Compound, fluorescent dye agent, kit, and cell detection method

Abstract

The purpose of the present invention is to provide a compound that can be suitably used in a fluorochrome or similar, a novel fluorochrome and kit for conveniently detecting cells, and a novel method for conveniently detecting cells or similar.　A compound represented by formula (1) or a salt thereof. (In formula (1), R1 is a substituted or unsubstituted C1-12 alkyl group or similar, R2 and R3 are each independently a substituted or unsubstituted C1-6 alkyl group or similar, R4 and R5 are each independently a C1-12 alkyl group or similar, n is an integer from 1 to 4, and a and b are each independently an integer from 0 to 4.)

Description

[Technical Field]

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

[0002] Various staining methods are used to detect cells and tissues, such as tumor cells. For example, hematoxylin-eosin (HE) staining is the most widely used staining method in pathological diagnosis and is considered the gold standard for many diagnostic methods. However, HE staining stains normal tissue as well as tumor cells, resulting in poor optical transparency, necessitating thin sectioning of tissue specimens. Because thin sections only provide two-dimensional information, determining the distribution of tumor cells requires the preparation and observation of numerous sections. Furthermore, preparing tissue specimens requires advanced skills, is tedious, and time-consuming.

[0003] Furthermore, HE staining can sometimes result in low contrast and unclear boundaries between tumor cells and normal tissue.

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

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

[0006] [Patent Document 1] Japanese Patent Application Publication No. 2018-201678 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a compound that can be suitably used as a fluorescent dye agent or the like.

[0008] Another object of the present invention is to provide a novel fluorescent dye agent and kit for easily detecting cells and the like.

[0009] Another object of the present invention is to provide a new method for easily detecting cells and the like. [Means for solving the problem]

[0010] The present inventors have discovered that cells and the like can be easily detected by applying a fluorescent dye agent containing a compound exhibiting solvatochromism to staining cells or tissues, and have completed the present invention.

[0011] That is, the present invention provides the following compounds:

[0012] [1] A compound represented by the following formula (1) or a salt thereof: [ka] (In formula (1), R 1 is a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and halogen. R 2 and R 3are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and halogen; R 2 and R 3 may together form a ring structure. R 4 and R 5 are each independently an alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, an aryl group having 5 to 12 carbon atoms, a heteroaryl group having 5 to 12 carbon atoms, a halogen atom, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and a halogen atom. n is an integer of 1 to 4. a and b are each independently an integer of 0 to 4.

[0013] [2] The compound or salt thereof according to [1], wherein n is 1 or 2.

[0014] [3] R 1 is a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, an i-butyl group, a sec-butyl group, or a t-butyl group, or a salt thereof.

[0015] [4] R 2 and R 3 are each independently Shi base The compound or salt thereof according to any one of [1] to [3], wherein the aryl group is a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, an i-butyl group, a sec-butyl group, or a t-butyl group, which is unsubstituted or substituted with a hydroxyl group, a sulfo group, an amido group, a halogen atom, or a group which is a salt thereof.

[0016] [5] R 2 and R3 is Carboxy Shi base The compound or salt thereof according to any one of [1] to [3], wherein the compound is a divalent cyclic hydrocarbon group having 3 to 12 carbon atoms, which is substituted or unsubstituted with a hydroxyl group, a sulfo group, an amide group, a halogen atom, or a group which is a salt thereof, and which is formed by combining these groups together with the carbon atoms to which they are bonded.

[0017] [6] R 4 and R 5 and each independently represent a methyl group, an ethyl group, a hydroxyl group, or an amino group, or a salt thereof.

[0018] [7] The compound or salt thereof according to any one of [1] to [6], which has an absorption maximum wavelength of 300 to 550 nm in 20 mM phosphate buffer (pH 7.4) at 25°C.

[0019] [8] The compound or salt thereof according to any one of [1] to [7], which has a molecular weight of 700 or less.

[0020] The present invention also provides the following fluorescent dye agents:

[0021] [9] A fluorescent dye agent comprising the compound according to any one of [1] to [8] or a salt thereof.

[0022]

[10] The fluorescent dye agent according to [9], which is used for detecting cell morphology.

[0023]

[11] The fluorescent dye agent according to [9], which is used for staining or visualizing biological samples.

[0024]

[12] The fluorescent dye agent according to any one of [9] to

[11] , which is used for fluorescent imaging.

[0025] The present invention also provides the following kit:

[0026]

[13] A kit comprising the fluorescent dye agent according to any one of [9] to

[12] .

[0027] The present invention also provides the following cell detection methods.

[0028]

[14] A method for detecting cells, comprising the step (1) of staining cells with a fluorescent dye agent containing the compound according to any one of [1] to [8] or a salt thereof.

[0029]

[15] The cell detection method according to

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

[0030]

[16] The cell detection method according to

[15] , wherein the step (2) is carried out using fluorescent imaging. [Effects of the Invention]

[0031] The compound of the present invention can be suitably used as a fluorescent dye agent or the like.

[0032] Furthermore, by using the fluorescent dye agent and kit of the present invention, cells and the like can be stained simply and quickly.

[0033] Furthermore, by using the cell detection method of the present invention, cells and the like can be detected easily. [Brief explanation of the drawings]

[0034] [Figure 1] 1 is a chart of 1H NMR spectrum measurement of Compound A in an example. [Figure 2] 1 is a chart of 13C NMR spectrum measurement of Compound A in an example. [Figure 3]1 shows charts of (left) absorption spectrum measurement and (right) fluorescence spectrum measurement (normalized) of compound A in an organic solvent in an example. [Figure 4] FIG. 1 is a diagram comparing the photostability of Compound A and Laurdan in an example. [Figure 5] 1 is a chart showing the fluorescence spectrum measurement of Compound A in a liposome composed of 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) and sphingomyelin (SM) / cholesterol (Chol) in an example. [Figure 6] 1 shows confocal fluorescence imaging images of human prostate cancer cells (PC3) stained with Compound A in an example. [Figure 7] 1 is a graph showing the toxicity evaluation (MTT test) of Compound A on human keratinocytes K16 (P4). [Figure 8] This is a two-photon fluorescence imaging image of compound A-stained human skin tissue (frozen tissue). [Figure 9] 1 is a chart of 1H NMR spectrum measurement of Compound B in an example. [Figure 10] 1 shows charts of (left) absorption spectrum measurement and (right) fluorescence spectrum measurement (normalized) of compound B in an organic solvent in an example. [Figure 11] 1 is a chart of 1H NMR spectrum measurement of Compound C in an example. [Figure 12] 1 shows charts of (left) absorption spectrum measurement and (right) fluorescence spectrum measurement (normalized) of compound C in an organic solvent in an example. DETAILED DESCRIPTION OF THE INVENTION

[0035] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to these embodiments.

[0036] [Compound] The compound of the present invention is a compound represented by the following formula (1) (hereinafter also referred to as "compound (1)") or a salt thereof. [ka] (In formula (1), R 1 is a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and halogen. R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and halogen; R 2 and R 3 may together form a ring structure. R 4 and R 5 are each independently an alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, an aryl group having 5 to 12 carbon atoms, a heteroaryl group having 5 to 12 carbon atoms, a halogen atom, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and a halogen atom. n is an integer of 1 to 4. a and b are each independently an integer of 0 to 4.

[0037] The compound (1) may be a compound exhibiting solvatochromism, and may be suitably used, for example, as a fluorescent dye agent, etc. In this specification, a compound exhibiting solvatochromism refers to a compound whose maximum absorption wavelength, maximum fluorescence wavelength, or both change depending on the polarity (hydrophobicity) of the surroundings of the compound.

[0038] The above compounds also include derivatives such as substitution products in which the structure is partially or completely substituted, and solvates such as hydrates, as appropriate.

[0039] In the above formula (1), R 1 is a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and halogen.

[0040] Above R 1 Examples of the alkyl group having 1 to 12 carbon atoms represented by the formula (I) include linear or branched alkyl groups, such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decanyl group, an n-undecanyl group, and an n-dodecanyl group. 1 Suitable examples of the alkyl group having 1 to 12 carbon atoms represented by the formula below include those having 2 to 10 carbon atoms, 3 to 9 carbon atoms, etc.

[0041] Above R 1 Examples of the alkenyl group having 2 to 12 carbon atoms represented by the formula (I) include linear or branched alkenyl groups, such as vinyl and allyl groups.

[0042] Above R 1 Examples of the alkynyl group having 2 to 12 carbon atoms represented by the formula (I) include linear or branched alkenyl groups, such as an ethynyl group and a propargyl group.

[0043] Also, the above R 1 Examples of the substituents of the alkyl group represented by the formula (I) include carboxyl groups. Shi base, a hydroxyl group, a sulfo group, an amide group, a halogen atom, or a group which is a salt thereof. When a plurality of substituents are present, they may be substituted singly or in combination of two or more.

[0044] Above R 1 From the viewpoint of improving water solubility and promoting dispersion into cell membranes and cells, the hydrophilic substituent represented by the formula (I) is preferably a group containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and halogen. In particular, from the viewpoint of suppressing reaction with biomolecules, the hydrophilic substituent preferably contains one or more atoms selected from the group consisting of a tertiary amino group, a quaternary ammonium group, and a carbonyl group (excluding an aldehyde group).

[0045] In the above formula (1), R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and halogen; R 2 and R 3 may together form a ring structure.

[0046] Above R 2 and R 3 Examples of the alkyl group having 1 to 6 carbon atoms represented by the formula (I) include linear or branched alkyl groups, such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, and an n-hexyl group.

[0047] Above R 2 and R 3 Examples of the alkenyl group having 2 to 12 carbon atoms represented by the formula (I) include linear or branched alkenyl groups, such as vinyl and allyl groups.

[0048] Above R 2 and R 3 Examples of the alkynyl group having 2 to 12 carbon atoms represented by the formula (I) include linear or branched alkenyl groups, such as an ethynyl group and a propargyl group.

[0049] Also, the above R 2 and R 3 and the like, each independently may be, for example, a carboxyl group. Shi base , a hydroxyl group, a sulfo group, an amide group, a halogen atom, or a group which is a salt thereof. When a plurality of substituents are present, they may be substituted singly or in combination of two or more.

[0050] Above R 2 and R 3 The hydrophilic substituent represented by the formula (I) is, for example, the above-mentioned R 1 Those described in the column of the hydrophilic substituents represented can be used similarly as appropriate.

[0051] In the above formula (1), the R 2 and R 3 may together form a ring structure. 2 and R 3 When these together form one ring structure, examples of the ring structure include a pyrrolidine ring structure, an imidazolidine ring structure, an oxazolidine ring structure, a piperidine ring structure, a piperazine ring structure, and a morpholine ring structure.

[0052] In the above formula (1), R 4 and R 5 are each independently an alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, an aryl group having 5 to 12 carbon atoms, a heteroaryl group having 5 to 12 carbon atoms, a halogen atom, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and a halogen atom.

[0053] Above R4 and R 5 Examples of the alkyl group having 1 to 12 carbon atoms represented by the formula (I) include linear or branched alkyl groups, such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decanyl group, an n-undecanyl group, and an n-dodecanyl group.

[0054] Above R 4 and R 5 Examples of the alkenyl group having 2 to 12 carbon atoms represented by the formula (I) include linear or branched alkenyl groups, such as vinyl and allyl groups.

[0055] Above R 4 and R 5 Examples of the alkynyl group having 2 to 12 carbon atoms represented by the formula (I) include linear or branched alkenyl groups, such as an ethynyl group and a propargyl group.

[0056] Above R 4 and R 5 Examples of the aryl group having 5 to 12 carbon atoms represented by the formula (I) include aromatic hydrocarbon groups having 5 to 12 carbon atoms, such as a phenyl group, a tolyl group, a xylyl group, a naphthyl group, a methylnaphthyl group, anthracyl, or indenyl.

[0057] Above R 4 and R 5 Examples of heteroaryl groups having 5 to 12 carbon atoms represented by the formula (I) include aromatic hydrocarbon groups having 5 to 12 carbon atoms, and include those in which one or more carbon atoms in the aromatic ring are substituted with an atom such as a nitrogen atom, an oxygen atom, a sulfur atom, etc. Examples include a pyrrolyl group, a pyridinyl group, an imidazolyl group, a thienyl group, a furanyl group, a pyrazolyl group, an oxazolyl group, and a thiazolyl group.

[0058] Above R 4 and R5 Examples of the halogen atom represented by the formula (I) include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0059] Above R 4 and R 5 The hydrophilic substituent represented by the formula (I) is, for example, the above-mentioned R 1 Those described in the column of the hydrophilic substituents represented can be used similarly as appropriate.

[0060] In the above formula (1), R 4 and R 5 are each independently, for example, a methyl group, an ethyl group, a hydroxyl group, or an amino group.

[0061] In the above formula (1), n ​​is an integer of 1 to 4.

[0062] In the above formula (1), a and b are each independently an integer of 0 to 4. When the sum of a and b is 2 or more, the R 4 and R 5 These may be the same or two or more may be combined.

[0063] Furthermore, examples of the compound (1) include, but are not limited to, the following embodiments.

[0064] (Example 1) R 1 is a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, R 4 and R 5each independently represents an alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, an aryl group having 5 to 12 carbon atoms, a heteroaryl group having 5 to 12 carbon atoms, a halogen atom, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and a halogen; n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4. (Example 2) R 1 is a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, R 4 and R 5 are each independently an alkyl group having 1 to 12 carbon atoms, n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4. (Embodiment Example 3) R 1 is a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, R 4 and R 5 are each independently an aryl group having 5 to 12 carbon atoms, n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4.

[0065] (Example 4) R 1 is a substituted or unsubstituted alkenyl group having 1 to 12 carbon atoms, R 2 and R 3are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, R 4 and R 5 each independently represents an alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, an aryl group having 5 to 12 carbon atoms, a heteroaryl group having 5 to 12 carbon atoms, a halogen atom, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and a halogen; n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4. (Example 5) R 1 is a substituted or unsubstituted alkenyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, R 4 and R 5 are each independently an alkyl group having 1 to 12 carbon atoms, n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4. (Example 6) R 1 is a substituted or unsubstituted alkenyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, R 4 and R 5 are each independently an aryl group having 5 to 12 carbon atoms, n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4.

[0066] (Embodiment Example 7) R 1is a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, and R 2 and R 3 Both form a ring structure, R 4 and R 5 each independently represents an alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, an aryl group having 5 to 12 carbon atoms, a heteroaryl group having 5 to 12 carbon atoms, a halogen atom, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and a halogen; n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4. (Embodiment Example 8) R 1 is a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, and R 2 and R 3 Both form a ring structure, R 4 and R 5 are each independently an alkyl group having 1 to 12 carbon atoms, n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4. (Example 9) R 1 is a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, and R 2 and R 3 Both form a ring structure, R 4 and R 5 are each independently an aryl group having 5 to 12 carbon atoms, n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4.

[0067] (Example 10) R 1 is a substituted or unsubstituted alkenyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, and R 2 and R 3 Both form a ring structure, R 4 and R 5 each independently represents an alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, an aryl group having 5 to 12 carbon atoms, a heteroaryl group having 5 to 12 carbon atoms, a halogen atom, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and a halogen; n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4. (Example 11) R 1 is a substituted or unsubstituted alkenyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, and R 2 and R 3 Both form a ring structure, R 4 and R 5 are each independently an alkyl group having 1 to 12 carbon atoms, n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4. (Example 12) R 1 is a substituted or unsubstituted alkenyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, and R 2 and R 3 Both form a ring structure, R 4 and R 5 are each independently an aryl group having 5 to 12 carbon atoms, n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4.

[0068] (Example 13) R 1 is a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, and R 2 and R 3 together form a ring structure, and the ring structure is a piperidine ring; R 4 and R 5 each independently represents an alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, an aryl group having 5 to 12 carbon atoms, a heteroaryl group having 5 to 12 carbon atoms, a halogen atom, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and a halogen; n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4. (Example 14) R 1is a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, and R 2 and R 3 together form a ring structure, and the ring structure is a piperidine ring; R 4 and R 5 are each independently an alkyl group having 1 to 12 carbon atoms, n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4. (Example 15) R 1 is a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, and R 2 and R 3 together form a ring structure, and the ring structure is a piperidine ring; R 4 and R 5 are each independently an aryl group having 5 to 12 carbon atoms, n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4.

[0069] (Example 16) R 1 is a substituted or unsubstituted alkenyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, and R 2 and R 3 together form a ring structure, and the ring structure is a piperidine ring; R 4 and R 5each independently represents an alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, an aryl group having 5 to 12 carbon atoms, a heteroaryl group having 5 to 12 carbon atoms, a halogen atom, or a hydrophilic substituent containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and a halogen; n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4. (Example 17) R 1 is a substituted or unsubstituted alkenyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, and R 2 and R 3 together form a ring structure, and the ring structure is a piperidine ring; R 4 and R 5 are each independently an alkyl group having 1 to 12 carbon atoms, n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4. (Example 18) R 1 is a substituted or unsubstituted alkenyl group having 1 to 12 carbon atoms, R 2 and R 3 are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, and R 2 and R 3 together form a ring structure, and the ring structure is a piperidine ring; R 4 and R 5 are each independently an aryl group having 5 to 12 carbon atoms, n is 1, A compound represented by the above formula (1) or a salt thereof, wherein a and b are each independently an integer of 0 to 4.

[0070] The absorption maximum wavelength of the compound is, for example, between 300 and 550 nm in 20 mM phosphate buffer (pH 7.4) at 25° C. The absorption maximum wavelength of the compound can be, for example, within a range between any two values ​​selected from the group consisting of 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, and 550 nm.

[0071] The maximum fluorescence wavelength of the above compound in methanol at 25°C is λ Met , the fluorescence maximum wavelength in n-heptane at 25 °C is λ Hep Then, the compound λ Met and λ Hep The difference between λ and λ is, for example, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, or 110 nm or more. Met and λ Hep The larger the difference between the λ and λ peaks, the easier it is to detect tumor tissue with high contrast compared to healthy tissue, which is preferable. Met and λ Hep The difference may be 300 nm or less, 200 nm or less, or 150 nm or less.

[0072] Furthermore, the two-photon absorption maximum wavelength of the compound is preferably between 600 and 1200 nm in 20 mM phosphate buffer (pH 7.4) at 25°C. A wavelength longer than 900 nm is particularly preferred. Such compounds can be excited with light of a wavelength that is not easily absorbed by biological materials in tissue, making them suitable for use in two-photon microscopy.

[0073] The molecular weight of the compound is, for example, 700 or less, and can be 650 or less, 600 or less, or 500 or less. The lower limit can be 100 or more, 150 or more, etc.

[0074] (Salts of compounds, etc.) Furthermore, the salt of the compound of the present invention refers to a compound in which part or all of the compound is in the form of a salt. For example, the amino group portion of the compound is an ammonium cation, and the salt is formed with a halogen ion, an organic cation, an inorganic cation, or the like as a counter anion. Furthermore, for example, the compound of the present invention may form a salt as a zwitterion having both a cation and an anion in the same molecule.

[0075] In the present invention, the above compounds also include derivatives produced by chemical changes of small parts within the molecule, including simple structural substitutions, adducts, hydrates, etc., and also include compounds known as analogs.

[0076] [Method for producing compounds] The compounds of the present invention can be produced by appropriately using known methods.

[0077] For example, the following compound A can be synthesized according to the following scheme. [ka]

[0078] Compound A can be synthesized, for example, by first substituting one bromo group of 1,6-dibromopyrene with a piperidinyl group using piperidine (compound 2 above), and then converting the other bromo group to a formyl group using N,N-dimethylformamide (compound 1 above) (see, for example, Y. Niko et al., Sci. Rep., 2016). Compound 2 can then be converted to an enone structure using 2-pentanone under alkaline conditions.

[0079] [Fluorescent dye agent] The fluorescent dye agent of the present invention contains the above-mentioned compound or a salt thereof.

[0080] By using the fluorescent dye agent of the present invention, cells and the like can be stained simply and quickly.

[0081] The above-mentioned fluorescent dye agent is used, for example, to detect cells (such as tumor cells) in tissues derived from living organisms.

[0082] The fluorescent dye agents are used, for example, for detecting cell morphology.

[0083] The fluorescent dye agents are used, for example, for staining or visualizing biological samples.

[0084] The fluorescent dye agent is used, for example, for fluorescent imaging.

[0085] The tissues and cells to which the fluorescent dye agent of the present invention is applied are not particularly limited, regardless of whether they are animal or plant, living or dead cells, and may be, for example, derived from a living organism, artificially synthesized, or cultured.

[0086] The living body is not particularly limited as long as it is a multicellular animal, and is preferably a mammal, and more preferably a human.

[0087] Examples of the tissues include skin, brain, spinal cord, esophagus, stomach, small intestine, large intestine, duodenum, rectum, liver, pancreas, gallbladder, urinary bladder, kidney, heart, spleen, thymus, prostate, uterus, ovaries, testes, breast, lung, bronchi, eyeball, nose, paranasal cavity, oral cavity, pharynx, salivary gland, thyroid gland, parathyroid gland, adrenal gland, muscle, bone marrow, blood vessel, nerve, lymph node, peritoneum, diaphragm, blood, etc. In one embodiment, the tissue to which the fluorescent dye agent of the present invention is applied is skin, for example, the epidermis or dermis, or a combination of both.

[0088] The fluorescent dye agent of the present invention can be applied to tissues isolated from living bodies by surgical procedures such as excision, resection, puncture, and blood sampling, or tissues obtained from feces, urine, sweat, and other body fluids.

[0089] In this specification, tissue derived from a living organism includes not only tissue separated from a living organism, but also the living organism itself or a part of a living organism that has not been separated from the living organism.

[0090] In one embodiment, the form of the tissue can be appropriately selected depending on the detection method, but may be, for example, an organ or tissue itself, or a thin section or three-dimensional fragment thereof.

[0091] Depending on the form of the tissue, the tissue may be subjected to treatments such as fixation with formalin or the like, paraffin embedding, deparaffinization, dehydration, and clearing.

[0092] The tumor cells detected by the fluorescent dye agent of the present invention may be benign or malignant (such as cancer or sarcoma cells), but are preferably malignant tumor cells. The type of tumor cells is not particularly limited, and examples include tumor cells occurring in the above-mentioned tissues.

[0093] In one embodiment, the tumor cells are cells derived from a skin tumor. Examples of such tumor cells include sweat gland tumors (e.g., extramammary Paget's disease, Paget's disease of the breast, eccrine porocarcinoma, microcystic adnexal carcinoma, and cutaneous mucinous carcinoma), malignant melanoma, epidermal and hair follicle tumors (e.g., basal cell carcinoma, squamous cell carcinoma, actinic keratosis, Bowen's disease, leukoplakia, and keratoacanthoma), nervous system tumors (e.g., Merkel cell carcinoma and malignant peripheral nerve sheath tumor), and mesenchymal tumors (e.g., dermatofibrosarcoma protuberans, solitary fibrous tumor, muscle tumors, liposarcoma, angiosarcoma, Kaposi's sarcoma, spindle cell hemangioendothelioma, heterogeneous fibroxanthomatosis, epithelioid sarcoma, synovial sarcoma, and undifferentiated pleomorphic cell sarcoma). In a specific embodiment, the tumor cells are cells derived from extramammary Paget's disease (Paget's cells) or malignant melanoma cells.

[0094] In the fluorescent dye agent of the present invention, the compound is not particularly limited as long as it is applicable to tissues and the like.

[0095] In one embodiment, the compound is capable of distributing to the cell membrane and intracellularly and secreted, and in a more particular embodiment, the compound is capable of inserting into the cell membrane.

[0096] Examples of the secretions include bile, hormones, digestive enzymes, exosomes, amyloids, and insulin.

[0097] In one embodiment, the compound exhibits strong fluorescence anisotropy at the cell membrane.

[0098] In one embodiment, the compound has the property of exhibiting lower fluorescence intensity outside the cell compared to the cell membrane and / or inside the cell. In a specific embodiment, the compound exhibits substantially no fluorescence outside the cell. Without being particularly limited thereto, for example, the fluorescence intensity outside the cell of the compound may be 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the fluorescence intensity inside the cell. Here, the comparison of the fluorescence intensity inside and outside the cell is performed by exciting cells derived from the tissue of interest of the present invention with light of a wavelength capable of exciting the compound, obtaining fluorescence microscope images under conditions that allow detection of light of the maximum fluorescence wavelength, and comparing the average signal intensities of the fluorescence inside and outside the cell.

[0099] The fluorescent dye agents of the present invention may be used alone or in combination of two or more.

[0100] Furthermore, the fluorescent dye agent of the present invention can be used in appropriate combination with the above-mentioned compounds or salts thereof as well as other components used in known fluorescent dye agents.

[0101] The other components may include, for example, a pH buffer, a surfactant, a salt, a solvent, a dye composition other than compound (1), and the like, either singly or in combination of two or more thereof.

[0102] Examples of pH buffers include one or more selected from the group consisting of tris(hydroxymethyl)aminomethane; Good's buffers (HEPES, MOPS, etc.); and pH buffers containing citric acid, acetic acid, lactic acid, oxalic acid, phthalic acid, imidazole, triethanolamine, diethanolamine, glycine, boric acid, phosphoric acid, or carbonate.

[0103] Examples of the solvent include one or more selected from the group consisting of water, ethanol, methanol, 2-propanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, acetone, ethyl acetate, tetrahydrofuran, and 1,2-dichloroethane, and the like.

[0104] The dye composition other than compound (1) is not particularly limited as long as it does not interfere with the detection of cells by compound (1). For example, nuclear staining dyes such as propidium iodide (PI), ethidium bromide, acridine orange, DAPI, and Hoechst are suitable because they do not interfere with the staining of the above-mentioned compound. Furthermore, dye compositions used in various tissue staining procedures, such as hematoxylin-eosin (HE) staining, Azan staining, Masson-Trichrome staining, Elastica-van Gieson staining, silver impregnation staining, Victoria blue staining, PAM staining, PTAH staining, Sudan III staining, Oil Red O staining, PAS staining, Alcian blue staining, toluidine blue staining, colloidal iron staining, mucicarmine staining, Congo red staining, Dylon staining, Grimelius staining, Fontana-Masson staining, Kossa staining, Berlin blue staining, Bodian staining, Klüver-Barrera staining, and Giemsa staining, can be used as dye compositions other than compound (1). These other dye compositions may be used singly or in combination of two or more.

[0105] The fluorescent dye agent of the present invention may be in the form of a solid such as a powder, or may be in the form of a liquid.

[0106] When tissue is stained with the fluorescent dye of the present invention, for example, compound (1) can be distributed in various parts of intracellular lipid membranes, and the fluorescence wavelength shifts depending on the phase (composition) of the lipid membrane, resulting in a change in fluorescence color. Therefore, by appropriately selecting the wavelength of the fluorescence to be detected, for example, changes in the phase of intracellular lipid membranes can be detected with high contrast.

[0107] Furthermore, when tissue is stained with the fluorescent dye agent of the present invention, for example, compound (1) may be distributed in both tumor cells and normal tissue cells, but the fluorescence wavelength of compound (1) in tumor cells is shifted from the fluorescence wavelength in normal tissue cells. Therefore, by appropriately selecting the wavelength of the fluorescence to be detected, tumor cells can be detected with high contrast from normal tissue. Thus, the fluorescent dye agent of the present invention can be used for the examination or diagnosis of tumors, particularly malignant tumors.

[0108] Furthermore, when applied to a living body, the fluorescent dye agent of the present invention can be used, for example, for diagnosing tumors, particularly malignant tumors. Furthermore, the fluorescent dye agent of the present invention may be applied to a living body before, during, or after tumor removal treatment in order to identify the extent of removal of a living body tumor, particularly a cancer, or to confirm whether or not any tumor remains.

[0109] 〔kit〕 The kit of the present invention comprises the above-mentioned compound or a salt thereof.

[0110] By using the kit of the present invention, cells and the like can be stained simply and quickly.

[0111] The components contained in the kit may be similarly, as appropriate, to those described above in the section on fluorescent dye agents.

[0112] The kit may be, for example, a combination of the compound or a salt thereof with reagents and instruments for staining or preparing tissue specimens.

[0113] In one embodiment, the kit also includes a reagent for preparing a staining solution. In a more specific embodiment, the reagent for preparing a staining solution may include, for example, one or a mixture of two or more selected from the group consisting of the above-mentioned pH buffers, surfactants, salts, solvents, and other dye compositions.

[0114] [Cell detection method] The cell detection method of the present invention comprises the step (1) of staining cells with a fluorescent dye agent containing the above-mentioned compound or a salt thereof.

[0115] Furthermore, by using the cell detection method of the present invention, cells and the like can be detected easily.

[0116] As the fluorescent dye agent in the step (1), the above-mentioned fluorescent dye agents can be suitably used in the same manner.

[0117] In addition, the descriptions of each component in the above section on fluorescent dye agents can be similarly adopted as appropriate.

[0118] In the step (1) above, the step of staining the cells can be carried out using materials and techniques used in known cell staining steps as appropriate.

[0119] Furthermore, the cell detection method of the present invention can include, after the above step (1), a step (2) of evaluation using fluorescence spectra measured in two or more types of solvents.

[0120] The fluorescent dye agent of the present invention may be a compound exhibiting the above-mentioned solvatochromism, and therefore its absorption maximum wavelength, fluorescence maximum wavelength, or both may change depending on the polarity (hydrophobicity) of the compound or the surroundings of the fluorescent dye agent. Since the fluorescent dye agent is used in the above step (2), fluorescence spectra measured in two or more solvents may be used to easily perform evaluations such as cell morphology detection, staining or visualization of biological samples, etc.

[0121] The step of evaluating using the fluorescence spectrum in the above step (2) can be performed by appropriately using a known method for measuring the fluorescence spectrum of a substance such as a compound or tissue.

[0122] Examples of the solvent that can be used in the above step (2) include one or more selected from the group consisting of water, ethanol, methanol, 2-propanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, acetone, ethyl acetate, tetrahydrofuran, and 1,2-dichloroethane, either singly or in combination.

[0123] Furthermore, the step (2) is preferably carried out using, for example, fluorescence imaging. For example, two-photon fluorescence imaging can also be suitably used.

[0124] In one embodiment, the method of the present invention is applied to tissues that are organs or organ systems themselves, or three-dimensional fragments thereof. In this embodiment, it is preferable to include a step of clearing the tissue before staining. Examples of tissue clearing methods include the TDE method, LUCID method, CLARITY method, PACT / PARS method, CUBIC method, 3DISCO method, Scale method, ScaleS method, SeeDB method, FocusClear method, Clear method, BABB method, iDISCO method, and uDISCO method. These clearing methods are described, for example, in Cell Chem. Biol. 2016, Vol. 23, 137-157 and Laser Photonics Rev. 2019, Vol. 13, 1800292.

[0125] In one embodiment, the method of the present invention is applied to a living organism itself, or a tissue that is part of a living organism and is not separated from the living organism.

[0126] In one embodiment, the method of the present invention is applied to thin sections, which may optionally be subjected to treatments commonly used in clinical examinations, such as fixation, dehydration, dealcoholization, paraffin infiltration, paraffin embedding, deparaffinization, immersion, and staining using the various tissue staining methods described above.

[0127] The staining is usually carried out by contacting a staining solution containing the compound with tissue, etc. The concentration of the compound in the staining solution is adjusted to, for example, 0.001 mg / mL or more, 0.01 mg / mL or more, 0.1 mg / mL or more, 0.2 mg / mL or more, 0.3 mg / mL or more, 0.4 mg / mL or more, 0.5 mg / mL or more, 0.6 mg / mL or more, 0.7 mg / mL or more, 0.8 mg / mL or more, 0.9 mg / mL or more, or 1 mg / mL or more relative to the total volume of the staining solution.

[0128] In addition, the concentration of the above compound in the staining solution is adjusted to, for example, 500 mg / mL or less, 200 mg / mL or less, 100 mg / mL or less, 50 mg / mL or less, 20 mg / mL or less, 10 mg / mL or less, 5 mg / mL or less, or 2 mg / mL or less relative to the total amount of the staining solution.

[0129] The temperature during dyeing is not particularly limited, but is, for example, 0 to 80°C, 4 to 50°C, 20 to 45°C, or 25 to 40°C, and preferably 35 to 42°C.

[0130] The time for which the staining solution is brought into contact with the tissue or the like is, for example, 1 minute or more, 10 minutes or more, 20 minutes or more, 1 hour or more, 2 hours or more, 1 day or more, or 2 days or more, for example, 14 days or less or 7 days or less.

[0131] In one embodiment, the time for which the staining solution is brought into contact with the tissue or the like is 0 to 40°C, for example, 12 hours or less, 6 hours or less, preferably 2 hours or less, more preferably 1 hour or less, even more preferably 30 minutes or less, still more preferably 10 minutes or less, or may be, for example, 1 minute or more, 2 minutes or more, 5 minutes or more, or 10 minutes or more.

[0132] Tissues stained with a staining solution containing the above-mentioned compound can be used directly for the morphological detection of cells or the detection of tumor cells, but optionally may be subjected to processing such as staining with other dye compositions before detection.

[0133] The method of the present invention may further include a step of detecting tumor cells. Detection of tumor cells can be performed, for example, by exciting the compound with light of an appropriate wavelength and detecting the emitted fluorescence. Detection can be performed using, for example, a confocal laser scanning microscope, and depending on the thickness of the section, a microscope capable of multiphoton excitation, such as a two-photon microscope, can also be used. For example, when 1-acetyl-6-piperidylpyrene (PK) is used as the compound, it is suitable for measurement using a two-photon microscope, as shown in the examples.

[0134] In one embodiment, cells such as tumor cells are detected by selecting a fluorescent light containing one particular wavelength that provides contrast between the tumor cells and cells of normal tissue, and measuring the fluorescent intensity.

[0135] In another embodiment, cells, such as tumor cells, are detected by multi-wavelength measurement, i.e., cells are detected by detecting fluorescence containing two or more different specific wavelengths and integrating the intensity of each fluorescence.

[0136] In one embodiment, when compound A in the examples is used as the compound, the detected fluorescence includes, for example, one or more wavelengths selected from the ranges of 350 to 750 nm, 550 to 700 nm, 600 to 700 nm, and 650 to 750 nm.

[0137] As described above in the section on fluorescent dye agents, the detection method of the present invention makes it possible to inspect and diagnose cells such as tumors, or to identify the extent of tumor removal.

[0138] In addition to merging, ratiometric analysis (a method of detecting fluorescence of two different wavelengths and calculating the ratio of their fluorescence intensities) is also effective as an image analysis that utilizes solvatochromism.

[0139] In addition to the above-mentioned fluorescence imaging, fluorescence lifetime imaging and the like can also be used as appropriate. [Example]

[0140] Next, the present invention will be specifically explained by way of examples, but the present invention is not limited to the following examples.

[0141] Example 1 (Synthesis of Compound A) Compound A was synthesized according to the following scheme. [ka]

[0142] To a known pyrene derivative, Compound 1 (100 mg, 0.32 mmol), and 2-pentanone (47 μL, 0.48 mmol), 1 M aqueous sodium hydroxide solution (1 mL) and dehydrated ethanol (6 mL) were added, and the resulting solution was heated and stirred at 60°C for 4 hours under an argon atmosphere.

[0143] Next, water was added to the above solution, and the resulting precipitate was collected by filtration.

[0144] The resulting precipitate was purified by silica gel column chromatography (dichloromethane:hexane=2:1) ​​and further recrystallized from acetonitrile to obtain the target compound A (PC) (yield: 20 mg, 16%).

[0145] ( 1 H-NMR analysis, and 13 C-NMR analysis) 1 H-NMR, and 13 The C-NMR analysis was performed using a nuclear magnetic resonance spectrometer (JMN-LA500 manufactured by JEOL Ltd.) The results obtained for Compound A are shown below and in Figures 1 and 2. · 1H NMR (500MHz, CDCl3): δ(ppm)=8.71(d,J=15.6Hz,1H),8.46(d,J=9.2Hz,1H),8.33(d,J=9.2H) z,1H),8.25(d,J=9.2Hz,1H),8.15(d,J=8.1Hz,1H),8.09(d,J=8.1Hz,1H),8.08(d,J=9.2Hz, 1H),8.03(d,J=9.2Hz,1H),7.74(d,J=8.1Hz,1H),7.01(d,J=15.6Hz,1H),3.22(s,4H),2.78 (t,J=7.4Hz,2H),1.93-1.95(m,4H),1.77-1.85(m,2H),1.73(s,2H),1.06(t,J=7.33Hz,1H). · 13 C NMR(CDCl3,TMS)δ(ppm)=14.11,18.12,24.67,26.84,43.49,55.21,117.53,120.49,124.26,124.48,124.71 ,125.09,125.68,126.02,126.31,126.51,126.57,127.66,128.75,130.80,133.21,139.25,150.33,200.49.

[0146] (High resolution mass spectrometry) High-resolution mass spectrometry was performed using a high-resolution mass spectrometer (JMS-700 manufactured by JEOL Ltd.) The results obtained for Compound A are shown below. HRMS (ESI + ),calcd for C 25 H 23 NO[M+Na] + 404.1985,found 404.1981.

[0147] Example 2 (Optical property measurement of compound A in organic solvent) The absorption spectrum and fluorescence spectrum of Compound A obtained in Example 1 were measured in each organic solvent.

[0148] Absorption and fluorescence spectra were measured using a UV-visible-near-infrared spectrophotometer (JASCO Corporation: V-670) and a spectrofluorometer (JASCO Corporation: FP6600), respectively. Fluorescence quantum yields were measured using an absolute PL quantum yield measurement system (Hamamatsu Photonics K.K.: C9920-02V). The concentration of Compound A in each solvent was 5 μM. The solvents used were toluene, dichloromethane, DMSO, and ethanol. The results are shown in Figure 3 and Table 1.

[0149] [Table 1]

[0150] As shown in Figure 3 and Table 1, the maximum absorption wavelength was almost the same for all solvents, but the maximum fluorescence wavelength differed depending on the solvent. More specifically, the fluorescence wavelength was longer in polar solvents, demonstrating fluorescent solvatochromism that is sensitive to solvent polarity.

[0151] Example 3 (Photostability evaluation of compound A) The photostability of Compound A obtained in Example 1 was evaluated.

[0152] The photostability of Compound A was compared with that of the commercially available solvatochromic dye Laurdan (solvent: toluene, dye concentration: 5 μM, excitation wavelength: 378 nm, detection wavelength: 483 nm). Laurdan is a compound with CAS number 74515-25-6. Fluorescence spectra were measured using the same equipment as in Example 2. The results are shown in Figure 4.

[0153] As shown in Figure 4, when the known Laurdan was used, the fluorescence intensity decreased over time, and for example, a significant decrease to 0.6 or less was observed 3000 seconds after excitation by UV irradiation. In contrast, when Compound A was used, the fluorescence intensity remained unchanged even 3000 seconds after excitation by UV irradiation.

[0154] Example 4 (Fluorescence spectrum measurement of compound A in lipid membrane) The fluorescence spectrum of Compound A obtained in Example 1 in a lipid membrane (liposome) was measured. More specifically, the fluorescence spectrum of Compound A in a liposome composed of 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) and sphingomyelin (SM) / cholesterol (Chol) was measured (Compound A concentration: 2 μM, lipid concentration: 200 μM). The fluorescence spectrum was measured using the same equipment as in Example 2. The results are shown in FIG. 5 and Table 2.

[0155] [Table 2]

[0156] As shown in Figure 5 and Table 2, the maximum fluorescence wavelength changed depending on the lipid composition. More specifically, a longer wavelength was observed in liposomes that were made of highly polar lipids and contained many water molecules in the membrane.

[0157] Example 5 (Fluorescence imaging of cells using compound A) Fluorescence imaging of cells was carried out using Compound A obtained in Example 1. More specifically, confocal fluorescence imaging was carried out on human prostate cancer cells (PC3) stained with Compound A in the example (Compound A concentration: 500 nM, excitation wavelength: 405 nm).

[0158] A confocal scanning laser microscope (Olympus, FV1000-D) was used for microscopic observation. Fluorescence was detected in the green (490-590 nm) and red (655-755 nm) channels using a 405 nm laser light source.

[0159] Cultured human prostate cancer cells were used for observation. Compound A (500 nM) was added to the cultured cells, which were then left at room temperature for 30 minutes, and then imaging was performed.

[0160] The results obtained are shown in FIG.

[0161] As shown in Figure 6, the fluorescence intensity detected in the green and red channels varied depending on the location of the cell. More specifically, strong fluorescence was observed in the green channel in the plasma membrane, which is rich in saturated phospholipids and cholesterol, which are considered to have low polarity and hydration, and in lipid droplets, which are rich in triglycerides and cholesterol esters. Similarly, in the merged image, the cell membrane and oil droplets had a strong green hue, while the intracellular membranes had a strong red hue.

[0162] Example 6 (Toxicity evaluation of compound A) Toxicity evaluation was carried out using fluorescent imaging of cells using Compound A obtained in Example 1. More specifically, toxicity evaluation (MTT test) of Compound A against human keratinocytes K16 (P4) was carried out.

[0163] Toxicity evaluation procedures were performed by adding compound A to the cells at a maximum concentration of 100 μM (maximum DMSO concentration = 0.5% v / v) in a medium containing 2-fold dilutions. After 24 hours, the medium was discarded and MTT reagent (5 μg / mL) was added. After 3 hours, 10% SDS reagent was added to the cells, and the absorbance at 570 nm was measured the following day.

[0164] The results obtained are shown in FIG.

[0165] 7, almost no cytotoxicity was observed even at a concentration of 100 μM of Compound A (cell viability: 93.9%). Note that the cell viability in the presence of 0.5% DMSO alone was 87.3%.

[0166] Example 7 (Human skin tissue imaging using compound A) Human skin tissue (frozen tissue) imaging was performed using Compound A obtained in Example 1. More specifically, two-photon fluorescence imaging was performed on human skin tissue (frozen tissue) stained with Compound A in the example (Compound A concentration: 10 μM, excitation wavelength: 960 nm, combined use of a clearing reagent (LUCID)).

[0167] A two-photon microscope, A1R MP+ (NIKON), was used for microscopic observation. For compound A, a 960 nm laser light source was used for excitation. Scanning was performed to a depth of 100 μm into the sample. A three-dimensional reconstruction image was obtained by processing the data using the accompanying software.

[0168] Human skin tissue was embedded in paraffin and then excised to prepare tissue sections. The sections (5 μm thick) were prepared using the procedure commonly used in histopathological examinations. Staining with Compound A staining solution was performed by deparaffinizing the tissue using a conventional method, followed by immersion in the staining solution at room temperature for several days to a week. In practice, sufficient staining was achieved by immersion in the staining solution at room temperature for 2 to 3 days.

[0169] The results obtained are shown in FIG.

[0170] As shown in Figure 8, Compound A, when used in combination with a clearing reagent, strongly stains the membranes of each cell that makes up human skin tissue, enabling the distinction of individual cell boundaries and clear visualization of tissue structure. More specifically, it is particularly effective in visualizing the stratum corneum structure, stratum lucidum, interkeratinocyte bridges in the stratum spinosum, and intraepidermal sweat duct (acrosyringium) structure.

[0171] Example 8 (Synthesis of Compound B) Compound B was synthesized according to the following scheme. [ka]

[0172] To a mixture of known compound 2 (200 mg, 0.54 mmol), tris(dibenzylideneacetone)dipalladium(0) (3 mg, 0.003 mmol), cataCXium® PtB (3.2 mg, 0.01 mmol), tetrabutylammonium chloride (150 mg, 0.54 mmol), and sodium bicarbonate (113 mg, 1.35 mmol), 3 mL of dehydrated DMF and methyl vinyl ketone (66 μL, 0.81 mmol) were added, and the solution was refluxed under an argon atmosphere for 12 hours with stirring.

[0173] The resulting solution was returned to room temperature, the resulting precipitate was filtered, and dichloromethane was added to the filtrate. The organic layer was washed with water and brine. Magnesium sulfate was added to the solution to dry it, and the magnesium sulfate was filtered off, and the organic solvent was removed using an evaporator.

[0174] The residue obtained above was purified by silica gel column chromatography (dichloromethane) and further recrystallized from acetonitrile to obtain the target compound B (yield: 23 mg, yield: 12%).

[0175] ( 1 H-NMR analysis) 1 The H-NMR analysis was performed using a nuclear magnetic resonance spectrometer (JMN-LA500 manufactured by JEOL Ltd.) The results obtained for Compound B are shown below and in FIG. · 1 H NMR(500MHz,CDCl3,TMS)δ:8.67(d,J=16.0,1H),8.46(d,J=9.1,1H),8.3 1(d,J=9.2,1H),8.25(d,J=8.2,1H),8.15(d,J=8.2,1H),8.09(d,J=8.1,1 H),8.09(d,J=9.2,1H),8.03(d,J=9.1,1H),7.75(d,J=8.1,1H),7.00(d, J=16.0,1H),3.21(br,4H),2.52(s,3H),1.91-1.94(m,4H),1.72(br,2H).

[0176] (High resolution mass spectrometry) High-resolution mass spectrometry was performed using a high-resolution mass spectrometer (JMS-700 manufactured by JEOL Ltd.) The results obtained for Compound B are shown below. ·HRMS(ESI),m / z:[M]calcd for C 25 H 23 NO,353.1780;found,353.1773.

[0177] Example 9 (Optical property measurement of compound B in organic solvent) The absorption spectrum and fluorescence spectrum of Compound B obtained in Example 8 in each organic solvent were measured.

[0178] The absorption spectrum and fluorescence spectrum were measured using an ultraviolet-visible-near-infrared spectrophotometer (JASCO Corporation: V-670) and a spectrofluorometer (JASCO Corporation: FP6600), respectively, as in Example 2. The fluorescence quantum yield was measured using an absolute PL quantum yield measurement device (Hamamatsu Photonics K.K.: C9920-02V). The concentration of Compound B was 5 μM in each solvent. The solvents used were toluene, dichloromethane, DMSO, and ethanol. The results are shown in FIG. 10.

[0179] As shown in Figure 10, the maximum absorption wavelength was almost the same for all solvents, but the maximum fluorescence wavelength differed depending on the solvent. More specifically, the fluorescence wavelength shifted to longer wavelengths in polar solvents, demonstrating fluorescent solvatochromism that is sensitive to solvent polarity.

[0180] Example 10 (Synthesis of Compound C) Compound C was synthesized according to the following scheme. [ka]

[0181] To the known compound 1 (50 mg, 0.16 mmol) and 2-undecanone (48 μL, 0.24 mmol), 1 M aqueous sodium hydroxide solution (0.5 mL) and dehydrated ethanol (3 mL) were added, and the solution was heated and stirred at 60°C for 4 hours under an argon atmosphere to obtain compound C.

[0182] Water was added to the resulting solution, and the resulting precipitate was collected by filtration.

[0183] The precipitate obtained above was purified by silica gel column chromatography (dichloromethane:hexane=2:1) ​​and further recrystallized from acetonitrile to obtain the target compound C (yield: 15 mg, 13%).

[0184] ( 1 H-NMR analysis) 1 The H-NMR analysis was performed using a nuclear magnetic resonance spectrometer (JMN-LA500 manufactured by JEOL Ltd.) The results obtained for Compound C are shown below and in FIG. · 1 H NMR(500MHz,CDCl3,TMS)δ:8.71(d,J=15.8,1H),8.45(d,J=9.1,1H),8.33(d,J=9.1,1H), 8.25(d,J=8.2,1H),8.15(d,J=8.2,1H),8.09(d,J=8.3,1H),8.07(d,J=9.2,1H),8.03(d,J =9.2,1H),7.74(d,J=8.3,1H),7.01(d,J=15.8,1H),3.21(br,4H),2.78(t,J=7.1,2H),1. 91-1.94(m,4H),1.73-1.79(m,2H),1.73(br,2H),1.27-1.41(m,12H),0.88(t,J=6.7,3H).

[0185] (High resolution mass spectrometry) High-resolution mass spectrometry was performed using a high-resolution mass spectrometer (JMS-700 manufactured by JEOL Ltd.) The results obtained for Compound C are shown below. HRMS(ESI), m / z:[MNa] +calcd for C 33 H 39 NO,488.2924;found,488.2924.

[0186] Example 11 (Optical property measurement of compound C in organic solvent) The absorption spectrum and fluorescence spectrum of Compound C obtained in Example 10 in each organic solvent were measured.

[0187] The absorption spectrum and fluorescence spectrum were measured using an ultraviolet-visible-near-infrared spectrophotometer (JASCO Corporation: V-670) and a spectrofluorometer (JASCO Corporation: FP6600), respectively, as in Example 2. The fluorescence quantum yield was measured using an absolute PL quantum yield measurement device (Hamamatsu Photonics K.K.: C9920-02V). The concentration of Compound C was 5 μM in each solvent. The solvents used were toluene, dichloromethane, DMSO, and ethanol. The results are shown in FIG. 12.

[0188] As shown in Figure 12, the maximum absorption wavelength was almost the same for all solvents, but the maximum fluorescence wavelength differed depending on the solvent. More specifically, the fluorescence wavelength shifted to longer wavelengths in polar solvents, demonstrating fluorescent solvatochromism that is sensitive to solvent polarity.

Claims

1. A compound represented by the following formula (1) or a salt thereof: 【Chemistry 1】 (In formula (1), R 1 is a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, or an alkynyl group having 2 to 12 carbon atoms. R 2 and R 3 are each independently one or more groups selected from the group consisting of a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, a tertiary amino group, a quaternary ammonium group, and a carboxy group; R 2 and R 3 may together form a ring structure. R 4 and R 5 are each independently one or more selected from the group consisting of an alkyl group having 1 to 12 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an alkynyl group having 2 to 12 carbon atoms, an aryl group having 5 to 12 carbon atoms, a heteroaryl group having 5 to 12 carbon atoms, a halogen atom, a tertiary amino group, a quaternary ammonium group, and a carboxy group. n is an integer of 1 to 2. a and b are each independently an integer of 0 to 4.

2. The compound or salt thereof according to claim 1 , wherein n is 1.

3. The R 1 The compound or salt thereof according to claim 1 or 2, wherein is a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, an i-butyl group, a sec-butyl group, or a t-butyl group.

4. R 2 and R 3 are each independently a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, an i-butyl group, a sec-butyl group, or a t-butyl group, which is unsubstituted or substituted with a carboxy group, a hydroxyl group, a sulfo group, an amide group, a halogen atom, or a group which is a salt thereof, or a salt thereof.

5. R 2 and R 3 is a divalent cyclic hydrocarbon group having 3 to 12 carbon atoms, which is unsubstituted or substituted with a carboxy group, a hydroxy group, a sulfo group, an amide group, a halogen atom, or a salt thereof, and which is formed by combining these groups together with the nitrogen atom to which they are bonded, or a salt thereof according to any one of claims 1 to 3,

6. R 2 and R 3 is the divalent cyclic hydrocarbon group having 3 to 12 carbon atoms, or a salt thereof according to claim 5.

7. R 4 and R 5 The compound or salt thereof according to any one of claims 1 to 6, wherein each of is independently a methyl group, an ethyl group, or an amino group.

8. The compound or salt thereof according to any one of claims 1 to 7, which has an absorption maximum wavelength of 300 to 550 nm in 20 mM phosphate buffer (pH 7.4) at 25°C.

9. The compound or salt thereof according to any one of claims 1 to 8, which has a molecular weight of 700 or less.

10. A fluorescent dye agent comprising the compound according to any one of claims 1 to 9 or a salt thereof.

11. The fluorescent dye agent according to claim 10, which is used for detecting cell morphology.

12. The fluorescent dye agent according to claim 10, which is used for staining or visualizing biological samples.

13. The fluorescent dye agent according to any one of claims 10 to 12, which is used for fluorescent imaging.

14. A kit comprising the fluorescent dye agent according to any one of claims 10 to 13.

15. A method for detecting cells, comprising the step (1) of staining cells with a fluorescent dye agent comprising the compound according to any one of claims 1 to 9 or a salt thereof.

16. The cell detection method according to claim 15, further comprising, after the step (1), a step (2) of evaluating using fluorescence spectra measured in two or more types of solvents.

17. The cell detection method according to claim 16, wherein step (2) is carried out using fluorescent imaging.

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