WO2016133218A1 - Phospha-fluorescein compound or salt thereof, and fluorescent dye using same - Google Patents

Abstract

A phospha-fluorescein compound represented by general formula (I), or a salt, hydrate or solvate of the phospha-fluorescein compound is capable of achieving a sufficient quantum yield, while having a maximum absorption wavelength and a maximum fluorescence wavelength within the range of 600-700 nm. This phospha-fluorescein compound, or a salt, hydrate or solvate thereof is also capable of sufficiently reducing HOMO. (In general formula (I), R¹ represents an optionally substituted aryl group; R² represents a hydrogen atom or an organic group; and R represents a group that is represented by general formula (II) (where, R³ represents an optionally substituted aryl group, an optionally substituted alkyl group, an optionally substituted alkenyl group or an optionally substituted alkynyl group; and R⁴ represents an optionally substituted alkyl group).)

Description

Phosphafluorescein compound or a salt thereof, or fluorescent dye using the same

The present invention relates to a phosphafluorescein compound or a salt thereof, or a fluorescent dye using the same.

Since light in the red to near-infrared region has high tissue permeability, a dye having absorption and fluorescence maximum wavelengths in this region (620 nm or more) has attracted attention in deep observation of living tissue. In this deep observation of living tissue, in order to suppress cell autofluorescence and suppress damage to cells, the fluorescence quantum yield is increased while having a fluorescence maximum wavelength at a position of about 600 to 700 nm. It is preferable to improve. In particular, among fluorescent dyes exhibiting water solubility, there are not so many dyes having a fluorescence maximum at 650 nm or more compared to green or yellow phosphors (dyes having a fluorescence maximum at 495 to 570 nm). These include, for example, π-extended xanthene dyes, silicon-substituted rhodamines (Si-rhodamines), fluorescein dyes, cyanine dyes (Cy5, etc.). For any fluorescent dye, practical examples have been reported for visualization of biological samples.

For example, a fluorescein dye has an absorption maximum wavelength of 491 nm, a fluorescence maximum wavelength of 510 nm, and a quantum yield of 0.85 (Non-Patent Document 1). Since it is necessary to exhibit absorption and fluorescence with respect to the wavelength, there is a demand for a dye having an absorption maximum and a fluorescence maximum at a wavelength of about 600 to 700 nm.

It is known that when the oxygen atom of the xanthene skeleton of this fluorescein dye is replaced with a silicon atom, the absorption maximum wavelength is 582 nm, the fluorescence maximum wavelength is 598 nm, and the quantum yield is 0.42, which can be shifted to the longer wavelength side. (Non-Patent Document 2). However, the effect is insufficient and further long wavelength shifts are necessary.

On the other hand, in order to further shift the absorption maximum wavelength and the fluorescence maximum wavelength, a compound having a structure in which the π conjugation of the xanthene skeleton is extended, although the absorption maximum wavelengths are 595 nm and less than 600 nm, the fluorescence maximum wavelength is 660 nm. Although it is also known that this dye can be set to nm, this dye has an extremely small quantum yield of 0.14, which is insufficient for bioimaging applications (Non-patent Document 3).

As described above, in general, a dye having an absorption maximum and a fluorescence maximum in a long wavelength region has a low quantum yield and is difficult to sufficiently increase. In addition, these fluorescent dyes have a high maximum occupied orbit (HOMO), and thus react with oxygen under light irradiation to give active oxygen species. Therefore, the stability to light is low.

For this reason, the present invention provides a compound (fluorescent dye) capable of obtaining a sufficient quantum yield and sufficiently reducing HOMO while having an absorption maximum wavelength and a fluorescence maximum wavelength in the range of 600 to 700 nm. ).

As a result of intensive investigations in view of the above object, the present inventors have developed a series of red fluorescent dyes (phosphafluorescein compounds) in which the oxygen atom at the 10-position of the xanthene ring portion of fluorescein is substituted with a phosphorus atom. This compound is anionized under neutral or alkaline conditions, and has a sufficiently high fluorescence quantum yield even under physiological conditions while having an absorption maximum wavelength and a fluorescence maximum wavelength of 600 to 700 nm. We found that rate was obtained. In addition, since this compound lowered the HOMO level due to the electronic effect of the phosphorus substituent, a remarkable stabilizing effect against light irradiation was also observed. The present invention has been completed by further research based on such knowledge. That is, the present invention includes the following configurations.

Item 1. General formula (1):

[Wherein, R ¹ represents an optionally substituted aryl group. R ² represents a hydrogen atom or an organic group. R is the general formula (1A) to (1C):

(In the formula, R ³ represents an optionally substituted aryl group, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group. R ⁴ represents a substituted group. Represents an alkyl group which may be substituted.)
It is group represented by these. ]
Or a salt, hydrate or solvate thereof.

Item 2. Item 2. The phosphafluorescein compound according to item 1, or a salt, hydrate or solvate thereof, wherein in the general formula (1), R is a group represented by the general formula (1A).

Item 3. The salt of the phosphafluorescein compound represented by the general formula (1) is represented by the general formula (2):

[Wherein, R ¹ and R are the same as defined above. ]
Item 3. The phosphafluorescein compound according to Item 1 or 2, or a salt, hydrate, or solvate thereof, having an anion represented by:

Item 4. The phosphafluorescein compound or a salt, hydrate or solvate thereof according to any one of Items 1 to 3, which has a maximum absorption wavelength at 600 to 700 nm.

Item 5. The phosphafluorescein compound according to any one of Items 1 to 4, having a maximum absorption wavelength at 600 to 700 nm and a fluorescence quantum yield of 0.25 to 0.60, or a salt, hydrate or Solvate.

Item 6. Item 6. A fluorescent dye containing the phosphafluorescein compound according to any one of Items 1 to 5 or a salt, hydrate or solvate thereof.

Item 7. Item 7. The fluorescent dye according to Item 6, which is a fluorescent dye for bioimaging.

Item 8. Item 8. The fluorescent dye according to Item 7, which is a fluorescent dye for cancer cell bioimaging.

Item 9. Item 9. A cell detection agent comprising the fluorescent dye according to any one of Items 6 to 8.

Item 10. Item 10. The cell detection agent according to Item 9, which is a cancer cell detection agent.

Item 11. Item 11. A cell bioimaging method using the fluorescent dye according to any one of Items 6 to 8, or the cell detection agent according to Item 9 or 10.

Item 12. Item 12. The bioimaging method according to Item 11, wherein the cell is a cancer cell.

The phosphafluorescein compound of the present invention or a salt, hydrate or solvate thereof has an absorption maximum wavelength and a fluorescence maximum wavelength of 600 to 700 nm because the oxygen atom at the 10-position of the xanthene ring portion of fluorescein is substituted with a phosphorus atom. In addition, a sufficiently high fluorescence quantum yield can be obtained even under physiological conditions. In particular, the absorption maximum wavelength of 627 nm in the examples is almost the same as the excitation wavelength of 633 nm of the HeNe laser normally provided in a laser microscope, so that the excitation efficiency as a fluorescent dye is extremely high.

In addition, while having an absorption maximum wavelength and a fluorescence maximum wavelength in the range of 600 to 700 nm, it has a high fluorescence quantum yield that was not found in conventional fluorescent dyes having a maximum wavelength in this region, which is necessary for bioimaging. The fluorescent dye concentration can be kept low, and damage to the living body can be greatly reduced.

Furthermore, the phosphafluorescein compound of the present invention or a salt thereof can reduce the HOMO level due to the electronic effect of the phosphorus substituent, and can dramatically improve the stability to light. It is possible to observe for a long time.

3 is a result of X-ray crystal structure analysis of a phosphafluorescein compound (compound POF) obtained in Example 1. FIG. 2 is an absorption spectrum and a fluorescence spectrum of the phosphafluorescein compound (compound POF) of the present invention when adjusted to pH 3, pH 7, and pH 9 prepared in Test Example 3. FIG. It is an absorption spectrum (upper figure) of the phosphafluorocein compound (compound POF) of the present invention at each pH, and a graph (lower figure) showing the relationship between relative absorption at 627 nm and pH. It is a graph (lower figure) which shows the relationship between the excitation spectrum (upper figure) of the phosphafluorocein compound (compound POF) of the present invention at each pH, the ratio of the excitation intensity at 627 nm to the excitation intensity at 532 nm, and the pH. . 6 is a graph showing the results of Test Example 5. (A) is a graph showing the absorbance maintenance rate at the absorption maximum wavelength when the phosphafluorescein compound of the present invention and known fluorescein compounds (TokyoGreen and TokyoMagenta) are used. (B) is a graph showing the absorbance maintenance rate at 630 nm of test solutions 7 to 10 prepared in Test Example 5. 6 is a photograph showing the results of cell incubation and imaging in Test Example 6. The left figure (a) is a Confocal fluorescence image obtained by excitation at 633 nm. The middle figure (b) is a bright field image. The right figure (c) is a merged image of (a) and (b). In the figure, the scale bar is 50 μm. 6 is a graph showing the results of cytotoxicity in Test Example 6.

1． Phosphafluorescein compound or a salt, hydrate or solvate thereof The phosphafluorescein compound of the present invention or a salt thereof has the general formula (1):

[Wherein, R ¹ represents an optionally substituted aryl group. R ² represents a hydrogen atom or an organic group. R is the general formula (1A) to (1C):

(In the formula, R ³ represents an optionally substituted aryl group, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group. R ⁴ represents a substituted group. Represents an alkyl group which may be substituted.)
It is group represented by these. ]
Or a salt thereof. The phosphafluorescein compound represented by the general formula (1) or a salt thereof is a novel compound not described in any literature.

In the general formula (1), as the aryl group represented by R ¹ , any of a monocyclic aryl group, a polycyclic aryl group, and a heteroaromatic ring group can be employed. For example, a phenyl group, an oligoaryl group (Naphthyl group, anthryl group, etc.), biphenyl group, terphenyl group, pyrenyl group, phenanthrenyl group, fluorenyl group, thienyl group, furyl group, pyridyl group and the like.

The substituent which the aryl group represented by R ¹ may have is not particularly limited, and is a hydroxyl group, a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), alkyl group (methyl group, ethyl group). Group, n-propyl group, etc.), formyl group, carboxyl group, ester group, amide group (amide group, methylamide group, dimethylamide group etc.), azido group (azidephenyl group etc.), alkynyl group (ethynyl group, propargyl group) Etc.), alkenyl groups (vinyl group, allyl group, etc.) and the like. The number of such substituents is not particularly limited and is preferably 0 to 6, more preferably 0 to 3.

Among them, R ¹ is preferably a substituted or unsubstituted monocyclic aryl group (substituted or unsubstituted phenyl group) from the viewpoint of further improving the fluorescence quantum yield and making it easier to recognize during bioimaging. A substituted monocyclic aryl group having a substituent at the position (substituted phenyl group) is more preferred, and an o-tolyl group is more preferred.

In the general formula (1), the organic group represented by R ² may be an alkyl group which may have a substituent, an acyl group which may have a substituent, or a substituent. Good cyclic ether groups and the like can be mentioned.

In the general formula (1), as the alkyl group as the organic group represented by R ² , both a linear alkyl group and a branched alkyl group can be employed.

As the straight-chain alkyl group, a straight-chain alkyl group having 1 to 6 carbon atoms (particularly 1 to 4) is preferable. Specific examples include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, and an n-pentyl group. Group, n-hexyl group and the like.

As the branched alkyl group, a branched alkyl group having 3 to 6 carbon atoms (particularly 3 to 5 carbon atoms) is preferable. Specifically, isopropyl group, isobutyl group, t-butyl group, s-butyl group, neopentyl group, Examples thereof include an isohexyl group and a 3-methylpentyl group.

The substituent that the alkyl group as the organic group represented by R ² may have is not particularly limited, and examples thereof include a hydroxyl group and a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.). . The number of such substituents is not particularly limited and is preferably 0 to 6, more preferably 0 to 3.

In the general formula (1), examples of the acyl group as the organic group represented by R ² include a methanoyl group and an ethanoyl group.

In the general formula (1), examples of the cyclic ether group as the organic group represented by R ² include a tetrahydropyranyl group and a tetrahydrofuranyl group.

The substituent that the cyclic ether as the organic group represented by R ² may have is not particularly limited, and is a hydroxyl group, a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), acyl group ( Methanoyl group, ethanoyl group, etc.), silyl group (trimethylsilyl group, triethylsilyl group, etc.) and the like. The number of such substituents is not particularly limited, but is preferably 0 to 2, more preferably 0 to 1.

Among them, R ² is preferably a hydrogen atom, an acyl group, or a substituted cyclic ether group, and more preferably a hydrogen atom, from the viewpoint of easy anionization and easy shift of the absorption maximum wavelength and the fluorescence maximum wavelength.

In the general formula (1) (general formulas (1A) to (1C)), among the groups represented by R, as the aryl group represented by R ³ , both a monocyclic aryl group and a polycyclic aryl group are employed. Examples thereof include a phenyl group, an oligoaryl group (naphthyl group, anthryl group, etc.), a biphenyl group, a terphenyl group, a pyrenyl group, a phenanthrenyl group, and a fluorenyl group.

The substituent which the aryl group represented by R ³ may have is not particularly limited, and is a hydroxyl group, a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), alkyl group (methyl group, ethyl group). Group, n-propyl group, etc.). The number of such substituents is not particularly limited, but is preferably 0 to 6, more preferably 0 to 3.

In the general formula (1) (general formulas (1A) to (1C)), among the groups represented by R, as the alkyl group represented by R ³ , either a linear alkyl group or a branched alkyl group can be employed. .

As the straight-chain alkyl group, a straight-chain alkyl group having 1 to 6 carbon atoms (particularly 1 to 4) is preferable. Specific examples include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, and an n-pentyl group. Group, n-hexyl group and the like.

As the branched alkyl group, a branched alkyl group having 3 to 6 carbon atoms (particularly 3 to 5 carbon atoms) is preferable. Specifically, isopropyl group, isobutyl group, t-butyl group, s-butyl group, neopentyl group, Examples thereof include an isohexyl group and a 3-methylpentyl group.

The substituent that the alkyl group as the organic group represented by R ² may have is not particularly limited, and examples thereof include a hydroxyl group and a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.). . The number of such substituents is not particularly limited, but is preferably 0 to 6, more preferably 0 to 3.

In the general formula (1) (general formulas (1A) to (1C)), among the groups represented by R, examples of the alkenyl group represented by R ³ include a vinyl group and an allyl group.

In the general formula (1) (general formulas (1A) to (1C)), among the groups represented by R, examples of the alkynyl group represented by R ³ include an alkynyl group and a propargyl group.

Depending on the type of R ³ , the electronic structure can be finely adjusted and the physical properties (solubility, cell permeability, etc.) can be adjusted.

In the general formula (1) (general formula (1C)), among the groups represented by R, as the alkyl group represented by R ⁴ , both a linear alkyl group and a branched alkyl group can be employed.

As the straight-chain alkyl group, a straight-chain alkyl group having 1 to 6 carbon atoms (particularly 1 to 4) is preferable. Specific examples include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, and an n-pentyl group. Group, n-hexyl group and the like.

As the branched alkyl group, a branched alkyl group having 3 to 6 carbon atoms (particularly 3 to 5 carbon atoms) is preferable. Specifically, isopropyl group, isobutyl group, t-butyl group, s-butyl group, neopentyl group, Examples thereof include an isohexyl group and a 3-methylpentyl group.

The substituent that the alkyl group as the organic group represented by R ² may have is not particularly limited, and examples thereof include a hydroxyl group and a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.). . The number of such substituents is not particularly limited, but is preferably 0 to 6, more preferably 0 to 3.

Among them, R has higher electron withdrawing properties, and it is easier to reduce the HOMO level (energy level of the highest occupied orbit) and LUMO level (energy level of the lowest unoccupied orbit). From the viewpoint of easily improving the stability, a group represented by the general formula (1A) is preferable.

Examples of the compound of the present invention that satisfies such conditions include, for example,

[Wherein, Ph represents a phenyl group. The same applies hereinafter. ]
A phosphafluorescein compound represented by the above, or a salt, hydrate or solvate thereof is preferred.

The compound of the present invention having such a structure has a high electron withdrawing property due to the presence of a phosphorus-containing group, and the HOMO level (energy level of the highest occupied orbit) and LUMO level (energy level of the lowest unoccupied orbit). ) Can be reduced. In particular, the LUMO level can be effectively reduced compared to the HOMO level, so the HOMO-LUMO energy gap when changed to an anion type is reduced to 2.00-2.50 eV, especially 2.25-2.48 eV. (B3LYP / 6-31 + G value obtained by quantum chemistry calculations performed at the G level). For this reason, it is also possible to shift the absorption maximum wavelength and the fluorescence maximum wavelength by longer wavelengths. HOMO and LUMO levels are measured by structural optimization using Gaussian 09 program.

As the compound of the present invention, even a neutral compound (compound represented by the general formula (1)) can have a fluorescence maximum wavelength of about 600 to 650 nm, particularly 610 to 640 nm. However, if the salt form of the compound represented by the general formula (1) is anionized, the absorption maximum wavelength is shifted to a wavelength longer than 600 to 700 nm (particularly about 600 to 650 nm). It is also possible to further shift the fluorescence maximum wavelength (about 650 to 700 nm, particularly about 655 to 670 nm) and further improve the fluorescence quantum yield (about 0.25 to 0.60, especially 0.35 to 0.50).

From such a viewpoint, the compound of the present invention is preferably a salt of the compound represented by the general formula (1), and the general formula (2):

[Wherein, R ¹ and R are the same as defined above. ]
It is more preferable to have an anion represented by:

In the general formula (2), R ¹ and R are the same as those described above.

Moreover, there is no restriction | limiting in particular as an ion (cation) which is paired with the anion shown by General formula (2), For example, metal salts, such as a sodium salt, potassium salt, calcium salt, magnesium salt; An organic ammonium salt such as triethylammonium.

In addition, the compound of the present invention may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention.

2． Method for producing phosphafluorescein compound or salt, hydrate or solvate thereof The phosphafluorescein compound of the present invention or a salt, hydrate or solvate thereof is not particularly limited, and examples thereof include silicon-substituted fluorescein compounds. It can be synthesized according to the method already reported for the production method (Chem. Commun. 2011, 47, 4162.) (except for changing the raw material compound).

Specifically, the following reaction formula 1:

[Wherein, R and R ¹ are the same as defined above. R ⁵ and R ⁶ are the same or different and represent a protecting group. X ¹ and X ² are the same or different and each represents a halogen atom. ]
Can be synthesized according to

In Reaction Scheme 1, the protecting group represented by R ⁵ and R ⁶ is not particularly limited as long as it is a group capable of protecting a hydroxyl group, and any group can be used. For example, an alkanoyl group (formyl group, C1-C4 alkanoyl groups such as acetyl group and propionyl), optionally substituted aralkyl groups (benzyl group, p-methoxybenzyl group, p-nitrobenzyl group, etc.), silyl groups (trimethylsilyl group, triethylsilyl group) Group, t-butyldimethylsilyl group, etc.), alkoxyalkyl group (methoxymethyl group etc.), tetrahydropyranyl (THP) group and the like. In the present invention, a silyl group is preferable and a t-butyldimethylsilyl group is more preferable from the viewpoint of protecting the hydroxyl group and facilitating the progress of the nucleophilic addition reaction.

In the reaction formula 1, as the halogen atom represented by X ¹ and X ² , any of a fluorine atom, a chlorine atom, a bromine atom and an iodine atom can be adopted, but from the viewpoint of easy progress of the cyclization reaction, a chlorine atom , A bromine atom, an iodine atom and the like are preferable, and a bromine atom is more preferable.

(2-1) Cyclization reaction In this step, for example, when a compound having a group represented by the general formula (1A) as R is to be obtained, a specific phosphorus compound and Except for using hydrogen peroxide, it can be carried out in the same manner as previously reported (Chem. Commun. 2011, 47, 4162.). Specifically, by reacting the compound (3) in the above reaction formula 1 and the organolithium compound in an organic solvent, an organophosphorus compound is added, and then hydrogen peroxide solution is added. The desired cyclization reaction can proceed. When obtaining a compound having a group represented by the general formula (1B) or (1C) as R, a desired raw material compound is used according to the substituent to be obtained instead of the hydrogen peroxide solution. It is preferable.

In addition, the compound (3) in the above reaction formula 1 can be a known or commercially available compound, or can be synthesized. In the case of synthesis, it can be synthesized by reacting 3-halogenated-N, N-diallylaniline obtained by reacting 3-halogenated aniline and allyl halide with formaldehyde.

The organic lithium compound is not particularly limited, and known compounds can be used. For example, ethyl lithium, n-propyl lithium, isopropyl lithium, n-butyl lithium, s-butyl lithium, t-butyl lithium, pentyl lithium, hexyl Alkyl lithium such as lithium; cycloalkyl lithium such as cyclohexyl lithium; aryl lithium such as phenyl lithium; and the like. Among these, in this step, alkyllithium is preferable and n-butyllithium is more preferable from the viewpoint of yield.

The organophosphorus compound is not particularly limited as long as a compound having a group represented by R can be obtained in this step, and known compounds can be used, for example, dihalogenated arylphosphine such as dichlorophenylphosphine and dibromophenylphosphine. Can be used.

The amount of the organolithium compound, organophosphorus compound and hydrogen peroxide used is not particularly limited, and from the viewpoint of yield and the like, 1 to 20 moles (especially 2 to 2 moles) of the organolithium compound per mole of the compound (3) It is preferable to use 0.1 to 10 mol (especially 0.5 to 5 mol) of the organic phosphorus compound. Further, it is preferable to use an excess amount of an aqueous solution of hydrogen peroxide.

As the organic solvent that can be used in this step, known ones can be adopted, and in this step, for example, cyclic ethers such as tetrahydrofuran and dioxane are preferable. The reaction conditions are preferably such that the reaction proceeds sufficiently. For example, the reaction can be carried out at −150 to 0 ° C., particularly −100 to −50 ° C. for 5 minutes to 12 hours, particularly 10 minutes to 6 hours.

(2-2) Oxidation reaction This step can be carried out in the same manner as previously reported (Chem. Commun. 2011, 47, 4162.). Specifically, an oxidation reaction can be caused to the compound (4) in the above reaction formula 1 using an oxidizing agent in an organic solvent.

The oxidizing agent is not particularly limited, and permanganate (such as potassium permanganate) can be used.

The amount of the oxidizing agent used is not particularly limited, and is preferably used in an amount of 1 to 10 mol (especially 2 to 5 mol) with respect to 1 mol of compound (4) from the viewpoint of yield and the like.

As the organic solvent that can be used in this step, a known one may be employed. In this step, for example, cyclic ethers such as tetrahydrofuran and dioxane are preferable. In addition, the same solvent as the said cyclization process can be used. The reaction conditions may be such that the reaction proceeds sufficiently. For example, the reaction conditions may be -50 to 100 ° C., particularly 0 to 50 ° C., 1 to 48 hours, particularly 2 to 24 hours.

(2-3) Deallylation Reaction This step can be performed in the same manner as previously reported (Chem. Commun. 2011, 47, 4162.). Specifically, 1,3-dimethylbarbituric acid or the like is added to the compound (6) in the above reaction formula 1 in the presence of a palladium catalyst (tetrakis (triphenylphosphine) palladium or the like) in an organic solvent. Can be used to cause a deallylation reaction.

The amount of the palladium catalyst and 1,3-dimethylbarbituric acid used is not particularly limited. From the viewpoint of yield and the like, 0.1 to 1 mol of palladium catalyst (especially 0.2 to 1 mol) per 1 mol of compound (5). 0.5 mol) and 2 to 50 mol (especially 5 to 30 mol) of 1,3-dimethylbarbituric acid are preferably used.

As the organic solvent that can be used in this step, a known one may be employed. In this step, for example, cyclic ethers such as tetrahydrofuran and dioxane are preferable. In addition, the same solvent as the said cyclization process can be used. The reaction conditions may be such that the reaction proceeds sufficiently. For example, the reaction conditions may be 1 to 96 hours, particularly 2 to 72 hours at 0 to 150 ° C., particularly 50 to 100 ° C.

(2-4) Conversion reaction This step can be carried out in the same manner as previously reported (Chem. Commun. 2011, 47, 4162.). Specifically, a conversion reaction to a hydroxyl group can be caused by using nitrous acid or a salt thereof for the compound (6) in the above reaction formula 1 in an organic solvent.

As nitrous acid or a salt thereof, in addition to nitrous acid, nitrite (sodium nitrite) or the like can be adopted.

The amount of nitrous acid or a salt thereof used is not particularly limited, but from the viewpoint of yield and the like, it is preferable to use 1 to 10 mol (particularly 2 to 5 mol) with respect to 1 mol of compound (6). .

As the organic solvent that can be used in this step, known ones can be adopted, and in this step, for example, cyclic ethers such as tetrahydrofuran and dioxane are preferable. In addition, the same solvent as the said cyclization process can be used. The reaction conditions may be such that the reaction proceeds sufficiently, for example, 50 to 200 ° C., particularly 100 to 150 ° C., 5 minutes to 5 hours, particularly 10 minutes to 2 hours.

(2-5) Protection This step can be performed in the same manner as previously reported (Chem. Commun. 2011, 47, 4162.). Specifically, the compound (7) in the above reaction formula 1 can be protected by a known method in an organic solvent. Any conventionally known methods and conditions can be adopted as the protection method and conditions.

(2-6) Nucleophilic addition reaction In this step, specifically, the compound (7) in the above reaction formula 1 is reacted with the Grignard reagent in an organic solvent, and then an oxidation reaction is performed using an oxidizing agent. This can cause a nucleophilic addition reaction.

As the Grignard reagent, those capable of introducing the desired R ¹ in the compound of the present invention are preferable, and organic magnesium represented by R ¹ MgX ³ (wherein R ¹ is the same as above, X ³ represents a halogen atom). Compounds are preferred.

As the halogen atom represented by X ³ , those described above can be adopted. The same applies to preferred embodiments.

Examples of Grignard reagents that satisfy these conditions include:

Etc.

There is no restriction | limiting in particular as an oxidizing agent, Hydrogen chloride (hydrochloric acid) etc. can be used.

The amount of Grignard reagent and oxidizing agent used is not particularly limited, and from the viewpoint of yield, etc., 1 to 10 moles (especially 2 to 5 moles) of Grignard reagent should be used per 1 mole of compound (5). Is preferred. The oxidizing agent is preferably used in an excess amount as an aqueous solution.

As the organic solvent that can be used in this step, known ones can be adopted, and in this step, for example, cyclic ethers such as tetrahydrofuran and dioxane are preferable. In addition, the same solvent as the said cyclization process can be used. The reaction conditions may be such that the reaction proceeds sufficiently. For example, the reaction conditions may be −50 to 100 ° C., particularly 0 to 50 ° C., 30 minutes to 10 hours, particularly 1 to 5 hours.

In this way, the phosphafluorescein compound represented by the general formula (1) can be obtained, and can be used through ordinary isolation and purification steps as necessary. The phosphafluorescein compound represented by the general formula (1) is a neutral compound under acidic conditions (for example, pH 1 to 5), but is made to be under neutral conditions or alkaline conditions (for example, pH 6 to 14). Thus, it can be easily converted into a salt having an anion represented by the general formula (2), and can be used through ordinary isolation and purification steps as necessary. As a result, the absorption maximum wavelength is shifted to a longer wavelength in the range of 600 to 700 nm (especially about 600 to 650 nm) and the fluorescence maximum wavelength is further shifted (about 650 to 700 nm, particularly 655 to 650 nm). It is possible to further improve the fluorescence quantum yield (about 0.25 to 0.60, especially 0.35 to 0.50).

In addition, in the above, although an example of the synthesis | combining method of the one aspect | mode of the phosphafluorescein compound of this invention was described, it is not limited to this manufacturing method, It can synthesize | combine with various synthesis methods. Other phosphafluorescein compounds can be synthesized by the same method.

3． Fluorescent dye and cell detection agent The fluorescent dye of the present invention contains the phosphafluorescein compound of the present invention or a salt thereof.

In the fluorescent dye of the present invention, the oxygen atom at the 10-position of the xanthene ring portion of fluorescein is substituted with a phosphorus atom, so that it has a maximum absorption wavelength and a maximum fluorescence wavelength in the range of 600 to 700 nm, but it is sufficient even under physiological conditions. A high fluorescence quantum yield is obtained. In particular, the absorption maximum wavelength of 627 nm in the examples is almost the same as the excitation wavelength of 633 nm of a HeNe laser that is normally equipped with a laser microscope, so that the excitation efficiency as a fluorescent dye is extremely high. In particular, the phosphafluorescein compound or a salt thereof of the present invention has high permeability into cells, and when a desired substituent is introduced as R ¹ , desired cells (for example, cancer cells such as HeLa cells) in vivo. ) Can be selectively localized. That is, only desired cells (for example, cancer cells such as HeLa cells) can emit light. In particular, the phosphafluorescein compound or a salt thereof of the present invention has a high fluorescence quantum that is not found in conventional fluorescent dyes that have an absorption maximum wavelength and a fluorescence maximum wavelength in the range of 600 to 700 nm but have a maximum wavelength in this region. Since it has a high yield, it can facilitate the bioimaging of desired cells (for example, cancer cells such as HeLa cells), and the fluorescent dye concentration required for bioimaging can be kept low, resulting in damage to the living body. Can be greatly reduced. Furthermore, the phosphafluorescein compound of the present invention or a salt thereof can reduce the HOMO level by the electronic effect of the phosphorus substituent, and can dramatically improve the stability to light. It is possible to observe for a long time.

The cell detection agent of the present invention (particularly a cancer cell detection agent such as HeLa cells) contains the phosphafluorescein compound of the present invention or a salt thereof, preferably dissolved in an organic solvent to form a solution, From the viewpoint of detecting more desired cells (for example, cancer cells such as HeLa cells) and color rendering (visualization in real time) the desired cells (for example, cancer cells such as HeLa cells) more selectively. The content of the fluorescein compound is preferably 1 × 10 ⁻⁸ to 1 × 10 ⁻⁴ mol / L, more preferably 1 × 10 ⁻⁷ to 1 × 10 ⁻⁵ mol / L. Thus, in this invention, compared with the conventional fluorescent pigment | dye, content of a phosphafluorescein compound can be restrained low.

When the fluorescent dye (phosphafluorescein compound) of the present invention is used as a solution containing the cell detection agent of the present invention, the organic solvent that can be used is not particularly limited, and both polar solvents and nonpolar solvents are used. it can.

Examples of polar solvents include ether compounds (tetrahydrofuran, anisole, 1,4-dioxane, cyclopentylmethyl ether, etc.), alcohols (methanol, ethanol, allyl alcohol, etc.), ester compounds (ethyl acetate, etc.), ketones (acetone, etc.) , Halogenated hydrocarbons (dichloromethane, chloroform), dimethyl sulfoxide, amide solvents (N, N-dimethylformamide, dimethylacetamide, 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, etc.) .

Examples of the nonpolar solvent include aliphatic organic solvents such as pentane, hexane, cyclohexane and heptane; aromatic solvents such as benzene, toluene, xylene and mesitylene.

As described above, the cell detection agent of the present invention is preferably in the form of a solution, but has a sufficiently high fluorescence quantum yield even under physiological conditions while having an absorption maximum wavelength and a fluorescence maximum wavelength at 600 to 700 nm. However, the pH is preferably about 5 to 11 and more preferably about 6.5 to 7.5 from the viewpoint of introduction into cells. Buffers (Hepes buffer, Tris buffer, tricine-sodium hydroxide buffer, phosphate buffer, phosphate buffered saline, etc.) are used to adjust the pH of the cell detection agent of the present invention. May be.

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

[Example 1]

The general operating melting point (mp) or decomposition temperature was measured with a Yanaco MP-S3 instrument (MP-S3). ¹ H, ¹³ C { ¹ H} and ³¹ P { ¹ H} NMR spectra were measured using JEOL AL-400 spectrometer (400 MHz for ¹ H, 100 MHz for ¹³ C and 161.70 MHz for ³¹ P), JEOL JNM-ECS400 ( CDCl ₃ , CD ₂ Cl ₂ or 400 MHz for ¹ H, 100 MHz for ¹³ C and 161.70 MHz for ³¹ P) or JEOL A-600 spectrometer (600 MHz for ¹ H and 150 MHz for ¹³ C) Measured in CD ₃ OD. The chemical shift of the ¹ H NMR spectrum is expressed in δ ppm using the residual proton of the solvent as an internal standard (CHCl ₃ δ 7.26, CH ₂ Cl ₂ δ 5.32, CD ₃ OD δ 3.31), and the ¹³ C NMR spectrum Chemical shifts were expressed in δ ppm using the solvent signal as an internal standard (CDCl ₃ δ77.16, CD ₂ Cl ₂ δ53.84, CD ₃ ODδ49.0). As the chemical shift value of the ³¹ P NMR spectrum, a signal of H ₃ PO ₄ (δ0.00) was used as an external standard. Mass spectra were measured by electrospray ionization (ESI) using a Bruker micrOTOF Focus spectrometry system using atmospheric pressure chemical ionization (APCI) or Thermo Fisher Scientific Exactive. Thin layer chromatography (TLC) was performed using a glass plate coated with silica gel 60F ₂₅₄ (Merck). Column chromatography was performed using neutral silica gel PSQ100B (Fuji Silysia Chemical) or silica gel 60 (Kanto Chemical). Preparative recycling HPLC was performed using a YMC LC-forte / R equipped with a reverse phase column (YMC-Actus Triart C18). Unless otherwise stated, all reactions were performed under a nitrogen atmosphere. Unless otherwise noted, solvents and reagents were used without purifying commercial products. Dehydrated THF and CH ₂ Cl ₂ were purchased from Kanto Chemical and purified by Glass Contour Solvent Systems. Bis (2-bromo-4-N, N-diallylaminophenyl) methane (compound 1) and TokyoMagenta were synthesized according to the previous report (Chem. Commun., 2011, 47, 4162-4164.), And TokyoGreen was reported (J. Am. Chem. Soc., 2005, 127, 4888-4894.).

Synthesis of Compound 2 Bis [2-bromo-4- (N, N-diallylamino) phenyl] methane (Compound 1) (1.01 g, 1.96 mmol) was dissolved in dehydrated THF (20 mL). A pentane solution of t-butyllithium (1.63 M, 4.80 mL, 7.82 mmol) was added dropwise at −78 ° C. over 20 minutes, and the mixture was stirred as it was for 1 hour. Dichlorophenylphosphine (PhPCl ₂ ; 0.318 mL, 2.34 mmol) was added dropwise over 25 minutes, and 1.5 hours after completion of the addition, a 30% H ₂ O ₂ solution (1.0 mL) was added at 0 ° C., and the mixture was stirred as it was for 1 hour. A sodium hypochlorite aqueous solution and a saturated sodium sulfite aqueous solution were added to the reaction mixture, followed by extraction with CH ₂ Cl ₂ . The combined organic layers were washed with saturated brine and then dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (CH ₂ Cl ₂ / methanol 20/1, Rf = 0.4) to obtain 761 mg (1.58 mmol, 81% yield) of Compound 2 as a yellow liquid. The spectral data of Compound 2 is as follows.
¹ H NMR (400MHz, CDCl ₃ ): δ7.47-7.42 (m, 4H), 7.39-7.31 (m, 3H), 7.18 (dd, J = 8.2 Hz, 6.2 Hz, 2H), 6.80 (bs, 2H ), 5.89-5.80 (m, 4H) 5.18-5.13 (m, 8H), 4.03-3.90 (m, 8H), 3.84 (d, J = 18Hz, 1H), 3.66 (d, J = 18 Hz, 3.6 Hz ¹³ C { ¹ H} NMR (100 MHz, CDCl ₃ ): δ 147.5 (d, J _CP = 13.3 Hz, C), 134.7 (d, J _CP = 104.1 Hz, C), 133.7 (s, CH), 131.1 (d, J _CP = 2.5 Hz, CH), 130.9 (d, J _CP = 9.9 Hz, CH), 129.8 (d, J _CP = 100.1 Hz, C), 129.3 (d, J _CP = 8.3 Hz, C), 129.0 (d, J _CP = 11.5 Hz, CH), 128.4 (d, J _CP = 12.4 Hz, CH), 116.4 (s, CH ₂ ), 115.8 (s, CH), 114.2 (d, _{. J CP = 8.2 Hz, CH} ), 52.9 (s, CH 2), 35.3 (d, J CP = 9.1 Hz, CH 2) 31 P {1 H} NMR (161.70MHz, CDCl 3):. δ 14.33 HRMS (ESI): m / z calcd. For C ₃₁ H ₃₃ N ₂ NaOP: 503.2228 ([M + Na] ⁺ ); found. 503.2224.

In addition, compound 2 was obtained by the following method. Bis [2-bromo-4- (N, N-diallylamino) phenyl] methane (Compound 1) (0.978 g, 1.89 mmol) in dehydrated THF (9 mL) at -78 ° C with s-butyl Lithium in cyclohexane and hexane (0.99 M, 4.00 mL, 3.96 mmol) was added dropwise over 5 minutes. After stirring for 1 hour, dichlorophenylphosphine (PhPCl ₂ ; 0.290 mL, 0.383 g, 2.14 mmol) was added dropwise over 10 minutes. After stirring at the same temperature for 3 hours, the solution was warmed to 0 ° C. and 30% H ₂ O ₂ solution (1.0 mL) was added. The mixture was stirred for 1 hour and quenched with aqueous sodium sulfite. The resulting solution was then extracted with ethyl acetate (AcOEt). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and filtered. Volatiles were removed under reduced pressure, and the resulting solid was purified by silica gel column chromatography (CH ₂ Cl ₂ / methanol 20/1, Rf = 0.4) to give 0.257 g (0.525 mmol) of Compound 2 as a yellow liquid. Yield 28%).

Compound 2 (761 mg, 1.58 mmol) was dissolved in CH ₂ Cl ₂ (16 mL) in the synthetic air of Compound 3 and Compound 4 . Chloranyl (1.19 g, 4.84 mmol) was added at room temperature, and the mixture was stirred as it was for 12.5 hours. The solvent was distilled off under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (CH ₂ Cl ₂ / methanol 20/1, R _f = 0.44) to obtain 304 mg (0.61 mmol, 39% yield) of compound 3 as a yellow liquid. The spectral data of Compound 3 is as follows.
¹ H NMR (400MHz, CDCl ₃ ): δ8.27 (dd, J = 9.0 Hz, 6.2 Hz, 2H), 7.59-7.54 (m, 2H), 7.39-7.32 (m, 3H), 7.14 (dd, J = 15 Hz, 2.6 Hz, 2H), 6.86 (dd, J = 9.0 Hz, 2.6 Hz, 2H), 5.82-5.74 (m, 4H), 5.15-5.09 (m, 8H), 4.08-3.93 (m, 8H ). ³¹ P { ¹ H} NMR (161.70 MHz, CDCl ₃ ): δ 6.84. HRMS (ESI): m / z calcd. For C ₃₁ H ₃₁ NaN ₂ O ₂ P: 517.2021 ([M + Na] ⁺ ) found. 517.2013.

Dehydrated 1,2-bis (diphenylphosphino) butane (464 mg, 1.08 mmol) dehydrated with tris (dibenzylideneacetone) dipalladium (Pd ₂ (dba) ₃ ; 50.4 mg, 0.055 mmol) Dissolved in dichloroethane (1.0 mL) and stirred at room temperature for 1 hour. A solution of compound 3 (71.0 mg, 0.144 mmol) and 1,3-dimethylbarbituric acid (265 mg, 1.70 mmol) in dehydrated dehydrated 1,2-dichloroethane (0.4 mL) The mixture was stirred at 80 ° C. for 2 days, and extracted with CH ₂ Cl ₂ . The combined organic layers were washed with saturated brine and then dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (CH ₂ Cl ₂ / methanol 10/1, R _f = 0.35) to obtain 31 mg (0.0927 mmol, yield 65%) of compound 4 as a yellow solid. The spectral data of Compound 4 is as follows.
Mp: 151 ° C. ¹ H NMR (400MHz, CD ₃ OD): δ 8.15 (dd, J = 8.8 Hz, 6.0 Hz, 2H), 7.60-7.42 (m, 5H), 6.99 (dd, J = 14.8 Hz, 2.2 Hz, 2H), 6.91 (dd, J = 8.8 Hz, 2.2 Hz, 2H). The signal corresponding to NH ₂ moiety was not observed. ¹³ C { ¹ H} NMR (100 MHz, CD ₃ OD): δ181.38 (d, J _CP = 9.0 Hz, C), 154.84 (d, J _CP = 13.3 Hz, C), 135.14 (d, J _CP = 97.6 Hz, C), 134.49 (d, J _CP = 108.3 Hz, C) , 133.29 (d, J _CP = 2.5 Hz, CH), 132.59 (d, J _CP = 10.7 Hz, CH), 131.64 (d, J _CP = 10.7 Hz, CH), 129.94 (d, J _CP = 13.3 Hz, . CH), 125.7 (d, J CP = 6.6 Hz, C), 118.57 (CH), 115.25 (d, J CP = 7.4 Hz, CH) 31 P {1 H} NMR (161.70MHz, CD 3 OD): δ 7.83. HRMS (APCI): m / z calcd. for C ₁₉ H ₁₆ N ₂ O ₂ P: 335.0944 ([M + H] ⁺ ); found. 335.0947.

In addition, compounds 3 and 4 were also obtained by the following method. To a CH ₂ Cl ₂ (112 mL) solution of Compound 2 (5.37 g, 11.2 mmol), chloranil (8.26 g, 33.6 mmol) was added in air. The mixture was stirred for 3 hours and quenched with aqueous sodium sulfite. The resulting solution was then extracted with CH ₂ Cl ₂ . The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and filtered. Volatiles were removed under reduced pressure, and the resulting solid was purified by silica gel column chromatography (CH ₂ Cl ₂ / methanol 20/1, Rf = 0.4) to give compound 3 as a yellow solid. Although some impurities could not be isolated, they were used as they were in the next step.

To a degassed 1,2-dichloroethane (370 mL) solution of compound 3 (4.39 g), tris (dibenzylideneacetone) dipalladium (Pd ₂ (dba) ₃ ; 1.34 g, 1.16 mmol), and 1,3- Dimethyl barbituric acid (6.39 g, 40.9 mmol) was added. The mixture was stirred at 80 ° C. for 24 hours. In order to proceed with the reaction, Pd ₂ (dba) ₃ (1.34 g, 1.16 mmol) and 1,3-dimethylbarbituric acid (6.39 g, 40.9 mmol) were further added to the obtained mixture. After stirring for a further 23 hours at 80 ° C., all volatiles were removed under reduced pressure. The obtained orange solid was purified by silica gel column chromatography (CHCl ₃ / methanol 10/1, R _f = 0.30) to obtain 2.39 g (7.15 mmol, yield 64%) of compound 4 as a yellow solid.

Compound 4 (52.0 mg, 0.156 mmol) was dissolved in 96% sulfuric acid (0.46 mL) in the synthetic air of compound 5 . NaNO ₂ (33.0 mg, 0.478 mmol) was added at 0 ° C., and the mixture was stirred as it was for 3 hours. The obtained reaction solution was added dropwise to an appropriate amount of ice and stirred at 110 ° C. for 0.5 hour. The obtained solid was filtered off, and the residue was washed with distilled water to obtain a crude product. The crude product was purified by silica gel column chromatography (CHCl ₃ / methanol 10/1, R _f = 0.25) to obtain 28.0 mg (0.832 mmol, yield 54%) of compound 5 as a yellow solid. The spectral data of Compound 5 is as follows.
¹ H NMR (400MHz, CD ₃ OD): δ8.33 (dd, J = 8.8 Hz, 6.0 Hz, 2H), 7.59-7.45 (m, 5H), 7.21 (dd, J = 14.2 Hz, 2.4 Hz, 2H ), 7.16 (dd, J = 8.8 Hz, 2.4 Hz, 2H) The signal corresponding to the OH moiety was not observed 13 C {1 H} NMR (100MHz, CD 3 OD):.. δ 181.48 (d, J CP = 9.1 Hz, C), 163.96 (d, J _CP = 13.2 Hz, C), 135.58 (d, J _CP = 97.5 Hz, C), 133.68 (d, J _CP = 3.3 Hz, CH), 133.64 (d, J _CP = 109.9 Hz, C), 133.32 (d, J _CP = 10.7 Hz, CH), 131.69 (d, J _CP = 10.7 Hz, CH), 130.20 (d, J _CP = 12.3 Hz, CH), 129.07 ( _{d, J CP = 7.4 Hz,} C), 121.56 (d, J CP = 1.6 Hz, CH), 117.60 (d, J CP = 6.6 Hz, CH). 31 P {1 H} NMR (161.70MHz, CD 3 OD): δ 6.44. HRMS (APCI): m / z calcd. For C ₁₉ H ₁₄ O ₄ P: 337.0624 ([M + H] ⁺ ); found. 337.0640.

In addition, compound 5 was also obtained by the following method. NaNO ₂ (171 mg, 2.48 mmol) was added in air at 0 ° C. to a 96% sulfuric acid (2.5 mL) solution of compound 4 (247 mg, 0.739 mmol) in air. After stirring for 3 hours, the mixture was slowly added dropwise to ice and stirred at 110 ° C. for 0.5 hours. The resulting precipitate was collected by filtration, washed with distilled water, and dispersed in methanol. The obtained dark brown precipitate was filtered, and the filtrate was concentrated under reduced pressure to obtain 158 mg (0.470 mmol, yield 64%) of Compound 5 as a yellow solid.

Synthesis of Compound 6 Compound 5 (195 mg, 0.580 mmol) and imidazole (197 mg, 2.89 mmol) were dissolved in dehydrated CH ₂ Cl ₂ (50 mL) and stirred for 15 minutes. A solution in which t-butylchlorodimethylsilane (505 mg, 3.35 mmol) was dissolved in dehydrated CH ₂ Cl ₂ (35 mL) was added dropwise to the solution containing Compound 5. After stirring for 3 hours, water was added and extracted with CH ₂ Cl ₂ . The combined organic layers were washed with saturated brine and then dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (CH ₂ Cl ₂ / ethyl acetate 30/1, R _f = 0.53) to obtain 265 mg (0.469 mmol, 81% yield) of Compound 6 as a colorless liquid. . The spectral data of Compound 6 is as follows.
¹ H NMR (400MHz, CDCl ₃ ): δ8.36 (dd, J = 8.7 Hz, 5.8 Hz, 2H), 7.59-7.53 (m, 2H), 7.46-7.36 (m, 5H), 7.10 (dd, J = 8.7 Hz, 2.4 Hz, 2H ), 0.95 (s, 18H), 0.21 (s, 6H), 0.20 (s, 6H) 13 C {1 H} NMR (100MHz, CDCl 3):. δ 181.08 (d, J _CP = 9.1 Hz, C), 160.57 (d, J _CP = 14.1 Hz, C), 135.55 (d, J _CP = 95.9 Hz, C), 133.60 (d, J _CP = 107.4 Hz, C), 132.08 ( d, J _CP = 10.7 Hz, CH), 131.99 (d, J _CP = 2.5 Hz, CH), 130.78 (d, J _CP = 10.7 Hz, CH), 129.64 (d, J _CP = 6.6 Hz, C), 128.87 (d, J _CP = 12.3 Hz, CH), 124.34 (d, J _CP = 2.5 Hz, CH), 121.91 (d, J _CP = 6.6 Hz, CH), 25.69 (s, CH ₃ ), 18.35 (s , C), -4.20 (s, CH 3), -4.30 (s, CH 3) 31 P {1 H} NMR (161.70 MHz, CDCl 3):.. δ 4.50 HRMS (APCI): m / z calcd. for C ₃₁ H ₄₁ NaO ₄ PSi ₂ : 587.2179 ([M + H] ⁺ ); found. 587.2167.

In addition, compound 6 was also obtained by the following method. Compound 5 (229 mg, 0.680 mmol) and imidazole (232 mg, 3.40 mmol) against dehydration CH ₂ Cl ₂ (25 mL) solution of, t- butyl chloro dimethyl silane (512 mg, 3.40 mmol) dehydrated CH ₂ of A solution of Cl ₂ (7 mL) was added. After stirring for 3 hours, water was added and the two layers were separated. The aqueous layer was extracted with CH ₂ Cl ₂ . The combined organic layers were dried over dehydrated sodium sulfate and filtered. After removing volatile substances under reduced pressure, the resulting mixture was purified by silica gel column chromatography (CH ₂ Cl ₂ / ethyl acetate 30/1, R _f = 0.53) to obtain Compound 354 mg as a colorless liquid. (0.627 mmol, yield 92%).

Compound POF Synthesis Compound 6 (230 mg, 0.407 mmol) was dissolved in dehydrated THF (70 mL). A THF solution of 2-methylphenylmagnesium bromide (1.03 M, 1.43 mL, 1.47 mmol) was added dropwise at room temperature, and the mixture was stirred for 4 hours. 1N aqueous HCl (50 mL) was added and stirred for 2 h, and the reaction mixture was extracted with CH ₂ Cl ₂ . The combined organic layers were washed with saturated brine and then dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated by silica gel column chromatography (CHCl ₃ / methanol 5/1 (TEA 10%), R _f = 0.48) and purified by HPLC to obtain 55 mg of a blue solid. The obtained solid was dissolved in toluene, put into a separatory funnel, 1N HCl aqueous solution was added, and the separatory funnel was shaken. The combined organic layers were washed with saturated brine and then dried over anhydrous sodium sulfate. Sodium sulfate was filtered off, and the filtrate was concentrated under reduced pressure to obtain Compound POF as a red solid in 30 mg (0.0730 mmol, 18% yield). The spectral data of the compound POF is as follows.
Mp.> 300 ° C. ¹ H NMR (400 MHz, CD ₃ OD): δ 7.76-7.36 (m, 8H), 7.27-7.13 (m, 3H), 7.03-6.94 (m, 2H), 6.58 (d, . J = 9.5 Hz, 2H) , 2.12 and 2.06 (two signals, 3H, tolyl-methyl group signals from two diastereoisomers) 13 C NMR (100 MHz, CD 3 OD): δ 140.0 (CH), 140.0 (CH), 138.3 (C), 138.2 (C), 137.9 (C), 137.7 (C), 137.6 (C), 137.3 (C), 137.2 (C), 137.1 (C), 134.1 (CH), 131.6 (CH), 131.6 (CH), 131.5 (CH), 131.4 (CH), 130.8 (CH), 130.5 (CH), 130.4 (CH), 130.4 (CH), 130.3 (CH), 129.9 (CH), 129.1 (CH), 127.2 (CH), 127.1 (CH ), 125.9 (C), 125.8 (C), 125.7 (C), 125.3 (CH), 19.7 (CH 3), 19.6 (CH 3). 31 P {1 H} NMR ( 161.70 MHz, CDCl ₃ ): δ 11.1, 11.0. HRMS (ESI): m / z calcd. For C ₂₆ H ₁₉ NaO ₃ P: 433.0970 ([M + H] ⁺ ); found. 433.0960.

In addition, the compound POF was also obtained by the following method. To a dehydrated THF (3 mL) solution of 2-bromotoluene (89 μL, 0.74 mmol), a cyclohexane and hexane solution (0.99 M, 0.90 mL, 0.89 mmol) of s-BuLi was added at −78 ° C. The mixture was stirred at −78 ° C. for 2 hours and a solution of compound 6 (139 mg, 0.246 mmol) in dehydrated THF (3 mL) was added slowly. The temperature was raised to room temperature and the reaction mixture was stirred for 1.5 hours. Thereafter, 0.5 M hydrochloric acid (20 mL) was added to quench the reaction. After stirring vigorously for 45 minutes, the mixture was diluted with water and CH ₂ Cl ₂ . The layers were separated and the aqueous layer was extracted 5 times with CH ₂ Cl ₂ . The combined organic layers were washed with brine and dried over Na ₂ SO ₄ . After filtration, volatiles were removed under reduced pressure and the resulting mixture was dispersed in CH ₂ Cl ₂ (10 mL). To the resulting yellow liquid, p-toluenesulfonic acid monohydrate (47 mg, 0.25 mmol) was added, and the resulting mixture was stirred at room temperature for 45 minutes to complete the dehydroxylation. The obtained deep red solution was directly purified by silica gel column chromatography (CH ₂ Cl ₂ / ethyl acetate 95/5). After removing the eluent under reduced pressure, a red liquid coagulated simultaneously with the addition of toluene was obtained. The resulting suspension was cooled to 0 ° C. and the resulting precipitate was collected by filtration and dried under vacuum to give the crude product as a vermilion solid. Then, it is purified by reverse phase HPLC (eluent: CH ₃ CN / H ₂ O mixed solvent containing 5 mM ammonium carbonate (mixing ratio 20/80 to 100/0) to obtain 34 mg of compound POF as a brown powder. (0.083 mmol, yield 34%).

[Example 2]

Under a nitrogen atmosphere, the compound POF (37 mg, 0.090 mmol) obtained in Example 1 was dissolved in 5 mL of dry pyridine. Acetic anhydride (85 μL, 0.90 mmol) was added at room temperature and the mixture was stirred for 3 hours. The solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography (CH ₂ Cl ₂ / ethyl acetate 3/1 to 2/1), then further purified by recrystallization from CH ₂ Cl ₂ / hexane. Then, 33 mg (0.073 mmol, yield 81%) of compound AcPOF was obtained as a yellow powder. The spectral data of the compound AcPOF is as follows.
Mp. 175-179 ° C. ¹ H NMR (400 MHz, CD ₂ Cl ₂ ): δ7.75-7.60 (m, 3H), 7.60-7.52 (m, 1H), 7.52-7.30 (m, 5H), 7.30 -7.06 (m, 3H), 7.06-6.91 (m, 2H), 6.26 (dd, J = 9.8, 1.8 Hz, 1H), 2.26 (s, 3H), 2.13 and 2.08 (two singlets, 3H, tolyl-methyl ^{. group signals from two diastereoisomers) 13} C NMR (150 MHz, CD 2 Cl 2): δ 184.3 (s, C), 184.2 (s, C), 168.9 (s, C), 153.0 (d, J CP = 14.4 Hz, C), 150.6 (d, J _CP = 7.2 Hz, C), 150.5 (d, J _CP = 10.1 Hz, C), 140.7 (d, J _CP = 90.5 Hz, C), 140.33 (d, J _CP = 90.5 Hz, CH), 139.7 (d, J _CP = 10.1 Hz, CH), 139.62 (d, J _CP = 8.6 Hz, CH), 137.2 (s, C), 137.2 (d, J _CP = 4.4 Hz, CH), 137.0 (d, J _CP = 2.9 Hz, CH), 136.5 (s, C), 136.4 (s, C), 136.4 (s, C), 135.0 (d, J _CP = 5.9 Hz, C), 134.9 (d, J _CP = 5.7 Hz, C), 134.3 (d, J _CP = 11.6 Hz, CH), 134.3 (d, J _CP = 10.1 Hz, CH), 133.7 (d, J _CP = 102.0 Hz, C ), 133.5 (d, J _CP = 107.7 Hz, C), 133.0 (d, J _CP = 89.1 Hz, C), 132.9 (s, CH), 131.0 (s, CH), 130.8 (d, J _CP = 10.1 Hz, CH), 130.8 (d, J _CP = 11.4 Hz, CH), 130.3 (s, CH ), 129.62 (CH), 129.61 (CH), 129.51 (CH), 129.46 (CH), 129.42 (CH), 129.39 (CH), 129.38 (d, J _CP = 8.6 Hz, C), 129.2 (s, CH ), 127.73 (d, J _CP = 5.9 Hz, C), 126.62 (s, CH), 126.59 (s, CH), 126.56 (s, CH), 125.94 (d, J _CP = 5.7 Hz, CH), 125.87 (d, J _CP = 7.2 Hz, CH), 21.2 (s, CH ₃ ), 19.8 (s, CH ₃ ). ³¹ P { ¹ H} NMR (162 MHz, CDCl ₃ ): δ5.8, 5.7. HRMS (ESI): m / z calcd. For C ₂₈ H ₂₁ NaO ₄ P: 475.1075 ([M + Na] ⁺ ); found. 475.1066.

[Example 3]

Synthesis of Compound 7 Bis [2-bromo-4- (N, N-diallylamino) phenyl] methane (Compound 1) (5.70 g, 11.0 mmol) was dissolved in dehydrated THF (110 mL). A pentane solution of t-butyllithium (1.61 M, 27.0 mL, 43.5 mmol) was added dropwise over 35 minutes at -78 ° C, and the mixture was stirred as it was for 2 hours. Dichlorophenylphosphine (PhPCl ₂ ; 1.80 mL, 13.2 mmol) was added dropwise over 90 minutes, and the mixture was stirred overnight after completion of the addition. S ₈ (705 mg) was added and stirred as such for 30 minutes. The reaction solvent was concentrated under reduced pressure, and the resulting mixture was extracted with dichloromethane. The combined organic layers were washed with saturated brine and then dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (CH ₂ Cl ₂ / hexane 1/1, R _f = 0.25) to give compound 7 as a yellow liquid in 2.84 mg (5.72 mmol, 52% yield). . The spectral data of Compound 7 is as follows.
¹ H NMR (400MHz, CD ₂ Cl ₂ ): δ 7.67 (dd, J = 16.4 Hz, 2.4 Hz, 2H), 7.36-7.29 (m, 5H), 7.17 (dd, J = 8.2 Hz, 6.4 Hz, 2H ), 6.80 (dd, J = 8.2 Hz, 2.4 Hz, 2H), 5.94-5.85 (m, 4H), 5.23-5.17 (m, 8H), 4.05-3.97 (m, 8H), 3.81 (d, J = 18 Hz, 1H), 3.56 (dd, J = 18 Hz, 3.4 Hz, 1H). ¹³ C { ¹ H} NMR (100 MHz, CD ₂ Cl ₂ ): δ 147.92 (d, J _CP = 14.9 Hz, C) , 136.04 (d, J _CP = 84.3 Hz, C), 134.12 (s, CH), 131.01 (d, J _CP = 2.5 Hz, CH), 130.62 (d, J _CP = 11.5 Hz, CH), 129.42 (d , J _CP = 81.8 Hz, C), 129. 32 (d, J _CP = 5.7 Hz, C), 129.09 (d, J _CP = 10.7 Hz, CH), 128.80 (d, J _CP = 12.4 Hz,), 116.50 (s, CH ₂ ), 115.84 (d, J _CP = 12.3 Hz, CH), 115.66 (d, J _CP = 2.5 Hz, CH), 53.44 (s, CH ₂ ), 35.77 (d, J _CP = 9.0 Hz, CH ₂ ). ³¹ P { ¹ H} NMR (161.70 MHz, CD ₂ Cl ₂ ): δ26.9. HRMS (APCI): m / z calcd.for C ₃₁ H ₃₄ N ₂ PS: 497.2175 ([M + H] ⁺ ); found. 497.2153.

Compound 7 (2.79 g, 5.62 mmol) was dissolved in CH ₂ Cl ₂ (56 mL) in the synthetic air of compound 8 . Chloranil (6.22 g, 25.3 mmol) was added at room temperature, and the mixture was stirred as it was for 8.5 hours. The solvent was distilled off under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (CH ₂ Cl ₂ , R _f = 0.45) to obtain 849 mg (1.66 mmol, yield 30%) of Compound 8 as a yellow liquid. The spectral data of Compound 8 is as follows.
¹ H NMR (400MHz, CD ₂ Cl ₂ ): δ 8.21 (dd, J = 9.0 Hz, 6.2 Hz, 2H), 7.60-7.54 (m, 2H), 7.36-7.30 (m, 5H), 6.88 (d, J = 9.0 Hz, 2H), 5.88-5.79 (m, 4H), 5.18-5.13 (m, 8H), 4.08-3.97 (m, 8H). ¹³ C { ¹ H} NMR (100MHz, CD ₂ Cl ₂ ) : δ 179.73 (d, J _CP = 8.3 Hz, C), 151.94 (d, J _CP = 14.1 Hz, C), 136.31 (d, J _CP = 83.5 Hz, C), 135.35 (d, J _CP = 80.9, C), 132.71 (s, CH), 131.35 (d, J _CP = 3.3 Hz, CH), 131. 20 (d, J _CP = 9.1 Hz, CH), 130.63 (d, J _CP = 11.5 Hz, CH) , 128.78 (d, J _CP = 12.4 Hz, CH), 124.30 (d, J _CP = 4.9 Hz, C), 117.10 (s, CH ₂ ), 115.27 (d, J _CP = 2.5 Hz, CH), 114.03 ( d, J _CP = 12.4 Hz, CH), 53.30 (s, CH ₂ ). ³¹ P { ¹ H} NMR (161.70 MHz, CD ₂ Cl ₂ ): δ 18.9. HRMS (APCI): m / z calcd. for C ₃₁ H ₃₁ N ₂ OPS: 510.1889 ([M] ⁺ ); found. 510.1870.

Synthetic compound 8 (800 mg, 1.57 mmol), 1,3-dimethylbarbituric acid (1.14 g, 7.30 mmol), tetrakis (triphenylphosphine) palladium (Pd (PPh ₃ ) ₄ ; 1.01 g, 0.874 mmol Was dissolved in degassed dehydrated 1,2-dichloroethane (16.0 mL), stirred at 85 ° C. for 3 days, and extracted with CH ₂ Cl ₂ . The combined organic layers were washed with saturated brine and then dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (CH ₂ Cl ₂ / methanol 30/1, R _f = 0.28) to obtain 312 mg (0.890 mmol, 57% yield) of compound 9 as a yellow solid. The spectral data of Compound 9 is as follows.
¹ H NMR (400 MHz, CD ₃ OD): δ8.13 (dd, J = 8.6 Hz, 5.8 Hz, 2H), 7.65-7.60 (m, 2H), 7.43-7.34 (m, 2H), 7.19 (dd , J = 17.2 Hz, 2.2 Hz, 2H), 6.85 (dd, J = 8.6 Hz, 2.2 Hz, 2H).
¹³ C { ¹ H} NMR (100 MHz, CD ₃ OD): δ 181.58 (d, J _CP = 8.3 Hz, C), 154.95 (d, J _CP = 14.9 Hz, C), 137.27 (d, J _CP = 80.1 Hz, C), 136.54 (d, J _CP = 83.4 Hz, C), 132.41 (d, J _CP = 3.3 Hz, CH), 132.25 (d, J _CP = 9.9 Hz, CH), 131.50 (d, J _CP = 10.7 Hz, CH), 129.58 (d, J _CP = 12.3 Hz, CH), 124.84 (d, J _CP = 4.9 Hz, C), 117.90 (d, J _CP = 2.5 Hz, CH), 116.40 (d _{, J CP = 10.7 Hz, CH} ) 31 P {1 H} NMR (161.70 MHz, CD 3 OD):.. δ 16.35 HRMS (APCI):. m / z calcd for C 19 H 16 N 2 OPS: 351.0715 ( [M + H] ⁺ ); found. 351.0725.

Compound 9 (101 mg, 0.288 mmol) was dissolved in sulfuric acid (0.83 mL) in the synthetic air of compounds 10 and 11 . Sodium nitrite (60.0 mg, 0.869 mmol) was added at 0 ° C., and the mixture was stirred as it was for 3 hours. The obtained reaction solution was added dropwise to an appropriate amount of ice and stirred at 110 ° C. for 30 minutes. The obtained solid was filtered off and the residue was washed with distilled water to obtain 87.0 mg of a crude product containing Compound 10 as a yellow solid. Compositional organisms obtained under a nitrogen atmosphere and imidazole (85.0 mg, 1.25 mmol) were dissolved in dehydrated CH ₂ Cl ₂ (21 mL) and stirred for 30 minutes. A solution in which t-butyldimethylchlorosilane (TBDMSCl; 230 mg, 1.52 mmol) was dissolved in dehydrated CH ₂ Cl ₂ (15 mL) was added dropwise to the solution containing Compound 10. After stirring for 4 hours, water was added and extracted with CH ₂ Cl ₂ . The combined organic layers were washed with saturated brine and then dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (CH ₂ Cl ₂ / hexane 3/2, R _f = 0.53) to give 98 mg (0.169 mmol, yield 59% (2 steps)) as a colorless liquid )Obtained. The spectral data of Compound 11 is as follows.
¹ H NMR (400MHz, CDCl ₃ ): δ8.34 (dd, J = 8.6 Hz, 5.8 Hz, 2H), 7.63-7.53 (m, 4H), 7.39-7.30 (m, 3H), 7.086 (ddd, J = 8.8 Hz, 2.5 Hz, 0.60 Hz, 2H), 0.95 (s, 18H), 0.23 (s, 6H), 0.22 (s, 6H). ³¹ P { ¹ H} NMR (161.70 MHz, CDCl ₃ ): δ17 .1.

Synthesis of Compound PSF Compound 11 (78.0 mg, 0.134 mmol) was dissolved in dehydrated THF (23 mL). A THF solution of 2-methylphenylmagnesium bromide (1.03 M, 0.50 mL, 0.515 mmol) was added dropwise at room temperature, and the mixture was stirred for 4 hours. 1N aqueous HCl (16 mL) was added and stirred for 2 h, and the reaction mixture was extracted with CH ₂ Cl ₂ . The combined organic layers were washed with saturated brine and then dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated by silica gel column chromatography (CHCl ₃ / methanol 5/1 (triethylamine 10%), R _f = 0.50) and then purified by HPLC to obtain 28 mg of a blue solid. The obtained solid was dissolved in toluene, placed in a separatory funnel, and extracted with 1N aqueous HCl. The combined organic layers were washed with saturated brine and then dried over anhydrous sodium sulfate. Sodium sulfate was filtered off, and the filtrate was concentrated under reduced pressure to obtain Compound PSF as a red-purple solid in 18 mg (0.0422 mmol, yield 32%). The spectral data of the compound PSF is as follows.
¹ H NMR (400MHz, CD ₂ Cl ₂ ): δ 7.73-7.60 (m, 4H), 7.54-7.35 (m, 16H), 7.13 (dd, J = 21 Hz, 7.4 Hz, 2H), 6.97-6.91 ( m, 4H), 6.56 (d, J = 9.2 Hz, 4H), 2.10 (s, 3H), 1.99 (s, 3H). (mixture of two rotamers) HRMS (APCI): m / z calcd. for C ₂₆ H ₂₀ O ₂ PS: 427.0916 ([M + H] ⁺ ); found. 427.0936.

[Test Example 1: LUMO and HOMO]
The phosphafluorescein compound obtained in Example 1 (position 10 is a phosphorus atom), a known fluorescein compound (position 10 is an oxygen atom; TokyoGreen; JACS, 2005, 127, 4888), and a known silicon-substituted fluorescein compound (position 10) For the silicon atom; TokyoMagenta; Chem. Commun. 2011, 47, 4162), the HOMO level and the LUMO level were calculated by structural optimization using the Gaussian 09 program. The calculation was performed at the B3LYP / 6-31 + G level. The results are shown in Table 1. As a result, it can be understood that the phosphafluorescein compound of the present invention can reduce LUMO and HOMO (particularly LUMO) as well as the energy gap as compared with conventional compounds. For this reason, it is expected that the stability to light is further improved.

[Test Example 2: X-ray crystal structure analysis]
About the phosphafluorescein compound obtained in Example 1 (position 10 is a phosphorus atom), a Rigaku Single Crystal CCD X-ray analyzer (FR-X microfocus generator, VariMax-Mo optical system, PILATUS 200K detector, Mo Kα irradiation (λ = 0.71070 ii)). 19043 reflections were measured in the 2θ angle range of 50 ° or less, of which 4694 were independent reflections (R _int = 0.0195). The structure was determined by the direct method (SIR-2003) and by the full matrix least squares method of F ² (SHELXL-2013). The crystal data is as follows. C ₂₆ H ₁₉ O ₃ P; FW = 410.38, crystal size 0.30 × 0.30 × 0.10 mm ³ , monoclinic, C2 / c, a = 16.295 (13) Å, b = 14.157 (18) Å, c = 18.637 (17) Å, β = 104.42 (2) °, V = 4164 (7) Å ³ , Z = 8, D _c = 1.309 g cm ^-3 , μ = 0.157 mm ^-1 , R ₁ = 0.0402 (I> 2σ (I) ), wR ₂ = 0.1095 (all data), GOF = 1.063. Cambridge Crystal Structure Database ID: CCDC 1435421. The results are shown in FIG.

[Test Example 3: Photophysical characteristics (1)]
The UV-vis absorption spectrum of the compound POF obtained in Example 1 was analyzed using a buffered aqueous solution containing 1% DMSO (pH = 3-5.5: citric acid and Na ₂ HPO ₄ aqueous solution, pH = 6-8: Na ₂ HPO). ₄ and aqueous NaH ₂ PO _4, pH = 9 ~ 11: with Na ₂ CO ₃ and the sample solution obtained by dissolving 10 ^-5 M aqueous NaHCO _3), by Shimadzu UV-3150 spectrometer, it was measured at a resolution 0.2 nm . The fluorescence spectrum was measured with a Hitachi F-4500 spectrometer with a resolution of 1 nm using a sample solution dissolved with 10 ⁻⁶ M. The excitation spectrum was measured with a Horiba SPEX Fluorolog 3 spectrofluorometer equipped with Hamamatsu PMA R5509-73 and cooling system C9940-01, using a sample solution dissolved with 10 ⁻⁶ M. The absolute fluorescence quantum yield was measured by Hamamatsu photonics PMA-11.

For example, when a pH 3 buffer aqueous solution is used, the phosphafluorescein compound obtained in Example 1 is dissolved in dimethyl sulfoxide (DMSO) as a solvent, and then the citric acid aqueous solution adjusted to pH 3 and Na ₂ HPO are used. A test solution in which the phosphafluorescein compound obtained in Example 1 was diluted to 7.4 × 10 ⁻⁶ M was prepared by diluting 100 times with a mixed aqueous solution of ₄ aqueous solutions (pH 3 test solution).

In the case of using a buffered aqueous solution of pH 7, after dissolving the phosphazene fluorescein compound obtained in Example 1 in dimethyl sulfoxide (DMSO), furthermore, Na ₂ HPO ₄ aqueous solution and Na ₂ HPO ₄ adjusted to pH 7 A test solution was prepared by diluting 100 times with a mixed aqueous solution of the aqueous solutions and dissolving the phosphafluorescein compound obtained in Example 1 so as to be 7.4 × 10 ⁻⁶ M (test solution of pH 7).

Furthermore, when using a pH 9 buffered aqueous solution, the phosphafluorescein compound obtained in Example 1 was dissolved in dimethyl sulfoxide (DMSO), and then a mixed aqueous solution of Na ₂ CO ₃ aqueous solution and NaHCO ₃ aqueous solution adjusted to pH 9 A test solution was prepared by diluting the phosphafluorescein compound obtained in Example 1 to 7.4 × 10 ⁻⁶ M (pH 9 test solution).

The results are shown in Figs. As a result, under the neutral or alkaline conditions, the absorption maximum wavelength and the fluorescence maximum wavelength can be made 600 to 700 nm, and the fluorescence quantum yield can be particularly improved. This is because, in the lower diagram of FIG. 3, the relative absorption at 627 nm increases with increasing pH and is almost the same in the neutral to alkaline region. In the lower diagram of FIG. 4, 627 nm with increasing pH. It can also be understood from the fact that the ratio of the excitation intensity at 532 nm to the excitation intensity at 532 nm is almost the same in the neutral to alkaline region. This behavior is considered to be due to the anionization of the phosphafluorescein compound of the present invention by increasing the pH.

[Test Example 4: Photophysical characteristics (2)]
The photophysical properties of test solutions 2 and 3 obtained in Test Example 3 above are reported values (TokyoGreen: JACS, 2005, 127, 4888, TokyoMagenda: Chem. Commun. 2011, 47, 4162, Nap-Fluorescein: Cytometry. 1989, 10, 151). The results are shown in Table 2. As a result, it can be understood that the phosphafluorescein compound of the present invention has the highest fluorescence quantum yield among compounds having an absorption maximum wavelength and a fluorescence maximum wavelength at 600 nm or more. In addition, the absorption maximum wavelength is maximum, and self-absorption of organelles and light damage to cells can be minimized in bioimaging.

[Test Example 5: Stability to light]
After dissolving the phosphafluorescein compound in dimethyl sulfoxide (DMSO), a mixed aqueous solution of Na ₂ HPO ₄ solution and Na ₂ HPO ₄ solution adjusted to pH 7 was added and diluted 100 times. A test solution 4 was obtained in which the obtained phosphafluorescein compound was dissolved to 7.4 × 10 ⁻⁶ M.

Apart from this, not the phosphafluorescein compound obtained in Example 1, but a known fluorescein compound (10-position oxygen atom; TokyoGreen; JACS, 2005, 127, 4888) and silicon-containing fluorescein compound (10-position silicon atom; Test solution 5 and test solution 6 were obtained in the same manner except that TokyoMagenta; Chem. Commun., 2010, 47, 4162.) was used.

The test solutions 4, 5 and 6 are irradiated with white light having a wavelength of 350 nm or more using a xenon lamp, and the absorption maximum wavelength (test solution 4: 627 nm; test solution 5: 491 nm; test solution 6: The absorbance maintenance rate at 582 nm was measured. The results are shown in FIG. In the figure, the upper line (along with POF) is the phosphafluorescein compound of the present invention, the lower line (along with TM) is a known silicon-containing fluorescein compound, and the line between them (along with TG) is known. It is a fluorescein compound.

In addition, the light stability of the phosphafluorescein compound was evaluated by the following method.

After the phosphafluorescein compound obtained in Example 1 and the known red fluorescent dyes Alexa Fluor (registered trademark) 633, Alexa Fluor (registered trademark) 647, and Cy5 were dissolved in dimethyl sulfoxide (DMSO), pH was diluted by adding a mixed aqueous solution of aqueous solution _of Na ₂ HPO ₄ and aqueous solution _of Na ₂ HPO ₄ adjusted to 7, by absorbance at 630 nm is adjusted to a concentration to be identical, the test solution 7, test solution 8. Test solution 9 and test solution 10 were obtained. Light for test solution 7, test solution 8, test solution 9, and test solution 10 with a 300 W xenon lamp (Asahi Spectroscopy MAX) fitted with a bandpass filter that transmits only light with a wavelength of 630 nm ± 10 nm Irradiation was performed, and the absorbance maintenance rate at 630 nm was measured. The results are shown in FIG. Phosphorfluorescein dye (POF) has higher light stability than Cy5, which is a well-known representative red fluorescent dye, and is known as Alexa Fluor (registered trademark) 633, which is known as a red fluorescent dye excellent in light stability. It was shown to have the same light stability as Alexa Fluor (registered trademark) 647.

[Test Example 6: Cell test]
Cell incubation and imaging HeLa cells (RIKEN Cell Bank, Japan) and RAW264.7 cells (JCRB Cell Bank, Japan), 10% fetal bovine serum (FBS, Gibco) and 1% antibiotic-antifungal (AA, Sigma) Incubated for 24 hours in an incubator with 5% CO ₂ /95% air at 37 ° C. in Dulbecco's modified Eagle's medium (DMEM, Sigma). Two days before imaging, HeLa cells and RAW264.7 cells were seeded in 35 mm glass bottom and 8-well glass bottom dishes, respectively. For staining experiments, HeLa cells were incubated for 10 minutes at 37 ° C. in 5 μM phosphate buffered saline (PBS) solution of the compound AcPOF of Example 2 and then rinsed twice with PBS. After changing the medium to DMEM without phenol red, cells were imaged with an excitation laser of 635 nm using an Olympus FV10i confocal fluorescence microscope. On the other hand, in the case of RAW264.7 cells, as the culture medium, the compound AcPOF of Example 2 is 5 μM, 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES; pH 7.4) is 10 mM, DMSO. Was used, and DMEM containing Pluronic F-127 0.02% was used. The cells were stained at 37 ° C. for 15 minutes, washed twice with DMEM, and each well was filled with a pH calibration buffer (Thermo Fisher Scientific) containing 10 μM valinomycin and 10 μM nigericin at pH 4.5 or 6.5. Cell images were obtained using TCS SP8 STED (Leica) equipped with HC PL APO 20 × / 0.75 IMM CORR CS2 lens. Cells were excited with a 627 nm (white light laser, 80 MHz, output power 70%, AOTF 50%) laser line, and fluorescence was recorded through filters at 485-527 nm and 635-750 nm.

The results are shown in FIG. From this result, it can be understood that the phosphafluorocein compound of the present invention does not permeate the nuclear membrane, but permeates the cell membrane and enters the cell, and can be used as a fluorescent dye of the cell (HeLa cell etc.). .

Cytotoxicity assessment HeLa cells were seeded in flat bottom 96-well plates (1 × 10 ⁴ cells / well) and cultured in DMEM containing 10% FBS at 37 ° C. in an incubator with 5% CO ₂ /95% air for 24 hours. . The medium was then replaced with medium having various concentrations of compound AcPOF of Example 2 (1 μM, 5 μM and 10 μM) and the cells were further incubated at 37 ° C. for 24 hours. After removing the medium, 3- (4,5-di-methylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) reagent is added to each well (final concentration 0.5 mg / mL) and the plate Was incubated for an additional 4 hours in a CO ₂ incubator. Excess MTT in tetrazolium was removed and the cells were washed once with PBS. Formazan crystals were solubilized in DMSO (100 μL / well) at room temperature for 30 minutes, and then the absorbance of each well was measured with SpectraMax i3 (Molecular Devices) at a wavelength of 535 nm.

The results are shown in FIG. In FIG. 7, the results represent the survival rate as a percentage relative to the case where no fluorescent dye was used. All data are shown by mean standard deviation (number of measurements n is 12). From this result, it can be understood that the phosphafluorocein compound of the present invention can cause cells to fluoresce within a range that can significantly reduce damage to the cells.

Claims (12)

1. General formula (1):
   

   

   [Wherein, R ¹ represents an optionally substituted aryl group. R ² represents a hydrogen atom or an organic group. R is the general formula (1A) to (1C):
   

   

   (In the formula, R ³ represents an optionally substituted aryl group, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group. R ⁴ represents a substituted group. Represents an alkyl group which may be substituted.)
   It is group represented by these. ]
   Or a salt, hydrate or solvate thereof.
2. The phosphafluorescein compound or a salt, hydrate or solvate thereof according to claim 1, wherein R is a group represented by the general formula (1A) in the general formula (1).
3. The salt of the phosphafluorescein compound represented by the general formula (1) is represented by the general formula (2):
   

   

   [Wherein, R ¹ and R are the same as defined above. ]
   The phosphafluorescein compound according to claim 1 or 2, or a salt, hydrate or solvate thereof, having an anion represented by:
4. The phosphafluorescein compound or a salt, hydrate or solvate thereof according to any one of claims 1 to 3, which has a maximum absorption wavelength at 600 to 700 nm.
5. The phosphafluorescein compound or a salt, hydrate or solvate thereof according to any one of claims 1 to 4, having a maximum absorption wavelength at 600 to 700 nm and a fluorescence quantum yield of 0.25 to 0.60. object.
6. A fluorescent dye comprising the phosphafluorescein compound according to any one of claims 1 to 5 or a salt, hydrate or solvate thereof.
7. The fluorescent dye according to claim 6, which is a fluorescent dye for bioimaging.
8. The fluorescent dye according to claim 7, which is a fluorescent dye for bioimaging of cancer cells.
9. A cell detection agent comprising the fluorescent dye according to any one of claims 6 to 8.
10. The cell detection agent according to claim 9, which is a cancer cell detection agent.
11. A cell bioimaging method using the fluorescent dye according to any one of claims 6 to 8 or the cell detection agent according to claim 9 or 10.
12. The bioimaging method according to claim 11, wherein the cell is a cancer cell.
    

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