Fluorescent labeling agent and fluorescent dye

Abstract

To provide a fluorescent dye and a fluorescent labeling agent, featuring superior accumulation in phospholipids and exhibiting high fluorescence intensity.SOLUTION: The present invention provides a fluorescent labeling agent containing a fluorescent dye represented by the formula (1). General formula (1): Q-Z-R1-R2-R3 (where Q is a residue of the fluorescent dye; Z is a direct bond, an alkylene group, or an arylene group; R1 is a direct bond, -O-, -OP(=O) R4-, -OC(=O)-, -OS(=O)2-, -OSiR5R6-, -C(=O)-, or -C(=O) NH-; R2 is one group selected from the group consisting of an alkylene group, an arylene group, and a heterocyclic group, or a group consisting of a combination of these groups; R3 is COOM1, NR7R8, or N+R9R10R11; where, R4 is H, a hydroxy group, an alkyl group, an aryl group, an alkoxy group, an aryloxy group, or a heterocyclic group; R5 and R6 each represent an alkyl group, an aryl group, or a heterocyclic group; R7-R11 each represent H, an alkyl group, an aryl group; M1 is a monovalent cation).SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] SUMMARY OF THE DISCLOSURE Embodiments of the present invention relate to fluorescent labeling agents and fluorescent dyes used in fluorescent labeling agents. [Background technology]

[0002] Bioimaging is a technique for visualizing proteins, cells, tissues, etc. in vivo. Bioimaging is widely used in the fields of biology and medicine, for example, to elucidate the functions of molecules and cells in vivo and in drug discovery research. Among these, the fluorescence bioimaging method is an imaging method that allows dynamic, multicolor, and highly sensitive observation of phenomena. In recent years, the fluorescence bioimaging method has also attracted attention as an imaging method that allows non-invasive diagnosis, and is expected to be applied in clinical settings, such as imaging diagnosis that places less of a burden on patients and real-time diagnosis during surgery.

[0003] The fluorescent bioimaging method is a method for visualizing a target using a fluorescent dye that specifically binds to a target substance or that accumulates at a target site. In the above method, the fluorescent dye is irradiated with light in the ultraviolet to near-infrared range and the fluorescence emitted from the fluorescent dye is detected.

[0004] The accumulation-type fluorescent bioimaging using accumulation at a target site has a simpler and faster labeling method than the binding-type fluorescent bioimaging using specific binding with a target substance. In addition, since the accumulation-type fluorescent bioimaging does not require specific binding with a target substance, it has the advantages of not requiring a waiting time until the fluorescence intensity stabilizes and minimizing the effect on the target substance.

[0005] Patent Documents 1 and 2 disclose an accumulating fluorescent dye that is characterized by accumulating in phospholipids forming cell membranes.

[0006] Phospholipids form the surfaces of various biological materials such as cells, liposomes, and extracellular vesicles. In recent years, imaging of minute materials containing phospholipids has been attracting attention, such as liposome imaging for drug delivery systems (DDS) and exosome imaging described in Non-Patent Document 1. When the fluorescent dyes disclosed in Patent Documents 1 and 2 were used to perform such in vitro and in vivo imaging, there was a problem that the fluorescence intensity was low. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2009-524580 A [Patent Document 2] JP 2008-209361 A [Non-patent literature]

[0008] [Non-Patent Document 1] Drug Delivery System, Vol. 29, No. 2, March 25, 2014, p. 116-124 Summary of the Invention [Problem to be solved by the invention]

[0009] In view of the above-mentioned circumstances, an embodiment of the present invention provides a fluorescent dye that has excellent accumulation in phospholipids and exhibits high fluorescence intensity, and in particular has a fluorescence intensity suitable for use as a fluorescent labeling agent for in vitro and in vivo imaging. [Means for solving the problem]

[0010] As a result of intensive research aimed at solving the above problems, the present inventors have discovered an excellent fluorescent dye and completed the present invention. That is, the present invention relates to the following embodiments. However, the present invention is not limited to the following embodiments and includes various embodiments.

[0011] One embodiment relates to a fluorescent labeling agent containing a fluorescent dye represented by the following general formula (1): General formula (1): QZ-R1-R2-R3 In the formula, Q represents a residue of a fluorescent dye. Z represents a direct bond, a substituted or unsubstituted alkylene group, or a substituted or unsubstituted arylene group. R1 represents a direct bond, -O-, -OP(=O)R4-, -OC(=O)-, -OS(=O)2-, -OSiR5R6-, -C(=O)-, or -C(=O)NH-. R2 represents one group selected from the group consisting of a substituted or unsubstituted alkylene group, a substituted or unsubstituted arylene group, and a substituted or unsubstituted heterocyclic group, or a group formed by combining these groups. R3 is COOM1, NR7R8, N + R9R 10 R 11 , -OM2, or -P(=O)(OM3)OM4. In the above, R4 represents a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, or a substituted or unsubstituted heterocyclic group. R5 and R6 each independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. R7~R 11 each independently represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group. M1, M2, M3, and M4 each independently represent a hydrogen atom or a monovalent cation.

[0012] In one embodiment, the fluorescent labeling agent is preferably a phospholipid-accumulating fluorescent labeling agent.

[0013] In one embodiment, the fluorescent dye preferably includes a phthalocyanine dye represented by the following general formula (2): [ka] In the formula, X1~X 16 each independently represents -Z-R1-R2-R3, a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted heterocyclic group, -AB, -SO3M5, or -COOM6. In the above, A represents a Group 16 element. B represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclic group. M5 and M6 each independently represent a monovalent cation. X1~X 16 Adjacent substituents may be linked to each other to form a ring. X 17 is -Z-R1-R2-R3, a hydroxyl group, a halogen atom, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, -OP(=O)X 18 X 19 , -OC(=O)X 20 , -OS(=O)2X 21 , or -OSiX 22 X 23 X 24 Represents. In the above, X 18 and X 19 each independently represents a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, or a substituted or unsubstituted heterocyclic group. X 20represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. X 21 represents a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. X 22 ~X 24 each independently represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. Y represents a divalent to pentavalent metal atom, and k is an integer. When Y is a divalent metal atom, k is 0. When Y is a trivalent metal atom, k is 1. When Y is a tetravalent or pentavalent metal atom, k is 2. However, in the above, X1 to X 17 At least one of is -Z-R1-R2-R3.

[0014] In one embodiment, the fluorescent labeling agent is X 17 is -Z-R1-R2-R3.

[0015] One embodiment relates to a compound represented by the following general formula (3): [ka] In the formula, X1~X 16 each independently represents -Z-R1-R2-R3, a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted heterocyclic group, -AB, -SO3M5, or -COOM6. In the above, A represents a Group 16 element. B represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclic group. M5 and M6 each independently represent a monovalent cation. X1~X 16Adjacent substituents may be linked to each other to form a ring. X 17 is -Z-R1-R2-R3, a hydroxyl group, a halogen atom, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, -OP(=O)X 18 X 19 , -OC(=O)X 20 , -OS(=O)2X 21 , or -OSiX 22 X 23 X 24 Represents. In the above, X 18 and X 19 each independently represents a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, or a substituted or unsubstituted heterocyclic group. X 20 represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. X 21 represents a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. X 22 ~X 24 each independently represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. Y represents a divalent to pentavalent metal atom, and k is an integer. When Y is a divalent metal atom, k is 0. When Y is a trivalent metal atom, k is 1. When Y is a tetravalent or pentavalent metal atom, k is 2. However, in the above, X1 to X 17 At least one of is -Z-R1-R2-R3, as follows: Z represents a direct bond, a substituted or unsubstituted alkylene group, or a substituted or unsubstituted arylene group. R1 represents a direct bond, -O-, -OP(=O)R4-, -OC(=O)-, -OS(=O)2-, -OSiR5R6-, -C(=O)-, or -C(=O)NH-. R2 represents one group selected from the group consisting of a substituted or unsubstituted alkylene group, a substituted or unsubstituted arylene group, and a substituted or unsubstituted heterocyclic group, or a group formed by combining these groups. R3 is COOM1, NR7R8, N + R9R 10 R 11 , -OM2, or -P(=O)(OM3)OM4. In the above, R4 represents a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, or a substituted or unsubstituted heterocyclic group. R5 and R6 each independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. R7 to R 11 each independently represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group. M1, M2, M3, and M4 each independently represent a hydrogen atom or a monovalent cation. Effect of the Invention

[0016] According to an embodiment of the present invention, by introducing a functional group that has a high accumulation tendency into phospholipids, it is possible to provide a fluorescent dye having a fluorescence intensity suitable for use as a fluorescent labeling agent for in vitro and in vivo imaging. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a graph showing the evaluation results of the fluorescence intensity of fluorescent labeling agents 1, 15, 19, 24, 25, 68, and 75. [Diagram 2] FIG. 2 is a fluorescence micrograph of cells labeled with fluorescent labeling agent 1. [Diagram 3]FIG. 3 is a fluorescent micrograph of cells labeled with fluorescent labeling agent 15. [Figure 4] FIG. 4 is a fluorescence micrograph of cells labeled with fluorescent labeling agent 19. [Diagram 5] FIG. 5 is a fluorescent micrograph of cells labeled with the fluorescent labeling agent 24. [Figure 6] FIG. 6 is a fluorescent micrograph of cells labeled with fluorescent labeling agent 25. [Figure 7] FIG. 7 is a fluorescent micrograph of cells labeled with fluorescent labeling agent 68. [Figure 8] FIG. 8 is a fluorescent micrograph of cells labeled with fluorescent labeling agent 75. [Figure 9] FIG. 9 is a fluorescent micrograph of cells labeled with the fluorescent labeling agent 42. [Figure 10] FIG. 10 is a fluorescent micrograph of cells labeled with fluorescent labeling agent 53. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments of the present invention are not limited to the following description and include various embodiments. A fluorescent labeling agent according to one embodiment of the present invention includes a compound represented by the following general formula (1): The compound represented by the following general formula (1) is a fluorescent dye. General formula (1): QZ-R1-R2-R3

[0019] In the formula, Q represents a residue of a fluorescent dye. In this specification, the fluorescent dye is a dye that emits fluorescence when irradiated with light in the ultraviolet region to the near infrared region (for example, light with a wavelength of 560 to 900 nm), and may be a known compound. The fluorescent dye is not particularly limited, and examples thereof include dyes such as fluoresceins, rhodamines, coumarins, cyanines, phthalocyanines, diketopyrrolopyrroles, boron dipyrromethenes (BODIPY), xanthenes, pyrenes, merocyanines, perylenes, acridines, stilbenes, pyrromethenes, acridines, and umbelliferones. In one embodiment, the compound (fluorescent dye) represented by the above general formula (1) may have, for example, the skeleton of the above-exemplified dye as the fluorescent dye residue Q. That is, the compound represented by the above general formula (1) may be a compound having a structure in which at least one substituent (functional group) represented by -Z-R1-R2-R3 is introduced into the skeleton of the above-exemplified dye.

[0020] In one embodiment, the fluorescent dye is preferably a phthalocyanine from the viewpoints of stability and fluorescent wavelength. In one embodiment, a compound (phthalocyanine dye) represented by the following general formula (2) can be suitably used as the fluorescent dye. In the formula, X1 to X 17 It is assumed that at least one of the following is a substituent represented by -Z-R1-R2-R3. When the fluorescent dye constituting the fluorescent labeling agent contains a compound represented by the following general formula (2), a fluorescent labeling agent having excellent durability can be easily obtained due to the phthalocyanine dye skeleton. In addition, light emission at a wavelength (e.g., 650 to 900 nm) suitable for in vitro and in vivo bioimaging can be easily obtained due to the phthalocyanine dye skeleton.

[0021] [ka]

[0022] In the fluorescent dye of the above embodiment, "-Z-R1-R2-R3" is a substituent having a hydrophilic group, and can enhance the accumulation of the fluorescent dye in phospholipids by electrostatic interaction with the hydrophilic group in the phospholipid. The specific structure of the above substituent is as follows.

[0023] Z represents a direct bond, a substituted or unsubstituted alkylene group, or a substituted or unsubstituted arylene group. In one embodiment, Z is preferably a direct bond.

[0024] R1 represents a direct bond, -O-, -OP(=O)R4-, -OC(=O)-, -OS(=O)2-, -OSiR5R6-, -C(=O)-, or -C(=O)NH-. In one embodiment, R1 is preferably -OP(=O)R4-, -OS(=O)2-, or -OSiR5R6-.

[0025] R2 represents one group selected from the group consisting of a substituted or unsubstituted alkylene group, a substituted or unsubstituted arylene group, and a substituted or unsubstituted heterocyclic group, or a group formed by combining these groups. In one embodiment, R2 is preferably a substituted or unsubstituted alkylene group, or a substituted or unsubstituted arylene group, or more preferably a substituted or unsubstituted alkylene group. In one embodiment, R2 is preferably an alkylene group. The main chain of the alkylene group preferably has 1 to 10 carbon atoms.

[0026] R3 is -COOM1, -NR7R8, or -N + R9R 10 R 11 In addition, R3 represents -OM2 or -P(=O)(OM3)OM4. In one embodiment, R3 is preferably -COOM1, -NR7R8, -OM2, or -P(=O)(OM3)OM4.

[0027] In the above, R4 represents a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, or a substituted or unsubstituted heterocyclic group. In one embodiment, R4 is preferably a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, or an unsubstituted aryl group.

[0028] R5 and R6 each independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. In one embodiment, R5 and R6 each independently represent a substituted or unsubstituted alkyl group or an unsubstituted aryl group. The alkyl group is more preferably a linear or branched alkyl group having 1 to 5 carbon atoms.

[0029] R7~R 11 Each of R7 to R7 independently represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group. 11 are preferably each independently a hydrogen atom or a substituted or unsubstituted alkyl group. The alkyl group is more preferably a linear or branched alkyl group having 1 to 5 carbon atoms.

[0030] M1, M2, M3, and M4 each independently represent a hydrogen atom or a monovalent cation. Examples of monovalent cations include alkali metals and quaternary amines. Examples of alkali metals include lithium, sodium, potassium, rubidium, and cesium. In one embodiment, M1, M2, M3, and M4 are each preferably a hydrogen atom.

[0031] X1~X 16each independently represents a hydrogen atom or a substituent selected from the group consisting of -Z-R1-R2-R3, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted heterocyclic group, -AB, -SO3M5, and -COOM6. In the above, M5 and M6 each independently represent a monovalent cation. Examples of the monovalent cation include alkali metals and quaternary amines. Examples of the alkali metal include lithium, sodium, potassium, rubidium, and cesium.

[0032] In one embodiment, X1 to X 16 It is preferable that at least one of, and preferably four or more of, the above-mentioned substituents are each independently a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or -AB.

[0033] In the above formula -AB, A represents a group 16 element. Examples of group 16 elements include oxygen, sulfur, selenium, and tellurium. In one embodiment, A is preferably oxygen, sulfur, or selenium. From the viewpoints of ease of synthesis and stability, oxygen or sulfur is more preferable. From the viewpoint of fluorescence intensity, oxygen is even more preferable. B represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclic group, each as described above. In one embodiment, B is preferably a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group. Therefore, in one embodiment, -AB is preferably -OR, -OAr, -SR, or -SAr. Here, R represents an alkyl group, and Ar represents an aryl group.

[0034] Y represents a divalent to pentavalent metal atom, and k is an integer. When Y is a divalent metal atom, k is 0, when Y is a trivalent metal atom, k is 1, and when Y is a tetravalent or pentavalent metal atom, k is 2. Examples of divalent metal atoms include Mg, Cu, and Zn. Examples of trivalent metal atoms include Al, Ga, and In. Examples of tetravalent metal atoms include Si, Mn, Sn, Cr, and Zr. Examples of pentavalent metal atoms include P. From the viewpoint of fluorescence intensity, Y is preferably Al, Si, or P, and more preferably Al. From the viewpoint of light resistance, Y is preferably Al or Si.

[0035] In one embodiment, X1 to X 16 In the above, adjacent substituents may be linked to each other to form a ring. The ring structure may be any of cycloalkyl, cycloalkene, aryl, and heteroaryl, and forms a condensed ring with an aromatic ring in the phthalocyanine skeleton. The ring structure may further have a substituent, or may be unsubstituted. The number of carbon atoms forming the ring structure may be 2 to 30, and is preferably in the range of 4 to 6. The ring is preferably a 5-membered or 6-membered ring. In one embodiment, it is preferred that adjacent substituents are linked to each other to form a phenylene group. In this case, they are linked to the aromatic ring in the phthalocyanine skeleton to form a naphthalene structure. In another embodiment, adjacent substituents may be linked to each other to form a ring containing a nitrogen atom. In this case, they are linked to the aromatic ring in the phthalocyanine skeleton to form, for example, an imidazole structure. The ring structure such as the naphthalene structure or the imidazole structure may further have a substituent such as an alkyl group or an aryl group.

[0036] X 17 is -Z-R1-R2-R3, a hydroxyl group, a halogen atom, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, -OP(=O)X 18 X 19 , -OC(=O)X 20 , -OS(=O)2X 21 , -OSiX 22 X23 X 24 In one embodiment, X 17 is preferably -Z-R1-R2-R3 or a hydroxyl group, where Z, R1, R2, and R3 are as described above.

[0037] In the above, X 18 and X 19 each independently represents a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, or a substituted or unsubstituted heterocyclic group. X 20 represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. X 21 represents a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. X 22 ~X 24 each independently represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group.

[0038] Here, the above R4 to R 11 , and X1 to X 24 The alkyl groups in are each independently selected. The alkyl groups may be substituted or unsubstituted. The alkyl group may be a linear or branched alkyl group. Specific examples include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, a dodecyl group, an octadecyl group, an isopropyl group, an isobutyl group, an isopentyl group, a 2-ethylhexyl group, a sec-butyl group, a tert-butyl group, a sec-pentyl group, a tert-pentyl group, a tert-octyl group, and a neopentyl group. The number of carbon atoms in the alkyl group is preferably within the range of 1 to 30. The number of carbon atoms is more preferably within the range of 1 to 20, and further preferably within the range of 1 to 10.

[0039] Examples of the substituent in the alkyl group include halogen atoms such as fluorine, chlorine, and bromine, hydroxyl groups, amino groups, nitro groups, formyl groups, cyano groups, and carboxyl groups, as well as the above-mentioned alkyl groups, aryl groups, cycloalkyl groups, and heterocyclic groups described below. In addition, when a part of the structure is substituted with an amide bond (-NHCO-), an ester bond (-COO-), an ether bond (-O-), a urea bond (-NHCONH-), or a urethane bond (-NHCOO-), the substituted portion is also considered to be a "substituent".

[0040] Therefore, the substituted alkyl group means an alkyl group substituted with the above-mentioned substituent. The substituted alkyl group may be an alkyl group substituted with one or more substituents. For example, specific examples of the alkyl group substituted with a halogen atom include a trifluoromethyl group, a 2,2,2-trifluoroethyl group, -(CF2)4CF3, -(CF2)5CF3, -(CF2)6CF3, -(CF2)7CF3, -(CF2)8CF3, a trichloromethyl group, and a 2,2-dibromoethyl group.

[0041] Specific examples of alkyl groups substituted with an amide bond include -CH2-CH2-CH2-NHCO-CH2-CH3, -CH2-CH(-CH3)-CH2-NHCO-CH2-CH3, -CH2-CH2-CH2-NHCO-CH2-CH3, -CH2-CH2-CH2-CH2-NHCO-CH2-CH3, -CH2-CH2-CH2-CH2-NHCO-CH2-CH(CH2-CH3)-CH2-CH2-CH2-CH3, -(CH2)5-NHCO-(CH2) 11 -CH3, -CH2-CH2-CH2-C(-NHCO-CH2-CH3)3, etc. The number of carbon atoms in the alkyl group substituted with an amide bond is preferably within the range of 2 to 30. The number of carbon atoms is more preferably within the range of 2 to 10, and further preferably within the range of 2 to 5.

[0042] Specific examples of alkyl groups substituted with an ester bond include -CH2-CH2-CH2-COO-CH2-CH3, -CH2-CH(-CH3)-CH2-COO-CH2-CH3, -CH2-CH2-CH2-OCO-CH2-CH3, -CH2-CH2-CH2-CH2-CH2-COO-CH2-CH(CH2-CH3)-CH2-CH2-CH2-CH3, -(CH2)5-COO-(CH2) 11 Examples include -CH3, -CH2-CH2-CH2-CH-(COO-CH2-CH3)2, etc. The number of carbon atoms in the alkyl group substituted with an ester bond is preferably within the range of 2 to 30. The number of carbon atoms is more preferably within the range of 2 to 10, and further preferably within the range of 2 to 5.

[0043] Specific examples of alkyl groups substituted with an ether bond include -CH2-O-CH3, -CH2-CH2-O-CH2-CH3, -CH2-CH2-CH2-O-CH2-CH3, -(CH2-CH2-O) n -CH3 (wherein n is an integer from 1 to 8), -(CH2-CH2-CH2-O) mExamples of the alkyl group substituted with an ether bond include, but are not limited to, -CH3 (where m is an integer of 1 to 5), -CH2-CH(CH3)-O-CH2-CH3-, and -CH2-CH-(OCH3)2. The number of carbon atoms in the alkyl group substituted with an ether bond is preferably within the range of 2 to 30. The number of carbon atoms is more preferably within the range of 2 to 10, and further preferably within the range of 2 to 5.

[0044] Specific examples of the alkyl group substituted with a urea bond (-NHCONH-) include -CH2-NHCONH-CH3, -CH2-CH2-NHCONH-CH2-CH3, -CH2-CH2-CH2-NHCONH-CH2-CH3, -(CH2-CH2-NHCONH) n -CH3 (where n is an integer from 1 to 8), -(CH2-CH2-CH2-NHCONH) m Examples of the alkyl group substituted with an ether bond include, but are not limited to, -CH3 (where m is an integer of 1 to 5), -CH2-CH(CH3)-NHCONH-CH2-CH3-, and -CH2-CH-(NHCONHCH3)2. The number of carbon atoms in the alkyl group substituted with an ether bond is preferably within the range of 2 to 30. The number of carbon atoms is more preferably within the range of 2 to 10, and further preferably within the range of 2 to 5.

[0045] Specific examples of alkyl groups substituted with a urethane bond include -CH2-CH2-CH2-NHCOO-CH2-CH3, -CH2-CH(-CH3)-CH2-NHCOO-CH2-CH3, -CH2-CH2-CH2-NHCOO-CH2-CH3, -CH2-CH2-CH2-CH2-NHCOO-CH2-CH3, -CH2-CH2-CH2-CH2-NHCOO-CH2-CH(CH2-CH3)-CH2-CH2-CH2-CH3, -(CH2)5-NHCOO-(CH2) 11 Examples include -CH3, -CH2-CH2-CH2-CH-(NHCOO-CH2-CH3)2, etc. The number of carbon atoms in the alkyl group substituted with an ester bond is preferably within the range of 2 to 30. The number of carbon atoms is more preferably within the range of 2 to 10, and further preferably within the range of 2 to 5.

[0046] Specific examples of an alkyl group substituted with two or more substituents selected from the group consisting of an amide bond (-NHCO-), an ester bond (-COO-), an ether bond (-O-), a urea bond (-NHCONH-), and a urethane bond (-NHCOO-) include -CH2-CH2-NHCO-CH2-CH2-O-CH2-CH(CH2-CH3)-CH2-CH2-CH2-CH3, -CH2-CH2-COO-CH2-CH2-O-CH2-CH2-NHCOO-CH2-CH(CH2-CH3)-CH2-CH2-CH2-CH3, and -CH2-CH2-NHCO-CH2(OCO-CH2)-CH2-. The number of carbon atoms in the alkyl group substituted with two or more substituents selected from an amide bond (-NHCO-), an ester bond (-COO-), an ether bond (-O-), a urea bond (-NHCONH-), and a urethane bond (-NHCOO-) is preferably within the range of 3 to 30. The number of carbon atoms is more preferably within the range of 3 to 10, and further preferably within the range of 3 to 5.

[0047] Above R4~R 11 , and X1 to X 24 Each aryl group in is independently selected. The aryl group may be substituted or unsubstituted. The aryl group may be a monocyclic or condensed polycyclic aryl group, such as a phenyl group, a 1-naphthyl group, a 2-naphthyl group, a p-biphenyl group, a m-biphenyl group, a 2-anthryl group, a 9-anthryl group, a 2-phenanthryl group, a 3-phenanthryl group, a 9-phenanthryl group, a 2-fluorenyl group, a 3-fluorenyl group, a 9-fluorenyl group, a 1-pyrenyl group, a 2-pyrenyl group, a 3-perylenyl group, an o-tolyl group, a m-tolyl group, a p-tolyl group, a 4-methylbiphenyl group, a terphenyl group, a 4-methyl-1-naphthyl group, a 4-tert-butyl-1-naphthyl group, a 4-naphthyl-1-naphthyl group, a 6-phenyl-2-naphthyl group, a 10-phenyl-9-anthryl group, a spirofluorenyl group, or a 2-benzocyclobutenyl group. The number of carbon atoms in the aryl group is preferably within the range of 6 to 18. The number of carbon atoms is more preferably within the range of 6 to 10. The substituents of the substituted aryl group may be the same as those exemplified as the substituents of the alkyl group above.

[0048] X1~X 16 The cycloalkyl groups in are each independently selected. The cycloalkyl groups may be substituted or unsubstituted. Examples of the cycloalkyl groups include a cyclopentyl group, a cyclohexyl group, a 2,5-dimethylcyclopentyl group, and a 4-tert-butylcyclohexyl group. The number of carbon atoms in the cycloalkyl group is preferably within a range of 3 to 12. The number of carbon atoms is more preferably within a range of 3 to 6. The substituents of the substituted cycloalkyl groups may be the same as those exemplified as the substituents in the alkyl groups.

[0049] X1~X 16 The alkenyl groups in are each independently selected. The alkenyl group may have a substituent or may be unsubstituted. Examples of the alkenyl group include linear or branched alkenyl groups. The alkenyl group generally refers to a group having one double bond in its structure, but in this specification, the alkenyl group may have multiple double bonds in its structure. Specific examples of the alkenyl group include vinyl group, 1-propenyl group, allyl group, 2-butenyl group, 3-butenyl group, isopropenyl group, isobutenyl group, 1-pentenyl group, 2-pentenyl group, 3-pentenyl group, 4-pentenyl group, 1-hexenyl group, 2-hexenyl group, 3-hexenyl group, 4-hexenyl group, and 1,3-butadienyl group. The number of carbon atoms in the alkenyl group is preferably within the range of 2 to 18. The number of carbon atoms is more preferably 2 to 10, and even more preferably 2 to 5. The substituents of the substituted alkenyl group may be the same as those exemplified as the substituents of the alkyl group above.

[0050] Above R2, R4~R 11 , and X1 to X 24 The heterocyclic groups in are each independently selected. The heterocyclic groups may be substituted or unsubstituted. Examples of the heterocyclic group include an aliphatic heterocyclic group and an aromatic heterocyclic group. Specific examples include a pyridyl group, a pyrazyl group, a piperidino group, a pyranyl group, a morpholino group, and an acridinyl group. In addition, groups represented by the following structural formulas are also included. The number of carbon atoms (the number of carbon atoms constituting the ring) of the heterocyclic group is preferably 4 to 12. The number of ring members is preferably 5 to 13.

[0051] [ka]

[0052] The substituent of the substituted heterocyclic group may be the same as the substituent exemplified for the alkyl group. Examples of the substituted heterocyclic group include a heterocyclic group, such as a 3-methylpyridyl group, an N-methylpiperidyl group, and an N-methylpyrrolyl group.

[0053] Above R4, X 18 , and X 19 The alkoxy groups in are each independently selected. The alkoxy groups can be substituted or unsubstituted. The alkoxy group may be a linear or branched alkoxy group. Specific examples include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a tert-butoxy group, a neopentyloxy group, a 2,3-dimethyl-3-pentyloxy group, an n-hexyloxy group, an n-octyloxy group, a stearyloxy group, and a 2-ethylhexyloxy group. The number of carbon atoms in the alkoxy group is preferably within the range of 1 to 6. The substituent of the substituted alkoxyl group may be the same as the substituent exemplified as the substituent of the alkyl group above.

[0054] The substituent of the substituted alkoxy group may be the same as the substituent exemplified in the alkyl group. Specific examples of the substituted alkoxy group include a trichloromethoxy group, a trifluoromethoxy group, a 2,2,2-trifluoroethoxy group, a 2,2,3,3-tetrafluoropropoxy group, a 2,2-bis(trifluoromethyl)propoxy group, a 2-ethoxyethoxy group, a 2-butoxyethoxy group, a 2-nitropropoxy group, and a benzyloxy group.

[0055] Above R4, X 18 , and X 19 The aryloxy groups in are each independently selected. The aryloxy groups may be substituted or unsubstituted. The aryloxy group may be a monocyclic or condensed polycyclic aryloxy group. Specific examples include a phenoxy group, a p-methylphenoxy group, a naphthyloxy group, and an anthryloxy group. The aryloxy group is preferably a monocyclic aryloxy group. In addition, an aryloxy group having 6 to 12 carbon atoms is preferable.

[0056] The substituent of the substituted aryloxy group may be the same as the substituent exemplified for the above aryl group. Examples of the substituted aryloxy group include p-nitrophenoxy group, p-methoxyphenoxy group, 2,4-dichlorophenoxy group, pentafluorophenoxy group, and 2-methyl-4-chlorophenoxy group.

[0057] The alkylene groups in Z and R2 are each independently selected. The alkylene group may have a substituent or may be unsubstituted. The alkylene group may be a divalent group obtained by removing one hydrogen atom from the alkyl group. Specific examples of the substituted or unsubstituted alkylene group include -CH2-CH2-, -CH2-CH2-CH2-NHCO-CH2-CH2-, -CH2-CH2-CH2-OCO-CH2-CH2-, -CH2-CH2-CH2-O-CH2-CH2-, and the like.

[0058] The arylene group in Z and R2 is selected independently. The arylene group may have a substituent or may be unsubstituted. The arylene group may be a divalent group obtained by removing one hydrogen atom from an aryl group. The arylene group preferably has 6 to 10 carbon atoms. In one embodiment, the arylene group may be a phenylene group or a naphthylene group. Specific examples of the substituted or unsubstituted arylene group include groups represented by the following structural formula.

[0059] [ka]

[0060] One embodiment of the present invention relates to a fluorescent labeling agent containing the above-mentioned fluorescent dye. This fluorescent labeling agent can be applied for fluorescent labeling in bioimaging in a wide range of fields from biochemical research to medical diagnosis. For example, it can be used for fluorescent labeling in fields such as genetic diagnosis, immunodiagnosis, medical development, regenerative medicine, environmental testing, biotechnology, and fluorescence testing.

[0061] In particular, in the fluorescent labeling agent of the above embodiment, the structure (substituent) represented by -Z-R1-R2-R3 in the fluorescent dye has a function of interacting with phospholipids. Therefore, it can be suitably used as a phospholipid-accumulating fluorescent labeling agent. The phospholipid-accumulating fluorescent labeling agent can be suitably used as a fluorescent labeling agent in cell membrane staining, exosome tracking, liposome imaging for drug delivery systems (DDS), and the like.

[0062] In the fluorescent labeling agent of the above embodiment, the concentration of the fluorescent dye is not particularly limited. For example, when handling cells, taking into consideration the effects on cell dysfunction and growth inhibition, etc., the concentration of the fluorescent dye is preferably low. In one embodiment, for example, the concentration of the fluorescent dye for 10,000 cells / well seeded on a 96-well plate is preferably 100 μM or less. The concentration is more preferably 50 μM or less, and even more preferably 10 μM or less. According to the fluorescent labeling agent of the above embodiment, imaging with high fluorescence intensity is possible even with a low concentration of the fluorescent dye due to its excellent accumulation in phospholipids. Therefore, even with a low concentration of, for example, 2 μM or less, more accurate detection can be performed.

[0063] The fluorescent labeling agent of the above embodiment may contain the fluorescent dye of the above embodiment, but may also contain other components as necessary. The other components may be components well known in the art, such as a solvent and an amphiphilic substance.

[0064] The solvent may be water or an organic solvent, and water is more preferable. Taking into consideration the solubility of the fluorescent dye, water and an organic solvent may be mixed and used. For example, the organic solvent is preferably ethanol or dimethyl sulfoxide (DMSO).

[0065] An amphipathic substance is a general term for a compound having a hydrophilic group and a hydrophobic group in one molecule. Specific examples include surfactants and phospholipids. Only one type of amphipathic substance may be used, or two or more types may be mixed and used. In the fluorescent labeling agent of the above embodiment, the amphipathic substance is not particularly limited, and any compound may be used as long as it can solubilize a water-insoluble fluorescent dye that emits fluorescence in the near infrared region in water. Specific examples of amphipathic substances that can be used are listed below, although they are not particularly limited.

[0066] Examples of the surfactant include a nonionic surfactant, a cationic surfactant, an anionic surfactant, and a polymer surfactant.

[0067] Examples of nonionic surfactants include polyoxyethylene sorbitan fatty acid esters such as Tween (registered trademark) 20, Tween (registered trademark) 40, Tween (registered trademark) 60, and Tween (registered trademark) 80; polyoxyethylene castor oil derivatives such as Cremophor (registered trademark) EL and Cremophor (registered trademark) RH60; 12-hydroxystearic acid-polyethylene glycol copolymers such as Solutol (registered trademark) HS 15; and octylphenol ethoxylates such as Triton (registered trademark) X-100 and Triton (registered trademark) X-114.

[0068] Examples of cationic surfactants include alkyl trimethyl ammonium salts such as stearyl trimethyl ammonium chloride and lauryl trimethyl ammonium chloride, alkyl pyridinium salts such as cetyl pyridinium chloride, alkyl quaternary ammonium salts such as distearyl dimethyl ammonium chloride, dialkyl dimethyl ammonium salts, and poly(N,N'-dimethyl-3,5-methylene piperidinium) chloride, alkyl dimethyl benzyl ammonium salts, alkyl isoquinolinium salts, dialkyl morphonium salts, polyoxyethylene alkyl amines, alkyl amine salts, polyamine fatty acid derivatives, amyl alcohol fatty acid derivatives, benzalkonium chloride, and benzethonium chloride.

[0069] Examples of anionic surfactants include sodium dodecyl sulfate, dodecylbenzenesulfonate, decylbenzenesulfonate, undecylbenzenesulfonate, tridecylbenzenesulfonate, and nonylbenzenesulfonate, as well as sodium, potassium, and ammonium salts thereof.

[0070] Examples of polymer surfactants include block copolymers such as polyvinyl alcohol, polyoxyethylene polyoxypropylene glycol, polyethylene glycol-polyalkyl, polyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, polyethylene glycol-polyglycolic acid, and polyethylene glycol-poly(lactide-glycolide).

[0071] In one embodiment, the fluorescent labeling agent of the above embodiment may contain one or more of the compounds exemplified above as an amphipathic substance. However, since the fluorescent labeling agent of the above embodiment has excellent accumulation properties at target sites such as phospholipids, it is possible to perform detection with high sensitivity without requiring an amphipathic substance.

[0072] In the fluorescent labeling agent of the above embodiment, the fluorescent dye preferably includes a phthalocyanine dye. The method of synthesizing the phthalocyanine dye is not particularly limited. For example, first, a dye having a phthalocyanine skeleton (phthalocyanine metal complex) is synthesized by a known method using a phthalonitrile derivative as a raw material. Next, a component having a substituent (-Z-R1-R2-R3) is added to the dye, and these are reacted while being heated and stirred in a dimethyl sulfoxide solvent. By such a reaction, a desired fluorescent dye can be obtained. As the component having the above substituent, for example, an acidic compound or a compound shown as an axial ligand in the examples described later can be used.

[0073] When the raw material phthalonitrile derivative has an asymmetric structure, the phthalocyanine is obtained as a mixture of isomers having different positions of the substituents. In the following description, only one example of the phthalocyanine structure is shown, but isomers having different positions of the substituents are not excluded.

[0074] Specific examples of the fluorescent dye according to one embodiment of the present invention include the following: However, the fluorescent dye according to the present invention is not limited to these.

[0075] [Table 1-1]

[0076] [Table 1-2]

[0077] [Table 1-3]

[0078] [Table 1-4]

[0079] Among the fluorescent dyes exemplified above, fluorescent dyes 1 to 37 each have a phthalocyanine dye skeleton (dye residue). Fluorescent dye 38 has a diketopyrrolopyrrole dye skeleton. Fluorescent dyes 39 and 40 have a xanthene dye skeleton. The fluorescent dye 41 is a fluorescent dye having a boron dipyrromethene dye skeleton (dye residue) having the following structure: For example, the substituents "-Z-R1-R2-R3" in the fluorescent dye 41 are Z -C2H4-, R1 -C(=O)NH-, R2 -C3H6-, and R3 -N(CH3)2.

[0080] [ka]

[0081] [Table 1-5]

[0082] [Table 1-6]

[0083] [Table 1-7]

[0084] [Table 1-8]

[0085] Of the fluorescent dyes exemplified above, the fluorescent dyes 42 to 59 each have a phthalocyanine dye skeleton. The fluorescent dye 60 is a fluorescent dye having a diketopyrrolopyrrole dye skeleton. The fluorescent dye 61 is a fluorescent dye having a xanthene dye skeleton. The fluorescent dye 62 is a fluorescent dye having a cyanine dye skeleton. The fluorescent dye 63 is a fluorescent dye having a borondipyrromethene dye skeleton.

[0086] Although not particularly limited, in one embodiment, the fluorescent labeling agent preferably contains a fluorescent dye having a phthalocyanine dye skeleton from the viewpoint of stability such as durability. EXAMPLES

[0087] The present invention will be described below based on examples, but the present invention is not limited thereto. In the examples, "parts" means "parts by mass".

[0088] (Mass spectrometry) The analysis was performed using a mass spectrometer (TOF-MS: autoflexII manufactured by Bruker Daltonics).

[0089] Fluorescent dyes [Production Example 1] <Method of producing compound A-1> Ammonia gas was introduced into a solution of 50 parts of quinoline and 1 part of anhydrous aluminum chloride, and 5 parts of 3-ethoxyphthalonitrile were added, and these were reacted at 180°C for 7 hours. After cooling the reaction solution to room temperature, 200 parts of methanol and 200 parts of 10% aqueous hydrochloric acid were added. Then, the precipitated solid was filtered and washed with 200 parts of water. The washed solid was dried at 80°C to obtain 4.7 parts of compound A-1 shown in Table 2 (yield 88.4%).

[0090] [Manufacturing Examples 2-11] <Method of producing compounds A-2 to A-10> Compounds A-2 to A-10 shown in Table 2 were each produced in the same manner as in the production of compound A-1, except that the 3-ethoxyphthalonitrile and anhydrous aluminum chloride used in the production of compound A-1 were replaced with the phthalonitrile derivatives and metal sources shown in Table 2. The phthalonitrile derivatives and metal sources were used in the same molar amounts as 3-ethoxyphthalonitrile and anhydrous aluminum chloride used in the production of compound A-1.

[0091] [Table 2-1]

[0092] [Table 2-2]

[0093] [Production Example 11] <Method of producing compound B-1> To a solution in which 3 parts of compound A-1 were dissolved in 10 parts of N-methyl-2-pyrrolidone (NMP), an aqueous solution in which 0.45 parts of potassium hydroxide were dissolved in 1 part of water was added in its entirety. These were reacted at 110°C for 7 hours. After the reaction solution was cooled to room temperature, 100 parts of water was added. Next, the precipitated solid was filtered and washed with 100 parts of water. The washed solid was dried at 80°C to obtain 2.9 parts of compound B-1 shown in Table 3 (yield 99.2%).

[0094] [Examples 12 to 15] <Method of producing compounds B-2 to B-5> Compounds B-2 to B-5 shown in Table 3 were each produced in the same manner as in the production of compound B-1, except that compound A-1 used in the production of compound B-1 was changed to compound A shown in Table 3. Compound A was used in the same molar amount as compound A-1 in the production of compound B-1.

[0095] [Table 3]

[0096] [Production Example 16] <Method of producing compound C-1> Ammonia gas was introduced into a solution of 50 parts of quinoline and 1 part of anhydrous aluminum chloride, and 3.8 parts of 3-ethoxyphthalonitrile and 1.1 parts of 4-fluorophthalonitrile were added. These were reacted at 180°C for 7 hours. After cooling the reaction solution to room temperature, 200 parts of methanol and 200 parts of 10% aqueous hydrochloric acid were added. The precipitated solid was then filtered and washed with 200 parts of water. The washed solid (crude product) was purified using a medium pressure preparative liquid chromatograph (Yamazen Smart Flash AKROS). The obtained purified product was dried at 80°C to obtain 1.6 parts of compound C-1 shown in Table 4 (yield 30.5%).

[0097] [Manufacturing Examples 17-19] <Method of producing compounds C-2 to C-4> Phthalocyanines C-2 to C-4 shown in Table 4 were each produced in the same manner as in the production of compound C-1, except that the anhydrous aluminum chloride used in the production of compound C-1 was changed to the metal source shown in Table 4. The metal source was used in the same molar amount as that of the anhydrous aluminum chloride used in the production of compound C-1.

[0098] [Table 4]

[0099] [Example 1] <Method of manufacturing fluorescent dye 1> One part of compound B-1 and 0.6 parts of 3-aminopropyldimethylethoxysilane were dissolved in pyridine, and the solution was refluxed at 115°C for 3 hours to obtain a reaction solution. After removing pyridine from the reaction solution using an evaporator, a mixed solution of 10 parts of ethanol and 50 parts of water was added. The precipitated solid was then filtered and washed with 50 parts of water. The washed solid was dried at 80°C to obtain 0.39 parts of fluorescent dye 1 shown in Table 1 (yield 33.7%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 848.64 (theoretical value 847.99), and it was identified to have the structure of fluorescent dye 1 shown in Table 1.

[0100] [Examples 2 to 5] <Methods of producing fluorescent dyes 2 to 5> Fluorescent dyes 2 to 5 shown in Table 1 were each produced in the same manner as in the production of fluorescent dye 1, except that compound B-1 used in the production of fluorescent dye 1 was changed to compound B shown in Table 5. Compound B was used in the same molar amount as compound B-1 in the production of fluorescent dye 1. The structures of the obtained fluorescent dyes 2 to 5 were identified by mass spectrometry, and it was confirmed that they had the structures shown in Table 1. Table 11 shows the results of mass spectrum analysis.

[0101] [Table 5]

[0102] [Example 6] <Method of manufacturing fluorescent dye 6> 0.7 parts of compound A-1 and 0.4 parts of 4-(3-aminopropyl)benzenesulfonic acid were dissolved in 50 parts of dimethyl sulfoxide, and 0.3 parts of 1,8-diazabicyclo[5.4.0]-7-undecene were added, and the mixture was reacted at 90°C for 5 hours. After cooling the reaction solution to room temperature, 100 parts of water and 10 parts of salt were added. The precipitated solid was then filtered and washed with 50 parts of water. The washed solid was dried at 80°C to obtain 0.36 parts of fluorescent dye 6 shown in Table 1 (yield 41.6%). As a result of mass spectrometry, a molecular ion peak was detected at m / z=916.57 (theoretical value 915.96), and the structure of fluorescent dye 6 shown in Table 1 was identified.

[0103] [Examples 7 to 14] <Method of manufacturing fluorescent dyes 7 to 14> Fluorescent dyes 7 to 14 shown in Table 1 were each produced in the same manner as in the production of fluorescent dye 6, except that compound A-1 and 4-(3-aminopropyl)benzenesulfonic acid used in the production of fluorescent dye 6 were replaced with compound A and an acidic compound shown in Table 6. Compound A and the acidic compound were used in the same molar amounts as compound A-1 and 4-(3-aminopropyl)benzenesulfonic acid in the production of fluorescent dye 6. The structures of the obtained fluorescent dyes 7 to 14 were identified by mass spectrometry, and it was confirmed that they had the structures shown in Table 1. Table 11 shows the results of mass spectrum analysis.

[0104] [Table 6]

[0105] [Example 15] <Method of manufacturing fluorescent dye 15> 0.5 parts of compound A-1 and 0.29 parts of (2-carboxyethyl)phenylphosphinic acid were dissolved in 20 parts of dimethyl sulfoxide, and the solution was reacted at 80°C for 8 hours. After cooling the reaction solution to room temperature, 50 parts of water and 10 parts of salt were added. The precipitated solid was then filtered and washed with 50 parts of water. The washed solid was dried at 80°C to obtain 0.46 parts of fluorescent dye 15 shown in Table 1 (yield 74.4%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 929.46 (theoretical value 928.88), and the structure of fluorescent dye 15 shown in Table 1 was identified.

[0106] [Examples 16 to 18] <Method of manufacturing fluorescent dyes 16 to 18> Fluorescent dyes 16 to 18 shown in Table 1 were each produced in the same manner as in the production of fluorescent dye 15, except that compound A-1 and (2-carboxyethyl)phenylphosphinic acid used in the production of fluorescent dye 15 were replaced with compound A and an acidic compound shown in Table 7. Compound A and the acidic compound were used in the same molar amounts as compound A-1 and (2-carboxyethyl)phenylphosphinic acid in the production of fluorescent dye 15. The structures of the obtained fluorescent dyes 16 to 18 were identified by mass spectrometry, and were confirmed to have the structures shown in Table 1. Table 11 shows the results of mass spectrum analysis.

[0107] [Table 7]

[0108] [Example 19] <Method of manufacturing fluorescent dye 19> 0.5 parts of fluorescent dye 1, 0.8 parts of methyl iodide, and 0.8 parts of potassium carbonate were dissolved in 50 parts of tetrahydrofuran, and the solution was reacted at 25°C for 5 hours. Tetrahydrofuran was removed from the reaction solution using an evaporator, and then 20 parts of tetrahydrofuran and 60 parts of water were added. The precipitated solid was then filtered and washed with 60 parts of water. The washed solid was dried at 80°C to obtain 0.21 parts of fluorescent dye 19 shown in Table 1 (yield 33.3%). As a result of mass spectrometry, a molecular ion peak was detected at m / z(positive)=892.25 (theoretical value 891.08), and it was identified to have the structure of fluorescent dye 19 shown in Table 1.

[0109] [Examples 20 to 23] <Method of manufacturing fluorescent dyes 20 to 23> Fluorescent dyes 20 to 23 shown in Table 1 were each produced in the same manner as in the production of fluorescent dye 19, except that the methyl iodide and fluorescent dye 1 used in the production method of fluorescent dye 19 were replaced with the iodide compounds and amines shown in Table 8. The iodide compounds and amines were used in the same molar amounts as the methyl iodide and fluorescent dye 1 used in the production of fluorescent dye 19. The structures of the obtained fluorescent dyes 20 to 23 were identified by mass spectrometry, and it was confirmed that they had the structures shown in Table 1. Table 11 shows the results of mass spectrum analysis.

[0110] [Table 8]

[0111] [Example 24] <Method of manufacturing fluorescent dye 24> 0.06 parts of fluorescent dye 1 and 0.007 parts of succinic anhydride were dissolved in 5 parts of N-methyl-2-pyrrolidone (NMP), and the solution was reacted at 90°C for 4 hours. After removing NMP from the reaction solution using a centrifugal evaporator, 5 parts of water were added. The precipitated solid was then filtered and washed with 5 parts of water. The washed solid was dried at 80°C to obtain 0.041 parts of fluorescent dye 24 shown in Table 1 (yield 61.1%). As a result of mass spectrometry, a molecular ion peak was detected at / z=949.07 (theoretical value 948.06), and the structure of fluorescent dye 24 shown in Table 1 was identified.

[0112] [Examples 25 to 30] <Method of manufacturing fluorescent dyes 25 to 30> Fluorescent dyes 25 to 30 shown in Table 1 were each produced in the same manner as in the production of fluorescent dye 24, except that the succinic anhydride and fluorescent dye 1 used in the production method of fluorescent dye 24 were replaced with the succinic anhydride derivatives and amines shown in Table 9. The succinic anhydride derivatives and amines were used in the same molar amounts as the succinic anhydride and fluorescent dye 1 used in the production of fluorescent dye 24. The structures of the obtained fluorescent dyes 25 to 30 were identified by mass spectrometry, and were confirmed to have the structures shown in Table 1. Table 11 shows the results of mass spectrum analysis.

[0113] [Table 9]

[0114] [Example 31] <Method of manufacturing fluorescent dye 31> 1.0 part of compound C-1 and 0.6 parts of (2-carboxyethyl)phenylphosphinic acid were dissolved in 50 parts of dimethyl sulfoxide, and 0.4 parts of 1,8-diazabicyclo[5.4.0]-7-undecene were added, and the solution was reacted at 90°C for 8 hours. The reaction solution was cooled to room temperature, and 100 parts of water were added. The precipitated solid was then filtered and washed with 50 parts of water. The obtained solid (crude product) was purified using a medium pressure preparative liquid chromatograph (Yamazen Smart Flash AKROS). The obtained purified product was dried at 80°C to obtain 0.72 parts of fluorescent dye 31 shown in Table 1 (yield 60.1%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 901.46 (theoretical value 900.82), and it was identified to have the structure of fluorescent dye 31 shown in Table 1.

[0115] [Examples 32 to 37] <Methods of producing fluorescent dyes 32 to 37> Fluorescent dyes 32 to 37 shown in Table 1 were each produced in the same manner as in the production of fluorescent dye 31, except that compound C-1 and (2-carboxyethyl)phenylphosphinic acid used in the production of fluorescent dye 31 were replaced with compound C and an acidic compound shown in Table 10. Compound C and the acidic compound were used in the same molar amounts as compound C-1 and (2-carboxyethyl)phenylphosphinic acid in the production of fluorescent dye 31. The structures of the obtained fluorescent dyes 32 to 37 were identified by mass spectrometry, and were confirmed to have the structures shown in Table 1. Table 11 shows the results of mass spectrum analysis.

[0116] [Table 10]

[0117] [Example 38] <Method of manufacturing fluorescent dye 38> 1.0 part of PigmentRED255 (Tokyo Chemical Industry Co., Ltd.), which is a diketopyrrolopyrrole dye, 0.6 parts of 4-bromobutyric acid, and 0.1 parts of sodium hydride (60%) were dissolved in 50 parts of N,N-dimethylformamide, and the solution was reacted at 90°C for 4 hours. After the reaction solution was cooled to room temperature, 100 parts of water was added. Next, the precipitated solid was filtered and washed with 50 parts of water. The washed solid (crude product) was purified using a medium pressure preparative liquid chromatograph (Smart Flash AKROS, Yamazen Co., Ltd.). The obtained purified product was dried at 80°C to obtain 0.70 parts of fluorescent dye 38 shown in Table 1 (yield 53.9%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 375.28 (theoretical value 374.40), and it was identified to have the structure of fluorescent dye 38 shown in Table 1.

[0118] [Example 39] <Method of manufacturing fluorescent dye 39> 1.0 part of 5-carboxyfluorescein (Tokyo Chemical Industry Co., Ltd.), 0.3 parts of N,N-dimethyl-1,3-propanediamine, and 0.1 parts of paratoluenesulfonic acid were dissolved in 50 parts of xylene, and the solution was reacted at 140°C for 24 hours. After the reaction solution was cooled to room temperature, xylene was removed from the reaction solution using an evaporator, and 50 parts of petroleum ether were added. Next, insoluble matter was removed by suction filtration, and then petroleum ether was removed using an evaporator to obtain a solid. The solid was dried at 80°C to obtain 0.57 parts of the fluorescent dye 39 shown in Table 1 (yield 46.6%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 461.37 (theoretical value 460.49), and it was identified to have the structure of the fluorescent dye 39 shown in Table 1.

[0119] [Example 40] <Method of manufacturing fluorescent dye 40> 1.0 part of Rhodamin B (Tokyo Chemical Industry Co., Ltd.), 0.2 parts of N,N-dimethyl-1,3-propanediamine, and 0.1 parts of paratoluenesulfonic acid were dissolved in 50 parts of xylene, and the solution was reacted at 140°C for 24 hours. After the reaction solution was cooled to room temperature, xylene was removed from the reaction solution using an evaporator, and 50 parts of petroleum ether were added. Next, insoluble matter was removed by suction filtration, and then petroleum ether was removed using an evaporator to obtain a solid. This solid was dried at 80°C to obtain 0.46 parts of fluorescent dye 40 shown in Table 1 (yield 39.1%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 564.02 (theoretical value 563.18), and it was identified to have the structure of fluorescent dye 40 shown in Table 1.

[0120] [Example 41] <Method of manufacturing fluorescent dye 41> 1.0 part of BDP FL (Tokyo Chemical Industry Co., Ltd.), a BODIPY dye, and 0.3 parts of N,N-dimethyl-1,3-propanediamine were dissolved in 50 parts of xylene, and the solution was reacted at 140°C for 24 hours. After the reaction solution was cooled to room temperature, xylene was removed from the reaction solution using an evaporator, and 50 parts of petroleum ether were added. Next, insoluble matter was removed by suction filtration, and then petroleum ether was removed using an evaporator to obtain a solid. The solid was dried at 80°C to obtain 0.38 parts of fluorescent dye 41 shown in Table 1 (yield 29.3%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 379.11 (theoretical value 378.27), and it was identified to have the structure of fluorescent dye 41 shown in Table 1.

[0121] [Table 11]

[0122] [Manufacturing Example 20] <Production method of compound A-11> Ammonia gas was introduced into a solution of 50 parts of quinoline and 1 part of anhydrous aluminum chloride, and 5 parts of 3,6-bis(phenylthio)phthalonitrile were added, and the mixture was reacted at 180°C for 7 hours. After cooling the reaction solution to room temperature, 200 parts of methanol and 200 parts of 10% aqueous hydrochloric acid were added. The precipitated solid was then filtered and washed with 200 parts of water. The washed solid was dried at 80°C to obtain compound A-11 shown in Table 12 (yield 72.8%).

[0123] [Examples 21 and 22] <Method of producing compounds A-12 and A-13> Compounds A-12 and A-13 shown in Table 12 were produced in the same manner as in the production of compound A-11, except that the 3,6-bis(phenylthio)phthalonitrile used in the production of compound A-11 was changed to the phthalonitrile derivative shown in Table 12. The phthalonitrile derivative was used in the same molar amount as that of 3,6-bis(phenylthio)phthalonitrile in the production of compound A-11.

[0124] [Table 12]

[0125] [Example 42] <Method of manufacturing fluorescent dye 42> 0.7 parts of compound A-1 and 0.4 parts of 1,2-ethylenediphosphonic acid were dissolved in 50 parts of dimethyl sulfoxide, and 0.3 parts of 1,8-diazabicyclo[5.4.0]-7-undecene were added, and the solution was reacted at 90°C for 5 hours. After cooling the reaction solution to room temperature, 100 parts of water and 10 parts of salt were added. The precipitated solid was then filtered and washed with 50 parts of water. The washed solid was dried at 80°C to obtain 0.42 parts of fluorescent dye 1 shown in Table 1 (yield 50.6%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 905.35 (theoretical value 905.21), and it was identified to have the structure of fluorescent dye 1 shown in Table 1.

[0126] [Examples 43-55] <Method of manufacturing fluorescent dye 43-55> Fluorescent dyes 2 to 14 shown in Table 1 were each produced in the same manner as in the production of fluorescent dye 6, except that compound A-1 and 1,2-ethylenediphosphonic acid used in the production of fluorescent dye 1 were replaced with compound A and an axial ligand shown in Table 13. Compound A and the axial ligand were used in the same molar amounts as compound A-1 and 1,2-ethylenediphosphonic acid in the production of fluorescent dye 1. The structures of the obtained fluorescent dyes 2 to 14 were identified by analysis using a mass spectrometer, and were confirmed to have the structures shown in Table 1. Table 3 shows the results of mass spectrum analysis.

[0127] [Table 13-1]

[0128] [Table 13-2]

[0129] [Table 13-3]

[0130] [Production Example 23] <Production of Compound D-1> 200 parts of sulfolane and 15.7 parts of 1,8-diazabicyclo[5,4,0]-7-undecene (DBU), 5 parts of 4-butylthio-1,3-diiminoisoindoline, and 8.8 parts of silicon tetrachloride were added, and the mixture was heated and stirred at 160 to 170 ° C for 8 hours. Then, the reaction solution was cooled to room temperature (25 ° C), and 200 parts of methanol were added. Then, the precipitate (solid) was filtered off, and the solid was washed with a mixed solution of methanol:water (mass ratio 4:1), and then dried to obtain 2.6 parts of compound D-1 shown in Table 14 (yield 63.6%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 751.65 (theoretical value 751.24), and it was confirmed that the compound had the structure of compound D-1 shown in Table 14.

[0131] [Table 14]

[0132] [Example 56] <Method of manufacturing fluorescent dye 56> 1.0 part of compound B-5 and 0.5 parts of 1,2-ethylenediphosphonic acid were dissolved in 50 parts of dimethyl sulfoxide, and 0.3 parts of 1,8-diazabicyclo[5.4.0]-7-undecene were added, and the mixture was reacted at 90°C for 5 hours. After cooling the reaction solution to room temperature, 100 parts of water and 10 parts of salt were added. The precipitated solid was then filtered and washed with 50 parts of water. The washed solid was dried at 80°C to obtain 0.52 parts of fluorescent dye 1 shown in Table 1 (yield 42.3%). As a result of mass spectrometry, a molecular ion peak was detected at m / z=923.47 (theoretical value 923.21), and the structure of fluorescent dye 56 shown in Table 1 was identified.

[0133] [Example 57] <Method of manufacturing fluorescent dye 57> Fluorescent dye 57 shown in Table 1 was produced in the same manner as in the production of fluorescent dye 56, except that compound B-5 used in the production method of fluorescent dye 1 was changed to compound D-1. Compound D-1 was used in the same molar amount as compound B-5 in the production of fluorescent dye 56. The structure of the obtained fluorescent dye 57 was identified by analysis using a mass spectrometer, and it was confirmed that it had the structure shown in Table 1. Table 15 shows the results of the mass spectrum analysis.

[0134] [Table 15]

[0135] [Example 58] <Method of manufacturing fluorescent dye 58> 0.7 parts of compound C-3 and 0.7 parts of 1,2-hexylenediphosphonic acid were dissolved in 50 parts of dimethyl sulfoxide, and 0.3 parts of 1,8-diazabicyclo[5.4.0]-7-undecene were added to the solution, and the mixture was reacted at 90°C for 5 hours. After the reaction solution was cooled to room temperature, 100 parts of water and 10 parts of salt were added. The precipitated solid was then filtered and washed with 50 parts of water. The washed solid was dried at 80°C to obtain 0.39 parts of fluorescent dye 1 shown in Table 1 (yield 42.3%). As a result of mass spectrometry, a molecular ion peak was detected at m / z=913.66 (theoretical value 913.24), and the structure of fluorescent dye 58 shown in Table 1 was identified.

[0136] [Example 59] <Method of manufacturing fluorescent dye 59> Fluorescent dye 59 shown in Table 1 was produced in the same manner as in the production of fluorescent dye 58, except that compound C-3 and 1,2-hexylenediphosphonic acid used in the production of fluorescent dye 58 were replaced with compound C-4 and ring substituents shown in Table 16. Compound C-4 was used in the same molar amount as compound C-3 in the production of fluorescent dye 17. The structure of the obtained fluorescent dye 59 was identified by analysis using a mass spectrometer, and it was confirmed that it had the structure shown in Table 1. Table 16 shows the results of the mass spectrum analysis.

[0137] [Table 16]

[0138] [Example 60] <Method of manufacturing fluorescent dye 60> 1.0 part of PigmentRED255 (Tokyo Chemical Industry Co., Ltd.), which is a diketopyrrolopyrrole dye, 0.6 parts of 3-aminopropylphosphonic acid, and 0.1 parts of sodium hydride (60% dispersion) were dissolved in 50 parts of N,N-dimethylformamide, and the solution was reacted at 90°C for 4 hours. After the reaction solution was cooled to room temperature, 100 parts of water were added. Next, the precipitated solid was filtered and washed with 50 parts of water. The washed solid (crude product) was purified using a medium pressure preparative liquid chromatograph (Yamazen Smart Flash AKROS). The obtained purified product was dried at 80°C to obtain 0.65 parts of fluorescent dye 60 shown in Table 1 (yield 45.5%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 411.52 (theoretical value 411.10), and it was identified to have the structure of fluorescent dye 60 shown in Table 1.

[0139] [Example 61] <Method of manufacturing fluorescent dye 61> 1.0 part of 5-carboxyfluorescein (Tokyo Chemical Industry Co., Ltd.), 0.7 parts of 3-aminopropylphosphonic acid, and 0.1 parts of paratoluenesulfonic acid were dissolved in 50 parts of xylene, and the solution was reacted at 140°C for 24 hours. After the reaction solution was cooled to room temperature, xylene was removed from the reaction solution using an evaporator, and 50 parts of petroleum ether were added. Next, insoluble matter was removed by suction filtration, and then petroleum ether was removed using an evaporator to obtain a solid. This solid was dried at 80°C to obtain 0.75 parts of the fluorescent dye 61 shown in Table 1 (yield 51.2%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 551.02 (theoretical value 551.18), and it was identified to have the structure of the fluorescent dye 61 shown in Table 1.

[0140] [Example 62] <Method of manufacturing fluorescent dye 62> 1.0 part of Cy5-NHS ester (Funakoshi), 0.5 parts of 3-aminopropanol, and 0.5 parts of triethylamine were dissolved in 50 parts of DMF, and the solution was reacted at room temperature for 12 hours. 50 parts of water were added to the reaction solution, and the precipitate (solid) was filtered and washed with water. The washed solid was dried at 80°C to obtain 0.70 parts of fluorescent dye 62 shown in Table 1 (yield 86.4%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 541.18 (theoretical value 541.36), and it was identified to have the structure of fluorescent dye 62 shown in Table 1.

[0141] [Example 63] <Method of manufacturing fluorescent dye 63> 1.0 part of BDP FL (Tokyo Chemical Industry Co., Ltd.), a BODIPY dye, and 0.3 parts of 3-aminopropanol were dissolved in 50 parts of xylene, and the solution was reacted at 140°C for 24 hours. After the reaction solution was cooled to room temperature, xylene was removed from the reaction solution using an evaporator, and 50 parts of petroleum ether were added. Next, the precipitated insoluble matter was removed by suction filtration, and then petroleum ether was removed from the reaction solution using an evaporator to obtain a solid. The solid was dried at 80°C to obtain 0.64 parts of the fluorescent dye 63 shown in Table 1 (yield 70.6%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 350.01 (theoretical value 350.18), and it was identified to have the structure of the fluorescent dye 63 shown in Table 1.

[0142] [Comparative Example 1] As comparative compound 1, compound A-1 was used.

[0143] [Comparative Example 2] As comparative compound 2, compound A-9 was used.

[0144] In Comparative Examples 3 to 11 described later, comparative compounds 3 to 11 shown in Table 17 were produced.

[0145] [Table 17]

[0146] [Comparative Example 3] <Production method of comparative compound 3> 7.0 parts of aluminum chloride, 39 parts of urea, 0.2 parts of ammonium molybdate, and 25 parts of trimellitic anhydride were dissolved in 40 parts of N-methyl-2-pyrrolidone (NMP), and the solution was stirred at 139°C for 9 hours. After cooling the reaction solution to room temperature, 100 parts of water were added. Then, the precipitated solid was filtered and washed with 50 parts of water. The washed solid was dried at 80°C to obtain 14.5 parts of comparative compound 3 shown in Table 17 (yield 59.3%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 751.84 (theoretical value 751.00), and it was identified to have the structure of comparative compound 3 shown in Table 17.

[0147] [Comparative Example 4] <Production method of comparative compound 4> Ammonia gas was introduced into a solution of 30 parts of quinoline and 0.7 parts of anhydrous aluminum chloride, and 1.5 parts of 3-ethoxyphthalonitrile and 2.1 parts of 4-octadecyloxyphthalonitrile were added, and the solution was reacted at 180 ° C for 7 hours. After cooling the reaction solution to room temperature, 200 parts of methanol and 200 parts of 10% aqueous hydrochloric acid were added. Next, the precipitated solid was filtered and washed with 200 parts of water. The washed solid (crude product) was purified using a medium pressure preparative liquid chromatograph (Yamazen Smart Flash AKROS). The obtained purified product was dried at 80 ° C to obtain 0.36 parts of comparative compound 4 shown in Table 17 (yield 12.6%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 976.44 (theoretical value 975.61), and it was identified to have the structure of comparative compound 4 shown in Table 17.

[0148] [Comparative Example 5] <Production method of comparative compound 5> 1.0 part of compound A-9 and 0.35 parts of triphenylsilanol were dissolved in 20 parts of dimethyl sulfoxide, and the solution was reacted at 80°C for 8 hours. After cooling the reaction solution to room temperature, 50 parts of water and 10 parts of salt were added. The precipitated solid was then filtered and washed with 50 parts of water. The washed solid was dried at 80°C to obtain 1.00 parts of comparative compound 5 shown in Table 17 (yield 80.5%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 1282.53 (theoretical value 1271.67), and it was identified to have the structure of comparative compound 5 shown in Table 17.

[0149] [Comparative Examples 6 to 9] <Production methods of comparative compounds 6 to 9> Comparative compounds 6 to 9 shown in Table 17 were produced in the same manner as in the production of comparative compound 5, except that compound A-9 and triphenylsilanol used in the production of comparative compound 5 were replaced with halogens and acidic compounds shown in Table 18. The halogens and acidic compounds were used in the same molar amounts as compound A-9 and triphenylsilanol in the production of comparative compound 5. The structures of the obtained comparative compounds 6 to 9 were identified by mass spectrometry and confirmed to have the structures shown in Table 17. Table 19 shows the results of mass spectrum analysis.

[0150] [Table 18]

[0151] [Comparative Example 10] <Production method of comparative compound 10> 2.0 parts of compound A-9 and 1.0 parts of paratoluenesulfonic acid were dissolved in 50 parts of dimethylsulfoxide, and 0.3 parts of 1,8-diazabicyclo[5.4.0]-7-undecene were added to the solution, and the mixture was reacted at 90°C for 5 hours. After cooling the reaction solution to room temperature, 100 parts of water were added. The precipitated solid was then filtered and washed with 50 parts of water. The washed solid was dried at 80°C to obtain 1.35 parts of comparative compound 10 shown in Table 17 (yield 60.0%). As a result of mass spectrometry, a molecular ion peak was detected at m / z=933.67 (theoretical value 932.74), and the structure of comparative compound 10 shown in Table 17 was identified.

[0152] [Comparative Example 11] <Production Method of Comparative Compound 11> One part of compound A-1 was added to a mixed solution of 9.2 parts of concentrated sulfuric acid and 5.5 parts of 25% fuming sulfuric acid, and the solution was heated and stirred at 50 ° C. for 4 hours. After cooling the reaction solution, it was added to 80 parts of ice, and the precipitate (solid) was filtered off. The filtered solid was suspended in 50 parts of tetrahydrofuran, and the precipitate was filtered off again. The filtered solid was washed with 50 parts of tetrahydrofuran, and the washed solid was dried to obtain 0.5 parts of a crude product. The crude product was purified using a medium pressure preparative liquid chromatograph (Yamazen Smart Flash AKROS) to obtain 0.2 parts of comparative compound 11 (yield 16.0%). As a result of mass spectrometry, a molecular ion peak was detected at m / z = 939.65 (theoretical value 940.75), and it was identified to have the structure of comparative compound 11 shown in Table 17.

[0153] The results of mass spectrum analysis of Comparative Compounds 3 to 11 prepared in Comparative Examples 3 to 11 are shown below. [Table 19]

[0154] [Comparative Example 12] XenoLight DIR (Sumitomo Pharma), a cyanine dye, was used as comparative compound 12. This compound corresponds to a conventional fluorescent labeling agent having a long-chain alkylene group, and accumulates in phospholipids via hydrophobic interactions.

[0155] [ka]

[0156] [Comparative Example 13] As comparative compound 13, Rhodamine B (Tokyo Chemical Industry Co., Ltd.) was used.

[0157] <ii>Dye solution [Example 64] <Preparation of dye solution 1> In 10 ml of dimethyl sulfoxide, 1.696 mg of fluorescent dye 1 was dissolved. This solution was filtered using a nylon membrane filter with a pore size of 0.2 μm, and then diluted 100 times with dimethyl sulfoxide to prepare dye solution 1 of fluorescent dye 1.

[0158] [Examples 65 to 126] <Preparation of dye solutions 2 to 63> Dye solutions 2 to 63 were each prepared in the same manner as dye solution 1, except that the fluorescent dye 1 and dimethyl sulfoxide used in the preparation of dye solution 1 were changed to the fluorescent dyes and solvents shown in Table 20. However, each fluorescent dye was used in the same molar amount as fluorescent dye 1, and the solvent was used in the same volume amount as dimethyl sulfoxide.

[0159] [Comparative Examples 12 to 24] <Preparation of dye solutions 64 to 76> Dye solutions 64 to 76 were each prepared in the same manner as dye solution 1, except that the fluorescent dye 1 and dimethyl sulfoxide used in the preparation of dye solution 1 were changed to the fluorescent dyes and solvents shown in Table 20. However, each fluorescent dye was used in the same molar amount as fluorescent dye 1, and the solvent was used in the same volume amount as dimethyl sulfoxide.

[0160] [Table 20-1]

[0161] [Table 20-2]

[0162] [Table 20-3]

[0163] <Evaluation of the fluorescence intensity of dye solutions> The fluorescence spectrum of each dye solution was measured using a fluorometer (JASCO Corporation, FP-6500). Furthermore, the fluorescence intensity was calculated by adding the fluorescence intensity within the range of the fluorescence wavelength shown in Table 22 to the measured value. In addition, the excitation light used at this time had a wavelength equivalent to the absorption maximum wavelength on the longest wavelength side of the dye.

[0164] <iii>Fluorescent Labeling Agents [Example 127] <Preparation of fluorescent labeling agent 1> 1.696 mg of fluorescent dye 1 was dissolved in 10 ml of dimethyl sulfoxide. After filtering through a nylon membrane filter with a pore size of 0.2 μm, the solution was diluted 100-fold with RPMI1640 medium to prepare fluorescent labeling agent 1 of fluorescent dye 1.

[0165] [Examples 128 to 189] <Preparation of fluorescent labeling agents 2 to 63> Fluorescent labeling agents 2 to 63 were each prepared in the same manner as for preparation of fluorescent labeling agent 1, except that the fluorescent dye 1 and dimethyl sulfoxide used in the preparation of fluorescent labeling agent 1 were changed to the fluorescent dyes and solvents shown in Table 21. However, each fluorescent dye was used in the same molar amount as fluorescent dye 1, and the solvent was used in the same volume amount as dimethyl sulfoxide.

[0166] [Comparative Examples 25 to 37] <Preparation of fluorescent labeling agents 64 to 76> Fluorescent labels 64 to 76 were each prepared in the same manner as for preparing fluorescent labeling agent 1, except that the fluorescent dye 1 and dimethyl sulfoxide used in the preparation of fluorescent labeling agent 1 were changed to the fluorescent dyes and solvents shown in Table 21. However, each fluorescent dye was used in the same molar amount as fluorescent dye 1, and the solvent was used in the same volume amount as dimethyl sulfoxide.

[0167] [Table 21-1]

[0168] [Table 21-2]

[0169] [Table 21-3]

[0170] <Cytotoxicity evaluation of fluorescent labeling agents> Human epidermoid carcinoma cells A431 were seeded in a 96-well plate (1 × 10 4 Then, the A431 cells were cultured for 24 hours in an incubator (37°C, 5% CO2-containing air, humidified environment) using RPMI1640 medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. Sigma-Aldrich RPM1-164- Medium was used as the RPMI1640 medium. After the above-mentioned culture, the medium was removed, and the fluorescent labeling agent prepared in Examples 83 to 123 and Comparative Examples 25 to 37 and RPMI1640 medium containing 1% dimethyl sulfoxide (DMSO medium solution) were added. These were left to stand in an incubator for 1 hour, and then washed with RPMI1640 medium. 10 μL of Cell Counting Kit-8 (Dojindo Chemical Industries, Ltd.) was added to each well, and the wells were left to stand in an incubator (37°C, 5% CO2-containing air, humidified environment) for 1 hour. Then, the absorbance at 450 nm was measured using a plate reader (TECAN, SPARK). The absorbance of each fluorescent labeling agent was calculated relative to the absorbance of the well to which DMSO medium solution was added, assuming it was 1, and evaluated based on the following criteria. If the evaluation was "P", it could be said that it did not show cytotoxicity. When calculating the relative absorbance of the fluorescent labeling agent, the value obtained by subtracting the absorbance before adding Cell Counting Kit-8 (Dojindo Chemical Industries) from the measured absorbance was used. The evaluation results are shown in Table 22. (Evaluation Criteria) P (pass): 0.8 or higher F (fail): less than 0.8

[0171] <Evaluation of fluorescence intensity of fluorescent labeling agents> Human epidermoid carcinoma cells A431 were seeded in a 96-well plate (1 × 10 4 Next, the A431 cells were cultured for 24 hours in an incubator (37°C, 5% CO2-containing air, humidified environment) using RPMI1640 medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. After the above-mentioned culture, the medium was removed, and the fluorescent labeling agent prepared in Examples 127 to 167 and Comparative Examples 25 to 37 was added, and the cells were left to stand in an incubator for 1 hour. Then, the cells were washed with RPMI1640 medium. Fluorescence intensity was evaluated within the range of fluorescence wavelength shown in Table 22 using a plate reader (TECAN, SPARK).

[0172] 1 shows the results of evaluating the fluorescence intensity of fluorescent labeling agents 1, 15, 19, 24, 25, 68, and 75. It was confirmed that fluorescent labeling agents 1, 15, 19, 24, and 25 (Examples), which are embodiments of the present invention, exhibit higher fluorescence intensity than fluorescent labeling agents 68 and 75 (Comparative Examples), which were prepared using comparative compounds.

[0173] <Evaluation of phospholipid accumulation of fluorescent dyes> The phospholipid accumulation of each dye was calculated using formula (1) from the fluorescence intensity integral value determined from the fluorescence spectrum of the dye solution and the fluorescence intensity obtained from the fluorescence intensity of the fluorescent labeling agent shown in Table 22. The relative value of the phospholipid accumulation of each fluorescent labeling agent was calculated when the phospholipid accumulation of comparative compound 12 was set to 1, and evaluated based on the following criteria. When the evaluation score was 3 or higher, it can be said that each fluorescent dye has good phospholipid accumulation. (Evaluation Criteria) 4: Phospholipid accumulation level 4 or higher 3: Phospholipid accumulation: 2 or more, less than 4 2: Phospholipid accumulation: 1 or more, less than 2 1: Phospholipid accumulation less than 1

[0174]

number

[0175] The evaluation results of phospholipid accumulation are shown in Table 22. It was confirmed that the fluorescent labeling agent according to the embodiment of the present invention (Example) exhibited higher phospholipid accumulation than the fluorescent labeling agent prepared using the comparative compound (Comparative Example).

[0176] <Evaluation of cell visibility> Human epidermoid carcinoma cells A431 were seeded in a 96-well plate (1 × 10 4 The A431 cells were cultured for 24 hours in an incubator (37°C, 5% CO2-containing air, humidified environment) using RPMI1640 medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. After the above-mentioned culture, the medium was removed, and the fluorescent labeling agent prepared in Examples 127 to 189 and Comparative Examples 25 to 37 was added, and the cells were allowed to stand in an incubator for 1 hour. Then, the cells were washed with RPMI1640 medium. Dark field images and fluorescent images of the cells were observed using a fluorescent microscope (Keyence Corporation, BZ-X800) equipped with excitation filters and fluorescent filters of appropriate wavelengths, and evaluated based on the following criteria. The evaluation results are shown in Table 22. (Evaluation Criteria) P (Pass): Clear F (Fail): Unclear

[0177] [Table 22-1]

[0178] [Table 22-2]

[0179] [Table 22-3]

[0180] The visibility evaluation results of cells labeled with fluorescent labeling agents 1, 15, 19, 24, 25, 68, and 75 are shown in Figures 2 to 8, respectively (magnification: 10x, fluorescence capture time: 1 second).

[0181] The results of evaluating the visibility of the cells labeled with the fluorescent labeling agents 42 and 53 are shown in FIG. 9 and FIG. 10, respectively (magnification: 40 times, fluorescence capture time: 1 second).

[0182] As is clear from a comparison of Figs. 7 and 8 corresponding to the comparative examples with Figs. 2 to 6, 9 and 10 corresponding to the examples, the fluorescent labeling agent according to the embodiment of the present invention was observed to exhibit higher fluorescence intensity due to the specific substituent. As such, it can be seen that the fluorescent labeling agent according to the embodiment of the present invention (Example) has superior cell accumulation compared to the comparative compound, thereby providing superior visibility. From the above, it has become clear that the fluorescent labeling agent according to the embodiment of the present invention has excellent properties as a fluorescent labeling agent.< / iii> < / ii>

Claims

1. A phospholipid-accumulating fluorescent labeling agent containing a water-insoluble fluorescent dye represented by the following general formula (1). General formula (1): Q-Z-R １ -R ２ -R ３ In the formula, Q represents a residue of the fluorescent dye, and -Z-R １ -R ２ -R ３ The symbol represents a group that binds to the residue of the fluorescent dye, and the number of -Z-R1 -R2 -R3 groups that bind to the residue of the fluorescent dye is 1. The residue Q of the fluorescent dye is a residue of a phthalocyanine dye, a diketopyrrolopyrrole dye, a xanthene dye, a borondipyrromethene dye, or a cyanine dye, represented by the following general formula (2). Z represents a directly bonded, substituted, or unsubstituted alkylene group, or a substituted or unsubstituted arylene group. R １ represents direct bond, -O-, -OP(=O)R ４ -, -OC(=O)-, -OS(=O) ２ -, -OSiR ５ R ６ -, -C(=O)-, or -C(=O)NH-. R ２ This represents one group selected from the group consisting of unsubstituted alkylene groups, unsubstituted arylene groups, and substituted or unsubstituted heterocyclic groups, or a group formed by a combination of these groups. R ３ is, -COOM １ , -NR ７ R ８ , -N ＋ R ９ R １０ R １１ I -, -OM ２ , or -P(=O)(OM ３ ) OM ４ It represents. The aforementioned R ４ This represents a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, or a substituted or unsubstituted heterocyclic group. The aforementioned R ５ and R ６ Each of these independently represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. The aforementioned R ７ R8 independently represents a hydrogen atom, a substituted or unsubstituted C1-C5 alkyl group, or a substituted or unsubstituted aryl group. R9 to R11 each independently represent a hydrogen atom, an unsubstituted alkyl group, and an unsubstituted aryl group. Said M １ M ２ M ３ , and M ４ Each of these independently represents a hydrogen atom or a monovalent cation. 【Chemistry 1】 In the formula, X １ ~X １６ These are, independently, -Z-R in the general formula (1) above. １ -R ２ -R ３ A group represented by , a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted heterocyclic group, -AB, or -COOM ６ It represents. In the above-AB, A represents a Group 16 element. B represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclic group. The aforementioned M6 represents a monovalent cation. X １ ~X １６ Adjacent substituents may be linked to each other to form a cycloalkane or cycloalkene ring. X １７ This is -Z-R in the general formula (1) above. １ -R ２ -R ３ The group represented by , hydroxyl group, halogen element, substituted or unsubstituted alkoxy group, substituted or unsubstituted aryloxy group, -OP(=O)X １８ X １９ , -OC(=O)X ２０ , -OS (=O) ２ X ２１ , or -OSix ２２ X ２３ X ２４ It represents. The aforementioned X １８ and X １９ Each of these independently represents a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, or a substituted or unsubstituted heterocyclic group. The aforementioned X ２０ This represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. The aforementioned X ２１ This represents a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. The aforementioned X ２２ ~X ２４ Each of these independently represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. Y represents a metal atom with a valency ranging from divalent to pentavalent, and k is an integer. When Y is a divalent metal atom, k is 0; when Y is a trivalent metal atom, k is 1; and when Y is a tetravalent or pentavalent metal atom, k is 2. However, X １ ~X １７ At least one of them is -Z-R in the general formula (1) １ -R ２ -R ３ (This is the base represented by [this symbol].)

2. In the group represented by -Z-R1 -R2 -R3 in the general formula (1), The phospholipid-accumulating fluorescent labeling agent according to claim 1, wherein R1 represents -O-, -OP(=O)R4-, -OC(=O)-, -OS(=O)2-, -OSiR5R6-, -C(=O)-, or -C(=O)NH-.

3. In the group represented by -Z-R1 -R2 -R3 in the general formula (1), Z represents a direct bond, R1 represents -OP(=O)R4-, -OS(=O)2-, or -OSiR5R6-, R2 represents an unsubstituted alkylene group or an unsubstituted arylene group. R3 represents -COOM1, -NR7R8, -OM2, or -P(=O)(OM3)OM4, The aforementioned R4 represents a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group. R5 and R6 each independently represent a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group. R7 and R8 each independently represent a hydrogen atom and an unsubstituted alkyl group having 1 to 5 carbon atoms. Each of the R9 to R11 elements independently represents either a hydrogen atom or an unsubstituted alkyl group. The fluorescent labeling agent according to claim 1, wherein M1, M2, M3, and M4 each independently represent a hydrogen atom.

4. X in the general formula (2) above １７ However, in the general formula (1) above, -Z-R １ -R ２ -R ３ A fluorescent labeling agent according to any one of claims 1 to 3, wherein the group is represented by .

5. A compound represented by the following general formula (3). 【Chemistry 2】 (In the formula, X １ ~X １６ These are, independently, -Z-R １ -R ２ -R ３ A group represented by , a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted heterocyclic group, -AB, or -COOM ６ It represents. In the above-AB, A represents a Group 16 element. B represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heterocyclic group. The aforementioned M6 represents a monovalent cation. The aforementioned X １ ~X １６ Adjacent substituents may be linked to each other to form a cycloalkane or cycloalkene ring. X １７ is, -Z-R １ -R ２ -R ３ The group represented by , hydroxyl group, halogen element, substituted or unsubstituted alkoxy group, substituted or unsubstituted aryloxy group, -OP(=O)X １８ X １９ , -OC(=O)X ２０ , -OS (=O) ２ X ２１ , or -OSix ２２ X ２３ X ２４ It represents. The aforementioned X １８ and X １９ Each of these independently represents a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, or a substituted or unsubstituted heterocyclic group. The aforementioned X ２０ This represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. Said X ２１ represents a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. The aforementioned X ２２ ~X ２４ Each of these independently represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. Y represents a metal atom with a valency ranging from divalent to pentavalent, and k is an integer. When Y is a divalent metal atom, k is 0; when Y is a trivalent metal atom, k is 1; and when Y is a tetravalent or pentavalent metal atom, k is 2. However, X １ ~X １７ at least one of which is a group represented by -Z-R １ -R ２ -R ３ is a group represented by In the formula, Z represents a directly bonded, substituted, or unsubstituted alkylene group, or a substituted or unsubstituted arylene group. R １ These are direct bonds, -O-, -OP(=O)R ４ -, -OC(=O)-, -OS(=O) ２ -, -OSir ５ R ６ It represents -, -C(=O)-, or -C(=O)NH-. R ２ This represents one group selected from the group consisting of unsubstituted alkylene groups, unsubstituted arylene groups, and substituted or unsubstituted heterocyclic groups, or a group formed by a combination of these groups. R ３ is, -COOM １ , -NR ７ R ８ , -N ＋ R ９ R １０ R １１ I -, -OM ２ , or -P(=O)(OM ３ ) OM ４ It represents. The aforementioned R ４ This represents a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, or a substituted or unsubstituted heterocyclic group. The aforementioned R ５ and R ６ Each of these independently represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. The aforementioned R ７ R8 independently represents a hydrogen atom, a substituted or unsubstituted C1-C5 alkyl group, or a substituted or unsubstituted aryl group. R9 to R11 each independently represent a hydrogen atom, an unsubstituted alkyl group, and an unsubstituted aryl group. Said M １ M ２ M ３ , and M ４ Each of these independently represents either a hydrogen atom or a monovalent cation.

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