JP2013245290A - Fluorescent dyestuff, fluorescent reagent, method for diagnosing affected part of living body, method for identifying target cell and method for detecting target substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescent dyestuff excellent in quantum yield whether a fluorescent wavelength is long or short; to provide a fluorescent reagent containing the fluorescent dyestuff; and to provide a method for diagnosing an affected part of a living body, a method for identifying a target cell and a method for detecting a target substance, the methods each using the fluorescent reagent.SOLUTION: Since each of benzoxanthones each having such a structure that one benzene ring is added to only one side of a xanthone skeleton and dibenzoxanthones each having such another structure that one benzene ring is added to both sides of the xanthone skeleton has generally the quantum yield higher than that of a linear type compound of each of naphtho fluoresceins each having such a different structure that one benzene ring is added to both sides of a xanthene skeleton and naphtho rhodamines, the state of a viable tissue can be observed excellently in diagnostic imaging of the affected site of the living body, and analytical precision of the target cell or the target substance can be improved in measurements with an instrument such as in flow cytometry by using the fluorescent dyestuff containing at least one of the benzoxanthones and the dibenzoxanthones.

Description

  The present invention relates to a fluorescent dye, a fluorescent reagent containing the fluorescent dye, a method for diagnosing a living body lesion using the fluorescent reagent, a method for specifying a target cell, and a method for detecting a target substance.

  Conventionally, a fluorescent reagent containing a fluorescent dye that absorbs light energy, such as fluorescein or rhodamine, is excited to return to the ground state and emits fluorescence when returning to the ground state, for example, is used as a labeling reagent. In addition, identification of target cells such as tumor cells and detection of target substances such as dioxins in instrumental measurements such as flow cytometry can be performed.

  Patent Document 1 discloses a fluorescent dye in which a benzene ring is added one by one on both sides of a xanthene skeleton of fluorescein to extend the conjugated system and shift the fluorescence wavelength to the longer wavelength side. According to this, the overlap between the fluorescence wavelength of the fluorescent dye and the fluorescence wavelength of the autofluorescence of the living body can be avoided, and the fluorescent dye can be favorably used as a living body imaging tool.

  The present applicant relates to a linear compound in which the condensation position of the benzene ring is [b, i] of xanthene among naphthofluoresceins and naphthorhodamines having a structure in which one benzene ring is added to both sides of the xanthene skeleton. A patent application has already been filed for the invention (Japanese Patent Application No. 2011-90109).

Japanese Patent No. 3955638 (Claim 7, page 12)

  By the way, when using a fluorescent reagent as a labeling reagent, it is an important factor that the quantum yield of the fluorescent dye is high and the fluorescence is bright regardless of whether the fluorescent wavelength is long or short. In diagnostic imaging, the state of the target biological tissue can be observed well, and in instrumental measurements such as flow cytometry, the analysis accuracy of target cells and target substances can be improved.

  In addition, it is preferably applicable to a multi-color imaging technique in which fluorescent dyes having a plurality of fluorescent wavelengths are excited by light having a single wavelength, whereby a plurality of fluorescent reagents have different colors only by being excited at a single wavelength. In this case, multiple types of targets can be diagnosed, specified or detected at the same time.

  It is also preferable to have a so-called fluorescence-off to fluorescence-on switching function that does not normally emit fluorescence, but emits fluorescence by reacting with a specific biological tissue, target cell, or target substance. Thus, by observing that a region that has not emitted fluorescence until now emits fluorescence, it is possible to accurately confirm that a specific biological tissue, target cell, or target substance is present.

  Therefore, the present invention mainly aims to provide a fluorescent dye having an excellent quantum yield regardless of whether the fluorescent wavelength is a long wavelength or a short wavelength, and to provide a fluorescent dye applicable to the technique of multicolor imaging. Another object of the present invention is to provide a fluorescent dye having a function of switching from fluorescence off to fluorescence on. Furthermore, still another object of the present invention is to provide a fluorescent reagent containing the fluorescent dye, a method for diagnosing a living body affected area using the fluorescent reagent, a method for identifying a target cell, and a method for detecting a target substance.

  The present invention relates to a compound represented by the following general formula (I) or a tautomer in which the lactone ring is opened, a compound represented by the following general formula (II), and a compound represented by the following general formula (III) And a fluorescent dye containing at least one compound selected from the group consisting of a compound represented by the following general formula (IV) and a compound represented by the following general formula (V).

In the formula, three benzene rings condensed at different positions indicated by broken lines on each of the two benzene rings of the xanthene-like skeleton are at least one of the two benzene rings of the xanthene-like skeleton. 1 shows that any one of the three benzene rings indicated by a broken line is condensed; X is an oxygen atom; Y ¹ , Y ² (Y ² is each benzene of xanthene-like skeleton) Each of the three benzene rings indicated by broken lines in the ring is a substituent when each ring is not condensed.) Is independently OH, OR ¹ or NR ² R ³ ; Z ¹ is independently A hydrogen atom, a halogen atom (F, Cl, Br or I), a part or all of carbon atoms of 1 to 5 is substituted or unsubstituted alkyl group with NR ² R ³ , Part of 5 Ku is an entirely substituted or unsubstituted alkyl group OR ^4, the following structural formula (a) or the following structural formula (b); Z ² each independently represent a hydrogen atom, a halogen atom (F, Cl, Br or I), the following structural formula (a), or the following structural formula (b); Z ³ and Z ⁴ are each independently a hydrogen atom, NR ² R ³ , OR ⁴ , or a carbon number of 1; Or a part or all of ˜5 are substituted or unsubstituted alkyl groups, acyl groups, the following structural formula (a) or the following structural formula (b) with a halogen atom (F, Cl, Br or I). In the above, each R ¹ is independently an alkyl group, a formyl group, an acyl group, a sugar residue, or an aromatic compound in which some or all of the carbon atoms are substituted or unsubstituted. Yes; R ² and R ³ are each independently a hydrogen atom or a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms; R ⁴ is independently A hydrogen atom or a part or all of 1 to 5 carbon atoms is a substituted or unsubstituted alkyl group.

In the formula, three benzene rings condensed at different positions indicated by broken lines in each of the two benzene rings of the xanthone-like skeleton are at least one of the two benzene rings of the xanthone-like skeleton. 1 shows that any one of the three benzene rings indicated by a broken line is condensed; X is an oxygen atom; Y ¹ , Y ² (Y ² is each benzene in the xanthone-like skeleton) Each of the three benzene rings indicated by broken lines in the ring is a substituent when each ring is not condensed.) Is independently OH, OR ¹ or NR ² R ³ ; Z ¹ is independently A hydrogen atom, a halogen atom (F, Cl, Br or I), a part or all of carbon atoms of 1 to 5 is substituted or unsubstituted alkyl group with NR ² R ³ , Part of 5 Ku is an entirely substituted or unsubstituted alkyl group OR ^4, the following structural formula (a) or the following structural formula (b); Z ² each independently represent a hydrogen atom, a halogen atom (F, Cl, Br or I), the following structural formula (a), or the following structural formula (b). In the above, each R ¹ is independently an alkyl group, a formyl group, an acyl group, a sugar residue, or an aromatic compound in which some or all of the carbon atoms are substituted or unsubstituted. Yes; R ² and R ³ are each independently a hydrogen atom or a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms; R ⁴ is independently A hydrogen atom or a part or all of 1 to 5 carbon atoms is a substituted or unsubstituted alkyl group.

In the formula, any of the three benzene rings condensed at different positions indicated by broken lines on one benzene ring of the xanthene-like skeleton is not condensed or any of the three benzene rings. One of the two 6-membered rings condensed at different positions indicated by broken lines on the other conjugated 6-membered ring of the xanthene-like skeleton, one of which is a condensed ring. A is an oxygen atom or N ⁺ R ² R ³ ; X is an oxygen atom; Y ¹ , Y ² (Y ² is one benzene ring of a xanthene-like skeleton) And each of the three benzene rings indicated by the broken lines in FIG. 4 is a substituent when each is not condensed.) Is independently OH, OR ¹ or NR ² R ³ ; Z ¹ is independently Hydrogen atoms, halogen atoms (F, Cl, Br or I), part or all of NR ² R ³ substituted or unsubstituted alkyl group having 1 to 5 carbon atoms, or unsubstituted some or all of 1 to 5 carbon atoms is substituted by OR ⁴ A substituted alkyl group, the following structural formula (a) or the following structural formula (b); each Z ² independently represents a hydrogen atom, a halogen atom (F, Cl, Br or I), or a structural formula (a Or R ⁵ is a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms or a partially or completely substituted or unsubstituted aryl. It is a group. In the above, each R ¹ is independently an alkyl group, a formyl group, an acyl group, a sugar residue, or an aromatic compound in which some or all of the carbon atoms are substituted or unsubstituted. Yes; R ² and R ³ are each independently a hydrogen atom or a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms; R ⁴ is independently A hydrogen atom or a part or all of 1 to 5 carbon atoms is a substituted or unsubstituted alkyl group.

In the formula, any of the three benzene rings condensed at different positions indicated by broken lines on one benzene ring of the xanthene-like skeleton is not condensed or any of the three benzene rings. One is fused; A is an oxygen atom or N ⁺ R ² R ³ ; X is an oxygen atom; Y ¹ , Y ² (Y ² is a xanthene-like skeleton) . one of the three benzene rings shown in the benzene ring by a broken line is a substituent when not fused all) are each independently, OH, be oR ¹ or NR ² R ^3; Z ¹ is Independently, a hydrogen atom, a halogen atom (F, Cl, Br or I), a part or all of carbon atoms of 1 to 5 is substituted or unsubstituted alkyl group with NR ² R ³ , carbon number Is a part or all of 1 to 5 is OR ⁴ A substituted or unsubstituted alkyl group, the following structural formula (a) or the following structural formula (b); each Z ² independently represents a hydrogen atom, a halogen atom (F, Cl, Br or I), It is the following structural formula (a) or the following structural formula (b); R ⁵ is a C1-C5 partially or fully substituted or unsubstituted alkyl group or part or all substituted Or it is an unsubstituted aryl group. In the above, each R ¹ is independently an alkyl group, a formyl group, an acyl group, a sugar residue, or an aromatic compound in which some or all of the carbon atoms are substituted or unsubstituted. Yes; R ² and R ³ are each independently a hydrogen atom or a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms; R ⁴ is independently A hydrogen atom or a part or all of 1 to 5 carbon atoms is a substituted or unsubstituted alkyl group.

In the formula, three benzene rings condensed at different positions indicated by broken lines on each of the two benzene rings of the xanthene-like skeleton are at least one of the two benzene rings of the xanthene-like skeleton. 1 shows that any one of the three benzene rings indicated by a broken line is condensed; X is an oxygen atom; Y ¹ , Y ² (Y ² is each benzene of xanthene-like skeleton) Each of the three benzene rings indicated by broken lines in the ring is a substituent when each ring is not condensed.) Is independently OH, OR ¹ or NR ² R ³ ; Z ¹ is independently A hydrogen atom, a halogen atom (F, Cl, Br or I), a part or all of carbon atoms of 1 to 5 is substituted or unsubstituted alkyl group with NR ² R ³ , Part of 5 Or all substituted with OR ⁴ or an unsubstituted alkyl group; Z ² each independently represents a hydrogen atom or a halogen atom (F, Cl, Br or I); R ⁶ , R ⁷ Are each independently a hydrogen atom, a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms, a partially or completely substituted or unsubstituted aryl group, a formyl group, The following structural formula (a), the following structural formula (b), or R ⁶ and R ⁷ together is the following structural formula (c) or the following structural formula (d). In the above, each R ¹ is independently an alkyl group, a formyl group, an acyl group, a sugar residue, or an aromatic compound in which some or all of the carbon atoms are substituted or unsubstituted. Yes; R ² and R ³ are each independently a hydrogen atom or a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms; R ⁴ is independently A hydrogen atom or a part or all of 1 to 5 carbon atoms is a substituted or unsubstituted alkyl group.

  The fluorescent dye preferably has a switch function of emitting fluorescence by reacting with a specific living tissue, target cell, or target substance.

  Moreover, this invention is a fluorescent reagent containing the said fluorescent pigment | dye.

  In addition, the present invention is an image diagnostic method for an affected area of a living body using the fluorescent reagent.

  In addition, the present invention is a method for identifying a target cell or detecting a target substance using the fluorescent reagent for instrumental measurement such as flow cytometry.

  In the image diagnosis, identification or detection method, it is preferable to use a combination of a plurality of types of fluorescent dyes whose excitation wavelengths are close to each other and whose fluorescence wavelengths are separated from each other.

  According to the present invention, regardless of whether the fluorescence wavelength is a long wavelength or a short wavelength, a fluorescent dye having an excellent quantum yield, a fluorescent reagent containing the fluorescent dye, a method for diagnosing a diseased part in a living body using the fluorescent reagent, and identification of a target cell A method and a method for detecting a target substance are provided. Therefore, it is possible to satisfactorily observe the state of the living tissue in the image diagnosis of the affected part of the living body, and to improve the analysis accuracy of the target cell and the target substance in the instrument measurement such as flow cytometry.

  In addition, according to the present invention, a fluorescent dye applicable to the technique of multi-color imaging, a fluorescent reagent containing the fluorescent dye, a living body disease diagnosis method using the fluorescent reagent, a target cell identification method, and a target substance detection method Is provided. Therefore, multiple types of targets can be diagnosed, specified, or detected at the same time by simply exciting with one wavelength.

  Further, according to the present invention, a fluorescent dye having a function of switching from fluorescence off to fluorescence on, a fluorescent reagent containing the fluorescent dye, a method for diagnosing a living diseased part using the fluorescent reagent, a method for identifying a target cell, and a target substance A detection method is provided. Therefore, by observing that a region that did not emit fluorescence until then emits fluorescence, it can be accurately confirmed that a specific biological tissue, target cell, or target substance is present.

  The present inventors have reported that benzoxanthones having a structure in which one benzene ring is added to only one side of the xanthone skeleton, and dibenzoxanthones having a structure in which one benzene ring is added to both sides of the xanthone skeleton have xanthene skeletons. It has been found that quantum yields are generally higher than those of linear compounds among naphthofluoresceins and naphthorhodamines having a structure in which one benzene ring is added on each side.

  In addition, the present inventors also added naphthofluoresceins and naphthorhodamines having a structure in which one benzene ring is added to only one side of the xanthene skeleton, and naphthofluorescein having a structure in which one benzene ring is added to both sides of the xanthene skeleton. Quantities of non-linear compounds among naphtholones and naphthorhodamines are higher than those of linear compounds among naphthofluoresceins and naphthorhodamines having a structure in which one benzene ring is added to each side of the xanthene skeleton. I found.

  The present invention has been completed on the basis of the above knowledge, the compound represented by the general formula (I) or the tautomer having the lactone ring opened, and the compound represented by the general formula (II) And at least one compound selected from the group consisting of the compound represented by the general formula (III), the compound represented by the general formula (IV), and the compound represented by the general formula (V) The purpose of the present invention is a dye, a fluorescent reagent containing the fluorescent dye, a method for diagnosing a living body using the fluorescent reagent, a method for identifying a target cell, and a method for detecting a target substance.

The compound represented by the general formula (I) has a xanthene-like skeleton, and those in which Y ¹ and Y ² are OH are naphthofluoresceins, and those in which Y ¹ and Y ² are NR ² R ³ are Naphthrodamines.

When Z ^{1 to} Z ⁴ are the structural formula (a) or the structural formula (b), introduction of the compound represented by the general formula (I) into the protein is promoted.

Among tautomers in which the lactone ring of the compound represented by the general formula (I) is opened, in the case of naphthofluoresceins, “—OH” of Y ¹ becomes “═O”, and the conjugated system extends. The fluorescence wavelength further shifts to the longer wavelength side than the compound represented by the general formula (I).

Among the tautomers in which the lactone ring of the compound represented by the general formula (I) is opened, in the case of naphthorhodamines, “—NR ² R ³ ” of Y ¹ is “= N ⁺ R ² R ³ ”. Thus, by extending the conjugated system, the fluorescence wavelength is further shifted to the longer wavelength side than the compound represented by the general formula (I).

  In the compound represented by the general formula (II), the lactone ring portion of the compound represented by the general formula (I) is substituted with “═O”.

The compound represented by the general formula (II) has a xanthone-like skeleton, and those in which Y ¹ and Y ² are OH are fluorescein-type naphthoxanthones, and Y ¹ and Y ² are NR ² R ³ . Those are rhodamine type naphthoxanthones.

  Of the compounds represented by the general formula (II), those having a structure in which one benzene ring is added to only one side of the xanthone-like skeleton are benzoxanthones, and one benzene ring is added to each side of the xanthone-like skeleton. Those of structure are dibenzoxanthones.

When Z ^{1 to} Z ² are structural formula (a) or structural formula (b), introduction of the compound represented by general formula (II) into a protein is promoted.

In the compound represented by the general formula (III) or the general formula (IV), “═O” of the compound represented by the general formula (II) is substituted with “—R ⁵ ”, and the conjugated system is represented by the general formula (I) and It extends from the compound represented by the general formula (II). Therefore, the fluorescence wavelength is further shifted to the longer wavelength side than the compounds represented by the general formulas (I) and (II).

Of the compounds represented by general formula (III) or general formula (IV), those in which A is an oxygen atom are fluorescein type naphthoxanthones derivatives, and those in which A is N ⁺ R ² R ³ are rhodamine types. These are naphthoxanthones derivatives.

When Z ^{1 to} Z ² are structural formulas (a) or (b), introduction of a compound represented by general formula (III) or general formula (IV) into a protein is promoted.

In the compound represented by the general formula (V), “—O” of the compound represented by the general formula (II) is replaced with “—R ⁶ , —R ⁷ ”.

Among the compounds represented by the general formula (V), those in which Y ¹ and Y ² are OH are fluorescein type naphthoxanthones derivatives, and those in which Y ¹ and Y ² are NR ² R ³ are rhodamine type. It is a naphthoxanthone derivative.

When R ⁶ and R ⁷ are structural formula (a) or structural formula (b), introduction of a compound represented by general formula (V) into a protein is promoted.

When R ⁶ and R ⁷ are, for example, a combination of a phenyl group and a formyl group, a structural formula (c), or a structural formula (d), a specific living tissue, target cell, or target substance by reaction with, easily one of ^{R 6} and ^{R 7} undergoes the elimination reaction, "-OH" and "= O" and of ^{Y 1,} or ^{Y 1} of ^"-NR 2 ^{R 3"} "= N ⁺ R ² R ³ ”and a derivative in which the conjugated system is longer than the compound represented by the general formula (V) is obtained. In this derivative, the fluorescence wavelength is further shifted to the longer wavelength side than the compound represented by the general formula (V). Alternatively, when the compound represented by the general formula (V) usually does not emit fluorescence, this derivative becomes fluorescent. That is, R ⁶ and R ⁷ function as switches that turn fluorescence on from off.

  The fluorescent dye according to the present embodiment has an excellent quantum yield regardless of whether the fluorescence wavelength is a long wavelength or a short wavelength. Therefore, by using the fluorescent dye according to the present embodiment, it is possible to satisfactorily observe the state of the living tissue in the image diagnosis of the affected part of the living body, and to improve the analysis accuracy of the target cell and the target substance in the instrument measurement such as flow cytometry. It is done.

  For example, a medicine (fluorescent reagent) containing the fluorescent dye according to the present embodiment is administered to a subject, and excitation light having a specific wavelength band is irradiated into the living body, and at that time, the fluorescent dye is contained in the living body. By taking an image of the fluorescence emitted by the drug and observing the fluorescence image, state observation (image diagnosis of the affected part of the living body) for distinguishing between a normal site and a lesion site in the living body is realized.

  Further, by using the labeling reagent (fluorescent reagent) containing the fluorescent dye according to the present embodiment for instrumental measurement such as flow cytometry, it is possible to accurately identify target cells such as tumor cells and detect target substances such as dioxins. It can be carried out.

  Among the fluorescent dyes according to the present embodiment, a plurality of types of fluorescent dyes whose excitation wavelengths are close to each other and whose fluorescent wavelengths are separated from each other are used in combination, so that a plurality of fluorescent lights can be obtained with light of one wavelength. The present invention can also be applied to multi-color imaging technology in which a fluorescent dye having a wavelength is excited. Since the plurality of fluorescent reagents emit light with different colors only by exciting at one wavelength, multiple types of targets can be diagnosed, specified or detected at the same time. There is also an advantage that the light source for excitation can be small.

  Among the fluorescent dyes according to the present embodiment, a function of switching from so-called fluorescence off to fluorescence on, which does not normally emit fluorescence, but emits fluorescence by reacting with a specific biological tissue, target cell or target substance. By observing that the region that did not fluoresce before emits fluorescence, it is possible to accurately confirm that a specific biological tissue, target cell, or target substance exists. Can be confirmed.

  Hereinafter, the present invention will be described in more detail through examples, but the present invention is not limited to these examples.

<Synthesis of benzoxanthones or dibenzoxanthones>
First, synthetic schemes (Scheme 1., Scheme 2-1., Scheme 2-2., Scheme 3.) are shown below. The starting materials are the compounds 1, 3, 5, and 7 shown in Scheme 1. The target substances are the compounds shown in Scheme 3. That is, (di) benzoxanthones 27, 29, 31, 33, 35, 37, 39, 41, 43. “MOM” represents a methoxymethyl group (CH ₃ OCH ₂ —).

[Scheme 1: Synthesis of aldehydes 2, 4, 6, 8]
(Synthesis of aldehyde 2)
Under a nitrogen atmosphere, Compound 1 (19.0 g, 68.6 mmol) was dissolved in anhydrous tetrahydrofuran (THF) (600 mL), cooled to −78 ° C., and n-butyllithium (n-BuLi) (1.6 M n-hexane solution). , 50.8 mL, 82.3 mmol) was added dropwise. After stirring at −78 ° C. for 45 minutes, anhydrous N, N-dimethylformamide (DMF) (8.0 mL, 102.8 mmol) was added dropwise. Stirring was continued for 18 hours, and the temperature was naturally raised and returned to room temperature. The reaction mixture was poured into 0.1 M aqueous hydrochloric acid and ethyl acetate, washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. After the desiccant was filtered off, the solvent was distilled off under reduced pressure. The residue was purified by column chromatography (hexane-ethyl acetate) to obtain aldehyde 2 (12.5 g) in a yield of 81%.

(Synthesis of aldehyde 4)
Compound 1 was changed to compound 3, and aldehyde 4 was synthesized in the same manner as synthesis of aldehyde 2. The yield was 64%.

(Synthesis of aldehyde 6)
Compound 1 was changed to compound 5, and aldehyde 6 was synthesized in the same manner as synthesis of aldehyde 2. The yield was 93%.

(Synthesis of aldehyde 8)
Compound 1 was changed to compound 7, and aldehyde 8 was synthesized in the same manner as synthesis of aldehyde 2. The yield was 81%.

[Scheme 2: Synthesis of ketone bodies 10, 12, 14, 16, 18, 20, 23, 25 via hydroxy bodies 9, 11, 13, 15, 17, 19, 22, 24]
(Synthesis of hydroxy 9)
Under a nitrogen atmosphere, compound 3 (1.85 g, 5.65 mmol) was dissolved in anhydrous THF (50 mL), cooled to −78 ° C., and n-BuLi (1.6 M n-hexane solution, 3.9 mL, 6.17 mmol) was added dropwise. did. After stirring at −78 ° C. for 1 hour, an anhydrous THF solution (8 mL) of aldehyde 2 (1.16 g, 5.14 mmol) was added dropwise. Stirring was continued for 5 hours, and the temperature was naturally raised and returned to room temperature. The reaction mixture was poured into 0.1 M aqueous hydrochloric acid and ethyl acetate, washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. After the desiccant was filtered off, the solvent was distilled off under reduced pressure. The residue was purified by column chromatography (hexane-ethyl acetate) to obtain hydroxy 9 (2.30 g) in a yield of 97%.

(Synthesis of Hydroxy Compound 11)
The compound 3 was changed to the compound 5, and the hydroxy compound 11 was synthesized in the same manner as the synthesis of the hydroxy compound 9. The yield was 81%.

(Synthesis of Hydroxy Compound 13)
The compound 3 was changed to the compound 7, and the hydroxy form 13 was synthesized in the same manner as the synthesis of the hydroxy form 9. The yield was 86%.

(Synthesis of hydroxy body 15)
The aldehyde body 2 was changed to the aldehyde body 4, and the hydroxy body 15 was synthesized in the same manner as the synthesis of the hydroxy body 9. The yield was 84%.

(Synthesis of Hydroxy Compound 17)
The aldehyde body 2 was changed to the aldehyde body 6 and the hydroxy body 17 was synthesized in the same manner as the synthesis of the hydroxy body 9. The yield was 95%.

(Synthesis of Hydroxy Compound 19)
The aldehyde form 2 was changed to the aldehyde form 8, and the hydroxy form 19 was synthesized in the same manner as the synthesis of the hydroxy form 9. The yield was 93%.

(Synthesis of Hydroxy Compound 22)
The compound 3 was changed to the compound 7, and the aldehyde compound 2 was changed to the aldehyde compound 6, and the hydroxy compound 22 was synthesized in the same manner as the synthesis of the hydroxy compound 9. The yield was 96%.

(Synthesis of Hydroxy Compound 24)
The compound 3 was changed to the compound 7, and the aldehyde form 2 was changed to the aldehyde form 8, and the hydroxy form 24 was synthesized in the same manner as the synthesis of the hydroxy form 9. The yield was 86%.

(Synthesis of ketone body 10)
Hydroxy compound 9 (1.85 g, 3.90 mmol) was dissolved in methylene chloride (80 mL), manganese dioxide (14.8 g, 17.0 mmol) was added, and the mixture was stirred at room temperature for 13 hours. The reaction solution was filtered through celite, and the filtrate was concentrated. The residue was purified by column chromatography (hexane-ethyl acetate) to obtain ketone body 10 (1.34 g) in a yield of 73%.

(Synthesis of ketone body 12)
The hydroxy body 9 was changed to the hydroxy body 11, and the ketone body 12 was synthesized in the same manner as the synthesis of the ketone body 10. The yield was 94%.

(Synthesis of ketone body 14)
The hydroxy body 9 was changed to the hydroxy body 13, and the ketone body 14 was synthesized in the same manner as the synthesis of the ketone body 10. The yield was 87%.

(Synthesis of ketone body 16)
The hydroxy body 9 was changed to the hydroxy body 15, and the ketone body 16 was synthesized in the same manner as the synthesis of the ketone body 10. The yield was 78%.

(Synthesis of ketone body 18)
The hydroxy body 9 was changed to the hydroxy body 17, and the ketone body 18 was synthesized in the same manner as the synthesis of the ketone body 10. The yield was 96%.

(Synthesis of ketone body 20)
The hydroxy body 9 was changed to the hydroxy body 19, and a ketone body 20 was synthesized in the same manner as the synthesis of the ketone body 10. The yield was 72%.

(Synthesis of ketone body 23)
The hydroxy body 9 was changed to the hydroxy body 22, and the ketone body 23 was synthesized in the same manner as the synthesis of the ketone body 10. The yield was 88%.

(Synthesis of ketone body 25)
The hydroxy body 9 was changed to the hydroxy body 24, and the ketone body 25 was synthesized in the same manner as the synthesis of the ketone body 10. The yield was 90%.

[Scheme 2: Synthesis of ketone body 21]
(Synthesis of ketone body 21)
Under a nitrogen atmosphere, compound 5 (400 mg, 1.23 mmol) is dissolved in anhydrous THF (3 mL), cooled to −78 ° C., and n-BuLi (1.6 M n-hexane solution, 0.799 mL, 1.28 mmol) is added dropwise. did. After stirring at −78 ° C. for 1 hour, the reaction solution was added dropwise to an anhydrous THF solution (1.5 mL) of dimethyl carbonate (47.9 mg, 0.53 mmol). Stirring was continued for 4 hours, and the temperature was naturally raised and returned to room temperature. The reaction mixture was poured into water and ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. After the desiccant was filtered off, the solvent was distilled off under reduced pressure. The residue was purified by column chromatography (hexane-ethyl acetate) to obtain ketone body 21 (162.2 mg) in a yield of 58%.

[Scheme 3: Synthesis of Deprotected Compounds 26, 28, 30, 32, 34, 36, 38, 40, 42 by Deprotection of Methoxymethyl Group and (Di) Benzoxanthones 27, 29, 31 by Cyclization Reaction , 33, 35, 37, 39, 41, 43]
(Synthesis of deprotected body 26)
The ketone body 10 (900.0 mg, 1.90 mmol) was dissolved in 1,4-dioxane (10 mL), 4 M hydrochloric acid-1,4-dioxane (14.3 mL, 57.1 mmol) was added at room temperature, and the mixture was stirred for 11 hours. . The solvent was distilled off under reduced pressure to obtain the deprotected product 26 (558.0 mg) in a yield of 99%.

(Synthesis of deprotected body 28)
The ketone body 10 was changed to the ketone body 12, and the deprotection body 28 was synthesized in the same manner as the synthesis of the deprotection body 26. The yield was 86%.

(Synthesis of deprotected body 30)
The ketone body 10 was changed to the ketone body 14, and the deprotection body 30 was synthesized in the same manner as the synthesis of the deprotection body 26. The yield was 94%.

(Synthesis of deprotected body 32)
The ketone body 10 was changed to the ketone body 16, and the deprotection body 32 was synthesized in the same manner as the synthesis of the deprotection body 26. The yield was 96%.

(Synthesis of deprotected body 34)
The ketone body 10 was changed to the ketone body 18, and the deprotection body 34 was synthesized in the same manner as the synthesis of the deprotection body 26. The yield was 98%.

(Synthesis of deprotected body 36)
The ketone body 10 was changed to the ketone body 20, and the deprotection body 36 was synthesized in the same manner as the synthesis of the deprotection body 26. The yield was 96%.

(Synthesis of deprotected body 38)
The ketone body 10 was changed to the ketone body 21, and the deprotection body 38 was synthesized in the same manner as the synthesis of the deprotection body 26. The yield was 97%.

(Synthesis of deprotected body 40)
The ketone body 10 was changed to the ketone body 23, and the deprotection body 40 was synthesized in the same manner as the synthesis of the deprotection body 26. The yield was 100%.

(Synthesis of deprotected body 42)
The ketone body 10 was changed to the ketone body 25, and the deprotected body 42 was quantitatively synthesized in the same manner as the synthesis of the deprotected body 26.

(Synthesis of benzoxanthones 27)
Deprotection body 26 (11.0 mg, 0.037 mmol), potassium carbonate (5.1 mg, 0.037 mmol), and distilled water (6 mL) were added to the pressure vessel, and the mixture was heated to 150 ° C. After 2.5 hours, the temperature was returned to room temperature, 1 M aqueous hydrochloric acid (0.3 mL) was added, and the mixture was extracted with ethyl acetate-methanol. Furthermore, it wash | cleaned one by one by water and a saturated salt solution, and dried with anhydrous sodium sulfate. After the desiccant was filtered off, the solvent was distilled off under reduced pressure to obtain benzoxanthones 27 quantitatively.

  The excitation wavelength and the fluorescence wavelength were λex: 358 nm (MeOH) and λem: 480 nm (MeOH). The quantum yield was φ: 6%.

Benzoxanthones 27: ¹ H-NMR (270 MHz, DMSO-d ₆ ) δ 10.96 (s, 1H), 10.45 (s, 1H), 8.50 (d, J = 8.9 Hz, 1H), 8.05 (d, J = 8.9 Hz, 1H), 7.97 (d, J = 8.9 Hz, 1H), 7.63 (d, J = 8.9 Hz, 1H), 7.29 (dd, J = 8.9 Hz, 2.4 Hz, 1H), 7.28 (d, J = 2.4 Hz, 1H), 7.09 (d, J = 2.4 Hz, 1H), 6.94 (dd, J = 8.9 Hz, 2.4 Hz, 1H).

(Synthesis of benzoxanthone 31)
The deprotection body 26 was changed to the deprotection body 30, and the benzoxanthones 31 were synthesized in the same manner as the synthesis of the benzoxanthones 27. The yield was 78%.

  The excitation wavelength and the fluorescence wavelength were λex: 360 nm (MeOH) and λem: 413 nm (MeOH). The quantum yield was φ: 17%.

Benzoxanthones 31: ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 10.94 (s, 1H), 10.44 (s, 1H), 8.52 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 8.8 Hz, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.30 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.29 (d, J = 2.4 Hz, 1H), 7.09 (d, J = 2.4 Hz, 1H), 6.95 (dd, J = 8.8 Hz, 2.4 Hz, 1H); ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ 174.6 , 163.6, 159.1, 157.3, 153.5, 138.4, 127.8, 124.8, 122.8, 121.5, 119.4, 117.1, 114.9, 114.7, 114.6, 110.2, 102.7.

(Synthesis of dibenzoxanthones 43)
The deprotected body 26 was changed to the deprotected body 42, and the dibenzoxanthone 43 was quantitatively synthesized in the same manner as the synthesis of the benzoxanthone 27.

  The excitation wavelength and the fluorescence wavelength were λex: 362 nm (MeOH) and λem: 421 nm (MeOH). The quantum yield was φ: 14%.

Dibenzoxanthones 43: IR (KBr) 3114, 1618, 1473, 1248, 858, 777, 733 cm ^-1 ; ¹ H-NMR (270 MHz, DMSO-d ₆ ) δ 10.60 (s, 2H), 8.84 (d , J = 8.6 Hz, 2H), 8.06 (d, J = 8.6 Hz, 2H), 7.74 (d, J = 8.6 Hz, 2H), 7.41 (dd, J = 8.6 Hz, 2.2 Hz, 2H), 7.37 ( d, J = 2.2 Hz, 2H); ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ 175.0, 159.3, 153.0, 138.4, 124.9, 123.5, 121.3, 119.7, 117.3, 115.6, 110.3.

(Synthesis of benzoxanthones 29)
Deprotector 28 (522.3 mg, 1.76 mmol), potassium carbonate (24.4 mg, 0.17 mmol) and distilled water (10 mL) were added to the pressure vessel, and the mixture was heated to 150 ° C. After 27 hours, the temperature was returned to room temperature, 1 M aqueous hydrochloric acid (1 mL) was added, and the solid was filtered to obtain benzoxanthones 29 (246.6 mg) in a yield of 50%.

  The excitation wavelength and the fluorescence wavelength were λex: 355 nm (MeOH) and λem: 493 nm (MeOH). The quantum yield was φ: 63%.

Benzoxanthones 29: ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 10.96 (s, 1H), 10.35 (s, 1H), 8.69 (s, 1H), 8.08 (d, J = 8.8 Hz, 1H ), 8.04 (d, J = 8.8 Hz, 1H), 7.80 (s, 1H), 7.20 (d, J = 2.4 Hz, 1H), 7.12 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.86 ( dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.83 (d, J = 2.4 Hz, 1H); ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ 175.5, 164.4, 158.4, 158.2, 152.6, 138.6 , 131.9, 128.5, 127.6, 124.6, 119.6, 118.5, 113.7, 113.6, 111.0, 107.4, 102.3.

(Synthesis of dibenzoxanthone 33)
The deprotected body 28 was changed to the deprotected body 32, and the dibenzoxanthone 33 was synthesized in the same manner as the synthesis of the benzoxanthone 29. The yield was 100%.

  The excitation wavelength and the fluorescence wavelength were λex: 380 nm (MeOH) and λem: 480 nm (MeOH). The quantum yield was φ: 0.01%.

Dibenzoxanthones 33: ¹ H-NMR (270 MHz, DMSO-d ₆ ) δ 10.21 (s, 2H), 9.49 (s, 2H), 8.23 (d, J = 8.6 Hz, 2H), 7.95 (d, J = 8.6 Hz, 2H), 7.51 (d, J = 8.6 Hz, 2H), 7.20 (d, J = 8.6 Hz, 2H); ¹³ C-NMR (68 MHz, DMSO-d ₆ ) δ 179.3, 158.7, 155.8 , 136.0, 132.1, 130.4, 124.4, 117.4, 114.2, 113.6, 109.3.

(Synthesis of dibenzoxanthones 35)
The deprotected body 28 was changed to the deprotected body 34, and the dibenzoxanthones 35 were synthesized in the same manner as the synthesis of the benzoxanthones 29. The yield was 86%.

  The excitation wavelength and the fluorescence wavelength were λex: 374 nm (MeOH) and λem: 532 nm (MeOH). The quantum yield was φ: 2%.

Dibenzoxanthones 35: ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 10.38 (s, 1H), 10.27 (s, 1H), 9.40 (d, J = 2.4 Hz, 1H), 8.79 (s, 1H ), 8.23 (d, J = 8.8 Hz, 1H), 8.12 (d, J = 8.8 Hz, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.90 (s, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 7.16 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.15 (dd, J = 8.8 Hz, 2.4 Hz, 1H); ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ 178.2, 159.4, 158.5, 158.3, 151.4, 138.3, 137.3, 132.9, 131.9, 130.7, 127.5, 125.0, 124.3, 119.9, 119.8, 117.4, 114.5, 111.9, 110.7 , 109.5, 107.4.

(Synthesis of dibenzoxanthones 37)
The deprotected body 28 was changed to the deprotected body 36, and the dibenzoxanthones 37 were synthesized in the same manner as the synthesis of the benzoxanthones 29. The yield was 92%.

  The excitation wavelength and the fluorescence wavelength were λex: 370 nm (MeOH) and λem: 476 nm (MeOH). The quantum yield was φ: 3%.

Dibenzoxanthones 37: ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 10.47 (s, 1H), 10.24 (s, 1H), 9.48 (d, J = 2.4 Hz, 1H), 8.58 (d, J = 8.8 Hz, 1H), 8.27 (d, J = 8.8 Hz, 1H), 8.11 (d, J = 8.8 Hz, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.33 (d, J = 8.8 Hz, 2.4 Hz, 1H), 7.32 (d, J = 2.4 Hz, 1H), 7.19 (dd, J = 8.8 Hz, 2.4 Hz, 1H); ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ 177.2, 159.1, 157.4, 151.9, 138.2, 136.4, 132.6, 130.6, 124.7, 124.6, 123.3, 121.6, 119.4, 117.8 , 116.8, 116.8, 114.5, 113.4, 110.3, 109.5 (one peak overlapped).

(Synthesis of dibenzoxanthone 39)
The deprotected body 28 was changed to the deprotected body 38, and the dibenzoxanthone 39 was synthesized in the same manner as the synthesis of the benzoxanthone 29. The yield was 31%.

  The excitation wavelength and the fluorescence wavelength were λex: 374 nm (MeOH) and λem: 529 nm (MeOH). The quantum yield was φ: 10%.

Dibenzoxanthones 39: ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 10.42 (s, 2H), 8.77 (s, 2H), 8.10 (d, J = 8.8 Hz, 2H), 7.80 (s, 2H ), 7.22 (d, J = 2.4 Hz, 2H), 7.13 (dd, J = 8.8 Hz, 2.4 Hz, 2H); ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ 176.8, 158.5, 152.5, 138.9 , 131.8, 127.9, 124.1, 119.1, 117.8, 110.3, 107.2.

(Synthesis of dibenzoxanthone 41)
The deprotected body 28 was changed to the deprotected body 40, and the dibenzoxanthone 41 was synthesized in the same manner as the synthesis of the benzoxanthone 29. The yield was 93%.

  The excitation wavelength and the fluorescence wavelength were λex: 360 nm (MeOH) and λem: 494 nm (MeOH). The quantum yield was φ: 45%.

Dibenzoxanthones 41: ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 10.52 (s, 1H), 10.42 (s, 1H), 8.77 (s, 1H), 8.56 (d, J = 8.8 Hz, 1H ), 8.13 (d, J = 8.8 Hz, 1H), 8.07 (s, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.33 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.30 (d, J = 2.4 Hz, 1H), 7.27 (d, J = 2.4 Hz, 1H), 7.17 (dd, J = 8.8 Hz, 2.4 Hz, 1H); ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ 176.1, 159.4, 158.5, 154.0, 152.2, 138.9, 138.5, 132.0, 127.3, 125.0, 122.4, 121.7, 119.9, 119.4, 118.8, 117.1, 114.1, 111.5, 110.4 , 107.5 (one peak overlapped).

  Table 1 summarizes the (di) benzoxanthones 27, 29, 31, 33, 35, 37, 39, 41, and 43 synthesized in this example.

<Comparison of quantum yield with linear naphthofluorescein>
Of the naphthofluoresceins having a structure in which one benzene ring is added to each side of the xanthene skeleton, the quantum yield of the linear compound (the following structural formula (e)) was φ: 0.17%.

  Therefore, the (di) benzoxanthones 27, 29, 31, 33, 35, 37, 39, 41, and 43, which are compounds according to the present invention, have a long or short fluorescence wavelength except for the dibenzoxanthones 33. Regardless of this, the quantum yield is superior to the compound of the structural formula (e). Therefore, by using the fluorescent dye according to the present invention, the state of the living tissue can be satisfactorily observed in the image diagnosis of the affected part of the living body, and the analysis accuracy of the target cell and the target substance can be improved in the instrument measurement such as flow cytometry. .

<Application to multi-color imaging technology>
Among the (di) benzoxanthones 27, 29, 31, 33, 35, 37, 39, 41, 43 which are compounds according to the present invention, for example, benzoxanthones 27, benzoxanthones 29, benzoxanthones 31 The dibenzoxanthones 41 and the dibenzoxanthones 43 have excitation wavelengths close to each other (λex: 355 to 362 nm) and fluorescence wavelengths separated from each other (λem: 413 nm, 421 nm, 480 nm, 493 nm, 494 nm). ). Therefore, by using these in combination, light of one wavelength (for example, light having λex in the vicinity of 358 to 359 nm) and a plurality of fluorescent wavelengths (λem having light in the range of 410 to 420 nm and light in the range of 480 to 490 nm) The present invention can be applied to a technique of multi-color imaging in which a seed) fluorescent dye is excited. Since a plurality of fluorescent reagents emit light in different colors only by excitation with one wavelength, multiple types of biological tissues, target cells or target substances can be diagnosed, identified or detected simultaneously. Further, there is an advantage that the number of light sources for excitation is small and small.

Claims (6)

A compound represented by the following general formula (I) or a tautomer in which the lactone ring is opened, a compound represented by the following general formula (II), a compound represented by the following general formula (III), and the following general formula A fluorescent dye comprising at least one compound selected from the group consisting of a compound represented by the formula (IV) and a compound represented by the following general formula (V).

[In the formula, three benzene rings condensed at different positions indicated by broken lines on each of the two benzene rings of the xanthene-like skeleton are at least one of the two benzene rings of the xanthene-like skeleton. It shows that any one of the three benzene rings indicated by a broken line is condensed; X is an oxygen atom; Y ¹ , Y ² (Y ² is each benzene ring of the xanthene-like skeleton) And each of the three benzene rings indicated by the broken lines in FIG. 4 is a substituent when each is not condensed.) Is independently OH, OR ¹ or NR ² R ³ ; Z ¹ is independently A hydrogen atom, a halogen atom (F, Cl, Br or I), a part or all of carbon atoms of 1 to 5 or an unsubstituted alkyl group substituted with NR ² R ³ , or a carbon number of 1 to 5 Part of or All substituted with OR ⁴ or an unsubstituted alkyl group, the following structural formula (a) or the following structural formula (b); each Z ² independently represents a hydrogen atom, a halogen atom (F, Cl, Br or I), the following structural formula (a), or the following structural formula (b); Z ³ and Z ⁴ are each independently a hydrogen atom, NR ² R ³ , OR ⁴ , or a carbon number of 1 to 5. Is an alkyl group, acyl group, the following structural formula (a), or the following structural formula (b), in which a part or all of is substituted with a halogen atom (F, Cl, Br or I). In the above, each R ¹ is independently an alkyl group, a formyl group, an acyl group, a sugar residue, or an aromatic compound in which a part or all of carbon atoms having 1 to 5 carbon atoms is substituted or unsubstituted; R ² and R ³ are each independently a hydrogen atom or a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms; R ⁴ is independently a hydrogen atom Or it is a substituted or unsubstituted alkyl group in which some or all of the carbon atoms are 1 to 5. ]

[In the formula, three benzene rings condensed at different positions indicated by broken lines in each of the two benzene rings of the xanthone-like skeleton are at least one of the two benzene rings of the xanthone-like skeleton. It shows that any one of the three benzene rings indicated by the broken line is condensed; X is an oxygen atom; Y ¹ , Y ² (Y ² is each benzene ring of the xanthone-like skeleton) And each of the three benzene rings indicated by the broken lines in FIG. 4 is a substituent when each is not condensed.) Is independently OH, OR ¹ or NR ² R ³ ; Z ¹ is independently A hydrogen atom, a halogen atom (F, Cl, Br or I), a part or all of carbon atoms of 1 to 5 or an unsubstituted alkyl group substituted with NR ² R ³ , or a carbon number of 1 to 5 Part of or All substituted with OR ⁴ or an unsubstituted alkyl group, the following structural formula (a) or the following structural formula (b); each Z ² independently represents a hydrogen atom, a halogen atom (F, Cl, Br or I), the following structural formula (a), or the following structural formula (b). In the above, each R ¹ is independently an alkyl group, a formyl group, an acyl group, a sugar residue, or an aromatic compound in which a part or all of carbon atoms having 1 to 5 carbon atoms is substituted or unsubstituted; R ² and R ³ are each independently a hydrogen atom or a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms; R ⁴ is independently a hydrogen atom Or it is a substituted or unsubstituted alkyl group in which some or all of the carbon atoms are 1 to 5. ]

[In the formula, any of the three benzene rings condensed at different positions indicated by broken lines on one benzene ring of the xanthene-like skeleton is not condensed or any one of the three benzene rings. One of the two 6-membered rings that are condensed at different positions indicated by broken lines in the other conjugated 6-membered ring of the xanthene-like skeleton. A is an oxygen atom or N ⁺ R ² R ³ ; X is an oxygen atom; Y ¹ , Y ² (Y ² is in one benzene ring of the xanthene-like skeleton) Each of the three benzene rings indicated by the broken lines is a substituent when all are not condensed.) Is independently OH, OR ¹ or NR ² R ³ ; Z ¹ is independently , Hydrogen atom, halogen atom (F, Cl, Br or I , Part or all of 1 to 5 carbon atoms NR ² R ³ substituted or unsubstituted alkyl group, the number or unsubstituted substituted some or all of the 1-5 by OR ⁴ carbon An alkyl group, the following structural formula (a) or the following structural formula (b); each Z ² independently represents a hydrogen atom, a halogen atom (F, Cl, Br or I), the following structural formula (a) or R ⁵ is a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms or a partially or completely substituted or unsubstituted aryl group having the following structural formula (b): is there. In the above, each R ¹ is independently an alkyl group, a formyl group, an acyl group, a sugar residue, or an aromatic compound in which a part or all of carbon atoms having 1 to 5 carbon atoms is substituted or unsubstituted; R ² and R ³ are each independently a hydrogen atom or a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms; R ⁴ is independently a hydrogen atom Or it is a substituted or unsubstituted alkyl group in which some or all of the carbon atoms are 1 to 5. ]

[In the formula, any of the three benzene rings condensed at different positions indicated by broken lines on one benzene ring of the xanthene-like skeleton is not condensed or any one of the three benzene rings. One is an oxygen atom or N ⁺ R ² R ³ ; X is an oxygen atom; Y ¹ , Y ² (Y ² is one of xanthene-like skeletons) . three benzene rings indicated by broken lines in the benzene ring of the substituent when not fused all) are each independently, OH, it is oR ¹ or NR ² R ^3; Z ¹ is Independently, a hydrogen atom, a halogen atom (F, Cl, Br or I), a part or all of carbon atoms having 1 to 5 carbon atoms or an unsubstituted alkyl group substituted with NR ² R ³ , 1 to 5 part or all replaced with OR ⁴ Or an unsubstituted alkyl group, the following structural formula (a) or the following structural formula (b); Z ² is independently a hydrogen atom, a halogen atom (F, Cl, Br or I), Structural formula (a) or the following structural formula (b); R ⁵ is a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms, or a partially or completely substituted carbon group, or An unsubstituted aryl group. In the above, each R ¹ is independently an alkyl group, a formyl group, an acyl group, a sugar residue, or an aromatic compound in which a part or all of carbon atoms having 1 to 5 carbon atoms is substituted or unsubstituted; R ² and R ³ are each independently a hydrogen atom or a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms; R ⁴ is independently a hydrogen atom Or it is a substituted or unsubstituted alkyl group in which some or all of the carbon atoms are 1 to 5. ]

[In the formula, three benzene rings condensed at different positions indicated by broken lines on each of the two benzene rings of the xanthene-like skeleton are at least one of the two benzene rings of the xanthene-like skeleton. It shows that any one of the three benzene rings indicated by a broken line is condensed; X is an oxygen atom; Y ¹ , Y ² (Y ² is each benzene ring of the xanthene-like skeleton) And each of the three benzene rings indicated by the broken lines in FIG. 4 is a substituent when each is not condensed.) Is independently OH, OR ¹ or NR ² R ³ ; Z ¹ is independently A hydrogen atom, a halogen atom (F, Cl, Br or I), a part or all of carbon atoms of 1 to 5 or an unsubstituted alkyl group substituted with NR ² R ³ , or a carbon number of 1 to 5 Part of or All are substituted or unsubstituted alkyl groups with OR ⁴ ; each Z ² is independently a hydrogen atom or a halogen atom (F, Cl, Br or I); R ⁶ and R ⁷ are Independently, a hydrogen atom, a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms, a partially or completely substituted or unsubstituted aryl group, a formyl group, the following structure The formula (a), the following structural formula (b), or R ⁶ and R ⁷ together is the following structural formula (c) or the following structural formula (d). In the above, each R ¹ is independently an alkyl group, a formyl group, an acyl group, a sugar residue, or an aromatic compound in which a part or all of carbon atoms having 1 to 5 carbon atoms is substituted or unsubstituted; R ² and R ³ are each independently a hydrogen atom or a partially or completely substituted or unsubstituted alkyl group having 1 to 5 carbon atoms; R ⁴ is independently a hydrogen atom Or it is a substituted or unsubstituted alkyl group in which some or all of the carbon atoms are 1 to 5. ]

  The fluorescent dye according to claim 1, wherein the fluorescent dye emits fluorescence by reacting with a specific biological tissue, target cell or target substance.   A fluorescent reagent comprising the fluorescent dye according to claim 1.   A method for diagnosing an image of an affected area using the fluorescent reagent according to claim 3.   A method for identifying a target cell or detecting a target substance, wherein the fluorescent reagent according to claim 3 is used for instrument measurement such as flow cytometry.   The diagnostic imaging, identification or detection method according to claim 4 or 5, wherein a plurality of types of fluorescent dyes having excitation wavelengths close to each other and fluorescence wavelengths spaced apart from each other are used in combination.