions, miniemulsions, ultrafine emulsions, or submicron emulsions.

[0108] The emulsions mentioned above are preferably, for example, emulsions with a diameter between 1 and 500 nm. The diameter may be within the range of any two values ​​selected from the group consisting of 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, and 475 nm. For example, a diameter of 10 to 75 nm is preferred. The diameter refers to the diameter measured using the hydrodynamic diameter (volume equivalent, CONTIN method). Furthermore, it is preferable that the emulsion maintains the desired diameter at temperatures ranging from refrigeration temperatures to body temperature.

[0109] The above-mentioned emulsion, for example, is an emulsion with a polydispersity index between 0.005 and 0.5, and preferably has a particle size distribution that allows for sufficient cell uptake. The above-mentioned polydispersity index may be within the range of any two values ​​selected from the group consisting of 0.01, 0.015, 0.02, 0.03, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, and 0.5. For example, the above-mentioned polydispersity index is preferably between 0.01 and 0.3. Note that the above-mentioned polydispersity index refers to the value measured by the hydrodynamic diameter (volume-converted, CONTIN method).

[0110] The above-mentioned surfactant can be any known surfactant as appropriate, and it is particularly preferable that it is one that can be used, for example, for the examination and analysis of living cells.

[0111] Examples of the above-mentioned surfactants include cationic surfactants, anionic surfactants, amphoteric surfactants, and nonionic surfactants. Examples of the above-mentioned surfactants include sodium lauryl sulfate, sulfosuccinate (sulfosuccinate hemiester), cocoamphocarboxyglycinate, potassium cetyl phosphate, sodium alkyl polyoxyethylene ether carboxylate, potassium benzalkonium chloride, alkylamidopropyl betaine, cetyl stearate ethoxylated alcohol, and sorbitan ethoxylate (20) monooleate Tween20. Examples of commercially available nonionic surfactants include Koliphor ELP and Solutol HS15. These may be used individually or in combination of two or more.

[0112] Furthermore, in the formation of the above-mentioned emulsions, a continuous phase of concentrated aqueous solutions of polyhydroxylated compounds, such as polyalcohols and polysaccharides, may be effective.

[0113] Examples of the polyalcohols and polysaccharides mentioned above include glycerin, xylitol, mannitol, sorbitol, glucose, fructose, saccharose, maltitol, glycerin (diglycerin, dimer compounds of bis(2,3-dihydroxypropyl) ether, solid water-soluble polyhydroxylated compounds such as sugars, and glycerin condensates such as triglycerin and tetraglycerin. These compounds may be used individually or in combination of two or more.

[0114] Furthermore, the above composition may contain other known components as needed. Examples of these other components include oil components, pH buffering agents, and the like.

[0115] Examples of the oil components mentioned above include hydrocarbon compounds and glycerides (glycerin fatty acid esters).

[0116] Examples of the above-mentioned pH buffers include phosphate buffer, tris(hydroxymethyl)aminomethane; Good's buffers (HEPES, MOPS, etc.); and pH buffers containing citric acid, acetic acid, lactic acid, oxalic acid, phthalic acid, imidazole, triethanolamine, diethanolamine, glycine, boric acid, phosphoric acid, or carbonic acid. These may be used individually or in combination of two or more.

[0117] Furthermore, in the above composition, the compound may be present in, for example, 0.01 to 20% by mass, or 0.015 to 19% by mass, or 0.02 to 18% by mass.

[0118] Furthermore, in the above composition, the surfactant can be, for example, 20 to 90% by mass, and may also be 30 to 85% by mass or 40 to 80% by mass.

[0119] Furthermore, in the above composition, the oil component may be, for example, 10 to 80% by mass, 15 to 70% by mass, or 20 to 60% by mass.

[0120] Generally, when attempting to administer large quantities of low-molecular-weight fluorescent substances into the bloodstream of living organisms such as mice, problems arise such as adsorption to the blood vessel walls, precipitation in the blood, and toxicity due to overdose. Furthermore, low-molecular-weight fluorescent substances are quickly excreted by the kidneys and therefore do not remain in the bloodstream for long. In contrast, the above-mentioned composition, even when administered to living organisms such as mice, can be made into a nanoemulsion or the like that encapsulates a large amount of the above-mentioned compound, making it difficult for the kidneys to excrete it and increasing its residence time in the blood.

[0121] [Method for manufacturing the composition] The compositions of the present invention can be manufactured using known methods as appropriate.

[0122] For example, the above composition (e.g., emulsion or micelle) can be obtained by stirring a composition containing the above compound (1) with an oil component added as needed.

[0123] Furthermore, in the production of the above composition, the anions of the above compound may be replaced prior to stirring.

[0124] [Fluorescent dyes] The fluorescent dye of the present invention comprises the above compound (1) or the above composition (1). It may also contain both the above compound and the above composition.

[0125] By using the fluorescent dye of the present invention, cells and other organisms can be stained simply and quickly.

[0126] The fluorescent dye of the present invention can be used as appropriate for staining in vivo (in vivo, etc.) or in vitro (in a test tube, etc.).

[0127] The above-mentioned fluorescent dyes are used, for example, to detect cells, tissues, or organs (e.g., tumor cells or cerebrovascular tissue) in tissues of living organisms.

[0128] The above-mentioned fluorescent dyes are used, for example, for morphological detection of cells, tissues, or organs.

[0129] The above-mentioned fluorescent dyes are used, for example, for staining or visualizing biological samples.

[0130] The above-mentioned fluorescent dyes are used, for example, for fluorescence imaging.

[0131] The above-mentioned fluorescent dyes may be used individually or in combination of two or more types.

[0132] Furthermore, the above-mentioned fluorescent dye can be used in combination with other components used in known fluorescent dyes, in addition to the above-mentioned compound.

[0133] The tissues and cells to which the fluorescent dye of the present invention is applied are not particularly limited, including animals and plants, living cells and dead cells, etc., and can be of biological origin or artificially synthesized or cultured.

[0134] The organism described above is not particularly limited as long as it is a multicellular animal, preferably a mammal, and more preferably a human.

[0135] The cells and tissues mentioned above include, for example, the skin (e.g., epidermis and dermis), brain, spinal cord, esophagus, stomach, small intestine, large intestine, duodenum, rectum, liver, pancreas, gallbladder, bladder, kidneys, heart, spleen, thymus, prostate, uterus, ovaries, testes, breasts, lungs, bronchi, eyeballs, nose, sinuses, oral cavity, pharynx, salivary glands, thyroid gland, parathyroid gland, adrenal gland, muscle, bone marrow, blood vessels, nerves, lymph nodes, peritoneum, diaphragm, blood, and the cells and tissues of these tissues.

[0136] In this specification, tissues derived from living organisms include not only tissues isolated from living organisms, but also living organisms themselves, or tissues that are parts of living organisms but have not been isolated from them.

[0137] In one embodiment, the morphology of the tissue can be appropriately selected depending on the detection method, but may be, for example, the organ or tissue itself, or thin sections or three-dimensional fragments thereof.

[0138] The other components mentioned above may include, for example, one or more of the following: pH buffers, surfactants, salts, solvents, and dye compositions separate from the compound and composition.

[0139] Examples of solvents include water, ethanol, methanol, 2-propanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, acetone, ethyl acetate, tetrahydrofuran, and 1,2-dichloroethane, either individually or in combination of two or more.

[0140] Furthermore, the fluorescent dye of the present invention may be in the form of a solid such as a powder, or in the form of a paste or liquid.

[0141] 〔kit〕 The kit of the present invention comprises the above compound (1) or the above composition (1). It may also contain both the above compound and the above composition.

[0142] By using the kit of the present invention, cells and other tissues can be stained simply and quickly.

[0143] The components included in the above kit can, for example, be similarly adopted from the description in the section on fluorescent dyes.

[0144] The above kit can also be made by combining the above compound with reagents and equipment for staining or tissue sample preparation, for example.

[0145] In one embodiment, the kit also includes reagents for preparing the staining solution. In a more specific embodiment, the reagents for preparing the staining solution may include, for example, one or more selected from the group consisting of the above-mentioned pH buffers, surfactants, salts, solvents, and other dye compositions.

[0146] [Methods for detecting cells] The present invention provides a method for detecting cells, tissues, or organs, comprising the step (1) of staining cells, tissues, or organs with the fluorescent dye containing the above compound (1) or the above composition (1). The method may also contain both the above compound and the above composition.

[0147] Furthermore, by using the detection method of the present invention, cells and other elements can be easily detected.

[0148] The detection method of the present invention can be used as appropriate, for example, in vivo (in a living organism, etc.) or in vitro (in a test tube, etc.).

[0149] In addition, in step (1) above, the term "fluorescent dye" also includes the case of a fluorescent dye containing the above compound and the above composition.

[0150] In step (1) above, the fluorescent dyes described above can be used as appropriate.

[0151] Furthermore, the descriptions of each component in the section on fluorescent dyes and other parts mentioned above can be adopted in a similar manner as appropriate.

[0152] In step (1) above, the step of staining cells can appropriately use materials and methods used in known cell staining and tissue staining steps.

[0153] Furthermore, the cell detection method of the present invention may include a step (2) after step (1) above, in which the target is evaluated using the measured fluorescence spectrum.

[0154] In the step (2) above, which involves evaluation using fluorescence spectra, methods for measuring the fluorescence spectra of known compounds, tissues, and other substances can be appropriately used.

[0155] Furthermore, examples of solvents that can be used in step (2) above include one or more selected from the group consisting of water, ethanol, methanol, 2-propanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, acetone, ethyl acetate, tetrahydrofuran, and 1,2-dichloroethane.

[0156] Furthermore, the cell detection method of the present invention may include a step (3) in which the target is evaluated using fluorescence imaging after step (1) above.

[0157] In step (3) above, the step of evaluation using fluorescence spectra can appropriately utilize methods for measuring the fluorescence spectra of known compounds, tissues, and other substances.

[0158] Furthermore, examples of solvents that can be used in step (3) above include one or more selected from the group consisting of water, ethanol, methanol, 2-propanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, acetone, ethyl acetate, tetrahydrofuran, and 1,2-dichloroethane.

[0159] Furthermore, the above-described fluorescence imaging can be suitably used, for example, in multiphoton excitation fluorescence imaging such as two-photon fluorescence imaging.

[0160] Furthermore, steps (2) and (3) above may be used in combination, and their order can be appropriately combined depending on the purpose.

[0161] In one embodiment, the above method is applied to the living organism itself, or to tissues or other parts of the living organism that have not been separated from the living organism.

[0162] The above staining is usually performed by contacting a staining solution containing the above compound or composition with tissue, etc. The concentration of the above compound or composition in the staining solution is adjusted to, for example, 0.001 mg / mL or more, 0.01 mg / mL or more, 0.1 mg / mL or more, 0.2 mg / mL or more, 0.3 mg / mL or more, 0.4 mg / mL or more, 0.5 mg / mL or more, 0.6 mg / mL or more, 0.7 mg / mL or more, 0.8 mg / mL or more, 0.9 mg / mL or more, or 1 mg / mL or more, relative to the total volume of the staining solution.

[0163] Furthermore, the concentration of the above compound or composition in the staining solution is adjusted to, for example, 500 mg / mL or less, 200 mg / mL or less, 100 mg / mL or less, 50 mg / mL or less, 20 mg / mL or less, 10 mg / mL or less, 5 mg / mL or less, or 2 mg / mL or less relative to the total volume of the staining solution.

[0164] The temperature during dyeing is not particularly limited, but for example, it may be 0-80°C, 4-50°C, 20-45°C, or 25-40°C, preferably 35-42°C.

[0165] The time the staining solution is in contact with the tissue, etc., is, for example, 1 minute or more, 10 minutes or more, 20 minutes or more, 1 hour or more, 2 hours or more, 1 day or more, or 2 days or more, for example, 14 days or less or 7 days or less.

[0166] In one embodiment, the time for contacting the tissue with the staining solution is, for example, 12 hours or less, 6 hours or less, preferably 2 hours or less, more preferably 1 hour or less, even more preferably 30 minutes or less, even more preferably 10 minutes or less, and also, for example, 1 minute or more, 2 minutes or more, 5 minutes or more, or 10 minutes or more.

[0167] Tissues stained with the above-mentioned compound can be used directly for morphological detection of cells or cell detection, but optionally, they may be treated with other dye compositions before detection.

[0168] The method of the present invention may further include a step of detecting specific target cells or the like.

[0169] In another embodiment, cell detection is performed by multi-wavelength measurement.

[0170] In one embodiment, when compound A in the example is used as the above compound, the detected fluorescence includes, for example, one or more wavelengths selected from the ranges of 650-800 nm, 660-790 nm, 670-780 nm, 660-770 nm, 670-760 nm, and 680-750 nm.

[0171] Furthermore, in addition to merging, ratiometric analysis (a method that detects fluorescence at two different wavelengths and takes the ratio of their fluorescence intensities) is also effective as an analysis using the fluorescent dye images of the present invention.

[0172] In addition to the fluorescence imaging described above, fluorescence lifetime imaging and other methods can also be used as appropriate. [Examples]

[0173] Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples.

[0174] [Example 1] (Synthesis of compound A) Compound A was synthesized according to the following scheme. [ka]

[0175] [Example 1-1] (Synthesis of 1,6-diethylhexylpyrene(a)) The synthesis of compound a was carried out according to the following scheme. [ka]

[0176] Zinc chloride (2.8 g, 21 mmol), (2-ethylhexyl)magnesium bromide solution (22 mL, 22 mmol), and super-anhydrous tetrahydrofuran (42 mL) were added to a 50 mL two-necked round-bottom flask, and the mixture was stirred at 0°C for 30 minutes under an argon atmosphere.

[0177] Next, 1,6-dibromopyrene (3.0 g, 8.3 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride-dichloromethane adduct (680 mg, 0.83 mmol) were added to the above reaction solution, and the mixture was stirred at room temperature for 3 hours.

[0178] Dichloromethane was added to the resulting reaction solution, and after washing twice with water and once with saturated saline solution, the mixture was further dehydrated with sodium sulfate and concentrated under reduced pressure. The resulting crude product was purified by silica gel chromatography (eluent: hexane) to obtain the target product a (compound a) (3.4 g, 95%) as a pale yellow solid.

[0179] ( 1 H-NMR analysis and 13 C-NMR analysis) below, 1 H-NMR analysis and 13 ¹³C-NMR analysis was performed using a nuclear magnetic resonance spectrometer (JEOL Ltd.: JMN-LA500). The results obtained for compound a are shown below and in Figure 1. · 1 H-NMR(500MHz,CDCl3,TMS)δ 8.20(d,J=9.2Hz,2H),8.06(d,J=7.6Hz,2H),8.04(d,J=9.2Hz,2H),7.80(d,J=7.6Hz,2H),3.28- 3.19(m,4H),1.88-1.86(m,2H),1.41-1.25(m,16H),0.93(t,J=7.4Hz,6H),0.87(t,J=7.2Hz,6H). · 13C-NMR(125MHz,CDCl3,TMS)δ 136.2,129.6,129.4128.5,127.2,125.6,124.2,122.9,41.6,38.3,32.9,29.0,26.0,23.3,14.3,11.0.

[0180] [Examples 1-2] (Synthesis of 1,6-diethylhexyl-3,8-dibromopyrene(b)) The synthesis of compound b was carried out according to the following scheme. [ka]

[0181] Compound a (3.4g, 7.9 mmol), N-bromosuccinimide (5.7g, 32 mmol), and N,N-dimethylformamide (40 mL) were added to a 50 mL round-bottom flask and heated under reflux at 140°C for 16 hours.

[0182] Water was added to the resulting reaction solution, and the organic layer was extracted three times with dichloromethane. The resulting organic layer was washed twice with water and once with saturated brine, then dehydrated with sodium sulfate and concentrated under reduced pressure. The resulting crude product was purified by silica gel chromatography (eluent: hexane) to obtain the target product b (compound b) (2.7 g, 57%) as a pale yellow solid.

[0183] ( 1 H-NMR analysis and 13 C-NMR analysis) The results obtained for compound b are shown below and in Figure 2. · 1 H-NMR(500MHz,CDCl3,TMS)δ 8.44(d,J=9.4Hz,2H),8.25(d,J=9.4Hz,2H),8.09(s,2H),3.25-3.15(m,4H),1.8 7-1.81(m,2H),1.42-1.26(m,16H),0.93(t,J=7.4Hz,6H),0.88(t,J=7.1Hz,6H). · 1313C-NMR (125 MHz, CDCl3, TMS) δ 137.7, 132.8, 128.8, 128.0, 126.2, 126.0, 124.2, 120.1, 41.6, 38.0, 32.8, 29.0, 25.9, 23.2, 14.3, 11.0.

[0184] [Example 1-3] (Synthesis of 1,6-diethylhexyl-3,8-diethanaldehyde pyrene (c)) The synthesis of Compound c was carried out as shown in the following scheme. [Chemical formula]

[0185] Compound b (2.7 g, 4.5 mmol), palladium(II) acetate (310 mg, 1.4 mmol), tetrabutylammonium diacetate (2.7 g, 9.1 mmol), potassium carbonate (940 mg, 6.8 mmol), and potassium chloride (340 mg, 4.5 mmol) were added to a 50 mL two-necked eggplant flask. Further, acrolein diethyl acetal (4.2 mL, 27 mmol) and ultra-dehydrated N,N-dimethylformamide (23 mL) were added, and the mixture was heated and stirred at 90 °C for 3 hours under an argon atmosphere.

[0186] Hydrochloric acid was added to the obtained reaction solution, and the organic layer obtained by extracting three times with dichloromethane was washed twice with water and once with saturated brine, further dehydrated using sodium sulfate, and concentrated under reduced pressure. The obtained crude product was purified by silica gel chromatography (developing solvent: dichloromethane:hexane = 9:1) to obtain the target product c (Compound c) (630 mg, 26%) as an orange solid. [[ID=2...]]

[0187] ( 1 [[ID=...]]1H-NMR analysis and 13 13C-NMR analysis) The results obtained for the above Compound c are shown below and in Figure 3. · 1H-NMR(500MHz,CDCl3,TMS)δ 9.94(d,J=7.7Hz,2H),8.64(d,J=15.6Hz,2H),8.55(d,J=9.6Hz,2H),8.38(d,J=9.6Hz,2H),8.14(s,2H),7.02(dd,J=7.7 Hz,15.6Hz,2H),3.21-3.31(m,4H),1.85-1.86(m,2H),1.25-1.43(m,16H),0.95(t,J=7.4Hz,6H),0.88(t,J=7.1Hz,6H). · 13 C-NMR(125MHz,CDCl3,TMS)δ 193.6,149.0,137.6,131.4,130.9,128.5,127.9,127.3,126.0,124.4,123.3,41.6,38.4,32.9,29.0,25.9,23.2,14.3,11.0.

[0188] [Examples 1-4] (Synthesis of compound A) Compound A was synthesized according to the following scheme. [ka]

[0189] Compound c (186 mg, 0.35 mmol), 1-(2-ethylhexyl)-4-methylpyridinium iodide (333 mg, 1.0 mmol), piperidine (0.2 mL), and chloroform (2 mL) were added to a 10 mL round-bottom flask and heated under reflux at 60 °C for 6 hours. After concentrating the solvent under reduced pressure, hot ethanol was added, and the precipitate was collected by suction filtration. The target product A (compound A) (58 mg, 14%) was obtained as a dark purple solid.

[0190] ( 1 H-NMR analysis) The results obtained for compound A are shown below and in Figure 4. · 1H-NMR(500MHz,DMSO-d6,TMS)δ 8.89(d,J=6.7Hz,4H),8.60(d,J=9.8Hz,2H),8.36(s,2H),8.34(d,J=9.8Hz,2H),8.31(d,J=15. 4Hz),8.16(d,J=6.7,4H),8.09(dd,J=15.4Hz,10.8Hz,2H),7.60(dd,J=15.4Hz,10.8Hz,2H),7.0 5(d,J=15.4Hz,2H),4.40(d,J=7.4Hz,4H),1.95-1.97(m,2H),1.82-1.84(m,2H),1.18-1.38(m, 32H),0.90(t,J=7.3Hz,6H),0.85(t,J=7.4Hz,6H),0.83(t,J=7.4Hz,6H),0.80(t,J=7.3Hz,6H).

[0191] (High resolution mass spectrometry) High-resolution mass spectrometry was performed using a high-resolution mass spectrometer (JEOL Ltd.: JMS-700). The results obtained for compound A are shown below. • HRMS(ESI)Calcd for C 66 H 90 N2 2+ :455.35,Found:455.35([M] 2+ ).

[0192] [Example 2] (Measurement of optical properties of compound A in an organic solvent) The absorption and fluorescence spectra of compound A obtained in Example 1 were measured in each organic solvent.

[0193] The absorption and fluorescence spectra were measured using a UV-Vis-Near-Infrared spectrophotometer (JASCO Corporation: V-670) and a spectrofluorometer (JASCO Corporation: FP6600), respectively. The fluorescence quantum yield was measured using an absolute PL quantum yield analyzer (Hamamatsu Photonics K.K.: C9920-02V). The concentration of compound A was 5 μM in each solvent. Ethanol and dimethyl sulfoxide were used as solvents. The results are shown in Figure 5 and Table 1.

[0194]

Table 1

[0195] 〔Example 3〕 (Preparation of Composition A and Measurement of Optical Properties) The preparation of Composition A (compound A - integrated nanoemulsion) was carried out as per the following scheme.

Chemical formula

[0196] Compound A (2.0 mg, 1.7 mmol) and lithium tetrakis(pentafluorophenyl)borate - ethyl ether complex (3.3 mg, 3.8 mmol) were weighed into a microtube, 300 μL of dichloromethane was added, and the mixture was stirred for 10 minutes (anion exchange).

[0197] 177 μL of the obtained reaction solution was taken and added to a microtube containing 23 mg of Labrafac CC and 27 mg of Cremophor ELP. The above - mentioned 177 μL of the reaction solution was added, and the mixture was stirred in a 90 °C water bath for 15 minutes. By using the surfactant Labrafac CC and the oil Cremophor ELP, spontaneous nano - emulsification occurred. After confirming that dichloromethane had volatilized, 230 μL of phosphate buffer (20 mM, pH = 7.2) was added, and the mixture was further stirred at 90 °C for 15 minutes to obtain the target Composition A (compound A - containing nanoemulsion). · Hydrodynamic diameter: 54 nm (20 °C, volume conversion, CONTIN method) · Polydispersity index: 0.09

[0198] 〔Example 4〕 (Measurement of Optical Properties of Compound A - Integrated Nanoemulsion) The absorption spectrum and fluorescence spectrum of Composition A obtained in Example 3 were measured.

[0199] The concentration of compound A in the particles of composition A was 20 μM. The measurement was performed in the nanoemulsion solution. The obtained results are shown in Figure 6. The one-photon maximum absorption wavelength was 553 nm, the maximum fluorescence wavelength was 711 nm, and the fluorescence quantum yield was 0.33.

[0200] As shown in Figure 6, composition A, which is a nanoemulsion, also exhibited excellent fluorescence properties similar to compound A, without losing its fluorescence due to intermolecular interactions or other factors in a high-density state.

[0201] [Example 5] (Two-photon excitation fluorescence imaging of cerebral blood vessels in living mice using composition A) Two-photon excitation fluorescence imaging of cerebral blood vessels in living mice was performed using composition A obtained in Example 3, following the procedure below.

[0202] A skull removal surgery was performed on a wild-type mouse (four weeks old, male) to create an observation window.

[0203] Next, 100 μL of composition A (dye concentration in solution: 0.88 mM) prepared in Example 3 was intravenously injected into the mice.

[0204] An excitation light of 1100 nm (210 mW below the objective) was used to illuminate the object using resonant scanning (frame rate: 30 fps, average: 8), and an image was acquired.

[0205] The results obtained are shown in Figure 7.

[0206] As shown in Figure 7, composition A was found to be able to clearly depict the cerebrovascular tissue structure of living mice, as well as tissues deeper than the surface.

Claims

1. A compound represented by the following formula (1). 【Chemistry 1】 (In formula (1), R ａ and R ｂ Each of them independently has R on the N atom. １ and R ２ These are pyridinium rings, indorene rings, or benzoindorene rings, respectively, that are substituted with these compounds. R １ and R ２ Each of these groups is independently selected from the group consisting of methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decyl group, n-dodecyl group, and 2-ethylhexyl group. R ｍ１ and R ｍ２ Each of these is independently a hydrogen atom, a methyl group, or an ethyl group. R ｎ１ and R ｎ２ is each independently a hydrogen atom, a methyl group, or an ethyl group. m and n are independent integers between 2 and 4. R ５ and R ６ Each of these is independently one or more selected from the group consisting of substituted or unsubstituted C1-C12 alkyl groups, C2-C12 alkenyl groups, C2-C12 alkynyl groups, C5-C12 aryl groups, C5-C12 heteroaryl groups, halogen atoms, tertiary amino groups, quaternary ammonium groups, and carbonyl groups. p and q are independent integers between 0 and 4. X S― This is an anion with the s-valence. s is either 1 or 2. t is either 1 or 2. However, when s is 1, t is 2, and when s is 2, t is 1.

2. The compound according to claim 1, wherein Ra and Rb are each independently pyridinium rings in which the N atom is substituted with R1 and R2, respectively.

3. The compound according to claim 1, represented by the following formula (2). 【Chemistry 2】 (In formula (2), R １ and R ２ Each of these groups is independently selected from the group consisting of methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decyl group, n-dodecyl group, and 2-ethylhexyl group. R ３ and R ４ Each of these is independently one or more selected from the group consisting of substituted or unsubstituted alkyl groups having 1 to 6 carbon atoms, alkenyl groups having 2 to 6 carbon atoms, alkynyl groups having 2 to 6 carbon atoms, tertiary amino groups, quaternary ammonium groups, and carbonyl groups. a and b are independent integers between 0 and 4. R ｍ１ and R ｍ２ Each of these is independently a hydrogen atom, a methyl group, or an ethyl group. R ｎ１ and R ｎ２ Each of these is independently a hydrogen atom, a methyl group, or an ethyl group. m and n are independent integers between 2 and 4. R ５ and R ６ Each of these is independently one or more selected from the group consisting of substituted or unsubstituted C1-C12 alkyl groups, C2-C12 alkenyl groups, C2-C12 alkynyl groups, C5-C12 aryl groups, C5-C12 heteroaryl groups, halogen atoms, tertiary amino groups, quaternary ammonium groups, and carbonyl groups. p and q are independent integers between 0 and 4. X S― This is an anion with the s-valence. s is either 1 or 2. t is either 1 or 2. However, when s is 1, t is 2, and when s is 2, t is 1.

4. The compound according to any one of claims 1 to 3, wherein m and n are each independently integers between 2 and 3.

5. The compound according to any one of claims 1 to 4, wherein m and n are 2.

6. The aforementioned R ３ and R ４ The compound according to claim 3, wherein each of them is independently a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decyl group, an n-dodecyl group, or a 2-ethylhexyl group, a hydroxyl group, or an amino group.

7. The aforementioned X S― Each of them is independent of Cl - , I － , Br － , OH － , monovalent organoboron anion, p-toluenesulfonate anion, methanesulfonate anion, trifluoromethanesulfonate anion, or (COO － ) ２ The compound according to any one of claims 1 to 6.

8. The aforementioned R ５ and R ６ The compound according to any one of claims 1 to 7, wherein each of them is independently a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decyl group, an n-dodecyl group, or a 2-ethylhexyl group, a hydroxyl group, or an amino group.

9. The compound according to any one of claims 1 to 8, wherein the absorption maximum wavelength is 400 to 700 nm in 20 mM phosphate buffer (pH 7.4) at 25°C.

10. The compound according to any one of claims 1 to 9, wherein the molecular weight is 500 to 3000.

11. A composition comprising the compound according to any one of claims 1 to 10 and a surfactant.

12. The composition according to claim 11, which is an emulsion or micelles.

13. A fluorescent dye comprising a compound according to any one of claims 1 to 9, or a composition according to claim 11 or 12.

14. The fluorescent dye according to claim 13, for use in the morphological detection of cells, tissues, or organs.

15. A fluorescent dye according to claim 13, for use in staining or visualizing biological samples.

16. A fluorescent dye according to any one of claims 13 to 15, for use in fluorescence imaging.

17. A kit comprising a fluorescent dye according to any one of claims 13 to 16.

18. A method for detecting cells, tissues, or organs, comprising the step (1) of staining cells, tissues, or organs with a fluorescent dye according to any one of claims 13 to 16.

19. The detection method according to claim 18, further comprising, after step (1), a step (2) of evaluating the target using the measured fluorescence spectrum.

20. The detection method according to claim 18, further comprising, after step (1), a step (3) of evaluating the target using fluorescence imaging.

21. The detection method according to claim 20, wherein the fluorescence imaging is multiphoton excitation fluorescence imaging.

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