Bisphosphoryl crosslinked stilbene compounds and oil droplet dyes

Abstract

To provide an oil droplet staining agent and an inner mitochondrial membrane staining agent which can estimate the composition of oil droplets and the membrane structure (membrane composition) of the inner mitochondrial membrane.SOLUTION: A bisphosphoryl-crosslinked stilbene compound is represented by general formula (1) [where Ar1 and Ar2 are the same or different to represent a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. R1 and R2 are the same or different to represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group. Y1 and Y2 are the same or different to represent a phosphine oxide group or the like].SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to bisphosphoryl crosslinked stilbene compounds and oil droplet dyes. [Background technology]

[0002] Oil droplet stains, such as those used for lipid droplets, are useful for observing the dynamics of lipid droplets and other oil droplets within cells. A wide variety of oil droplet stains are commercially available, including the light-resistant lipid droplet stain LAQ1. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] ACS Materials Lett. 2021, 3, 1, 42-49 [Overview of the project] [Problems that the invention aims to solve]

[0004] It is known that the composition of triglycerides constituting lipid droplets differs depending on the cell type. Furthermore, it is thought that the characteristics of lipid droplets differ even within the same cell. However, conventional lipid droplet stains have not been able to evaluate the characteristics of individual lipid droplets, making it difficult to identify the constituent components of lipid droplets or estimate their composition. For example, triglycerides in lipid droplets can be broadly classified into two types: triacylglycerols and cholesterol esters, but it has been difficult to evaluate the differences in the composition of these two types. Similarly, it has also been difficult to evaluate the differences in the membrane structure (membrane composition) of the inner mitochondrial membrane.

[0005] The present invention aims to solve the above-mentioned conventional problems and provides an oil droplet stain and a mitochondrial inner membrane stain that can estimate the composition of oil droplets and the membrane structure (membrane composition) of the mitochondrial inner membrane. [Means for solving the problem]

[0006] As a result of intensive studies in view of the above problems, the present inventors have found that when the aromatic ring on the phosphorus atom has no substituent, a compound having a specific bisphosphoryl cross-linked stilbene skeleton can not only stain oil droplets, but can also identify the tissues possessed by the oil droplets by fluorescence lifetime and distinguish them from each other, so that the composition of the oil droplets can be estimated. Further, the present inventors have also found that when the aromatic ring on the phosphorus atom of this specific bisphosphoryl cross-linked stilbene skeleton has a substituent, it can identify the tissues present in the inner mitochondrial membrane and can estimate the membrane structure (membrane composition) of the inner mitochondrial membrane. Based on such findings, the present inventors have further conducted research and completed the present invention. That is, the present invention includes the following configurations.

[0007] Item 1. General formula (1):

[0008] [Chemical formula] [In the formula, Ar 2 , , 2 , 2 , , 1 , , , 2 , 1 , 1 , , 1 , 2 ,

[0009] , and Ar 2 are the same or different and represent a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. R 1 and R 2 are the same or different and represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group. However, R 1 and R 2 may together form a ring with the adjacent nitrogen atom. R 1 and / or R 2 and / or R 2 2 may together form a ring with the adjacent nitrogen atom. Y 1 and Y 2 are the same or different and are of general formula (2):

[0009] [Chemical formula] (Ar 3 This indicates an unsubstituted aromatic hydrocarbon ring or an unsubstituted heteroaromatic ring. Y 3 (This represents an oxygen atom or a sulfur atom.) This indicates the group represented by [ ]. A bisphosphoryl-crosslinked stilbene compound represented by the formula.

[0010] Item 2. Said Ar 1 The bisphosphoryl-bridged stilbene compound described in item 1, wherein the stilbene is a substituted or unsubstituted polycyclic aromatic hydrocarbon ring.

[0011] Item 3. Said Ar 2 The bisphosphoryl-bridged stilbene compound according to item 1 or 2, wherein the stilbene is a substituted or unsubstituted polycyclic aromatic hydrocarbon ring.

[0012] Item 4. Said Ar 3 However, the bisphosphoryl-bridged stilbene compound described in any one of items 1 to 3 is an unsubstituted polycyclic aromatic hydrocarbon ring.

[0013] Item 5. The aforementioned Y 3 However, the oxygen atom is a bisphosphoryl-bridged stilbene compound as described in any one of items 1 to 4.

[0014] Item 6. A fluorescent dye containing a bisphosphoryl crosslinked stilbene compound as described in any one of items 1 to 5.

[0015] Item 7. An oil droplet stain containing a bisphosphoryl crosslinked stilbene compound as described in any one of items 1 to 5.

[0016] Item 8. An oil droplet staining agent as described in Item 7, for identifying tissue within an oil droplet by fluorescence lifetime.

[0017] Section 9. General formula (3):

[0018] [ka] [In the formula, Ar 1 and Ar 2 These are identical or different, representing a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. R 1 and R 2 These terms, whether identical or distinct, represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group. However, R 1 and R 2 They may come together to form a ring with adjacent nitrogen atoms. 1 and / or R 2 is Ar 2 They may also form a ring together with adjacent nitrogen atoms. Y 4 and Y 5 They are the same or different, and general formula (4):

[0019] [ka] (Ar 4 This indicates a substituted aromatic hydrocarbon ring or a substituted heteroaromatic ring. Y 6 (This represents an oxygen atom or a sulfur atom.) This indicates the group represented by [ ]. A mitochondrial inner membrane staining agent containing a bisphosphoryl crosslinked stilbene compound represented by [formula].

[0020] Item 10. A mitochondrial inner membrane staining agent according to item 8 or 9, which identifies tissue present in the mitochondrial inner membrane by fluorescence lifetime.

[0021] Item 11. A method for estimating the composition of an oil droplet using a bisphosphoryl crosslinked stilbene compound described in any one of items 1 to 5, a fluorescent dye described in item 6, or an oil droplet stain described in item 7 or 8.

[0022] Section 12. A method for estimating the membrane structure of the mitochondrial inner membrane using the mitochondrial inner membrane staining agents described in Section 9 or 10. [Effects of the Invention]

[0023] The bisphosphoryl crosslinked stilbene compound of the present invention can identify and distinguish the constituent components inside an oil droplet, and therefore can be used as an oil droplet dyeing agent that can estimate the composition of the oil droplet.

[0024] Since the composition of triglycerides constituting lipid droplets is known to differ depending on the cell type, the bisphosphoryl-crosslinked stilbene compound of the present invention can also be used to identify the cell type.

[0025] Furthermore, the mitochondrial inner membrane staining agent of the present invention can identify tissue present in the mitochondrial inner membrane and estimate the membrane structure (membrane composition) of the mitochondrial inner membrane. [Brief explanation of the drawing]

[0026] [Figure 1] This is a fluorescence lifetime imaging (FLIM) image of HeLa cells using MitoPBRed (Example 1). The image on the right is a magnified view of the image on the left. [Figure 2] The left figure shows fluorescence lifetime imaging (FLIM) images of HeLa cells using MitoPBRed (Example 1), and the right figure shows carbonyl cyanide-m-chlorophenylhydrazone (CCCP) after treatment with CCCP. [Figure 3] The fluorescence lifetimes of MitoPBRed (Example 1) before and after treatment with carbonyl cyanide-m-chlorophenylhydrazone (CCCP) are shown. [Figure 4] The results of staining various cells using fluorescence lifetime imaging (FLIM) with LipiCo (Example 2) are shown. The left figure is a fluorescence lifetime imaging (FLIM) image, the middle figure is a bright-field image, and the right figure is a histogram showing the number of pixels in the fluorescence lifetime imaging (FLIM) images sorted by fluorescence lifetime. In the middle figure of Figure 4, the scale bar is 10 μm. [Figure 5] The results of staining HuH-7 cells using fluorescence lifetime imaging (FLIM) with LipiCo (Example 2) are shown. [Figure 6] The following shows the fluorescence lifetime results when HuH-7 cells were stained using fluorescence lifetime imaging (FLIM) immediately after and 6 hours after cholesterol addition. The left figure is a histogram showing the number of pixels in fluorescence lifetime imaging (FLIM) images sorted by fluorescence lifetime immediately after cholesterol addition, and the right figure is a histogram showing the number of pixels in fluorescence lifetime imaging (FLIM) images sorted by fluorescence lifetime 6 hours after cholesterol addition. [Modes for carrying out the invention]

[0027] In this specification, "comprise" is a concept that also includes "consist essentially of" and "consist of."

[0028] In this specification, when a range is expressed as "A to B", it means A or greater and B or less, unless otherwise specified.

[0029] 1. Bisphosphoryl-crosslinked stilbene compounds The bisphospholyl crosslinked stilbene compound of the present invention has general formula (1):

[0030] [ka] [In the formula, Ar 1 and Ar 2 These are identical or different, representing a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. R 1 and R 2 These terms, whether identical or distinct, represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group. However, R 1and R 2 They may come together to form a ring with adjacent nitrogen atoms. 1 and / or R 2 is Ar 2 They may also form a ring together with adjacent nitrogen atoms. Y 1 and Y 2 They are the same or different, and general formula (2):

[0031] [ka] (Ar 3 This indicates an unsubstituted aromatic hydrocarbon ring or an unsubstituted heteroaromatic ring. Y 3 (This represents an oxygen atom or a sulfur atom.) This indicates the group represented by [ ]. It is represented as follows.

[0032] Furthermore, the mitochondrial inner membrane staining agent of the present invention has the general formula (3):

[0033] [ka] [In the formula, Ar 1 and Ar 2 These are identical or different, representing a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. R 1 and R 2 These terms, whether identical or distinct, represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group. However, R 1 and R 2 They may come together to form a ring with adjacent nitrogen atoms. 1 and / or R 2 is Ar 2 They may also form a ring together with adjacent nitrogen atoms. Y 4 and Y 5 They are the same or different, and general formula (4):

[0034] [ka] (Ar 4 This indicates a substituted aromatic hydrocarbon ring or a substituted heteroaromatic ring. Y 6 (This represents an oxygen atom or a sulfur atom.) This indicates the group represented by [ ]. It contains a bisphosphoryl crosslinked stilbene compound represented by [the formula shown].

[0035] In other words, in the present invention, the bisphosphoryl-crosslinked stilbene compound represented by general formula (1) is a novel compound and can be used as an oil droplet staining agent that can identify tissue within oil droplets and thus estimate the composition of the oil droplets. The bisphosphoryl-crosslinked stilbene compound represented by general formula (1) can be used as a mitochondrial inner membrane staining agent that can identify tissue within the mitochondrial inner membrane and thus estimate the membrane structure of the mitochondrial inner membrane.

[0036] The bisphosphoryl-bridged stilbene compound represented by general formula (1) has an oxygen atom bonded to the phosphorus atom and a ring Ar 4 Depending on the position, general formulas (1A) and (1B):

[0037] [ka] [In the formula, Ar 1 and Ar 2 These are identical or different, representing a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. Ar 3a and Ar 3b These are identical or distinct, representing an unsubstituted aromatic hydrocarbon ring or an unsubstituted heteroaromatic ring. R 1 and R 2 These terms, whether identical or distinct, represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group. However, R1 and R 2 They may come together to form a ring with adjacent nitrogen atoms. 1 and / or R 2 is Ar 2 They may also form a ring together with adjacent nitrogen atoms. Y 3a and Y 3b These represent either the same or different oxygen or sulfur atoms. This includes all bisphosphoryl-crosslinked stilbene compounds represented by [the formula].

[0038] The trans-bisphosphoryl crosslinked stilbene compound represented by the above general formula (1A) and the cis-bisphosphoryl crosslinked stilbene compound represented by the above general formula (1B) both exhibit different fluorescence lifetimes depending on the internal structure of the oil droplet (e.g., triacylglycerol and cholesterol esters within the lipid droplet). Therefore, it is possible to identify and distinguish between the internal structure of the oil droplet, e.g., triacylglycerol and cholesterol esters, and estimate the composition of the oil droplet. Furthermore, since the composition of neutral fats constituting lipid droplets is known to differ depending on the cell type, the fluorescence lifetimes of the above-mentioned bisphosphoryl crosslinked stilbene compounds also differ. Thus, both the trans-bisphosphoryl crosslinked stilbene compound represented by the above general formula (1A) and the cis-bisphosphoryl crosslinked stilbene compound represented by the above general formula (1B) can also be used to identify the cell type based on their fluorescence lifetime.

[0039] Furthermore, the bisphosphoryl crosslinked stilbene compound represented by the general formula (1) described above, by having an amino group or substituted amino group which is an electron-donating group, can be given environmental responsiveness and can also achieve a longer absorption peak wavelength, thereby reducing phototoxicity to intracellular organelles.

[0040] Furthermore, the bisphosphoryl-bridged stilbene compound represented by the general formula (1) described above has a bisphosphoryl-bridged stilbene skeleton, and therefore MitoPB Yellow (Y in general formula (1)) 2It is possible to impart lightfastness such that the absorption intensity hardly decreases even under strong light irradiation that would cause photobleaching of a compound (which has a bis(4-methoxyphenyl)methylene group).

[0041] Furthermore, the bisphosphoryl-bridged stilbene compound represented by general formula (3) has an oxygen atom bonded to the phosphorus atom and a ring Ar 4 Depending on the position, general formulas (3A) and (3B):

[0042] [ka] [In the formula, Ar 1 and Ar 2 These are identical or different, representing a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. Ar 4a and Ar 4b This indicates a substituted aromatic hydrocarbon ring or a substituted heteroaromatic ring. R 1 and R 2 These terms, whether identical or distinct, represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group. However, R 1 and R 2 They may come together to form a ring with adjacent nitrogen atoms. 1 and / or R 2 is Ar 2 They may also form a ring together with adjacent nitrogen atoms. Y 6a and Y 6b These represent either the same or different oxygen or sulfur atoms. This includes all bisphosphoryl-crosslinked stilbene compounds represented by [the formula].

[0043] Both the transbisphosphoryl-bridged stilbene compound represented by the general formula (3A) and the cisbisphosphoryl-bridged stilbene compound represented by the general formula (3B) exhibit different fluorescence lifetimes depending on the tissue within the mitochondrial inner membrane. Therefore, it is possible to identify and distinguish between the tissues within the mitochondria, and thus estimate the membrane structure of the mitochondrial inner membrane.

[0044] Furthermore, the bisphosphoryl-crosslinked stilbene compounds described above have an Ar ring. 4 When a functional group is attached to it, it is possible to control organelle localization and dispersibility, and it can be used as a mitochondrial inner membrane staining agent. Therefore, bisphosphoryl-bridged stilbene compounds represented by general formula (3) are compounds suitable for repeated observation in vivo using super-resolution microscopy such as stimulated release inhibition (STED) imaging.

[0045] Furthermore, the bisphosphoryl crosslinked stilbene compound represented by the general formula (3) described above, by having an amino group or substituted amino group which is an electron-donating group, can be given environmental responsiveness and can also achieve a longer absorption peak wavelength, thereby reducing phototoxicity to mitochondria.

[0046] Furthermore, the bisphosphoryl-bridged stilbene compound represented by the general formula (3A) described above has a bisphosphoryl-bridged stilbene skeleton, and therefore MitoPB Yellow (Y in general formula (3)) 5 It is possible to impart lightfastness such that the absorption intensity hardly decreases even under strong light irradiation that would cause photobleaching of a compound (which has a bis(4-methoxyphenyl)methylene group).

[0047] In general formulas (1), (1A) and (1B), (3), (3A) and (3B), Ar 1As the aromatic hydrocarbon ring represented by , either a monocyclic aromatic hydrocarbon ring or a polycyclic aromatic hydrocarbon ring can be adopted. For example, a benzene ring can be cited as the monocyclic aromatic hydrocarbon ring, and naphthalene ring, anthracene ring, phenanthrene ring, fluorene ring, pyrene ring, triphenylene ring, etc. can be cited as the polycyclic aromatic hydrocarbon ring.

[0048] Ar 1 The aromatic hydrocarbon ring represented by may have a substituent. Examples of the substituent include, for example, an alkyl group described later, a cycloalkyl group described later, an aryl group described later, a heteroaryl group described later, an alkenyl group (vinyl group, propenyl group, etc.), an alkynyl group (ethynyl group, 1-propynyl group, etc.), a carbonyl group, a cyano group, a nitro group, -OR 3 (R 3 (where R represents an organic group) and the like. When having a substituent, the number of substituents is preferably, for example, 1 to 6, and more preferably 1 to 3.

[0049] In General Formulas (1), (1A), and (1B), (3), (3A), and (3B), Ar 1 Examples of the heteroaromatic ring represented by include, as the monocyclic heteroaromatic ring, a pyridine ring, a pyrazine ring, etc., and as the polycyclic heteroaromatic ring, an indole ring, an isoindole ring, a benzimidazole ring, a quinoline ring, an isoquinoline ring, a quinoxaline ring, etc.

[0050] Ar 1 The heteroaromatic ring represented by may have a substituent. Examples of the substituent include, for example, an alkyl group described later, a cycloalkyl group described later, an aryl group described later, a heteroaryl group described later, an alkenyl group (vinyl group, propenyl group, etc.), an alkynyl group (ethynyl group, 1-propynyl group, etc.), a carbonyl group, a cyano group, a nitro group, a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), -OR 3 (R 3 (where R represents an organic group) and the like. When having a substituent, the number of substituents is preferably, for example, 1 to 6, and more preferably 1 to 3.

[0051] Ar1 In terms of oil droplet staining, identification of the tissues inside the oil droplets, cell identification, mitochondrial inner membrane staining, identification of the tissues of the mitochondrial inner membrane, structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, light resistance, etc., substituted or unsubstituted aromatic hydrocarbon rings are preferred, and polycyclic aromatic hydrocarbon rings are preferred.

[0052] Note that Ar 1 When it is a substituted or unsubstituted polycyclic aromatic hydrocarbon ring, the bisphosphoryl-bridged stilbene compound represented by the general formula (1) is the general formulas (1C) and (1D):

[0053] [Chemical formula] [In the formula, Ar 2 , Y 1 , Y 2 , R 1 and R 2 are the same as described above. Ar 5 represents a substituted or unsubstituted aromatic hydrocarbon ring. ] Any of the bisphosphoryl-bridged stilbene compounds represented by the formula can be adopted, and the bisphosphoryl-bridged stilbene compound represented by the general formula (3) is the general formulas (3C) and (3D):

[0054] [Chemical formula] [In the formula, Ar 2 , Y 4 , Y 5 , R 1 and R 2 are the same as described above. Ar 5 represents a substituted or unsubstituted aromatic hydrocarbon ring. ] Any of the bisphosphoryl-bridged stilbene compounds represented by the formula can be adopted.

[0055] The bisphosphoryl-bridged stilbene compound represented by the general formula (1C) has a group Y 3 bonded to the phosphorus atom and a ring Ar 3Depending on the position, the general formulas (1C1) and (1C2):

[0056] [ka] [In the formula, Ar 2 Ar 3a Ar 3b Ar 5 , R 1 , R 2 , Y 3a and Y 3b This is the same as above. This includes all bisphosphoryl-crosslinked stilbene compounds represented by [the formula].

[0057] Furthermore, the bisphosphoryl-bridged stilbene compound represented by general formula (1D) has a group Y bonded to the phosphorus atom. 3 and ring Ar 3 Depending on the position, the general formulas (1D1) and (1D2) are:

[0058] [ka] [In the formula, Ar 2 Ar 4a Ar 4b Ar 5 , R 1 , R 2 , Y 6a and Y 6b This is the same as above. This includes all bisphosphoryl-crosslinked stilbene compounds represented by [the formula].

[0059] The bisphosphoryl-bridged stilbene compound represented by the general formula (3C) has a group Y bonded to the phosphorus atom. 6 and ring Ar 4 Depending on the position, the general formulas (3C1) and (3C2):

[0060] [ka] [In the formula, Ar 2 Ar 4a Ar 4bAr 5 , R 1 , R 2 , Y 6a and Y 6b This is the same as above. This includes all bisphosphoryl-crosslinked stilbene compounds represented by [the formula].

[0061] Furthermore, bisphosphoryl-bridged stilbene compounds represented by the general formula (3D) have a group Y bonded to the phosphorus atom. 6 and ring Ar 4 Depending on the position, the general formulas (3D1) and (3D2) are:

[0062] [ka] [In the formula, Ar 2 Ar 4a Ar 4b Ar 5 , R 1 , R 2 , Y 6a and Y 6b This is the same as above. This includes all bisphosphoryl-crosslinked stilbene compounds represented by [the formula].

[0063] In the general formulas (1C), (1C1), (1C2), (1D), (1D1), (1D2), (3C), (3C1), (3C2), (3D), (3D1), and (3D2), Ar 5 The aromatic hydrocarbon ring represented by can be either a monocyclic or polycyclic aromatic hydrocarbon ring. For example, a monocyclic aromatic hydrocarbon ring is a benzene ring, and polycyclic aromatic hydrocarbon rings include naphthalene rings, anthracene rings, phenanthrene rings, fluorene rings, pyrene rings, and triphenylene rings. Of these, the benzene ring is particularly preferred from the viewpoints of oil droplet staining, identification of tissues inside oil droplets, cell identification, mitochondrial inner membrane staining, identification of tissues in the mitochondrial inner membrane, structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, and light resistance.

[0064] Ar 5The aromatic hydrocarbon ring represented by may have substituents. Substituents include, for example, alkyl groups, cycloalkyl groups, aryl groups, heteroaryl groups, alkenyl groups (vinyl group, propenyl group, etc.), alkynyl groups (ethynyl group, 1-propynyl group, etc.), carbonyl groups, cyano groups, nitro groups, and -OR groups. 3 (R 3 Examples include (where represents an organic group). When substituents are present, the number of substituents is preferably 1 to 6, and more preferably 1 to 3.

[0065] In the general formulas (1C), (1C1), (1C2), (1D), (1D1), (1D2), (3C), (3C1), (3C2), (3D), (3D1), and (3D2), Ar 5 Examples of heteroaromatic rings represented by include, as monocyclic heteroaromatic rings, pyridine rings and pyrazine rings, and as polycyclic heteroaromatic rings, indole rings, isoindole rings, benzimidazole rings, quinoline rings, isoquinoline rings and quinoxaline rings.

[0066] Ar 5 The heteroaromatic ring represented by may have substituents. Substituents include, for example, alkyl groups, cycloalkyl groups, aryl groups, heteroaryl groups, alkenyl groups (vinyl group, propenyl group, etc.), alkynyl groups (ethynyl group, 1-propynyl group, etc.), carbonyl groups, cyano groups, nitro groups, halogen atoms (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), and -OR 3 (R 3 Examples include (where represents an organic group). When substituents are present, the number of substituents is preferably 1 to 6, and more preferably 1 to 3.

[0067] Note that Ar 1 Compared to cases where the compound is a substituted or unsubstituted monocyclic aromatic hydrocarbon ring, bisphosphoryl-bridged stilbene compounds represented by general formulas (1C), (1C1), (1C2), (3C), (3C1), or (3C2) exhibit different absorption ε and fluorescence Φ FFurthermore, the bisphosphoryl cross-linked stilbene compounds represented by general formulas (1C), (1C1), or (1C2) are more selective in staining oil droplets, and the bisphosphoryl cross-linked stilbene compounds represented by general formulas (3C), (3C1), or (3C2) can cause the target mitochondrial inner membrane to fluoresce more strongly and clearly.

[0068] Also, Ar 1 Compared to cases where the compound is a substituted or unsubstituted monocyclic aromatic hydrocarbon ring, bisphosphoryl-crosslinked stilbene compounds represented by general formulas (3D), (3D1), or (3D2) can achieve longer absorption and fluorescence peak wavelengths. Furthermore, bisphosphoryl-crosslinked stilbene compounds represented by general formulas (1C), (1C1), or (1C2) are more selective in staining oil droplets, while bisphosphoryl-crosslinked stilbene compounds represented by general formulas (3C), (3C1), or (3C2) are more selective in staining the inner mitochondrial membrane.

[0069] From the above, Ar 1 The structure allows for diversity in optical properties, and depending on the required properties, Ar 1 It is preferable to adjust the structure as appropriate.

[0070] In general formulas (1), (2), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1), (1D2), (3), (4), (3A), (3B), (3C), (3C1), (3C2), (3D), (3D1), and (3D2), Ar 2 Ar 3 Ar 4 Ar 4a and Ar 4bThe aromatic hydrocarbon ring represented by can be either a monocyclic or polycyclic aromatic hydrocarbon ring. For example, a monocyclic aromatic hydrocarbon ring is a benzene ring, and polycyclic aromatic hydrocarbon rings include naphthalene rings, anthracene rings, phenanthrene rings, fluorene rings, pyrene rings, and triphenylene rings. Of these, the benzene ring is particularly preferred from the viewpoint of oil droplet staining, identification of components inside oil droplets, cell identification, structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, and light resistance.

[0071] Ar 2 The aromatic hydrocarbon ring represented by may have substituents. Substituents include, for example, alkyl groups, cycloalkyl groups, aryl groups, heteroaryl groups, alkenyl groups (vinyl group, propenyl group, etc.), alkynyl groups (ethynyl group, 1-propynyl group, etc.), carbonyl groups, cyano groups, nitro groups, and -OR groups. 3 (R 3 Examples include (where represents an organic group). When substituents are present, the number of substituents is preferably 1 to 6, and more preferably 1 to 3.

[0072] Ar 3 The aromatic hydrocarbon ring shown is one that has no substituents. That is, Ar 3 Examples include the unsubstituted aromatic hydrocarbon rings mentioned above.

[0073] Ar 4 Ar 4a and Ar 4b The aromatic hydrocarbon ring shown has substituents. That is, Ar 4 Ar 4a and Ar 4b Examples include the aromatic hydrocarbon ring described above, having 1 to 6 (preferably 1 to 3) of the substituents described above.

[0074] In general formulas (1), (2), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1), (1D2), (3), (4), (3A), (3B), (3C), (3C1), (3C2), (3D), (3D1), and (3D2), Ar 2 Ar 3 Ar 4 Ar 4a and Ar 4b Examples of heteroaromatic rings represented by include, as monocyclic heteroaromatic rings, pyridine rings and pyrazine rings, and as polycyclic heteroaromatic rings, indole rings, isoindole rings, benzimidazole rings, quinoline rings, isoquinoline rings and quinoxaline rings.

[0075] Ar 2 The heteroaromatic ring represented by may have substituents. Substituents include, for example, alkyl groups, cycloalkyl groups, aryl groups, heteroaryl groups, alkenyl groups (vinyl group, propenyl group, etc.), alkynyl groups (ethynyl group, 1-propynyl group, etc.), carbonyl groups, cyano groups, nitro groups, halogen atoms (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), and -OR 3 (R 3 Examples include (where represents an organic group). When substituents are present, the number of substituents is preferably 1 to 6, and more preferably 1 to 3.

[0076] Ar 3 The heteroaromatic ring represented by is one that does not have substituents. That is, Ar 3 Examples include the unsubstituted heteroaromatic rings mentioned above.

[0077] Ar 4 Ar 4a and Ar 4b The heteroaromatic ring shown is one that has substituents. That is, Ar 4 Ar 4a and Ar 4b Examples include the heteroaromatic ring described above, having 1 to 6 (preferably 1 to 3) of the substituents described above.

[0078] Ar2 From the viewpoints of oil droplet staining, identification of tissues within oil droplets, cell identification, mitochondrial inner membrane staining, identification of tissues within the mitochondrial inner membrane, structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, and photoresistance, substituted or unsubstituted aromatic hydrocarbon rings are preferred, and substituted or unsubstituted monocyclic aromatic hydrocarbon rings (substituted or unsubstituted benzene rings) are more preferred.

[0079] Ar 3 From the viewpoints of oil droplet staining, identification of tissues within oil droplets, cell identification, mitochondrial inner membrane staining, identification of tissues in the mitochondrial inner membrane, structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, and light resistance, unsubstituted aromatic hydrocarbon rings are preferred, and unsubstituted monocyclic aromatic hydrocarbon rings (unsubstituted benzene rings) are more preferred.

[0080] Ar 4 Ar 4a and Ar 4b From the viewpoints of oil droplet staining, identification of tissues within oil droplets, cell identification, mitochondrial inner membrane staining, identification of tissues in the mitochondrial inner membrane, structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, and light resistance, substituted aromatic hydrocarbon rings are preferred, and substituted monocyclic aromatic hydrocarbon rings (substituted benzene rings) are more preferred.

[0081] In general formulas (1), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1), (1D2), (3), (3A), (3B), (3C), (3C1), (3C2), (3D), (3D1), and (3D2), R 1 and R 2 The alkyl group represented by can be either a linear alkyl group or a branched alkyl group. Examples include C1-10 alkyl groups (especially C1-6 alkyl groups) such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, and tert-butyl group.

[0082] R 1 and R 2The alkyl group represented by may have substituents. Examples of substituents include halogen atoms, cycloalkyl groups as described below, aryl groups as described below, heteroaryl groups as described below, cyano groups, nitro groups, and the like. When substituents are present, the number of substituents is preferably 1 to 6, and more preferably 1 to 3.

[0083] In general formulas (1), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1), (1D2), (3), (3A), (3B), (3C), (3C1), (3C2), (3D), (3D1), and (3D2), R 1 and R 2 Examples of cycloalkyl groups represented by include C3-10 cycloalkyl groups (especially C4-8 cycloalkyl groups) such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl groups.

[0084] R 1 and R 2 The cycloalkyl group represented by may have substituents. Examples of substituents include halogen atoms, the alkyl group described above, the aryl group described below, the heteroaryl group described below, the cyano group, the nitro group, and so on. When substituents are present, the number of substituents is preferably 1 to 6, and more preferably 1 to 3.

[0085] In general formulas (1), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1), (1D2), (3), (3A), (3B), (3C), (3C1), (3C2), (3D), (3D1), and (3D2), R 1 and R 2 The aryl group represented by can be any of the following: monocyclic aryl group, fused aryl group, or polycyclic aryl group. For example, the phenyl group is a monocyclic aryl group; the naphthyl group, anthracenyl group, phenantrenyl group, fluorenyl group, pyrenyl group, triphenylenyl group, etc., are fused aryl groups; and C6-18 aryl groups (especially C6-14 aryl groups) such as biphenyl group and terphenyl group are examples of polycyclic aryl groups.

[0086] R 1 and R 2 The aryl group represented by may have substituents. Examples of substituents include halogen atoms, the alkyl group mentioned above, the aryl group mentioned above, the heteroaryl group described later, cyano group, nitro group, etc. When substituents are present, the number of substituents is preferably 1 to 6, and more preferably 1 to 3.

[0087] In general formulas (1), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1), (1D2), (3), (3A), (3B), (3C), (3C1), (3C2), (3D), (3D1), and (3D2), R 1 and R 2 The heteroaryl group represented by can be either a monocyclic heteroaryl group or a fused heteroaryl group. For example, monocyclic heteroaryl groups include pyrrolyl, thienyl, furanyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, piperidyl, pyridyl, and pyrazyl groups, while fused heteroaryl groups include indolyl, isoindolyl, benzimidazolyl, quinolyl, isoquinolyl, and quinoxalyl groups.

[0088] R 1 and R 2 The heteroaryl group represented by may have substituents. Examples of substituents include the halogen atom, alkyl group, aryl group, heteroaryl group, cyano group, nitro group, etc. When substituents are present, the number of substituents is preferably 1 to 6, and more preferably 1 to 3.

[0089] Among them, R 1 and R 2From the viewpoint of easily imparting environmental responsiveness, easily extending the absorption peak wavelength, and easily identifying cells, substituted or unsubstituted (hetero)aryl groups are preferred, substituted or unsubstituted aryl groups are more preferred, and unsubstituted aryl groups are even more preferred.

[0090] Note, R 1 and R 2 They may come together and form a ring with adjacent nitrogen atoms. That is, -NR 1 R 2 The base represented by,

[0091] [ka] It may also be a base represented by, for example.

[0092] Also, R 1 and / or R 2 is Ar 2 It may also form a ring with adjacent nitrogen atoms, that is, Ar 2 -NR 1 R 2 The structure represented by

[0093] [ka] They are equivalent.

[0094] In this case, Ar 2 In -NR 1 R 2 The substitutional position to which the group represented by is attached is not particularly restricted.

[0095] Ar 1 Ar 2 Ar 4 Ar 4a Ar 4b , and Ar 5 When the aromatic hydrocarbon ring and heteroaromatic ring shown have substituents, the substituent is -OR 3 In R 3 The organic group represented by is not particularly limited, and may be an alkyl group, a polyethylene glycol group, or a derivative group thereof (-(C2H4O) m R 7 Examples include:

[0096] Ar 1 Ar 2 Ar 4 Ar 4a Ar 4b , and Ar 5 When the aromatic hydrocarbon ring and heteroaromatic ring shown have substituents, the substituent is -OR 3 In R 3 The alkyl group shown can be either a linear alkyl group or a branched alkyl group, but it is preferable to select an appropriate group depending on the object to be stained.

[0097] R 3 The alkyl group represented by may have substituents. Examples of substituents include epoxy groups, phosphorus-containing groups, carboxyl groups, and alkoxycarbonyl groups (-COOR 8 ), amide group or its derivative group (-CONHR 9 Examples include:

[0098] R 3 There are no particular restrictions on the phosphorus-containing group used as a substituent on the alkyl group represented by , and from the viewpoint of easily controlling organelle localization and dispersibility, general formula (5): -P + R 4 n X 1 (4-n) - (5) [In the formula, R 4These are identical or different, and represent substituted or unsubstituted (hetero)aryl groups. X 1 These represent halogen atoms, either identical or distinct. n represents an integer between 1 and 3. Examples of groups represented by the following are given.

[0099] In general formula (5), R 4 The aryl group represented by can be any of the following: monocyclic aryl group, fused aryl group, or polycyclic aryl group. For example, the phenyl group is a monocyclic aryl group; the naphthyl group, anthracenyl group, phenantrenyl group, fluorenyl group, pyrenyl group, triphenylenyl group, etc., are fused aryl groups; and C6-18 aryl groups (especially C6-14 aryl groups) such as biphenyl group and terphenyl group are examples of polycyclic aryl groups.

[0100] R 4 The aryl group represented by may have substituents. Examples of substituents include halogen atoms, the alkyl group mentioned above, the aryl group mentioned above, the heteroaryl group described later, cyano group, nitro group, etc. When substituents are present, the number of substituents is preferably 1 to 6, and more preferably 1 to 3.

[0101] In general formula (5), R 4 The heteroaryl group represented by can be either a monocyclic heteroaryl group or a fused heteroaryl group. For example, monocyclic heteroaryl groups include pyrrolyl, thienyl, furanyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, piperidyl, pyridyl, and pyrazyl groups, while fused heteroaryl groups include indolyl, isoindolyl, benzimidazolyl, quinolyl, isoquinolyl, and quinoxalyl groups.

[0102] R 4The heteroaryl group represented by may have substituents. Examples of substituents include the halogen atom, alkyl group, aryl group, heteroaryl group, cyano group, nitro group, etc. When substituents are present, the number of substituents is preferably 1 to 6, and more preferably 1 to 3.

[0103] Among them, R 4 From the viewpoint of easily controlling organelle localization and dispersibility, substituted or unsubstituted aryl groups are preferred, with unsubstituted aryl groups being more preferred.

[0104] In general formula (5), X 1 Examples of halogen atoms represented by include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms. From the viewpoint of easily controlling organelle localization and dispersibility, chlorine atoms, bromine atoms, and iodine atoms are preferred, chlorine atoms, bromine atoms are more preferred, and bromine atoms are even more preferred.

[0105] In general formula (5), n is preferably an integer between 1 and 3, more preferably 2 or 3, and even more preferably 3, from the viewpoint of easily controlling organelle localization and dispersion.

[0106] R 3 Alkoxycarbonyl group (-COOR) as substituent of alkyl group shown 8 ) is R 8 As long as it contains an alkyl group (methyl group, ethyl group, n-propyl group, isopropyl group, etc.), it can be used without any particular limitations. Examples include methoxycarbonyl group, ethoxycarbonyl group, n-propoxycarbonyl group, isopropoxycarbonyl group, etc.

[0107] R 3 An amide group or its derivative group (-CONHR) as a substituent of the alkyl group shown. 9 ) is R 8It can be used without particular limitation as long as it contains a hydrogen atom or an alkyl group (methyl group, ethyl group, n-propyl group, isopropyl group, etc.), for example, an amide group, N-methylamide group, N-ethylamide group, Nn-propylamide group, N-isopropylamide group, etc.

[0108] In general formula (5), R 3 Polyethylene glycol group or its derivative group (-(C2H4O)) m R 7 ) is R 7 It can be used without particular limitations as long as it contains a hydrogen atom or an alkyl group (methyl group, ethyl group, n-propyl group, isopropyl group, etc.) and m is an integer from 1 to 100, for example, -(C2H4O) m CH3 can be preferably used.

[0109] R that satisfies the above conditions 3 Specifically, the organic groups represented by are:

[0110] [ka] [In the formula, R 8 and R 9 The same applies as above. Ph indicates a phenyl group. k is an integer from 5 to 15. m is an integer from 2 to 100. These are some examples.

[0111] Examples of bisphosphoryl-crosslinked stilbene compounds represented by general formula (1) that satisfy the above conditions include,

[0112] [ka]

[0113] [ka] These are some examples.

[0114] Furthermore, examples of bisphosphoryl-crosslinked stilbene compounds represented by general formula (3) that satisfy the above conditions include,

[0115] [ka]

[0116] [ka] Examples include bisphosphoryl-crosslinked stilbene compounds represented by formulas such as R. 5 and R 6 They are the same or different,

[0117] [ka] [In the formula, Ph represents a phenyl group. m represents an integer from 1 to 100.] Examples include the above. In particular, the bisphosphoryl crosslinked stilbene compounds shown in the examples described later are preferred.

[0118] The method for producing the bisphosphoryl-crosslinked stilbene compound described above is not particularly limited.

[0119] Ar 4 is a substituent, for example, -OR 3 (R 3 Bisphosphoryl-bridged stilbene compounds having a group represented by (where indicates an organic group) can be synthesized according to previously reported methods (Organic Letters (2020), 22(8), 3185-3189, Journal of Organic Chemistry (2015), 80(8), 3790-3797, etc.).

[0120] Also, Ar 4Bisphosphoryl-bridged stilbene compounds in which the compound is unsubstituted can be synthesized according to the previously reported method (Chem. Asian J., 13, 1616-1624 (2018)), for example, according to the examples described below. Compounds not described in the examples described below can also be synthesized in a similar manner, for example, by using an appropriate compound.

[0121] 3. Fluorescent dyes, oil droplet stains, and mitochondrial inner membrane stains The fluorescent dye of the present invention contains the above-mentioned bisphosphoryl crosslinked stilbene compound.

[0122] The fluorescent dye of the present invention has excellent lightfastness because it has a bisphosphoryl-crosslinked stilbene skeleton.

[0123] Furthermore, when using a bisphosphoryl-crosslinked stilbene compound represented by general formula (1), the fluorescence lifetime differs depending on the internal structure of the oil droplet (e.g., triacylglycerol and cholesterol esters within the lipid droplet). Therefore, it is possible to identify and distinguish between the internal structure of the oil droplet, such as triacylglycerol and cholesterol esters, and estimate the composition of the oil droplet. In addition, since the composition of neutral fats constituting lipid droplets is known to differ depending on the cell type, the fluorescence lifetime of the above-mentioned bisphosphoryl-crosslinked stilbene compound also differs, and the cell type can be identified by the fluorescence lifetime. Taking triacylglycerol and cholesterol esters in lipid droplets as an example, the above-mentioned bisphosphoryl-crosslinked stilbene compound exhibits a fluorescence where the fluorescence lifetime increases with increasing cholesterol ester content. Therefore, the fluorescence lifetime increases with higher cholesterol levels. For this reason, it is possible to estimate the composition of triacylglycerol and cholesterol esters in lipid droplets from the fluorescence lifetime of the above-mentioned bisphosphoryl-crosslinked stilbene compound, and it is expected that insights can be gained into the processes by which lipid droplets store cholesterol. Furthermore, the bisphosphoryl-crosslinked stilbene compound described above can strongly fluoresce oil droplets such as intracellular fat globules (lipid droplets) while suppressing fluorescence from tissues other than oil droplets. By suppressing fluorescence from tissues other than oil droplets, the bisphosphoryl-crosslinked stilbene compound can significantly increase the fluorescence intensity of oil droplets compared to other tissues. As a result, even small oil droplets (especially intracellular fat droplets) can be detected with high sensitivity, and fluorescence staining of other tissues can be suppressed. Therefore, the fluorescent dye of the present invention using the bisphosphoryl-crosslinked stilbene compound represented by general formula (1) is useful in that it can be used as an oil droplet staining agent (especially a lipid droplet staining agent). In addition, by using such a fluorescent dye of the present invention, it is possible to estimate the composition of oil droplets and identify the type of cell.

[0124] Furthermore, when using a bisphosphoryl-crosslinked stilbene compound represented by general formula (3), the fluorescence lifetime differs depending on the structure of the mitochondrial inner membrane (e.g., phosphatidylcholine (PC), cholesterol esters, phosphatidylethanolamine (PE), cardiolipin (Cl), etc. in the mitochondrial inner membrane). Therefore, it is possible to distinguish between the structures of the mitochondrial inner membrane, such as phosphatidylcholine (PC) and cholesterol esters, which have long fluorescence lifetimes and tend to form a homogeneous phase, and phosphatidylcholine (PC) and cholesterol esters, which have short fluorescence lifetimes and tend to form a heterogeneous phase, as well as phosphatidylethanolamine (PE), cardiolipin (Cl), etc., and to estimate the membrane structure (membrane composition) of the mitochondrial inner membrane. Moreover, the bisphosphoryl-crosslinked stilbene compound represented by general formula (3) can strongly fluoresce the mitochondrial inner membrane and suppress the fluorescence of other tissues. By suppressing the fluorescence of tissues other than the mitochondrial inner membrane, the bisphosphoryl-crosslinked stilbene compound represented by general formula (3) can make the fluorescence intensity of the mitochondrial inner membrane significantly greater than that of other tissues. Therefore, mitochondrial inner membrane tissue can be detected with high sensitivity even at small sizes, and furthermore, the fluorescence staining of other tissues can be suppressed. For this reason, the fluorescent dye of the present invention, which uses a bisphosphoryl crosslinked stilbene compound represented by general formula (3), is useful in that it can be used as a mitochondrial inner membrane staining agent. In addition, by using such a fluorescent dye of the present invention, the membrane structure (membrane composition) of the mitochondrial inner membrane can be estimated.

[0125] The fluorescent dye (oil droplet staining agent or mitochondrial inner membrane staining agent) of the present invention contains the above-mentioned bisphosphoryl-bridged stilbene compound or its solvate. Its usage form is not particularly limited. For example, it can be dissolved in an organic solvent to form a solution. At this time, even in tissues with small-sized oil droplets or mitochondrial inner membranes, it is easy to detect (stain) with high sensitivity, easy to suppress the fluorescence of tissues other than oil droplets and mitochondrial inner membranes, easy to distinguish the tissues inside the oil droplets and mitochondrial inner membranes, and easy to distinguish cells. From this perspective, the content of the above-mentioned bisphosphoryl-bridged stilbene compound is preferably 10 nmol / L to 10 μmol / L, and more preferably 100 nmol / L to 5 μmol / L. In particular, even if the content is reduced to a small amount such as 100 nmol to 500 nmol, it is possible to detect tissues of oil droplets (especially intracellular lipid droplets) and mitochondrial inner membranes with high sensitivity. Thus, in the present invention, since the content of the bisphosphoryl-bridged stilbene compound can be kept low, it is easy to suppress damage to living cells.

[0126] When the fluorescent dye (oil droplet staining agent or mitochondrial inner membrane staining agent) of the present invention is made into a solution containing the above-mentioned bisphosphoryl-bridged stilbene compound, from the perspective of being easy to fluoresce oil droplets (especially lipid droplets) with high sensitivity, easy to distinguish the components inside the oil droplets, and easy to distinguish cells, a non-polar solvent is preferred as the organic solvent that can be used.

[0127] Examples of non-polar solvents include aliphatic organic solvents such as pentane, hexane, cyclohexane, and heptane; aromatic solvents such as benzene, toluene, xylene, and mesitylene; aliphatic halogenated hydrocarbons such as dichloromethane and dichloroethane, etc.

[0128] When the fluorescent dye (oil droplet staining agent or mitochondrial inner membrane staining agent) of the present invention is in the form of a solution, it is easy to use in living cells, and even with a small size, it is easy to detect the structure of oil droplets (especially lipid droplets in cells) and mitochondrial inner membranes with high sensitivity, easy to suppress the fluorescence staining of other tissues, easy to identify the internal structure of oil droplets and mitochondrial inner membranes, and easy to identify cells. From this perspective, the pH is preferably about 5 to 11, and more preferably about 6.5 to 7.5. To adjust the pH of the oil droplet staining agent of the present invention, a buffer (such as a HEPES buffer, a Tris buffer, a Tris-HCl buffer, a phosphate buffer, a phosphate buffered saline, etc.) can also be used.

Examples

[0129] Based on the examples, the present invention will be specifically described, but the present invention is not limited thereto.

[0130] Unless otherwise specified, all reactions were carried out under a nitrogen atmosphere. Unless otherwise specified, commercially available solvents and reagents were used without purification.

[0131] In the examples, the fluorescence lifetime was measured by a Hamamatsu Picosecond Fluorescence Measurement System C4780 (PLP-10 laser). The fluorescence lifetime imaging microscopy (FLIM) images were acquired by a Leica TCS SP8 STED 3X and imaged by FLIMfit.

[0132] Example 1: MitoPBRed

[0133]

Chemical formula

[0134] 2-Bromo-3-iodonaphthalene was synthesized according to the previously reported method (Synthesis, 2005, 5, 798-803).

[0135] (10-bromo-n-decyl)triphenylphosphonium bromide was synthesized according to a previously reported method (Chem, Commn, 2014, 50, 15366-15369).

[0136] (Reaction 1)

[0137] [ka] 2-bromo-3-iodonaphthalene (5.4 g, 16.2 mmol), bis(triphenylphosphine)palladium dichloride (PdCl2(PPh3)2; 231 mg, 0.33 mmol), CuI (62 mg, 0.33 mmol), and triethylamine (Et3N; 70 mL) were added to a nitrogen-purged flask and strictly degassed by freeze-degassing. Trimethylsilylacetylene (2.47 mL) was added by syringe and stirred overnight at room temperature. The mixture was filtered to remove insoluble compounds, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane) to obtain 2-bromo-3-[2-(trimethylsilyl)ethynyl]naphthalene as a yellow liquid (4.3 g, yield 86%). 1 H NMR (500 MHz, CDCl3) δ 8.05 (d, J = 19.9 Hz, 2H), 7.77-7.70 (m, 2H), 7.51-7.48 (m, 2H), 0.31 (s, 7H).

[0138] To a solution of the obtained 2-bromo-3-[2-(trimethylsilyl)ethynyl]naphthalene (4.2 g, 14.0 mmol) in CH3OH / tetrahydrofuran (1 / 1, v / v, 140 mL), K2CO3 (5.8 g, 42.0 mmol) was added and the mixture was stirred at room temperature for 8 hours. The product was extracted with CH2Cl2 (40 mL), washed with 1 M HCl (100 mL), and then dried over Na2SO4. The solvent was removed under reduced pressure to obtain 2-bromo-3-ethynylnaphthalene as a brown solid (3.1 g, 97% yield). 1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 8.6 Hz, 2H), 7.80-7.71 (m, 2H), 7.56-7.48 (m, 2H), 3.40 (s, 1H).

[0139] A suspension of 2-bromo-4-chloro-1-iodobenzene (3.98 g, 12.5 mmol), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4; 1.45 g, 13.0 mmol), and CuI (47.7 mg, 0.25 mmol) in triethylamine (150 mL) was degassed, and the 2-bromo-3-ethynylnaphthalene (2.9 g, 12.5 mmol) obtained above was added to it. After stirring overnight at room temperature, the mixture was diluted with toluene (150 mL), filtered, and insoluble compounds were removed. The resulting filtrate was concentrated under reduced pressure, recrystallized from ethanol, and purified to obtain 2-bromo-3-((2-bromo-4-chlorophenyl)ethynyl)naphthalene (compound 1) as a white solid (4.4 g, yield 85%). 1 H NMR (500 MHz, CDCl3) δ 8.12 (d, J = 5.5 Hz, 2H), 7.84-7.77 (m, 1H), 7.76-7.72 (m, 1H), 7.66 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.54-7.49 (m, 2H), 7.31 (dd, J = 8.3, 2.0 Hz, 1H). 13 C NMR (125 MHz, CDCl3) δ 134.98, 134.23, 133.97, 133.76, 132.47, 131.75, 131.24, 127.97, 127.80, 127.65, 127.08, 126.99, 126.03, 123.93, 122.17, 121.63, 93.61, 91.03.

[0140] (Reaction 2)

[0141] [ka] To a solution of 2-bromo-3-((2-bromo-4-chlorophenyl)ethynyl)naphthalene (compound 1; 1.0 g, 2.38 mmol) in anhydrous tetrahydrofuran (anhydrous THF; 20 mL), a solution of tert-butyllithium (tBuLi) in n-pentane (1.6 M, 6.1 mL, 9.7 mmol) was added dropwise over 0.5 hours at -78°C. After stirring at -78°C for 2 hours, chloro-N,N-diethylamino-(4-methoxyphenyl)phosphine (compound 2; 1.23 g, 5.0 mmol) was added, and the resulting mixture was stirred at -78°C for 0.5 hours, then kept at 0°C for 30 minutes. After cooling again to -78°C, PCl3 (1.3 mL, 15 mmol) was added, and the mixture was raised to room temperature. After stirring at room temperature for 17 hours, water (1 mL) was added to the mixture. Next, an aqueous solution of H2O2 (30%, 0.8 mL) was added at 0°C, and after stirring for 30 minutes, an aqueous solution of Na2SO3 (10%, 50 mL) was added at 0°C. The reaction mixture was extracted with ethyl acetate (siRNA; 200 mL), the organic layer was washed with saline solution (30 mL), dried with anhydrous Na2SO4, and filtered. The obtained filtrate was concentrated under reduced pressure, and the resulting mixture was purified by silica gel column chromatography (CH2Cl2 / acetone = 1 / 10 to 1 / 1) to obtain 469 mg of trans-PO-Na2 as a greenish-yellow solid (yield 34%) and 540 mg of cis-PO-Na2 as a greenish-yellow solid (39%). Trans-PO-Na2: 1 H NMR (400 MHz, CDCl3) δ 8.11 (dd, J = 11.5, 1.8 Hz, 1H), 7.84-7.72 (m, 7H), 7.60 (dt, J = 11.3, 1.4 Hz, 1H), 7.56-7.45 (m, 2H), 7.42-7.34 (m, 2H), 7.02-6.92 (m, 4H), 3.80 (d, J = 0.8 Hz, 6H). 31 P NMR (162 MHz, CDCl3) δ 32.54, 32.32, 31.04, 30.82. HRMS (ESI): m / z calcd. For C32 H 24 ClO4P2: 569.0833 ([M+H] + ); found: 569.0826。 Cis-PO-Na2: 1 1H NMR (400 MHz, CDCl3) δ 8.14 (dd, J = 11.5, 1.7 Hz, 1H), 7.87 (d, J = 3.3 Hz, 1H), 7.80 (t, J = 8.2 Hz, 2H), 7.69 - 7.59 (m, 5H), 7.59 - 7.48 (m, 2H), 7.48 - 7.41 (m, 2H), 6.97 - 6.91 (m, 4H), 3.82 (d, J = 1.3 Hz, 6H). 31 31P NMR (162 MHz, CDCl3) δ 31.00 (d, J = 37.0 Hz), 29.78 (d, J = 37.0 Hz). HRMS (ESI): m / z calcd. For C 32 H 24 ClO4P2: 569.0833 ([M+H] + ); found: 569.0827。

[0142] (Reaction 3)

[0143]

Chem.

[0144] (Reaction 4)

[0145] [ka] To a solution of the obtained trans-PO-Naphox(2,3)M (90 mg, 0.128 mmol) in anhydrous CH2Cl2 (8 mL), BBr3 (0.28 mL, 2.56 mmol) was slowly added dropwise at -78°C. After stirring at -78°C for 1 hour, the mixture was slowly heated to room temperature over 4 hours. Water (5 mL) was added at 0°C, and the mixture was extracted with ethyl acetate (Â; 20 mL × 2). After drying the organic layer over Na2SO4, the crude product was purified by column chromatography (CH3OH / CH2Cl2 = 1 / 20) to obtain trans-PO-Na3 as a red solid (80 mg, yield 92%). 1 H NMR (400 MHz, CH3OH-d4) δ8.19 (dd, J = 11.7, 1.9 Hz, 1H), 7.95-7.84 (m, 2H), 7.78 (d, J = 3.4 Hz, 1H), 7.73-7.63 (m, 4H), 7.63-7.52 (m, 2H), 7.36-7.26 (m, 5H), 7.20 (dt, J = 11.4, 2.0 Hz, 1H), 7.16-7.03 (m, 7H), 6.98-6.91 (m, 4H). 31 P NMR (162 MHz, CH3OH-d4) δ 36.29 (d, J = 38.0 Hz), 34.71 (d, J = 38.4 Hz). HRMS (ESI): m / z calcd. For C 42 H 30 NO4P2: 673.1650 ([M+H] + ); found: 674.1641.

[0146] (Reaction 5)

[0147] [ka] The obtained trans-PO-Na3 (30 mg, 0.0445 mmol), (10-bromodecyl)triphenylphosphonium bromide (25 mg, 0.0445 mmol), and K2CO3 (123 mg, 0.89 mmol) were added to anhydrous dimethylformamide (anhydrous DMF; 3.6 mL) and stirred at room temperature for 24 hours. Then, epibromohydrin (12.2 mg, 0.089 mmol) was added at room temperature, and the mixture was stirred at 50°C for 14 hours. After cooling to room temperature, water (20 mL) was added, and the mixture was extracted with CH2Cl2 (20 mL). The organic layer was washed twice with water (20 mL), dried over anhydrous Na2SO4, and filtered. After concentrating the filtrate under reduced pressure, the resulting solid was purified by silica gel column chromatography (CH3OH / CH2Cl2 = 1 / 30-1 / 10) to obtain trans-PO-NaphoxA1 / A2 as an orange-red solid (13 mg, yield 24%). HRMS (ESI): m / z calcd. For C 73 H 67 NO5P3: 1130.4227 ([M-Br] + ); found: 1130.4229.

[0148] Example 2: LipiCo

[0149] [ka] In Example 1, instead of reacting 2-bromo-3-((2-bromo-4-chlorophenyl)ethynyl)naphthalene (compound 1) with compound 2, a compound that is not methoxy-substituted:

[0150] [ka] The reaction proceeds in the same manner as in Reactions 1-2 of Example 1, except for reacting compound 1 with compound 1, to obtain the target compound LipiCo:

[0151] [ka] I obtained it.

[0152] To a solution of 2-bromo-3-((2-bromo-4-chlorophenyl)ethynyl)naphthalene (compound 1; 1.27 g, 3.02 mmol) obtained in Reaction 1 of Example 1 in anhydrous tetrahydrofuran (anhydrous THF; 25 mL), a solution of tert-butyllithium (tBuLi) in n-pentane (1.6 M, 7.7 mL, 12.3 mmol) was added dropwise at -78°C. After stirring at -78°C for 2 hours, chloro-N,N-diethylaminophenylphosphine (compound 4; 1.24 mL, 6.30 mmol) was added, and the resulting mixture was stirred at -78°C for 0.5 hours, then kept at 0°C for 0.5 hours. After cooling again to -78°C, PCl3 (1.6 mL, 18.3 mmol) was added, and the mixture was raised to room temperature. After stirring at room temperature for 19 hours, water (1 mL) was added to the mixture. Next, an aqueous solution of H2O2 (30%, 50 mL) was added at 0°C, the reaction mixture was extracted with ethyl acetate (₹), the organic layer was washed with saline solution, dried with anhydrous Na2SO4, and filtered. The obtained filtrate was concentrated under reduced pressure, and the resulting mixture was purified by silica gel column chromatography (CHCl3, trans-6 Rf=0.15, cis-6 Rf=0.08) to obtain 837 mg (1.64 mmol, yield 55%) of trans-6 and 482 mg (0.95 mmol, 31%) of cis-6 as a yellowish-green solid. Trans-6: 1 H NMR (400 MHz, CDCl3): δ 8.16 (d, J = 12.0 Hz, 1 H), 7.93-7.83 (m, 5 H), 7.81 (d, J = 9.6 Hz, 2 H), 7.65-7.39 (m, 11 H). 31 P{ 1 H} NMR (202 MHz, CDCl3): δ 32.2 (d, J = 38.8 Hz), 30.8 (d, J = 38.8 Hz). HRMS (ESI): m / z calcd. for C 30 H 19 ClNaO2P2: 531.0441 ([M+Na] +); found. 531.0437. Sys-6: 1 H NMR (400 MHz, CDCl3): δ 8.18 (dd, J = 11.4 Hz, 1.8 Hz, 1 H), 7.91 (d, J = 3.7 Hz, 1 H), 7.82 (t, J = 8.2 Hz, 2 H), 7.76-7.65 (m, 6 H), 7.60-7.45 (m, 9 H). 31 P{ 1 H} NMR (202 MHz, CDCl3): δ 30.7 (d, J = 39.0 Hz), 29.6 (d, J = 39.0 Hz). HRMS (ESI): m / z calcd. for C 30 H 19 ClNaO2P2: 531.0441 ([M+Na] + ); found. 531.0436.

[0153] The obtained trans-6 (219 mg, 0.43 mmol), diphenylamine (259 mg, 1.53 mmol), palladium acetate (Pd(OAc)2; 15 mg, 0.07 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (Xphos; 26 mg, 0.05 mmol), and K2CO3 (314 mg, 2.27 mmol) were added to anhydrous toluene (25 mL) and stirred under reflux under a nitrogen atmosphere for 38 hours. After cooling to room temperature, an aqueous solution of HCl (1 M) was added, and the mixture was extracted three times with toluene. The combined organic layer was washed with saline solution, dried over anhydrous Na2SO4, and then filtered. After concentrating the filtrate under reduced pressure, the resulting solid was subjected to silica gel column chromatography (CH2Cl2~7 / 1 CH2Cl2 / acetone, Rf=0.6 (in CH2Cl2 / acetone)). The product was further purified by preparative gel permeation chromatography to obtain LipiCo (91.1 mg, 0.142 mmol, yield 33%) as a red solid. 1H NMR (500 MHz, acetone-d6): δ 8.25 (d, J = 10.9 Hz, 1H), 8.00 (t, J = 7.2 Hz, 2H), 7.87 (dd, J = 12.3 Hz, 7.2 Hz, 5H), 7.65 (q, J = 7.8 Hz, 3H), 7.60-7.55 (m, 5H), 7.39-7.33 (m, 5H), 7.23 (d, J = 11.5 Hz, 1H), 7.15 (t, J = 7.2 Hz, 6H), 7.08 (d, J = 8.6 Hz, 1H). 31 P{ 1 H} NMR (202 MHz, CDCl3): δ 32.9 (d, J = 33.3 Hz), 31.1 (d, J = 33.3 Hz). HRMS (ESI): m / z calculus for C 42 H 29 NNaO2P2: 664.1566([M+Na] + ); found. 664.1559.

[0154] Test Example 1: Staining of the inner mitochondrial membrane HeLa cells were cultured in DMEM medium containing 500 nM MitoPBRed (Example 1), 10% FBS, 0.5% DMSO, and 0.05% pluronic F127 in a 5 vol. % CO2 incubator at 37°C for 2 hours. After washing the cells three times with DMEM(-), the medium was replaced with DMEM(+), and the mitochondrial inner membrane was observed by fluorescence lifetime imaging (FLIM) using a Leica TCS SP8 STED 3X microscope. The excitation wavelength was 490 nm, the fluorescence wavelength was 550-750 nm, and the amplitude was 20 MHz. The results are shown in Figure 1 and the left panel of Figure 2.

[0155] Next, HeLa cells were cultured in DMEM medium containing 500 nM MitoPBRed, 10% FBS, 0.5% DMSO, and 0.05% pluronic F127 in a 5 vol. % CO2 incubator at 37°C for 2 hours. The cells were washed three times with DMEM(-), then replaced with DMEM(+), and subsequently, carbonyl cyanide-m-chlorophenylhydrazone (CCCP) was added to a concentration of 10 μM. The cells were then incubated at 37°C for 3 hours. Similarly, the results observed by fluorescence lifetime imaging microscopy (FLIM) are shown in the right panel of Figure 2, and the difference in fluorescence lifetime of MitoPBRed before and after CCCP addition is shown in Figure 3. Note that the right panel of Figure 2 shows the same region as the left panel of Figure 2.

[0156] Based on the above results, the homogeneous liquid phase consists of domains rich in phosphatidylcholine (PC), which tend to be more hydrophobic and have a longer fluorescence lifetime, and is therefore shown in red in Figure 1 and the upper left of Figure 2. On the other hand, the heterogeneous liquid phase consists of domains rich in phosphatidylethanolamine (PE) and cardiolipin (Cl), which tend to be more hydrophilic and have a shorter fluorescence lifetime, and is therefore shown in blue in the upper left of Figure 1. Even within the same mitochondrial inner membrane, it is assumed that the homogeneous liquid phase has higher levels of phosphatidylcholine (PC) and cholesterol esters, while the heterogeneous liquid phase has lower levels of phosphatidylethanolamine (PE) and cardiolipin (Cl), depending on the region. Furthermore, it is speculated that after treatment with carbonyl cyanide-m-chlorophenylhydrazone (CCCP), the levels of phosphatidylcholine (PC) and cholesterol esters increase due to the degradation of unsaturated phospholipids by ROS, leading to a stiffening of the mitochondrial inner membrane and an increase in fluorescence lifetime.

[0157] Test Example 2: Staining of lipid droplets in various cells One day prior to imaging, each cell was treated with 400 μM oleic acid. The cells were cultured for 3 hours in DMEM medium containing 500 nM LipiCo (Example 2), 10% FBS, and 0.1% DMSO. After washing three times with DMEM(-), the medium was replaced with DMEM(+), and intracellular lipid droplets were observed using fluorescence lifetime imaging (FLIM) with a Leica TCS SP8 STED 3X. The excitation wavelength was 488 nm, the fluorescence wavelength was 550-650 nm, and the amplitude was 20 MHz.

[0158] The results are shown in Figure 4. In Figure 4, the left panel shows a fluorescence lifetime imaging (FLIM) image, the middle panel shows a bright-field image, and the right panel shows a histogram of the number of pixels in the fluorescence lifetime imaging (FLIM) images sorted by fluorescence lifetime. In the middle panel of Figure 4, the scale bar is 10 μm. These results show that fluorescence lifetimes differ depending on the cell type, suggesting that cells can be identified by these differences in fluorescence lifetime.

[0159] Here, taking HuH-7 cells as an example, tissues with different fluorescence lifetimes were found depending on the location. In addition to Figure 4, Figure 5 shows the results of staining HuH-7 cells using the same method. From Figure 5, it can be seen that even within a single cell, the fluorescence lifetime showed a non-uniform distribution ranging from 5.5 nanoseconds to 6.8 nanoseconds depending on the tissue. Note that in Figure 4, measurements were taken in the range of 6.0 to 8.5 nanoseconds, whereas in Figure 5, measurements were taken in the range of 6.5 to 8.0 nanoseconds.

[0160] By utilizing the unique properties of LipiCo (Example 2), it is possible to observe the metabolic accumulation process of cholesterol in lipid droplets of hepatocytes.

[0161] HuH-7 cells were stained in the same manner as described above, except that cholesterol was added to 500 nM LipiCo (Example 2) to a concentration of 170 μM. Figure 6 shows the fluorescence lifetime results when HuH-7 cells were stained using fluorescence lifetime imaging (FLIM) immediately after cholesterol addition and 6 hours later. The left panel of Figure 6 shows the fluorescence lifetime results immediately after cholesterol addition, and the right panel of Figure 6 shows the fluorescence lifetime results 6 hours after cholesterol addition.

[0162] These results show that adding cholesterol increases the fluorescence lifetime by increasing the culture time. In this case, the fluorescence lifetime is widely distributed, suggesting that cholesterol is not uniformly incorporated into all lipid droplets, but rather that each lipid droplet plays a different role in the metabolic process.

Claims

1. General formula (1): 【Chemistry 1】 [In the formula, Ar １ and Ar ２ These are identical or different, and represent an unsubstituted aromatic hydrocarbon ring. R １ and R ２ These are the same or different, representing a hydrogen atom, an unsubstituted alkyl group, an unsubstituted cycloalkyl group, or an unsubstituted aryl group. Y １ and Y ２ They are the same or different, and general formula (2): 【Chemistry 2】 (Ar ３ This indicates an unsubstituted benzene ring. Y ３ (This represents an oxygen atom or a sulfur atom.) This indicates the group represented by [ ]. A bisphosphoryl-crosslinked stilbene compound represented by the formula.

2. The above-mentioned Ar １ The bisphosphoryl crosslinked stilbene compound according to claim 1, wherein the above-mentioned Ar is an unsubstituted polycyclic aromatic hydrocarbon ring.

3. The Ar ２ The bisphosphoryl-bridged stilbene compound according to claim 1 or 2, wherein the ring is an unsubstituted polycyclic aromatic hydrocarbon ring.

4. The aforementioned Y ３ The bisphosphoryl-bridged stilbene compound according to any one of claims 1 to 3, wherein the atom is an oxygen atom.

5. A fluorescent dye containing a bisphosphoryl crosslinked stilbene compound according to any one of claims 1 to 4.

6. An oil droplet stain containing the bisphosphoryl crosslinked stilbene compound described in any one of claims 1 to 4.

7. An oil droplet staining agent according to claim 6, which identifies tissue within an oil droplet by fluorescence lifetime.

8. General formula (3): 【Transformation 3】 [In the formula, Ar １ and Ar ２ These are identical or different, and represent an unsubstituted aromatic hydrocarbon ring. R １ and R ２ These are the same or different, representing a hydrogen atom, an unsubstituted alkyl group, an unsubstituted cycloalkyl group, or an unsubstituted aryl group. Y ４ and Y ５ They are the same or different, and the general formula (4): 【Chemistry 4】 (Ar ４ R represents a benzene ring having a substituent, and the substituent represents an alkyl group, a cycloalkyl group, an aryl group, a heteroaryl group, an alkenyl group, an alkynyl group, a carbonyl group, a cyano group, a nitro group, a halogen atom, or -OR3, where R3 represents an alkyl group, a polyethylene glycol group, or a derivative thereof. Y ６ (This represents an oxygen atom or a sulfur atom.) This indicates the group represented by [ ]. A mitochondrial inner membrane staining agent containing a bisphosphoryl crosslinked stilbene compound represented by [formula].

9. A mitochondrial inner membrane staining agent according to claim 7 or 8, which identifies tissue present in the mitochondrial inner membrane by fluorescence lifetime.

10. A method for estimating the composition of an oil droplet, which utilizes the fact that the fluorescence lifetime differs depending on the internal structure of the oil droplet, with the fluorescence lifetime increasing as the cholesterol content increases, and uses a bisphosphoryl crosslinked stilbene compound according to any one of claims 1 to 4, a fluorescent dye according to claim 5, or an oil droplet staining agent according to claim 6 or 7 to identify the internal structure of the oil droplet.

11. A method for estimating the membrane structure of a mitochondrial inner membrane, which utilizes the fact that the fluorescence lifetime differs depending on the tissue of the mitochondrial inner membrane, and uses the mitochondrial inner membrane staining agent described in claim 8 or 9 to identify the tissue of the mitochondrial inner membrane and estimate its membrane structure.

Images