JP6675758B2 - Phosphafluorescein compound or salt thereof, or fluorescent dye using the same - Google Patents

Description

  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, dyes having absorption and fluorescence maximum wavelengths in this region (620 nm or more) have attracted attention in deep observation of living tissue. In this deep observation of living tissue, in order to suppress auto-fluorescence of cells and to prevent damage to cells, it is necessary to reduce the fluorescence quantum yield while having a fluorescence maximum wavelength at a position of about 600 to 700 nm. Preferably, it is improved. In particular, among the water-soluble fluorescent dyes, dyes having a fluorescence maximum at 650 nm or more are not so many as compared with green or yellow phosphors (dyes having a fluorescence maximum at 495 to 570 nm). These include, for example, xanthene dyes expanded by π, silicon-substituted rhodamine (Si-rhodamine), fluorescein dyes, cyanine dyes (such as Cy5), and the like. Practical examples of any of the fluorescent dyes have been reported in visualizing biological samples.

  For example, a fluorescein-based 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, a dye having an absorption maximum and a fluorescence maximum at a wavelength of about 600 to 700 nm is required.

  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 a longer wavelength shift is required.

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

JACS, 2005, 127, 4888. Chem. Commun. 2011, 47, 4162. Cytometry, 1989, 10, 151.

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

  Therefore, the present invention provides a compound (fluorescent dye) that can obtain a sufficient quantum yield and sufficiently reduce HOMO while having an absorption maximum wavelength and a fluorescence maximum wavelength at 600 to 700 nm. ).

  In view of the above object, as a result of intensive studies, 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 site of fluorescein has been substituted with a phosphorus atom. This compound has an absorption maximum wavelength and a fluorescence maximum wavelength at 600 to 700 nm by anionization under neutral or alkaline conditions, but has sufficiently high fluorescence quantum yield under physiological conditions. Rate was obtained. In addition, since the HOMO level of this compound was lowered due to the electronic effect of the phosphorus substituent, a remarkable stabilizing effect on light irradiation was observed. The present invention has been completed by further research based on such findings. That is, the present invention includes the following configurations.

  Item 1. General formula (1):

[In the formula, R ¹ represents an optionally substituted aryl group. R ² represents a hydrogen atom or an organic group. R is a 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 Represents an alkyl group which may be substituted.)
Is a group represented by ]
Or a salt, hydrate or solvate thereof.

  Item 2. Item 2. The phosphafluorescein compound or a salt, hydrate or solvate thereof according to item 1, 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 above. ]
Item 3. The phosphafluorescein compound or a salt, hydrate or solvate thereof according to Item 1 or 2, having an anion represented by the formula:

  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 or a salt, hydrate or a phosphafluorescein compound according to any one of Items 1 to 4, which has a maximum absorption wavelength at 600 to 700 nm, and has a fluorescence quantum yield of 0.25 to 0.60. 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 bioimaging cancer cells.

  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 detecting agent according to Item 9, which is a cancer cell detecting agent.

  Item 11. Item 10. A bioimaging method for cells, 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 cells are cancer cells.

  The phosphafluorescein compound of the present invention or a salt, hydrate, or solvate thereof has the absorption maximum wavelength and the fluorescence maximum wavelength of 600 to 700 nm because the oxygen atom at the 10-position of the xanthene ring site of fluorescein is substituted with a phosphorus atom. And a sufficiently high fluorescence quantum yield can be obtained under physiological conditions. In particular, since the absorption maximum wavelength of 627 nm in the examples substantially coincides with the excitation wavelength of 633 nm of the HeNe laser normally provided in a laser microscope, 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 available in conventional fluorescent dyes having a maximum wavelength in this region, so that it is necessary for bioimaging. The fluorescent dye concentration can be kept low, and the damage to the living body can be greatly reduced.

  Furthermore, the phosphafluorescein compound of the present invention or a salt thereof can lower the HOMO level by the electronic effect of the phosphorus substituent, and can dramatically improve the stability to light. Observation can be performed over a long period of time.

2 is a result of X-ray crystal structure analysis of the phosphafluorescein compound (compound POF) obtained in Example 1. 9 shows an absorption spectrum and a fluorescence spectrum of the phosphafluorescein compound (compound POF) of the present invention when the pH was adjusted to pH 3, pH 7, and pH 9 prepared in Test Example 3. FIG. 2 is an absorption spectrum (upper figure) of the phosphafluorocein compound of the present invention (compound POF) at each pH, and a graph (lower figure) showing the relationship between relative absorption at 627 nm and pH. It is an excitation spectrum (upper figure) of the phosphafluorescein compound (compound POF) of the present invention at each pH, and a graph (lower figure) showing a relation between a ratio of an excitation intensity at 627 nm and an excitation intensity at 532 nm and pH. . 9 is a graph showing the results of Test Example 5. (A) is a graph showing the retention of absorbance 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 retention of absorbance at 630 nm of Test Solutions 7 to 10 prepared in Test Example 5. 9 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 diagram (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. 9 is a graph showing the results of cytotoxicity of Test Example 6.

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

[In the formula, R ¹ represents an optionally substituted aryl group. R ² represents a hydrogen atom or an organic group. R is a 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 Represents an alkyl group which may be substituted.)
Is a group represented by ]
Or a salt thereof. The phosphafluorescein compound represented by the general formula (1) or a salt thereof is a novel compound not described in the 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 that the aryl group represented by R ¹ may have is not particularly limited, and may be a hydroxyl group, a halogen atom (such as a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), or an alkyl group (a methyl group, an ethyl group). Group, n-propyl group, etc.), formyl group, carboxyl group, ester group, amide group (amide group, methylamide group, dimethylamide group, etc.), azide group (azidophenyl 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 from 0 to 6, more preferably from 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 facilitating recognition in bioimaging. A substituted monocyclic aryl group having a substituent at the position (substituted phenyl group) is more preferred, and an o-tolyl group is still more preferred.

In the general formula (1), as the organic group represented by R ² , an alkyl group which may have a substituent, an acyl group which may have a substituent, and a group which has a substituent Examples include a good cyclic ether group.

In the general formula (1), as the alkyl group as the organic group represented by R ² , any of a straight-chain alkyl group and a branched-chain alkyl group can be employed.

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

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

The substituent which the alkyl group as the organic group represented by R ² may have is not particularly limited, and examples thereof include a hydroxyl group, a halogen atom (such as a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom). . The number of such substituents is not particularly limited, and is preferably from 0 to 6, more preferably from 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 which the cyclic ether as the organic group represented by R ² may have is not particularly limited, and may be a hydroxyl group, a halogen atom (such as a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), or an acyl group ( Methanoyl group, ethanoyl group, etc.), silyl group (trimethylsilyl group, triethylsilyl group, etc.). Although the number of such substituents is not particularly limited, 0 to 2 is preferable, and 0 to 1 is more preferable.

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 easily anionizing and easily shifting the absorption maximum wavelength and the fluorescence maximum wavelength to long wavelengths.

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 (such as a naphthyl group and an anthryl group), a biphenyl group, a terphenyl group, a pyrenyl group, a phenanthrenyl group, and a fluorenyl group.

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

In the general formula (1) (general formulas (1A) to (1C)), among the groups represented by R, the alkyl group represented by R ³ may be any of a straight-chain alkyl group and a branched-chain alkyl group. .

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

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

The substituent which the alkyl group as the organic group represented by R ² may have is not particularly limited, and examples thereof include a hydroxyl group, a halogen atom (such as a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom). . The number of such substituents is not particularly limited, but is preferably 0 to 6, and 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) (the general formula (1A) ~ (1C)), among the groups represented by R, the alkynyl group represented by R ^3, for example, an alkynyl group and a propargyl group.

Depending on the type of R ^3, tweaks and physical properties (solubility, cell permeability, etc.) of the electronic structure it is also possible to adjust the.

In the general formula (1) (the general formula (1C)), among the groups represented by R, the alkyl group represented by R ^4, may employ either straight-chain alkyl groups and branched chain alkyl groups.

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

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

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

  Above all, R has a higher electron-withdrawing property and is more likely to reduce the HOMO level (energy level of the highest occupied orbital) and the LUMO level (energy level of the lowest unoccupied orbital). From the viewpoint of easily improving the stability, a group represented by the general formula (1A) is preferable.

  As the compound of the present invention satisfying such conditions, for example,

[In the formula, Ph represents a phenyl group. The same applies hereinafter. ]
And the like, or a salt, hydrate or solvate thereof.

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

  In addition, 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 of the compound represented by the general formula (1) is anionized, the absorption maximum wavelength is shifted to a longer wavelength within a range of 600 to 700 nm (particularly, about 600 to 650 nm). It is also possible to further increase the fluorescence quantum yield (about 0.25 to 0.60, especially 0.35 to 0.50) by further shifting the fluorescence maximum wavelength to a longer wavelength (about 650 to 700 nm, particularly about 655 to 670 nm).

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

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

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

  The ion (cation) paired with the anion represented by the general formula (2) is not particularly limited, and examples thereof include metal salts such as sodium salts, potassium salts, calcium salts, and magnesium salts as base addition salts; ammonium salts Organic ammonium salts such as triethylammonium.

  Further, the compound of the present invention may exist as a hydrate or a solvate, and these substances are all included in the scope of the present invention.

2． Method for producing phosphafluorescein compound or salt thereof, hydrate or solvate The phosphafluorescein compound of the present invention or a salt, hydrate or solvate thereof is not particularly limited, for example, a silicon-substituted fluorescein compound. It can be synthesized according to the method reported in the previous report (Chem. Commun. 2011, 47, 4162.) (similarly except that the starting compounds are changed).

  Specifically, the following reaction formula 1:

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

In Reaction Scheme 1, the protecting groups represented by R ⁵ and R ⁶ are not particularly limited as long as they can protect a hydroxyl group, and any of them can be used. For example, alkanoyl groups (formyl group, An alkanoyl group having 1 to 4 carbon atoms such as acetyl group and propionyl), an aralkyl group which may be substituted (benzyl group, p-methoxybenzyl group, p-nitrobenzyl group, etc.), silyl group (trimethylsilyl group, triethylsilyl) 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 and the like are more preferable, from the viewpoint of protecting the hydroxyl group and facilitating the progress of the nucleophilic addition reaction.

In Reaction Formula 1, the halogen atom represented by X ¹ and X ² can be any of a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, but from the viewpoint of the ease 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 it is intended to obtain a compound having a group represented by the general formula (1A) as R, instead of using a silicon compound as a raw material, 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, in an organic solvent, after reacting the compound (3) in the above reaction formula 1 with an organolithium compound, an organic phosphorus compound is added, and then hydrogen peroxide solution is added. The desired cyclization reaction can proceed. When it is intended to obtain 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. Is preferred.

  In addition, as the compound (3) in the above Reaction Scheme 1, a known or commercially available compound can be used, or it 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 with allyl halide and formaldehyde.

  The organic lithium compound is not particularly limited, and known compounds can be employed.Examples include ethyl lithium, n-propyl lithium, isopropyl lithium, n-butyl lithium, s-butyl lithium, t-butyl lithium, pentyl lithium, and hexyl. Alkyl lithium such as lithium; cycloalkyl lithium such as cyclohexyl lithium; aryl lithium such as phenyl lithium and the like. Of these, in the present step, from the viewpoint of yield, alkyl lithium is preferable, and n-butyl lithium is more preferable.

  The organic phosphorus 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 employed.For example, dichlorophenylphosphine, dihalogenated arylphosphine such as dibromophenylphosphine, etc. Can be used.

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

  As the organic solvent that can be used in this step, a known organic solvent can be employed. 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, at -150 to 0 ° C, particularly at -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, in an organic solvent, an oxidation reaction can be caused to the compound (4) in the above reaction formula 1 by using an oxidizing agent.

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

  The amount of the oxidizing agent to be used is not particularly limited, and is preferably 1 to 10 mol (particularly 2 to 5 mol) per 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 organic solvent may be employed. In this step, for example, cyclic ethers such as tetrahydrofuran and dioxane are preferable. Note that the same solvent as in the cyclization step can be used. The reaction conditions may be such that the reaction proceeds sufficiently, and may be, for example, -50 to 100 ° C, particularly 0 to 50 ° C, for 1 to 48 hours, particularly 2 to 24 hours.

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

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

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

(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 with respect to the compound (6) in the above reaction formula 1 in an organic solvent.

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

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

  As the organic solvent that can be used in this step, a known organic solvent can be used. In this step, for example, cyclic ethers such as tetrahydrofuran and dioxane are preferable. Note that the same solvent as in the cyclization step can be used. The reaction conditions can be such that the reaction proceeds sufficiently, for example, at 50 to 200 ° C., particularly 100 to 150 ° C., for 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. As the method and conditions for protection, any of conventionally known methods and conditions can be adopted.

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

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

The halogen atom represented by X ^3, can be adopted as described above. The same applies to preferred specific examples.

  Grignard reagents satisfying such conditions include, for example,

And the like.

  The oxidizing agent is not particularly limited, and hydrogen chloride (hydrochloric acid) or the like can be used.

  The amounts of the Grignard reagent and the oxidizing agent are not particularly limited, and from the viewpoint of yield and the like, 1 to 10 mol (particularly 2 to 5 mol) of the Grignard reagent is used per 1 mol of the 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, a known organic solvent can be used. In this step, for example, cyclic ethers such as tetrahydrofuran and dioxane are preferable. Note that the same solvent as in the cyclization step can be used. The reaction conditions may be such that the reaction proceeds sufficiently, and may be, for example, -50 to 100 ° C, particularly 0 to 50 ° C, for 30 minutes to 10 hours, particularly 1 to 5 hours.

  Thus, the phosphafluorescein compound represented by the general formula (1) can be obtained, and if necessary, can be used through ordinary isolation and purification steps. The phosphafluorescein compound represented by the general formula (1) is a neutral compound under acidic conditions (for example, pH 1 to 5), but under neutral conditions or alkaline conditions (for example, pH 6 to 14). Thus, the compound can be easily converted to a salt having an anion represented by the general formula (2), and can be used through ordinary isolation and purification steps as necessary. Thereby, the absorption maximum wavelength is shifted to a longer wavelength range of 600 to 700 nm (particularly about 600 to 650 nm), and the fluorescence maximum wavelength is further shifted to a longer wavelength (about 650 to 700 nm, especially 655 to 650 nm). (About 670 nm), and the fluorescence quantum yield can be further improved (about 0.25 to 0.60, particularly 0.35 to 0.50).

  Note that, in the above, an example of a synthesis method of one embodiment of the phosphafluorescein compound of the present invention has been described. However, the present invention is not limited to this production method, and can be synthesized by various synthesis methods. Further, other phosphafluorescein compounds can be synthesized by the same method.

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

Since the fluorescent dye of the present invention has replaced the oxygen atom at the 10-position of the xanthene ring site of fluorescein with a phosphorus atom, it has an absorption maximum wavelength and a fluorescence maximum wavelength at 600 to 700 nm, but still has sufficient under physiological conditions. A high fluorescence quantum yield is obtained. In particular, since the absorption maximum wavelength of 627 nm in the examples almost coincides with the excitation wavelength of 633 nm of the HeNe laser usually provided with a laser microscope, the excitation efficiency as a fluorescent dye is extremely high. In particular, phosphazene fluorescein compound or a salt thereof of the present invention, together with high permeability into cells, is introduced the desired substituent as R ^1, cancer cells such as the desired cells (e.g., HeLa cells in vivo ) Can be selectively localized. That is, it is possible to emit light only from desired cells (for example, cancer cells such as HeLa cells). In particular, the phosphafluorescein compound of the present invention or a salt thereof has a maximum fluorescence wavelength and a maximum fluorescence wavelength in the range of 600 to 700 nm, but has a high fluorescence quantum not found in conventional fluorescent dyes having a maximum wavelength in this region. The high yield facilitates bioimaging of desired cells (e.g., cancer cells such as HeLa cells), and reduces the concentration of the fluorescent dye required for bioimaging, thereby damaging 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. Observation can be performed over a long period of 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, but is 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 rendering the desired cells (for example, cancer cells such as HeLa cells) in a more selective manner (visualization in real time), the host of the present invention can be used. The content of the fluorescein compound is preferably from 1 × 10 ⁻⁸ to 1 × 10 ⁻⁴ mol / L, more preferably from 1 × 10 ⁻⁷ to 1 × 10 ⁻⁵ mol / L. Thus, in the present invention, the content of the phosphafluorescein compound can be suppressed lower than that of the conventional fluorescent dye.

  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 either a polar solvent or a nonpolar solvent can be used. it can.

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

  Examples of the non-polar solvent include aliphatic organic solvents such as pentane, hexane, cyclohexane, and heptane; and 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 maximum absorption wavelength and a maximum fluorescence wavelength of 600 to 700 nm, but has a sufficiently higher fluorescence quantum yield under physiological conditions. From the viewpoint of introduction into cells, the pH is preferably about 5 to 11, and more preferably about 6.5 to 7.5. In order to adjust the pH of the cell detection agent of the present invention, a buffer (Hepes buffer, Tris buffer, Tricine-sodium hydroxide buffer, phosphate buffer, phosphate buffered saline, etc.) or the like is used. May be.

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

  [Example 1]

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 obtained from JEOL AL-400 spectrometer (400 MHz for ¹ H, 100 MHz for ¹³ C and 161.70 MHz for ³¹ P), JEOL JNM-ECS400 ( Using 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), use CDCl ₃ , CD ₂ Cl ₂ or 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 signal of the solvent as an internal standard (CDCl ₃ δ 77.16, CD ₂ Cl ₂ δ 53.84, CD ₃ OD δ 49.0). As the chemical shift value of the ³¹ P NMR spectrum, the signal of H ₃ PO ₄ (δ0.00) was used as an external standard. The mass spectrum was measured by an electrospray ionization method (ESI) using an atmospheric pressure chemical ionization method (APCI) or a Thermo Fisher Scientific Exactive using a Bruker micrOTOF Focus spectrometry system. Thin layer chromatography (TLC) was performed using glass plates 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 recycle HPLC was performed using a YMC LC-forte / R equipped with a reversed phase column (YMC-Actus Triart C18). Unless otherwise noted, all reactions were performed under a nitrogen atmosphere. Unless otherwise noted, solvents and reagents were used without purification from 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 previously 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). At -78 ° C, a pentane solution of t-butyllithium (1.63 M, 4.80 mL, 7.82 mmol) was added dropwise over 20 minutes, and the mixture was stirred for 1 hour. Dichlorophenylphosphine (PhPCl ₂ ; 0.318 mL, 2.34 mmol) was added dropwise over 25 minutes, 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 for 1 hour. An aqueous sodium hypochlorite solution and a saturated aqueous sodium sulfite solution were added to the reaction mixture, and the mixture was extracted with CH ₂ Cl ₂ . The combined organic layer was washed with a saturated saline solution, and then dried with dehydrated sodium sulfate. The sodium sulfate was filtered off, 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, yield 81%) of compound 2 as a yellow liquid. The spectrum data of compound 2 are as follows.
¹ H NMR (400 MHz, 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 = 18 Hz, 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 (161.70 MHz, 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. To a solution of bis [2-bromo-4- (N, N-diallylamino) phenyl] methane (compound 1) (0.978 g, 1.89 mmol) in dehydrated THF (9 mL) was added s-butyl at -78 ° C. A solution of 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 heated to 0 ° C., and a 30% H ₂ O ₂ solution (1.0 mL) was added. The mixture was stirred for 1 hour and quenched with aqueous sodium sulfite. Thereafter, the resulting solution was extracted with ethyl acetate (AcOEt). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and filtered. The volatile substance was removed under reduced pressure, and the obtained solid was purified by silica gel column chromatography (CH ₂ Cl ₂ / methanol 20/1, Rf = 0.4) to give Compound 2 as a yellow liquid in an amount of 0.257 g (0.525 mmol). , Yield 28%).

Compound 2 (761 mg, 1.58 mmol) was dissolved in CH ₂ Cl ₂ (16 mL) in a synthetic air of compound 3 and compound 4 . Chloranil (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, yield 39%) of compound 3 as a yellow liquid. The spectrum data of compound 3 are as follows.
¹ H NMR (400 MHz, CDCl ₃ ): δ 8.27 (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.

Tris (dibenzylideneacetone) dipalladium (Pd ₂ (dba) ₃ ; 50.4 mg, 0.055 mmol), 1,4-bis (diphenylphosphino) butane (464 mg, 1.08 mmol) degassed dehydrated 1,2- Dissolved in dichloroethane (1.0 mL) and stirred at room temperature for 1 hour. The obtained solution was prepared by dissolving Compound 3 (71.0 mg, 0.144 mmol) and 1,3-dimethylbarbituric acid (265 mg, 1.70 mmol) in degassed dehydrated 1,2-dichloroethane (0.4 mL). After stirring at 80 ° C. for 2 days, the mixture was extracted with CH ₂ Cl ₂ . The combined organic layer was washed with a saturated saline solution, and then dried with dehydrated sodium sulfate. The sodium sulfate was filtered off, 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 spectrum data of compound 4 are as follows.
Mp: 151 ° C. ¹ H NMR (400 MHz, 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 (100MHz, 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 (161.70 MHz, 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 obtained by the following method. Compound 2 (5.37 g, 11.2 mmol) against CH ₂ Cl ₂ (112 mL) solution of was added chloranil (8.26 g, 33.6 mmol) and in air. The mixture was stirred for 3 hours and quenched with aqueous sodium sulfite. Thereafter, the resulting solution was extracted with CH ₂ Cl _2. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and filtered. The volatile substance was removed under reduced pressure, and the obtained solid was purified by silica gel column chromatography (CH ₂ Cl ₂ / methanol 20/1, Rf = 0.4) to obtain 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 solution of compound 3 (4.39 g) in 1,2-dichloroethane (370 mL) was added 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. To progress 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 resulting mixture. After stirring at 80 ° C. for a further 23 hours, 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 synthesis 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 the mixture was stirred at 110 ° C for 0.5 hour. The obtained solid was separated by filtration, 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 give 28.0 mg (0.832 mmol, yield 54%) of compound 5 as a yellow solid. The spectrum data of compound 5 are as follows.
¹ H NMR (400 MHz, CD ₃ OD): δ 8.33 (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 (161.70 MHz, 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 obtained by the following method. To a solution of compound 4 (247 mg, 0.739 mmol) in 96% sulfuric acid (2.5 mL) in air was added NaNO ₂ (171 mg, 2.48 mmol) at 0 ° C. in air. After stirring for 3 hours, the mixture was slowly dropped onto ice, and stirred at 110 ° C. for 0.5 hour. The resulting precipitate was collected by filtration, washed with distilled water, and dispersed in methanol. The resulting 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.

Compound 6 (195 mg, 0.580 mmol) of Compound 6 and imidazole (197 mg, 2.89 mmol) were dissolved in dehydrated CH ₂ Cl ₂ (50 mL) and stirred for 15 minutes. A solution of t-butylchlorodimethylsilane (505 mg, 3.35 mmol) 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 layer was washed with a saturated saline solution, and then dried with dehydrated sodium sulfate. The sodium sulfate was filtered off, 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, yield 81%) of compound 6 as a colorless liquid. . The spectrum data of compound 6 are as follows.
¹ H NMR (400 MHz, CDCl ₃ ): δ 8.36 (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 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 Cl ₂ (7 mL) solution 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 the 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 give 354 mg of compound 6 as a colorless liquid. (0.627 mmol, 92% yield).

Compound POF Synthesis Compound 6 (230 mg, 0.407 mmol) was dissolved in dehydrated THF (70 mL). At room temperature, a THF solution of 2-methylphenylmagnesium bromide (1.03 M, 1.43 mL, 1.47 mmol) was added dropwise, and the mixture was stirred for 4 hours. 1N HCl aqueous solution (50 mL) was added, stirred for 2 hours, and the reaction mixture was extracted with CH ₂ Cl ₂ . The combined organic layer was washed with a saturated saline solution, and then dried with dehydrated sodium sulfate. The sodium sulfate was filtered off, 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 then purified by HPLC to obtain 55 mg of a blue solid. The obtained solid was dissolved in toluene, placed in a separatory funnel, 1N HCl aqueous solution was added, and the separatory funnel was shaken. The combined organic layer was washed with a saturated saline solution, and then dried with dehydrated sodium sulfate. The sodium sulfate was separated by filtration, and the filtrate was concentrated under reduced pressure to obtain 30 mg (0.0730 mmol, 18% yield) of the compound POF as a red solid. The spectrum data of compound POF are 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, compound POF was obtained by the following method. To a solution of 2-bromotoluene (89 μL, 0.74 mmol) in dehydrated THF (3 mL) was added a solution of s-BuLi in cyclohexane and hexane (0.99 M, 0.90 mL, 0.89 mmol) 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 dry THF (3 mL) was added slowly. The temperature was raised to room temperature, and the reaction mixture was stirred for 1.5 hours. Thereafter, the reaction was quenched by the addition of 0.5 M hydrochloric acid (20 mL). After vigorous stirring for 45 min, 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, the volatiles were removed under reduced pressure, and the resulting mixture was dispersed in _{CH 2 Cl 2 (10 mL)} . To the obtained yellow liquid, p-toluenesulfonic acid monohydrate (47 mg, 0.25 mmol) was added, and the obtained mixture was stirred at room temperature for 45 minutes to complete dehydroxylation. The resulting 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 was obtained which solidified simultaneously with the addition of toluene. 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. Thereafter, the compound POF was purified as a brown powder by purifying with reverse phase HPLC (eluent: CH ₃ CN / H ₂ O mixed solvent containing 5 mM ammonium carbonate at a mixing ratio of 20/80 to 100/0) to give 34 mg of brown powder. (0.083 mmol, 34% yield).

  [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), and then further purified by recrystallization from CH ₂ Cl ₂ / hexane. Then, 33 mg (0.073 mmol, yield 81%) of the compound AcPOF was obtained as a yellow powder. The spectrum data of the compound AcPOF are 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). At −78 ° C., a pentane solution of t-butyllithium (1.61 M, 27.0 mL, 43.5 mmol) was added dropwise over 35 minutes, and the mixture was stirred for 2 hours. Dichlorophenylphosphine (PhPCl ₂ ; 1.80 mL, 13.2 mmol) was added dropwise over 90 minutes, and after the addition was completed, stirring was performed overnight. S ₈ (705 mg) was added, and the mixture was stirred for 30 minutes. The reaction solvent was concentrated under reduced pressure, and the resulting mixture was extracted with dichloromethane. The combined organic layer was washed with a saturated saline solution, and then dried with dehydrated sodium sulfate. The sodium sulfate was filtered off, 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 obtain 2.84 mg (5.72 mmol, yield 52%) of compound 7 as a yellow liquid. . The spectrum data of compound 7 are as follows.
¹ H NMR (400 MHz, 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 synthesis air of compound 8 . Chloranil (6.22 g, 25.3 mmol) was added at room temperature, and the mixture was stirred 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 spectrum data of compound 8 are as follows.
¹ H NMR (400 MHz, 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 of compound 9 (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 layer was washed with a saturated saline solution, and then dried with dehydrated sodium sulfate. The sodium sulfate was filtered off, 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, yield 57%) of compound 9 as a yellow solid. The spectrum data of compound 9 are as follows.
¹ H NMR (400 MHz, CD ₃ OD): δ 8.13 (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 for 3 hours. The obtained reaction solution was added dropwise to an appropriate amount of ice, and the mixture was stirred at 110 ° C for 30 minutes. The obtained solid was separated by filtration, and the residue was washed with distilled water to obtain 87.0 mg of a crude product containing Compound 10 as a yellow solid. Under a nitrogen atmosphere, the resulting composition 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 of t-butyldimethylchlorosilane (TBDMSCl; 230 mg, 1.52 mmol) 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 layer was washed with a saturated saline solution, and then dried with dehydrated sodium sulfate. The sodium sulfate was filtered off, 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 of compound 11 as a colorless liquid (0.169 mmol, 59% yield (two steps)). )Obtained. The spectrum data of the compound 11 are as follows.
¹ H NMR (400 MHz, CDCl ₃ ): δ 8.34 (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.

Compound PSF Synthesis Compound 11 (78.0 mg, 0.134 mmol) was dissolved in dehydrated THF (23 mL). At room temperature, a THF solution of 2-methylphenylmagnesium bromide (1.03 M, 0.50 mL, 0.515 mmol) was added dropwise, and the mixture was stirred for 4 hours. 1N HCl aqueous solution (16 mL) was added, the mixture was stirred for 2 hours, and the reaction mixture was extracted with CH ₂ Cl ₂ . The combined organic layer was washed with a saturated saline solution, and then dried with dehydrated sodium sulfate. The sodium sulfate was filtered off, 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 HCl aqueous solution. The combined organic layers were washed with a saturated saline solution and dried over anhydrous sodium sulfate. The sodium sulfate was filtered off, and the filtrate was concentrated under reduced pressure to obtain 18 mg (0.0422 mmol, yield 32%) of a compound PSF as a red-purple solid. The spectrum data of compound PSF are as follows.
¹ H NMR (400 MHz, 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 (10-position is a phosphorus atom) obtained in Example 1, a known fluorescein compound (an oxygen atom at 10-position; TokyoGreen; JACS, 2005, 127, 4888), and a known silicon-substituted fluorescein compound (10-position HOMO and LUMO levels of silicon atoms; TokyoMagenta; Chem. Commun. 2011, 47, 4162) were calculated by structural optimization using the Gaussian 09 program. Calculations were performed at the B3LYP / 6-31 + G level. Table 1 shows the results. As a result, it can be understood that the phosphafluorescein compound of the present invention can reduce LUMO and HOMO (particularly LUMO) and also reduce the energy gap as compared with the conventional compound. Therefore, it is expected that the stability to light is further improved.

[Test Example 2: X-ray crystal structure analysis]
For the phosphafluorescein compound (phosphorus atom at position 10) obtained in Example 1, 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 Å)). 19043 reflections were measured in the range where the 2θ angle was 50 ° or less, of which 4694 were independent reflections (R _int = 0.0195). Structure was determined by direct methods (SIR-2003), were determined by full matrix least squares 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 properties (1)]
The UV-vis absorption spectrum of the compound POF obtained in Example 1 was measured using an aqueous buffer solution containing 1% DMSO (pH = 3 to 5.5: citric acid and Na ₂ HPO ₄ aqueous solution, pH = 6 to 8: Na ₂ HPO ₄ and NaH ₂ PO ₄ aqueous solution, pH = 9 to 11: Na ₂ CO ₃ and NaHCO ₃ aqueous solution) were measured with a Shimadzu UV-3150 spectrometer at a resolution of 0.2 nm using a sample solution in which 10 ⁻⁵ M was dissolved. . The fluorescence spectrum was measured with a Hitachi F-4500 spectrometer having a resolution of 1 nm using a sample solution in which 10 ⁻⁶ M was dissolved. The excitation spectrum was measured with a Horiba SPEX Fluorolog 3 spectrofluorometer equipped with Hamamatsu PMA R5509-73 and a cooling system C9940-01, using a sample solution in which 10 ^-6 M was dissolved. Absolute fluorescence quantum yield was measured with Hamamatsu photonics PMA-11.

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

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 the phosphafluorescein compound obtained in Example 1 at a concentration of 7.4 × 10 ⁻⁶ M by diluting the aqueous solution 100-fold with a mixed aqueous solution (pH 7 test solution).

Furthermore, when a buffered aqueous solution of pH 9 is used, the phosphafluorescein compound obtained in Example 1 is dissolved in dimethyl sulfoxide (DMSO), and then a mixed aqueous solution of an aqueous solution of Na ₂ CO _{3 and an} aqueous solution of NaHCO ₃ adjusted to pH 9 Was diluted 100 times to prepare a test solution in which the phosphafluorescein compound obtained in Example 1 was dissolved to a concentration of 7.4 × 10 ⁻⁶ M (test solution at pH 9).

  The results are shown in FIGS. As a result, under neutral or alkaline conditions, the maximum absorption wavelength and the maximum fluorescence wavelength could be set to 600 to 700 nm, and the fluorescence quantum yield could be particularly improved. This is because the relative absorption at 627 nm increases with increasing pH in the lower part of FIG. 3 and is almost the same in the neutral to alkaline region, and the lower part of FIG. 4 shows that the relative absorption at 627 nm increases 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 increases and is almost the same in the neutral to alkaline region. This behavior is considered to be due to the fact that the phosphafluorescein compound of the present invention is anionized by increasing the pH.

[Test Example 4: Photophysical properties (Part 2)]
The photophysical properties of the test liquids 2 and 3 obtained in the above Test Example 3 were measured using reported values (Tokyo Green: JACS, 2005, 127, 4888, Tokyo Magenda: Chem. Commun. 2011, 47, 4162, Nap-Fluorescein: Cytometry). , 1989, 10, 151). Table 2 shows the results. 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. Further, the maximum absorption wavelength is maximum, and in bioimaging, self-absorption of organelles and damage to cells by light can be minimized.

[Test Example 5: Stability to light]
After dissolving the phosphazene fluorescein compound in dimethyl sulfoxide (DMSO), and diluted 100-fold by adding a mixed aqueous solution of aqueous solution _of Na ₂ HPO ₄ and aqueous solution _of Na ₂ HPO ₄ adjusted to pH 7, in Example 1 Test solution 4 was obtained in which the obtained phosphafluorescein compound was dissolved to a concentration of 7.4 × 10 ⁻⁶ M.

  Apart from this, instead of the phosphafluorescein compound obtained in Example 1, a known fluorescein compound (oxygen atom at position 10; Tokyo Green; JACS, 2005, 127, 4888) and a silicon-containing fluorescein compound (silicon atom at position 10; 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 liquids 4, 5 and 6 were irradiated with white light having a wavelength of 350 nm or more using a xenon lamp, and the absorption maximum wavelength (test liquid 4: 627 nm; test liquid 5: 491 nm; test liquid 6: The retention of absorbance at 582 nm) was measured. The results are shown in FIG. In the figure, the upper line (also described as POF) is a phosphafluorescein compound of the present invention, the lower line (also described as TM) is a known silicon-containing fluorescein compound, and the line between these (also described as TG) is a known compound. It is a fluorescein compound.

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

After dissolving 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 in dimethyl sulfoxide (DMSO), 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 liquid 9 and test liquid 10 were obtained. For test solution 7, test solution 8, test solution 9 and test solution 10, light was applied to a 300 W xenon lamp (Asahi Spectroscopy MAX) with a bandpass filter that transmits only light with a wavelength of 630 nm ± 10 nm. Irradiation was performed, and the retention of absorbance at 630 nm was measured. The results are shown in FIG. Phosphafluorescein dye (POF) has higher photostability than Cy5, which is a known representative red fluorescent dye, and is known as Alexa Fluor (registered trademark) 633 and a red fluorescent dye having excellent light stability. It was shown to have comparable photostability to Alexa Fluor® 647.

[Test Example 6: Cell test]
Cell incubation and imaging
HeLa cells (RIKEN Cell Bank, Japan) and RAW264.7 cells (JCRB Cell Bank, Japan) were subjected to Dulbecco's modified with 10% fetal bovine serum (FBS, Gibco) and 1% antibiotic-antifungal (AA, Sigma). Incubated in Eagle's medium (DMEM, Sigma) at 37 ° C. in a 5% CO ₂ /95% air incubator for 24 hours. 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 in a 5 μM solution of the compound AcPOF of Example 2 in phosphate buffered saline (PBS) at 37 ° C. for 10 minutes, and then rinsed twice with PBS. After replacing the medium with DMEM containing no 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 a culture medium, 5 μM of the compound AcPOF of Example 2, 10 mM of 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES; pH 7.4), DMSO And 1% of Pluronic F-127 and DMEM containing 0.02% of Pluronic F-127 were used. After the cells were stained at 37 ° C. for 15 minutes and washed twice with DMEM, each well was filled with a pH calibration buffer (Thermo Fisher Scientific) containing 10 μM of valinomycin and 10 μM of nigericin and having a pH of 4.5 or 6.5. Cell images were obtained using a TCS SP8 STED (Leica) equipped with HC PL APO 20 × / 0.75 IMM CORR CS2 lens. Cells were excited with a laser line at 627 nm (white light laser, 80 MHz, output power 70%, AOTF 50%), and fluorescence was recorded through filters 485-527 nm and 635-750 nm.

  FIG. 6 shows the results. From these results, it can be understood that the phosphafluorocein compound of the present invention can penetrate the cell membrane, penetrate into cells, and be used as a fluorescent dye for cells (HeLa cells, etc.), though the nuclear membrane did not permeate. .

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

FIG. 7 shows the results. In FIG. 7, the results show the survival rate as a percentage when no fluorescent dye was used. All data are expressed as the mean standard deviation (the number of measurements n is 12). From these results, it can be understood that the phosphafluorescein compound of the present invention can cause cells to fluoresce within a range that can significantly reduce damage to the cells.

Claims (13)

General formula (1):

[In the formula, R ¹ represents an optionally substituted aryl group. R ² represents a hydrogen atom , an alkyl group which may have a substituent, an acyl group which may have a substituent, or a cyclic ether group which may have a substituent . R is a general formula (1A) to (1B):

(Wherein, R ³ is an optionally substituted aryl group, an optionally substituted alkenyl group,
Or an alkynyl group which may be substituted. )
Is a group represented by ]
Or a salt, hydrate or solvate thereof. General formula (1):

[In the formula, R ¹ represents an optionally substituted aryl group. R ² represents a hydrogen atom , an alkyl group which may have a substituent, an acyl group which may have a substituent, or a cyclic ether group which may have a substituent . R is a general formula (1A) to (1C):

(In the formula, R ³ represents an aryl group which may be substituted. R ⁴ represents an alkyl group which may be substituted.)
Is a group represented by ]
Or a salt, hydrate or solvate thereof. The phosphafluorescein compound according to claim 1 or 2 , wherein R is a group represented by the general formula (1A) in the general formula (1), or a salt, hydrate or solvate thereof. 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 above. ]
The phosphafluorescein compound or a salt, hydrate or solvate thereof according to any one of claims 1 to 3 , which has an anion represented by the following formula: Claim 1 phosphazene fluorescein compound or a salt thereof according to any one of 4, a fluorescent dye containing a hydrate or solvate. The fluorescent dye according to claim 5 , which is a fluorescent dye for bioimaging. The fluorescent dye according to claim 6 , which is a fluorescent dye for bioimaging cancer cells. A cell detection agent comprising the fluorescent dye according to any one of claims 5 to 7 . General formula (1):

[In the formula, R ¹ represents an optionally substituted aryl group. R ² represents a hydrogen atom , an alkyl group which may have a substituent, an acyl group which may have a substituent, or a cyclic ether group which may have a substituent . R is a general formula ( 1A) to (1C):

(Wherein, 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 is substituted Represents an alkyl group which may be substituted.)
Is a group represented by ]
A cell detection agent comprising a phosphafluorescein compound represented by the formula: or a salt, hydrate or solvate thereof. The cell detection agent according to claim 8 or 9 , which is a cancer cell detection agent. The cell detecting agent according to any one of claims 8 to 10 , which is a real-time visualizing agent for cells. A method for bioimaging cells using the fluorescent dye according to any one of claims 5 to 7 or the cell detection agent according to any one of claims 8 to 11 . The bioimaging method according to claim 12 , wherein the cells are cancer cells.

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