JP2022090883A - Bisphosphoryl cross-linked stilbene compound - Google Patents

Abstract

To provide a compound that can stain intracellular organelle using a long wavelength excitation wavelength capable of reducing phototoxicity to intracellular organelle and that has even better light resistance as compared with MitoPB Yellow.SOLUTION: A bisphosphoryl cross-linked stilbene compound, for example, the compound trans-PO-BphoxM having the following structural formula is shown.SELECTED DRAWING: None

Description

The present invention relates to bisphosphoryl crosslinked stilbene compounds.

Fluorescent organic compounds with high fluorescence quantum yields are important as light emitting materials for organic EL devices or fluorescent dyes for biofluorescence imaging, and the examples reported so far are listed in both basic research and application. I have no time.

However, many of the conventionally known fluorescent organic compounds gradually decompose and fade when light irradiation is continued. For example, as a fluorescent probe, Alexa Fluor dye, ATTO dye, and the like are well-known as a dye group having improved light resistance, but even when these are used, a super-resolution microscope such as stimulated emission suppression (STED) imaging can be used. It is difficult to observe repeatedly. For this reason, the observation target of the most advanced fluorescence microscope technology is currently limited, and improvement of the light resistance of the fluorescent dye is required.

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

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

However, in super-resolution microscopy such as STED imaging, in the case of MitoPB Yellow, a STED laser with a wavelength of 660 nm was used as the excitation wavelength, so phototoxicity to intracellular small organs such as mitochondria was observed. Was being. Therefore, it is required to extend the excitation wavelength to a longer wavelength and reduce phototoxicity to organelles. In addition, MitoPB Yellow showed overwhelming light resistance that surpassed that of other existing fluorescent dyes, but it did not know that the required characteristics for light resistance remained, and MitoPB Yellow was exposed to strong light such as photobleaching. Is also required to be able to reduce photobleaching.

The present invention attempts to solve the above-mentioned conventional problems, and stains organelles using a long-wavelength excitation wavelength capable of reducing phototoxicity to organelles. It is an object of the present invention to provide a compound which can be obtained and has further excellent light resistance as compared with MitoPB Yellow.

As a result of diligent research in view of the above problems, the present inventors have been able to stain intracellular organelles, and the compound having a specific bisphosphoryl cross-linked stylben skeleton is excellent in light resistance and induction. It has been found that the absorption intensity hardly decreases even when irradiated with a powerful laser beam used for a super-resolution microscope such as emission suppression (STED) imaging. Based on such findings, the present inventors have further studied 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）：

[During the ceremony,
Ar ¹ 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 be combined to form a ring with adjacent nitrogen atoms. R ¹ and / or R ² may be combined with Ar ² to form a ring with adjacent nitrogen atoms.
Y ¹ and Y ² are the same or different, and 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。

(Ar ³ indicates a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring.
R ³ represents a hydrogen atom or an organic group.
^Y3 represents an oxygen atom or a sulfur atom. )
Indicates a group represented by. ]
A bisphosphoryl cross-linked stilbene compound represented by.

Item 2. Item 2. The bisphosphoryl crosslinked stilbene compound according to Item ¹ , wherein R3 is a substituted or unsubstituted alkyl group.

Item 3. Item 2. The bisphosphoryl crosslinked 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 crosslinked stilbene compound according to any one of Items 1 to 3.

Item 5. An intracellular organelle stain containing the bisphosphoryl crosslinked stilbene compound according to any one of Items 1 to 3.

Item 6. Suppression of induced release of organelles (STED) using the bisphosphoryl cross-linked stylben compound according to any one of Items 1 to 3, the fluorescent dye according to Item 4, or the intracellular organelle staining agent according to Item 5. ) Imaging method.

The bisphosphoryl cross-linked stilbene compound of the present invention has a longer absorption peak wavelength than that of MitoPB Yellow, and can stain intracellular organelles using a STD laser having a long wavelength such as 775 nm. Therefore, it is possible to reduce the phototoxicity to organelles in cells, and it is also useful as a light-resistant electronic material for which a long absorption peak wavelength is required.

Further, the bisphosphoryl cross-linked stilbene compound of the present invention is extremely excellent in light resistance, and hardly fades even by intense light irradiation such that MitoPB Yellow is photofaded.

Therefore, the bisphosphoryl cross-linked stilbene compound of the present invention is a fluorescent dye suitable for repeatedly observing intracellular organelles with a super-resolution microscope such as STED imaging.

The ultraviolet-visible absorption spectrum, fluorescence spectrum, absolute fluorescence quantum yield and appearance photograph of the cis-PO-BfoxM of Example 3 and the trans-PS-BfoxC4 of Example 8 in various solvents are shown. The ultraviolet-visible absorption spectrum, the fluorescence spectrum, the absolute fluorescence quantum yield, and the appearance photograph of the Lance-PO-Naphox (1, 2) M of Example 4 in various solvents are shown. The light resistance of Trans-PO-Bfox M of Example 1 and Mito-PB hellom of Comparative Example 1 in acetonitrile (MeCN) is shown. (A) Ultraviolet-visible absorption spectra of Mito-PB hello M of Comparative Example 1 after various lapses of time. (B) Maintenance rate (A / A ₀ ) of absorbance (A) after a lapse of a predetermined time of Mito-PB hello M of Comparative Example 1. (C) Ultraviolet-visible absorption spectra of Trans-PO-BfoxM of Example 1 after various lapses of time. (D) The maintenance rate (A / A ₀ ) of the absorbance (A) of the trans-PO-BfoxM of Example 1 after a lapse of a predetermined time. The light resistance of cis-PO-BfoxM of Example 3 and trans-PO-Nafox (2,3) M of Example 5 in acetonitrile (MeCN) is shown. (A) Ultraviolet-visible absorption spectra of cis-PO-BfoxM of Example 3 after various lapses of time. (B) Maintenance rate (A / A ₀ ) of absorbance (A) after a lapse of a predetermined time of cis-PO-BfoxM of Example 3. (C) Ultraviolet-visible absorption spectra of Trans-PO-Naphox (2,3) M of Example 5 after various lapses of time. (D) The maintenance rate (A / A ₀ ) of the absorbance (A) of the trans-PO-Naphox (2,3) M of Example 5 after a predetermined time has elapsed. The light resistance of Trans-PO-Naphox (1, 2) M of Example 4 in acetonitrile (MeCN) is shown. (A) Ultraviolet-visible absorption spectra of trans-PO-Naphox (1, 2) M of Example 4 after various lapses of time. (B) The maintenance rate (A / A ₀ ) of the absorbance (A) of the trans-PO-Naphox (1, 2) M of Example 4 after a predetermined time has elapsed. Irradiated with light of 515 ± 5 nm in a dimethyl sulfoxide / water mixed solvent (DMSO / _H2O ) of trans-PO-Naphox (2,3) M of Example 5 and Mito-PB hydrogen of Comparative Example 1. Shows the light resistance of the case. (A) Ultraviolet-visible absorption spectra of Mito-PB hello M of Comparative Example 1 after various lapses of time. (B) Maintenance rate (A / A ₀ ) of absorbance (A) after a lapse of a predetermined time of Mito-PB hello M of Comparative Example 1. (C) Ultraviolet-visible absorption spectra of Trans-PO-Naphox (2,3) M of Example 5 after various lapses of time. (D) The maintenance rate (A / A ₀ ) of the absorbance (A) of the trans-PO-Naphox (2,3) M of Example 5 after a predetermined time has elapsed. The light resistance when irradiated with light of 450 ± 5 nm in the dimethyl sulfoxide / water mixed solvent (DMSO / _H2O ) of cis-PO-BphoxM of Example 3 is shown. (A) Ultraviolet-visible absorption spectrum after various time lapses when irradiated with light having an irradiation intensity of 2050 W / m ² . (B) Maintenance rate (A / A ₀ ) of absorbance (A) after a lapse of a predetermined time when irradiated with light having an irradiation intensity of 2050 W / m ² . (C) Ultraviolet-visible absorption spectrum after various time lapses when irradiated with light having an intensity of 97% of the maximum intensity of the LED light. (D) The maintenance rate (A / A ₀ ) of the absorbance (A) after a lapse of a predetermined time when irradiated with light having an intensity of 97% of the maximum intensity of the LED light. The light resistance when irradiated with light of 450 ± 5 nm in the dimethyl sulfoxide / water mixed solvent (DMSO / _H2O ) of trans-PO-Naphox (2,3) M of Example 5 is shown. (A) Ultraviolet-visible absorption spectrum after various time lapses when irradiated with light having an irradiation intensity of 2050 W / m ² . (B) Maintenance rate (A / A ₀ ) of absorbance (A) after a lapse of a predetermined time when irradiated with light having an irradiation intensity of 2050 W / m ² . (C) Ultraviolet-visible absorption spectrum after various time lapses when irradiated with light having an intensity of 97% of the maximum intensity of LED light. (D) The maintenance rate (A / A ₀ ) of the absorbance (A) after a lapse of a predetermined time when irradiated with light having an intensity of 97% of the maximum intensity of the LED light. The light resistance when irradiated with light of 450 ± 5 nm in the dimethyl sulfoxide / water mixed solvent (DMSO / _H2O ) of Mito-PB hydrogen of Comparative Example 1 is shown. (A) Ultraviolet-visible absorption spectrum after various time lapses when irradiated with light having an irradiation intensity of 2050 W / m ² . (B) Maintenance rate (A / A ₀ ) of absorbance (A) after a lapse of a predetermined time when irradiated with light having an irradiation intensity of 2050 W / m ² . (C) Ultraviolet-visible absorption spectrum after various time lapses when irradiated with light having an intensity of 97% of the maximum intensity of the LED light. (D) The maintenance rate (A / A ₀ ) of the absorbance (A) after a lapse of a predetermined time when irradiated with light having an intensity of 97% of the maximum intensity of the LED light. 6 is a fluorescence imaging photograph of cells of Test Example 6 by Trans-PO-Bfox A1 / A2 of Example 2 and Mito Tracker Deep Red of Comparative Example 2. 6 is a fluorescence imaging photograph of cells of Test Example 6 by Trans-PO-Nafox A1 / A2 of Example 6 and Mito Tracker Deep Red of Comparative Example 2. 3 is a fluorescence imaging photograph of cells of Test Example 7 by Trans-PO-BfoxA1 / A2 of Example 2. 6 is a fluorescence imaging photograph of cells of Test Example 7 by Trans-PO-Nafox A1 / A2 of Example 6. Trans-PO-BfoxM of Example 1, Sith-PO-BfoxM of Example 3, Trans-PO-Nafox (1,2) M of Example 4, Trans-PO-Nafox (2,3) of Example 5. 3 is a fluorescence imaging photograph of cells of Test Example 9 by M and cis-PO-Naphox (2,3) M of Example 7.

As used herein, "comprise" is a concept that also includes "consist essentially of" and "consist of".

In the present specification, when the range is represented by "A to B", it means A or more and B or less unless otherwise specified.

1. 1. Bisphosphoryl crosslinked stilbene compound The bisphosphoryl crosslinked stilbene compound of the present invention has 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）：

[During the ceremony,
Ar ¹ 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 be combined to form a ring with adjacent nitrogen atoms. R ¹ and / or R ² may be combined with Ar ² to form a ring with adjacent nitrogen atoms.
Y ¹ and Y ² are the same or different, and 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。

(Ar ³ indicates a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring.
R ³ represents an organic group.
^Y3 represents an oxygen atom or a sulfur atom. )
Indicates a group represented by. ]
It is a compound represented by.

This bisphosphoryl cross-linked stilbene compound has the general formulas (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。

[During the ceremony,
Ar ¹ , Ar ² , Ar ^3a and Ar ^3b are identical or different and represent substituted or unsubstituted aromatic hydrocarbon rings or substituted or unsubstituted heteroaromatic rings.
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 be combined to form a ring with adjacent nitrogen atoms. R ¹ and / or R ² may be combined with Ar ² to form a ring with adjacent nitrogen atoms.
R ³ represents an organic group.
Y ^3a and Y ^3b are the same or different and indicate an oxygen atom or a sulfur atom. ]
Any of the bisphosphoryl cross-linked stilbene compounds represented by is included.

Both the transbisphosphoryl-bridged stillben compound represented by the general formula (1A) and the cisbisphosphoryl-bridged stillben compound represented by the general formula (1B) have absorption peak wavelengths as compared with MitoPB Yellow. Since the wavelength is lengthened and the intracellular small organs can be stained by using a long wavelength STED laser such as 775 nm, the phototoxicity to the intracellular small organs can be reduced and the length of the absorption peak wavelength can be reduced. It is a compound that is also useful as a light-resistant electronic material that is required to have a wavelength, and is also extremely excellent in light resistance. be.

As described above, the bisphosphoryl crosslinked stylben compound of the present invention has -OR ³ groups, which are functional groups, bonded to the ring Ar ³ , so that it is possible to control organelle localization and dispersibility. It is possible to bind to various organelles and stain various organelles. At this time, as shown in Examples described later, the bisphosphoryl cross-linked stilbene compound selectively exists at a desired site of the organelle, and is therefore useful for grasping the appearance shape of the organelle. Is. For example, when R ³ is a substituted alkyl group containing a phosphonium group, it can be used as a stain for mitochondrial membranes, and when R ³ is an alkyl group such as a methyl group, it can be used as a stain for lipid droplets. Can be used as. Therefore, since it can be used as an intracellular organelle stain, the bisphosphoryl cross-linked stilbene compound of the present invention is repeatedly observed in vivo with a super-resolution microscope such as stimulated emission suppression (STED) imaging. It is a suitable compound for.

Further, the bisphosphoryl crosslinked stylben compound of the present invention has an amino group or a substituted amino group which is an electron donating group, so that it can impart environmental responsiveness and can also achieve a longer absorption peak wavelength. Phototoxicity to intracellular small organs can be reduced.

Furthermore, the bisphosphoryl-crosslinked stilbene compound of the present invention has a bisphosphoryl-crosslinked stilbene skeleton, and thus imparts light resistance to such an extent that the absorption intensity is hardly reduced even by intense light irradiation such that MitoPB Yellow is photofaded. It is possible.

In the general formulas (1), (1A) and (1B), as the aromatic hydrocarbon ring represented by Ar ¹ , either a monocyclic aromatic hydrocarbon ring or a polycyclic aromatic hydrocarbon ring can be adopted, for example. 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, and a triphenylene ring.

The aromatic hydrocarbon ring represented by Ar ¹ may have a substituent. Examples of the substituent include 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.), and an alkynyl group (ethynyl group, 1-propynyl group). Etc.), carbonyl group, cyano group, nitro group and the like. When having a substituent, the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

In the general formulas (1), (1A) and (1B), examples of the heteroaromatic ring represented by Ar ¹ include a pyridine ring, a pyrazine ring and the like as a monocyclic heteroaromatic ring, and a polycyclic heteroaromatic ring. Examples thereof include an indole ring, an isoindole ring, a benzoimidazole ring, a quinoline ring, an isoquinoline ring, a quinoxaline ring and the like.

The heteroaromatic ring represented by Ar ¹ may have a substituent. Examples of the substituent include 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.), and an alkynyl group (ethynyl group, 1-propynyl group). Etc.), carbonyl group, cyano group, nitro group, halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.) and the like. When having a substituent, the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

As Ar ¹ , substituted or unsubstituted aromatic hydrocarbon rings are preferable from the viewpoints of structural stability, absorption peak wavelength, phototoxicity to intracellular small organs, light resistance, etc., and polycyclic aromatic rings are particularly preferable from the viewpoint of light resistance. Hydrocarbon rings are preferred.

When Ar ¹ is a substituted or unsubstituted polycyclic aromatic hydrocarbon ring, the bisphosphoryl crosslinked stilbene compound of the present invention has 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 described above.
Ar ⁴ represents a substituted or unsubstituted aromatic hydrocarbon ring. ]
Any of the bisphosphoryl cross-linked stilbene compounds represented by is used.

The bisphosphoryl cross-linked stilbene compound represented by the general formula (1C) has the general formulas (1C1) and (1C2): depending on the positions of the oxygen atom and the ring Ar ³ bonded to the phosphorus atom.

［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 described above. ]
Any of the bisphosphoryl cross-linked stilbene compounds represented by is included.

Further, the bisphosphoryl crosslinked stilbene compound represented by the general formula (1D) has 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 described above. ]
Any of the bisphosphoryl cross-linked stilbene compounds represented by is included.

In the general formulas (1C), (1C1), (1C2), (1D), (1D1) and (1D2), the aromatic hydrocarbon rings represented by Ar ⁴ include monocyclic aromatic hydrocarbon rings and polycyclic rings. Any of the aromatic hydrocarbon rings can be adopted, for example, a benzene ring is mentioned as a monocyclic aromatic hydrocarbon ring, and a naphthalene ring, an anthracene ring, a phenanthrene ring, a fluorene ring, a pyrene ring, etc. as a polycyclic aromatic hydrocarbon ring. Examples include the triphenylene ring. Of these, the benzene ring is particularly preferable from the viewpoints of structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, light resistance, and the like.

The aromatic hydrocarbon ring represented by Ar ⁴ may have a substituent. Examples of the substituent include 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.), and an alkynyl group (ethynyl group, 1-propynyl group). Etc.), carbonyl group, cyano group, nitro group and the like. When having a substituent, the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

In the general formulas (1C), (1C1), (1C2), (1D), (1D1) and (1D2), the heteroaromatic ring represented by Ar ⁴ is, for example, a pyridine ring as a monocyclic heteroaromatic ring. Examples thereof include a pyrazine ring and the like, and examples of the polycyclic heteroaromatic ring include an indole ring, an isoindole ring, a benzoimidazole ring, a quinoline ring, an isoquinoline ring and a quinoxaline ring.

The heteroaromatic ring represented by Ar ⁴ may have a substituent. Examples of the substituent include 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.), and an alkynyl group (ethynyl group, 1-propynyl group). Etc.), carbonyl group, cyano group, nitro group, halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.) and the like. When having a substituent, the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

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 (1C), (1C1) or (1C2) is ε in absorption. And Φ _F in fluorescence is even better, and it is possible to make the organelles of interest emit more intense and clearer light.

Further, as compared with the case where Ar ¹ is a substituted or unsubstituted monocyclic aromatic hydrocarbon ring, the bisphosphoryl cross-linked stilbene compound represented by the general formula (1D), (1D1) or (1D2) has an absorption peak wavelength. And it is possible to make the fluorescence peak wavelength longer.

From the above, it is possible to give diversity to the optical physical properties by the structure of Ar ¹ , and it is preferable to appropriately adjust the structure of Ar ¹ according to the required physical characteristics.

In the general formulas (1), (2), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1) and (1D2), Ar ² , Ar ³ , Ar. As the aromatic hydrocarbon ring represented by ^3a and Ar ^3b , either a monocyclic aromatic hydrocarbon ring or a polycyclic aromatic hydrocarbon ring can be adopted, and for example, a benzene ring is mentioned as the monocyclic aromatic hydrocarbon ring. Examples of the polycyclic aromatic hydrocarbon ring include a naphthalene ring, an anthracene ring, a phenylene ring, a fluorene ring, a pyrene ring, and a triphenylene ring. Of these, the benzene ring is particularly preferable from the viewpoints of structural stability, absorption peak wavelength, phototoxicity to intracellular organelles, light resistance, and the like.

The aromatic hydrocarbon ring represented by Ar ² may have a substituent. Examples of the substituent include 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.), and an alkynyl group (ethynyl group, 1-propynyl group). Etc.), carbonyl group, cyano group, nitro group and the like. When having a substituent, the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

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

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 later, a cycloalkyl group described later, an aryl group described later, a heteroaryl group described later, an alkenyl group (vinyl group, propenyl group, etc.), and an alkynyl group (ethynyl group, 1-propynyl group). Etc.), carbonyl group, cyano group, nitro group, halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.) and the like. When having a substituent, the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

As Ar ³ , Ar ^3a and Ar ^3b , substituted or unsubstituted aromatic hydrocarbon rings are preferable from the viewpoints of structural stability, absorption peak wavelength, phototoxicity to intracellular small organs, light resistance and the like, and substituted or unsubstituted. A monocyclic aromatic hydrocarbon ring is more preferable.

As the alkyl group represented by ^R1 and ^R2 in the general formulas (1), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1) and (1D2). Can be either a linear alkyl group or a branched alkyl group, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, or a tert-butyl group. C1-10 alkyl groups such as C1-10 alkyl groups (particularly C1-6 alkyl groups) can be mentioned.

The alkyl groups represented by R ¹ and R ² may have a substituent. Examples of the substituent include a halogen atom, a cycloalkyl group described later, an aryl group described later, a heteroaryl group described later, a cyano group, a nitro group and the like. When having a substituent, the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

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

The cycloalkyl groups represented by R ¹ and R ² may have a substituent. Examples of the substituent include a halogen atom, the above-mentioned alkyl group, a below-mentioned aryl group, a below-mentioned heteroaryl group, a cyano group, a nitro group and the like. When having a substituent, the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

As the aryl group represented by ^R1 and ^R2 in the general formulas (1), (1A), (1B), (1C), (1C1), (1C2), (1D), (1D1) and (1D2). For example, any of a monocyclic aryl group, a fused aryl group and a polycyclic aryl group can be adopted. For example, a phenyl group can be mentioned as a monocyclic aryl group, and a naphthyl group, an anthracenyl group or a phenanthrenyl group can be used as a condensed aryl group. , Fluolenyl group, Pyrenyl group, Triphenylenyl group and the like, and examples of the polycyclic aryl group include C6-18 aryl groups (particularly C6-14 aryl groups) such as biphenyl group and terphenyl group.

The aryl group represented by R ¹ and R ² may have a substituent. Examples of the substituent include a halogen atom, the above-mentioned alkyl group, the above-mentioned aryl group, a heteroaryl group described later, a cyano group, a nitro group and the like. When having a substituent, the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

In the general formula (1), as the heteroaryl group represented by R ¹ and R ² , either a monocyclic heteroaryl group or a condensed heteroaryl group can be adopted, and for example, a pyrrolyl group or thienyl as a monocyclic heteroaryl group can be adopted. Examples include a group, a furanyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an oxazolyl group, a piperidyl group, a pyridyl group, a pyrazil group, and examples of the condensed heteroaryl group include an indolyl group, an isoindryl group, a benzoimidazolyl group, a quinolyl group, and an isoquinolyl group. , Kinoxalyl group and the like.

The heteroaryl group represented by R ¹ and R ² may have a substituent. Examples of the substituent include the halogen atom, the alkyl group, the aryl group, the heteroaryl group, the cyano group, the nitro group and the like. When having a substituent, the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

Among them, as R ¹ and R ² , substituted or unsubstituted (hetero) aryl groups are preferable, and substituted or unsubstituted aryl groups are more preferable, from the viewpoint of easily imparting environmental responsiveness and easily lengthening the absorption peak wavelength. Preferred, unsubstituted aryl groups are even more preferred.

In addition, R ¹ and R ² may be combined to form a ring together with adjacent nitrogen atoms. 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。

It may be a group represented by such as.

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

等であってもよい。

And so on.

In this case, in Ar ² , the substitution position to which 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), either a linear alkyl group or a branched chain alkyl group can be adopted as the alkyl group represented by R3 ^, but it is preferable to select an appropriate group depending on the dyeing target.

The alkyl group represented by R ³ may have a substituent. The substituent is preferably a functional group so as to facilitate control of organoella localization and dispersibility, for example, an epoxy group, a phosphorus-containing group, a carboxy group, an alkoxycarbonyl group (-COOR ⁸ ), and an amide. Examples thereof include a group or a derivative group thereof (-CONHR ⁹ ).

The phosphorus-containing group as a substituent of the alkyl group represented by R ³ is not particularly limited, and from the viewpoint of easily controlling the organelle localization and dispersibility, the general formula (3):
-P ⁺ R ⁴ _n X ¹ _(4-n) ^- (3)
[In the formula, ^R4 is the same or different and represents a substituted or unsubstituted (hetero) aryl group.
X ¹ is the same or different and represents a halogen atom.
n represents an integer of 1 to 3. ]
The group represented by is preferable.

In the general formula (3), as the aryl group represented by ^R4 , for example, any monocyclic aryl group, condensed ring aryl group or polycyclic aryl group can be adopted, and for example, a phenyl group is mentioned as the monocyclic aryl group. Examples of the fused aryl group include a naphthyl group, an anthrasenyl group, a phenanthrenyl group, a fluorenyl group, a pyrenyl group, a triphenylenyl group and the like, and a polycyclic aryl group includes a C6-18 aryl group such as a biphenyl group and a terphenyl group (particularly C6). -14 aryl group).

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

In the general formula (3), as the heteroaryl group represented by ^R4 , either a monocyclic heteroaryl group or a condensed heteroaryl group can be adopted. For example, as the monocyclic heteroaryl group, a pyrrolyl group, a thienyl group, or a furanyl group can be adopted. Examples include a group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an oxazolyl group, a piperidyl group, a pyridyl group, a pyrazil group, etc. And so on.

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

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

In the general formula (3), examples of the halogen atom represented by X ¹ include a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, and the like. A bromine atom, an iodine atom and the like are preferable, a chlorine atom, a bromine atom and the like are more preferable, and a bromine atom is further preferable.

In the general formula (3), n is preferably an integer of 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 (-COOR ⁸ ) as a substituent of the alkyl group represented by R ³ is particularly high if it contains an alkyl group (methyl group, ethyl group, n-propyl group, isopropyl group, etc.) as R ⁸ . It can be used without limitation, and examples thereof include a methoxycarbonyl group, an ethoxycarbonyl group, an n-propoxycarbonyl group, and an isopropoxycarbonyl group.

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

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

Specifically ^, the organic group represented by R3 that satisfies the above conditions is specifically.

［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, R ⁸ and R ⁹ are the same as described above. Ph represents a phenyl group. k represents an integer of 5 to 15. m represents an integer from 2 to 100. ]
And so on.

Examples of the bisphosphoryl crosslinked stilbene compound represented by the general formula (1) satisfying the above conditions include, for example.

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、

Examples thereof include a bisphosphoryl cross-linked stilbene compound represented by. ^In these equations, R5 and R6 are the ^same or different.

［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. m represents an integer from 1 to 100. ]
And so on. In particular, the bisphosphoryl cross-linked stilbene compound shown in Examples described later is preferable.

2. 2. Method for Producing Bisphosphoryl Crosslinked Stilbene Compound The method for producing the phosphole compound of the present invention is not particularly limited. For example, 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 described above. ]
Using the bisphosphoryl crosslinked stilbene compound represented by the above as a starting material, the general formula (5):
R ³ X ² (5)
[In the formula, R ³ is the same as described above. X ² represents a halogen atom. ]
It can be obtained by reacting with a compound represented by.

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

In the general formula (5), examples of the halogen atom represented by X ² include a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like, preferably a chlorine atom, a bromine atom, an iodine atom and the like, and a chlorine atom and a bromine atom. Etc. are more preferable, and a bromine atom is further preferable.

Bases can also be used in the reaction, if desired. Examples of the base include potassium fluoride, cesium fluoride, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, sodium acetate, potassium acetate, calcium acetate and the like. Potassium carbonate is preferred from the viewpoint of yield and ease of synthesis. When a base is used, the amount used is preferably 2 to 30 mol, more preferably 10 to 20 mol, per 1 mol of compound (4A) or compound (4B) from the viewpoint of ease of synthesis, yield and the like. preferable.

The reaction can usually be carried out in the presence of a reaction solvent. Examples of the reaction solvent 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; dimethylformamide and the like. The amide solvent and the like are mentioned, and the amide solvent is preferable, and the dimethylformamide is more preferable, from the viewpoint of ease of synthesis, yield and the like. These reaction solvents may be used alone or in combination of two or more.

As the reaction atmosphere, an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.) can be usually adopted. The reaction temperature can be any of heating, normal temperature and cooling, and is usually preferably 0 to 100 ° C, more preferably 10 to 70 ° C. The reaction time is not particularly limited, and is preferably a time during which the reaction proceeds sufficiently.

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

In the above, an example of a method for synthesizing one aspect of the bisphosphoryl crosslinked stilbene compound of the present invention has been described, but the present invention is not limited to this production method and can be synthesized by various synthetic methods.

3. 3. Fluorescent dye and intracellular small organ stain The fluorescent dye of the present invention contains the above-mentioned bisphosphoryl cross-linked stilbene compound of the present invention.

Since the fluorescent dye of the present invention has a bisphosphoryl crosslinked stilbene skeleton, it has excellent light resistance ^, and various reactive groups can be introduced into R3. Therefore, 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, vesicles, cell membranes, etc.). Therefore, the fluorescent dye of the present invention is suitable for repeated observation in a living body with a super-resolution microscope (particularly, an induced emission suppression (STED) microscope) such as induced emission suppression (STED) imaging.

When the bisphosphoryl cross-linked stylben compound of the present invention is used as an intracellular small organ stain (particularly a lipid droplet stain, a mitochondrial stain, a lysosome stain, an endoplasmic reticulum stain, a cell membrane stain, etc.), the inside of the cell of the present invention is used. Small organ stains (particularly lipid droplet stains, mitochondrial stains, lysosome stains, endoplasmic reticulum stains, cell membrane stains, etc.) contain the bisphosphoryl cross-linked stylben compound of the present invention, but the culture medium (Dulbecco's). It is preferable to dissolve it in modified Eagle's medium (DMEM, etc.) to make a solution, which makes intracellular small organs (particularly lipid droplets, mitochondria, lysosomes, endoplasmic reticulum, cell membrane, etc.) stronger while improving light resistance. From the viewpoint of staining, the content of the bisphosphoryl cross-linked stylben compound of the present invention is preferably 1 to 10000 nmol / L, more preferably 10 to 1000 nmol / L.

As described above, the intracellular organelle stain (particularly lipid droplet stain, mitochondria stain, lysosomal stain, endoplasmic reticulum stain, cell membrane stain, etc.) of the present invention is preferably in the form of a solution, but in vivo. When observing, the pH is preferably about 5 to 11, more preferably about 6.5 to 7.5. In order 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 described with reference to Examples, but the present invention is not limited thereto.

Unless otherwise restricted, all reactions were carried out in 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 CH ₂ Cl ₂ were purchased from Kanto Chemical Co., Ltd. and purified by Glass Contour Solvent Systems.

In addition, 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 the previous report (Chem. Asian J., 13, 1616-1624 (2018)).

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

1,2-Epoxy-6-bromohexane was synthesized according to the previous report (Angew. Chem. Int. Ed. 2019, 58, 1727-1731).

(4-Bromo-n-butyl) triphenylphosphonium bromide was synthesized according to the previous report (J. Am. Chem. Soc. 2015, 137, 6837-6843).

(10-bromo-n-decyl) triphenylphosphonium bromide was synthesized according to the previously reported (International Publication No. 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 meta-chloroperbenzoic acid.

Dichloromethane (8 mL) of metachloroperbenzoic acid (purity 70%, 1.8 g, 10.4 mmol) with stirring in a solution of 9-bromonon-1-ene (1.0 g, 4.87 mmol) in dichloromethane (8 mL). ) Was dropped. Then, the mixture was stirred at 25 ° C. for 16 hours to precipitate metachloroperbenzoic acid. The reaction is filtered to remove the white precipitate, extracted with dichloromethane (DCM; 20 mL x 2), washed with saturated NaHCO ₃ (20 mL), water (20 mL) and brine (20 mL) and 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, yield 83%).
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) in a nitrogen-purged flask. , 0.33 mmol), triethylamine (Et ₃ N; 70 mL) was added, and the mixture was strictly degassed by the freeze degassing method. Trimethylsilylacetylene (2.47 mL) was added via 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, CDCl ₃ ) 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).

K ₂ CO ₃ in the obtained 2-bromo-3- [2- (trimethylsilyl) ethynyl] naphthalene (4.2 g, 14.0 mmol) CH ₃ OH / tetrahydrofuran (1/1, v / v, 140 mL) solution. (5.8 g, 42.0 mmol) was added, 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).
¹ H NMR (400 MHz, CDCl ₃ ) 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).

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. A 7 mg, 0.25 mmol) triethylamine (150 mL) suspension was degassed, to which 2-bromo-3-ethynylnaphthalene (2.9 g, 12.5 mmol) obtained above was added. After stirring overnight at room temperature, the mixture was diluted with toluene (150 mL) and filtered to remove insoluble compounds. The obtained filtrate was concentrated under reduced pressure, recrystallized from ethanol and purified, and 2-bromo-3-((2-bromo-4-chlorophenyl) ethynyl) naphthalene (Compound 1) was added to 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

In an anhydrous tetrahydrofuran (anhydrous THF; 20 mL) solution of 2-bromo-3-((2-bromo-4-chlorophenyl) ethynyl) naphthalene (Compound 1; 1.0 g, 2.38 mmol) obtained in Synthesis Example 2 An n-pentane solution of tert-butyllithium (tBuLi) (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 kept at 0 ° C. for 30 minutes. Then, after cooling to −78 ° C. again, PCl ₃ (1.3 mL, 15 mmol) was added to raise the temperature of the mixture to room temperature. After stirring at room temperature for 17 hours, 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 after stirring for 30 minutes, 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), the organic layer was washed with brine (30 mL), dried over anhydrous Na ₂ SO ₄ , and then filtered. After concentrating the obtained filtrate under reduced pressure, the obtained mixture was purified by silica gel column chromatography (CH ₂ Cl ₂ / acetone = 1/10 to 1/1) to obtain trans-PO-Na2 as a green-yellow solid. 469 mg (yield 34%) and 540 mg (39%) of cis-PO-Na2 as a green-yellow solid were obtained.
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).
³¹ P 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.
Sith-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).
³¹ P 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-BfoxM

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), K2 CO 3 (} 801 mg, 5.8 mmol) was added to anhydrous toluene (60 mL), and the mixture was stirred at 80 ° C. for 20 hours under a nitrogen atmosphere. did. Then, it was cooled to room temperature, and the insoluble compound was 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-BfoxM (520 mg, yield) as a red solid. The rate was 83%).
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).
³¹ P 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-BfoxA1 / A2

BBr ₃ (0.85 mL, 8.3 mmol) was slowly added dropwise to the anhydrous CH ₂ Cl ₂ (21 mL) solution of trans-PO-BfoxM (270 mg, 0.40 mmol) obtained in Example 1 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 x 2). After drying the organic layer with 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.

Trans-PO-B2 (30 mg, 0.048 mmol), (10-bromo-n-decyl) triphenylphosphonium bromide (27 mg, 0.048 mmol) and K2 CO _{3 (} 133 mg, 0.96 mmol) obtained above. Was added to anhydrous dimethylformamide (anhydrous DMF; 3.6 mL), and the mixture was 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 the mixture was extracted 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 then filtered. The filtrate is concentrated under reduced pressure, and the obtained solid is purified by silica gel column chromatography (CH ₃ OH / CH ₂ Cl ₂ = 1 / 30-1 / 20) to form a red solid, trans-PO-BfoxA1 / A2. Was obtained (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: Sith-PO-BfoxM

As in Example 1, cis-PO-BfoxM (yield 78%) was obtained as in Example 1 except that cis-PO-B1 was used instead of trans-PO-B1 as a starting 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).
³¹ P 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 K2 CO 3 (} 160 mg, 1.16 mmol) were added to anhydrous toluene (12 mL) and stirred at 90 ° C. for 24 hours under a nitrogen atmosphere. .. Then, it was cooled to room temperature, and the insoluble compound was 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), and trans-PO-Naphox (1, 2) was used as a red solid. ) M (120 mg, 88% yield) was obtained.
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 = 5.5 Hz, 6H).
³¹ P 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

Trans-PO-Na2 (90 mg, 0.158 mmol), diphenylamine (133 mg, 0.79 mmol), palladium acetate (Pd (OAc) ₂ ; 2.1 mg, 0.0095 mmol), 2-dicyclohexyl obtained in Synthesis Example 3 Phosphino- ₂ ', 4', 6'-triisopropylbiphenyl (Xphos; 9 mg, 0.019 mmol) and K2 CO ₃ (131 mg, 0.948 mmol) were added to anhydrous toluene (10 mL), and 90 under a nitrogen atmosphere. The mixture was stirred at ° C for 24 hours. Then, it was cooled to room temperature, and the insoluble compound was 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), and trans-PO-Naphox (2,3) was purified as a red solid. ) M (100 mg, 90% yield) was obtained.
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).
³¹ P 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-NafoxA1 / A2

BBr ₃ (0.28 mL, 2.56 mmol) was added to the anhydrous CH ₂ Cl ₂ (8 mL) solution of trans-PO-Naphox (2,3) M (90 mg, 0.128 mmol) obtained in Example 5. It 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 x 2). After drying the organic layer with 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-Na 3 as a red solid (80 mg, 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).
³¹ P 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 resulting trans-PO-Na3 (30 mg, 0.0445 mmol), (10-bromodecyl) triphenylphosphonium bromide (25 mg, 0.0445 mmol), and K2 CO _{3 (} 123 mg, 0.89 mmol) were mixed with anhydrous dimethylformamide (10-bromodecyl) (25 mg, 0.0445 mmol). Anhydrous DMF (3.6 mL) was added, and the mixture was 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 CH ₂ Cl ₂ (20 mL). The organic layer was washed twice with water (20 mL), dried over anhydrous Na ₂ SO ₄ , and then filtered. After concentrating the filtrate under reduced pressure, the obtained solid was purified by silica gel column chromatography (CH ₃ OH / CH ₂ Cl ₂ = 1 / 30-1 / 10) to form a trans-PO-NaphoxA1 / as an orange-red solid. A2 was obtained (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: Sis-PO-Naphox (2,3) M

As in Example 5, cis-PO-Nafox was used as the starting compound except that the cis-PO-Na2 obtained in Synthesis Example 3 was used instead of the trans-PO-Na2 obtained in Synthesis Example 3. (2,3) M (yield 81%) was obtained.
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).
³¹ P 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: Transformer-PS-BfoxA1 / A2

The solution of the mixture of trans-PO-BphoxM (40.0 mg, 0.061 mmol) and Lawesson's reagent (99 mg, 0.246 mmol) obtained in Example 1 in anhydrous toluene (5 mL) was refluxed for 20 hours. After cooling this solution to room temperature and removing the solvent under reduced pressure, the obtained solid was purified by silica gel column chromatography (CH ₂ Cl ₂ / hexane = 1 / 2-1 / 1), and 34 g (yield). 81%) of trans-PS-BfoxM and 8 mg (19% yield) of cis-PS-BfoxM were obtained as orange solids.
Transformer-PS-BfoxM:
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).
³¹ P 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.
Sith-PS-BfoxM:
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).
³¹ P 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.

BBr ₃ (0.08 mL, 0.79 mmol) was added dropwise at −78 ° C. to the solution of trans-PS-BfoxM (27 mg, 0.039 mmol) obtained above in anhydrous CH ₂ Cl ₂ (2 mL). did. The solution was stirred at −78 ° C. for 1 hour and then the mixture was slowly warmed to room temperature in 4 hours. At 0 ° C., the reaction was quenched with water (2 mL) and extracted with ethyl acetate (20 mL x 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, yield 79%).

Trans-PS-BfoxA1 / A2 (yield 17%) was used as the starting compound in the same manner as in Example 2 except that the trans-PS-B1 obtained above was used instead of the trans-PO-B2. Obtained.

Comparative Example 1: Mito-PB hello M

Mito-PB hellom of Comparative Example 1 was synthesized according to the previously reported (PNAS, 2019, 116, 15817-15822).

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

Test Example 1: Photophysical properties For cis-PO-BfoxM of Example 3, lance-PO-Nafox (1, 2) M of Example 4, and trans-PS-BfoxC4 of Example 8, toluene, dichloromethane (DCM) or The ultraviolet-visible absorption spectrum, fluorescence spectrum, absolute fluorescence quantum yield, etc. when dissolved in acetonitrile (MeCN) were measured. I took a photo of the exterior. The results of Examples 3 and 8 are shown in FIG. 1, the results of Example 4 are shown in FIG. 2, and each optical property data is shown in Table 1.

From the above, the bisphosphoryl crosslinked stilbene compound of the present invention can absorb light in the visible light region (particularly about 450 to 550 nm) in any of various solvents, and fluorescence of various wavelengths depending on the solvent. Was able to emit. In particular, the fluorescence was extremely small in a polar solvent such as acetonitrile, suggesting that it hardly fluoresces in the cytoplasm. Therefore, it is suggested that hydrophobic tissues such as phospholipid membranes and lipid droplets can be stained with high contrast.

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

Next, the light resistance of each sample at the maximum absorption wavelength was evaluated. Specifically, an LED light (2050 W / m ² ) equipped with a bandpass filter that transmits light of 515 ± 5 nm irradiates light with a wavelength of 515 ± 5 nm, and the maximum absorption wavelength immediately after irradiation (after 0 hours). (Example 1 is 460 nm, Example 3 is 487 nm, Example 4 is 513 nm, Example 5 is 482 nm, Comparative Example ₁ is 487 nm). The maintenance rate (A / A ₀ ) of A) was evaluated. The results of Examples 1 and 1 are shown in FIGS. 3 (b) and 3 (d), the results of Examples 3 and 5 are shown in FIGS. 4 (b) and (d), and the results of Example 4 are shown in FIG. 5 (b). ).

As a result, the bisphosphoryl cross-linked stilbene compound of the present invention had remarkably excellent light resistance as compared with Mito-PB hello, and the absorbance was hardly reduced.

Test Example 3: Light resistance (1 in DMSO / _H2O )
Trans-PO-Naphox (2,3) M of Example 5 and Mito-PB hellom of Comparative Example 1 were dissolved in a dimethyl sulfoxide / water mixed solvent (volume ratio 1: 1) at 25 ° C. The concentration at this time was adjusted so that the absorbance at 515 nm was about the same (0.1). An LED light equipped with a bandpass filter that transmits light of 515 ± 5 nm is irradiated with light having a wavelength of 515 ± 5 nm at an irradiation intensity of 2050 W / m ² , and the ultraviolet-visible absorption spectrum is measured after various lapses of time. rice field. The result of Comparative Example 1 is shown in FIG. 6 (a), and the result of Example 5 is shown in FIG. 6 (c).

Next, the light resistance at the maximum absorption wavelength was evaluated. Specifically, an LED light equipped with a bandpass filter that transmits light of 515 ± 5 nm is irradiated with light having a wavelength of 515 ± 5 nm at an irradiation intensity of 2050 W / m ² , and is maximized immediately after irradiation (after 0 hours). The absorbance (A ₀ ) at the absorption wavelength (506 nm in Example 5 and 473 nm in Comparative Example 1) was set to 1.00, and the retention rate (A / A ₀ ) of the absorbance (A) after a lapse of 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 (Part 2 in DMSO / _H2O )
Dimethyl sulfoxide / water mixed solvent (volume ratio 1) for cis-PO-Bfox M of Example 3, trans-PO-Nafox (2,3) M of Example 5 and Mito-PB ywllow M of Comparative Example 1 at 25 ° C. 1) Dissolved in. The concentration at this time was adjusted so that the absorbance at 450 nm was about the same (0.1). An LED light equipped with a bandpass filter that transmits light of 450 ± 5 nm, and irradiates light with a wavelength of 450 ± 5 nm at an irradiation intensity of 2050 W / m ² or 97% of the maximum intensity of the LED light for various times. The ultraviolet visible absorption spectrum after the lapse was measured. The result of the irradiation intensity of 2050 W / m ² of Example 3 is shown in FIG. 7 (a), the result of the intensity of 97% of the maximum intensity of the LED light of Example 3 is shown in FIG. 7 (c), and the irradiation intensity of Example 5 is shown. The result of 2050 W / m ² is shown in FIG. 8 (a), the result of the intensity of 97% of the maximum intensity of the LED light of Example 5 is shown in FIG. 8 (c), and the result of the irradiation intensity of Comparative Example 1 is 2050 W / m ² . 9 (a) shows the result of the intensity of 97% of the maximum intensity of the LED light of Comparative Example 1 in FIG. 9 (c).

Next, the light resistance at the maximum absorption wavelength was evaluated. Specifically, it is an LED light equipped with a bandpass filter that transmits light of 450 ± 5 nm, and irradiates light with a wavelength of 450 ± 5 nm at an irradiation intensity of 2050 W / m ² or 97% of the maximum intensity of LED light. The absorbance (A ₀ ) at the maximum absorption wavelength (509 nm in Example 3, 504 nm in Example 5, 473 nm in Comparative Example 1) immediately after irradiation (0 hours later) is set to 1.00, and the absorbance after a predetermined time has elapsed. The maintenance rate (A / A ₀ ) of (A) was evaluated. The result of the irradiation intensity of 2050 W / m ² of Example 3 is shown in FIG. 7 (b), the result of the intensity of 97% of the maximum intensity of the LED light of Example 3 is shown in FIG. 7 (d), and the irradiation intensity of Example 5 is shown. The result of 2050 W / m ² is shown in FIG. 8 (b), the result of 97% of the maximum intensity of the LED light of Example 5 is shown in FIG. 8 (d), and the result of the irradiation intensity of 2050 W / m ² of Comparative Example 1 is shown. 9 (b) shows the result of the intensity of 97% of the maximum intensity of the LED light of Comparative Example 1 in FIG. 9 (d).

Test Example 5: Preparation of cells (1)
HeLa cells (RIKEN Cell Bank, Japan) at 37 ° C., Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 10% fetal bovine serum (FBS, Gibco) and 1% antibiotic-antifungal agent (AA, Sigma). Incubated in a 5% by volume CO ₂ incubator for 24 hours. Three days prior to imaging, cells (5 × 10 ⁴ ) were seeded on a glass bottom dish.

Test Example 6: Fluorescence Imaging of Cells (Part 1)
In the trans-PO-BfoxA1 / A2 staining experiment of Example 2, HeLa cells were cultivated in DMEM medium containing 500 nM trans-PO-BfoxA1 / A2, 1% dimethyl sulfoxide and 0.1% PlusnicF-127. The cells were cultured at 37 ° C. for 2 hours in a volume% CO ₂ incubator. The cells were washed 3 times with DMEM (-), replaced with DMEM (-), and observed using a super-resolution microscope TCS SP8 STED (manufactured by Leica). At this time, the excitation wavelength is preferably 470 to 540 nm, preferably 488 to 515 nm, and a fluorescence signal in the range of 550 to 750 nm is detected. The results are shown in FIG.

The same was performed in the staining experiments of Trans-PO-Nafox A1 / A2 of Example 6 and Mito Tracker Deep Red of Comparative Example 2. The results are shown in FIGS. 10 and 11.

Test Example 7: Fluorescence Imaging of Cells (Part 2)
In the trans-PO-BfoxA1 / A2 staining experiment of Example 2, HeLa cells were cultivated in DMEM medium containing 500 nM trans-PO-BfoxA1 / A2, 1% dimethyl sulfoxide and 0.1% PlusnicF-127. The cells were cultured at 37 ° C. for 2 hours in a volume% CO ₂ incubator. The cells were washed 3 times with DMEM (-), replaced with DMEM (-), and imaged using a super-resolution microscope TCS SP8 STED (manufactured by Leica).

For confocal imaging, a super-resolution microscope TCS SP8 STED was used, and the excitation wavelength was preferably 470 to 540 nm, optimally 488 to 515 nm, and a fluorescence signal in the range of 550 to 750 nm was detected. On the other hand, STED imaging used a 775 nm STD laser (pulse laser) to obtain a super-resolution image, and detected a signal of 550 to 750 nm.

The same was performed in the staining experiment of trans-PO-NafoxA1 / A2 of Example 6.

The results of Trans-PO-BfoxA1 / A2 of Example 2 are shown in FIG. 12, and the results of Trans-PO-NafoxA1 / A2 of Example 6 are shown in FIG.

Test Example 8: Preparation of cells (Part 2)
HeLa cells (RIKEN Cell Bank, Japan) were treated with oleic acid (400 nM) in a 5% by volume CO ₂ incubator for 24 hours and the medium was replaced. HeLa cells (RIKEN Cell Bank, Japan) at 37 ° C., Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 10% fetal bovine serum (FBS, Gibco) and 1% antibiotic-antifungal agent (AA, Sigma). Incubated in a 5% by volume CO ₂ incubator for 24 hours. Three days prior to imaging, cells (5 × 10 ⁴ ) were seeded on a glass bottom dish.

Test Example 9: Fluorescence Imaging of Cells (Part 3)
In the trans-PO-BfoxM staining experiment of Example 1, HeLa cells were subjected to 5% by volume CO ₂ in DMEM medium containing 500 nM trans-PO-BfoxM, 1% dimethyl sulfoxide and 0.1% Pluonic F-127. The cells were cultured at 37 ° C. for 2 hours in an incubator. The cells were washed 3 times with DMEM (-), replaced with DMEM (-), and observed using a super-resolution microscope TCS SP8 STED and FV-10 (manufactured by Olympus). At this time, the excitation wavelength is preferably 470 to 540 nm, preferably 488 to 515 nm, and a fluorescence signal in the range of 550 to 750 nm is detected.

Example 3 cis-PO-BfoxM, Example 4 trans-PO-Naphox (1,2) M, Example 5 trans-PO-Nafox (2,3) M and Example 7 cis-PO- The same was performed in the staining experiment of Naphox (2,3) M.

The results are shown in FIG.

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

Claims (6)

General formula (1):

[During the ceremony,
Ar ¹ 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 be combined to form a ring with adjacent nitrogen atoms. R ¹ and / or R ² may be combined with Ar ² to form a ring with adjacent nitrogen atoms.
Y ¹ and Y ² are the same or different, and the general formula (2):

(Ar ³ indicates a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring.
R ³ represents an organic group.
Y ³ represents an oxygen atom or a sulfur atom. )
Indicates a group represented by. ]
A bisphosphoryl cross-linked stilbene compound represented by. The bisphosphoryl crosslinked stilbene compound according to claim ¹ , wherein R3 is a substituted or unsubstituted alkyl group. The bisphosphoryl crosslinked 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 crosslinked stilbene compound according to any one of claims 1 to 3. An intracellular organelle stain containing the bisphosphoryl crosslinked stilbene compound according to any one of claims 1 to 3. Induced release of organelles using the bisphosphoryl cross-linked stylben 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. Suppression (STED) imaging method.

Images