Compound, fluorescent dye agent, kit, method for detecting cell membrane, and staining material

Abstract

To provide a compound suitable for use in a fluorescent dye agent or the like.SOLUTION: A compound has a squaraine structure, a carbocyanine structure, a merocyanine structure, or a polycyclic aromatic structure, and includes at least one structure (1) represented by formula (1).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a fluorescent dye agent used for detecting cells, a compound that can be used therefor, a kit, a method for detecting cell membranes, and staining materials, etc. [Background technology]

[0002] Various staining methods are used to detect cells and tissues, such as tumor cells. For example, hematoxylin-eosin (HE) staining is the most widely used method in pathological diagnosis and is the gold standard in many diagnostic methods. However, HE staining stains normal tissues as well as tumor cells, resulting in poor light transmission, making it necessary to slice the tissue specimen thinly. Since sliced ​​specimens only have two-dimensional information, it is necessary to prepare and observe many slices in order to determine the distribution range of tumor cells. In addition, preparation of tissue specimens requires advanced skills and is a complicated and time-consuming process.

[0003] Furthermore, with HE staining, the boundary between tumor cells and normal tissue may be unclear due to low contrast in some cases.

[0004] In addition, a method is known in which specific tissues are stained with antibodies bound to fluorescent dyes, radionuclides, metal particles, etc. to obtain high-contrast images of tumor cells. However, because the antibody moiety does not pass through the cell membrane, treatment to increase membrane permeability is required, which makes the procedure complicated. In addition, the reagents are expensive.

[0005] Meanwhile, a method for detecting skin diseases has been reported that utilizes the difference in Raman scattering between abnormal and normal tissues by irradiating light without using dyes for staining (see, for example, Patent Document 1). However, it is considered difficult to identify tumor areas at the cellular level using the Raman scattering method.

[0006] In addition, most of the conventional fluorescent probes capable of staining cell membranes (plasma membranes) have been used in in vitro experiments using cultured cells, and there are currently very few examples of their use in in vivo experiments using living organisms, especially mice. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2018-201678 A Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a compound that can be suitably used as a fluorescent dye agent or the like.

[0009] Another object of the present invention is to provide a novel fluorescent dye agent and kit for easily detecting cells, cell membranes (plasma membranes), and the like.

[0010] Another object of the present invention is to provide a new method for simply detecting cells, cell membranes (plasma membranes), and the like.

[0011] Another object of the present invention is to provide new staining materials such as lipid bilayers, oil droplets, or protein aggregates that exist in cells or that model the cellular environment. [Means for solving the problem]

[0012] The inventors have newly created novel compounds and discovered that by applying fluorescent dye agents containing these to stain cells, cell membranes, and tissues, cells, cell membranes, etc., can be easily detected, thereby completing the present invention.

[0013] That is, the present invention provides the following compounds.

[0014] [1] A compound having a squaraine structure, a carbocyanine structure, a merocyanine structure, or a polycyclic aromatic structure, and having at least one structure (1) represented by the following formula (1): [ka] (In formula (1), R 1 represents a substituted or unsubstituted alkyl group having 4 to 30 carbon atoms, an alkenyl group having 4 to 30 carbon atoms, or an alkynyl group having 4 to 30 carbon atoms. R 2 R is a substituted or unsubstituted methylene group. 2 When multiple are present, they may be the same or different. X - is a monovalent anion. X - When more than one is present, each group is the same or different. Y + is a monovalent cation. Y + When more than one is present, each group is the same or different. n2 is an integer of 1 to 10. When n2 is 2 or more, each group is the same or different. R 3 R is a substituted or unsubstituted methylene group. 2 When multiple are present, they may be the same or different. n3 is an integer of 1 to 10. When n3 is 2 or more, each group is the same or different. L represents a linker. * indicates the site of attachment.)

[0015] [2] At least one X - is -SO3 - , or -COO - The compound according to [1],

[0016] [3] At least one Y + is H + , an alkali metal cation, or an ammonium cation.

[0017] [4] The compound according to any one of [1] to [3], wherein the compound contains one or two of the above structures (1) in the molecule.

[0018] [5] The compound according to any one of [1] to [4], wherein L is a substituted or unsubstituted hydrocarbon group having 1 to 20 carbon atoms (including those partially substituted with heteroatoms).

[0019] [6] The compound according to any one of [1] to [5], wherein n2 is an integer of 1 to 4.

[0020] [7] The compound according to any one of [1] to [6], wherein n3 is an integer of 1 to 4.

[0021] [8] The compound according to any one of [1] to [7], wherein the polycyclic aromatic structure is a naphthalene structure, anthracene structure, phenanthrene structure, pyrene structure, tetracene structure, triphenylene structure, perylene structure, or fluorene structure.

[0022] [9] The compound according to any one of [1] to [8], having a structure (2) represented by the following formula (2): [ka] [ka] (In formula (2), R 4 are each independently an 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, and a halogen. Each n4 is independently an integer of 0 to 4. When n4 is 2 or more, R 4are the same or different. L represents a linker. R is a structure (3) represented by the above formula (3). R 1 , R 2 , X - , Y + , n2, R 3 , n3, * are the same as in formula (1).

[0023]

[10] The compound according to any one of [1] to [8], having a structure (4) represented by the following formula (4): [ka] [ka] (In formula (4), R 5 are each independently an 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, and a halogen. Each n5 is independently an integer of 0 to 4. When n5 is 2 or more, R 5 are the same or different. Each 1 is independently an integer of 1 to 4. Z - is an anion. L represents a linker. R is a structure (3) represented by the above formula (3). R 1 , R 2 , X - , Y + , n2, R 3 , n3, * are the same as in formula (1).

[0024]

[11] The compound according to any one of [1] to [8], having a structure (5) represented by the following formula (5): [ka] [ka] (In formula (5), n6 is an integer of 1 to 4. When n is 2 or more, each group is the same or different. n7 is an integer of 0 to 4. When n2 is 2 or greater, each group is the same or different. R 7 and R 8 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms; R 7 and R 8 may together form a ring structure. R 7 , or R 8 , and contains at least one hydrophilic substituent selected from the group consisting of a carboxyl group, a hydroxyl group, a sulfo group, an amino group, an amide group, a halogen atom, and a salt thereof. When there are two or more hydrophilic substituents, the respective groups may be the same or different. m is an integer of 1 to 4. When m is 2 or more, each group is the same or different. R 6 are each independently an 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, and a halogen. Each a is independently an integer of 0 to 4. When a is 2 or more, R 6 are the same or different. L represents a linker. R is a structure (3) represented by the above formula (3). R 1 , R 2 , X - , Y + , n2, R 3, n3, * are the same as in formula (1).

[0025]

[12] The compound according to any one of [1] to [8], having a structure (6) represented by the following formula (6): [ka] [ka] (In formula (6), R 9 represents a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, or an alkynyl group having 2 to 12 carbon atoms. R 10 R is a substituted or unsubstituted methylene group. 10 When multiple are present, they may be the same or different. n8 is an integer of 1 to 4. When n8 is 2 or greater, each group is the same or different. n9 is an integer of 0 to 4. When n9 is 2 or greater, each group is the same or different. n10 is an integer of 0 to 4. When n10 is 2 or more, each group is the same or different. R 6 are each independently an 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, and a halogen. Each a is independently an integer of 0 to 4. When a is 2 or more, R 6 are the same or different. R 7 and R 11 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms; R 7 and R 11 may be joined together to form a ring structure. 11has one hydrogen atom replaced by a -LR group. R 7 , R 9 , R 11 or a plurality thereof, at least one hydrophilic substituent selected from the group consisting of a carboxyl group, a hydroxyl group, a sulfo group, an amino group, an amide group, a halogen atom, and a salt thereof. When there are two or more hydrophilic substituents, each group may be the same or different. m is an integer of 1 to 4. When m is 2 or more, each group is the same or different. L represents a linker. R is a structure (3) represented by the above formula (3). R 1 , R 2 , X - , Y + , n2, R 3 , n3, * are the same as in formula (1).

[0026] The present invention also provides the following fluorescent dye agents, etc.

[13] A fluorescent dye agent comprising the compound according to any one of [1] to

[12] .

[0027]

[14] The fluorescent dye agent according to

[13] , which is for detecting the morphology of cells and / or tissues.

[0028]

[15] The fluorescent dye agent according to

[13] , which is for morphological detection of cells and / or tissues in a living body.

[0029]

[16] The fluorescent dye agent according to

[13] , which is used for staining or visualizing a biological sample.

[0030]

[17] The fluorescent dye agent according to

[13] , which is used for staining or visualizing cell membranes in biological tissue.

[0031]

[18] The fluorescent dye agent according to

[13] , which is for use in fluorescent imaging.

[0032]

[19] The fluorescent dye agent according to

[13] , which is for in vivo observation.

[0033] The present invention also provides the following kit.

[0034]

[20] A kit comprising the compound according to any one of [1] to

[12] .

[0035] The present invention also provides the following cell detection methods.

[0036] [twenty one] A method for detecting a cell membrane, comprising the step (1) of staining the cell membrane with a fluorescent dye agent containing the compound according to any one of [1] to

[12] .

[0037] [twenty two] The cell membrane detection method described in

[21] , further comprising, after the above step (1), a step (2) of evaluating using fluorescence spectra measured in two or more types of solvents.

[0038] [twenty three] The above step (2) is carried out using fluorescence imaging. A method for detecting a cell membrane described in

[22] .

[0039] The present invention also provides the following dyeing material.

[0040] [twenty four] A material for staining lipid bilayer oil droplets or protein aggregates present in cells or modeling a cellular environment, comprising the compound according to any one of [1] to

[12] . Effect of the Invention

[0041] The compound of the present invention can be suitably used as a fluorescent dye agent or the like.

[0042] Furthermore, by using the fluorescent dye agent and kit of the present invention, cells and the like can be stained clearly, simply and quickly.

[0043] Furthermore, by using the detection method for cells, etc. of the present invention, lipid bilayers, oil droplets, protein aggregates, etc. present in cells or modeled on the cellular environment can be clearly, simply, and suitably stained.

[0044] Furthermore, by using the staining material of the present invention, lipid bilayers, oil droplets, protein aggregates, and the like that exist in cells or that model the cellular environment can be clearly, simply, and quickly stained. [Brief description of the drawings]

[0045] [Figure 1] 1 is a chart of 1H NMR spectrum measurement (500 MHz, DMSO-d6) of Compound 1 in an Example. [Diagram 2] 1 is a chart of a 1H NMR measurement spectrum (500 MHz, CDCl3) of compound 2 in an example. [Diagram 3] 1 is a chart of 1H NMR spectrum measurement (500 MHz, CDCl3) of compound 3 in an example. [Figure 4] 1 is a chart of 1H NMR spectrum measurement (500 MHz, MeOD) of compound A in an example. [Diagram 5] 1 is a chart of 1H NMR spectrum measurement (500 MHz, CDCl3) of compound 5 in an example. [Figure 6] 1 is a chart of 1H NMR spectrum measurement (500 MHz, DMSO) of compound 6 in an example. [Figure 7] 1 is a chart of 1H NMR spectrum measurement (500 MHz, DMSO) of compound 7 in an example. [Figure 8] 1 is a chart of 1H NMR spectrum measurement (500 MHz, DMSO) of compound 8 in an example. [Figure 9]1 is a chart of 1H NMR spectrum measurement (500 MHz, MeOD) of compound 4 in an example. [Figure 10] 1 is a chart of 1H NMR spectrum measurement (500 MHz, CDCl3) of Compound 9 in an example. [Figure 11] 1 is a chart of 1H NMR spectrum measurement (500 MHz, MeOD) of compound B in an example. [Figure 12] Absorption spectra of Compound A (left) and Compound B (right) in the presence of an artificial lipid bilayer (liposome) in the embodiment. Yellow: Absorption spectrum when only the dye is added to the phosphate buffer. Red: Absorption spectrum in the presence of liposomes forming an Ld phase made of DOPC. Blue: Absorption spectrum in the presence of liposomes forming an Lo phase made of SM / Chol. Green: Chart of absorption spectrum in the presence of liposomes forming an Lo phase made of DPPC / Chol. In the figure for Compound A (left), the order of the maximum absorbance is red, blue, green, and yellow. In the figure for Compound B (right), the order of the maximum absorbance is red, blue, green, and yellow. Dye concentration: 0.4 μM, lipid concentration: 80 μM, phosphate buffer used was 20 mM, pH=7.2. [Figure 13] Fluorescence spectrum charts of compounds A (left) and B (right) in the presence of an artificial lipid bilayer (liposome) in an embodiment. Yellow: Fluorescence spectrum when only dye is added to phosphate buffer. Red: Fluorescence spectrum in the presence of liposomes forming Ld phase made of DOPC. Blue: Fluorescence spectrum in the presence of liposomes forming Lo phase made of SM / Chol. Green: Fluorescence spectrum in the presence of liposomes forming Lo phase made of DPPC / Chol. In the figure of compound A (left), the maximum values ​​of emission intensity are blue, red, green, and yellow in descending order. In the figure of compound B (right), the maximum values ​​of emission intensity are red, blue, green, and yellow in descending order. Dye concentration: 0.4 μM, lipid concentration: 80 μM, excitation wavelength: 630 nm, phosphate buffer used was 20 mM in concentration and pH=7.2. [Figure 14] 1 is a chart of the 1H NMR spectrum (500 MHz, CDCl3) of a mixture of compounds 11a and 11b in an example. [Figure 15] 1 is a chart of the 1H NMR spectrum (500 MHz, CDCl3) of compound 12 in an example. [Figure 16] 1 is a chart of the 1H NMR spectrum (500 MHz, DMSO-d6) of compound 13 in an example. [Figure 17] 1 is a chart of the 1H NMR spectrum (500 MHz, DMSO-d6) of compound C in an example. [Figure 18] 1 is a chart of the 1H NMR spectrum (500 MHz, CDCl3) of a mixture of compounds 14a and 14b in an example. [Figure 19] 1 is a chart of the 1H NMR spectrum (500 MHz, CDCl3) of compound 15 in an example. [Figure 20] 1 is a chart of the 1H NMR spectrum (500 MHz, DMSO-d6) of compound 16 in an example. [Figure 21] 1 is a chart of the 1H NMR spectrum (500 MHz, DMSO-d6) of compound 21 in an example. [Figure 22] FIG. 1 is a chart of the absorption (solid line) and fluorescence (dotted line) spectra of compounds C (red) and D (green) in DMSO in an example. In the absorption spectrum (solid line), the solid line with the maximum value on the relatively long wavelength side is red. In the fluorescence spectrum (dotted line), the dotted line with the maximum value on the relatively long wavelength side is green. Dye concentration: 10 μM (absorption spectrum), 5 μM (fluorescence spectrum). Excitation wavelength: 400 nm. [Diagram 23] Fluorescence spectra of compounds C (red) and D (green) in 20 mM phosphate buffer in the examples. The line where the maximum value of the fluorescence spectrum is on the relatively long wavelength side is green. Dye concentration: 5 μM. Excitation wavelength: 400 nm. [Figure 24]1 is a chart of the fluorescence spectrum of compound C in the presence of an artificial lipid bilayer (liposome) in an example. Green: Fluorescence spectrum in the presence of liposomes forming a Lophase made of SM / Chol. Red: Fluorescence spectrum in the presence of liposomes forming an Ldphase made of DOPC. Yellow: Fluorescence spectrum in the presence of liposomes forming an Ldphase made of DOPC / Chol. The maximum emission intensity is green, yellow, and red in descending order. Lipid concentration: 200 μM, dye concentration: 2 μM. Excitation wavelength: 400 nm. [Diagram 25] 1 is a chart of the fluorescence spectrum of compound D in the presence of an artificial lipid bilayer (liposome) in an example. Green: Fluorescence spectrum in the presence of liposomes forming a Lophase made of SM / Chol. Red: Fluorescence spectrum in the presence of liposomes forming an Ldphase made of DOPC. Yellow: Fluorescence spectrum in the presence of liposomes forming an Ldphase made of DOPC / Chol. Red, green, and yellow are shown in descending order of maximum emission intensity. Lipid concentration: 200 μM, dye concentration: 2 μM. Excitation wavelength: 400 nm. [Figure 26] 1 shows a multiphoton fluorescence imaging image of a living mouse tissue in an embodiment. The image shows bone marrow of a skull stained with a dye having squaraine as a core structure. It can be seen that the membrane of the bone marrow tissue and the membrane of blood cells running through the blood vessels in the bone marrow are stained. [Figure 27] 1 is a multiphoton fluorescence imaging image of a living mouse tissue in an embodiment. The image is an image of skin tissue stained with a dye having a carbocyanine core structure and observed. The cell membrane of the cells in the skin tissue is observed. [Figure 28] 1 shows multiphoton fluorescence imaging images of tissues of a living mouse in an example, in which skin tissue was stained with a dye having pyrene as a core structure, and image analysis was performed using a ratiometric method. [Figure 29] 1 shows multiphoton fluorescence imaging images of tissues of a living mouse in an example, in which skin tissue was stained with a dye having pyrene as a core structure, and image analysis was performed using a ratiometric method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Hereinafter, the embodiments of the present invention will be described in detail, but the present invention is not limited to these embodiments.

[0047] [Compound] The compound of the present invention is a compound (hereinafter also referred to as "compound (1)") that has a squaraine structure, a carbocyanine structure, a merocyanine structure, or a polycyclic aromatic structure (hereinafter also referred to as "core structure"), and has at least one structure (1) represented by the following formula (1) (hereinafter also referred to as "side chain structure (1)"). [ka] (In formula (1), R 1 represents a substituted or unsubstituted alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 30 carbon atoms, or an alkynyl group having 2 to 30 carbon atoms. R 2 R is a substituted or unsubstituted methylene group. 2 When multiple are present, they may be the same or different. X - is a monovalent anion. X - When more than one is present, each group is the same or different. Y + is a monovalent cation. Y + When more than one is present, each group is the same or different. n2 is an integer of 1 to 10. When n2 is 2 or more, each group is the same or different. R 3 R is a substituted or unsubstituted methylene group. 2 When multiple are present, they may be the same or different. n3 is an integer of 1 to 10. When n3 is 2 or more, each group is the same or different. L represents a linker. * indicates the site of attachment.)

[0048] The compound (1) may be a compound exhibiting solvatochromism, and may be suitably used, for example, as a fluorescent dye agent, etc. In this specification, a compound exhibiting solvatochromism refers to a compound whose absorption maximum wavelength or fluorescence maximum wavelength, or both, change with a change in the polarity (hydrophobicity) of the surroundings of the compound.

[0049] The compound (1) has a core structure in the molecule, which is a squaraine structure, a carbocyanine structure, a merocyanine structure, or a polycyclic aromatic structure. Each of the above structures is a site that mainly plays a central role in fluorescence emission.

[0050] The squaraine structure, carbocyanine structure, merocyanine structure, and polycyclic aromatic structure as the core structure may have any of the known structures as long as they have the side chain structure (1).

[0051] Examples of the polycyclic aromatic structure include a naphthalene structure, an anthracene structure, a phenanthrene structure, a pyrene structure, a tetracene structure, a triphenylene structure, a perylene structure, and a fluorene structure.

[0052] In the compound (1), at least one of the side chain structures (1) is bonded to the core structure via the linker site L in the molecule.

[0053] The compound (1) has at least one structure (1) (side chain structure (1)) represented by the following formula (1). [ka] (In formula (1), R 1 represents a substituted or unsubstituted alkyl group having 4 to 30 carbon atoms, an alkenyl group having 430 carbon atoms, or an alkynyl group having 4 to 30 carbon atoms. R 2 R is a substituted or unsubstituted methylene group. 2 When multiple are present, they may be the same or different. X - is a monovalent anion. X - When more than one is present, each group is the same or different. Y + is a monovalent cation. Y + When more than one is present, each group is the same or different. n2 is an integer of 1 to 10. When n2 is 2 or more, each group is the same or different. R 3 R is a substituted or unsubstituted methylene group. 2 When multiple are present, they may be the same or different. n3 is an integer of 1 to 10. When n3 is 2 or more, each group is the same or different. L represents a linker. * indicates the site of attachment.)

[0054] The above compound (1) also includes derivatives such as substitution products in which the structure is partially or entirely substituted, and solvates such as hydrates.

[0055] In the above formula (1), R 1 represents a substituted or unsubstituted alkyl group having 4 to 30 carbon atoms, an alkenyl group having 4 to 30 carbon atoms, or an alkynyl group having 4 to 30 carbon atoms.

[0056] Above R 1 Examples of the alkyl group having 4 to 30 carbon atoms represented by the formula (I) include linear or branched alkyl groups, such as an n-butyl group, an iso-butyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decanyl group, an n-undecanyl group, and an n-dodecanyl group.

[0057] Above R 1 The alkenyl group having 4 to 30 carbon atoms and represented by the following formula (1) includes linear or branched alkenyl groups. The above alkenyl groups also include those in which a part of the alkenyl group has a carbon-carbon double bond.

[0058] Above R1 The alkynyl group having 4 to 30 carbon atoms and represented by the following formula (1) includes linear or branched alkynyl groups. The above alkynyl groups also include those in which a part of the alkynyl group has a carbon-carbon triple bond.

[0059] In addition, the above R 1 Examples of the substituent of the alkyl group represented by the formula (I) include a carboxyl group, a hydroxyl group, a sulfo group, an amino group, an amide group, a halogen atom, and salts thereof. When a plurality of substituents are present, they may be substituted alone or in combination of two or more kinds.

[0060] In the above formula (1), for example, depending on the purpose and application, at least one R 1 It is preferable that the alkyl group contains a substituted or unsubstituted linear alkyl structure having 8 to 20 carbon atoms.

[0061] In the above formula (1), R 2 R is a substituted or unsubstituted methylene group. 2 When multiple are present, they may be the same or different.

[0062] In addition, the above R 2 Examples of the substituent of the methylene group represented by the formula (1) include a carboxyl group, a hydroxyl group, a sulfo group, an amino group, an amide group, a halogen atom, and salts thereof. When a plurality of substituents are present, they may be substituted singly or in combination of two or more.

[0063] In the above formula (1), n2 is an integer of 1 to 10. When n2 is 2 or more, each group is the same or different. A preferable example of n2 is 1 to 4.

[0064] In the above formula (1), X - is a monovalent anion. X - When multiple X are present, each group may be the same or different. - Any known monovalent anion can be appropriately used.

[0065] Above X - The monovalent anion represented by the formula is, for example, -SO3 - , or -COO - In the above formula (1), at least one X - is -SO3 - , or -COO - It is preferable that:

[0066] In the above formula (1), Y + is a monovalent cation. Y + When multiple Y groups are present, each group may be the same or different. + Any known monovalent cation can be appropriately used.

[0067] Above Y + The monovalent cation represented by the formula: + , alkali metal cation, or ammonium cation. + is H + , an alkali metal cation, or an ammonium cation.

[0068] In the above formula (1), R 3 R is a substituted or unsubstituted methylene group. 2 When multiple are present, they may be the same or different.

[0069] Above R 3 The substituted or unsubstituted methylene group represented by the above R 2 Examples of the substituted or unsubstituted methylene group include

[0070] In the above formula (1), n3 is an integer of 1 to 10. When n3 is 2 or more, each group is the same or different. A preferable example of n3 is 1 to 4.

[0071] In the above formula (1), L represents a linker.

[0072] L is, for example, a substituted or unsubstituted hydrocarbon group having 1 to 20 carbon atoms (including those partially substituted with heteroatoms).

[0073] The substituted or unsubstituted hydrocarbon group having 1 to 20 carbon atoms represented by L is 1 Among the groups in the formula (I), examples include substituted or unsubstituted alkyl groups having 1 to 20 carbon atoms, alkenyl groups having 2 to 20 carbon atoms, and alkynyl groups having 2 to 20 carbon atoms. The above-mentioned hydrocarbon groups also include those partially substituted with heteroatoms, and examples thereof include those containing a triazole ring.

[0074] In the above formula (1), * indicates a bond site. The bond is, for example, a covalent bond.

[0075] In the above formula (1), the above structure (1) may be present in, for example, one to four units depending on the intended use or application, but it is preferable that one or two units are present.

[0076] The above compound (1) is preferably a compound having a structure (2) represented by the following formula (2) (hereinafter, also referred to as "compound (2)"). [ka] [ka] (In formula (2), R 4 are each independently an 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, and a halogen. Each n4 is independently an integer of 0 to 4. When n4 is 2 or more, R 4 are the same or different. L represents a linker. R is a structure (3) represented by the above formula (3) (hereinafter also referred to as "side chain structure (3)"). R 1 , R 2 , X - , Y + , n2, R 3 , n3, * are the same as in formula (1).

[0077] The compound (2) is a compound containing a squaraine structure as a core structure. For details regarding each group in the compound (2), please refer to the following and other descriptions in this specification as appropriate.

[0078] In the compound (2), at least one of the side chain structures (3) is bonded to the core structure via the linker site L in the molecule.

[0079] In the above formula (2), R 4 are each independently an 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, and a halogen.

[0080] Above R 4 Examples of the alkyl group having 1 to 12 carbon atoms represented by the formula (I) include linear or branched alkyl groups, such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an iso-butyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decanyl group, an n-undecanyl group, and an n-dodecanyl group.

[0081] Above R 4 Examples of the alkenyl group having 2 to 12 carbon atoms represented by the formula (I) include linear or branched alkenyl groups, such as a vinyl group and an allyl group.

[0082] Above R 4 Examples of the alkynyl group having 2 to 12 carbon atoms represented by the following formula include linear or branched alkenyl groups, such as an ethynyl group and a propargyl group.

[0083] Above R 4 Examples of the aryl group having 5 to 12 carbon atoms represented by the formula (I) 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, anthracyl, or indenyl.

[0084] Above R 4 Examples of the heteroaryl group having 5 to 12 carbon atoms represented by the formula (1) include aromatic hydrocarbon groups having 5 to 12 carbon atoms, and include those in which one or more carbon atoms in the aromatic ring are substituted with an atom such as a nitrogen atom, an oxygen atom, a sulfur atom, etc. Examples of the heteroaryl group include a pyrrolyl group, a pyridinyl group, an imidazolyl group, a thienyl group, a furanyl group, a pyrazolyl group, an oxazolyl group, and a thiazolyl group.

[0085] Above R 4 Examples of the halogen atom represented by the formula (I) include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0086] Above R 4 The hydrophilic substituent represented by the formula (I) is, for example, selected from the group consisting of a carboxyl group, a hydroxyl group, a sulfo group, an amide group, a halogen atom, and salts thereof. When there are two or more hydrophilic substituents, the respective groups may be the same or different.

[0087] In the above formula (2), R 4 and are each independently, for example, a methyl group, an ethyl group, or a hydroxyl group.

[0088] In the above formula (2), n4 is independently an integer of 0 to 4. When n4 is 2 or more, R 4 are the same or different. Also, R 4can be on any ring in the ring.

[0089] In the above formula (2), L represents a linker. L is the same as in formula (1).

[0090] In the above formula (2), R is a structure (3) represented by the above formula (3).

[0091] In the above formulas (2) and (3), R 1 , R 2 , X - , Y + , n2, R 3 , n3, and * are the same as in equation (1) etc.

[0092] The above compound (1) is preferably a compound having a structure (4) represented by the following formula (4) (hereinafter, also referred to as "compound (4)"). [ka] [ka] (In formula (4), R 5 are each independently an 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, and a halogen. Each n5 is independently an integer of 0 to 4. When n5 is 2 or more, R 5 are the same or different. Each 1 is independently an integer of 1 to 4. Z - is an anion. L represents a linker. R is a structure (3) represented by the above formula (3). R 1 , R 2 , X - , Y +, n2, R 3 , n3, * are the same as in formula (1).

[0093] The compound (4) is a compound containing a carbocyanine structure as a core structure. For details regarding each group in the compound (4), see below and other descriptions in this specification as appropriate.

[0094] In the compound (4), at least one of the side chain structures (3) is bonded to the core structure via the linker site L in the molecule.

[0095] In the above formula (4), R 5 are each independently an 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, and a halogen.

[0096] R 5 is, for example, the above R in the above formula (2) 4 Groups similar to those shown below can be used.

[0097] Each n5 is independently an integer of 0 to 4. When n5 is 2 or more, R 5 are the same or different. Also, R 5 can be on any ring in the ring.

[0098] In the above formula (4), each 1 is independently an integer of 1 to 4. Preferred examples of the above 1 include 1 and 2.

[0099] In the above formula (4), the structure (3) represented by the above formula (3) is the same as the side chain structure (3) of the above compound (2).

[0100] In the above formulas (4) and (3), R 1 , R 2 , X - , Y+ , n2, R 3 ,n3,*,L,Z - are the same as equation (1), etc.

[0101] The compound (1) is preferably a compound having a structure (5) represented by the following formula (5) (hereinafter, also referred to as "compound (5)"). [ka] [ka] (In formula (5), n6 is an integer of 1 to 4. When n is 2 or more, each group is the same or different. n7 is an integer of 0 to 4. When n2 is 2 or greater, each group is the same or different. R 7 and R 8 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms; R 7 and R 8 may together form a ring structure. R 7 , or R 8 , and contains at least one hydrophilic substituent selected from the group consisting of a carboxyl group, a hydroxyl group, a sulfo group, an amino group, an amide group, a halogen atom, and a salt thereof. When there are two or more hydrophilic substituents, the respective groups may be the same or different. m is an integer of 1 to 4. When m is 2 or more, each group is the same or different. R 6 are each independently an 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, and a halogen. Each a is independently an integer of 0 to 4. When a is 2 or more, R 6are the same or different. L represents a linker. R is a structure (3) represented by the above formula (3). R 1 , R 2 , X - , Y + , n2, R 3 , n3, * are the same as in formula (1).

[0102] The compound (5) is a compound containing a pyrene structure as a core structure. For details regarding each group in the compound (5), see below and other descriptions in this specification as appropriate.

[0103] In the compound (5), at least one of the side chain structures (3) is bonded to the core structure via the linker site L in the molecule.

[0104] In the above formula (5), n6 is an integer of 1 to 4. When n6 is 2 or more, each group is the same or different. In addition, the group (group including a side chain structure) can be on any ring in the ring.

[0105] In the above formula (5), n6 is an integer of 1 to 4. When n is 2 or more, each group is the same or different.

[0106] In the above formula (5), n7 is an integer of 0 to 4. When n2 is 2 or greater, each group is the same or different.

[0107] In the above formula (5), R 7 and R 8 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms; R 7 and R 8 may together form a ring structure.

[0108] In the above formula (5), the R 7 and R 8 may form a ring structure together.7 and R 8 When these together form one ring structure, examples of the ring structure include a pyrrolidine ring structure, an imidazolidine ring structure, an oxazolidine ring structure, a piperidine ring structure, a piperazine ring structure, a morpholine ring structure, etc. In addition, the ring structure may have the hydrophilic substituent.

[0109] In the above formula (5), R 7 , or R 8 , and contains at least one hydrophilic substituent selected from the group consisting of a carboxyl group, a hydroxyl group, a sulfo group, an amino group, an amide group, a halogen atom, and a salt thereof. When there are two or more hydrophilic substituents, the respective groups may be the same or different.

[0110] In addition, in the above formula (5), for example, at least one pair of R 7 and R 8 and preferably form one pyrrolidine ring or one piperidine ring together.

[0111] In the above formula (5), m is an integer of 1 to 4. When m is 2 or more, each group may be the same or different. In addition, the group may be on any ring in the ring.

[0112] In the above formula (5), R 6 are each independently an 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, and a halogen.

[0113] R 6 is, for example, the above R in the above formula (2) 4 Groups similar to those shown below can be used.

[0114] In the above formula (5), each a is independently an integer of 0 to 4. When a is 2 or more, R 6are the same or different. Also, R 6 can be on any ring in the ring.

[0115] R is a structure (3) represented by the above formula (3).

[0116] In the above formula (5), the structure (3) represented by the above formula (3) is the same as the side chain structure (3) of the above compound (2).

[0117] In the above formulas (5) and (3), R 1 , R 2 , X - , Y + , n2, R 3 , n3, *, and L are the same as in equation (1) etc.

[0118] The compound (1) is preferably a compound having a structure (6) represented by the following formula (6) (hereinafter, also referred to as "compound (6)"). [ka] [ka] (In formula (6), R 9 represents a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, or an alkynyl group having 2 to 12 carbon atoms. R 10 R is a substituted or unsubstituted methylene group. 10 When multiple are present, they may be the same or different. n8 is an integer of 1 to 4. When n8 is 2 or greater, each group is the same or different. n9 is an integer of 0 to 4. When n9 is 2 or greater, each group is the same or different. n10 is an integer of 0 to 4. When n10 is 2 or more, each group is the same or different. R 6are each independently an 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, and a halogen. Each a is independently an integer of 0 to 4. When a is 2 or more, R 6 are the same or different. R 7 and R 11 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms; R 7 and R 11 may be joined together to form a ring structure. 11 has one hydrogen atom replaced by a -LR group. R 7 , R 9 , R 11 or a plurality thereof, at least one hydrophilic substituent selected from the group consisting of a carboxyl group, a hydroxyl group, a sulfo group, an amino group, an amide group, a halogen atom, and a salt thereof. When there are two or more hydrophilic substituents, each group may be the same or different. m is an integer of 1 to 4. When m is 2 or more, each group is the same or different. L represents a linker. R is a structure (3) represented by the above formula (3). R 1 , R 2 , X - , Y + , n2, R 3 , n3, * are the same as in formula (1).

[0119] The compound (6) is a compound containing a pyrene structure as a core structure. For details regarding each group in the compound (6), see below and other descriptions in this specification as appropriate.

[0120] In the compound (6), at least one of the side chain structures (3) is bonded to the core structure via the linker site L in the molecule.

[0121] In the above formula (6), R 9 represents a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, or an alkynyl group having 2 to 12 carbon atoms.

[0122] Above R 9 Examples of the alkyl group having 1 to 12 carbon atoms represented by the formula (I) include linear or branched alkyl groups, such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an iso-butyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decanyl group, an n-undecanyl group, and an n-dodecanyl group.

[0123] Above R 9 Examples of the alkenyl group having 2 to 12 carbon atoms represented by the formula (I) include linear or branched alkenyl groups, such as a vinyl group and an allyl group.

[0124] Above R 9 Examples of the alkynyl group having 2 to 12 carbon atoms represented by the following formula include linear or branched alkenyl groups, such as an ethynyl group and a propargyl group.

[0125] In addition, the above R 9 Examples of the substituent of the alkyl group represented by the formula (I) include a carboxyl group, a hydroxyl group, a sulfo group, an amide group, a halogen atom, and salts thereof. When a plurality of substituents are present, they may be substituted alone or in combination of two or more kinds.

[0126] Above R 9From the viewpoint of improving water solubility and promoting dispersion into cell membranes and cells, or from the viewpoint of improving transdermal absorbability, the hydrophilic substituent represented by the formula (I) is preferably a group containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and halogen. In particular, from the viewpoint of suppressing reaction with biomolecules, the hydrophilic substituent preferably contains, for example, a carbonyl group (excluding aldehyde groups).

[0127] In the above formula (6), for example, at least one R 9 is preferably a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, an i-butyl group, a sec-butyl group, a t-butyl group, an ethynyl group, a propargyl group, or a 1,2,3-triazole or 1,2,4-triazole group unsubstituted or substituted with an alkyl group having 1 to 4 carbon atoms.

[0128] In the above formula (6), R 10 R is a substituted or unsubstituted methylene group. 10 When multiple are present, they may be the same or different.

[0129] Above R 10 The substituted or unsubstituted methylene group represented by the above R 2 Examples of the substituted or unsubstituted methylene group include

[0130] In the above formula (6), n8 is an integer of 1 to 4. When n8 is 2 or greater, each group is the same or different.

[0131] In the above formula (6), n9 is an integer of 0 to 4. When n9 is 2 or greater, each group is the same or different.

[0132] In the above formula (6), n10 is an integer of 0 to 4. When n10 is 2 or more, each group is the same or different.

[0133] In the above formula (6), R 7 and R 11are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms; R 7 and R 11 may be joined together to form a ring structure. 11 has one hydrogen atom replaced by a -LR group.

[0134] In the above formula (6), the R 7 and R 11 may form a ring structure together. 7 and R 11 When both of them form one ring structure, the ring structure may be, for example, a pyrrolidine ring structure, an imidazolidine ring structure, an oxazolidine ring structure, a piperidine ring structure, a piperazine ring structure, a morpholine ring structure, etc. Also, the ring structure may have the hydrophilic substituent. Also, in this case, one hydrogen atom is substituted with a -LR group.

[0135] In the above formula (6), R 7 , R 9 , R 11 or a plurality thereof, at least one hydrophilic substituent selected from the group consisting of a carboxyl group, a hydroxyl group, a sulfo group, an amide group, a halogen atom, and salts thereof. When there are two or more hydrophilic substituents, each group may be the same or different.

[0136] In the above formula (6), m is an integer of 1 to 4. When m is 2 or more, each group is the same or different. In addition, the group (group including a side chain structure) can be on any ring in the ring.

[0137] In the above formula (6), the structure (3) represented by the above formula (3) is the same as the side chain structure (3) of the above compound (2).

[0138] In the above formula (6), R 1 , R 2 , X - , Y + , n2, R 3 ,n3,*,R6 , a are the same as in equations (1), (5), etc.

[0139] The absorption maximum wavelength of the compound (1) is preferably, for example, between 300 and 1200 nm in a 20 mM phosphate buffer (pH 7.4) at 25° C. The absorption maximum wavelength of the compound may be, for example, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 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, 600 nm, 650 nm, 700 nm, 750 nm, 760 nm, 770 nm, 780 nm, 790 nm, 800 nm, 810 nm, 820 nm, 830 nm, 840 nm, 850 nm, 860 nm, 870 nm, 880 nm, 890 nm, 900 nm, 910 nm, 920 nm, 930 nm, 940 nm, 950 nm, 960 nm, 970 nm, 980 nm, 990 nm, 1000 nm, 1010 nm, 1020 nm, 1030 nm, 1040 nm, 1050 nm, 1060 nm, 1070 nm, 1080 nm, 1090 nm, 1100 nm, 1110 nm, 1120 nm, 1130 nm, 1140 nm, 1150 nm, 1160 nm, 117 Suitable examples include wavelengths within a range between any two values ​​selected from the group consisting of 750 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, and 1200 nm.

[0140] In addition, when the above compound exhibits solvatochromism, the maximum fluorescence wavelength of the above compound in methanol at 25°C is λ Met The maximum fluorescence wavelength in n-heptane at 25°C is λ Hep Then, the compound λ Met and λ Hep The difference between λ and λ is preferably, for example, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, or 110 nm or more. Met and λ Hep The larger the difference between the λ and λ β of the compound, the easier it is to detect tumor tissue with high contrast relative to healthy tissue, or cancer cells with high contrast relative to normal cells, and this is preferable. Met and λ Hep The difference may be 300 nm or less, 200 nm or less, or 150 nm or less.

[0141] The two-photon absorption maximum wavelength of the compound is preferably, for example, between 600 and 1600 nm in 20 mM phosphate buffer (pH 7.4) at 25° C. In particular, a wavelength longer than 900 nm is more preferable. Such a compound can be excited with light of a wavelength that is not easily absorbed by biological materials in tissues, and is therefore suitable for use in two-photon microscopy.

[0142] In addition, the above compound has excellent water solubility. The above compound preferably has a concentration in water of, for example, 1 nM to 100 mM. The concentration of the above-mentioned compound in water can be set to, for example, 1 nM, 2 nM, 3 nM, 5 nM, 10 nM, 15 nM, 20 nM, 30 nM, 50 nM, 100 nM, 200 nM, 300 nM, 500 nM, 700 nM, 1 μM, 2 μM, 3 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 50 μM, 100 μM, 200 μM, 300 μM, 500 μM, 700 μM, 800 μM, 1 mM, 2 mM, 3 mM, 5 mM, 10 mM, 20 mM, 30 mM, 50 mM, 80 mM, or 100 mM, depending on the purpose and application. The concentration of the above compounds in water can be, for example, 100 mM, 80 mM, 50 mM, 30 mM, 20 mM, 10 mM, 5 mM, 3 mM, 2 mM, 1 mM, 700 μM, 500 μM, 300 μM, 200 μM, 100 μM, 50 μM, 30 μM, 20 μM, 15 μM, 10 μM, 5 μM, 3 μM, 2 μM, 1 μM, 700 nM, 500 nM, 300 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, or 10 nM, depending on the purpose and application. The above upper and lower limits may be any combination. When multiple compounds are used in combination, the concentration of the compounds is the total amount. Furthermore, when used for imaging of cells and / or tissues, the above compounds are preferably used so that their concentrations in water are within the above numerical ranges.

[0143] In addition, in the case of tissue imaging such as skin, the compound preferably has a concentration of 100 μM to 1 mM in water. The concentration of the compound in water in the above-mentioned use can be, for example, 100 μM, 200 μM, 300 μM, 500 μM, 700 μM, or 800 μM, depending on the purpose of use and the purpose of use. The concentration of the compound in water can be, for example, 1 mM, 700 μM, 500 μM, 300 μM, 200 μM, 100 μM, 50 μM, 30 μM, 20 μM, 15 μM, 10 μM, 5 μM, 3 μM, 2 μM, 1 μM, 700 nM, 500 nM, 300 nM, or 200 nM, depending on the purpose of use and the purpose of use. The upper limit and lower limit may be any combination. When a plurality of compounds are used in combination, the concentration of the compounds is the total amount of the compounds. When the compound is used for tissue imaging, the compound is preferably used so that the concentration of the compound in water falls within the above-mentioned numerical range.

[0144] In addition, in the case of cell imaging, the compound preferably has a concentration of 1 nM to 100 μM in water. The concentration of the compound in water in the case of the above-mentioned use can be, for example, 1 nM, 2 nM, 3 nM, 5 nM, 10 nM, 15 nM, 20 nM, 30 nM, 50 nM, 100 nM, 200 nM, 300 nM, 500 nM, 700 nM, 1 μM, 2 μM, 3 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, or 50 μM, etc., as a lower limit, depending on the purpose and use. The upper limit of the concentration of the compound in water can be, for example, 100 mM, 80 mM, 50 mM, 30 mM, 20 mM, 10 mM, 5 mM, 3 mM, 2 mM, 1 mM, 700 μM, 500 μM, 300 μM, 200 μM, 100 μM, 50 μM, 30 μM, 20 μM, 15 μM, 10 μM, 5 μM, 3 μM, 2 μM, 1 μM, 700 nM, 500 nM, 300 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, or 10 nM, depending on the purpose and application of the compound. The upper limit and lower limit may be any combination. When a plurality of compounds are used in combination, the concentration of the compounds is the total amount. In addition, when used for cell imaging, it is preferable to use the compound in water so that the concentration is within the above numerical range.

[0145] The molecular weight of the compound is, for example, 3000 or less, and may be 2800 or less, 2600 or less, 2500 or less, 2400 or less, 2200 or less, 2000 or less, 1800 or less, 1500 or less, 1300 or less, 1000, 900 or less, 800 or less, 700 or less, 650 or less, 600 or less, or 500 or less. The lower limit may be 100 or more, 150 or more, etc.

[0146] In the present invention, the above compounds also include derivatives produced by chemical changes of small parts in the molecule, simple structural substitutions, adducts, hydrates, etc., and also include compounds called analogues. [Method of producing compounds] The compounds of the present invention may be produced by appropriately using known methods other than the methods described in the present invention.

[0147] For example, the following compounds A to D, which correspond to the above compounds (2), (4) to (6), respectively, can be synthesized according to each of schemes 1 to 5 described in the Examples.

[0148] [Fluorescent dye agent] The fluorescent dye agent of the present invention contains the above compound.

[0149] By using the fluorescent dye agent of the present invention, cell membranes (or cells), biological tissues, etc. can be stained simply and quickly.

[0150] The above-mentioned fluorescent dye agent is used, for example, for detection of cells (eg, tumor cells, etc.) and / or tissues (eg, skin tissue, etc.) in tissues derived from a living organism.

[0151] The fluorescent dye agent is used, for example, for morphological detection of cells and / or tissues. For example, it can be suitably used for morphological detection of tumor cells or skin cancer cells. In addition, the fluorescent dye agent is suitably used, for example, for morphological detection of cells and / or tissues in a living body.

[0152] The fluorescent dye agent is used, for example, for staining or visualizing a biological sample. The fluorescent dye agent is also preferably used, for example, for staining or visualizing a cell membrane in a biological tissue.

[0153] The fluorescent dye agent is used, for example, for fluorescent imaging.

[0154] The fluorescent dye agent is used, for example, for in vivo observation. The fluorescent dye agent can be particularly suitably used in in vivo observation, and can easily and quickly perform clear imaging that has not been seen before. The fluorescent dye agent uses the compound with excellent water solubility, and therefore can perform in vivo observation without causing problems such as precipitation in blood vessels by introducing the fluorescent dye agent into the living body itself, which has been very difficult to do in the past, for example, by intravenous injection.

[0155] The fluorescent dye agent is used, for example, for transdermal absorption. The fluorescent dye agent has high skin tissue permeability and can be particularly suitably used in a system for transdermal absorption and observation, and can easily and quickly perform clear imaging that has not been seen in the past.

[0156] The fluorescent dye agent is used, for example, for intravenous administration or oral administration, etc. The fluorescent dye agent has high water solubility and can be particularly suitably used in a system in which the agent is administered to the body and observed, and can easily and quickly perform clear imaging not seen in the past.

[0157] The tissues and cells to which the fluorescent dye agent of the present invention is applied are not particularly limited, regardless of whether they are animal or plant, living or dead cells, and may be, for example, derived from a living organism, artificially synthesized or cultured.

[0158] The living body is not particularly limited as long as it is a multicellular animal, and is preferably a mammal, and more preferably a human.

[0159] Examples of the tissues include skin, brain, spinal cord, esophagus, stomach, small intestine, large intestine, duodenum, rectum, liver, pancreas, gallbladder, urinary bladder, kidney, heart, spleen, thymus, prostate, uterus, ovaries, testes, breasts, lungs, bronchi, eyes, nose, paranasal sinuses, oral cavity, pharynx, salivary glands, thyroid, parathyroid, adrenal glands, muscles, bone marrow, blood vessels, nerves, lymph nodes, peritoneum, diaphragm, blood, etc. In one embodiment, the tissue to which the fluorescent dye agent of the present invention is applied is skin, for example, the epidermis or dermis, or a combination of both.

[0160] The fluorescent dye agent of the present invention can be applied to tissues isolated from a living body by surgical procedures such as excision, resection, puncture, blood sampling, etc., or tissues obtained from feces, urine, sweat, and other body fluids, etc.

[0161] In this specification, tissue derived from a living organism includes not only tissue separated from a living organism, but also the living organism itself or a part of a living organism that has not been separated from the living organism.

[0162] In one embodiment, the form of the tissue may be appropriately selected depending on the detection method, and may be, for example, an organ or tissue itself, or a thin section or three-dimensional fragment thereof.

[0163] Depending on the form of the tissue, for example, the tissue may have been subjected to treatments such as fixation with formalin or the like, paraffin embedding, deparaffinization, dehydration, and transparency treatment.

[0164] In one embodiment, the tumor cells are cells derived from skin tumors, such as sweat gland tumors (extramammary Paget's disease, breast Paget's disease, eccrine porocarcinoma, microcystic adnexal carcinoma, cutaneous mucinous carcinoma, etc.), malignant melanoma, epidermal / hair follicular tumors (basal cell carcinoma, squamous cell carcinoma, actinic keratosis, Bowen's disease, leukoplakia, keratoacanthoma, etc.), nervous system tumors (Merkel cell carcinoma, malignant peripheral nerve sheath tumor, etc.), mesenchymal tumors (dermatofibrosarcoma protuberans, solitary fibrous tumor, muscle tumors, liposarcoma, angiosarcoma, Kaposi's sarcoma, spindle cell hemangioendothelioma, heterogeneous fibroxanthomas, epithelioid sarcoma, synovial sarcoma, undifferentiated pleomorphic cell sarcoma, etc.), etc.

[0165] In the fluorescent dye agent of the present invention, the above compound is not particularly limited as long as it is applicable to tissues and the like.

[0166] In one embodiment, the compound is capable of distributing to cell membranes and intracellular and secreted compartments, and in a more particular embodiment, the compound is capable of inserting into cell membranes.

[0167] Examples of the secretions include bile, hormones, digestive enzymes, exosomes, amyloids, insulin, etc.

[0168] In one embodiment, the compounds exhibit strong fluorescence anisotropy at the cell membrane.

[0169] In one embodiment, the compound has a property that the fluorescence intensity is lower outside the cell than in the cell membrane and / or inside the cell. In a particular embodiment, the compound does not substantially show fluorescence outside the cell. For example, the fluorescence intensity outside the cell of the compound may be, but is not limited to, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the fluorescence intensity inside the cell. Here, the comparison of the fluorescence intensity inside and outside the cell is performed by exciting cells derived from the tissue of interest of the present invention with light of a wavelength capable of exciting the compound, obtaining a fluorescence microscope image under conditions that allow detection of light of the maximum fluorescence wavelength, and comparing the average signal intensity of the fluorescence inside and outside the cell.

[0170] The fluorescent dyes of the present invention may be used alone or in combination of two or more kinds.

[0171] In addition to the above compounds, the fluorescent dye agent of the present invention can also be used in appropriate combination with other components used in known fluorescent dye agents.

[0172] The other components may include, for example, a pH buffer, a surfactant, a salt, a solvent, a dye composition other than compound (1), and the like, either alone or in combination of two or more thereof.

[0173] Examples of pH buffers include one or a combination of two or more selected from the group consisting of 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.

[0174] Examples of the solvent include one selected from the group consisting of water, ethanol, methanol, 2-propanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, acetone, ethyl acetate, tetrahydrofuran, 1,2-dichloroethane, and phosphate buffer, or a combination of two or more selected therefrom. Among these, preferred examples include water, an aqueous solution, and a phosphate buffer.

[0175] When the fluorescent dye agent is made into a solution, for example, the concentration of compound (1) is preferably 1 μM to 1 mM relative to the entire fluorescent dye agent, and may be 2 μM to 800 μM, 3 μM to 700 μM, 5 μM to 600 μM, 10 μM to 500 μM, 30 μM to 450 μM, 50 μM to 400 μM, 100 μM to 350 μM, or 200 μM to 300 μM depending on the purpose and application. When multiple types of compound (1) are used in combination, the concentration of compound (1) is the total amount. The fluorescent dye agent has high water solubility, and thus can easily and quickly perform clear imaging and the like that has not been seen before.

[0176] In the case where the fluorescent dye agent is a solid, for example, the compound (1) is preferably 0.1% by mass to 100% by mass relative to the entire fluorescent dye agent, and may be 1% by mass to 95% by mass, 3% by mass to 90% by mass, 5% by mass to 85% by mass, 10% by mass to 80% by mass, 15% by mass to 75% by mass, 20% by mass to 70% by mass, 30% by mass to 60% by mass, or 40% by mass to 50% by mass depending on the purpose and application. When a plurality of kinds of compounds (1) are used in combination, the amount of the compounds (1) is the total amount. The fluorescent dye agent has high water solubility, and thus can easily and quickly perform clear imaging and the like that has not been seen before.

[0177] The dye composition other than compound (1) is not particularly limited as long as it does not interfere with the detection of cells by compound (1). For example, nuclear staining dyes such as propidium iodide (PI), ethidium bromide, acridine orange, DAPI, and Hoechst are less likely to interfere with the staining of the above-mentioned compounds and can be suitably used. In addition, as the dye composition other than compound (1), for example, dye compositions used for various tissue staining such as hematoxylin-eosin (HE) staining, azan staining, Masson-trichrome staining, Elastica-van Gieson staining, silver impregnation staining, Victoria blue staining, PAM staining, PTAH staining, Sudan III staining, Oil Red O staining, PAS staining, Alcian blue staining, toluidine blue staining, colloidal iron staining, mucicarmine staining, Congo red staining, Dylon staining, Grimelius staining, Fontana-Masson staining, Kossa staining, Berlin blue staining, Bodian staining, Kluver-Barrera staining, and Giemsa staining can be used. These other dye compositions may be used either individually or in combination of two or more.

[0178] Furthermore, the fluorescent dye agent of the present invention may be in the form of a solid, such as a powder, or a liquid.

[0179] When a tissue is stained with the fluorescent dye of the present invention, for example, compound (1) may be distributed in each part of the intracellular lipid membrane, but the fluorescence wavelength may shift depending on the phase (composition) of the lipid membrane, and a change in the fluorescence color may occur. Therefore, by appropriately selecting the wavelength of the fluorescence to be detected, for example, a change in the composition or phase state of the intracellular lipid membrane can be detected with high contrast.

[0180] Furthermore, when tissue is stained with the fluorescent dye agent of the present invention, for example, compound (1) may be distributed in both tumor cells and normal tissue cells, but the fluorescence wavelength of compound (1) in tumor cells may be shifted from the fluorescence wavelength in normal tissue cells. Therefore, by appropriately selecting the wavelength of fluorescence to be detected, tumor cells can be detected with high contrast from normal tissue. The fluorescent dye agent of the present invention can be used for the examination or diagnosis of tumors, particularly malignant tumors.

[0181] Furthermore, when applied to a living body, the fluorescent dye agent of the present invention can be used, for example, for diagnosing a tumor, particularly a malignant tumor. Furthermore, the fluorescent dye agent of the present invention may be applied to a living body before, during, or after a tumor removal treatment in order to specify the extent of removal of a tumor, particularly a cancer, or to check whether any tumor is left behind.

[0182] The fluorescent dye agent of the present invention may be, for example, a reagent used in clinical tests, basic research, etc., as well as a drug or quasi-drug.

[0183] [Materials for dyeing] The staining material of the present invention is for staining lipid bilayers, oil droplets or protein aggregates present in cells or modeling the cellular environment, and contains the above-mentioned compound.

[0184] The staining material of the present invention is capable of suitably staining, for example, protein aggregates such as amyloid.

[0185] The dyeing materials may be appropriately applied to the matters described in the above-mentioned Compound (1) to Fluorescent Dye Agents.

[0186] Regarding the dyeing material, for the components other than the above compounds and their salts, new or known means can be appropriately used depending on the purpose and application of use.

[0187] 〔kit〕 The kit of the present invention comprises the above-mentioned compound.

[0188] By using the kit of the present invention, cells and the like can be stained simply and quickly.

[0189] The components contained in the kit may be similarly, as appropriate, to those described above in the section on fluorescent dye agents.

[0190] The above kit can also be prepared by combining the above compound with reagents and instruments for staining or preparing tissue specimens, for example.

[0191] In one embodiment, the kit further includes a reagent for preparing a staining solution. In a more specific embodiment, the reagent for preparing a staining solution may include, for example, one or a mixture of two or more selected from the group consisting of the above-mentioned pH buffer, surfactant, salt, solvent, and another dye composition.

[0192] [Method for detecting cells and cell membranes] The method for detecting a cell membrane of the present invention comprises the step (1) of staining a cell with a fluorescent dye agent containing the above-mentioned compound.

[0193] The cell detection method of the present invention also includes a step (1') of staining cells with a fluorescent dye agent containing the compound. In this specification, the matters related to the step (1) can be appropriately adopted in the same manner as in the step (1').

[0194] Furthermore, by using the cell detection method of the present invention, cells and the like can be detected easily.

[0195] As the fluorescent dye agent in the above step (1), the above-mentioned fluorescent dye agents can be suitably used in the same manner.

[0196] In addition, the descriptions of each component in the above section on fluorescent dye agents can be similarly adopted as appropriate.

[0197] The step of staining the cell membrane in the above step (1) can be carried out by appropriately using materials and techniques used in known cell staining steps.

[0198] Furthermore, the cell detection method of the present invention can include, after the above step (1), a step (2) of evaluating using fluorescence spectra measured in two or more types of solvents.

[0199] When the fluorescent dye agent of the present invention is a compound exhibiting the above-mentioned solvatochromism, its absorption maximum wavelength or fluorescence maximum wavelength, or both, change depending on the change in polarity (hydrophobicity) around the compound or the fluorescent dye agent. Since the above-mentioned step (2) employs a step using such a fluorescent dye agent, it is possible to easily perform, for example, morphological detection of cell membranes (or cells, etc.) and / or tissues, or staining or visualization of biological samples, or other evaluation, using the fluorescence spectra measured in two or more types of solvents.

[0200] The step of evaluating using the fluorescence spectrum in the above step (2) can appropriately use a known method for measuring the fluorescence spectrum of a substance such as a compound or tissue.

[0201] Examples of the solvent that can be used in the above step (2) include one or more solvents selected from the group consisting of water, ethanol, methanol, 2-propanol, dimethylsulfoxide, N,N-dimethylformamide, acetonitrile, acetone, ethyl acetate, tetrahydrofuran, and 1,2-dichloroethane.

[0202] Moreover, the above step (2) is preferably performed using, for example, fluorescence imaging. For example, two-photon fluorescence imaging may also be suitably used.

[0203] In one embodiment, the method of the present invention is applied to tissues that are organs or organs themselves or three-dimensional fragments thereof. In this embodiment, it is preferable to include a step of making the tissue transparent before staining. Examples of tissue transparentization methods include the TDE method, the LUCID method, the CLARITY method, the PACT / PARS method, the CUBIC method, the 3DISCO method, the Scale method, the ScaleS method, the SeeDB method, the FocusClear method, the Clear method, the BABB method, the iDISCO method, and the uDISCO method. These transparentization methods are described, for example, in Cell Chem.Biol.2016,Vol.23,137-157 and Laser Photonics Rev.2019,Vol.13,1800292.

[0204] In one embodiment, the method of the present invention is applied to a living organism or a part of a living organism, such as a tissue, that is not separated from the living organism.

[0205] The method of the present invention uses a fluorescent dye agent containing the above-mentioned compound, which has excellent water solubility, and therefore enables in vivo observation by introducing the agent into the living body itself, for example by intravenous injection, without causing problems such as precipitation within blood vessels, which was previously very difficult to do.

[0206] In one embodiment, the method of the present invention is applied to a thin section, which may optionally be subjected to processes such as fixation, dehydration, dealcoholization, paraffin infiltration, paraffin embedding, deparaffinization, immersion, and staining using the various tissue staining methods described above, which are commonly used in clinical examinations.

[0207] The staining is usually carried out by contacting a staining solution containing the compound with tissue etc. The concentration of the compound 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 amount of the staining solution.

[0208] In addition, the concentration of the above compound 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 amount of the staining solution.

[0209] The temperature during dyeing is not particularly limited, but is, for example, 0 to 80°C, 4 to 50°C, 20 to 45°C, or 25 to 40°C, and preferably 35 to 42°C.

[0210] The time for contacting the staining solution 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.

[0211] In one embodiment, the time for contacting the staining solution with the tissue or the like is 0 to 40°C, 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 may be, for example, 1 minute or more, 2 minutes or more, 5 minutes or more, or 10 minutes or more.

[0212] Tissues stained with a staining solution containing the above-mentioned compound can be used as is for the morphological detection of cells and / or tissues, the detection of tumor cells, etc., but may optionally be subjected to processing such as staining with other dye compositions before detection.

[0213] The method of the present invention may further include a step of detecting tumor cells, etc. The detection of tumor cells is carried out, for example, by exciting the compound with light of an appropriate wavelength and detecting the emitted fluorescence. For detection, for example, a confocal laser scanning microscope can be used, and depending on the thickness of the slice, a microscope capable of multiphoton excitation, such as a two-photon microscope, can be used. For example, when 1-acetyl-6-piperidylpyrene (PK) is used as the compound, it is suitable for measurement by a two-photon microscope, as shown in the examples.

[0214] In one embodiment, cells, such as tumor cells, are detected by selecting a fluorescent light containing one particular wavelength that provides contrast between the tumor cells and cells of normal tissue, and measuring the fluorescent intensity.

[0215] In another embodiment, detection of cells, such as tumor cells, is performed by multi-wavelength measurement, i.e., cells are detected by detecting fluorescence containing two or more different specific wavelengths and integrating the intensity of each fluorescence.

[0216] In one embodiment, when compound C and compound D in the examples are used as the above compounds, the detected fluorescence includes one or more wavelengths selected from the ranges of, for example, 350 to 750 nm, 550 to 700 nm, 600 to 700 nm, and 650 to 750 nm.

[0217] As described above in the section on fluorescent dye agents, the detection method of the present invention makes it possible to inspect and diagnose cells such as tumors, or to specify the extent of tumor removal.

[0218] In addition to merging, ratiometric analysis (a method of detecting fluorescence of two different wavelengths and taking the ratio of their fluorescence intensities) is also an effective image analysis that utilizes solvatochromism.

[0219] In addition to the above-mentioned fluorescence imaging, fluorescence lifetime imaging and the like can also be used as appropriate. EXAMPLES

[0220] Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples.

[0221] ( 1 H-NMR analysis) In the embodiment 1 The H-NMR analysis was performed using a nuclear magnetic resonance spectrometer (JMN-LA500 manufactured by JEOL Ltd.) The results obtained for each compound are shown in Figures 1 to 11 and 14 to 21.

[0222] (UV-Visible absorption spectrum measurement, Fluorescence spectrum measurement) The ultraviolet-visible absorption spectrum was measured using a spectrofluorometer (V670, manufactured by JASCO Corporation), and the fluorescence spectrum was measured using a spectrofluorometer (FP6600, manufactured by JASCO Corporation).

[0223] (Synthesis of Compound A) Compound A was synthesized according to the following scheme. [ka] Scheme 1. Synthesis of compound A

[0224] [ka] Scheme 2. Synthesis of compound 4

[0225] Example 1 Synthesis of compound 1 4-Pentyn-1-ol (1 mL, 10.8 mmol) was added to a two-necked eggplant flask, and the flask was filled with argon. Then, dehydrated dichloromethane (54 mL) was added, and the solution was cooled to -78 ° C. Trifluoromethanesulfonic anhydride (3.56 mL, 21.6 mmol) and dehydrated pyridine (1.74 mL, 21.6 mmol) were added to the cooled solution, and the mixture was stirred at -78 ° C for 2 hours, and then stirred at room temperature overnight. The reaction solution was filtered to remove impurities that were not dissolved in dichloromethane. The filtrate was then concentrated under reduced pressure, and isolation and purification were performed by short silica gel column chromatography (dichloromethane: hexane = 1: 4). As a result, the target compound 1 was obtained as a colorless to light brown liquid compound (722 mg, 31%, Figure 1). 1 H NMR(500MHz,DMSO)δ 4.28ppm(t,2H,J=6.3Hz),2.87ppm(t,1H,J=2.8Hz),2.23ppm(td,2H,J=2.8Hz,6.3Hz),1.81ppm(quin,2H,J=6.3Hz).

[0226] Example 2 Synthesis of compound 2 2,3,3-trimethylindolenine (257 μL, 1.6 mmol) was added to a two-necked eggplant flask, and the flask was filled with an argon atmosphere. Then, dehydrated dichloroethane (8 mL) and the obtained compound 1 (519 mg, 2.4 mmol) were added and stirred at 70 ° C overnight. The reaction solution was concentrated under reduced pressure, and then isolated and purified by medium pressure preparative column chromatography (dichloromethane:methanol = 100:0 to 96:4). As a result, the target compound 2 was obtained as a brown liquid compound (420 mg, 70%, Figure 2). 1 H NMR(500MHz,CDCl3)δ 7.73-7.69ppm(m,1H),7.60-7.52ppm(m,3H),4.70ppm(t,2H,J=7.6Hz),2.92ppm(s,3H),2.40ppm( td,2H,J=6.6Hz,2.5Hz),2.17ppm(quin,2H,J=6.6Hz),2.11ppm(t,1H,J=2.5Hz),1.59ppm(s,6H).

[0227] Example 3 Synthesis of compound 3 The obtained compound 2 (420 mg, 1.12 mmol), 3,4-diethoxy-3-cyclobutene-1,2-dione (75 μL, 0.51 mmol), 1-butanol (10 mL), and pyridine (10 mL) were added to a recovery flask and heated at 120°C overnight under reflux. The mixture was concentrated under reduced pressure using an evaporator, and the recovery flask was connected to a vacuum tube to remove the remaining solvent. Methanol was added to the solid matter remaining in the recovery flask and filtered, and the filter cake was washed with hot methanol and hot hexane. As a result, the target compound 3 was obtained as a green powdery solid (150 mg, 50%, Figure 3). 1H NMR(500MHz,CDCl3)δ 7.35ppm(d,2H,J=7.3Hz),7.31ppm(t,2H,J=7.5Hz,7.9Hz),7.15ppm(t,2H,J=7.3Hz,7.5Hz),7.10ppm(d,2H,J=7.9Hz),5.96ppm (s,2H),4.14ppm(br,4H),2.34ppm(td,4H,J=6.6Hz,2.6Hz),2.14ppm(br,2H),2.04ppm(quin,4H,J=6.6Hz),1.77ppm(br,12H).

[0228] Example 4 Synthesis of Compound A Copper sulfate pentahydrate (7 mg, 0.028 mmol), sodium ascorbate (0.039 mmol), and water (300 μL) were added to the microtube and stirred until the solution turned orange. Then, the obtained compound 3 (30 mg, 0.057 mmol), the obtained compound 4 (52 mg, 0.125 mmol), dimethylformamide (1 mL), and the aqueous solution in the microtube were all added to the eggplant flask and stirred at 90 ° C for 40 minutes. The reaction solvent was concentrated using a convenience evaporator, and isolation and purification were performed by reverse phase column chromatography (water:methanol = 1:1 to 1:3). As a result, the target compound A was obtained as a blue solid (14 mg, 17%, Figure 4). 1 The H NMR spectrum was complicated and accurate assignment was not possible. The presence of peaks derived from the triazole ring and peaks specific to the squaraine skeleton indicated that the reaction was proceeding, and mass spectrometry confirmed that it was the target product. Compound A also showed high solubility (>5mg / mL) in phosphate buffer (20mM, pH=7.2). 1H NMR(500MHz,MeOD)δ 7.89ppm(s,2H),7.43ppm(d,2H,J=7.5Hz),7.34ppm(t,2H,J=7.8Hz),7.20-7.15ppm(m,4H),5.95ppm(s,2H),4.45ppm(q,4H,J=6. 3Hz),4.19ppm(br,4H,),3.47-3.40ppm(m,4H),3.21ppm(t,2H,J=7.8Hz),3.16ppm(t,2H,J=7.1Hz),2.97ppm(t,2H,J=7.8Hz),2.9 3-2.86ppm(m,6H),2.80-2.74ppm(m,4H),2.45ppm(t,2H,J=7.1Hz),2.35ppm(t,2H,J=7.1Hz),2.25-2.05ppm(m,12H),2.02-1.93p pm(m,4H),1.73ppm(s,12H),1.67-1.60ppm,(m,2H),1.55-1.40ppm(m,6H),1.34-1.17ppm(m,60H),0.86ppm(quin,10H,J=7.1Hz). MS-ESI(m / z):[M] 2- calculated for 681.3929(C 74 H 110 N 10 O 10 S2),found 681.3898.

[0229] Example 5 Synthesis of compound 5 Sodium iodide (6.7 g, 44.8 mmol) was added to a recovery flask and the atmosphere was changed to argon. 4-tert-butyl bromobutanoate (5 g, 22.4 mmol) and acetone (56 mL) were added and stirred at room temperature for two hours. Water was added to the reaction solution and it was extracted with ethyl acetate. The obtained organic layer was washed once with saturated saline and the remaining water was removed with sodium sulfate. As a result, the target compound 5 was obtained as a yellow liquid (5.7 g, 94%, Figure 5). 1H NMR(500MHz,CDCl3)δ 3.12ppm(t,2H,J=6.9Hz),2.31ppm(t,2H,J=7.2Hz),2.04ppm(quin,2HJ=6.9Hz,7.2Hz),1.41ppm(s,9H,).

[0230] Example 6 Synthesis of compound 6 The obtained compound 5 (1g, 3.7mmol), sodium azide (600mg, 9.3mmol), and dimethylformamide (12mL) were added to a recovery flask and stirred at 80℃ for one hour. Water was added to the reaction solution, and it was extracted with ethyl acetate. The obtained organic phase was washed three times with a large amount of water, and the remaining water was removed with sodium sulfate. As a result, the target compound 6 was obtained as a colorless and transparent liquid (677mg, quant, Figure 6). 1 H NMR(500MHz,DMSO)δ 3.29ppm(t,2H,J=6.9Hz),2.23ppm(t,2H,J=7.2Hz)1.69ppm(quin,2H,J=6.9Hz,7.2Hz),1.36ppm,(s,9H).

[0231] Synthesis of compound 7 The obtained compound 6 (667 mg, 3.65 mmol) and dichloromethane (7.3 mL) were added to a recovery flask and cooled in an ice bath. Trifluoroacetic acid (7.3 mL) was added and stirred at 0°C for one hour. Toluene (20 mL) was added to the reaction solution and concentrated under reduced pressure using an evaporator. As a result, the target compound 7 was obtained as a colorless, transparent liquid (504 mg, quant, Figure 7). 1 H NMR(500MHz,DMSO)δ 3.30ppm(t,2H,J=6.9Hz),2.25ppm(t,2H,J=7.2Hz),1.70ppm(quin,2H,J=6.9Hz,7.2Hz).

[0232] Synthesis of compound 8 Dodecylamine (2 g, 10.8 mmol), 1,3-propane sultone (1.6 g, 12.9 mmol), and acetone (30 mL) were added to a recovery flask and stirred at 60°C overnight. After vacuum concentration, the residue was washed several times with both acetic acid and acetone to remove unreacted dodecylamine and propane sultone. After this washing, the product obtained was recrystallized from acetone with a minimum amount of methanol added. As a result, the target compound 8 was obtained as a white powder (2 g, 60%, Figure 8). 1 H NMR(500MHz,DMSO)δ 2.95ppm(t,2H,J=6.9Hz),2.88ppm(t,2H,J=7.6Hz),2.55ppm,(t,2H,J=6.9Hz),1.89-1 .82ppm(m,2H),1.51-1.44ppm(m,2H),1.28-1.16ppm(m,18H),0.82ppm(t,3H,J=6.8Hz).

[0233] Synthesis of compound 4 Compound 7 (50 mg, 0.39 mmol) and diisopropylethylamine (132 μL, 0.77 mmol) were added to a recovery flask and the mixture was placed in an argon atmosphere. Dehydrated dimethylformamide (9.5 mL) was added and stirred at room temperature for five minutes, after which a solution of compound 8 (125 mg, 0.41 mmol) and diisopropylethylamine (132 μL, 0.77 mmol) in dehydrated dimethylformamide (9.5 mL) was added and stirred at room temperature overnight. The reaction solution was filtered to remove unreacted compound 8, and dimethylformamide was removed using a convenience evaporator. Saturated saline was then added to the recovery flask and the mixture was extracted with ethyl acetate. Water remaining in the organic phase obtained with magnesium sulfate was removed. Finally, the mixture was isolated and purified by medium pressure preparative silica gel column chromatography (dichloromethane:methanol = 100:0 to 90:10). As a result, the target compound 4 was obtained as a brown viscous solid (41 mg, 25%, Figure 9). 1H NMR(500MHz,MeOD)δ 3.53-3.48ppm(m,2H),3.40-3.31ppm(m,4H),2.87-2.80ppm(m,2H)2.51-2.45ppm(m,2H),2.06-1.96ppm (m,2H),1.92-1.83ppm(m,2H),1.67-1.48ppm(m,2H),1.38-1.21ppm(m,18H),0.88ppm(t,3H,J=6.7Hz).

[0234] (Synthesis of Compound B) Compound B was synthesized according to the following scheme. [ka] Scheme 3. Synthesis of compound B

[0235] Example 10 Synthesis of compound 9 Compound 2 (200 mg, 0.53 mmol), malonaldehyde dianilide hydrochloride (69 mg, 0.27 mmol), sodium acetate (44 mg, 0.53 mmol), and acetic anhydride (2.7 mL) were added to a recovery flask and stirred at 100°C for three hours. Water was added to the reaction solution, and it was extracted with dichloromethane. The obtained organic phase was washed with 1M aqueous perchloric acid solution, and residual water was removed with magnesium sulfate. Then, it was isolated and purified by medium pressure preparative silica gel column chromatography (dichloromethane:methanol = 100:0 to 95:5). As a result, the target compound 9 was obtained as a blue solid (65 mg, 21%, Figure 10). 1 H NMR(500MHz,CDCl3)δ 7.97ppm(t,2H,J=13.1Hz),7.50ppm(d,2H,8.1Hz),7.40-7.27ppm(m,6H),7.22ppm(t,2H,J=7.3Hz),7.17ppm(d,2H,J=8.5Hz),7.0 8ppm(t,1H,J=7.8Hz),4.17ppm(brs,4H),2.42-2.34ppm(m,4H),2.22ppm(t,2H,J=2.5Hz),2.08-1.98ppm(m,4H),1.72ppm(s,12H).

[0236] Example 11 Synthesis of Compound B Copper sulfate pentahydrate (8 mg, 0.032 mmol), sodium ascorbate (0.044 mmol), and water (375 μL) were added to the microtube and stirred until the solution turned orange. Next, compound 3 (38 mg, 0.064 mmol), compound 4 (59 mg, 0.141 mmol), dimethylformamide (1.25 mL), and the aqueous solution in the microtube were all added to the eggplant flask and stirred at 90 ° C for 40 minutes. The reaction solvent was concentrated using a convenience evaporator and filtered through Celite using a mixed solvent of dichloromethane:methanol = 1:1. The filtrate was concentrated and isolated and purified by size exclusion column chromatography (dichloromethane:methanol = 1:1). As a result, the target compound B was obtained as a blue solid (40 mg, 41%, Figure 11). It was confirmed to be the target by mass spectrometry. Compound B also showed high solubility (>5 mg / mL) in phosphate buffer (20 mM, pH=7.2). 1 H NMR(500MHz,MeOD)δ 8.20ppm(br,2H),8.15-7.84ppm(br,2H),7.45ppm(d,2H,J=6.9Hz),7.37ppm(t,2H,J=7.8Hz),7 .30-7.19ppm(m,4H),6.61ppm(br,1H),6.28ppm(br,2H),4.46ppm(br,4H),4.18ppm(br,4H),3.4 1ppm(br,4H),3.20ppm,(br,2H),2.97-2.69ppm(m,8H),2.50-2.32ppm(m,4H),2.17ppm(br,8H), 1.97ppm(br,4H),1.68ppm(s,12H),1.47ppm(br,4H),1.21ppm,(br,38H),0.88-0.83ppm(m,6H). MS-ESI(m / z):[M] - calculated for 1321.8190(C 73 H 113 N 10 O8S2),found 13218164.

[0237] Example 12 Absorption and fluorescence spectral behavior of compound A and compound B in the presence of artificial lipid bilayer membranes Three types of liposomes were prepared, one forming two types of ordered phases (Lo phase, lipids: sphingomyelin-cholesterol (SM / Chol), dipalmitoylphosphatidylcholine-cholesterol (DPPC / Chol)) and one forming a disordered phase (Ld phase, lipids: dioleoylphosphatidylcholine (DOPC)). The liposomes were dispersed in phosphate buffer, and dye was added at a lipid:dye molar ratio of 200:1. After incubation at 37°C for 30 minutes, the absorption and fluorescence spectra were measured. The results are shown in Tables 1 and 2 and Figures 12 and 13.

[0238] [Table 1]

[0239] [Table 2]

[0240] (Synthesis of Compound C) Compound C was synthesized according to the following scheme. [ka] Scheme 4. Synthesis of compound C

[0241] Example 13 (Synthesis of Compound 11a and Compound 11b) 1-Bromopyrene (2.00 g, 7.11 mmol) was added to a two-necked eggplant flask, and the flask was filled with argon. Dehydrated dichloromethane (36 mL) was added and cooled in an ice bath. Aluminum chloride (1.04 g, 7.82 mmol) was added to this solution and stirred for 15 minutes, and propionyl chloride (0.744 mL, 8.53 mmol) was added dropwise and stirred at room temperature for 12 hours. The reaction was stopped by adding water, and the mixture was extracted with dichloromethane. The obtained organic layer was washed with saturated saline, and residual water was removed with magnesium sulfate. The organic layer was concentrated under reduced pressure, and then separated and purified by medium pressure preparative silica gel column chromatography (hexane:dichloromethane = 75:25 → 70:30). As a result, a mixture of the target compounds 11a and 11b was obtained as a yellow solid (1.64 g, 68%, Figure 14). 1 H NMR (500MHz, CDCl3, TMS): δ 8.96-8.05(m,8H),3.27-3.22(m,2H),1.38-1.34(m,3H).

[0242] Example 14 Synthesis of compound 12 A mixture of compound 11a and compound 11b (1.53 g, 4.54 mmol), cesium carbonate (4.40 g, 13.6 mmol), palladium (II) acetate (101 mg, 0.450 mmol), RuPhos (424 mg, 0.910 mmol), and a stirrer were added to a two-necked eggplant flask, and after attaching a reflux condenser, the flask was filled with argon. Ethyl 4-piperidinecarboxylate (1.05 mL, 6.81 mmol) and toluene (23 mL) with argon bubbling were added, and the mixture was heated and stirred in an oil bath at 100 ° C for 20 hours. Water was added to the reaction solution, and the mixture was extracted with dichloromethane. The obtained organic layer was washed with saturated saline, and the remaining water was removed with magnesium sulfate. The organic layer was concentrated under reduced pressure, and then separated and purified by silica gel column chromatography (hexane: dichloromethane = 80: 20). As a result, the target product 12 was obtained as a yellow solid (164.2 mg, 17%, FIG. 15). 1H NMR(500MHz,CDCl3,TMS):δ 8.75(d,J=9.5Hz,1H),8.48(d,J=9.1Hz,1H),8.28(d,J=8.1Hz,1H),8.16 (d,J=8.1Hz,1H),8.10(dd,J=8.4,9.5Hz,2H),8.04(d,J=9.1Hz,1H),7.75 (d,J=8.4Hz,1H),4.23(q,J=7.2Hz,2H),3.56-3.53(m,2H),3.25(q,J=7. 2Hz,2H),3.00(m,2H),2.57(m,1H),2.23-2.17(m,4H),1.38-1.31(m,6H).

[0243] Example 15 Synthesis of compound 13 Compound 12 (53.3 mg, 0.129 mmol) was added to a recovery flask. A 0.25 M ethanol solution of potassium hydroxide (2.58 mL) was added to the same flask, and the mixture was stirred in a 40°C oil bath for 21 hours. Dilute hydrochloric acid was then added dropwise to stop the reaction, and the mixture was extracted with dichloromethane. The resulting organic layer was washed with saturated saline, and residual water was removed with magnesium sulfate. The organic layer was concentrated under reduced pressure, and then separated and purified by medium-pressure preparative silica gel chromatography (dichloromethane → dichloromethane:methanol = 88:12). As a result, the target product 13 was obtained as a yellow solid (25.2 mg, 58%, Figure 16). 1 H NMR(500MHz,DMSO-d6):δ 8.57(d,J=9.3Hz,1H),8.45(d,J=8.1Hz,1H),8.40(d,J=9.3Hz,1H),8.26(d,J=8.2Hz,1H),8.25(d,J=8.2Hz,1H),8.18(d,J=7.9Hz,1H), 8.16(d,J=7.9Hz,1H),7.83(d,J=8.1Hz,1H),3.45(t,2H),3.25(q,J=7.2Hz,2H),2.95(m,2H),2.10-1.90(m,4H),1.19(t,J=7.2Hz,3H).

[0244] Example 16 Synthesis of compound C Compound 13 (87.4 mg, 0.23 mmol) and comu (102.8 mg, 0.24 mmol) were added to a recovery flask, and the flask was filled with argon. Dehydrated DMF (5.6 mL) and DIEPA (78.2 μL, 0.46 mmol) were added, and the mixture was stirred at room temperature for 45 minutes. Next, C12Sul (73.6 mg, 0.24 mmol) was added to another two-neck recovery flask, and the flask was filled with argon. Dehydrated DMF (6 mL) and DIEPA (78.2 μL, 0.46 mmol) were added, and the mixture was stirred at room temperature. This solution was added to the recovery flask containing compound 13, and the mixture was stirred at room temperature for 27.5 hours. The reaction solution was concentrated under reduced pressure, and separation and purification were performed by medium pressure preparative silica gel chromatography (dichloromethane → dichloromethane:methanol = 92:8). As a result, the target product 16 was obtained as a yellow solid (148.9 mg, 81%, Figure 17). Compound C also showed high solubility (>5 mg / mL) in phosphate buffer (20 mM, pH=7.2). *C12Sul:K.Sakai,et.al.Colloids Surf.A Physicochem.Eng.Asp.2009,333,26-31. 1 H NMR(500MHz,DMSO-d6):δ 8.56(d,J=9.5Hz,1H),8.43(d,J=9.3Hz,1H),8.41(d,J=8.2Hz,1H),8.24(d d,J=8.4Hz,8.2Hz,2H),8.18(brs,1H),8.15(d,J=9.5Hz,1H),7.84(d,J=8. 4Hz,1H),3.47(brs,4H),3.37-3.18(m,8H),3.12-3.01(m,7H),2.07-2.05( m,2H),1.88-1.80(m,4H),1.55(brs,1H),1.46(m,2H),1.24-1.18(m,35H), 0.83- 0.78 (m, 3H). HRMS(ESI)Calcd for C 40 H 53 N2O5S - :673.3681,Found:673.3659([M] - ).

[0245] (Synthesis of Compound D) Compound D was synthesized according to the following scheme. [ka] Scheme 5. Synthesis of compound D

[0246] Example 17 Synthesis of Compound 14a and Compound 14b 1-Bromopyrene (1.00g, 3.56mmol) was added to a two-necked eggplant flask, and the flask was filled with argon. Dehydrated dichloromethane (15mL) was added and cooled in an ice bath. Aluminum chloride (1.19g, 8.89mmol) was added to this solution and stirred for 20 minutes, and then succinic acid monoethyl chloride (1.25mL, 8.89mmol) was added dropwise and stirred at room temperature for 13 hours. Water was added while cooling in an ice bath to stop the reaction, and the reaction solution was washed with water and saturated saline, and residual water was removed with magnesium sulfate. The organic layer was concentrated under reduced pressure, and then separation and purification were performed by medium pressure preparative silica gel column chromatography (hexane:dichloromethane = 70:30 → 60:40). As a result, a mixture of the target compounds 14a and 14b was obtained as a yellow solid (824.4mg, 57%, Figure 18). 1 H NMR (500MHz, CDCl3, TMS): δ 8.99-8.05(m,8H),4.24-4.19(m,2H),3.55-3.52(m,2H),2.94-2.91(m,2H),1.32-1.25(m,3H).

[0247] Example 18 Synthesis of compound 15 A mixture of compound 14a and compound 14b (785.5 mg, 1.92 mmol), cesium carbonate (875.8 mg, 2.69 mmol), palladium (II) acetate (21.6 mg, 0.096 mmol), and RuPhos (89.6 mg, 0.19 mmol) were added to a two-necked eggplant flask, and a reflux condenser was attached to create an argon atmosphere in the flask. Toluene (9.5 mL) with argon bubbling was added, and the mixture was heated and stirred for 20 minutes in an oil bath at 100 ° C., and piperidine (0.23 mL, 2.30 mmol) was added dropwise and heated under reflux for 22.5 hours. The reaction solution was filtered, and the filtrate obtained was concentrated under reduced pressure, and then dichloromethane was added. The solution was washed with water and saturated saline, and the remaining water was removed with magnesium sulfate. The organic layer was concentrated under reduced pressure, and then separation and purification were performed by medium pressure preparative silica gel column chromatography (hexane: dichloromethane = 60: 40 → 50: 50). Furthermore, in order to separate the target product 15 from its isomers, isomer separation was performed using recycle GPC, resulting in the target product 15 being obtained as a yellow solid (255.0 mg, 32%, FIG. 19). 1 H NMR(500MHz,CDCl3,TMS):δ 8.79(d,J=9.4Hz,1H),8.51(d,J=9.2Hz,1H),8.36(d,J=8.1Hz,1H),8.16(d, J=8.1Hz,1H),8.10(dd,J=8.3,9.4Hz,2H),8.04(d,J=9.2Hz,1H),7.76(d,J= 8.3Hz,1H),4.20(q,J=7.2Hz,2H),3.54(t,J=6.6Hz,2H),3.22(brs,4H),2.9 0(t,J=6.6Hz,2H),1.95-1.91(m,4H),1.72(brs,2H),1.29(t,J=7.2Hz,3H).

[0248] Example 19 Synthesis of compound 16 A 0.25M potassium hydroxide solution (6mL) dissolved in ethanol, compound 15 (82.6mg, 0.20mmol), and two drops of water were added to a recovery flask and stirred in a 40°C oil bath for 6 hours. Dilute hydrochloric acid was then added dropwise to stop the reaction, and the mixture was extracted with dichloromethane. The resulting organic layer was washed with saturated saline, and residual water was removed with magnesium sulfate. The organic layer was concentrated under reduced pressure, and then separated and purified by medium pressure preparative silica gel chromatography (dichloromethane → dichloromethane:methanol = 90:10). As a result, the target product 16 was obtained as a yellow solid (quant., Figure 20). 1 H NMR(500MHz,DMSO-d6):δ 8.59(d,J=9.2Hz,1H),8.48(d,J=8.1Hz,1H),8.41(d,J=9.2Hz,1H),8.26(dd,J=8.2Hz,8.2Hz,2H),8.17(dd,J=9.2Hz,9.3Hz ,2H),7.83(d,J=8.1Hz,1H),3.43(t,J=6.2Hz,2H),3.13(brs,4H),2.70(t,J=6.2Hz,2H),1.86-1.84(m,4H),1.64(brs,2H).

[0249] Example 20 Synthesis of Compound D The obtained compound 16 (56.1 mg, 0.15 mmol) and comu (68.5 mg, 0.16 mmol) were added to a recovery flask, and the flask was filled with argon. Anhydrous DMF (3.5 mL) and DIEPA (51.0 μL, 0.30 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. Next, C12Sul (52.1 mg, 0.17 mmol) was added to another two-neck recovery flask, and the flask was filled with argon. Anhydrous DMF (3.8 mL) and DIEPA (51.0 μL, 0.30 mmol) were added, and the mixture was stirred at room temperature. This solution was added to the recovery flask containing compound 16, and the mixture was stirred at room temperature for 25 hours. The reaction solution was concentrated under reduced pressure, and separation and purification were performed by medium pressure preparative silica gel chromatography (dichloromethane → dichloromethane:methanol = 90:10). As a result, the target product 6 was obtained as a yellow solid (100.6 mg, 83%, Figure 21). Compound D also showed high solubility (>5 mg / mL) in phosphate buffer (20 mM, pH = 7.2). *C12Sul:K.Sakai,et.al.Colloids Surf.A Physicochem.Eng.Asp.2009,333,26-31. 1 H NMR(500MHz,DMSO-d6):δ 8.55-8.52(m,1H),8.45-8.41(m,2H),8.24-8.22(m,2H),8.15(d,J=9.2Hz,1H),8.11-8.09(brs,1H), 7.82(d,J=8.1Hz,1H),3.91(brs,2H),3.44(brs,1H),3.37(brs,2H),3.29(brs,2H),3.23-3.20(m,2H ),3.13-3.05(brs,12H),2.82-2.81(brs,2H),2.44-2.33(brs,6H),1.86-1.85(m,6H),1.75(brs,1H) ,1.66(m,2H),1.53(brs,1H),1.40(brs,1H),1.21-1.17(m,22H),0.81-0.79(m,3H);HRMS(ESI)Calcd for C 40 H 53 N2O5S - :673.3681,Found:673.3667([M] - ).

[0250] Example 21 Absorption and fluorescence spectral behavior of compounds C and D in organic solvents Table 3 and Figure 22 show the results of measuring the absorption (solid line) and fluorescence (dotted line) spectra of Compound C and Compound D in DMSO. Dye concentration: 10 μM (absorption spectrum), 5 μM (fluorescence spectrum). Excitation wavelength: 400 nm.

[0251] [Table 3]

[0252] Example 22 Fluorescence spectrum behavior of compounds C and D in phosphate buffer solution Table 4 and Figure 23 show the results of measuring the fluorescence spectra of Compound C and Compound D in 20 mM phosphate buffer. Dye concentration: 5 μM. Excitation wavelength: 400 nm.

[0253] [Table 4]

[0254] Example 23 Fluorescence behavior of Compound C and Compound D in the presence of artificial lipid bilayer membrane Three types of liposomes were prepared, one forming an ordered phase (Lo phase, lipid: sphingomyelin-cholesterol (SM / Chol)) and the other forming a disordered phase (Ld phase, lipid: dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine-cholesterol (DOPC / Chol)). The liposomes were dispersed in a phosphate buffer solution, and dye was added to the solution at a lipid:dye molar ratio of 100:1, and the fluorescence spectrum was measured. The results are shown in Table 5 and Figures 24-25.

[0255] [Table 5]

[0256] Example 24 Transcutaneous staining and fluorescent imaging of living mouse tissues using compounds A to D Using compounds A to D obtained in each Example, transdermal staining and fluorescence imaging of live mouse skin tissue was performed. More specifically, multiphoton fluorescence imaging of live mouse tissue stained with compounds A to D in the Examples was performed (aqueous solutions of compounds A to D at a concentration of 10 mg / mL were administered to the mouse vein. Excitation wavelength: 1100 nm for compound A, and 920 nm for compounds B to D). Since compounds A to D of the present invention are all highly water-soluble, it was possible to administer a 10 mg / mL aqueous solution to the mouse vein in this manner.

[0257] A two-photon microscope A1R MP+ (NIKON) was used for the microscopic observation. In the measurement, a 960 nm laser light source was used for excitation of compound A. Scanning was performed to a depth of 200 μm in the sample. A three-dimensional reconstruction image was obtained by processing the obtained data with the attached software.

[0258] Sufficient staining was achieved by anesthetizing mice and immersing their hind toes in the above staining solution at room temperature for 2 to 3 hours or administering it into the bloodstream.

[0259] The results are shown in Figure 26 for compound A, Figure 27 for compound B, Figure 28 for compound C, and Figure 29 for compound D, respectively.

[0260] As shown in Figures 26 to 29, it was confirmed that all of Compounds A to D strongly stained the membranes of cells constituting mouse tissues, and were able to distinguish the boundaries of individual cells and clearly depict the tissue structure. In addition, Compounds C and D showed fluorescent solvatochromism, making ratiometric analysis possible.

Claims

1. A compound having a squaraine structure, a carbocyanine structure, a merocyanine structure, or a polycyclic aromatic structure, and having at least one structure (1) represented by the following formula (1): 【Chemistry 1】 (In formula (1), R １ represents a substituted or unsubstituted alkyl group having 4 to 30 carbon atoms, an alkenyl group having 4 to 30 carbon atoms, or an alkynyl group having 4 to 30 carbon atoms. R ２ is a substituted or unsubstituted methylene group. ２ When multiple are present, they may be the same or different. X － is a monovalent anion. － When more than one is present, each group is the same or different. Y ＋ is a monovalent cation. ＋ When more than one is present, each group is the same or different. n2 is an integer from 1 to 10. When n2 is 2 or more, each group is the same or different. R ３ is a substituted or unsubstituted methylene group. ２ When multiple are present, they may be the same or different. n3 is an integer of 1 to 10. When n3 is 2 or more, each group is the same or different. L represents a linker. * indicates the binding site.)

2. At least one X － is -SO ３ － , or -COO － 2. The compound of claim 1,

3. At least one Y ＋ H ＋ , an alkali metal cation, or an ammonium cation.

4. The compound according to claim 1, wherein the molecule contains one or two of the structure (1).

5. 2. The compound according to claim 1, wherein L is a substituted or unsubstituted hydrocarbon group having 1 to 20 carbon atoms (including those partially substituted with heteroatoms).

6. The compound according to claim 1, wherein n2 is an integer from 1 to 4.

7. The compound according to claim 1, wherein n3 is an integer from 1 to 4.

8. The compound according to claim 1 , wherein the polycyclic aromatic structure is a naphthalene structure, anthracene structure, phenanthrene structure, pyrene structure, tetracene structure, triphenylene structure, perylene structure, or fluorene structure.

9. The compound according to claim 1 , having a structure (2) represented by the following formula (2): 【Chemistry 2】 【Chemistry 3】 (In formula (2), R ４ are each independently an 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, and a halogen. Each n4 is independently an integer from 0 to 4. When n4 is 2 or more, R ４ are the same or different. L represents a linker. R is a structure (3) represented by the above formula (3). R １ , R ２ , X － , Y ＋ , n2, R ３ , n3, and * are the same as in formula (1).

10. The compound according to claim 1 , having a structure (4) represented by the following formula (4): 【Chemistry 4】 【Chemistry 5】 (In formula (4), R ５ are each independently an 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, and a halogen. Each n5 is independently an integer of 0 to 4. When n5 is 2 or more, R ５ are the same or different. Each 1 is independently an integer of 1 to 4. Z － is an anion. L represents a linker. R is a structure (3) represented by the above formula (3). R １ , R ２ , X － , Y ＋ , n2, R ３ , n3, and * are the same as in formula (1).

11. The compound according to claim 1 , having a structure (5) represented by the following formula (5): 【Chemistry 6】 【Chemistry 7】 (In formula (5), n6 is an integer of 1 to 4. When n is 2 or more, each group is the same or different. n7 is an integer of 0 to 4. When n2 is 2 or more, each group is the same or different. R ７ and R ８ are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms; R ７ and R ８ may be joined together to form a ring structure. R ７ , or R ８ , and at least one hydrophilic substituent selected from the group consisting of a carboxyl group, a hydroxyl group, a sulfo group, an amino group, an amide group, a halogen atom, and a salt thereof. When there are two or more hydrophilic substituents, the respective groups may be the same or different. m is an integer of 1 to 4. When m is 2 or more, each group is the same or different. R ６ are each independently an 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, and a halogen. Each a is independently an integer of 0 to 4. When a is 2 or more, R ６ are the same or different. L represents a linker. R is a structure (3) represented by the above formula (3). R １ , R ２ , X － , Y ＋ , n2, R ３ , n3, and * are the same as in formula (1).

12. The compound according to claim 1, having a structure (6) represented by the following formula (6): 【Chemistry 8】 【Chemistry 9】 (In formula (6), R ９ is a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, or an alkynyl group having 2 to 12 carbon atoms. R １０ is a substituted or unsubstituted methylene group. １０ When multiple are present, they may be the same or different. n8 is an integer of 1 to 4. When n8 is 2 or more, each group is the same or different. n9 is an integer of 0 to 4. When n9 is 2 or more, each group is the same or different. n10 is an integer of 0 to 4. When n10 is 2 or more, each group is the same or different. R ６ are each independently an 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, and a halogen. Each a is independently an integer of 0 to 4. When a is 2 or more, R ６ are the same or different. R ７ and R １１ are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms; R ７ and R １１ may be joined together to form a ring structure. １１ has one hydrogen atom replaced by a -LR group. R ７ , R ９ , R １１ or a plurality thereof, at least one hydrophilic substituent selected from the group consisting of a carboxyl group, a hydroxyl group, a sulfo group, an amino group, an amide group, a halogen atom, and a salt thereof. When there are two or more hydrophilic substituents, each group may be the same or different. m is an integer of 1 to 4. When m is 2 or more, each group is the same or different. L represents a linker. R is a structure (3) represented by the above formula (3). R １ , R ２ , X － , Y ＋ , n2, R ３ , n3, and * are the same as in formula (1).

13. A fluorescent dye agent comprising the compound according to any one of claims 1 to 12.

14. The fluorescent dye agent according to claim 13, which is for detecting the morphology of cells and / or tissues.

15. The fluorescent dye agent according to claim 13, which is for detecting the morphology of cells and / or tissues in a living body.

16. 14. The fluorescent dye agent of claim 13 for staining or visualizing a biological sample.

17. The fluorescent dye agent according to claim 13, which is for staining or visualizing cell membranes in biological tissue.

18. The fluorescent dye agent according to claim 13, which is for use in fluorescent imaging.

19. The fluorescent dye agent according to claim 13, which is for in vivo observation.

20. A kit comprising a compound according to any one of claims 1 to 12.

21. A method for detecting a cell membrane, comprising the step (1) of staining the cell membrane with a fluorescent dye agent comprising the compound according to any one of claims 1 to 12.

22. The method for detecting a cell membrane according to claim 21, further comprising, after the step (1), a step (2) of evaluating the cell membrane using fluorescence spectra measured in two or more types of solvents.

23. The method for detecting a cell membrane according to claim 22, wherein step (2) is carried out using fluorescent imaging.

24. A material for staining lipid bilayer oil droplets or protein aggregates present in cells or modeling a cellular environment, comprising the compound according to any one of claims 1 to 12.

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