JP7716735B2 - Phosphonium compounds, and their salts, hydrates and solvates - Google Patents

Description

The present invention relates to phosphonium compounds, as well as their salts, hydrates, and solvates.

Molecules that emit strong light in the red to near-infrared region not only reduce phototoxicity to cells, but are also extremely useful for deep biological imaging. To date, dyes that emit light in the red to near-infrared region and have a phosphaxanthene skeleton are known (see, for example, Patent Document 1).

On the other hand, from a more general perspective, increasing the diversity of absorption and emission properties of fluorescent dyes will also lead to an expansion of their range of applications.

International Publication No. 2016/133218

The present invention was made to solve the above-mentioned problems, and aims to provide a compound that exhibits strong luminescence in the red to near-infrared region.

With the above objective in mind, and as a result of extensive research, the inventors have discovered that phosphonium compounds having a specific phosphaxanthene skeleton exhibit strong emission in the red to near-infrared region. Among such phosphonium compounds, phosphonium fluorescein compounds are negative solvatochromic fluorescent dyes whose absorption and fluorescence maximum wavelengths shift to shorter wavelengths as the polarity of the solvent increases. The present invention was completed through further research based on this finding. Specifically, the present invention encompasses the following configurations:

Item 1. General formula (1):

[Wherein, ^Y1 represents an oxygen atom or a substituted or unsubstituted amino cation. ^Y2 represents an oxygen anion or a substituted or unsubstituted amino group. ^R1 and ^R2 are the same or different and represent a substituted or unsubstituted (hetero)aryl group, or a substituted or unsubstituted alkyl group. ^R3 and ^R4 are the same or different and represent a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted alkoxy group, provided that at least one of ^R3 and ^R4 is a substituted or unsubstituted alkyl group, or a substituted or unsubstituted alkoxy group. ^R5 represents a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, or a substituted or unsubstituted aryl group. ^R6 , ^R7 , ^R8 , and ^R9 are the same or different and represent a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a sulfonic acid group. At least one selected from the group consisting of R ⁶ and Y ¹ , R ⁷ and Y ² , R ⁸ and Y ¹ , and R ⁹ and Y ² may combine to form a substituted or unsubstituted alkylene group.]
A phosphonium compound, or a salt, hydrate or solvate thereof, containing an ion represented by the following formula:

Item 2. General formula (1A):

[Wherein ^R1 , ^R2 , ^R3 , ^R4 and ^R5 are the same as defined above. ^R6a , ^R7a , ^R8a and ^R9a are the same or different and represent a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a sulfonic acid group. ^R10 , ^R11 , ^R12a and ^R13a are the same or different and represent a substituted or unsubstituted alkyl group. At least one selected from the group consisting of ^R6a and ^R10 , ^R7a and ^R13a , ^R8 and ^R11 , and ^R9a and ^R12a may be joined together to form a substituted or unsubstituted alkylene group.]
Item 2. The phosphonium compound according to Item 1, or a salt, hydrate or solvate thereof, containing a phosphonium rhodamine cation represented by the following formula:

Item 3. The phosphonium compound, or a salt, hydrate, or solvate thereof, according to Item 2, having an absorption maximum wavelength in water of 690 to 730 nm.

Item 4. General formula (1B):

[In the formula, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are the same as defined above. R ^6b , R ^7b , R ^8b and R ^9b are the same or different and represent a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a sulfonic acid group.]
Item 2. The phosphonium compound according to Item 1, or a salt, hydrate or solvate thereof, which is a phosphonium fluorescein compound represented by the following formula:

Item 5. The phosphonium compound, or a salt, hydrate, or solvate thereof, according to Item 4, having an absorption maximum wavelength in water of 630 to 660 nm.

Item 6. General formula (1C):

[Wherein ^R1 , ^R2 , ^R3 , ^R4 , and ^R5 are the same as defined above. ^R6c , ^R7c , ^R8c , and ^R9c are the same or different and represent a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a sulfonic acid group. ^R12c and ^R13c are the same or different and represent a substituted or unsubstituted alkyl group. At least one selected from the group consisting of ^R7c and ^R13c , and ^R9c and ^R12c may together form a substituted or unsubstituted alkylene group.]
Item 2. The phosphonium compound according to Item 1, or a salt, hydrate or solvate thereof, containing a phosphonium rhodol cation represented by the following formula:

Item 7. The phosphonium compound, or a salt, hydrate, or solvate thereof, according to Item 6, having an absorption maximum wavelength in water of 660 to 690 nm.

Item 8. The phosphonium compound or a salt, hydrate or solvate thereof according to any one of Items 1 to 7, wherein R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are all hydrogen atoms.

Item 9. The phosphonium compound or a salt, hydrate, or solvate thereof according to any one of Items 1 to 8, wherein ^{R 1} and R ² are the same or different and each represent a substituted or unsubstituted aryl group.

Item 10. A fluorescent dye containing the phosphonium compound or its salt, hydrate, or solvate described in any one of items 1 to 9.

Item 11. A compound comprising the phosphonium compound or its salt, hydrate, or solvate described in any one of items 1 to 9, wherein the phosphonium compound or its salt is represented by general formula (1B):

[In the formula, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are the same as defined above. R ^6b , R ^7b , R ^8b and R ^9b are the same or different and represent a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a sulfonic acid group.]
A solvatochromic fluorescent dye which is a phosphonium fluorescein compound represented by the formula:

Item 12. The solvatochromic fluorescent dye according to Item 11, which is a negative solvatochromic fluorescent dye.

The present invention provides a compound that exhibits strong luminescence in the red to near-infrared region.

Of these, phosphonium rhodamine compounds and phosphonium rhodol compounds are cationic compounds, and are expected to be able to selectively visualize negatively charged mitochondria and other structures within cells. In particular, phosphonium rhodamine compounds are fluorescent dyes with unprecedented structural characteristics, possessing a dication, and are expected to be able to selectively visualize negatively charged mitochondria and other structures within cells.

In addition, phosphonium fluorescein compounds have a cation and an anion in the same molecule, making them fluorescent dyes with unprecedented structural characteristics and negative solvatochromism.

FIG. 1 is a schematic diagram illustrating positive and negative solvent effects. 1 is an absorption spectrum and a fluorescence spectrum of the phosphonium fluorescein compound (Example 1) in water. 1 is an absorption spectrum and a fluorescence spectrum of a phosphonium rhodol compound (Example 3) in water. 1 shows the absorption and fluorescence spectra of the phosphonium rhodamine compound (Example 4) in water. 1 shows fluorescence spectra of the phosphonium fluorescein compound (Example 1) in various solvents.

In this specification, the term "containing" is a concept that encompasses all of "comprise," "consist essentially of," and "consist only of."

In addition, in this specification, when a numerical range is indicated as "A to B," it means A or more and B or less.

As used herein, the term "(hetero)aryl group" refers to an aryl group or a heteroaryl group.

1. Phosphonium Compound or Salt, Hydrate, or Solvate Thereof The phosphonium compound or salt, hydrate, or solvate thereof of the present invention is a phosphonium compound represented by the general formula (1):

[Wherein, ^Y1 represents an oxygen atom or a substituted or unsubstituted amino cation. ^Y2 represents an oxygen anion or a substituted or unsubstituted amino group. ^R1 and ^R2 are the same or different and represent a substituted or unsubstituted (hetero)aryl group, or a substituted or unsubstituted alkyl group. ^R3 and ^R4 are the same or different and represent a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted alkoxy group, provided that at least one of ^R3 and ^R4 is a substituted or unsubstituted alkyl group, or a substituted or unsubstituted alkoxy group. ^R5 represents a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, or a substituted or unsubstituted aryl group. ^R6 , ^R7 , ^R8 , and ^R9 are the same or different and represent a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a sulfonic acid group. At least one selected from the group consisting of R ⁶ and Y ¹ , R ⁷ and Y ² , R ⁸ and Y ¹ , and R ⁹ and Y ² may combine to form a substituted or unsubstituted alkylene group.]
It contains an ion represented by the formula:

In the general formula (1), ^Y1 is an oxygen atom or a substituted or unsubstituted amino cation, and ^Y2 is an oxygen anion or a substituted or unsubstituted amino group.

Among these, when Y ¹ is a substituted or unsubstituted amino cation and Y ² is a substituted or unsubstituted amino group, the compound represented by the general formula (1A):

[Wherein ^R1 , ^R2 , ^R3 , ^R4 and ^R5 are the same as defined above. ^R6a , ^R7a , ^R8a and ^R9a are the same or different and represent a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a sulfonic acid group. ^R10 , ^R11 , ^R12a and ^R13a are the same or different and represent a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted (hetero)aryl group. At least one selected from the group consisting of ^R6a and ^R10 , ^R7a and ^R13a , ^R8 and ^R11 , and ^R9a and ^R12a may be joined together to form a substituted or unsubstituted alkylene group.]
or a salt, hydrate or solvate thereof.

When Y ¹ is an oxygen atom and Y ² is an oxygen anion, the compound represented by the general formula (1B):

[In the formula, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are the same as defined above. R ^6b , R ^7b , R ^8b and R ^9b are the same or different and represent a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a sulfonic acid group.]
or a salt, hydrate or solvate thereof.

When Y ¹ is an oxygen atom and Y ² is a substituted or unsubstituted amino group, the compound represented by the general formula (1C):

[Wherein ^R1 , ^R2 , ^R3 , ^R4 , and ^R5 are the same as defined above. ^R6c , ^R7c , ^R8c , and ^R9c are the same or different and represent a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a sulfonic acid group. ^R12c and ^R13c are the same or different and represent a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted (hetero)aryl group. At least one selected from the group consisting of ^R7c and ^R13c , and ^R9c and ^R12c may together form a substituted or unsubstituted alkylene group.]
The compound is a phosphonium rhodol compound containing a phosphonium rhodol cation represented by the formula:

That is, in general formula (1), the substituted or unsubstituted amino cation represented by ^Y1 means =(NR ¹⁰ R ¹¹ ) ⁺ , and the substituted or unsubstituted amino group represented by ^Y2 means -NR ^12a R ^13a or -NR ^12c R ^13c . Details of R ¹⁰ , R ¹¹ , R ^12a , NR ^12c , R ^13a and R ^13c will be described later.

Thus, the phosphonium compounds or salts, hydrates or solvates thereof of the present invention include three types of compound groups depending on the types of ^Y1 and ^Y2 .

In particular, phosphonium rhodamine compounds or their salts, hydrates, or solvates, and phosphonium rhodol compounds or their salts, hydrates, or solvates, are cationic compounds with a cation on the phosphorus atom of the xanthene skeleton, and are expected to accumulate in negatively charged mitochondria and other cells, enabling selective visualization. In particular, phosphonium rhodamine compounds or their salts, hydrates, or solvates are dicationic compounds with cations at two locations: on the phosphorus atom of the xanthene skeleton and on the nitrogen atom that constitutes the substituted or unsubstituted amino cation. These fluorescent dyes have unprecedented structural characteristics, and are expected to easily accumulate in negatively charged mitochondria and other cells, enabling particularly selective visualization.

In addition, phosphonium fluorescein compounds or their salts, hydrates, or solvates have a cation on the phosphorus atom of the xanthene skeleton and an anion on the oxygen anion, and because they have a cation and anion in the same molecule, they are fluorescent dyes with unprecedented structural characteristics and exhibit negative solvatochromism.

In general formulas (1), (1A), (1B), and (1C), the aryl group represented by ^R1 and ^R2 may be any of a monocyclic aryl group, a fused ring aryl group, and a polycyclic aryl group. Examples thereof include aryl groups having 6 to 18 carbon atoms (particularly aryl groups having 6 to 14 carbon atoms), such as a phenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, a biphenyl group, a terphenyl group, a fluorenyl group, a pyrenyl group, and a triphenylenyl group.

The aryl groups represented by ^R1 and ^R2 may have a substituent. Examples of the substituent include, but are not limited to, a hydroxyl group, a halogen atom described below, the aryl group described above, a heteroaryl group described below, an alkyl group described below, an alkoxy group described below, an alkenyl group described below, and an alkynyl group described below. When an aryl group has a substituent, the number of the substituents is not particularly limited, and is preferably 1 to 5, and more preferably 1 to 3.

In general formulas (1), (1A), (1B), and (1C), the heteroaryl group represented by ^R1 and ^R2 may be either a monocyclic heteroaryl group or a polycyclic heteroaryl group, and examples thereof include a pyrrolidyl group, a pyrrolyl group, a tetrahydrothienyl group, a thienyl group, an oxolanyl group, a furanyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an oxazolyl group, a piperidyl group, a pyridyl group, a pyrazyl group, an indolyl group, an isoindolyl group, a benzimidazolyl group, a quinolyl group, an isoquinolyl group, and a quinoxalyl group.

The heteroaryl groups represented by ^R1 and ^R2 may have a substituent. Examples of the substituent include, but are not limited to, a hydroxyl group, a halogen atom described below, the aryl group described above, the heteroaryl group described above, an alkyl group described below, an alkoxy group described below, an alkenyl group described below, and an alkynyl group described below. When the heteroaryl group has a substituent, the number of the substituents is not particularly limited, and is preferably 1 to 6, and more preferably 1 to 3.

In the above general formulas (1), (1A), (1B) and (1C), the alkyl groups represented by R ¹ and R ² may be either linear or branched alkyl groups.

Straight-chain alkyl groups are preferably those having 1 to 6 carbon atoms (particularly 1 to 4 carbon atoms), and specific examples include methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl groups.

Preferably, the branched-chain alkyl group is one having 3 to 6 carbon atoms (especially 3 to 5 carbon atoms), and specific examples include isopropyl, isobutyl, tert-butyl, sec-butyl, neopentyl, isohexyl, and 3-methylpentyl groups.

The alkyl groups represented by ^R1 and ^R2 may have a substituent. The substituent that the alkyl group may have is not particularly limited, and examples thereof include a hydroxyl group, a halogen atom described below, the aryl group described above, the heteroaryl group described above, an alkoxy group described below, an alkenyl group described below, and an alkynyl group described below. When the alkyl group has a substituent, the number of the substituents is not particularly limited, and is preferably 1 to 6, and more preferably 1 to 3.

Among these, from the viewpoint of molecular stability and the like, R ¹ and R ² are preferably substituted or unsubstituted aryl groups, substituted or unsubstituted monocyclic alkyl groups, etc., and more preferably substituted or unsubstituted aryl groups.

In the above general formulas (1), (1A), (1B) and (1C), the alkyl groups represented by ^R3 and ^R4 can be those described above. The same applies to the type and number of substituents.

In the above general formulae (1), (1A), (1B) and (1C), the alkoxy group represented by ^R3 and ^R4 is not particularly limited, and examples thereof include alkoxy groups having 1 to 6 carbon atoms (particularly 1 to 4 carbon atoms), such as a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butyloxy group, an isobutyloxy group, a sec-butyloxy group and a tert-butyloxy group.

The alkoxy groups represented by ^R3 and ^R4 may have a substituent. The substituent that the alkoxy group may have is not particularly limited, and examples thereof include a hydroxyl group, a halogen atom described below, the aryl group described above, the heteroaryl group described above, the alkoxy group described above, the alkenyl group described below, and the alkynyl group described below. When the alkoxy group has a substituent, the number of the substituents is not particularly limited, and is preferably 1 to 6, and more preferably 1 to 3.

At least one of ^R3 and ^R4 is a substituted or unsubstituted alkyl group, or a substituted or unsubstituted alkoxy group. If both ^R3 and ^R4 are hydrogen atoms, the stability of the compound is poor. From the viewpoint of compound stability, it is preferable that both ^R3 and ^R4 are substituted or unsubstituted alkyl groups, or substituted or unsubstituted alkoxy groups.

In the above general formulas (1), (1A), (1B) and (1C), the alkyl group and aryl group represented by ^R5 can be those described above. The same applies to the type and number of substituents.

In the general formulae (1), (1A), (1B) and (1C), examples of the halogen atom represented by R ⁵ include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.

In the general formulae (1), (1A), (1B) and (1C), examples of the alkenyl group represented by ^R5 include alkenyl groups having 2 to 6 carbon atoms (particularly 2 to 4 carbon atoms), such as a vinyl group, an allyl group, a 1-butenyl group and a 2-butenyl group.

The alkenyl group represented by ^R5 may have a substituent. The substituent that the alkenyl group may have is not particularly limited, and examples thereof include a hydroxyl group, the halogen atom described above, the aryl group described above, the heteroaryl group described above, the alkoxy group described above, the alkenyl group described above, and the alkynyl group described below. When the alkenyl group has a substituent, the number of the substituents is not particularly limited, and is preferably 1 to 6, and more preferably 1 to 3.

In the general formulae (1), (1A), (1B) and (1C), examples of the alkynyl group represented by ^R5 include alkynyl groups having 2 to 6 carbon atoms (particularly 2 to 4 carbon atoms), such as an ethynyl group, a 1-propynyl group, a propargyl group, a 1-butynyl group and a 2-butynyl group.

The alkynyl group represented by ^R5 may have a substituent. The substituent that the alkynyl group may have is not particularly limited, and examples thereof include a hydroxyl group, the halogen atom, the aryl group, the heteroaryl group, the alkoxy group, the alkenyl group, and the alkynyl group. When the alkynyl group has a substituent, the number of the substituents is not particularly limited, and is preferably 1 to 6, and more preferably 1 to 3.

From the viewpoint of ease of synthesis, ^R5 is preferably a hydrogen atom.

In general formula (1), the halogen atoms and alkyl groups represented by ^R6 , ^R7 , ^R8 , and ^R9 can be those described above. The same applies to the type and number of substituents. The same also applies to the halogen atoms and alkyl groups represented by ^R6a , ^R7a , ^R8a , and ^R9a in general formula (1A), the halogen atoms and alkyl groups represented by ^R6b , ^R7b , ^R8b , and ^R9b in general formula (1B), and the halogen atoms and alkyl groups represented by ^R6c , ^R7c , ^R8c , and ^R9c in general formula (1C).

In general formula (1), R ⁶ , R ⁷ , R ⁸ and R ⁹ are preferably hydrogen atoms from the viewpoints of ease of synthesis, maximum absorption wavelength, maximum fluorescence wavelength, solvatochromism, etc. For the purpose of improving photostability, R ⁶ , R ⁷ , R ⁸ and R ⁹ can also be halogen atoms. The same applies to R ^6a , R ^7a , R ^8a and R ^9a in general formula (1A), R ^6b , R ^7b , R ^8b and R ^9b in general formula (1B), and R ^6c , R ^7c , R ^8c and R ^9c in general formula (1C).

In the general formula (1), the substituted or unsubstituted amino cation represented by Y ¹ means =(NR ¹⁰ R ¹¹ ) ^{2 +} , as also described in the general formula (1A).

The alkyl groups represented by R ¹⁰ and R ¹¹ can be those described above, as can the types and numbers of the substituents.

When ^R10 and ^R11 are (hetero)aryl groups, the fluorescence intensity is reduced, and when they are hydrogen atoms, they are removed when the compound is converted into a quinoid structure, resulting in a loss of cationicity. However, when they are alkyl groups, the fluorescence intensity is not impaired.

In general formula (1), it is preferable to employ a substituted or unsubstituted amino cation as ^Y1 when a phosphonium rhodamine compound, a salt thereof, a hydrate or a solvate thereof is intended, and it is preferable to employ an oxygen atom when a phosphonium fluorescein compound, a salt thereof, a hydrate or a solvate thereof, or a phosphonium rhodol compound, a salt thereof, a hydrate or a solvate thereof is intended.

In general formula (1), the substituted or unsubstituted amino group represented by Y ² means —NR ^12a R ^13a or —NR ^12c R ^13c , as also described in general formulae (1A) and (1C).

The alkyl groups represented by R ^12a , R ^12c , R ^13a and R ^13c can be those described above, as can the types and numbers of the substituents.

When R ^12a , R ^12c , R ^13a and R ^13c are (hetero)aryl groups, the fluorescence intensity is reduced, and when they are hydrogen atoms, they are removed when the compound is converted into a quinoid structure, resulting in a loss of cationicity. However, when they are alkyl groups, the fluorescence intensity is not impaired.

In general formula (1), it is preferable to use a substituted or unsubstituted amino group for ^Y2 when a phosphonium rhodamine compound, a salt thereof, a hydrate or a solvate thereof, or a phosphonium rhodol compound, a salt thereof, a hydrate or a solvate thereof is the target, and it is preferable to use an oxygen anion when a phosphonium fluorescein compound, a salt thereof, a hydrate or a solvate thereof is the target.

In the above general formula (1), ^R6 and ^Y1 , ^R7 and ^Y2 , ^R8 and ^Y1 , and ^R9 and ^Y2 may together form a substituted or unsubstituted alkylene group. Also, in the above general formula (1A), ^R6a and ^R10 , ^R7a and ^R13a , ^R8 and ^R11 , and ^R9 and ^R12a may together form a substituted or unsubstituted alkylene group. Also, in the above general formula (1C), ^R7c and ^R13c , and ^R9c and ^R12c may together form a substituted or unsubstituted alkylene group.

Such alkylene groups include alkylene groups having 1 to 6 carbon atoms (particularly 2 to 4 carbon atoms), such as methylene, ethylene, trimethylene, and tetramethylene. The alkylene group may have a substituent. Examples of the substituent include, but are not limited to, a hydroxyl group, the halogen atoms described above, the aryl groups described above, and the heteroaryl groups described above. If the alkylene group has a substituent, there is no particular restriction on the number of substituents; 1 to 6 is preferred, and 1 to 3 is more preferred.

However, from the viewpoint of further improving water solubility and further suppressing nonspecific adsorption during imaging, it is preferable that none of ^R6 and ^Y1 , ^R7 and ^Y2 , ^R8 and ^Y1 , and ^R9 and ^Y2 are bonded to each other to form a substituted or unsubstituted alkylene group.

Examples of the phosphonium compounds of the present invention that satisfy these conditions include:

[In the formula, Ph represents a phenyl group.]
or a salt, hydrate or solvate thereof.

The phosphonium compounds of the present invention can also exist in the form of salts. In the case of phosphonium fluorescein compounds represented by general formula (1B), since they contain a cation and an anion within the molecule, they can form intramolecular salts (zwitterions). On the other hand, in the case of phosphonium rhodamine compounds represented by general formula (1A) and phosphonium rhodol compounds represented by general formula (1C), examples of acid addition salts include mineral acid salts such as hydrochloride, sulfate, and nitrate; and organic acid salts such as p-toluenesulfonate, methanesulfonate, maleate, and oxalate. In addition, salts with amino acids such as glycine may also be formed.

The phosphonium compounds of the present invention may also exist as hydrates or solvates, and all of these substances are included within the scope of the present invention.

Such phosphonium compounds or salts, hydrates, or solvates of the present invention, for example, phosphonium rhodamine compounds represented by general formula (1A) or salts, hydrates, or solvates thereof, preferably have an absorption maximum wavelength of 690 to 730 nm, particularly 695 to 725 nm, and a fluorescence maximum wavelength of 730 to 770 nm, particularly 735 to 765 nm, in water. Furthermore, phosphonium fluorescein compounds represented by general formula (1B) or salts, hydrates, or solvates thereof preferably have an absorption maximum wavelength of 630 to 660 nm, particularly 635 to 655 nm, and a fluorescence maximum wavelength of 670 to 700 nm, particularly 675 to 695 nm, in water. Furthermore, in the case of a phosphonium rhodol compound represented by general formula (1C) or a salt, hydrate, or solvate thereof, it is preferable that in water the absorption maximum wavelength is 660 to 690 nm, particularly 665 to 685 nm, and the fluorescence maximum wavelength is 700 to 730 nm, particularly 695 to 725 nm.

When the phosphonium compound or its salt, hydrate, or solvate (fluorescent dye of the present invention) of the present invention is a phosphonium fluorescein compound or its salt, hydrate, or solvate, it is a group of compounds (solvatochromic fluorescent dyes) whose maximum absorption wavelength and maximum fluorescence wavelength change depending on the polarity of the solvent. For example, in a nonpolar solvent such as toluene, which has very little solvent polarity, the maximum absorption wavelength is preferably 670 to 700 nm, particularly 675 to 695 nm, and the maximum fluorescence wavelength is preferably 720 to 750 nm, particularly 725 to 745 nm.

Solvatochromic fluorescent dyes are expected to expand the range of applications. Solvatochromic fluorescent dyes are widely used in fluorescent probes because they can distinguish between the polarity of the surrounding environment. However, while traditionally used solvatochromic fluorescent dyes have wavelengths whose maximum absorption and fluorescence wavelengths change depending on the polarity of the solvent, they have the problem of having extremely low fluorescence quantum yields in highly polar solvents such as water.

Solvatochromic fluorescent dyes are divided into those with a positive solvent effect and those with a negative solvent effect. Most solvatochromic fluorescent dyes have a positive solvent effect, and as shown in the left diagram of Figure 1, the absorption and fluorescence maximum wavelengths shift toward longer wavelengths as the polarity of the solvent increases. Conversely, solvatochromic fluorescent dyes with a negative solvent effect typically have their absorption and fluorescence maximum wavelengths shift toward shorter wavelengths as the polarity of the solvent increases, as shown in the right diagram of Figure 1. Cell imaging requires a high fluorescence quantum yield in highly polar solvents such as water, so negative solvatochromic fluorescent dyes with a negative solvent effect, as in the present invention, are useful.

From the above, it is possible to shift the maximum absorption wavelength and maximum fluorescence wavelength to longer wavelengths, and to further increase the solvent dependence of the maximum absorption wavelength and maximum fluorescence wavelength. Furthermore, in the case of a phosphonium fluorescein compound or a salt, hydrate, or solvate thereof, the maximum absorption wavelength and maximum fluorescence wavelength can be particularly increased when the solvent is an aqueous solution such as water or PBS buffer solution. In other words, among the fluorescent dyes of the present invention, a phosphonium fluorescein compound or a salt, hydrate, or solvate thereof can also be described as a negative solvatochromic fluorescent dye, in which the maximum absorption wavelength and maximum fluorescence wavelength become shorter as the polarity of the solvent increases.

As such, the phosphonium compounds or salts, hydrates, or solvates thereof of the present invention are useful not only as academic research reagents such as various stains and fluorescent dyes, but also as detection reagents for diagnostic devices, such as fluorescent dyes with specific excitation and fluorescence wavelengths that match the device specifications.

2. Method for Producing a Phosphonium Compound, or a Salt, Hydrate, or Solvate Thereof The phosphonium compound, or a salt, hydrate, or solvate thereof of the present invention is not particularly limited. For example, taking a phosphonium fluorescein compound represented by general formula (B) as an example, it can be produced by the following reaction scheme 1:

[In the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ^6b , R ^7b , R ^8b , and R ^9b are the same as defined above. ^{R 14} and R ¹⁵ may be the same or different and represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted acyl group, or a substituted or unsubstituted alkoxycarbonyl group. X ⁻ represents a counter ion.]
can be synthesized according to the following:

In Reaction Scheme 1, the alkyl groups represented by R ¹⁴ and R ¹⁵ can be those mentioned above. The same applies to the type and number of substituents.

In Reaction Scheme 1, examples of the acyl group represented by R ¹⁴ and R ¹⁵ include alkanoyl groups having 1 to 6 carbon atoms, such as formyl, acetyl, and propionyl.

The acyl group represented by R ¹⁴ and R ¹⁵ may have a substituent. The substituent that the acyl group may have is not particularly limited, and examples thereof include a hydroxyl group, the halogen atom, the aryl group, the heteroaryl group, the alkoxy group, the alkenyl group, and the alkynyl group. When the acyl group has a substituent, the number of the substituents is not particularly limited, and is preferably 1 to 6, and more preferably 1 to 3.

In Reaction Scheme 1, examples of the alkoxycarbonyl group represented by R ¹⁴ and R ¹⁵ include alkoxycarbonyl groups having 1 to 6 carbon atoms, such as a methoxycarbonyl group, an ethoxycarbonyl group, an n-propoxycarbonyl group, and an isopropoxycarbonyl group.

The alkoxycarbonyl group represented by R ¹⁴ and R ¹⁵ may have a substituent. The substituent that the alkoxycarbonyl group may have is not particularly limited, and examples thereof include a hydroxyl group, the halogen atom, the aryl group, the heteroaryl group, the alkoxy group, the alkenyl group, and the alkynyl group. When the alkoxycarbonyl group has a substituent, the number of the substituents is not particularly limited, and is preferably 1 to 6, and more preferably 1 to 3.

In Reaction Scheme 1, examples of the counter ion represented by X ⁻ include chloride ion (Cl ⁻ ), bromide ion (Br ⁻ ), iodide ion (I ⁻ ), nitrate ion (NO ₃ ⁻ ), trifluoromethylacetate ion (CF ₃ COO ⁻ ), methanesulfonate ion, toluenesulfonate ion, trifluoromethanesulfonate ion, tetrafluoroborate ion, and hexafluorophosphate ion.

The compound represented by general formula (2) used as a raw material can be a known or commercially available product. The compound represented by general formula (2) can be synthesized, for example, according to the method described in Philos Trans A Math Phys Eng Sci. 2017 Aug 28; 375 (2101): 20170007.

Note that phosphonium rhodol compounds and phosphonium rhodamine compounds can also be synthesized in a similar manner by selecting an appropriate substrate instead of the substrate compound (2) represented by general formula (2).

Compound (2) → Compound (3)
The lithium compound is not particularly limited, and known compounds can be used, including, for example, alkyllithiums such as ethyllithium, n-propyllithium, isopropyllithium, n-butyllithium, sec-butyllithium, tert-butyllithium, n-pentyllithium, and n-hexyllithium; cycloalkyllithiums such as cyclohexyllithium; and aryllithiums such as phenyllithium. Of these, in this step, from the viewpoints of yield and ease of synthesis, alkyllithiums are preferred, and n-butyllithium is more preferred. These lithium compounds can be used alone or in combination of two or more.

There are no particular restrictions on the amount of the lithium compound used, but from the standpoint of yield, ease of synthesis, etc., it is preferably 0.8 to 5.0 moles, and more preferably 1.0 to 3.0 moles, per mole of compound (2).

There are no particular restrictions on the zinc compound, but zinc halides such as zinc chloride and zinc bromide are preferred from the standpoints of yield, ease of synthesis, etc.

There are no particular restrictions on the amount of the zinc compound used, but from the standpoints of yield, ease of synthesis, etc., it is preferably 0.1 to 5 moles, and more preferably 0.5 to 2 moles, per mole of compound (2).

Zinc compounds can also form complexes using ligands. In this case, the ligand compound can be added and the complex can be formed in the system, or the complex can be formed and then added to the system.

Examples of such ligands include 2,2'-bipyridyl, 4,4'-(tert-butyl)bipyridyl, 4,4'-bis(trifluoromethyl)-2,2'-bipyridyl, 5,5'-bis(trifluoromethyl)-2,2'-bipyridyl, 6,6'-bis(trifluoromethyl)-2,2'-bipyridyl, 4,4'-bis(methoxycarbonyl)-2,2'-bipyridyl, 4,4'-dimethyl-2,2'-bipyridyl, 5,5'-dimethyl-2,2'-bipyridyl, 4,4'-dimethoxy-2,2'-bipyridyl, 4,4'-dicyano-2,2'-bipyridyl, phenanthroline, 2,2'-bipyrimidyl, 1,4-di Examples include azabicyclo[2.2.2]octane, tetramethylethylenediamine (TMEDA), 1,1'-bis(diphenylphosphino)ferrocene, diphenylphosphinomethane, 1,2-bis(diphenylphosphino)ethane, 1,3-bis(diphenylphosphino)propane, 1,5-bis(diphenylphosphino)pentane, 1,2-bis(dicyclohexylphosphino)ethane, 1,3-(dicyclohexylphosphino)propane, 1,2-bis(di-tert-butylphosphino)ethane, 1,3-bis(di-tert-butylphosphino)propane, and 1,2-bis(diphenylphosphino)benzene. These ligands can be used alone or in combination of two or more.

There are no particular restrictions on the amount of the ligand used, but from the standpoint of yield, ease of synthesis, etc., 0.1 to 5 moles per mole of zinc compound is preferred, and 0.5 to 2 moles is more preferred.

The organic solvent that can be used in this step is not particularly limited, but preferred examples include cyclic ether compounds such as tetrahydrofuran and dioxane.

Reaction conditions are preferably such that the reaction proceeds sufficiently. For example, the reaction atmosphere is preferably an oxidizing atmosphere (e.g., an oxygen gas atmosphere, air, etc.), the reaction temperature is preferably room temperature (approximately 20 to 30°C), and the reaction time is preferably 5 minutes to 96 hours, particularly 10 minutes to 72 hours.

After the reaction is complete, purification can be performed according to standard methods if necessary, and the next step can then be carried out.

Compound (3) → Compound (1B)
Examples of Lewis acids include boron tribromide, aluminum tribromide, boron trichloride, aluminum trichloride, iodotrimethylsilane, etc. These Lewis acids can be used alone or in combination of two or more.

It is generally preferable to use an excess amount of Lewis acid relative to compound (3), and the amount of Lewis acid used is preferably 2 to 10 moles, and more preferably 3 to 8 moles, per mole of compound (3).

The reaction can usually be carried out in the presence of a reaction solvent. Usable reaction solvents include, for example, aromatic hydrocarbons such as benzene, toluene, and xylene; aliphatic halogenated hydrocarbons such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; and hydrocarbons such as pentane, hexane, heptane, and cyclohexane. From the standpoints of ease of synthesis and yield, aliphatic halogenated hydrocarbons are preferred, and dichloromethane is more preferred. These reaction solvents can be used alone or in combination of two or more.

The reaction atmosphere is typically an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.). The reaction can be carried out under heating, at room temperature, or under cooling, and is typically carried out at a temperature between -50 and 100°C, and more preferably between 0 and 50°C. There are no particular restrictions on the reaction time, but it is preferably set to a time that allows the reaction to proceed sufficiently.

After the reaction is complete, the phosphonium fluorescein compound of the present invention or its salt, hydrate, or solvate can be obtained by purifying the reaction mixture according to conventional methods, if necessary.

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

Synthesis Example 1: 4-bromo-3-(diphenylphosphino)anisole

In the formula, Me represents a methyl group, iPr represents an isopropyl group, Ph represents a phenyl group, and THF represents tetrahydrofuran.

Isopropylmagnesium bromide was added dropwise to a solution of 4-bromo-3-iodoanisole in anhydrous tetrahydrofuran (THF) at -20°C. After stirring at -20°C for 1 hour, diphenylphosphine chloride was added, and the resulting mixture was stirred at -20°C for 1 hour. The mixture was warmed to room temperature and stirred overnight. The reaction was quenched by adding a saturated aqueous solution of ammonium chloride, and the aqueous phase was extracted three times with ethyl acetate (EtOAc). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was subjected to silica gel column chromatography (hexane/ethyl acetate (EtOAc) = 10:1 to 5:1 as eluent) followed by purification by recrystallization from a hexane/dichloromethane solvent system at room temperature to obtain 4-bromo-3-(diphenylphosphino)anisole (4.40 g, 11.9 mmol, 59%) as a white solid.

Synthesis Example 2

In the formula, Me represents a methyl group, Ph represents a phenyl group, ^nBu represents an n-butyl group, and THF represents tetrahydrofuran.

To a solution of 4-bromo-3-(diphenylphosphino)anisole (3.71 g, 10 mmol) obtained in Synthesis Example 1 in anhydrous tetrahydrofuran (THF; 25 mL) was slowly added a 1.58 M hexane solution of n-butyllithium ( ^nBuLi ; 7 mL, 11 mmol) at −78°C, and the resulting mixture was stirred at the same temperature for 1 hour. A solution of Weinreb amide of 4-methoxybenzoic acid (1.95 g, 10 mmol) in tetrahydrofuran (THF; 8 mL) was slowly added. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction was quenched by adding saturated aqueous ammonium chloride solution, and the aqueous phase was extracted three times with ethyl acetate (EtOAc). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluent: hexane/ethyl acetate (EtOAc) = 5:1 to 3:1) to give the diaryl ketone compound (2.27 g, 5.3 mmol, 53%) as a yellowish solid.

Synthesis Example 3

In the formula, Me represents a methyl group, Ph represents a phenyl group, and THF represents tetrahydrofuran.

A solution of the diaryl ketone compound (2.13 g, 5 mmol) from Synthesis Example 2 dissolved in 25 mL of anhydrous tetrahydrofuran (THF) was added to a 2,6-xylylmagnesium bromide/lithium chloride solution (1 M, 10 mL). The cooling bath was removed, and the reaction mixture was stirred at room temperature for 2 hours and at 70°C for 2 days. The reaction was quenched by adding saturated aqueous ammonium chloride, and the aqueous phase was extracted three times with ethyl acetate (EtOAc). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (hexane/ethyl acetate (EtOAc) = 10:1 to 5:1 as eluent) to obtain the triaryl alcohol compound (1.08 g, 2 mmol, 38%) as a white solid.

Synthesis Example 4

In the formula, Me represents a methyl group, Ph represents a phenyl group, Ac represents an acetyl group, TFA represents trifluoroacetic acid, and ^{X -} represents a counter ion.

To a solution of the triaryl alcohol compound (1.08 g, 2 mmol) obtained in Synthesis Example 3 in acetic anhydride (8 mL) was slowly added trifluoroacetic acid (TFA; 0.5 mL, 6 mmol) at room temperature, and the resulting mixture was stirred at the same temperature for 1 hour. Water was then added, and the aqueous phase was extracted three times with dichloromethane. The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (chloroform/methanol (MeOH) = 5:1 as eluent) to obtain the corresponding phosphonium salt (1.26 g, 2 mmol, 99%) as a white solid.

Synthesis Example 5

In the formula, Me represents a methyl group, Ph represents a phenyl group, ^nBuLi represents n-butyllithium, TMEDA represents tetramethylethylenediamine, and X ^- represents a counter ion.

To a solution of the phosphonium salt (0.83 g, 2.1 mmol) obtained in Synthesis Example 4 in anhydrous tetrahydrofuran (THF; 10 mL) was slowly added 1.58 M n-butyllithium ( ^nBuLi ; 1.58 mL, 2.5 mmol) and zinc chloride/tetramethylethylenediamine (TMEDA) complex (0.53 g, 2.1 mmol) at −78°C, and the resulting mixture was stirred for 1 hour under an argon atmosphere. After replacing the argon with oxygen, stirring was maintained. The reaction was quenched by adding saturated aqueous ammonium chloride, and the aqueous phase was extracted three times with dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (chloroform/methanol (MeOH) = 10:1 to 5:1 as eluent) to obtain the phosphonium alcohol compound (0.81 g, 1.3 mmol, 60%) as a white solid.

Example 1: Phosphonium Fluorescein

In the formula, Me represents a methyl group. Ph represents a phenyl group.

To a solution of the phosphonium alcohol compound obtained in Example 1 (0.1 g, 0.15 mmol) in anhydrous dichloromethane (CH ₂ Cl ₂ ; 2 mL), a 1.0 M dichloromethane solution of boron tribromide (BBr ₃ ; 0.8 mL, 0.8 mmol) was slowly added at −78° C., and the resulting mixture was stirred at the same temperature for 30 minutes. The mixture was warmed to room temperature and stirred for 24 hours. The reaction was quenched by adding water, and the aqueous phase was extracted four times with dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (chloroform/methanol (MeOH) = 10:1 as eluent) to obtain the phosphonium fluorescein compound (0.03 g, 0.06 mmol, 41%) as a red solid.
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.88-7.80 (2H, m), 7.74-7.66 (8H, m), 7.29 (1H, t, J = 7.6 Hz), 7.14 (2H, d, J = 7.6 Hz), 6.86 (2H, dd, J = 10.0, 8.0 Hz), 6.79 (2H, dd, J = 19.2, 2.4 Hz), 6.49 (2H, dd, J = 10.0, 2.4 Hz), 1.93 (6H, s).

Example 2: Phosphonium Fluorescein

In the formula, Ph represents a phenyl group.

Synthesis was carried out according to the procedures of Synthesis Examples 3 to 5 and Example 1, except that the 2,6-xylylmagnesium bromide/lithium chloride solution used in Synthesis Example 3 was replaced with a 2-tolylmagnesium bromide/lithium chloride solution.
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.88-7.79 (2H, m), 7.75-7.62 (8H, m), 7.39 (1H, t, J = 7.0 Hz), 7.31 (1H, t, J = 7.0 Hz), 7.30 (1H, d, J = 7.0 Hz), 7.07 (1H, d, J = 7.0 Hz), 6.87 (2H, dd, J = 9.8, 8.0 Hz), 6.80 (2H, dd, J = 18.4, 2.4 Hz), 6.47 (2H, dd, J = 9.8, 2.4 Hz), 1.95 (3H, s).

Synthesis Example 6

In the formula, Me represents a methyl group. Ph represents a phenyl group.

To a solution of 4-bromo-3-(diphenylphosphino)anisole (1.23 g, 3.3 mmol) obtained in Synthesis Example 1 in anhydrous tetrahydrofuran (THF; 11 mL) was slowly added a 1.58 M hexane solution of n-butyllithium ( ^nBuLi ; 7 mL, 3.6 mmol) at −78°C, and the resulting mixture was stirred at the same temperature for 1 hour. A solution of the Weinreb amide of 4-dimethylaminobenzoic acid (0.63 g, 3 mmol) in tetrahydrofuran (THF; 3 mL) was slowly added. The mixture was allowed to warm to room temperature overnight. The reaction was quenched by adding saturated aqueous ammonium chloride solution, and the aqueous phase was extracted three times with ethyl acetate. The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by silica gel chromatography (eluent: hexane/ethyl acetate = 5:1 to 1:1) gave the corresponding diaryl ketone compound (0.51 g, 1.16 mmol, 39%) as a yellowish solid.

Synthesis Example 7

In the formula, Me represents a methyl group. Ph represents a phenyl group.

A solution of the diaryl ketone compound (0.51 g, 1.16 mmol) obtained in Synthesis Example 6 dissolved in 24 mL of anhydrous tetrahydrofuran (THF) was added to a 2-tolylmagnesium bromide/lithium chloride solution (0.3 M, 12 mL) at 0°C. The reaction mixture was stirred at room temperature for 2 hours and at 70°C overnight. The reaction was quenched by adding saturated aqueous ammonium chloride, and the aqueous phase was extracted three times with ethyl acetate. The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The product was purified by silica gel chromatography (hexane/ethyl acetate = 5:1 to 1:1 as eluent) to obtain the corresponding triarylmethanol compound (0.11 g, 0.21 mmol, 18%) as a white solid.

Synthesis Example 8

In the formula, Me represents a methyl group, Ph represents a phenyl group, Ac represents an acetyl group, and TFA represents trifluoroacetic acid.

To a solution of the triarylmethanol compound (0.11 g, 0.21 mmol) obtained in Synthesis Example 7 in acetic anhydride (Ac ₂ O; 1 mL), trifluoroacetic acid (TFA; 0.1 mL, 1 mmol) was slowly added at room temperature, and the resulting mixture was stirred at the same temperature for 2 hours. Water was then added, and the aqueous phase was extracted three times with dichloromethane. The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The product was purified by silica gel chromatography (chloroform/methanol = 10:1 to 5:1 as eluent) to obtain the corresponding phosphonium salt (0.1 g, 0.2 mmol, 95%) as a white solid.

Synthesis Example 9

In the formula, Me represents a methyl group, Ph represents a phenyl group, ^nBuLi represents n-butyllithium, TMEDA represents tetramethylethylenediamine, and THF represents tetrahydrofuran.

To a solution of the phosphonium salt (0.10 g, 0.20 mmol) obtained in Synthesis Example 8 in anhydrous tetrahydrofuran (THF; 2 mL) was slowly added 1.58 M n-butyllithium ( ^nBuLi ; 1.58 mL, 0.24 mmol) and zinc chloride/tetramethylethylenediamine (TMEDA) complex (0.05 g, 0.2 mmol) at −78°C, and the resulting mixture was stirred for 1 hour under an argon atmosphere. After replacing the argon with oxygen, stirring was maintained. The reaction was quenched by adding saturated aqueous ammonium chloride, and the aqueous phase was extracted three times with dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (chloroform/methanol (MeOH) = 10:1 to 5:1 as eluent) to obtain the corresponding phosphonium alcohol compound (0.07 g, 0.13 mmol, 65%).

Example 3: Phosphonium rhodol

In the formula, Me represents a methyl group. Ph represents a phenyl group.

To a solution of the phosphonium alcohol (0.07 g, 0.13 mmol) obtained in Synthesis Example 9 in anhydrous dichloromethane (CH ₂ Cl ₂ ; 1.3 mL), a 1.0 M solution of boron tribromide (BBr ₃ ; 0.67 mL, 0.67 mmol) in dichloromethane was slowly added at −78° C., and the resulting mixture was stirred at the same temperature for 30 minutes. The mixture was warmed to room temperature and stirred for 24 hours. The reaction was quenched by adding water, and the aqueous phase was extracted four times with dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (chloroform/methanol (MeOH) = 10:1 as eluent) to obtain the corresponding phosphonium rhodol (0.06 g, 0.1 mmol, 75%) as a blue solid.
¹ H NMR (400 MHz, CD ₃ OD) δ 7.99-7.67 (10H, m), 7.46-7.37 (3H, m), 7.22 (1H, d, J = 7.6 Hz), 7.19-6.93 (7H, m), 6.77 (1H, dd, J = 16.0, 2.4 Hz), 2.90 (6H, s), 1.60 (3H, s).

Synthesis Example 10

In the formula, Boc represents a tert-butyloxycarbonyl group, Me represents a methyl group, and Ph represents a phenyl group.

To a solution of N-Boc-N-methyl-4-bromo-3-(diphenylphosphino)aniline (1.03 g, 2.2 mmol) in anhydrous tetrahydrofuran (THF; 7 mL) was slowly added a 1.56 M hexane solution of n-butyllithium ( ^nBuLi ; 1.41 mL, 2.2 mmol) at −78°C, and the resulting mixture was stirred at the same temperature for 1 hour. A solution of N-Boc-N-methylaminobenzoic acid Weinreb amide (0.64 g, 2.2 mmol) in tetrahydrofuran (THF; 3 mL) was slowly added. The mixture was allowed to warm to room temperature overnight. The reaction was quenched by adding saturated aqueous ammonium chloride, and the aqueous phase was extracted three times with ethyl acetate. The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography (eluent: hexane/ethyl acetate = 3:1 to 1:1) to give the corresponding diaryl ketone compound (0.78 g, 1.25 mmol, 57%) as a yellow solid.

Synthesis Example 11

In the formula, Boc represents a tert-butyloxycarbonyl group. Ph represents a phenyl group.

A solution of the diaryl ketone compound (0.78 g, 1.25 mmol) obtained in Synthesis Example 10 dissolved in 24 mL of anhydrous tetrahydrofuran (THF) was added to a 2,6-xylylmagnesium bromide/lithium chloride solution (0.2 M, 12 mL) at 0°C. The reaction mixture was stirred at room temperature for 2 hours and at 70°C overnight. The reaction was quenched by adding saturated aqueous ammonium chloride, and the aqueous phase was extracted three times with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The product was purified by silica gel chromatography (hexane/ethyl acetate = 5:1 to 1:1 as eluent) to obtain the corresponding triarylmethanol compound (0.41 g, 0.55 mmol, 44%) as a white solid.

Synthesis Example 12

In the formula, Boc represents a tert-butyloxycarbonyl group, Ph represents a phenyl group, and TFA represents trifluoroacetic acid.

To a solution of the triarylmethanol compound (0.4 g, 0.55 mmol) from Synthesis Example 10 in anhydrous dichloromethane (2 mL), trifluoroacetic acid (TFA; 0.21 mL, 2.8 mmol) was slowly added at room temperature, and the resulting mixture was stirred at the same temperature for 3 days. Water was then added, and the aqueous phase was extracted three times with dichloromethane. The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification was performed by silica gel chromatography (chloroform/methanol = 10:1 to 5:1 as eluent) to obtain the corresponding phosphonium salt (0.31 g, 0.5 mmol, 90%) as a green solid.

Example 4: Phosphonium rhodamine

In the formula, Ph represents a phenyl group, Me represents a methyl group, and DMF represents dimethylformamide.

To a solution of the phosphonium salt (0.07 g, 0.11 mmol) obtained in Synthesis Example 12 in anhydrous dimethylformamide (DMF; 0.5 mL) was slowly added potassium carbonate (0.05 g, 0.33 mmol) and iodomethane (15 μL, 0.24 mmol) at room temperature, and the resulting mixture was stirred at 80 °C for 3 days. An equal amount of iodomethane was added every other day. Water was then added, and the aqueous phase was washed five times with ethyl acetate. A 2M aqueous solution of sodium tetrafluoroborate was then added, and the mixture was extracted three times with dichloromethane. The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Recrystallization from dichloromethane/diethyl ether afforded the corresponding phosphonium rhodamine compound (10 mg, 0.01 mmol, 12%) as a green solid.
¹ H NMR (400 MHz, CD ₃ OD) δ 8.05-7.95 (6H, m), 7.89-7.82 (4H, m), 7.45-7.36 (3H, m), 7.29-7.22 (4H, m), 7.14 (2H, dd, J = 9.8, 2.6 Hz), 3.37 (12H, s), 1.90 (6H, s).

Test Example 1: Photophysical Properties The absorption and fluorescence spectra of a phosphonium fluorescein compound (Example 1), a phosphonium rhodol compound (Example 3), and a phosphonium rhodamine compound (Example 4) were measured. For the measurement of the phosphonium fluorescein compound (Example 1), a sample solution prepared by dissolving 5.0 × 10 ^{−5 M of the phosphonium fluorescein compound (Example 1) in water (pH 7.1) was used. For the measurement of the phosphonium rhodol compound (Example 3), a sample solution prepared by dissolving 5.0 × 10 −5 M of the phosphonium rhodol compound (Example 3) in water (pH 7.0) containing 10% by mass of dimethyl sulfoxide (DMSO) was used. For the measurement of the phosphonium rhodamine compound (Example 4), a sample solution prepared by dissolving 1.0 × 10 −5 M} of the phosphonium rhodamine compound (Example 4) in water (pH 5.3) was used. Measurements were performed using a Shimadzu UV-3150 spectrometer. Fluorescence spectra were measured using each sample solution with a Hitachi F-4500 spectrometer. The results are shown in Figures 2 to 4. In Figures 2 to 4, the left side shows the absorption spectrum and the right side shows the fluorescence spectrum.

Test Example 2: Evaluation of solvent dependence of fluorescence The absorption spectrum and fluorescence spectrum of the phosphonium fluorescein compound (Example 1) were measured in various solvents. Methanol, acetonitrile, dimethyl sulfoxide, dichloromethane, and toluene were used as solvents, and the concentration of the phosphonium fluorescein compound (Example 1) was set to 5.0 × 10 ^-5 M. The absorption spectrum and fluorescence spectrum were measured in the same manner as in Test Example 1 above. The results are shown in Figure 5 and Table 1.

Claims (12)

General formula (1):
[Wherein, ^Y1 represents an oxygen atom or a substituted or unsubstituted amino cation. ^Y2 represents an oxygen anion or a substituted or unsubstituted amino group. ^R1 and ^R2 are the same or different and represent a substituted or unsubstituted (hetero)aryl group, or a substituted or unsubstituted alkyl group. ^R3 and ^R4 are the same or different and represent a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted alkoxy group, provided that at least one of ^R3 and ^R4 is a substituted or unsubstituted alkyl group, or a substituted or unsubstituted alkoxy group. ^R5 represents a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, or a substituted or unsubstituted aryl group. ^R6 , ^R7 , ^R8 , and ^R9 are the same or different and represent a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a sulfonic acid group. At least one selected from the group consisting of R ⁶ and Y ¹ , R ⁷ and Y ² , R ⁸ and Y ¹ , and R ⁹ and Y ² may combine to form a substituted or unsubstituted alkylene group.]
A phosphonium compound, or a salt, hydrate or solvate thereof, containing an ion represented by the following formula: General formula (1A):
[Wherein ^R1 , ^R2 , ^R3 , ^R4 and ^R5 are the same as defined above. ^R6a , ^R7a , ^R8a and ^R9a are the same or different and represent a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a sulfonic acid group. ^R10 , ^R11 , ^R12a and ^R13a are the same or different and represent a substituted or unsubstituted alkyl group. At least one selected from the group consisting of ^R6a and ^R10 , ^R7a and ^R13a , ^R8 and ^R11 , and ^R9 and ^R12a may be joined together to form a substituted or unsubstituted alkylene group.]
The phosphonium compound according to claim 1, or a salt, hydrate or solvate thereof, containing a phosphonium rhodamine cation represented by the following formula: The phosphonium compound, or a salt, hydrate, or solvate thereof, according to claim 2, having an absorption maximum wavelength in water of 690 to 730 nm. General formula (1B):
[In the formula, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are the same as defined above. R ^6b , R ^7b , R ^8b and R ^9b are the same or different and represent a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a sulfonic acid group.]
The phosphonium compound or a salt, hydrate or solvate thereof according to claim 1, which is a phosphonium fluorescein compound represented by the formula: The phosphonium compound, or a salt, hydrate, or solvate thereof, according to claim 4, having an absorption maximum wavelength in water of 630 to 660 nm. General formula (1C):
[Wherein ^R1 , ^R2 , ^R3 , ^R4 , and ^R5 are the same as defined above. ^R6c , ^R7c , ^R8c , and ^R9c are the same or different and represent a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a sulfonic acid group. ^R12c and ^R13c are the same or different and represent a substituted or unsubstituted alkyl group. At least one selected from the group consisting of ^R7c and ^R13c , and ^R9c and ^R12c may together form a substituted or unsubstituted alkylene group.]
The phosphonium compound according to claim 1, or a salt, hydrate or solvate thereof, containing a phosphonium rhodol cation represented by the following formula: The phosphonium compound, or a salt, hydrate, or solvate thereof, according to claim 6, having an absorption maximum wavelength in water of 660 to 690 nm. The phosphonium compound, or a salt, hydrate or solvate thereof, according to any one of claims 1 to 7, wherein R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are all hydrogen atoms. The phosphonium compound, or a salt, hydrate, or solvate thereof, according to any one of claims 1 to 8, wherein R ¹ and R ² are the same or different and each represent a substituted or unsubstituted aryl group. A fluorescent dye containing the phosphonium compound or its salt, hydrate, or solvate according to any one of claims 1 to 9. The present invention relates to a phosphonium compound or a salt, hydrate, or solvate thereof according to any one of claims 1 to 9, wherein the phosphonium compound or the salt thereof is represented by general formula (1B):
[In the formula, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are the same as defined above. R ^6b , R ^7b , R ^8b and R ^9b are the same or different and represent a hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl group, or a sulfonic acid group.]
A solvatochromic fluorescent dye which is a phosphonium fluorescein compound represented by the formula: The solvatochromic fluorescent dye according to claim 11, which is a negative solvatochromic fluorescent dye.