JP7705648B2 - Bisphosphoryl-bridged stilbene compounds - Google Patents

Description

The present invention relates to bisphosphoryl-bridged stilbene compounds.

Fluorescent organic compounds with high fluorescence quantum yields are important as light-emitting materials for organic electroluminescence devices or fluorescent dyes for biofluorescence imaging, and numerous examples have been reported in both basic research and applications.

However, many of the conventionally known fluorescent organic compounds gradually decompose and fade when exposed to light for a long period of time. For example, Alexa Fluor dyes and ATTO dyes are well-known fluorescent probes that are dyes with enhanced light resistance, but even when these are used, it is difficult to repeatedly observe them with a super-resolution microscope such as stimulated emission depletion (STED) imaging. For this reason, the observation targets of cutting-edge fluorescent microscope technology are currently limited, and there is a demand for improving the light resistance of fluorescent dyes.

Among these conventional techniques, a phosphole compound with a specific structure called "MitoPB Yellow" is known as a fluorescent dye with excellent light resistance (see, for example, Non-Patent Document 1).

PNAS 2019, vol. 116, no. 32, 15817-15822.

However, in super-resolution microscopes such as stimulated emission depletion (STED) imaging, MitoPB Yellow uses a STED laser with a wavelength of 660 nm as the excitation wavelength, and phototoxicity has been observed for intracellular organelles such as mitochondria. For this reason, there is a demand to reduce phototoxicity for intracellular organelles by lengthening the excitation wavelength. In addition, MitoPB Yellow has demonstrated overwhelming light resistance that surpasses other existing fluorescent dyes, but the requirements for light resistance are endless, and there is a demand for the ability to reduce photobleaching even when exposed to light strong enough to cause photobleaching of MitoPB Yellow.

The present invention aims to solve the above-mentioned problems of the prior art, and aims to provide a compound that can stain intracellular organelles using a long-wavelength excitation wavelength that can reduce phototoxicity to intracellular organelles, and that has superior light resistance compared to MitoPB Yellow.

In view of the above problems, the inventors have conducted extensive research and found that a compound having a specific bisphosphoryl-bridged stilbene skeleton can stain intracellular organelles, has excellent light resistance, and shows almost no decrease in absorption intensity even when irradiated with a strong laser beam used in super-resolution microscopes such as stimulated emission depletion (STED) imaging. Based on this finding, the inventors have conducted further research and completed the present invention. That is, the present invention includes the following configurations.

Item 1. General formula (1):

［式中、
Ａｒ^１及びＡｒ^２は同一又は異なって、置換若しくは非置換芳香族炭化水素環又は置換若しくは非置換複素芳香環を示す。
Ｒ^１及びＲ^２は同一又は異なって、水素原子、置換若しくは非置換アルキル基、置換若しくは非置換シクロアルキル基、又は置換若しくは非置換（ヘテロ）アリール基を示す。
ただし、Ｒ^１及びＲ^２は一緒になって隣接する窒素原子とともに環を形成してもよい。Ｒ^１及び／又はＲ^２はＡｒ^２と一緒になって隣接する窒素原子とともに環を形成してもよい。
Ｙ^１及びＹ^２は同一又は異なって、一般式（２）：

[In the formula,
Ar ¹ and Ar ² are the same or different and each 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 each represents 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 ^R2 may be joined together to form a ring with the adjacent nitrogen atom, and ^R1 and/or ^R2 may be joined together with ^Ar2 to form a ring with the adjacent nitrogen atom.
Y ¹ and Y ² are the same or different and are represented by the general formula (2):

（Ａｒ^３は置換若しくは非置換芳香族炭化水素環又は置換若しくは非置換複素芳香環を示す。
Ｒ^３は水素原子又は有機基を示す。
Ｙ^３は酸素原子又は硫黄原子を示す。）
で表される基を示す。］
で表される、ビスホスホリル架橋スチルベン化合物。

( ^Ar3 represents a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring.
^R3 represents a hydrogen atom or an organic group.
^Y3 represents an oxygen atom or a sulfur atom.
It represents a group represented by the following formula:
A bisphosphoryl-bridged stilbene compound represented by the formula:

Item 2. The bisphosphoryl-bridged stilbene compound according to Item 1, wherein R ³ is a substituted or unsubstituted alkyl group.

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

Item 4. A fluorescent dye containing the bisphosphoryl-bridged stilbene compound described in any one of items 1 to 3.

Item 5. A staining agent for intracellular organelles, comprising the bisphosphoryl-bridged stilbene compound according to any one of items 1 to 3.

Item 6. A method for imaging intracellular organelles using the bisphosphoryl-bridged stilbene compound described in any one of items 1 to 3, the fluorescent dye described in item 4, or the intracellular organelle stain described in item 5.

The bisphosphoryl-bridged stilbene compound of the present invention has a longer absorption peak wavelength than MitoPB Yellow, and can stain intracellular organelles using a long-wavelength STED laser, for example, 775 nm. This reduces phototoxicity to intracellular organelles, and is also useful as a light-resistant electronic material for which a longer absorption peak wavelength is required.

In addition, the bisphosphoryl-bridged stilbene compound of the present invention has excellent light resistance and hardly fades even when exposed to strong light that causes photobleaching of MitoPB Yellow.

Therefore, the bisphosphoryl-bridged stilbene compounds of the present invention are suitable fluorescent dyes for repeated observation of intracellular organelles using super-resolution microscopy, such as stimulated emission depletion (STED) imaging.

The ultraviolet-visible absorption spectra, fluorescence spectra, absolute fluorescence quantum yields, and appearance photographs of cis-PO-BphoxM of Example 3 and trans-PS-BphoxC4 of Example 8 are shown in various solvents. The ultraviolet-visible absorption spectrum, fluorescence spectrum, absolute fluorescence quantum yield, and appearance photographs of Lance-PO-Naphox(1,2)M in various solvents are shown. 1 shows the light resistance in acetonitrile (MeCN) of trans-PO-BphoxM of Example 1 and Mito-PB yellow M of Comparative Example 1. (a) UV-visible absorption spectrum of Mito-PB yellow M of Comparative Example 1 after various times. (b) Maintenance rate (A/A ₀ ) of absorbance (A) after a predetermined time of Mito-PB yellow M of Comparative Example 1. (c) UV-visible absorption spectrum of trans-PO-BphoxM of Example 1 after various times. (d) Maintenance rate (A/A ₀ ) of absorbance (A) after a predetermined time of trans-PO-BphoxM of Example 1. 1 shows the light resistance in acetonitrile (MeCN) of cis-PO-BphoxM of Example 3 and trans-PO-Naphox(2,3)M of Example 5. (a) UV-visible absorption spectrum of cis-PO-BphoxM of Example 3 after various times. (b) Maintenance rate (A/A ₀ ) of absorbance (A) of cis-PO-BphoxM of Example 3 after a predetermined time. (c) UV-visible absorption spectrum of trans-PO-Naphox(2,3)M of Example 5 after various times. (d) Maintenance rate (A/A ₀ ) of absorbance (A) of trans-PO-Naphox(2,3)M of Example 5 after a predetermined time. The light resistance of trans-PO-Naphox (1,2) M of Example 4 in acetonitrile (MeCN) is shown. (a) UV-visible absorption spectrum of trans-PO-Naphox (1,2) M of Example 4 after various time periods. (b) Maintenance rate (A/A ₀ ) of absorbance (A) of trans-PO-Naphox (1,2) M of Example 4 after a predetermined time period. 1 shows the light resistance of trans-PO-Naphox (2,3) M of Example 5 and Mito-PB yellow M of Comparative Example 1 when irradiated with light of 515±5 nm in a dimethyl sulfoxide/water mixed solvent (DMSO/H ₂ O). (a) UV-visible absorption spectrum of Mito-PB yellow M of Comparative Example 1 after various times. (b) Maintenance rate (A/A ₀ ) of absorbance (A) after a predetermined time for Mito-PB yellow M of Comparative Example 1. (c) UV-visible absorption spectrum of trans-PO-Naphox (2,3) M of Example 5 after various times. (d) Maintenance rate (A/A ₀ ) of absorbance (A) after a predetermined time for trans-PO-Naphox (2,3) M of Example 5. 1 shows the light resistance of cis-PO-BphoxM of Example 3 when irradiated with light of 450±5 nm in a dimethyl sulfoxide/water mixed solvent (DMSO/H ₂ O). (a) UV-visible absorption spectrum after various times when irradiated with light of an intensity of 2050 W/m ^2. (b) Maintenance rate (A/A 0 ) of absorbance (A) after a predetermined time when irradiated with light of an intensity of 2050 W/m ^2. (c) UV-visible absorption spectrum after various times when irradiated with light of an intensity of 97% of the maximum intensity of the LED light. (d) Maintenance rate (A/A ₀ ) of absorbance (A) after a predetermined time when irradiated with light of an intensity of ₉₇ % of the maximum intensity of the LED light. 1 shows the light resistance of trans-PO-Naphox(2,3)M of Example 5 when irradiated with light of 450±5 nm in a dimethylsulfoxide/water mixed solvent (DMSO/H ₂ O). (a) UV-visible absorption spectrum after various times when irradiated with light of 2050 W/m ² intensity. (b) Maintenance rate (A/A ₀ ) of absorbance (A) after a predetermined time when irradiated with light of 2050 W/m ² intensity. (c) UV-visible absorption spectrum after various times when irradiated with light of 97% of the maximum intensity of LED light. (d) Maintenance rate (A/A ₀ ) of absorbance (A) after a predetermined time when irradiated with light of 97% of the maximum intensity of LED light. 1 shows the light resistance of Mito-PB ywllow M of Comparative Example 1 when irradiated with light of 450±5 nm in a dimethyl sulfoxide/water mixed solvent (DMSO/H ₂ O). (a) UV-visible absorption spectrum after various times when irradiated with light of 2050 W/m ² intensity. (b) Maintenance rate (A/A ₀ ) of absorbance (A) after a predetermined time when irradiated with light of 2050 W/m ² intensity. (c) UV-visible absorption spectrum after various times when irradiated with light of 97% of the maximum intensity of LED light. (d) Maintenance rate (A/A ₀ ) of absorbance (A) after a predetermined time when irradiated with light of 97% of the maximum intensity of LED light. 1 shows fluorescent imaging photographs of the cells of Test Example 6 using trans-PO-BphoxA1/A2 of Example 2 and Mito Tracker Deep Red of Comparative Example 2. 1 shows fluorescent imaging photographs of the cells of Test Example 6 using trans-PO-Naphox A1/A2 of Example 6 and Mito Tracker Deep Red of Comparative Example 2. 13 is a fluorescent imaging photograph of the cells of Test Example 7 using trans-PO-BphoxA1/A2 of Example 2. 13 is a fluorescent imaging photograph of the cells of Test Example 7 using trans-PO-NaphoxA1/A2 of Example 6. Fluorescence imaging photographs of the cells of Test Example 9 using trans-PO-BphoxM of Example 1, cis-PO-BphoxM of Example 3, trans-PO-Naphox(1,2)M of Example 4, trans-PO-Naphox(2,3)M of Example 5, and cis-PO-Naphox(2,3)M of Example 7.

In this specification, "comprise" is a concept that also encompasses "consist essentially of" and "consist only of."

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

1. Bisphosphoryl-bridged stilbene compound The bisphosphoryl-bridged stilbene compound of the present invention is represented by the general formula (1):

［式中、
Ａｒ^１及びＡｒ^２は同一又は異なって、置換若しくは非置換芳香族炭化水素環又は置換若しくは非置換複素芳香環を示す。
Ｒ^１及びＲ^２は同一又は異なって、水素原子、置換若しくは非置換アルキル基、置換若しくは非置換シクロアルキル基、又は置換若しくは非置換（ヘテロ）アリール基を示す。
ただし、Ｒ^１及びＲ^２は一緒になって隣接する窒素原子とともに環を形成してもよい。Ｒ^１及び／又はＲ^２はＡｒ^２と一緒になって隣接する窒素原子とともに環を形成してもよい。
Ｙ^１及びＹ^２は同一又は異なって、一般式（２）：

[In the formula,
Ar ¹ and Ar ² are the same or different and each 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 each represents 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 ^R2 may be joined together to form a ring with the adjacent nitrogen atom, and ^R1 and/or ^R2 may be joined together with ^Ar2 to form a ring with the adjacent nitrogen atom.
Y ¹ and Y ² are the same or different and are represented by the general formula (2):

（Ａｒ^３は置換若しくは非置換芳香族炭化水素環又は置換若しくは非置換複素芳香環を示す。
Ｒ^３は有機基を示す。
Ｙ^３は酸素原子又は硫黄原子を示す。）
で表される基を示す。］
で表される化合物である。

( ^Ar3 represents a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring.
^R3 represents an organic group.
^Y3 represents an oxygen atom or a sulfur atom.
It represents a group represented by the following formula:
It is a compound represented by the formula:

The bisphosphoryl-bridged stilbene compounds can be represented by the general formulae (1A) and (1B) depending on the position of the oxygen atom bonded to the phosphorus atom and the ring ^Ar3 :

［式中、
Ａｒ^１、Ａｒ^２、Ａｒ^３ａ及びＡｒ^３ｂは同一又は異なって、置換若しくは非置換芳香族炭化水素環又は置換若しくは非置換複素芳香環を示す。
Ｒ^１及びＲ^２は同一又は異なって、水素原子、置換若しくは非置換アルキル基、置換若しくは非置換シクロアルキル基、又は置換若しくは非置換（ヘテロ）アリール基を示す。
ただし、Ｒ^１及びＲ^２は一緒になって隣接する窒素原子とともに環を形成してもよい。Ｒ^１及び／又はＲ^２はＡｒ^２と一緒になって隣接する窒素原子とともに環を形成してもよい。
Ｒ^３は有機基を示す。
Ｙ^３ａ及びＹ^３ｂは同一又は異なって、酸素原子又は硫黄原子を示す。］
で表されるビスホスホリル架橋スチルベン化合物がいずれも包含される。

[In the formula,
Ar ¹ , Ar ² , Ar ^3a and Ar ^3b are the same or different and each represents 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 each represents 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 ^R2 may be joined together to form a ring with the adjacent nitrogen atom, and ^R1 and/or ^R2 may be joined together with ^Ar2 to form a ring with the adjacent nitrogen atom.
^R3 represents an organic group.
Y ^3a and Y ^3b are the same or different and each represents an oxygen atom or a sulfur atom.
The present invention also includes any bisphosphoryl-bridged stilbene compound represented by the following formula:

The trans-bisphosphoryl-bridged stilbene compound represented by the above general formula (1A) and the cis-bisphosphoryl-bridged stilbene compound represented by the general formula (1B) both have a longer absorption peak wavelength than MitoPB Yellow, and can stain intracellular organelles using a long-wavelength STED laser such as 775 nm, thereby reducing phototoxicity to intracellular organelles. They are also useful as light-resistant electronic materials that require longer absorption peak wavelengths. Furthermore, they have excellent light resistance and hardly fade even when exposed to strong light that causes photobleaching of MitoPB Yellow.

In this way, since the bisphosphoryl-bridged stilbene compound of the present invention has a functional group, -OR ³ group, bonded to the ring Ar ³ , it is possible to control the organelle localization and dispersibility, and it is possible to bind to various intracellular organelles and stain various intracellular organelles. In this case, as will be shown in the examples described later, the bisphosphoryl-bridged stilbene compound is selectively present at a desired site of an intracellular organelle, and is useful for grasping the external shape of the intracellular organelle. For example, when R ³ is a substituted alkyl group containing a phosphonium group, it can be used as a staining agent for mitochondrial membranes, and when R ³ is an alkyl group such as a methyl group, it can be used as a staining agent for lipid droplets. Therefore, since it can be used as an intracellular organelle staining agent, the bisphosphoryl-bridged stilbene compound of the present invention is a compound suitable for repeated observation in vivo with a super-resolution microscope such as stimulated emission depletion (STED) imaging.

In addition, the bisphosphoryl-bridged stilbene compound of the present invention has an amino group or a substituted amino group, which is an electron-donating group, and thus imparts environmental responsiveness and can achieve a longer absorption peak wavelength, thereby reducing phototoxicity to intracellular organelles.

Furthermore, the bisphosphoryl-bridged stilbene compound of the present invention has a bisphosphoryl-bridged stilbene skeleton, which makes it possible to impart light resistance to the extent that the absorption intensity is hardly reduced even when exposed to light strong enough to cause photobleaching of MitoPB Yellow.

In general formulae (1), (1A), and (1B), the aromatic hydrocarbon ring represented by ^Ar1 may be either a monocyclic aromatic hydrocarbon ring or a polycyclic aromatic hydrocarbon ring. For example, an example of the monocyclic aromatic hydrocarbon ring is a benzene ring, and an example of the polycyclic aromatic hydrocarbon ring is a naphthalene ring, an anthracene ring, a phenanthrene ring, a fluorene ring, a pyrene ring, a triphenylene ring, etc.

The aromatic hydrocarbon ring represented by ^Ar1 may have a substituent. Examples of the substituent include an alkyl group described below, a cycloalkyl group described below, an aryl group described below, a heteroaryl group described below, 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, etc. When the aromatic hydrocarbon ring has a substituent, the number of the substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

In the general formulae (1), (1A), and (1B), examples of the heteroaromatic ring represented by Ar ¹ include, as monocyclic heteroaromatic rings, a pyridine ring and a pyrazine ring, and as polycyclic heteroaromatic rings, an indole ring, an isoindole ring, a benzimidazole ring, a quinoline ring, an isoquinoline ring, and a quinoxaline ring.

The heteroaromatic ring represented by Ar ¹ may have a substituent. Examples of the substituent include an alkyl group described below, a cycloalkyl group described below, an aryl group described below, a heteroaryl group described below, 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.), and the like. When the heteroaromatic ring has a substituent, the number of the substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

As Ar ¹ , from the viewpoints of structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, light resistance, and the like, a substituted or unsubstituted aromatic hydrocarbon ring is preferred, and from the viewpoint of light resistance in particular, a polycyclic aromatic hydrocarbon ring is preferred.

When Ar ¹ is a substituted or unsubstituted polycyclic aromatic hydrocarbon ring, the bisphosphoryl-bridged stilbene compound of the present invention is represented by the general formulas (1C) and (1D):

［式中、Ａｒ^２、Ｙ^１、Ｙ^２、Ｒ^１及びＲ^２は前記に同じである。
Ａｒ^４は置換又は非置換芳香族炭化水素環を示す。］
で表されるビスホスホリル架橋スチルベン化合物のいずれも採用できる。

In the formula, Ar ² , Y ¹ , Y ² , R ¹ and R ² are the same as defined above.
^Ar4 represents a substituted or unsubstituted aromatic hydrocarbon ring.
Any of the bisphosphoryl-bridged stilbene compounds represented by the following formula (I) can be used.

The bisphosphoryl-bridged stilbene compound represented by the general formula (1C) can be represented by the general formulas (1C1) and (1C2) depending on the position of the oxygen atom bonded to the phosphorus atom and the ring ^Ar3 :

［式中、Ａｒ^２、Ａｒ^３ａ、Ａｒ^３ｂ、Ａｒ^４、Ｒ^１、Ｒ^２、Ｒ^３ａ、Ｒ^３ｂ、Ｙ^３ａ及びＹ^３ｂは前記に同じである。］
で表されるビスホスホリル架橋スチルベン化合物がいずれも包含される。

[In the formula, Ar ² , Ar ^3a , Ar ^3b , Ar ⁴ , R ¹ , R ² , R ^3a , R ^3b , Y ^3a and Y ^3b are the same as above.]
The present invention also includes any bisphosphoryl-bridged stilbene compound represented by the following formula:

The bisphosphoryl-bridged stilbene compound represented by the general formula (1D) can be represented by the general formulas (1D1) and (1D2) depending on the position of the oxygen atom bonded to the phosphorus atom and the ring ^Ar3 :

［式中、Ａｒ^２、Ａｒ^３ａ、Ａｒ^３ｂ、Ａｒ^４、Ｒ^１、Ｒ^２、Ｒ^３ａ、Ｒ^３ｂ、Ｙ^３ａ及びＹ^３ｂは前記に同じである。］
で表されるビスホスホリル架橋スチルベン化合物がいずれも包含される。

[In the formula, Ar ² , Ar ^3a , Ar ^3b , Ar ⁴ , R ¹ , R ² , R ^3a , R ^3b , Y ^3a and Y ^3b are the same as above.]
The present invention also includes any bisphosphoryl-bridged stilbene compound represented by the following formula:

In the general formulae (1C), (1C1), (1C2), (1D), (1D1) and (1D2), the aromatic hydrocarbon ring represented by ^Ar4 may be either a monocyclic aromatic hydrocarbon ring or a polycyclic aromatic hydrocarbon ring, and examples of the monocyclic aromatic hydrocarbon ring include a benzene ring, and examples of the polycyclic aromatic hydrocarbon ring include a naphthalene ring, an anthracene ring, a phenanthrene ring, a fluorene ring, a pyrene ring, a triphenylene ring, etc. Among these, a benzene ring is particularly preferred from the viewpoints of structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, light resistance, etc.

The aromatic hydrocarbon ring represented by ^Ar4 may have a substituent. Examples of the substituent include an alkyl group described below, a cycloalkyl group described below, an aryl group described below, a heteroaryl group described below, 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, etc. When the aromatic hydrocarbon ring has a substituent, the number of the substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

In the general formulae (1C), (1C1), (1C2), (1D), (1D1), and (1D2), examples of the heteroaromatic ring represented by ^Ar4 include, as monocyclic heteroaromatic rings, a pyridine ring and a pyrazine ring, and as polycyclic heteroaromatic rings, an indole ring, an isoindole ring, a benzimidazole ring, a quinoline ring, an isoquinoline ring, and a quinoxaline ring.

The heteroaromatic ring represented by ^Ar4 may have a substituent. Examples of the substituent include an alkyl group described below, a cycloalkyl group described below, an aryl group described below, a heteroaryl group described below, 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.), and the like. When the heteroaromatic ring has a substituent, the number of the substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

In addition, compared with the case where Ar ¹ is a substituted or unsubstituted monocyclic aromatic hydrocarbon ring, the bisphosphoryl-bridged stilbene compounds represented by the general formula (1C), (1C1) or (1C2) have superior ε in absorption and Φ _F in fluorescence, and are capable of making the target intracellular organelles emit light more intensely and clearly.

In addition, compared with the case where Ar ¹ is a substituted or unsubstituted monocyclic aromatic hydrocarbon ring, the bisphosphoryl-bridged stilbene compound represented by the general formula (1D), (1D1) or (1D2) can shift the absorption peak wavelength and the fluorescence peak wavelength to longer wavelengths.

From the above, it is possible to provide a variety of optical properties depending on the structure of Ar ¹ , and it is preferable to appropriately adjust the structure of Ar ¹ according to the required physical properties.

In general formulae (1), (2), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1) and (1D2), the aromatic hydrocarbon rings represented by ^Ar2 , ^Ar3 , ^Ar3a and ^Ar3b may be either monocyclic aromatic hydrocarbon rings or polycyclic aromatic hydrocarbon rings, and examples of the monocyclic aromatic hydrocarbon ring include a benzene ring, and examples of the polycyclic aromatic hydrocarbon ring include a naphthalene ring, anthracene ring, phenanthrene ring, fluorene ring, pyrene ring, triphenylene ring, etc. Among these, a benzene ring is particularly preferred from the viewpoints of structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, light resistance, etc.

The aromatic hydrocarbon ring represented by ^Ar2 may have a substituent. Examples of the substituent include an alkyl group described below, a cycloalkyl group described below, an aryl group described below, a heteroaryl group described below, 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, etc. When the aromatic hydrocarbon ring has a substituent, the number of the substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

In general formulae (1), (2), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1), and (1D2), examples of the heteroaromatic ring represented by Ar ² , Ar ³ , Ar ^3a , and Ar ^3b include, as monocyclic heteroaromatic rings, a pyridine ring, a pyrazine ring, and the like, and as polycyclic heteroaromatic rings, an indole ring, an isoindole ring, a benzimidazole ring, a quinoline ring, an isoquinoline ring, a quinoxaline ring, and the like.

The heteroaromatic rings represented by Ar ³ , Ar ^3a and Ar ^3b may have a substituent. Examples of the substituent include an alkyl group described below, a cycloalkyl group described below, an aryl group described below, a heteroaryl group described below, 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.), etc. When the heteroaromatic ring has a substituent, the number of the substituents is, for example, preferably 1 to 6, more preferably 1 to 3.

From the viewpoints of structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, light resistance, and the like, Ar ³ , Ar ^3a and Ar ^3b are preferably substituted or unsubstituted aromatic hydrocarbon rings, more preferably substituted or unsubstituted monocyclic aromatic hydrocarbon rings.

In general formulas (1), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1) and (1D2), the alkyl group represented by R ¹ and R ² may be either a straight-chain alkyl group or a branched-chain alkyl group, and examples thereof include C1-10 alkyl groups (particularly 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.

The alkyl groups represented by ^R1 and ^R2 may have a substituent. Examples of the substituent include a halogen atom, a cycloalkyl group described below, an aryl group described below, a heteroaryl group described below, a cyano group, and a nitro group. When the alkyl group has a substituent, the number of the substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

In general formulas (1), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1) and (1D2), examples of the cycloalkyl group represented by ^R1 and ^R2 include C3-10 cycloalkyl groups (particularly C4-8 cycloalkyl groups) such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group and a cycloheptyl group.

The cycloalkyl group represented by ^R1 and ^R2 may have a substituent. Examples of the substituent include a halogen atom, the alkyl group described above, an aryl group described below, a heteroaryl group described below, a cyano group, and a nitro group. When the cycloalkyl group has a substituent, the number of the substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

In general formulae (1), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1) and (1D2), the aryl group represented by ^R1 and ^R2 may be, for example, any of a monocyclic aryl group, a fused aryl group and a polycyclic aryl group. For example, an example of the monocyclic aryl group is a phenyl group, an example of the fused aryl group is a naphthyl group, an anthracenyl group, a phenanthrenyl group, a fluorenyl group, a pyrenyl group, a triphenylenyl group and the like, and an example of the polycyclic aryl group is a C6-18 aryl group (particularly a C6-14 aryl group) such as a biphenyl group or a terphenyl group.

The aryl group represented by ^R1 and ^R2 may have a substituent. Examples of the substituent include a halogen atom, the alkyl group described above, the aryl group described above, a heteroaryl group described below, a cyano group, and a nitro group. When the aryl group has a substituent, the number of the substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

In general formula (1), the heteroaryl group represented by ^R1 and ^R2 may be either a monocyclic heteroaryl group or a fused ring heteroaryl group. Examples of the monocyclic heteroaryl group include a pyrrolyl group, a thienyl group, a furanyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an oxazolyl group, a piperidyl group, a pyridyl group, and a pyrazyl group. Examples of the fused ring heteroaryl group include an indolyl group, an isoindolyl group, a benzimidazolyl group, a quinolyl group, an isoquinolyl group, and a quinoxalyl group.

The heteroaryl group represented by ^R1 and ^R2 may have a substituent. Examples of the substituent include the above-mentioned halogen atom, the above-mentioned alkyl group, the above-mentioned aryl group, the above-mentioned heteroaryl group, a cyano group, a nitro group, etc. When the heteroaryl group has a substituent, the number of the substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

Among these, from the viewpoints of easily imparting environmental responsiveness and easily shifting the absorption peak wavelength to a longer wavelength, ^R1 and ^R2 are preferably substituted or unsubstituted (hetero)aryl groups, more preferably substituted or unsubstituted aryl groups, and even more preferably unsubstituted aryl groups.

In addition, R ¹ and R ² may be taken together to form a ring with the adjacent nitrogen atom. That is, the group represented by -NR ¹ R ² is

等で表される基であってもよい。

and the like.

In addition, R ¹ and/or R ² may be combined with Ar ² to form a ring with the adjacent nitrogen atom. That is, the structure represented by Ar ² -NR ¹ R ² is

等であってもよい。

etc.

In this case, the substitution position in Ar ² where the group represented by --NR ¹ R ² is bonded is not particularly limited.

In the general formula (2), the organic group represented by R ³ is not particularly limited, and examples thereof include an alkyl group, a polyethylene glycol group or a derivative group thereof (—(C ₂ H ₄ O) _m R ⁷ ).

In the general formula (2), the alkyl group represented by ^R3 may be either a straight-chain alkyl group or a branched-chain alkyl group, but it is preferable to select an appropriate group depending on the object to be dyed.

The alkyl group represented by ^R3 may have a substituent. The substituent is preferably a functional group so as to facilitate control of organelle localization and dispersibility, and examples thereof include an epoxy group, a phosphorus-containing group, a carboxy group, an alkoxycarbonyl group ( ^-COOR8 ), an amide group or a derivative group thereof ( ^-CONHR9 ), etc.

The phosphorus-containing group as a substituent of the alkyl group represented by ^R3 is not particularly limited, and from the viewpoint of easy control of organelle localization and dispersibility, a phosphorus-containing group represented by the general formula (3):
-P ⁺ R ⁴ _n X ¹ _(4-n) ^- (3)
In the formula, R ⁴ may be the same or different and represents a substituted or unsubstituted (hetero)aryl group.
X ^{1 '} s may be the same or different and each represent a halogen atom.
n represents an integer of 1 to 3.
A group represented by the following formula is preferred.

In general formula (3), the aryl group represented by ^R4 may be, for example, any of a monocyclic aryl group, a condensed aryl group, and a polycyclic aryl group. For example, an example of the monocyclic aryl group is a phenyl group, an example of the condensed aryl group is a naphthyl group, an anthracenyl group, a phenanthrenyl group, a fluorenyl group, a pyrenyl group, a triphenylenyl group, and the like, and an example of the polycyclic aryl group is a C6-18 aryl group (particularly a C6-14 aryl group) such as a biphenyl group or a terphenyl group.

The aryl group represented by ^R4 may have a substituent. Examples of the substituent include a halogen atom, the alkyl group described above, the aryl group described above, the heteroaryl group described below, a cyano group, a nitro group, etc. When the aryl group has a substituent, the number of the substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

In general formula (3), the heteroaryl group represented by ^R4 may be either a monocyclic heteroaryl group or a fused ring heteroaryl group. Examples of the monocyclic heteroaryl group include a pyrrolyl group, a thienyl group, a furanyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an oxazolyl group, a piperidyl group, a pyridyl group, and a pyrazyl group. Examples of the fused ring heteroaryl group include an indolyl group, an isoindolyl group, a benzimidazolyl group, a quinolyl group, an isoquinolyl group, and a quinoxalyl group.

The heteroaryl group represented by ^R4 may have a substituent. Examples of the substituent include the above-mentioned halogen atoms, the above-mentioned alkyl groups, the above-mentioned aryl groups, the above-mentioned heteroaryl groups, a cyano group, a nitro group, etc. When the heteroaryl group has a substituent, the number of the substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

Among these, from the viewpoint of easy control of organelle localization and dispersibility, ^R4 is preferably a substituted or unsubstituted aryl group, and more preferably an unsubstituted aryl group.

In the general formula (3), examples of the halogen atom represented by ^X1 include a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, and the like. From the viewpoint of easy control of organelle localization and dispersibility, a chlorine atom, a bromine atom, an iodine atom, and the like are preferred, a chlorine atom, a bromine atom, and the like are more preferred, and a bromine atom is even more preferred.

In general formula (3), n is preferably an integer from 1 to 3, more preferably 2 or 3, and even more preferably 3, from the viewpoint of easy control of organelle localization and dispersibility.

The alkoxycarbonyl group ( ^-COOR8 ) as a substituent of the alkyl group represented by ^R3 can be used without any particular limitation as long as ^R8 contains an alkyl group (such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, etc.), and examples thereof include a methoxycarbonyl group, an ethoxycarbonyl group, an n-propoxycarbonyl group, an isopropoxycarbonyl group, etc.

The amide group or its derivative group (-CONHR ⁹ ) as the substituent of the alkyl group represented by R ³ can be used without any particular limitation as long as R ⁸ contains a hydrogen atom or an alkyl group (such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, etc.), and examples thereof include an amide group, an N-methylamide group, an N-ethylamide group, an N-n-propylamide group, an N-isopropylamide group, etc.

In general formula (2), the polyethylene glycol group or its derivative group (-(C ₂ H ₄ O) _m R ⁷ ) represented by R ³ can be used without any particular limitation as long as R ⁷ contains a hydrogen atom or an alkyl group (such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, etc.) and m is an integer of, for example, 1 to 100. For example, -(C ₂ H ₄ O) _m CH ₃ can be preferably used.

Specific examples of the organic group represented by ^R3 that satisfies the above conditions include:

［式中、Ｒ^８及びＲ^９は前記に同じである。Ｐｈはフェニル基を示す。ｋは５～１５の整数を示す。ｍは２～１００の整数を示す。］
等が挙げられる。

[In the formula, ^R8 and ^R9 are the same as defined above. Ph represents a phenyl group. k represents an integer of 5 to 15. m represents an integer of 2 to 100.]
etc.

Examples of bisphosphoryl-bridged stilbene compounds represented by general formula (1) that satisfy the above conditions include:

で表されるビスホスホリル架橋スチルベン化合物が挙げられる。なお、これらの式中、Ｒ^５及びＲ^６は、同一又は異なって、

In these formulas, R ⁵ and R ⁶ may be the same or different and each may be represented by the following formula:

［式中、Ｐｈはフェニル基を示す。ｍは１～１００の整数を示す。］
等が挙げられる。特に、後述の実施例において示されるビスホスホリル架橋スチルベン化合物が好ましい。

[In the formula, Ph represents a phenyl group, and m represents an integer of 1 to 100.]
In particular, the bisphosphoryl-bridged stilbene compounds shown in the examples below are preferred.

2. Method for Producing Bisphosphoryl-Bridged Stilbene Compound The method for producing the phosphole compound of the present invention is not particularly limited. For example, a compound represented by the general formula (4A) or (4B):

［式中、Ａｒ^１、Ａｒ^２、Ａｒ^３ａ、Ａｒ^３ｂ、Ｒ^１、Ｒ^２、Ｙ^３ａ及びＹ^３ｂは前記に同じである。］
で表されるビスホスホリル架橋スチルベン化合物を出発物質として、一般式（５）：
Ｒ^３Ｘ^２ （５）
［式中、Ｒ^３は前記に同じである。Ｘ^２はハロゲン原子を示す。］
で表される化合物と反応させることにより、得ることができる。

[In the formula, Ar ¹ , Ar ² , Ar ^3a , Ar ^3b , R ¹ , R ² , Y ^3a and Y ^3b are the same as defined above.]
Starting from a bisphosphoryl-bridged stilbene compound represented by the general formula (5):
^R3X2 ( ⁵ )
[In the formula, ^R3 is the same as above. ^X2 represents a halogen atom.]
It can be obtained by reacting with a compound represented by the formula:

The bisphosphoryl-bridged stilbene compounds represented by the above general formula (4A) or (4B) can be synthesized, for example, according to a previously reported method (Chem. Asian J., 13, 1616-1624 (2018)).

In the general formula (5), examples of the halogen atom represented by ^X2 include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Of these, a chlorine atom, a bromine atom, and an iodine atom are preferred, a chlorine atom, a bromine atom, and the like are more preferred, and a bromine atom is even more preferred.

In the reaction, a base can be used as necessary. Examples of bases include potassium fluoride, cesium fluoride, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, sodium acetate, potassium acetate, calcium acetate, etc., and in this step, potassium carbonate is preferred from the viewpoints of yield and ease of synthesis. When a base is used, the amount used is preferably 2 to 30 moles, more preferably 10 to 20 moles, per mole of compound (4A) or compound (4B) from the viewpoints of ease of synthesis, yield, etc.

The reaction can be carried out in the presence of a reaction solvent. Examples of reaction solvents that can be used include aromatic hydrocarbon solvents such as benzene, toluene, and xylene; aliphatic halogenated hydrocarbon solvents such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; nitrile solvents such as acetonitrile; and amide solvents such as dimethylformamide. From the viewpoints of ease of synthesis and yield, amide solvents are preferred, and dimethylformamide is more preferred. These reaction solvents can be used alone or in combination of two or more.

The reaction atmosphere may usually be an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.). The reaction may be carried out under heating, at room temperature, or under cooling, and is usually preferably at a temperature of 0 to 100°C, more preferably 10 to 70°C. There are no particular limitations on the reaction time, but it is preferably a time that allows the reaction to proceed sufficiently.

After the reaction is complete, the bisphosphoryl-bridged stilbene compound of the present invention can be obtained by purifying the compound according to a conventional method, if necessary.

The above describes an example of a method for synthesizing one embodiment of the bisphosphoryl-bridged stilbene compound of the present invention, but the compound is not limited to this manufacturing method and can be synthesized by various synthesis methods.

3. Fluorescent Dye and Intracellular Organelle Stain The fluorescent dye of the present invention contains the above-mentioned bisphosphoryl-bridged stilbene compound of the present invention.

The fluorescent dye of the present invention has excellent light resistance due to the presence of a bisphosphoryl-bridged stilbene skeleton, and various reactive groups can be introduced into R ^3. For this reason, it can be used as an intracellular organelle stain (particularly, a lipid droplet stain, a mitochondrial stain, etc.) for staining intracellular organelles (particularly, lipid droplets, mitochondria, lysosomes, endoplasmic reticulum, cell membrane, etc.), and the fluorescent dye of the present invention is suitable for repeated observation in vivo with a super-resolution microscope (particularly, a stimulated emission depletion (STED) microscope) such as stimulated emission depletion (STED) imaging.

When the bisphosphoryl-bridged stilbene compound of the present invention is used as an intracellular organelle stain (particularly a lipid droplet stain, a mitochondrial stain, a lysosome stain, an endoplasmic reticulum stain, a cell membrane stain, etc.), the intracellular organelle stain (particularly a lipid droplet stain, a mitochondrial stain, a lysosome stain, an endoplasmic reticulum stain, a cell membrane stain, etc.) of the present invention contains the bisphosphoryl-bridged stilbene compound of the present invention, but it is preferable to dissolve it in a culture medium (Dulbecco's modified Eagle's medium; DMEM, etc.) to form a solution. From the viewpoint of more strongly staining intracellular organelles (particularly lipid droplets, mitochondria, lysosomes, endoplasmic reticulum, cell membrane, etc.) while further improving light resistance, the content of the bisphosphoryl-bridged stilbene compound of the present invention is preferably 1 to 10,000 nmol/L, more preferably 10 to 1,000 nmol/L.

As described above, the intracellular organelle staining agents of the present invention (particularly lipid droplet staining agents, mitochondrial staining agents, lysosomal staining agents, endoplasmic reticulum staining agents, cell membrane staining agents, etc.) are preferably in the form of a solution, but when observing in vivo, the pH is preferably about 5 to 11, and more preferably about 6.5 to 7.5. To adjust the pH, a buffer (Hepes buffer, Tris buffer, Tricine-sodium hydroxide buffer, phosphate buffer, phosphate buffered saline, etc.) can also be used in combination.

The present invention will be specifically explained based on examples, but the present invention is not limited to these.

Unless otherwise specified, all reactions were carried out under a nitrogen atmosphere. Unless otherwise specified, commercially available solvents and reagents were used without purification. However, anhydrous toluene, anhydrous tetrahydrofuran (THF), anhydrous dimethylformamide (DMF) and anhydrous _CH2Cl2 were purchased from Kanto Chemical Co. _, Ltd. and purified by Glass Contour Solvent Systems.

Also, chloro(N,N-diethylamino)(4-methoxyphenyl)phosphine and the following compounds (trans-PO-B1, cis-PO-B1, trans-PO-Na1 and cis-PO-Na1):

は、既報（Chem. Asian J., 13, 1616-1624 (2018)）にしたがって合成した。

was synthesized according to a previous report (Chem. Asian J., 13, 1616-1624 (2018)).

2-Bromo-3-iodonaphthalene was synthesized as previously reported (Synthesis, 2005, 5, 798-803).

1,2-Epoxy-6-bromohexane was synthesized as previously reported (Angew. Chem. Int. Ed. 2019, 58, 1727-1731).

(4-Bromo-n-butyl)triphenylphosphonium bromide was synthesized as previously reported (J. Am. Chem. Soc. 2015, 137, 6837-6843).

(10-Bromo-n-decyl)triphenylphosphonium bromide was synthesized according to a previous report (WO 2016/155679).

Synthesis Example 1: 1,2-epoxy-9-bromononane

式中、ｍ－ＣＰＢＡはメタクロロ過安息香酸を示す。

In the formula, m-CPBA represents metachloroperbenzoic acid.

To a stirred solution of 9-bromonon-1-ene (1.0 g, 4.87 mmol) in dichloromethane (8 mL) was added dropwise a solution of metachloroperbenzoic acid (70% purity, 1.8 g, 10.4 mmol) in dichloromethane (8 mL). The mixture was then stirred at 25° C. for 16 hours to precipitate metachloroperbenzoic acid. The reaction was filtered to remove a white precipitate, extracted with dichloromethane (DCM; 20 mL×2), washed with saturated NaHCO ₃ (20 mL), water (20 mL) and brine (20 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane=1/10) to give 1,2-epoxy-9-bromononane as a colorless oil (900 mg, 83% yield).
¹ H NMR (400 MHz, CDCl ₃ ) δ 3.40 (t, J = 6.9 Hz, 2H), 2.93-2.87 (m, 1H), 2.77-2.72 (m, 1H), 2.46 (dd, J = 5.0, 2.7 Hz, 1H), 1.91-1.78 (m, 2H), 1.55-1.30 (m, 10H).

Synthesis Example 2: 2-bromo-3-((2-bromo-4-chlorophenyl)ethynyl)naphthalene

2-Bromo-3-iodonaphthalene (5.4 g, 16.2 mmol), bis(triphenylphosphine)palladium dichloride (PdCl ₂ (PPh ₃ ) ₂ ; 231 mg, 0.33 mmol), CuI (62 mg, 0.33 mmol), and triethylamine (Et ₃ N; 70 mL) were added to a nitrogen-purged flask and rigorously degassed by freeze-degassing. Trimethylsilylacetylene (2.47 mL) was added via syringe and stirred at room temperature overnight. 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 give 2-bromo-3-[2-(trimethylsilyl)ethynyl]naphthalene (4.3 g, 86% yield) as a yellow liquid.
^1H 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).

To a solution of the obtained 2-bromo-3-[2-(trimethylsilyl)ethynyl]naphthalene (4.2 g, 14.0 mmol) in CH ₃ OH/tetrahydrofuran (1/1, v/v, 140 mL) was added K ₂ CO ₃ (5.8 g, 42.0 mmol) and the mixture was stirred at room temperature for 8 hours. The product was extracted with CH ₂ Cl ₂ (40 mL), washed with 1 M HCl (100 mL), and then dried over Na ₂ SO _4. The solvent was removed under reduced pressure to give 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).

A suspension of 2-bromo-4-chloro-1-iodobenzene (3.98 g, 12.5 mmol), tetrakis(triphenylphosphine)palladium (Pd(PPh ₃ ) 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 the degassed suspension. After stirring at room temperature overnight, the mixture was diluted with toluene (150 mL) and filtered to remove insoluble compounds. The obtained filtrate was concentrated under reduced pressure and then purified by recrystallization from ethanol 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, CDCl ₃ ) 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, CDCl ₃ ) 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.

Synthesis Example 3: trans-PO-Na2 and cis-PO-Na2

To a solution of 2-bromo-3-((2-bromo-4-chlorophenyl)ethynyl)naphthalene (compound 1; 1.0 g, 2.38 mmol) obtained in Synthesis Example 2 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 at −78° C. over 1.5 hours. The resulting mixture was stirred at −78° C. for 0.5 hours and then maintained at 0° C. for 30 minutes. Thereafter, the mixture was cooled again to −78° C., and PCl ₃ (1.3 mL, 15 mmol) was added, and the mixture was warmed to room temperature. The mixture was stirred at room temperature for 17 hours, and then water (1 mL) was added to the mixture. Then, an aqueous solution of H ₂ O ₂ (30%, 0.8 mL) was added at 0° C., and the mixture was stirred for 30 minutes, after which an aqueous solution of Na ₂ SO ₃ (10%, 50 mL) was added at 0° C. The reaction mixture was extracted with ethyl acetate (EtOAc; 200 mL), and the organic layer was washed with brine (30 mL), dried over anhydrous Na ₂ SO ₄ , and then filtered. The filtrate was concentrated under reduced pressure, and the mixture was purified by silica gel column chromatography (CH ₂ Cl ₂ /acetone = 1/10 to 1/1) to obtain 469 mg (yield 34%) of trans-PO-Na2 as a greenish yellow solid and 540 mg (39%) of cis-PO-Na2 as a greenish yellow solid.
Trans-PO-Na2:
¹ H NMR (400 MHz, CDCl ₃ ) 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).
^31P NMR (162 MHz, CDCl ₃ ) δ 32.54, 32.32, 31.04, 30.82.
HRMS (ESI): m/z calcd. For C ₃₂ H ₂₄ ClO ₄ P ₂ : 569.0833 ([M+H] ⁺ ); found: 569.0826.
cis-PO-Na2:
¹ H NMR (400 MHz, CDCl ₃ ) 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).
^31P NMR (162 MHz, CDCl ₃ ) 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 ClO ₄ P ₂ : 569.0833 ([M+H] ⁺ ); found: 569.0827.

Example 1: trans-PO-BphoxM

Trans-PO-B1 (0.5 g, 0.96 mmol), diphenylamine (0.82 g, 4.86 mmol), palladium acetate (Pd(OAc) ₂ ; 13 mg, 0.058 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (Xphos; 50 mg, 0.105 mmol), and K ₂ CO ₃ (801 mg, 5.8 mmol) were added to anhydrous toluene (60 mL) and stirred at 80°C under a nitrogen atmosphere for 20 hours. The mixture was then cooled to room temperature and insoluble compounds were removed by filtration. After removing the solvent under reduced pressure, the resulting solid was purified by silica gel column chromatography (acetone/CH ₂ Cl ₂ = 1/15-1/10) to obtain trans-PO-BphoxM (520 mg, yield 83%) as a red solid.
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.80-7.68 (m, 4H), 7.64-7.57 (m, 1H), 7.45-7.35 (m, 2H), 7.33-7.30 (m, 2H), 7.29-7.21 (m, 5H), 7.09-7.03 (m, 6H), 7.00-6.95 (m, 5H), 3.83 (s, 6H).
^31P NMR (162 MHz, CDCl ₃ ) δ 32.97.
HRMS (ESI): m/z calcd. For C ₄₀ H ₃₂ NO ₄ P ₂ : 652.1801 ([M+H] ⁺ ); found: 652.1793.

Example 2: trans-PO-BphoxA1/A2

To a solution of trans-PO-BphoxM (270 mg, 0.40 mmol) obtained in Example 1 in anhydrous CH ₂ Cl ₂ (21 mL), BBr ₃ (0.85 mL, 8.3 mmol) was slowly added dropwise at −78° C. After stirring at −78° C. for 1 hour, the temperature was slowly raised to room temperature over 4 hours. Water (5 mL) was added at 0° C., and the mixture was extracted with ethyl acetate (EtOAc; 20 mL×2). After drying the organic layer over Na ₂ SO ₄ , the crude product was purified by column chromatography (CH ₃ OH/CH ₂ Cl ₂ =1/20) to obtain trans-PO-B2 as a red solid (0.22 g, yield 88%).
¹ H NMR (400 MHz, CH ₃ OH-d ₄ ) δ 7.71-7.53 (m, 6H), 7.47-7.41 (m, 1H), 7.38 (dd, J = 7.4, 2.3 Hz, 1H), 7.35-7.25 (m, 5H), 7.21-7.04 (m, 8H), 6.96-6.91 (m, 4H).
HRMS (ESI): m/z calcd. For C ₃₈ H ₂₇ NNaO ₄ P ₂ : 646.1308 ([M+Na] ⁺ ); found: 646.1301.

The trans-PO-B2 (30 mg, 0.048 mmol), (10-bromo-n-decyl)triphenylphosphonium bromide (27 mg, 0.048 mmol) and K ₂ CO ₃ (133 mg, 0.96 mmol) obtained above were added to anhydrous dimethylformamide (anhydrous DMF; 3.6 mL) and stirred at room temperature for 12 hours. Then, epibromohydrin (13.1 mg, 0.096 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 extraction was performed with CH ₂ Cl ₂ (20 mL). The organic layer was washed twice with water (20 mL), dried over anhydrous Na ₂ SO ₄ , and filtered. The filtrate was concentrated under reduced pressure, and the resulting solid was purified by silica gel column chromatography (CH ₃ OH/CH ₂ Cl ₂ =1/30-1/20) to give trans-PO-BphoxA1/A2 as a red solid (15 mg, yield 27%).
HRMS (ESI): m/z calcd. For C ₆₉ H ₆₅ NO ₅ P ₃ : 1080.4070 ([M-Br] ⁺ ); found: 1080.4042.

Example 3: cis-PO-BphoxM

Cis-PO-BphoxM (yield 78%) was obtained in the same manner as in Example 1, except that cis-PO-B1 was used instead of trans-PO-B1 as the raw material compound.
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.65-7.53 (m, 5H), 7.43 (d, J = 4.9 Hz, 2H), 7.35-7.20 (m, 7H), 7.10-7.02 (m, 6H), 7.00 (dd, J = 8.3, 2.2 Hz, 1H), 6.92 (d, J = 8.2 Hz, 4H), 3.82 (d, J = 0.6 Hz, 6H).
^31P NMR (162 MHz, CDCl ₃ ) δ 31.69.
HRMS (ESI): m/z calcd. For C ₄₀ H ₃₁ NNaO ₄ P ₂ : 674.1621 ([M+Na] ⁺ ); found: 674.1615.

Example 4: trans-PO-Naphox(1,2)M

Trans-PO-Na1 (110 mg, 0.19 mmol), diphenylamine (163 mg, 0.968 mmol), palladium acetate (Pd(OAc) ₂ ; 2.6 mg, 0.0116 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (Xphos; 11 mg, 0.0232 mmol), and K ₂ CO ₃ (160 mg, 1.16 mmol) were added to anhydrous toluene (12 mL) and stirred at 90°C under nitrogen atmosphere for 24 hours. The mixture was then cooled to room temperature and insoluble compounds were removed by filtration. After removing the solvent under reduced pressure, the obtained solid was purified by silica gel column chromatography (acetone/CH ₂ Cl ₂ = 1/20-1/10) to obtain trans-PO-Naphox(1,2)M (120 mg, yield 88%) as a red solid.
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.96 (d, J = 8.3 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.85-7.72 (m, 5H), 7.52 (dd, J = 8.3, 2.8 Hz, 1H), 7.46-7.38 (m, 2H), 7.35-7.27 (m, 2H), 7.27-7.21 (m, 4H), 7.11-7.02 (m, 6H), 7.01-6.98 (m, 1H), 6.98-6.91 (m, 4H), 3.80 (d, J = 5.5Hz, 6H).
^31P NMR (162 MHz, CDCl ₃ ) δ 34.14 (d, J = 40.0 Hz), 32.96 (d, J = 39.9 Hz).
HRMS (ESI): m/z calcd. For C ₄₄ H ₃₄ NO ₄ P ₂ : 702.1958 ([M+H] ⁺ ); found: 702.1948.

Example 5: trans-PO-Naphox(2,3)M

The trans-PO-Na2 (90 mg, 0.158 mmol) obtained in Synthesis Example 3, diphenylamine (133 mg, 0.79 mmol), palladium acetate (Pd(OAc) ₂ ; 2.1 mg, 0.0095 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (Xphos; 9 mg, 0.019 mmol), and K ₂ CO ₃ (131 mg, 0.948 mmol) were added to anhydrous toluene (10 mL) and stirred at 90°C under a nitrogen atmosphere for 24 hours. The mixture was then cooled to room temperature and insoluble compounds were removed by filtration. After removing the solvent under reduced pressure, the obtained solid was purified by silica gel column chromatography (acetone/CH ₂ Cl ₂ =1/15-1/10) to obtain trans-PO-Naphox(2,3)M (100 mg, yield 90%) as a red solid.
¹ H NMR (400 MHz, CDCl ₃ ) δ 8.08 (dd, J = 11.5, 1.9 Hz, 1H), 7.84-7.73 (m, 7H), 7.48 (dtd, J = 16.1, 7.0, 1.0 Hz, 2H), 7.35 (dt, J = 11.3, 2.0 Hz, 1H), 7.30-7.21 (m, 5H), 7.11-7.03 (m, 6H), 7.02-6.93 (m, 5H), 3.82 (d, J = 2.7 Hz, 6H).
^31P NMR (162 MHz, CDCl ₃ ) δ 33.21 (d, J = 37.4 Hz), 31.35 (d, J = 37.3 Hz).
HRMS (ESI): m/z calcd. For C ₄₄ H ₃₄ NO ₄ P ₂ : 702.1958 ([M+H] ⁺ ); found: 702.1955.

Example 6: Trans-PO-Naphox A1/A2

To a solution of trans-PO-Naphox(2,3)M (90 mg, 0.128 mmol) obtained in Example 5 in anhydrous CH ₂ Cl ₂ (8 mL), BBr ₃ (0.28 mL, 2.56 mmol) was slowly added dropwise at −78° C. After stirring at −78° C. for 1 hour, the temperature was slowly raised to room temperature over 4 hours. Water (5 mL) was added at 0° C., and the mixture was extracted with ethyl acetate (EtOAc; 20 mL×2). After drying the organic layer over Na ₂ SO ₄ , the crude product was purified by column chromatography (CH ₃ OH/CH ₂ Cl ₂ =1/20) to obtain trans-PO-Na3 as a red solid (80 mg, yield 92%).
¹ H NMR (400 MHz, CH ₃ OH-d ₄ ) 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).
^31P NMR (162 MHz, CH ₃ OH-d ₄ ) 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 NO ₄ P ₂ : 673.1650 ([M+H] ⁺ ); found: 674.1641.

The obtained trans-PO-Na3 (30 mg, 0.0445 mmol), (10-bromodecyl)triphenylphosphonium bromide (25 mg, 0.0445 mmol), and K ₂ CO ₃ (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 extraction with CH ₂ Cl ₂ (20 mL) was performed. The organic layer was washed twice with water (20 mL), dried over anhydrous Na ₂ SO ₄ , and filtered. The filtrate was concentrated under reduced pressure, and the resulting solid was purified by silica gel column chromatography (CH ₃ OH/CH ₂ Cl ₂ =1/30-1/10) to give trans-PO-Naphox A1/A2 as an orange-red solid (13 mg, yield 24%).
HRMS (ESI): m/z calcd. For C ₇₃ H ₆₇ NO ₅ P ₃ : 1130.4227 ([M-Br] ⁺ ); found: 1130.4229.

Example 7: cis-PO-Naphox(2,3)M

Cis-PO-Naphox(2,3)M (yield 81%) was obtained in the same manner as in Example 5, except that cis-PO-Na2 obtained in Synthesis Example 3 was used as a raw material compound instead of trans-PO-Na2 obtained in Synthesis Example 3.
¹ H NMR (400 MHz, CDCl ₃ ) δ 8.10 (dd, J = 11.5, 1.8 Hz, 1H), 7.86-7.74 (m, 3H), 7.71-7.60 (m, 4H), 7.57-7.51 (m, 1H), 7.50-7.44 (m, 1H), 7.40-7.31 (m, 2H), 7.28-7.23 (m, 3H), 7.12-7.00 (m, 7H), 6.93 (ddd, J = 9.0, 2.3, 1.1 Hz, 4H), 3.83 (d, J = 3.8 Hz, 6H).
^31P NMR (162 MHz, CDCl ₃ ) δ 31.72 (d, J = 37.5 Hz), 30.11 (d, J = 37.9 Hz).
HRMS (ESI): m/z calcd. For C ₄₄ H ₃₃ NNaO ₄ P ₂ : 724.1777 ([M+Na] ⁺ ); found: 724.1765.

Example 8: Trans-PS-BphoxA1/A2

A solution of a mixture of trans-PO-BphoxM (40.0 mg, 0.061 mmol) obtained in Example 1 and Lawesson's reagent (99 mg, 0.246 mmol) in anhydrous toluene (5 mL) was refluxed for 20 hours. After cooling the solution to room temperature and removing the solvent under reduced pressure, the resulting solid was purified by silica gel column chromatography (CH ₂ Cl ₂ /hexane=1/2-1/1) to obtain 34 g (yield 81%) of trans-PS-BphoxM and 8 mg (yield 19%) of cis-PS-BphoxM as orange solids.
Trans-PS-BphoxM:
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.86-7.74 (m, 4H), 7.60 (dd, J = 12.8, 7.1 Hz, 1H), 7.52-7.46 (m, 1H), 7.44-7.38 (m, 1H), 7.37-7.29 (m, 3H), 7.26-7.21 (m, 4H), 7.11-7.03 (m, 6H), 7.00-6.91 (m, 5H), 3.82 (s, 6H).
^31P NMR (162 MHz, CDCl ₃ ) δ 37.47.
HRMS (ESI): m/z calcd. For C ₄₀ H ₃₁ NNaO ₂ P ₂ S ₂ : 706.1164 ([M+Na] ⁺ ); found: 706.1151.
cis-PS-BphoxM:
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.85-7.75 (m, 4H), 7.62-7.57 (m, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.36-7.30 (m, 3H), 7.26-7.21 (m, 4H), 7.09-7.03 (m, 6H), 6.99-6.91 (m, 5H), 3.82 (s, 6H).
^31P NMR (162 MHz, CDCl ₃ ) δ 36.91.
HRMS (ESI): m/z calcd. For C ₄₀ H ₃₁ NNaO ₂ P ₂ S ₂ : 706.1164 ([M+Na] ⁺ ); found: 706.1165.

To a solution of the trans-PS-BphoxM obtained above (27 mg, 0.039 mmol) in anhydrous CH ₂ Cl ₂ (2 mL) was added BBr ₃ (0.08 mL, 0.79 mmol) dropwise at −78° C. After stirring the solution at −78° C. for 1 h, the mixture was slowly warmed to room temperature over 4 h. The reaction was quenched with water (2 mL) at 0° C. and extracted with ethyl acetate (20 mL×2). The orange layer was separated and dried over Na ₂ SO _4. The crude product was purified by column chromatography (CH ₃ OH/CH ₂ Cl ₂ =1/20) to obtain trans-PS-B1 as an orange-red solid (20 mg, 79% yield).

Trans-PS-BphoxA1/A2 (yield 17%) was obtained in the same manner as in Example 2, except that the raw material compound used was the trans-PS-B1 obtained above instead of trans-PO-B2.

Comparative Example 1: Mito-PB yellow M

Mito-PB yellow M in Comparative Example 1 was synthesized according to a previous report (PNAS, 2019, 116, 15817-15822).

Comparative example 2: Mito Tracker Deep Red
Commercially available Mito-Tracker Deep Red was used as the compound of Comparative Example 2.

Test Example 1: Optical Properties For cis-PO-BphoxM of Example 3, lance-PO-Naphox(1,2)M of Example 4, and trans-PS-BphoxC4 of Example 8, measurements were made of the ultraviolet-visible absorption spectrum, fluorescence spectrum, absolute fluorescence quantum yield, etc. when dissolved in toluene, dichloromethane (DCM) or acetonitrile (MeCN). Appearance photographs were taken. The results of Examples 3 and 8 are shown in FIG. 1, the results of Example 4 in FIG. 2, and the optical property data of each are shown in Table 1.

From the above, the bisphosphoryl-bridged stilbene compound of the present invention can absorb light in the visible light region (especially about 450 to 550 nm) in any of a variety of solvents, and can emit fluorescence of various wavelengths depending on the solvent. In particular, the fluorescence was extremely small in polar solvents such as acetonitrile, suggesting that there is almost no fluorescence in the cytoplasm. This suggests that it is possible to stain hydrophobic tissues such as phospholipid membranes and lipid droplets with high contrast.

Test Example 2: Light resistance (in acetonitrile)
Trans-PO-BphoxM of Example 1, cis-PO-BphoxM of Example 3, trans-PO-Naphox(1,2)M of Example 4, trans-PO-Naphox(2,3)M of Example 5, and Mito-PB yellow M of Comparative Example 1 were dissolved in acetonitrile (MeCN) at 25°C. The concentration was adjusted so that the absorbance at 515 nm was about the same (0.1). Light with a wavelength of 515±5 nm was irradiated with an LED light (2050 W/m ² ) equipped with a bandpass filter that transmits light of 515±5 nm, and the ultraviolet-visible absorption spectrum was measured after various times. The results of Example 1 and Comparative Example 1 are shown in Figures 3(a) and (c), the results of Examples 3 and 5 are shown in Figures 4(a) and (c), and the result of Example 4 is shown in Figure 5(a).

Next, the light resistance at the maximum absorption wavelength was evaluated for each sample. Specifically, light of 515±5 nm wavelength was irradiated using an LED light (2050 W/m ² ) equipped with a bandpass filter that transmits light of 515±5 nm, and the absorbance (A 0 ) at the maximum absorption wavelength (460 nm for Example 1, 487 nm for Example 3, 513 nm for Example 4, 482 nm for Example 5, and 487 nm for Comparative Example 1) immediately after irradiation (0 hours later) was set to 1.00, and the maintenance rate (A/A ₀ ) of the absorbance (A ₎ after a predetermined time was evaluated. The results of Example 1 and Comparative Example 1 are shown in Figures 3(b) and (d), the results of Examples 3 and 5 are shown in Figures 4(b) and (d), and the result of Example 4 is shown in Figure 5(b).

As a result, the bisphosphoryl-bridged stilbene compound of the present invention has significantly better light resistance than Mito-PB yellow, and the absorbance hardly decreases at all.

Test Example 3: Light resistance (in DMSO/H ₂ O Part 1)
Trans-PO-Naphox (2,3) M of Example 5 and Mito-PB yellow M of Comparative Example 1 were dissolved in a dimethyl sulfoxide/water mixed solvent (volume ratio 1:1) at 25°C. The concentration was adjusted so that the absorbance at 515 nm was approximately the same (0.1). Light with a wavelength of 515±5 nm was irradiated with an LED light equipped with a bandpass filter that transmits light of 515±5 nm at an irradiation intensity of 2050 W/ ^m2 , and the ultraviolet-visible absorption spectrum was measured after various times. The results of Comparative Example 1 are shown in Figure 6(a), and the results of Example 5 are shown in Figure 6(c).

Next, the light resistance at the maximum absorption wavelength was evaluated. Specifically, light with a wavelength of 515±5 nm was irradiated at an irradiation intensity of 2050 W/ ^m2 using an LED light equipped with a bandpass filter that transmits light with a wavelength of 515±5 nm, and the absorbance (A ₀ ) at the maximum absorption wavelength (506 nm for Example 5 and 473 nm for Comparative Example 1) immediately after irradiation (0 hours) was set to 1.00, and the maintenance rate (A/A ₀ ) of the absorbance (A) after a predetermined time was evaluated. The results of Comparative Example 1 are shown in FIG. 6(b), and the results of Example 5 are shown in FIG. 6(d).

Test Example 4: Light resistance (in DMSO/H ₂ O, Part 2)
Cis-PO-BphoxM of Example 3, trans-PO-Naphox(2,3)M of Example 5, and Mito-PB ywllow M of Comparative Example 1 were dissolved in a dimethylsulfoxide/water mixed solvent (volume ratio 1:1) at 25°C. The concentration was adjusted so that the absorbance at 450 nm was approximately the same (0.1). Using an LED light equipped with a bandpass filter that transmits light of 450±5 nm, light of a wavelength of 450±5 nm was irradiated at an irradiation intensity of 2050 W/ ^m2 or 97% of the maximum intensity of the LED light, and the ultraviolet-visible absorption spectrum was measured after various times. The results for Example 3 at an irradiation intensity of 2050 W/ ^m2 are shown in FIG. 7(a), the results for Example 3 at an intensity of 97% of the maximum intensity of the LED light are shown in FIG. 7(c), the results for Example 5 at an irradiation intensity of 2050 W/ ^m2 are shown in FIG. 8(a), the results for Example 5 at an intensity of 97% of the maximum intensity of the LED light are shown in FIG. 8(c), the results for Comparative Example 1 at an irradiation intensity of 2050 W/ ^m2 are shown in FIG. 9(a), and the results for Comparative Example 1 at an intensity of 97% of the maximum intensity of the LED light are shown in FIG. 9(c).

Next, the light resistance at the maximum absorption wavelength was evaluated. Specifically, light with a wavelength of 450±5 nm was irradiated with an LED light equipped with a bandpass filter that transmits light of 450±5 nm at an irradiation intensity of 2050 W/ ^m2 or an intensity of 97% of the maximum intensity of the LED light, and the absorbance (A 0 ) at the maximum absorption wavelength (509 nm for Example 3, 504 nm for Example 5, and 473 nm for Comparative Example 1) immediately after irradiation ( ₀ hours) was set to 1.00, and the maintenance rate (A/A ₀ ) of the absorbance (A) after a predetermined time was evaluated. The results for Example 3 at an irradiation intensity of 2050 W/ ^m2 are shown in FIG. 7(b), the results for Example 3 at an intensity of 97% of the maximum intensity of the LED light are shown in FIG. 7(d), the results for Example 5 at an irradiation intensity of 2050 W/ ^m2 are shown in FIG. 8(b), the results for Example 5 at an intensity of 97% of the maximum intensity of the LED light are shown in FIG. 8(d), the results for Comparative Example 1 at an irradiation intensity of 2050 W/ ^m2 are shown in FIG. 9(b), and the results for Comparative Example 1 at an intensity of 97% of the maximum intensity of the LED light are shown in FIG. 9(d).

Test Example 5: Cell preparation (part 1)
HeLa cells (RIKEN Cell Bank, Japan) were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 10% fetal bovine serum (FBS, Gibco) and 1% antibiotic-antimycotic (AA, Sigma) at 37°C in a 5% _CO2 incubator for 24 hours. Three days before imaging, cells (5 x ¹⁰⁴ ) were seeded onto glass-bottom dishes.

Test Example 6: Fluorescence imaging of cells (part 1)
In the staining experiment of trans-PO-BphoxA1/A2 in Example 2, HeLa cells were cultured in DMEM medium containing 500 nM trans-PO-BphoxA1/A2, 1% dimethyl sulfoxide, and 0.1% Pluonic F-127 in a 5% by volume _CO2 incubator for 2 hours at 37°C. The cells were washed three times with DMEM(-), replaced with DMEM(-), and observed using a super-resolution microscope TCS SP8 STED (Leica). In this case, the excitation wavelength is preferably 470 to 540 nm, and optimally 488 to 515 nm, and a fluorescent signal in the range of 550 to 750 nm was detected. The results are shown in FIG. 10.

The same procedure was carried out for staining experiments using trans-PO-Naphox A1/A2 in Example 6 and Mito Tracker Deep Red in Comparative Example 2. The results are shown in Figures 10 and 11.

Test Example 7: Fluorescence imaging of cells (part 2)
In the staining experiment of trans-PO-BphoxA1/A2 in Example 2, HeLa cells were cultured in DMEM medium containing 500 nM trans-PO-BphoxA1/A2, 1% dimethyl sulfoxide, and 0.1% Pluonic F-127 in a 5% by volume CO ₂ incubator at 37°C for 2 hours. The cells were washed three times with DMEM(-), replaced with DMEM(-), and imaged using a super-resolution microscope TCS SP8 STED (Leica).

For confocal imaging, a super-resolution microscope TCS SP8 STED was used, with an excitation wavelength of 470-540 nm being preferred, with 488-515 nm being optimal, and fluorescent signals in the range of 550-750 nm being detected. On the other hand, for STED imaging, a 775 nm STED laser (pulsed laser) was used to obtain super-resolution images, and signals in the range of 550-750 nm were detected.

The same procedure was followed for the staining experiment of trans-PO-NaphoxA1/A2 in Example 6.

The results for trans-PO-BphoxA1/A2 in Example 2 are shown in Figure 12, and the results for trans-PO-NaphoxA1/A2 in Example 6 are shown in Figure 13.

Test Example 8: Preparation of cells (part 2)
HeLa cells (RIKEN Cell Bank, Japan) were treated with oleic acid (400 nM) for 24 hours in a 5% _CO2 incubator, and the medium was replaced. HeLa cells (RIKEN Cell Bank, Japan) were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 10% fetal bovine serum (FBS, Gibco) and 1% antibiotic-antimycotic (AA, Sigma) at 37°C for 24 hours in a 5% _CO2 incubator. Three days before imaging, cells (5 x ¹⁰⁴ ) were seeded on glass-bottom dishes.

Test Example 9: Fluorescence imaging of cells (part 3)
In the staining experiment of trans-PO-BphoxM in Example 1, HeLa cells were cultured in a DMEM medium containing 500 nM trans-PO-BphoxM, 1% dimethyl sulfoxide, and 0.1% Pluonic F-127 in a 5% by volume _CO2 incubator for 2 hours at 37°C. The cells were washed three times with DMEM(-) and then replaced with DMEM(-), and observed using super-resolution microscopes TCS SP8 STED and FV-10 (Olympus). In this case, the excitation wavelength is preferably 470 to 540 nm, and optimally 488 to 515 nm, and a fluorescent signal in the range of 550 to 750 nm was detected.

The same procedure was carried out for staining experiments of cis-PO-BphoxM in Example 3, trans-PO-Naphox(1,2)M in Example 4, trans-PO-Naphox(2,3)M in Example 5, and cis-PO-Naphox(2,3)M in Example 7.

The results are shown in Figure 14.

As a result, the bisphosphoryl-bridged stilbene compound of the present invention was able to selectively stain lipid droplets and mitochondria by modifying ^R3 .

Claims (6)

General formula (1):

[In the formula,
Ar ¹ and Ar ² are the same or different and each represent a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring.
^R1 and ^R2 are the same or different and each represents 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 ^R2 may be joined together to form a ring with the adjacent nitrogen atom. ^R1 and/or ^R2 may be joined together with ^Ar2 to form a ring with the adjacent nitrogen atom.
Y ¹ and Y ² are the same or different and are represented by the general formula (2):

( ^Ar3 represents a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring.
^R3 represents a substituted or unsubstituted alkyl group, or a polyethylene glycol group or a derivative thereof .
^Y3 represents an oxygen atom or a sulfur atom.
It represents a group represented by the following formula:
A bisphosphoryl-bridged stilbene compound represented by the formula: 2. The bisphosphoryl-bridged stilbene compound according to claim 1, wherein ^R3 is a substituted or unsubstituted alkyl group. The bisphosphoryl-bridged stilbene compound according to claim 1 or 2, wherein Ar ¹ is a substituted or unsubstituted polycyclic aromatic hydrocarbon ring. A fluorescent dye containing the bisphosphoryl-bridged stilbene compound according to any one of claims 1 to 3. An intracellular organelle staining agent containing the bisphosphoryl-bridged stilbene compound according to any one of claims 1 to 3. A method for imaging intracellular organelles using the bisphosphoryl-bridged stilbene compound according to any one of claims 1 to 3, the fluorescent dye according to claim 4, or the intracellular organelle stain according to claim 5.

Images